Preface The crop improvement in horticultural crops through conventional breeding programmes has been limited due to the unique nature of these crops. Recent developments in the area of Molecular Biology and Biotechnology hold great promise in the field of horticulture in improving the yield and quality of horticultural produce, tolerance of plants to biotic and abiotic stress, prolonging the shelf lire and so on. Developments so far achieved in this field in the country have been impressive. National Symposium on “Biotechnological Interventions for Improvement of Horticultural Crops — Issues and Strategies” was organized with this background at KAU, Main Campus, Vellanikkara during January 10-12, 2005 to discuss the current scenario and formulate future strategies for biotechnological interventions for improvement of horticultural crops. This publication consists of the full text of the lead and selected contributed papers presented in the various technical sessions of the symposium and the abstracts have been arranged under these heads. The sessions are (i) Tissue culture research in horticultural crops-new perspectives. (ii) Molecular markers and their exploitation. (iii) Molecular cloning of genes. (iv) Genetic modification for crop improvement. (v) Plant-microbe interaction. (vi) Environmental and Industrial Biotechnology and (vii) Bioinformatics and Biosafety issues of transgenic crops. Expert editorial guidance provided by Dr. G.S.L.H.V. Prasada Rao, Associate Dean is gratefully acknowledged. We also extend our grateful appreciation to Dr. Asha Shankar, Dr. Jayashree Krishnankutty, and the P.G. students of CPBMB for their whole hearted help in editing the presented papers.

ABSTRACT Genetic improvement of crop plants through conventional plant breeding has made tremendous contributions to the breakthrough in the global agricultural production. Recently, an array tools and techniques in the field of molecular biology has become available for supplementing the conventional genetic approaches. Consequently, new integrated approaches are being designed. One of the approaches employs molecular markers for genome mapping, gene tagging and marker assisted selection (MAS). With the complete sequencing of whole plant-genomes and a large number of random cDNAs in many different crop species, newer opportunities are emerging. This presentation is an attempt to briefly describe the current applications of molecular markers and to highlight their future prospects. Molecular markers Molecular markers are heritable differences in nucleotide sequences of DNA at corresponding position on homologous chromosomes of two different individuals, which follow a simple Mendelian pattern of inheritance. These differences are detected employing two basic techniques: (a) Southern blot hybridization and (b) Polymerase chain reaction (PCR). Restriction Fragment Length Polymorphism (RFLP) is the first molecular marker system to be conceived and developed. It is based on the southern blot technique. Markers such as random amplified polymorphic DNA (RAPD), sequence tagged site (STS) and amplified fragment length polymorphism (AFLP) are generated by enzymatic amplification of DNA by extension of oligo nucleotide primers and therefore, are based on PCR. Single/simple nucleotide polymorphism (SNP) markers are based on DNA sequence differences as revealed by sequencing and assayed by various techniques.

ABSTRACT The process of developing new crop varieties involves many steps and takes several years. In recent times, biotechnological advances have helped in reducing these considerably. Apart from the techniques of haploid culture and genetic engineering, one of the tools which is making it easier for scientists to select plant traits and develop new varieties is the use of molecular markers. Molecular markers can be used for (a) characterization of germplasm (b) varietal identification and clonal fidelity testing (c) assessment of genetic diversity (d) validation of genetic relationships and (e) marker- assisted selection. These have widespread applications in management of genetic resources as well as in breeding programmes. Marker-assisted selection has practically revolutionized breeding programmes. Traditionally, plant breeders have selected plants based on their visible or measurable traits called the phenotype. But this process can be difficult especially in the case of multigenic or quantitative traits. Molecular markers can be used to trace linkages with traits of importance. In the area of horticulture, this is a very useful tool in tree breeding, where the breeder has to wait for several years for phenotypic expression of the desired trait. A number of DNA marker technologies are now available and rapid advances are being made in the development of more sensitive marker technologies. The techniques involved, the various applications of DNA marker technologies in horticultural crops and future perspectives in the national context have been discussed.

ABSTRACT The coastal ecosystem happens to be one of the most productive ecosystems. This ecosystem has been under different kinds of threats such as climate change and consequent rise in sea level, increase in population and depletion of natural resources. All this has given rise to an increase in the level of abiotic stresses such as salinity, alkalinity, and drought affecting the agricultural productivity in the coastal region. Salinization is posing an increasing problem in coastal and agricultural areas reducing plant productivity and yield. Salinity is one of the major abiotic stresses decreasing the plant productivity. Salinity is a significant factor limiting agricultural productivity and affecting about 9x 108 hectares worldwide. Poor quality of water for irrigation and current unsustainable irrigation practices has significantly resulted in salinization and other forms of soil damage. About one third of all irrigated land is affected by salt due to secondary salinization. It is estimated that 50% of all arable land will be salinized by the year 2050. The problem of secondary salinization is also becoming more serious as it represents loss of highly productive lands. Added to this is the problem of coastal salinity where intrusion of seawater into arable land due to erosion of coastal habitat has rendered it unsuitable for cultivation. Salt-affected soils (~11 million ha in South and Southeast Asia) are found along coastlines or in inland areas where evaporation is greater than precipitation.

ABSTRACT Of the total production of the crop yield, only 63 per cent is available for human consumption. The remaining is lost in more or less equal proportions to weeds, pests and diseases. Prior to genetic engineering, the exchange of DNA material was possible only between individual organisms of the same species. In the field of agriculture for instance, farmers have continuously modified naturally occurring species through seed selection and controlled breeding. With the advent of the recombinant DNA technology, it has however been possible to identify specific genes associated with desirable traits in one organism and transfer those genes within and beyond the boundaries of species into other organisms. 1. Genes for herbicide resistance Modern agriculture has developed a range of effective herbicides to tackle the effects of weeds on crop yield. Some of the most useful of these are broad-spectrum herbicides because they are active against a wide range of weeds. However, these can only be used at times when the crop is not itself vulnerable to herbicide action. The development of herbicide-resistant crops therefore offers the opportunity to spray crops at the most effective time to kill weed species without damaging the crop plants. By definition, herbicides are much more toxic to plants than to animals. Therefore, it is not surprising that they generally affect ‘plant-specific’ biological processes. The herbicidal activity of many herbicides has been found to result from the specific inhibition of a single enzyme/protein. The following are four distinct strategies for engineering herbicide resistance: i. Overexpression of the target protein, ii. Mutation of the target protein, iii. Detoxification of the herbicide, using a single gene from a foreign source and iv. Enhanced plant detoxification.

Introduction Increasing production of cereal crops, oil seed crops, and horticultural crops is becoming more important than ever before owing to increasing population and changing food habits of the people. The modern tools of biotechnology are being exploited to the fullest throughout the world for developing transgenic crop plants with improved traits. Already about 20 countries in the world are growing transgenic crops, predominately with traits such herbicide tolerance and resistance to insect-pests. However, there is a constant fear of pollen flow from theses transgenics to the neighbouring non-transgenic fields or to their weedy or wild relatives. It has become a matter of great concern to all biotechnologists to develop alternative strategies to prevent the pollen flow. The best alternative strategy available today to circumvent the nuclear- transformation associated bottlenecks in developing tenable crop transgenics is the chloroplast transformation technology. This technology allows introduction and insertion of foreign genes into the chloroplast genome, called plastome. Chloroplasts are the site for photosynthesis in plants mostly present in leaves. In general called plastids, they are also present in other parts of the plant. For example, chromoplasts in coloured leaves and fruits, amyloplasts in starch storing organs, leucoplats in roots, and etioplasts in dark-grown seedlings. The plastids arise from proplastids - the undifferentiated form of plastids present in most plant cells. Just like nucleus, these plastids (and also mitochondria) contain their own genetic material on the chloroplast genome.

Introduction The common mycorrhizal association in most of the plants is the arbuscular type occurring in the majority of agricultural and horticultural crops. Arbuscular mycorrhizae (AM) are formed by non-septate Zygomycetous fungi belonging to the genera Glomus, Gigaspora, Acaulospora, Sclerocystis, Entrophospora, Scutellospora, Archaeospora and Paraglomus in the order Glomales. These fungi are obligate symbionts and have not been cultured on nutrient media. These endophytes are not host specific although evidence is growing that certain endophytes may form preferential association with certain host plants. Several investigations indicted that even in unsterile soils plants do respond to inoculation with efficient strains of arbuscular mycorrhizae. Numerous experiments have been conducted in different parts of the world confirming this growth improvement. The increased growth of mycorrhizal plants is favoured in soils with low to moderate fertility. More specifically, soils favouring mycorrhizal growth stimulation usually have one or more essential nutrients, especially phosphorus, in limiting concentrations. The mechanism of improved plant growth caused by mycorrhizal inoculation has been investigated by many workers. Greater soil exploration by mycorrhizal roots as means of increasing phosphate uptake is well established. They also improved the uptake other diffusion-limited elements like Zn, Cu, etc. The other beneficial effects are their role in the biological control of root pathogens, biological nitrogen fixation, hormone production and greater ability to withstand water stress (Bagyaraj, 1984).

ABSTRACT The Plant Growth Promoting Rhizobacteria (PGPR) is a group of rhizosphere-colonizing bacteria producing substances which increase the growth of plants and/or protect plants against pathogens. The use of PGPR has become rather common practice and is on the rise in many regions of the world. Inoculation with PGPR also aims at reducing the application of potent ially pollut ing chemical fertilisers and pesticides. Biocont rol microorganisms have unquestionable potential for managing plant diseases, insect pests and nematodes besides increasing crop productivity. Interest in biological control has recently been intensified because of imminent bans on effective pesticides, widespread development of pesticide resistance in insect pests and plant pathogens and a general need for more sustainable pest control strategies. Active and long-term colonization of the root surface of the plant and effective expression of disease-suppressive traits are generally considered prerequisites for successful suppression of horticultural crop diseases by rhizobacteria. The important mechanism of biocontrol by PGPR involve competition and production of antibiotics such as 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin, phenazine-1-carboxylic acid (PCA), 2-hydroxy phenazines and phenazine-1-carboxamide (PCN). Siderophores, including pyochelin and pyoverdine which chelate iron and other metals also contribute to disease suppression by conferring a competitive advantage to biocontrol agents for the limited supply of essential trace minerals in natural habitats. Induced systemic resistance (ISR) is a state of increased defensive capacity developed by the plant when appropriately stimulated, through activation of latent resistance mechanisms induced by diverse agents including rhizobacteria. Microplants are first grown in vitro under aseptic conditions devoid of microorganisms. They are then transferred to a post vitrum substrate for acclimatization. By creating a beneficial rhizosphere in microplants at an early stage, the plants could be better protected against biotic and abiotic stress that occurs in the greenhouse or in the field. The beneficial rhizosphere can be created by biotization of microplants with PGPR. The search for PGPR to control postharvest decay of fruit crops has been actively pursued and promising strategies have been practiced nowadays. Biological control of postharvest diseases of perishable horticultural crops with PGPR is complex and involves a number of mechanisms to suppress the development of the disease. PGPR, being a potential candidate in disease management through multiple mode of action, it becomes highly imperative to know its different role in the management of plant pathogens.

ABSTRACT Commercial propagation using tissue culture techniques started in seventies for orchids in USA. Since then, the science and technology of tissue culture have advanced particularly after the development of the most widely used medium by Murashighe and Skoog (1972). The science has led to the development of the fascinating area of gene transfer. Today, micropropagation protocols are available for almost all horticultural crops. But, there are certain grey areas where research need to be intensified to solve the problems confronting certain difficult to propagate species belonging to plantation and spice sectors. The major area that needs to be addressed is recalcitrance.

Waste is not actually waste but is the by-product of various activities of human life. The unclean handling of these wastes creates problem for public health departments and local bodies. The classification of waste depends on the agencies that classify these wastes according to the area of interest and method of disposal viz. medical field, agricultural field, local bodies, industries etc. In the present paper, the emphasis is given to the classification based on agricultural activities which includes a) Agricultural marketing waste b) Meat and fish marketing waste.

ABSTRACT Techniques could be standardized for the induction, initiation, maturation and germination of somatic embryoids from the nucellar tissue isolated from 30-45 day old fruits of mono embryonic mango variety Neelum and poly embryonic variety Vellari Manga. The treatment half strength Murashige and Skoog basal medium containing 2,4-D 5.0 mgl-1, GA3 5.0 mgl-1, glutamine 400 mgl-1, coconut water 200 mll-1, activated charcoal 500 mgl-1, sucrose 60 gl-1and agar gl-1was found to be the best in induction of embryogenic callus. The treatment 2,4-D 4.0 mgl-1 + BA 1.0 mgl-1+ GA3 5.0 mgl-1in half strength MS basal medium supplemented with glutamine 400 mgl-1, casein hydrolysate 500 mgl-1, sucrose 60.0 gl-1, coconut water 200 mll-1and agar 6.0 gl-1was the best in initiating somatic embryos from the induced nucellus of the three varieties. In the variety Neelum BA (8.0 and 16.0 mgl-1) and kinetin (8.0 to 32.0 mgl-1) were as effective as 2,4-D in inducing somatic embryogenesis. Polyembryonic variety Vellari Manga showed better response to induction treatments than the mono embryonic varieties. B5 major salts with MS minor salts in combination with 40 gl-1sucrose, 10 gl-1PVP, 200 mll-1coconut water, 5.0 mgl-1 ABA, 100 mgl-1 casein hydrolysate and 6.0 gl-1agar were the best suited for the maturation of the somatic embryos. The positive influence of cobalt chloride, casein hydrolysate and coconut water on the germination of embryoids of both mono and poly embryonic mango varieties could be well established. A basal medium consisting of B5 major salts and MS minor salts supported the highest percentage of germination of somatic embryoids in Neelum and Vellari Manga. However, abnormalities in germination were observed in most of the cases. Excellent germination of somatic embryos was observed in a liquid medium consisting of half strength B5 macro salts and full strength MS micro salts and supplemented with GA3 1.0 mgl-1+ glutamine 400 mgl-1 + sucrose 30gl-1.

ABSTRACT Microbial contamination is perceived as the most serious threat faced by plant tissue culture research laboratories and commercial production units. A culture is often viewed as contaminated when obvious microbial growth incited by fungi, yeast or bacteria is noticed on the culture medium. On the other hand, there can be situations when microbes may be present in a covert or latent form with serious side effects on the cultures. The present investigations were taken up with the objectives of elucidating the cause of poor growth, lack of regeneration response or modification of rooting observed with visibly clean cultures of different horticultural crops. Apparently clean cultures of grape, banana, watermelon, eggplant, gerbera and chrysanthemum of recent origin or after long-term in vitro culturing were used in this study. Indexing the medium of such cultures using fungal/ yeast and bacteriological media brought out the covert association of one or more bacterial types with varying proportions of incidence in such cultures. Indexing of tissue revealed bacterial presence in different parts of in vitro plantlets. In some instances, bacteria showed endophytic colonization while being not detectable in the tissue culture medium subsequent to a disinfection or antibiotic treatment. Such bacterial association appeared to be the cause of in vitro decline observed with long-term micropropagated cultures, lack of reproducibility of tissue culture protocols, failure of in vitro rooting etc. besides being a threat to in vitro gene banks and germplasm exchange.

ABSTRACT The totipotency of triploid endosperm tissue is exploited in many vegetatively propagated crops. Hevea brasiliensis, the most important commercial source of natural rubber, is a highly heterozygous perennial tree with a long breeding cycle. Conventional method of triploid production by crossing a diploid with an artificially induced tetraploid is a lengthy and laborious process. Hence in vitro regeneration of plants from endosperm tissue provides an easy and direct approach for the production of large number of triploids. Hevea fruits at different developmental stages were collected and sterilized using 0.1% HgCl2 for 15 minutes. Seeds were dissected out and the embryos and inner integuments were removed. Endosperm is cut into small pieces and cultured over MS medium supplemented with various levels of sucrose, phytagel and growth regulators viz BA, Kin thidiazuron, 2,4-D, IBA, NAA, zeatin & GA. Organic supplements viz. yeast extract, casein hydrolysate, coconut milk and banana powder were used for callus and embryo induction. Optimum callus induction was obtained in media supplemented with 2,4-D (2.0 mg -1) and Kinetin (3.0 mg-1). On further subculture of the primary calli to medium containing zeatin (1.0 mg -1), embryogenic calli could be obtained. Embryos were induced in MS medium supplemented with ABA (0.3 mg -1) , Kin (0.3 mg -1) and GA (0.5 mg -1). Maturation, germination and bipolar differentiation of the embryos were achieved in MS medium devoid of growth regulators. Root tips from the germinating embryos were subjected to cytological analysis and found to be triploids (3n=54). Regeneration from Hevea endosperm is being reported for the first time. The results indicates that like many other crops, Hevea endosperm responds well under in vitro conditions and endosperm culture could be utilized as a viable technique for triploid plant production.

ABSTRACT Immature seeds of Garcinia indica were excised from the green fruits and cultured on Woody Plant Medium (WPM) with a combination of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with BAP (0.5-5.0 mg/l) alone or in combination with NAA 90.5 mg/l) with 80 per cent frequency within a period of 2-3 weeks. Subculture of primary embryos on maturation medium containing BAP (3.0 mg/l) supplemented with IAA (0.5-1.0 mg/l) and / or Kinetin (1.0 mg/l) gave rise to clusters of secondary somatic embryos. In the subsequent subculture on hormone free half strength WPM the embryo clusters germinated with repetitive secondary embryogenesis. About 70 per cent somatic embryos germinated into complete plantlets, which were successfully transferred to the green house conditions.

ABSTRACT The investigation was carried out to develop protocol for micropropagation of acid lime (Citrus aurantifolia S) var. Sai-Sharbati. Shoot tip and nodal segment explants were obtained from axenically grown seedlings having 21 to 30 days age. Direct regeneration of shoots, roots and whole plant without intervention of callus was obtained from both the explants on MS basal media. In both the explants 100 per cent shoot proliferation was induced in nutrient medium MS + BA 0.25 mg/l + ME 200 mg/l. The shoot proliferation rate was more (5.0) in nodal segment as compare to the shoot tip explant (4.33). Regenerated shoots of more than 2.5 cm length with minimum of 4 leaves were transferred for rooting. Half strength MS medium supplemented with IBA 1.0 mg/l was best for root induction and root initiation within 15.66 days. More than 75 per cent plantlets were survived after hardening period of four weeks.

ABSTRACT Donor-recipient protoplast fusion vis-à-vis cybridization is an effective technique to transfer plasmon-coded traits in cultivated tetraploid (2n=4x=48) potato (Solanum tuberosum L.) from its diploid (2n=2x=24) cross-incompatible wild relatives. However, the method, although attractive, is difficult in terms of not only inactivation of nuclear and plasmonic genomes of the participating donor and recipient protoplasts, but also post- fusion culture and regeneration of putative cybrids. Here, we report an improved method for donor-recipient protoplast fusion and subsequent post-fusion culture and regeneration of electrofused protoplasts in potato. In the present experiment, Solanum verrucosum Schlechtd. (2n=2x=24) accessions CPC 1339 (CGN No. 17774) and HAW 2246 (CGN No. 17766) containing sensitive [Trs] plasmon for tetrad-type cytoplasmic male sterility were used as donors and S. tuberosum subsp. tuberosum cvs Kufri Ashoka, Kufri Chipsona-1 and Professor Broekema as recipients. For nuclear inactivation, the in vitro-cultured donor plants were exposed to 1000 Gy of unfiltered γ-rays before protoplast preparation using a revised protoplast isolation medium (PIM) containing 2.0 % Cellulase ‘Onozuka’ RS and 0.25 % Macerozyme R-10. The recipient protoplasts were treated with antimetabolite iodoacetate (1.0 mM ) for 45 min at 10 °C for chondriome inactivation. The protoplasts were aligned in an amplitude of 300 Vcm-1 for 0.5-1.0 s before being electrofused by one DC pulse of 600-800 Vcm-1 for 40 μs using a Cell Electrofusion Processor FPH 01001 (Bioelectronics Corporation, Troy, Michigan). The fused protoplasts were immobilized in alginate-plates, and cultured on modified VKM medium containing 0.3 M glucose at about 400 mOsmol (pH 5.6). After 8-10 weeks, the developing protocalluses were dissociated from alginate-plates and cultured for regeneration on MS medium supplemented with varying concentrations of zeatin/ zeatin riboside and gibberellic acid. The protocalluses derived from electrofusion between donor S. verrucosum accession CPC 1339 and recipient S. tuberosum cv. Kufri Chipsona-1 could be successfully regenerated into plantlets. Morphological as well as molecular-genetic characterization of regenerated microplants, both for nuclear and chondriome genomes, confirmed the putative cybridity as well as established the extent and pattern of mitochondrial recombination following donor-recipient protoplast fusion. The results have been discussed in relation to donor-recipient protoplast fusion approach and its potential application in potato genetic improvement.

ABSTRACT Jasmine is one of the economically most important flower crops cultivated for its fragrant flowers and valuable essential oil. In vitro studies to explore the possibility of regeneration of plantlets from different explants and to undertake biochemical analysis to detect the flavour compounds if present in the in vitro cultures were taken up with local varieties of Jasminum grandiflorum. Among the different explants tried, leaf bits were found ideal for callus induction, followed by petals and shoot tips. MS medium with 2, 4-D at 1.25 ppm was the suitable medium for callus induction in leaf explants. Miller medium with NAA at 1.25 ppm and BAP at 1.25 ppm was the suitable medium for callus induction in petal explants collected from 18 days old buds of local varieties of Jasminum grandiflorum. Among the different media combinations tried, MS medium supplemented with 0.2 ppm NAA and 0.3 ppm GA2 is the best medium for callus induction in shoot apex explants of 10mm size. Direct regeneration of plants was obtained in 10 per cent of shoot apex explants cultured in MS medium supplemented with 0.2 ppm GA1 and 3 ppm BAP in 60 days. A maximum of 95 per cent embryogenicity is induced in leaf callus transferred to MS basal medium supplemented with 1.5 per cent sucrose from MS medium with 2, 4-D at 1.0 ppm. The peroxidase assay indicated that embryogenic calli had six isoperoxidases while the non-embryogenic had only two. Biochemical analysis of the callus samples showed high Phenylalanine ammonia lyase and phenol indicating the initiation of phenolic pathway, which branches out and ultimately leads to the synthesis of flavour compounds in callus.

ABSTRACT Recognizing the need to apply plant tissue culture tools for generating planting materials of important spice crops especially vanilla, the Spices Board started Biotech Production Unit at its head quarters building at Kochi with an annual production potential of 8 lakhs plantlets. Complete tissue culture protocols were developed and scaled up for large scale production of vanilla and other spices viz. large cardamom, small cardamom and tree spices. The laboratory is equipped with all modern facilities and is established in an area of about 2000 sft. Department of Biotechnology, Govt. of India also participated in the venture by sanctioning a project entitled “Regional centre for large scale production of tissue culture plantlets of vanilla and other spices” with a financial out lay of Rs 95 lakhs equally shared by DBT, New Delhi and Spices Board, Cochin. At present there is a stock of over 3.00 lakh vanilla cultures in the lab and already 1.25 lakh plant lets were sent for hardening. The Spices Board, in coll aboration with “Kudumbashree”, a poverty eradication mission of Govt. of Kerala, extend short term training in tissue culture techniques to undergraduates from ‘Below Poverty Line’ (BPL) families and engage them as long term trainees in the Biotech Production Unit. The Board also coordinates and extends support to Kudumbashree members for, establishing and maintaining hardening facilities for TC plantlets thus providing means for empowering women from BPL families.

ABSTRACT Astudy was undertaken to evaluate the performance of tissue culture derived plants vis a vis open pollinated seedlings of cardamom (Elettaria cardamomum Maton). A total of 102.5 ha (units) covering 50 ha in Kerala, 37.5 ha in Karnataka and 15 ha in Tamil Nadu was selected for the present study out of which each demonstration plot (unit) had an area of one hectare in which 80% of the plants are tissue cultured and 20% are open pollinated. Elite cardamom clones were subjected to in vitro multiplication and their respective open pollinated seedlings were compared. The planting was initiated in 1989 and completed with 1991 in all the zones identified. Plant growth characters such as number of tillers, number of bearing tillers and panicles per clump were recorded and compared. The yield was recorded in 1993-94 and 94-95 seasons after its stabilisation. Results indicated that tissue culture plants performed better compared to open pollinated seedlings with regard to growth attributes and yield irrespective of the seasons and zones. In Kerala region tissue culture plants exhibited a yield increase of 24.3 and 36% over open pollinated seedlings for the 1989 and 1990 plantings respectively. Similar trend was observed in Tamil Nadu for 1989 and 1990 planting where the percentage increase in yield became 130 and 21 respectively. In Kerala, the yield performance of tissue culture plants and open pollinated seedlings was on par during 1990 planting but in the subsequent year (1991) tissue culture plants showed 24% increase in yield compared to their respective open pollinated ones. Further, the study enabled to identify cardamom clones suited to in various zones of the cardamom tract. Accordingly 14 clones with a yield potential of over 750 kg/ha under moderate management were identified and this indicated that cardamom clones are highly location specific in their performance.

ABSTRACT Vanilla planifolia Andrews - the high value, exotic spice crop has recently gained wide acceptance in the southern and northeastern states of the country. Being an orchid, germination of naked embryo is limited and this favours genetic uniformity. An attempt was made to induce genetic variability in Vanilla through biotechnology tools for its exploitation in crop improvement programmes. In vitro embryo culture and mutagenesis were applied for the purpose. Plantlets were multiplied, hardened, planted out and evaluated for morphological, biochemical and molecular markers. Great interclonal variability was observed in the in vitro derived plantlets. The intraclonal variability was very low in tissue culture derived regenerants. Isozyme and RAPD assay confirmed genotypic variation. Six superior clones were identified and distributed to farmers for further evaluation.

ABSTRACT Dissected shoot tips from healthy suckers of banana var. Meitei Hei (local variety) were inoculated on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators viz., NAA and BAP. After 15 days of incubation 50% of the shoot tips turned green in the medium with 5.0 mgl-1 BAP. A maximum of 85.7% of the explants produced phenolic exudates in MS + 5.0 mgl-1 BAP. After one month the explants were subcultured in fresh medium with the respective concentrations and combinations of plant growth regulators. After 75 days of incubation an average of 2.20 microshoots were induced in 78.57% of the explants in MS + 5.0 mgl-1 BAP. The regeneration of shoots, leaves and roots was more proportionate in MS + 0.1 mgl-l NAA + 0.5 mgl-1 BAP. Under the treatment the microshoots had an average height of 1.19 cm with 4.13 leaves and 2.40 roots. The multiple microshoots were separated and subcultured on MS + 0.1 mgl-l NAA + 0.5 mgl-1 BAP for proper development. After 120 days the completely regenerated plantlets were hardened under green house environment.

ABSTRACT Vanilla (Vanilla planifolia Andrews) is a high value spice crop having immense potential for cultivation in India as a source for natural vanillin. Though the crop was introduced way back in 1835, its cultivation gained momentum only in the recent past due to various reasons. Lack of sufficient good quality planting materials is one of the constraints for expanding the area under the crop. Conventionally vanilla is propagated by vine cuttings, but this method is inadequate to have large number of planting materials in a short span of time. So, micropropagation was attempted and proved to be highly successful. In the present study, performance of vanilla plants raised from tissue culture plantlets and vegetative cuttings was assessed on large-scale in planters’ field covering a total area of 111 ha in Kerala(68ha), Karnataka(28ha) and Tamilnadu(15ha) with the financial assistance of the Department of Biotechnology, New Delhi under the project entitled “Tissue culture Vanilla : Product Plan“. Disease free elite accessions of vanilla identified by the Indian Cardamom Research Institute (Spices Board), Myladumpara from its germplasm repository were used as mother plant source for tissue culture multiplication and also for raising the nursery for producing vegetative cuttings. The field planting for the demonstration plots was carried out during 1996-1999 on a cluster approach basis having 400 plants in each unit or plot of 0.25 ha. Accordingly a total of 444 evaluation plots were laid out under the project covering the aforesaid three states. Out of the 400 plants in a plot, 80% were nursery hardened grown up tissue culture plants and the balance( 20% )vegetative cuttings of comparable length. Observations on growth and yield attributes such as vine length, number of leaves per vine, internodal length, number of beans per vine, bean size, yield etc. revealed that performance of tissue culture plants is on par with that of conventional plants raised from vegetative cuttings of comparable length. It is also observed that if good management practices are adopted, tissue culture plants perform better at the full bearing stage of the plant. This proves that tissue culture plants of vanilla can be popularized as a cost effective and faster source of planting materials compared to conventional vegetative cuttings. As the demonstration plots were distributed under varied agroclimatic situations, the influence of different weather parameters on yield was also ascertained. Further, the project could convince the farmers about the comparable performance of tissue culture plants and that is quite evident from the ever increasing request for tissue culture vanilla plants received by the Spices Board.

ABSTRACT Direct shoot organogenesis from several explant types excised from in vitro grown plants of local elite tomato cv. PKM1 was studied in response to zeatin, BAP, IAA and NAA. Cotyledonary leaves were found to be the most responsive explants. High shoot bud induction rate (>25 shoot buds/cotyledonary explant) was obtained in the presence of 2 mg/l zeatin. The organogenic calli were subcultured onto the same shoot bud induction medium for shoot development. Among different growth regulators tested, zeatin @ 2 mg/ l induced more number of shoots per explant compared to BAP, IAA and NAA combinations. Rooting was efficiently induced on half strength MS basal medium supplemented with 0.5 mg/l IAA. The effects of various growth hormones on tomato regeneration on rooting were discussed.

ABSTRACT The present experiment was conducted in order to develop a protocol for rapid in vitro propagation of pomegranate Cv. Mridula. Shoot tip and nodal segment explants selected from mature plant were cultured in MS medium supplemented with NAA and BAP in different concentration and combinations. To control phenolic browning, sub- culturing of cultures was under taken at an interval of first and third day after inoculation. The MS medium supplemented with NAA 0.4 mg/l + BAP 1.0 mg/l showed highest percentage of shoot differentiation with 77.77 per cent and 81.25 per cent response from shoot tip and nodal segment explant respectively. In the same media, earlier shoot initiation as well as more number of multiple shoots/culture were obtained. The shoots when elongated up to 2.5 - 3.0 cm length were transferred for rooting in the ½ strength MS media supplemented with auxins. There was no significant difference for root induction among the type of auxin used either NAA, IBA or both in combination. The concentration of auxins 0.5 mg/l was found to be optimum. Rooted plantlets after hardening in green house gave 49 per cent survival.

ABSTRACT Papaya (Carica papaya L.) is an important crop in the tropics and subtropics for export of fruits and above all, for local consumption. Cultivation of papaya faces with problems due to its heterozygosity, dioecious nature and susceptibility to a large number of viral diseases. Crossing between two hermaphrodites will produce the highest ratio of fruit bearing female trees, which is of homozygous dominant andromonoecious types which are always letha l due to po st fertilization barriers leading to their abortion. The gynodioecious varieties of papaya have the M1M1 forms while selfing and hence for explant selection , gynodioecious varieties like Sunrise solo and CO7 were prefered Embryo culture is one of the earliest forms of in vitro culture applied to solve practical problems and is probably the tissue culture techniques that has proven of greatest value to breeders. Ovule culture is practised in a case when small (or) young embryos that abort at early stages of development and are difficult to isolate. This necessiates the development of a protocol for initiation, multiplication and regeneration of ovular callus of papaya.

ABSTRACT Shoot organogenesis from several explant types excised from in vitro grown plants of local elite brinjal (Solanum melongena L.) cv. Co2 was studied in response to the supplementation of thidiazuron (TDZ), BAP, IAA and NAA. Cotyledons and leaves were found to be the most responsive explants. High shoot bud induction rate (>50 shoot buds/explant) was achieved in the presence of 0.044 mg/l TDZ. The organogenic calli were subcultured in the same shoot bud induction medium for shoot development. Among different growth hormones, TDZ @0.044 mg/l induced more number of shoots per explant compared to BAP, IAA and NAA combinations. Efficient rooting was observed on half strength MS medium supplemented with 0.5 mg/l IAA. Higher percentage of rooting was achieved only when the well developed shoots were directly transferred to soil.

ABSTRACT Immature zygotic embryos were isolated from field grown papaya fruits harvested at 90-105 d post-anthesis. The embryos were placed on half-strength Murashige Skoog (MS) medium supplemented with 2, 4-D (2.0 mg l-1) and cultured under dark for a period of 2 months. The frequency of callus formation was around 66%. After 2 months, calli were transferred onto a half-strength MS basal medium and cultured for 3 months with frequent subculturing (once in 3 wk) on the same medium. The frequency of somatic embryo formation was around 36.6%. The somatic embryos on MS medium supplemented with BAP (0.4 mg l-1) and NAA (0.02 mg l-1) germinated and produced shoots. The emerging shoots of 2-3 cm height with 2-3 trilobed leaves were placed onto an MS basal liquid medium with IBA (1.0 mg l-1) for root induction. The plants developed with shoots and roots were planted in a pot-mixture (sand:soil:FYM) containing VAM.

ABSTRACT Aprotocol has been developed for the induction, maturation and germination of somatic embryos from leaf explants of Hevea brasiliensis (clone RRII 105). Leaf explants were cultured with their adaxial sides on modified Murashige and Skoog (MS) medium supplemented with different combinations of phytohormones such as 2,4-D & BA, NAA & BA as well as 2,4-D, BA and NAA. Compact calli could be developed from the cut ends of the explants on media containing 2,4-D (1.5 mg/l) and BA (1.0 mg/l) whereas pale yellow friable calli was obtained on media which contained NAA (0.2 mg/l) along with 2,4-D (1.2 mg/l) and BA (1.0 mg/l). The calli formed were detached from the explants and sub cultured for proliferation in medium containing reduced auxin (0.4 mg/l 2,4-D) and slightly increased level of sucrose (40 g/l). Proliferated calli were then sub cultured for embryo induction in modified MS medium supplemented with different auxins and cytokinins. Embryo induction could be achieved in modified MS medium containing BA (2.0 mg/l), GA (1.0 mg/l) and NAA (0.2 mg/l) and maturation occurred in WPM medium containing BA (0.3 mg/l), TDZ (0.5 mg/l) and GA (1.5mg/l). The cotyledonary stage embryos developed into plantlets on transfer to hormone free ½ MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Hevea which has not been reported previously. This explant could be used further as an ideal target tissue for Agrobacterium mediated genetic transformation in Hevea.

ABSTRACT In vitro culture induced genetic variation constitutes an important source of variability for the improvement in ginger (Zingiber officinale Rosc.). Hence attempts were made to standardize indirect method of in vitro plantlet regeneration in ginger using various explants like sprouted bud, shoot tip, leaf and pseudo stem. Explants from two cultivars of ginger viz. Maran and Rio-de-janeiro were cultured in Murashige and Skoog (MS) medium supplemented with various levels of 2,4-D and in combination with different levels of BA. Cultures were incubated under two different culture conditions viz. light and dark. Callusing was observed in all the explants except mature leaf in MS medium supplemented with 2,4- D alone (1 - 2 mg l-1) and in combination with BAP (0.5 - 3.0 mg l-1). Shoot morphogenesis was achieved from one month old calli in MS medium supplemented with BAP (3.0 mg l-1). Regenerated shoots were subcultured to medium of same composition for further shoot proliferation and rooting. Plantlets produced were hardened and planted out. From the present investigation, highly efficient protocol for indirect organogenesis in ginger could be developed and the response of two cultivars at various stages of morphogenesis could be studied in detail.

ABSTRACT In the studies on callus induction and somatic embryogenesis in acid lime (Citrus aurantifolia S.) var Sai-Sharbati, different ex-plants and different combinations of growth regulators (NAA, IBA and BA) in MS basal media were tried, the results indicated that cotyledonary segment was the best explant for callus induction among root tip hypocotyl stem, leaf, epicotyl stem and shoot tip explants tested of. The callus grown from epicotyl and hypocotyl stem explant was greenish white and compact. Whereas callus derived from cotylidonary segment and leaf segment was green and friable . Root tip and shoot tip did not exhibit callusing. The callus derived from epicotyl stem and hypocptyl stem explant responded shoot bud formation in MS medium supplemented with NAA 0.2mg/l + BA 0.5mg/l with or without ME 100mg/l. In somatic embryogenesis, full grown callus derived from epicotyl stem explant induced embryo formation within 28.33 days and more shoot bud (6.33) formation.

ABSTRACT The present experiment was conducted to standardize the composition of nutrient media for callus regeneration. Shoot tip, internodal segment, leaf and hypocotyls of seedling grown under controlled condition were used as explants for callus induction. The study revealed that explants collected from younger seedlings showed greater proliferation of callus as compared to those collected from older one. The basal MS medium as well as WP medium fortified with different concentrations of auxins and cytokinins were tried. Initial better results of profuse callus growth with green colour and compact texture having regenerative capacity were obtained by using leaf explants. In all the treatments, the callus initiation occurred at the cut ends and then spread over the surface of the explant as a result of injury. The level of auxins presumable rises and quiescent cells were stimulated to divide rapidly producing callus. The leaf segment explant induced the callus after 9.50 days in medium WPM + NAA 2.5 mg/l with maximum callus growth (976.75 mg) with green, friable callus which was difficult to subculture. White and friable callus occurred within 12.00 days in MS + 2, 4 - D 5 mg/l. The green and compact callus having vigorous growth was observed in MS + NAA 2.5 mg/l within 12.75 days when subcultured on different regeneration media proved recalcitrant for shoot regeneration. On the regeneration medium the callus turned brown while some callus proliferates to produce white growth around it and dried later on.

ABSTRACT In vitro multiplication studies on two peach cvs namely Prabhat and Flordasun grown widely under agroclimatic conditions of Haryana were carried out using nodal and shoot tip explants. Amongst various media modification, M medium containing 1.5 mg-1 BAP was found the best for shoot induction from both the explants. However, shoot tip showed better response in comparison to nodal explants. Shoot multiplication was highest on M7medium (MS+ 6 mg l-1 BAP). Cultivar response did not differ significantly. Rooting was obtained on M medium containing 0.5 mg l-1 NAA.

ABSTRACT Bacopa monnieri (L.) or Herpestis monniera, commonly known as brahmi, purifies the blood, and is useful in scabies, leucoderma, syphilis, diarrhoea and pyresis. As Brahmi is a major ingredient in several ayurvedic preparations, there is growing demand for steady supply of planting materials. Conventional propagation methods are less efficient than in vitro propagation. However, there are only a few reports on the in vitro propagation of this plant. Therefore, an attempt was made to evolve an improved protocol for the in vitro propagation of Bacopa. Shoot tips and nodal buds along with a portion of the stem excised from field grown disease free plants were taken as the explants. After three weeks of establishment on MS basal medium, the sterile cultures were sub-cultured on MS medium supplemented with different concentrations of BA (0.5,1.0 and 1.5mg l-1). Rooting was induced on in vitro proliferated shoots by culturing on MS basal medium with NAA 1.0 mg l-1 and IBA 0.5 mg l-1. Treatments with BA 1.0 mg l-1 and IAA 1.5 mg l-1 recorded the maximum number of shoots (40.9 and 35.2, respectively). The height of the shoots was also significantly influenced by the concentrations of IAA (1.5 or 1.0 mg l-1) in combination with BA (1.0 mg l-1), recording 5.4 and 5.5 cm. The shoots with an average length of 3.0 to 4.0 cm were kept for rooting. NAA (1.0 mg l-1) in combination with IBA (0.1, 0.5, 1.0 or 1.5 mg l-1) produced significantly greater number of roots. The length of the roots was also greater in this treatment. The plantlets were transferred to pots in a greenhouse and later to the field with ninety per cent survival.

ABSTRACT Aquilaria agallocha Roxb. (Thymelaeceae), otherwise known as ‘Eagle wood’ is a large evergreen tree restricted to the eastern parts of India and Bhutan. The tree is valuable for its fragrant wood - ‘Agar wood’ which is tremendously used in the perfumery and incense stick industries. Lack of rapid natural regeneration and over exploitation has reduced its population drastically. Micropropagation offers a rapid means for clonal multiplication and conservation of this endangered species and attempts were made to standardize available protocol for in vitro multiplication. Media and conditions were standardized for culture establishment and propagule multiplication. Multiple shoots were induced in vitro from nodal segments of 10 yr old trees having enhanced axillary branching. Culture establishmnet was strongly influenced by the nature of the explant and the season of collecting the explants. Nodal segments collected during the period March- April from current season shoots showed a better response (90 %) in half strength Murashige and Skoog medium supplemented with 1.0 mg l-1benzyl adenine and 0.1 mg l-1kinetin. The rudimentary multiple shoots induced, proliferated in the basal media supplemented with benzyl adenine (0.1 mg l-1), naphthalene acetic acid (0.5 mg l-1) and coconut water (10%). This media also supported further multiplication in serial subculturing with a multiplication rate of 1:6. There exists no earlier report on in vitro multiplication of this tree species valued in perfumery.

ABSTRACT Kattupadavalam (Trichosanthes cucumerina) is regarded as a laxative and blood purifier, considered beneficial in the treatment of skin diseases. Its entire supply being met from wild, unscrupulous exploitation has led to erosion of genetic resources of this crop. Hence it is imperative that in vitro trechniques are employed to conserve this valuable medicinal species and to evolve feasible protocols for large scale multiplication of this crop. Nodal cultures were established from two months old seedlings of Trichosanthes cucumerina and Murashige and Skoog (MS) medium, supplemented with BA @ 0.5 mg l-1. Subculture of initiated shoots in the same medium at intervals of three weeks registered enhanced release of axillary buds producing 21 shoots from a single culture at the end of fifth subculture cycle. Elongated shoots were rooted in half strength MS medium, following a pulse treatment with IBA @ 1000 mg l-1. A single stage root hardening process, wherein, rooted plantlets were subsequently transferred to half strength semisolid MS medium supplemented with 2 per cent sucrose, improved success rate. The rooted plantlets planted out in sterilized media and subjected to a hardening process in a phased manner, survived well.

ABSTRACT Vitex trifolia L. (Verbenaceae) is known for its medicinal property. Micropropagation can provide the opportunity to obtain large number of homogeneous plants and there are no reports of micropropagation of this species. So, we attempted for in vitro propagation of this species and described here is an efficient and rapid propagation method. This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2- isopentenyladenine (2 - iP) (0.25-10.0 mM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 mM BAP. Shoot cultures was established repeatedly by subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 mM naphthaleneacetic acid (NAA). Rooted plantlets were transferred to pots containing autoclaved soil and vermiculite mixture (1:1) that showed 90 % survival when transferred to outdoor. Plants transferred to the field have established themselves in the soil and are growing well. The regenerated plants did not show any immediately detectable phenotypic variation. Natural stands of Vitex trifolia are fast disappearing in India because of indiscriminate collection and over exploitation and the outlined procedure here offers a potential system for mass multiplication and conservation of Vitex trifolia.

ABSTRACT Rubia cordifolia is an effective blood purifier in indigenous medicine. Habitat destruction and indescriminate harvesting is posing threat to its survival necessitating concerted efforts for conservation. The present study is an attempt to standardise in vitro multiplication protocol, a basic step for in vitro conservation, in Rubia cordifolia. Nodal explants cultured in MS basal medium supplemented with BA 1mg l -1 and IAA 0.2 mgl-1 induced axillary bud break. The in vitro formed shoots developed roots on MS basal medium supplemented with IBA 1mg l -1. The plantlets were ready for plant out in 100 days of culture initiation and established well on planting out.

ABSTRACT Centella asiatica (Linn.) previously Hydrocotyle asiatica belongs to the family Apiaceae.It is commonly called gotu kola and is rich in triterpene glycosides such as asiaticoside and madecassoside. Health claims range from enhanced memory and longevity, to the treatment of leprosy. It is also used in the treatment of various skin diseases, ulceration, chronic rheumatism, malaria, fever, epilepsy and enlargement of glands. Inspite of its wide range of uses,it is still collected from the wild and commercial cultivation is lacking. A good tissue culture protocol therefore would be useful for commercial propagation and thereby saving the plant from extinction. Of the various explants, nodal segment gave the best response. For direct shoot regeneration from nodal segments, BAP 2.0 mgl-1, and BAP 1.0 mgl-1 with Kinetin 1.0 mgl-1 was found optimum. Callusing was observed in nodal segments and leaf bits when 3.0 mgl-1 NAA along with 0.3 mgl-1 BAP was used. ½ MS medium supplemented with IBA 0.5 mgl-1 and NAA 0.2 mgl-1 induced rhizogenesis of the regenerated shoots. Rooted plantlets survived well in a combination of pot mixture (1 sand: 1 soil: 1 FYM) and Vermiculite in the ratio of 1: 1.

ABSTRACT In vitro propagation of Plumbago rosea was attempted. One year old plants were used as the source of explants. Nodal explants were inoculated on half strength MS medium supplemented with 2,4-D 1.0 mg l-1. Induction of callus was observed three weeks after inoculation. The calli were sub-cultured on half strength MS medium supplemented with BA (0.5, 2.0, 3.0 and 4.0 mg l-1) and IAA (0.0 and 1.0 mg l-1). Eight treatments with six replications were tried. Benzyl adenine 2.0 mg l-1 promoted the proliferation of shoots (23.2). Rooting of micro shoots was obtained on MS medium supplemented with IBA 1.5 mg l-1. The plantlets were then transferred to pots containing sand/soil mixture (1:1) and kept in a mist chamber. The survival rate was found to be 90 per cent.

ABSTRACT Ipomoea mauritiana (L.) belonging to the Family Convolvulaceae is an important medicinal plant. The roots, leaves and flowers possess medicinal properties. It is used in indigenous medicine as a tonic, aphrodisiac, galactogogue, diuretic, carminative, expectorant and appetizer. It is useful in syphilis, inflammation, leprosy, diseases of blood and in the improvement of voice and complexion. Tender nodal portions from one year old disease free plants grown in pots were used as the source of explants. The explants were inoculated on half strength MS medium containing 3.0 per cent sucrose and 0.6 per cent agar supplemented with 2,4-D 0.5 mg l-1. All the explants showed callus initiation after three weeks. The contamination free, one week old callus cultures were used for testing the effect of the different treatments. The treatments comprised of different combinations of BA (0, 1.0, 2.0, 4.0 and 8.0 mg l-1) and NAA (0 and 0.1 mg l-1) added to half strength MS medium. The highest rate of shoot regeneration was obtained on half strength MS medium supplemented with BA 4.0 mg l-1. Rooting of the proliferated shoots could be induced on the same basal medium supplemented with IBA 1.0 mg l-1 and NAA 1.0 mg l-1. The regenerated plants were hardened in a humidity chamber and successfully transferred to pots, with 90 per cent survival.

ABSTRACT In vitro clonal propagation of Smilax zeylanica Linn was attempted. Axillary buds from shoot tip and single node explants were induced to proliferate on half strength MS basal medium supplemented with BA (0.5, 1.0 and 2.0 mg l-1) and IAA (1.0 and 2.0 mg l-1). Multiple shoots (6.0 - 10.0 per explant) developed from the axillary buds in about four weeks time in all the treatments tried. However, the highest rate of multiple shoot production was in the medium containing BA 1.0 mg l-1 and IAA 1.0 mg l-1. Maximum shoot length was also observed in the same medium. Rooting (83.3 per cent) of micro shoots could be induced on MS supplemented with NAA 0.5 mg l-1. Individual rooted plantlets could be successfully transferred to soil and established in the field with 80 per cent survival.

ABSTRACT Aloe vera is one of the oldest known therapeutic herb belongs to the family Liliaceae commonly known as guar patta. Gel from fleshy leaves of Aloe vera is rich in 200 nutrients which are used in healing the burns, wounds, gastric ulcers, treatment of diabetes and also used in normal functioning of the liver. Aloe vera is a desert plant which needs water in very less quantity. As Aloe vera is second in ranking in drug production in the world. Government of India is also trying to promote Aloe vera as a crop in dry and semi dry regions of India. As it needs very less and cheaper inputs, Aloe farming promises higher economic gains to farmers. But as the plant grows vegetatively the quality planting material is a problem. Tissue culture provides an opportunity in production of quality seedlings. To optimize the regeneration system in Aloe vera, MS based media were tried to induce shoot formation in cultured shoot tip explants. 6-10 cm long offshoots were collected. Shoot tip explants were cut vertically after sterilization in laminar hood and cultured on MS basal media supplemented with various growth regulators. A total of 23 media were tried for shoot regeneration. All the cultures were incubated under fluorescent light. Different concentrations of BAP (2, 5, 10 mg/l) were used and MS medium with 2 mg/l or 5 mg/l BAP exhibited similar response while BAP at 10 mg/l showed poor shoot formation. TDZ when used at 1 mg/l concentration improved percent shoot formation marginally. Multiple shoots were observed on TDZ containing medium and BAP (2.0 mg/l) supplemented medium in 12-15 days. Multiple shoots were obtained in 12 days when 2 mg/l BAP was added along with 0.18 mg/l IAA. The number of multiple shoots ranged from 4 -15 per explant. The regenerated plants developed roots on MS medium supplemented with 0.1mg/l NAA.

ABSTRACT Successful in vitro clonal propagation of mature tree of Lasoda (Cordia myxa Roxb) has been achieved through single node explant. Both types of organogenesis of shoot and root formation were obtained by placing nodal explant on shoot induction medium and subsequently on rooting medium. More than 90% culture were responsive for in-vitro axillary shoot proliferation on MS media supplemented with 30g Sucrose 8.0 g agar and a combination of 2.0 mg BA and 0.1 mg NAA per litre. After culture period of 6-8 week, the nodal explants with micro shoot were rooted on the MS media supplemented with30 g Sucrose + 3g activated charcol and different level of NAA (0, 1, 3 and 6 mg/ litre). The season and position of explant on the mother plant was also shown to influence in vitro performance of the regeneration in terms of establishment of aseptic culture, percentage of responsive explants and intensity of leaching of growth inhibitory phenolic chemicals.

ABSTRACT Asimple and reproducible protocol for the rapid propagation of Clitoria ternatea L., a flowering garden climber cum medicinally valuable plant, was developed through somatic embryogenesis from the leaf explants. Leaf segments excised from 12 days old in vitro raised seedlings on inducing callus by culturing in MS medium containing 0.5 mgl-12,4-D and 0.1 mgl-1 BAP and sub-culturing callus to MS medium containing 1.0 mgl-1 BAP, produced numerous somatic embryos within 30 - 45 days. Embryos germinated and produced plantlets with intact shoot and root when they were cultured in MS medium devoid of any hormones and supplemented with GA2 0.1 to 1.0 mgl-1. Elongation of plantlets could be achieved on hormone free MS medium in about 3 - 4 weeks. Well- grown plants on transfer to pots, containing sterile sand, in a growth chamber and three weeks later to the greenhouse gave about 40 per cent survival.

ABSTRACT Astudy was conducted to establish the regeneration capacity of pseudobulb segments of a Dendrobium hybrid orchid (D. nobile L. x D. chrysotoxum L.) in vitro on full strength (MS) and half strength (½ MS) Murashige and Skoog medium. Pseudobulb segments having 0.3-0.5 cm length cut from the in vitro maintained hybrid orchid were inoculated on both media supplemented with different concentrations and combinations of NAA, BAP and Kn. On MS medium the pseudobulb segments failed to respond, however, in ½ MS medium the explants responded well. Responding explants were maximum (35%) on the medium ½MS + 0.1 mgl-1 NAA + 0.5 mgl-1 BAP. An average of two (2) shoot buds per explant were regenerated on ½ MS + 2.0 mgl-1Kn and ½ MS + 0.1 mgl-1 NAA + 1.0 mgl-1 BAP. The regenerated plantlets grew healthily on subculture after separation from the pseudobulb segments.

ABSTRACT Considering the difficulties faced during ex vitro establishment of orchid plants the present investigation was undertaken to elicit information on the influence of different sucrose levels in the in vitro rooting medium and by subjecting the plants to different levels of triazole at the time of planting out in order to overcome the mortality percentage at the time of ex vitro establishment. Among the growth parameters observed viz. crop growth rate, net assimilation rate, and relative growth rate was found to be high in plants treated with 40 g/l of sucrose concentration and also at the triazole level of 5 mg/l. The photosynthetic rates were observed to increase meanwhile the transpiration rate was declined in plants grown under the above treatment levels of sucrose and triazole. The maximum survival percentage was observed when the in vitro plants grown in rooting medium with 40g/l of sucrose which is attributed to the influence of sucrose concentration on morphological characters studied viz. plant height, number of leaves per shoot and number of roots per shoot. Triazole treatment of plantlets at the time of planting out helped in better survival percentage which is the whole sum effect of increase in number of leaves per shoot, number of roots per shoot of plants grown under the triazole level of 5 mg/l. Where as the height of the plant was inversely proportional to triazole concentration,the biometric aspects studied viz root length, total fresh and dry weight of the plants with the above mentioned treatments were found to be superior. The present study envisages the measures to be taken during in vitro propagation and ex vitro establishment of tissue cultured plants in order to over come the field mortality.

ABSTRACT The mass production of orchids through in vitro approach is now practising on a commercial scale. But, in most cases, it is being done via an intermediate callus which may lead to variability in the progenies. A study was made to standardize a protocol for rapid mericloning of tropical orchid dendrobium cv. sonia from the shoot tip explant without an intermediate callus so as to produce true to type progenies. The efficiency of production of clones through agar gelled medium and shake cultures were compared. The shoot tip explants produced an average of 6-8 multiple shoots on agar gelled (0.8% w/v) MS medium containing 3% sucrose as carbon source supplemented with a hormonal combination of 0.75 mg l-1 NAA and 2.5 mg l-1 KIN without an intervening callus, within a period of 3 - 4 months. When the shoot tip explants were cultured in agitated ( 80 - 100 rpm) liquid Vacin and Went ( VW) medium supplemented with 3% sucrose, 2.0 - 2.5 mg l-1 BA and 0.5 - 1.0 mg l-1 NAA could induce a large number of protocorms within a period of two weeks. Agitated liquid MS medium supplemented with 0.5 mg l -1 IBA and 2 mg l-1 BA or 0.5 mg l -1 2,4 - D, 0.5 mg l-1 GA2 and 3.0 mg l-1 BA was identified as the rapid multiplication medium of the protocorms. About 60 - 80 protocorms could be produced from a single protocorm within a period of 3 weeks. Transfer of these protocorms to agar gelled (0.8%w/v) MS medium containing 2% sucrose supplemented with 20 - 25% coconut milk (CM) was found ideal for plantlet formation of size 1.0 - 1.5 cm within a period of three weeks. Sub culturing of the plantlets to the same medium resulted in rapid profuse rooting. Transfering the rooted plants into bottles containing sterile coconut husk provided with half MS medium without sucrose and hormones was sufficient for in vitro hardening. By this two stage culturing technique, about 50 - 60 true to type clones can be produced from a single shoot apex within a period of 3 months.

ABSTRACT Myriophyllum aquaticum (Velloso) Verdcourt, commonly known as ‘parrot feather milfoil’ is the aquatic plant belonging to the family Haloragaceae. The plant gets the name ‘parrot feather’ from its feather-like leaves arranged around the stem in whorls of 4 to 6 giving it the value of a beautiful aquarium plant. It is a commercially valuable imported aquatic plant used in aquarium plant trade. These plants are exclusively females and as such no seeds are produced. Moreover, current macropropagation techniques are insufficient to produce sufficient propagules for the domestic and export market. Tissue culture techniques offer great opportunities in this regard and attempts were made for micropropagation of this valuable species. A viable protocol has been outlined for the in vitro mass multiplication of Myriophyllum aquaticum. Shoot regeneration was induced from nodal segments and leaves in half strength Murashige and Skoog (1962) medium supplemented with 1.0 mg l-1 benzyl adenine. Incorporation of 0.2 mg l-1 kinetin along with benzyl adenine in the culture medium resulted in a marked increase in the frequency (1:20) of multiple shoot formation. The in vitro raised plantlets were successfully rooted, hardened and planted out. The protocol is ready for commercialization.

ABSTRACT Multiple shoot induction from the cotyledonary node explants of Leucaena leucocephala was studied in presence of various concentrations of TDZ. With the increase in concentration of TDZ from 0.045 μM to 0.227 μM the number of regenerated shoots increased. However, the length of individual shoot decreased. Further increase in TDZ concentration upto 22.71 μM resulted in the formation shoot clusters. Presence of TDZ in the medium inhibited shoot elongation. Transfer of the regenerated shoots to ½ x MS medium supplemented with 0.54 μM NAA ensured elongation of the shoots, which also developed roots. Rooted plantlets were transferred to coco peat for further development and hardening. This highly efficient regeneration protocol may find application in the genetic transformation of this leguminous tree species.

ABSTRACT The tomato TYLCV tolerant line MP-1 which has high efficiency of crossability & dwarf mutant dg/dg (Manapal) were choosen for the study of enhanced production of plantlets through tissue culture. The cotyledonary explants, hypocotyl sections & decapitated seedlings were taken to assess the regeneration efficiency of the explants as a base for efficient genetic transformation. The decapitated seedlings regenerated much faster (9.3 days) putting forth new sprouts of shootlets which were then transferred to rooting medium. Whereas cotyledonary leaves showed regeneration of plantlets (18.3 days) which is almost double the duration of decapped seedlings. The hypocotyl sections proliferated into callus & regenerated in a much slower rate (27 days). Hence, the faster rate of regeneration from decapitated seedlings served as a efficient base for enhanced production of plantlets during transformation studies. Also the number of harvest of new sprouts (2.3 regenerations) from decapitated seedlings is an added advantage for deriving more plantlets from single seedlings.

ABSTRACT The tissue culture derived black pepper plants of variety Panniyur 4 were compared with conventional propagules for its vegetative growth and field performance. The in vitro derived plants recorded better rooting, survival rate and shoot growth than the conventional propagules. More than 90% of the TC derived plants recovered after primary hardening and 99% recovered after secondary hardening, while it was only 70% in conventional propagules at both the hardening stages. The secondary roots developed in TC derived plants were observed to be far more superior in number and length. The initial growth rate in terms of plant height, number and size of leaves was much better in TC derived plants. The TC derived plants developed numerous bearing shoots - laterals - in the first year of growth and started bearing in the first year itself. The performance was superior to conventional propagules in which production of laterals and bearing begins much later. Thus the performance of TC derived plants was rated as superior to conventional propagules.

ABSTRACT The in vitro culture studies in crop plants show that there is a differential response of various explants to different concentrations of auxins and cytokinins. This indicate that hormonal requirement is specific to the genotype and developmental stage of the explant. Therefore standardisation of media and explants were done for in vitro callus culture and plant regeneration in L. esculentum and L. pimpinellifolium at the Department of Olericulture, College of Horticulture during 2000-2002. The source materials selected for the study were L. esculentum var. Sakthi and L. pimpinellifolium. The explants were leaf segments (0.5 cm2) from fully opened leaves, nodal segments (0.5 cm) and internodal segments (1.0 cm). The media consisted of the basal MS and ½ MS media along with different auxins and cytokinins either singly or in combination. The auxins used were NAA (0, 1, 2 and 3 mg l-1 and 2,4-D (0.5, 1, 1.5 and 2 mg l-1) and the cytokinins used were BA and kinetin (1, 2, 3, 4 and 5 mg l-1).

ABSTRACT In Kerala, the commercial cultivation of tomato is limited due to the bacterial wilt disease caused by the soil borne pathogen Ralstonia solanacearum. The release of bacterial wilt resistant varieties like Sakthi, Mukthi and Anagha has increased the area under tomato. But, these varieties are susceptible to tomato leaf curl virus diseases caused by Germini group virus and transmitted by white fly (Bemisia tabaci). The disease incidence varies from 10.77% to 90.5% and the yield loss is tremendous. Unless the tomato variety resistant to ToLCV disease is resistant to bacterial wilt also, it cannot be recommended for cultivation in the State. The somaclonal variations have been well exploited in developing disease resistant varieties in many crops. Therefore an experiment was formulated to study the somaclonal variations and to screen the somaclones for resistance to tomato leaf curl virus disease in L. esculentum var. Sakthi and L. pimpinellifolium. The somaclones of Sakthi and L. pimpinellifolium along with the parental lines were planted in earthen pots containing potting mixture with bacterial wilt sick soil. These were screened for resistance to ToLCV disease by artificial inoculation of the vector Bemisia tabaci inside the cages. The observations were recorded on ex vitro survival, biometric characters and reaction to ToLCV and other major diseases. The ex vitro survival was 46.2% in Sakthi and 36.6% in L. pimpinellifolium. The somaclones of Sakthi showed wide variation for plant height and spread compared to that of L. pimpinellifolium. Some of the somaclones exhibited ‘gigantism’ with potato leaved plants. The yield of the somaclones of Sakthi were higher than that of parental line.

ABSTRACT Cycas is the largest genus among cycads - the surviving remnants of an ancient line of plants of the early Mesozoic period. The IUCN has classified more than half of the 182 species of Cycadales as endangered, vulnerable or rare. The use of in vitro techniques could permit a way to conserve many of the endangered species. None of the in vitro studies has been successful in establishing a functional protocol for artificial propagation of cycads. The objective of the present investigation is to explore the possibilities of in vitro plant regeneration from megagametophyte explants of Cycas circinalis - the only species of Cycas grown in Kerala. Megagametophyte from the ovules of ten-year-old Cycas circinalis plant was used as explant. Standard tissue culture techniques were adopted for the in vitro culturing of the explant. The basal medium involved major salts of B5 and minor salts of MS media. Glutamine, casein hydrolysate, coconut water and megagametophyte extract were used as supplements. The plant growth regulators - Kn, BAP, 2iP, IAA, NAA and 2,4- D - were incorporated alone and in combinations. Highest percentage of shoot induction and highest average number of shoot buds per culture were observed in PGR combination involving Kn + BAP along with one of the auxins (2,4-D or NAA or IAA). Shoot buds developed did not show further growth even after one year. The protocol employed in the present investigation may lead to multiple shoot proliferation and help in large-scale propagation of Cycas circinalis.

ABSTRACT To study the influence of season on explant collection, axillary buds from mature trees of three elite genotypes of tamarind viz., Sour pulped type, Red pulped type, and Sweet pulped type (Thailand) were collected monthwise from September, 2002 to August, 2003, sterilized properly and cultured in MS medium supplemented with BAP (1, 1.5, 2, 2.5 and 3 mgl-1) + GA 0.5 mgl-1 + NAA 0.1 mgl-1. The studies revealed that, the bud break percentage was highly dependent on the season of explant collection. In case of Sour pulped type, bud break was recorded during the month of April, May and June. Maximum per cent bud break (50.00 per cent) was recorded in May and June but least number of days were required for bud break (12.33) in April month. In Red pulped type, the bud break was observed during February to June. The axillary buds collected and cultured during the month of February recorded the best results with respect to the per cent bud break (100 per cent) and least number of days taken for bud break (7.12 days). In Sweet pulped (Thailand) type, bud break was recorded during the months of February to June. The axillary buds collected and cultured in the month of may recorded the best results with respect to the percent bud break (87.50 per cent) but March showed least number of days for bud break. Nodal segments cultured on MS + BAP 2 mgl-1 + GA 0.5 mgl-1 + NAA 0.5 mgl-1 gave maximum and early bud break in three genotypes, but sprouted shoots showed abscission of leaflets and died later on.

ABSTRACT Wood apple is a hardy tree grown in arid and semiarid regions of the country. It is generally propagated through seeds. The traditional methods like budding and inarch grafting require 9 to 12 months for the root stock to come buddable size and thus 1½ to 2 years to become ready for transplanting. The present study was initiated to prepare a grafts within eight months by adopting the micrografting technique. The seeds were sown in the month of April in polybags filled with FYM, sand and soil in equal proportions with a pinch of blitox. The micrografting was done on 1½, 2, 2½ and 3 month old seedling root stock having a diameter of 0.13 to 0.15 cm, 0.17 to 0.19 cm, 0.20 to 0.22 cm and 0.23 to 0.26 cm, respectively. The seedling root stocks were decapitated with the surgical blade and 1 to 2 cm deep split was made. The scion micro bud (1 to 2 cm length and 1 to 2 mm width) of previous season’s growth was inserted in the split. The micropipette cap (0-200 μl) was inserted on the grafted portion. After sprouting of the micro bud, the micro pipette cap was removed. The study revealed that, micro bud grafting success was significantly influenced by the root stock age. Early bud take was observed when the micrografting was done on 2½ month (10.73 days) and 3-month (11.00 days) old root stock. The percent bud take on 3-month old root stock was significantly higher (99.33 per cent) than the rest of root stocks. Micrografting done on 3-month old seedling root stocks in June-July is suggested for quick multiplication of woodapple.

ABSTRACT Attempts were made to induce variability by irradiation of axillary buds of rose cv. Folklore followed by in vitro culture. Axillary bud explants collected at different growth stages were subjected to gamma irradiation at different doses (20, 30 and 40 Gray). The in vitro induced micro shoots were sub cultured on a rooting medium (M.S. basal medium supplemented with IAA 1.00 mg/l + NAA 1.00 mg/l + activated charcoal 500 mg/l). The rooting percentage was the lowest in explants irradiated with 40 Gy. Rooting efficiency was severely affected when buds were collected at vegetative phase. Significant interaction between bud stages and gamma rays was noticed for days to root initiation and number of roots per culture. Gamma irradiation at 30 and 40 Gy significantly delayed root initiation at all stages of buds exposed. Significant reduction in number of roots/ culture, and root length was observed at higher doses of gamma exposure. The reduced rooting efficiency may be due to inhibition of cell division on account of chromosome damage.

ABSTRACT Rose (Rosa hybrida Linn.) is universally acclaimed as the ‘Queen of Flowers’. But this woody plant is considered less amenable to in vitro culture as compared to herbaceous plants. This is due to the fact that the establishment rose explants is difficult due to heavy phenolic exudation. In the present study, this problem was solved by a very simple and cost effective method. The cut ends of the explant were sealed with wax after thorough sterilization. Ten different explant collection periods with 20 different hormonal combinations in MS culture medium were taken for establishment. The winter dormancy period (Mid- December) was considered best for explant collection and showed 85% survival. Amongst the hormonal combinations 5mg/l BA coupled with 0.05mg/l NAA was considered the best. The average number of leaves (7.5) and average shoot length (7.3 cm) was also highest in this treatment, 15 days after culture establishment.

ABSTRACT Haploids are of great importance in breeding programmes for the production of homozygous diploid lines. This technique reduces the time span in developing new varieties and fixes the traits including recessive ones in the homozygous condition. The haploid embryos or doubled haploid plants could be used in mutation breeding, genetic engineering, biochemical and physiological studies. Isolated microspore culture for the production of haploids has not yet been reported in Hevea brasiliensis. Hence the present work was initiated with the view of producing haploid plants by the culture of isolated pollen grains. The flower bud of the clone RRII 105 at the late uninucleate to early binucleate stage was used for culture initiation. Different pretreatment solutions were tried at different temperatures (4°C, 27°C and 33°C) with the macronutrients of N6 media. Three kinds of callus induction media were used viz. N6, WPM and Hunters medium supplemented with myoinositol (200 mg-1), glutamate (150 mg-1), phenyl acetic acid (7.0 mg-1), sucrose (70 g-1) and growth regulators. Induction medium include moving liquid, static liquid and semi- solid medium. The ovary co-culture was also tried in the induction medium to increase the frequency of callus formation. Pretreatment of the anther with 0.3 M mannitol at 33°C promoted the division of microspores. Static liquid cultures improved the formation of multicellular microspores compared to semi-solid or moving liquid cultures. N6 basal medium supplemented with 2.0 mg/l 2.4-D and 1.0 mg/l KIN induced microcalli formation. When the ovary co-culture was included in the callus induction medium, the rate of cell proliferation was increased considerably and the duration of callus formation was reduced from 3 months to 14 days. The results of the present study throws light towards the possibility of regenerating doubled haploids in future.

ABSTRACT Protoplast isolation of Piper nigrum L.and Piper colubrinum Link. was attempted using axenic leaves at two different osmotic potentials viz. 0.60 and 0.65 molar (M) osmoticum. In Piper nigrum, highest protoplast yield (6.90 x 104 protoplasts ml-1) was observed in the enzyme combination 1.40 per cent cellulase and 0.310 per cent pectinase while in Piper colubrinum, highest protoplast yield (13.0 x 104 protoplasts ml-1) was observed in the enzyme combination 1.0 per cent cellulase and 0.217 per cent pectinase during 21 h of digestion. In both the species, 0.6 M osmoticum was found to be optimum to maintain the osmotic potential of the isolation solution. Filtration-centrifugation technique was found to be superior in purifying the protoplasts of Piper species compared to sucrose floatation method. Centrifugation at 1000 rpm for three minutes was found to be best in purifying Piper nigrum protoplasts. For purifying Piper colubrinum protoplasts, 600 rpm for three minutes was found optimum. The protocol developed for protoplast isolation in Piper species can be used for biotechnological means like somatic hybridization between the two and gene transfer to develop foot rot resistant black pepper lines.

ABSTRACT Mucuna pruriens (L.) DC. is one of the important medicinal plants that contains L DOPA (L-3,4 dihydroxy phenylalanine) which is the precussor for the neurotransmitter dopamine that plays an important role in curing Parkinson?s disease. To accommodate the huge demand for L DOPA, in vitro production of the drug using cell cultures is felt essential. Hence, an experiment was carried out with completely randomized design with seven treatments viz., T0 - Control; T1 - MS + 2 mg l-1 NAA; T2 - MS + 2 mg l-1 IAA; T3 - MS + 2 mg l-1 2,4-D; T4 - MS + 2 mg l-1 Kinetin; T5 - MS + 2 mg l-1 BA; T6 - MS + 1 mg l-1 IAA + 1 mg l-1 BA and 3 replications. Among the various explants investigated stem bits registered the least days (12.38) for callus induction. The stem bits responded well with better callusability (78.57 per cent), relative growth rate (2.57) and callus index (249.16). MS medium + 2 mg l-1 2,4-D was found to be the best for inducing callus in various explants namely stem bits, leaf bits and root bits. L DOPA accumulation was more in cell suspension cultured in liquid MS medium with 4 per cent sucrose + 1 mg l-1 IAA + 1 mg l-1 BA and that was confirmed by means of HPLC.

ABSTRACT Ophiorrhiza mungos is a rich source of the potent antineoplastic therapeutic agent Camptothecin, which is effective in the complete remission of lung, breast, uterine and cervical cancer. With increasing demand, most of the plants have been indiscriminately exploited from their natural habitat for the isolation of this valuable therapeutic agent. The biotechnological application such as plant tissue culture seems to be a viable option for the production of this high value therapeutic compound without destroying the natural flora. For this purpose various tissue culture techniques have been established to enhance the production of this compound from different parts of O. mungos. The present study revealed that the Physical elicitation with varying doses of irradiation is significantly higher (0.87mg/ g dry weight) than that obtained by chemical elicitation using methyle jasmonate (0.41mg/ g dry weight) compared to control. Biotic elicitors like chitin, chitosan, fungal and bacterial extracts were also used and considerable increase in the production of Camptothecin was obtained in all these cases. Permeabilization with Tween 20 found to be enhancing the exudation of camptothecin (0.021 mg/ml) compared to control (0.0003mg/ml). Hence, the present study gives an alternative for large scale production of camptothecin without affecting the natural flora in a cost effective method.

ABSTRACT Tinospora cordifolia, a versatile medicinal species with antipyretic and immunomodulatory properties is one of the indigenous sources of the therapeutically valuable alkaloid, berberine. However, the plant contains only meagre amounts of this alkaloid. Hence upgradation of the content of berberine in Tinospora, through biotechnological interventions assumes importance. Four week old leaf, petiole and stem derived calli of two ecotypes of Tinospora viz., Vellanikkara ecotype and Madurai ecotype, subcultured on to Murashige and Skoog (MS) medium, supplemented with various growth regulators, were incubated in light and screened for the presence of berberine employing thin layer chromatography. MS medium with NAA @ 2 mg l-1 supplemented with BA or kinetin, each at 2 mg l-1, was identified as the basal production medium for in vitro production of berberine, yielding 7.55 μg and 7.36 μg berberine respectively, per gram of calli. Calli produced from stem segments registered maximum amount of berberine compared to leaf and petiole derived callus cultures. Increasing levels of sucrose in basal medium did not have a favourable influence on the expression of berberine in the test calli. Reducing nitrate and phosphate content of the basal medium to half their strength resulted in enhanced berberine production in in vitro cultures of both the experimental ecotypes. Favourable influence of precursor feeding on upgrading the content of berberine in leaf and stem derived calli of both the ecotypes of the experimental species was also confirmed in the study.

ABSTRACT Cassava, sweet potato and yams are root and tuber crops propagated through vegetative parts. The CTCRI is maintaining a large collection of (3985 accessions) these crop germplasm in field gene banks. Studies were conducted to transfer these collections to in vitro storage so as to clean off systemic infection and protect them from loss due to biotic and abiotic stress. Suitable media protocols, method of surface sterilization, inoculation and incubation were developed for these different crop species in order to establish initial cultures, for their multiplication and storage under slow growth. Meristem, shoot buds and tuber sprouts were used as propagules for culture initiation.Murashige and Skoog (1962) medium was found suitable for all the species studied with variations required in the concentration of growth regulators (NAA, BA and GA ) for meristem culture and micropropagation. Slow growth of cultures was induced using osmotic retardants like mannitol and sorbitol. Addenda like silver nitrate and activated charcoal was found effective in reducing leaf chlorosis and preventing leaf shedding, thereby contributing to culture life and regeneration capacity. The slow growth media developed were effective in prolonging the subculture period to 18 months (cassava), 10 months (sweet potato), and 20 months (yams). The studies have helped in establishing 400 accessions of cassava, 272 accessions of sweet potato and 166 accessions of yams in In vitro Active Gene Bank. The results are presented and discussed.

ABSTRACT Cultivated turmeric consists of fertile short duration types with a chromosome number of 2n=84. In vivo crossing was done between two short duration types VK 70 and VK76 to develop a high yielding variety with high curcumin and curing percentage. Seed set and seed development was obtained by in vivo stigmatic pollination. Hybrid seed germination was obtained under in vitro in dark on moist filter paper and the seedlings were micropropagated to establish a population of six plantlets /seedling with in a period of 10 months. The multiple shoots were obtained in the medium of half strength MS + 3 per cent sucrose+ BA 2.5 mg l-1+ NAA 0.5 mgl-1. Coupling conventional breeding with tissue culture reduces the breeding time.

ABSTRACT Protoplast isolation in orchids is considered to be a valuable tool for introduction of desirable traits through somatic cell fusion. The present work on the protoplast in Dendrobium var.sonia is undertaken with a view to standardize protocol for isolation and regeneration. Tender leaf mesophyll tissues of one-month-old Dendrobium var.sonia were used as explant for protoplast isolation. The leaf tissues were treated with enzyme mixture of cellulase 1per cent (Onozuka -R from Trichoderma viride) and macerase 0.25per cent (from Aspergillus niger) for 16 hours. Protoplast from the Dendrobium var. sonia was successfully isolated and regeneration of shoots and roots were obtained in MS medium containing BA 5mg l-1+NAA 1mg l-1.The plantlets were hardened in a span of five months.

ABSTRACT Aprotocol for in vitro clonal multiplication of Dendrobium var. Betty Ho is reported. Aseptic shoot tip explants cultured in MS medium containing Benzyl Adenine (5 mg l-1) and casein hydrolysate (1000 mg l-1) recorded a maximum multiplication rate of 10 fold in 30 days. Explant cultured in LS) medium supplemented with 1-napthalene acetic acid (0.1 mg l-1) and Kinetinetin (1 mg l-1) developed a maximum of 8.3 shoots in 30 days. Shoots thus obtained were rooted on MS medium supplemented with BA (0. 1mg l-1 ) and NAA (0.5mg l-1). Rooted plantlets after hardening were transferred to green house.

ABSTRACT In cocoa (Theobroma cacao L.) there are successful reports on the production of shoots from nodal segments under in vitro,the rooting was always inconsistent and mortality during hardening stage was very high. In the present study 20-30 per cent rooting was obtained in half strength MS supplemented with activated charcoal. Maximum rooting was obtained when the shoots were pretreated with IBA 5000 mg l-1 for three seconds followed by culturing in the media.

ABSTRACT Plants as a source of medicine are of special importance in the countries like India which has well developed traditional system of medicine which derive more than 90 per cent of medicaments from higher plants. Several of these also constitute important ingredients of Allopathic medicines. Most of the requirements for the medicinal plants are met from wild sources. But due to the changing ecological factors in the forests and unserupulous collection of medicinal plants, the wealth of medicinal plants is getting depleted and some types have already become extinct. The collection of these plants from the forests cannot cope up with the ever increasing and changing demand from the pharmaceutical, perfumery and cosmetic industry. In order to provide regular and sustained supply of medicinal plants, it is essential now to domesticate and systematically cultivate these plants for which large scale availability of planting material has to be ensured. In this context, in vitro multiplication protocols were standardised for rare, valuable and endangered medicinal plants such as Holostemma ada-kodien, Trichopus zylanicus, Aristolichia indica and Terminalia chebula. The study indicates the feasiblity of rapid multiplication of these plant species using in vitro techniques and the standardised protocols could be used to generate large number of true to type protocols within a short time.

ABSTRACT Medicinal plants are the most important source of life saving drugs for the majority of the world’s population. Therefore the biotechnological tools are important to select, multiply and conserve the critical genotypes of medicinal plants. With the increase in the demand for the crude drugs, the plants are being overexploited, threatening the survival of many rare species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. Maintenance of germplasm in the green house or in the field has many difficulties and so there is a need for the in vitro conservation of these plant germplasm. In the present paper we report the in vitro conservation of two medicinal plants and each of it has its own medicinal value. The tuberous roots of Holostemma are medicinally important and are utilized as a major ingredient of the drug ‘jivanthi’. Tylophora is used as a herbal medicine and the plant has been traditionally used as a folk remedy for the treatment of bronchial asthma, bronchitis and dermatitis. Shoot buds of these plants were cultured in Murashige and Skoog’s (1962) medium with different strengths and with different concentrations of sucrose and mannitol. The different treatments induced varied response with respect to the slowing of growth of cultures. Sucrose was found essential in combination with mannitol to induce maximum reduction in the growth of the plant tissues. The cultures could be maintained without sub culturing for over one year and resumed normal growth on transfer to mannitol free medium. This method of conservation has many advantages.

ABSTRACT Cashew (Anacardium occidentale L.) is a woody perennial tree of tropical origin, belonging to the family Anacardiaceae. Cashew is being cultivated in India, Brazil and Africa countries. It is well known for its food, noon food and medicinal uses. In vitro clonal propagation of cashew, which is an effective method for large-scale multiplication of trees was tried. Cashew plantlets were regenerated from shoot explants of 2 - 3 year old grafted plants, which were regularly sprayed with Bavistin 0.1 per cent at weekly intervals. Among the different surface sterilization treatments used, a combination of antioxidants, antibiotics and fungicides was found to be better with around 40 per cent contamination free cultures being obtained. MS medium containing full strength macro elements supplemented with Kinetin (5 mg l -1), NAA (1mg l -1) and Brassinolide (0.1 mg l -1) was found to be best for shoot proliferation and shoot elongation. Micro shoots were rooted in vitro at a frequency of 70-80 per cent, when cultured for 4-8 days in quarter MS liquid medium supplemented with IBA (1mg l -1) after the pulse treatment with IBA (100 mg l -1) for two minutes. The average number of roots ranged from 3 - 5 with an average length of 1.63 cm. About 50 - 60 per cent of the rooted microshoots survived wearing and produced healthy and vigorously growing plants, which were transferred successfully to the field.

ABSTRACT Asuccessful micro grafting technique in Cashew (Anacardium occidentale L.) was developed using in vitro germinated seedling as root stock and shoots from stage II cultures as scions. Grafting success was dependent on the method of grafting, size of the scion and age of the root stock. Among the methods tried, side and cleft grafting were found to be suitable. Due to the ease of operation and faster union, side grafting was preferred. High levels of graft union were obtained when 2 - 3 cm long shoots from stage II cultures were grafted on to 8 - 9 days old in vitro raised root stocks in full strength MS (Murashige and Skoog) medium supplemented with Kinetin (2.5 mg l-1), NAA (0.5 mg l-1) and Brassinolide (1mg l-1). The very high growth of grafted scion was obtained, when the micro grafts were transferred into MS medium containing full strength macroelements supplemented with Kinetin (5mg l-1), NAA (1mg l-1) and Brassinolide (1mg l-1). The micro grafts after a process of hardening could be successfully transplanted into soil. Micro grafting techniques standardized could be used for rejuvenation of shoot explants of mature tree.

ABSTRACT In vitro pollination and fertilization could be a tool for heterosis breeding of triploid turmeric cultivars, which are almost non-seed setting naturally. Investigations were undertaken to optimize media combinations for the development of in vitro pollinated ovules to mature seed .The half strength MS medium supplemented with auxins and cytokinins was superior to full strength MS. The supplementation of ½ strength MS medium with either auxins or cytokinins at lower concentrations induced some ovule development. The ovule development was more when auxins and cytokinins together were given. The combination of ½ MS +3% sucrose +NAA 0.5mg l -1 +BAP 1mg l-1 showed maximum ovule swelling in cultures. The combinations of BAP 4mg l-1 with IAA 0.2mg l-1 produced callusing instead of ovule swelling.

ABSTRACT Lack of high yielding varieties, variation among progenies through seedling propagation and transmission of viral diseases through sucker propagation together contribute to the decline in productivity of large cardamom (Amomum subulatum Roxb). To increase the productivity of this crop, planting material raised through tissue culture techniques from few selections having higher productivity was considered. From the study, it was revealed that TC plants were superior to conventionally propagated plants due to vigorous growth during juvenile phase, precocity in the yield and higher productivity compared to that of open pollinated seedlings.

ABSTRACT Tamarind, an economically important multipurpose tropical tree, is a species of choice for sustainable development of wasteland due to its hardy nature and adaptability to various agroclimatic conditions. However, there are very few reports on in vitro regeneration of tamarind. In an attempt to induce caulogenic response, effect of different cytokinins on nodal explants from mature trees was tested. The study extended to seedling explants, revealed that 6-Benzylaminopurine (0.08 - 44.39μM) and Zeatin (0.09 - 9.12μM) induced multiple shoots with callusing at the base of explant. Kinetin (0.09 - 9.29μM) and 6-γ,γ- dimethylallylaminopurine (2iP) (0.09-9.84μM) remained ineffective for multiplication of shoots and callusing was noted at the cut end of explant in contact of medium. Thidiazuron (TDZ) (0.09- 9.08μM) failed to induce morphogenic response and triggered dedifferentiation in the basal cut end. This led to profuse callusing in the explant and restricted morphogenesis. To avoid wound in the explant, tamarind seeds were cultured in medium containing TDZ. Seedlings were germinated in medium with or without Thidiazuron (4.54, 9.08, 13.12, 18.16μM). Differentiation of the apical meristem to shoot was restricted and proliferation of the cotyledonary node meristem was triggered. Large number of meristematic buds appeared in a radial pattern around the node. The meristematic activity subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. These buds differentiated to form shoot primordia and subsequently to shoots in medium devoid of growth regulator. Plants developed by micrografting of these shoots on seedling derived rootstocks, survived in soil.

Introduction At the Banana Research Station, Kannara activities focused on Musa germplasm conservation and evaluation are in progress. The aim of the project is to collect and conserve wild and cultivated germplasm representative of the diversity existing in the Musa genepool for evaluation and subsequent use in breeding programmes and to make available selected superior material to the farmers of Kerala. The center presently holds a Musa germplasm of 256 accessions in its field gene bank. These accessions have been characterized and evaluated as per INIBAP/IPGRI Musa descriptors and a database developed. In a vegetatively propagated crop like banana, in vitro culture is widely employed in germplasm introduction and conservation and is an essential requirement for the safe movement of germplasm (Sharrock and Engels, 1996). By this method, germplasm material is readily propagated, thereby facilitating the dissemination of disease free plants. Since 2001, efforts have been made at Banana Research Station, Kannara to introduce forty new exotic accessions employing in vitro culture for conservation and evaluation. These are material from the global Musa germplasm collection maintained by International Network for the Improvement of Banana and Plantain (INIBAP), France and supplied by National Bureau of Plant Genetic Resources (NBPGR), New Delhi.

ABSTRACT Young buds of ginger (Zingiber officinale Rosc.) were isolated and cultured on MS media with BA 3 mg l-1 induced shoot organogenesis. Young buds from aseptically grown cultures were evaluated for the effectiveness on embryogenic callus production on different concentration of auxin in combination with cytokinin. Among the plant growth hormones tested a combination of 2, 4-D 1 mg l-1 and BA 0.5 mg l-1 was most effective in inducing and maintaining embryogenic cultures. The somatic embryo germinated on half strength MS medium supplemented with 3 per cent w/v sucrose and 3 mg l-1 BA.

ABSTRACT Central Plantation Crops Research Institute at Kasaragod maintains largest collection of coconut germplasm accessions representing different geographical regions. So far, morphological and isozyme markers were employed for germplasm characterization. To overcome the difficulties associated with the morphological markers, DNA based molecular markers are presently employed for germplasm characterization. In the present study, Inter Simple Sequence Repeat (ISSR) markers are utilized to estimate the genetic diversity among South East Asian germplasm accessions. Nineteen ISSR primers were used to amplify 8 germplasm accessions (4 palms per accession referred as population). The PCR products were electrophoresed in 1.80 % agarose gels. Binary data were analyzed using the software POPGENE ver. 1.32. Dendrogram was constructed based on Nei’s unbiased measure of genetic distance. Nineteen ISSR primers detected a total of 85 polymorphic markers across 32 individuals belonging to eight populations. The average genetic distance among the eight populations ranged from 0.7908 to 0.9327 with a mean of 0.8540. Of the 28 pair wise comparisons the accessions Laguna Tall and Kongthienyong Tall (KTYT) showed the highest genetic identity (0.9327). The lowest identity (0.7908) was between San Roman Tall (SNRT) and Philippines Dalig Tall (PDLT). The Shannon’s index ranged from 0.1615 to 0.299. Diversity parameters viz. number of effective alleles, gene diversity, number of polymorphic markers were calculated for each population. The dendrogram constructed based on the genetic distance revealed clustering of Kongthienyong Tall (KTYT) and Laguna Tall (LAGT) while Philippines Dalig Tall (PDLT) was positioned separately. In the genetic improvement programmes the diverse accessions can be used in hybridization for increased heterosis.

ABSTRACT DNA based molecular marker technologies have been used extensively to study the genetic relationships and to develop markers for identification of cultivars/varieties in various crops. The present study is an attempt to assess the variability and relatedness among 49 varieties/ accessions of black pepper (Piper nigrum L) based on the data generated by both RAPD and AFLP analyses. The similarity matrix was subjected to cluster analysis and dendrogram generated using the software Ntsyspc 2.1. The dendrogram revealed an average similarity of 63 per cent among accessions. Two selections from the variety Karimunda, namely Sreekara and Subhakara, together formed a single cluster with almost 92 per cent similarity. The dissimilarity observed between the varieties Panniyur 1 and Panniyur 3 the progenies of the same parentage, Uthirankotta and Cheriyakanyakadan was only 18 per cent. Based on RAPD analysis 34 varieties were grouped into 5 clusters. Distinct clustering was not observed in AFLP analysis. The results obtained were correlated with the taxonomical interrelationships reported for these varieties. The overall study has thus helped in understanding the relationship among the local cultivars and released varieties.

ABSTRACT Grape is a commercially cultivated fruit crop in India. As a result of large-scale introduction, the origin and authenticity of many grape varieties is unclear and the subject of some controversy. Many of the varieties were mixed up and now have led to confusion regarding their correct identification. To explore the genetic relationships between different grape varieties and to analyze their diversity, molecular marker technique has proved to be useful. In the present study, 34 grape varieties have been characterized using Inter Simple Sequence Repeat (ISSR) markers. Out of 93 ISSR primers screened initially, 11-showed good polymorphism. Total 174 bands were obtained, out of which 145 were polymorphic. The similarity indices were calculated using DICE coefficient and NTSYS Pc 2.1 software. Cluster analysis resulted in the formation of two main clusters. One, of the varieties belonging to Vitis vinifera and other of V. labrusca. Varieties belonging to V. vinifera appeared more diverse by forming distinct sub-clusters based on their colour, flavour and seeds. Out of 34 varieties screened, 10 varieties are grouped in white berries cluster and 15 in coloured (red /black) berries cluster. Three white varieties- Italia, Queen of Vineyard and Thompson seedless are grouped into cluster of coloured varieties. The cluster of labrusca varieties showed homogeneity and had five varieties except Dakh, which belongs to vinifera. It may be wrong introduction. Concord separates initially from all other varieties. Incidentally, Concord is a pure selection from V. labrusca, while varieties like Bangalore blue, Black Muscat, Catawba and Muzzafar Nagar in labrusca group may be the hybrids of labrusca x vinifera. The current study thus revealed that genetic relationship among grape cultivars could be assessed using ISSR markers.

ABSTRACT It is essential to prospect wild Zingiber species for soft rot resistance, since the cultivated ginger is monotonously susceptible to this disease. We evaluated the genetic diversity of Zingiber species and their response to Pythium aphanidermatum, the causal agent of soft rot. Diversity of 21 cultivars and three wild accessions of Z. officinale, 84 accessions of five wild species: Z. zerumbet, Z. cernuum, Z. neesanum, Z. purpureum and Z. wightianum and one accession of Hedichium flavescens were examined using AFLP markers. No polymorphism was detected at more than 500 AFLP loci screened in ginger cultivars, which to our knowledge, is the most extreme case of genetic narrowness in a species. Analysis revealed high variability in Z. cernuum and relatively low variations in other species. Strong correspondence was evident between the genetic diversity of a species and its mode of propagation. Selected accessions of Zingiber species were inoculated with Pythium zoospores and the disease progression was scored. The response to Pythium varied from total susceptibility in Z. cernuum and Z. officinale to hardy behavior among Z. zerumbet accessions. The present study provided an estimate of genetic diversity of Zingiber species common in Kerala. Species relationships and population genetic structure were discussed. The monotypic nature of ginger cultivars may hinder the possibility of their identification based on their nucleic acid profiles. The present study identified Z. zerumbet as a potential source of soft rot resistance, which will trigger off the application of genomics tools to access this trait in order to genetically engineer soft rot resistance in ginger.

ABSTRACT In last twenty years, molecular biology has revolutionized conventional plant breeding techniques in many areas. The use of molecular markers in conventional breeding techniques has also improved the accuracy of selection and allowed breeders to produce varieties with combined traits that were difficult before the advent of DNA technology. Among vegetables, onion (Allium cepa L.) is the most important cash crop of India. Onion productivity in India is much lower (11.38 tones / ha.) compared to other countries (44.1 tones / ha. in Japan). Purple blotch disease (PBD) caused by Alternaria porri (Ellis) Ciferri decreases the yields by up to 50 percent. Genetically resistant onion varieties are the most economical, simple and eco-friendly mode of disease management. The advent of mo- lecular markers provided the opportunity to understand the genetic resistance more pre- cisely. In this paper we present our successful efforts in developing PCR based markers linked to purple blotch disease resistance, so that such markers can be easily integrated into PBD resistance breeding program in onion. Onion germplasm was screened for PBD resistance. Line PBR 287 was identified as a good source of resistance and the genetics of inheritance of resistance in this line was worked out. The protocols for isolating good quality DNA and PCR amplification conditions were standardized. 37 polymorphic mark- ers were identified between resistant and susceptible parents. Crosses were made between resistant and susceptible parents. Parents, F2 and individual F2 progenies were subjected for analysis of genetics of resistance and RAPD marker analysis. F2 individuals segregated in 3:1 ratio for resistance. DNA from resistant and susceptible F2 individuals was pooled separately for Bulked Segregant Analysis (BSA). BSA identified one RAPD marker of 900bp being linked to resistance. Further Chi-square test for detecting the deviation of segregation of this 900bp marker confirmed segregation with classical Mendalian ratio 3:1 for a monogenic trait. Identified marker is of great practical utility to the plant breeders to screen large number of populations for purple blotch disease resistance at the early stages of the crop even in absence of the pathogen.

ABSTRACT The investigation aimed at to examine the diversity among nineteen wild and cultivated germplasms of the genus Vigna namely, V. trichuriensis, V. hainiana, V. radiata, V. aconitifolia, V. minima, V. sinensis, V. radiata var. sublobata, V. radiata var.setulosa, V. umbellata, and allopolyploid of BR3x T9 at DNA level follwing Random Amplified Polymorphic DNA (RAPD) technique. Ten random decamer primers were used in amplification reactions, five of that were reproducible and clear. The five primers constituted 55 bands of, which 9 are polymorphic. The similarity index based on 0 and 1 matrix of banding was calculated and dendrogram following UPGMA was constructed. The germplasms formed altogether 4 clusters on the basis of 0.375 linkage distance. The first and second cluster consisted of single genotype of V. hainiana. and V. minima respectively. The third cluster comprised wild and cultivated types of V. sinensis. The 4th cluster on the otherhand, consisted of genotypes belonging to V. radiata, V. radiata var. sublobata, V. mungo, V. radiata var. setulosa, V. trichuriensis and allopolyploid of BR3 and T9. The wild forms of different species were found to exist in the same cluster as their cultivated form. The three mungbean genotypes namely B105 (V. radiata), B1 (V. radiata) and BR3 (V. radiata )are crossable within themselves and they belonged to the cluster -IV.The intercluster cross between different clusters would like to generate new genotypes with wide variability. Based on limited range of accessions tasted in the present investigation, the approach holds promise for the classification of Vigna germplasm, identification of Vigna and marker assisted breeding in Vigna.

ABSTRACT DNA fingerprinting is increasingly being used for identification of varieties, quantification of similarity between varieties and study of genetic diversity available in the population. This is all the more important in crops where morphological variability is less or one variety is known by different names or different varieties are known by same name. DNA fingerprinting using RAPD method has been standardized for tapioca. Dellaporta method has been used for extraction of DNA and amplification was done following William’s method. This technique has been used for molecular characterisation of accessions and identification of duplicates in the germplasm maintained at Central Tuber Crops Research Institute, Thiruvananthapuram. This technology has been extended for DNA fingerprinting of released varieties and also some of the elite breeding lines. Eleven varieties released from the institute were fingerprinted using a few primers, which showed high polymorphism in tapioca. One single primer gave distinct banding pattern for all the 11 varieties and it was capable of earmarking each variety. Individual variety was finger printed for 20 different primers and the results were documented. This technique is being extended to promising land races as well. DNA fingerprinting will be useful for protecting Plant Breeders’ Right as well as Farmers’ Right in the new Patent Regime.

ABSTRACT Hevea brasiliensis, the main source of natural rubber (NR) is commercially propagated through bud grafting. Since Hevea brasiliensis is a cross-pollinated tree crop, seeds obtained are highly heterozygous. Earlier, plantations were grown from unselected seeds. But due to poor yield and large tree - to - tree variations in growth and yield in such plantations bud grafting with desirable scions is now being adopted for commercial planting. Genetically divergent seedlings grown from open pollinated seeds are used as rootstocks to graft the selected scion. This can lead to stock - scion interactions. Large intraclonal variations observed in growth and yield of bud-grafted clones of Hevea are attributed to the genetic heterogeneity of the rootstocks. Monoclonal and polyclonal seed gardens have been established in Kanya Kumari District of Tamil Nadu for producing good quality seeds. Monoclonal seeds are collected from monoclonal seed gardens where only one clone is planted in a vast area without any interference from pollen grains from other clones. Polyclonal seeds are collected from polyclonal seed gardens where different clones are planted in an area and the seeds are fertilized by pollen grains from all the clones. It is a general assumption that seedlings grown from polyclonal seeds may be highly heterogeneous than seedlings grown from monoclonal seeds. The objective of the present study was to find out the reliability of this assumption by studying the genetic variations existing among the polyclonal and monoclonal seedlings. RAPD analysis of leaf samples from polyclonal and monoclonal viz, RRIM 600, PB 86 and GT1 seedlings was carried out using ten Operon arbitrary decamer primers that gave informative amplification. The results showed that there was no significant difference in the genetic variations among the polyclonal and monoclonal seedlings.

ABSTRACT India is the largest producer of banana, contributing to 17.3 per cent of the global production. In Kerala, it is the leading fruit crop, being cultivated in an area of 99,412 ha with an annual production of 7,31,650 tonnes. Biodiversity in the cultivars of banana, which consist of triploids, is complex especially in Nendran. Nendran belonging to Musa AAB plantain subgroup is the leading banana cultivar of the Kerala. in Kerala, several landraces are available, cultivated in different parts, known in different local names. Twelve Nendran ecotypes of banana belonging to AAB genomic group were collected from Banana Research Station, Kannara, Banana Nursery Farm, Palode and Instructional Farm, College of Agriculture, Vellayani to ana lyse genetic divergence using RAPD marker and morphological traits. Out of the forty one decamer primers screened for RAPD analysis, 34 could produce amplification. Twenty five primers showed high level of polymorphism. Finally, six of the most promising primers viz., OPA-01, OPA-03, OPA-13, OPB-04, OPB-10 and OPB-12 were used for RAPD analysis. Statistical analysis for the morphological traits and RAPD marker were conducted using the software programme NTSYS pc version 2.02e. The matrix average taxonomical distances (similarity coefficient) among the ecotypes ranged from 0.59 to 1.00 in RAPD profiles and morphological profiles (dissimilarity coefficient) ranged from 2.82 to 11.21. In the dendrogram (RAPD profiles), Koonoor Ethan showed the maximum genetic divergence. Quintal banana and Zanzibar came under a single clusters at a distance of 0.79 and clustered together at a distance of 0.82. Both the ecotypes showed next higher genetic divergence among the Nendran ecotypes. In the case of morphological traits, the same trend could be observed. Koonoor Ethan showed the maximum genetic divergence at a distance of 11.21 and Quintal banana showed next higher genetic divergence at a distance of 9.11. Koonoor Ethan, Quintal banana and Zanzibar were distinct ecotypes among the Nendran ecotypes of Kerala

ABSTRACT Random Amplified Polymorphic DNA (RAPD) markers were used for characterizing eleven banana (Musa AAB Plantain Subgroup) clones using 40 decamer primers. Eight primers, which produced reproducible banding patterns, yielded 42 scorable bands with an average of 5.25 bands per primer. The amplification products ranged in size from 400 to 1500 bp. A genetic similarity matrix was constructed using the Jaccard’s coeffecient method. The pair wise similarity coefficient values varied between 0.3333 and 0.9355. Based on the coefficients, distances between the clones were computed using SYSTAT software package. In the dendrogram constructed by the nearest neighbour (single- link) method (Krzanowski, 1988), all the eleven clones were found grouped under five clusters. Attu Nendran, Changanasseri Nendran, Changazhikodan, Kaliethan, Koonoor Ethan and Quintal Banana formed the largest cluster. Manjeri Nendran and Myndoli formed the second cluster. Padalamurian, Mysore Ethan and Zanzibar formed three separate clusters. Zanzibar, belonging to the Horn Plantain group was different from the rest of the clones.

ABSTRACT The study was conducted to fingerprint and to analyze genetic diversity among 20 genotypes. of Psidium guajava and two species P. friedrichsthalianum and P. cattleianum using random amplified polymorphic DNA. Genomic DNA was isolated using modified CTAB method and polymerase chain reaction was carried out using standard protocols. In all 41 primers were used, 39 of which amplified 376 alleles with 347 polymorphic alleles. All the primers discriminated between two and more genotypes. Primers produced 2-16 alleles. The size of amplified DNA alleles ranged from 0.12-2.2 kilobase pairs. Polymorphic index content value was found to be maximum for primer OPB-12 (0.916). In the dendrogram all the 22 genotypes were grouped into two major clusters. Cluster 1 comprised of Spear Acid, Apple Colour and P. friedrichsthalianum, while Cluster 2 constituted rest of the 19 genotypes. Thus, RAPD markers detected a high level of polymorphism and can be used for breeding of improved guava varieties.

ABSTRACT An effort was made to characterize twelve ber genotypes using RAPD markers. Of the twenty random decamer primers tested, 17 generated polymorphic bands, while rest of the primers showed monomorphic bands. The primers that amplified all the 12 genotypes were OPC-20, OPD-1, OPD-3 and OPD-4. The size of amplified DNA fragments varied from 0.56-4.26 kilobase pair. In total, 109 bands were obtained, of which 85 bands were polymorphic, while 24 bands were monomorphic. For the cultivars tested between 2-9 bands were obtained for each primer with an average of 5.45 bands per primer. The highest number of bands (9) were generated by OPA-4 and OPD-3. With un-weighted group method using arithmetic mean (UPGMA) cluster analysis, the twelve ber genotypes fell into four major clusters. The pair wise dissimilarities of ber genotypes showed that genotypes G-9 and G-8 were close to each other. Maximum dissimilarity was observed between the genotypes G-8 and G-12.

ABSTRACT The discovery of polymerase chain reaction method of amplification of DNA is an important milestone in molecular genetics and used for multiple purpose such as construction of linkage maps, marker saturation of specific genome regions, analysis of genetic diversity, molecular phylogeny and cultivar identifications (Williams et al; 1990, Demeke, Adams and Chibbar; 1992, Welsh and Maclelland; 1990;, Obara-Okeyo and Kako; 1998, Sawhoon Lin et al; 1998 and Grunger et al; 1998.). 15 species of three morphologically distinct groups of Orchids belonging to the subfamily Epidendroideae and tribe Dendroebieae and Cymbideae were studied using random amplified polymorphic DNA (RAPD) technique. Genetic variability and relation between these 3 genera and 15 species were estimated based on band sharing and cluster analysis. A total of 227 distinct major RAPD bands of which 97% were polymorphic were generated from 15 arbitrary primers. The number of bands generated per primer ranged from 3 (Primer A-10) to 22 (Primer K-10 and N-17) with a mean of 14.8 bands per primer. Out of 227 bands generated 222 bands (97.8%) where polymorphic while only 5 bands were monomorphic. The primer resolving power was found to range between 2.00 (prime A-10) and 13.38 (primer k-10) when all the species were studied were taken into account. The molecular analysis grouped all the species into 5 groups. The polymorphic patterns generated by RAPD profiles showed different degrees of genetic relationship among the species studied and the RAPD marker were found to be an useful tool for detecting genetic variation within the species of three important genera of Orchids.

ABSTRACT Genetic relationships were estimated among twenty five ashgourd (Benincasa hispida (Thunb.) Cogn.) landraces of different types using PCR-based random amplified polymorphic DNA (RAPD) markers. The molecular survey of the ashgourd germplasm by RAPD using three random primers resulted in twenty scorable bands (average of 6.66 bands per primer) of which two were monomorphic and rest, 18 were polymorphic (90.0%). Pair-wise genetic similarities among the landraces determined using Jaccard’s coefficient ranged from 0.14 to 1.00. The dendrogram based on clustering matrix separated the landraces with morphologically distinct smooth textured fruits from waxy textured fruits and the former group showed less divergence than the latter. With in the group of waxy textured fruits, limited variation was detected among landraces with small and medium sized fruits. Further, morphologically similar landraces with large fruits form distinct clusters with in the major clusters. The study demonstrated the usefulness of the RAPD marker analysis in effectively assessing the genetic relationship present among the ashgourd landraces examined.

Introduction Genetic markers have assisted in genomic characterization of various crops species (Rafalski and Tingey, 1993). Most of the genetic marker systems rely upon DNA sequence polymorphisms (Staub et al., 1996). Each system has unique advantages and disadvantages based on cost, experimental objectives and type of information desired. Regardless of its inherent methodology, the effective use of any marker system for genetic analysis requires reliability and consistency. The random amplified polymorphic DNA i.e. RAPD (Williams et al., 1990) or arbitrarily primed polymerase chain reaction (Welsh and McClelland, 1990) marker identifies DNA sequence polymorphisms. In this technique, segments of genomic DNA are amplified in a polymerase chain reaction i.e. PCR (Mullis et al., 1986) with random primers followed by agarose gel electrophoresis. This is simple, relatively inexpensive and is amenable to high throughput investigation. RAPD markers are usually dominant and polymorphisms are observed as the presence or the absence of DNA bands of a particular size. RAPDs are common molecular approaches employed in plant breeding, genotypic differentiation, gene linkage analysis, assigning evolutionary and taxonomic affinities and relatedness and DNA fingerprinting analysis (Lin et al., 1996).

ABSTRACT Genetic variability was assessed among twenty five distinct drumstick (Moringa oleifera Lam.) landraces using PCR based random amplified polymorphic DNA (RAPD) markers. Genomic DNA extracted from tender leaves was used for RAPD analysis. Thirty five primers were screened and based on the number of bands three primers were selected. The primers produced 34 bands, of which 58 % (20 bands) were polymorphic. The data were analysed using NTSYS pc 2.1 software. The similarity matrix constructed using Jaccard’s coefficient method was subjected to cluster analysis using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and dendrogram was generated. The pair wise coefficient values in the genetic similarity matrix among the landraces varied between 0.28 and 1.00. The dendrogram based on the matrix separated the landraces with distinct morphological characters and it was noted that the cluster formation was not fully in agreement with the geographical location. The study showed the use of RAPD markers in detecting genetic variability present among the drumstick landraces studied.

ABSTRACT There is a wide variability in the contents of the commercially important chemical compounds of Centella asiatica depending on the source / ecotypes of the plants. The glycosides found in Indian plants include brahmoside, brahminoside and minor amounts of asiaticosides, which vary in their relative contents depending on the ecotypes. An RAPD marker assisted study of ten accessions, collected from different geographical locations of Kerala, viz., Parumala (Kottayam District), Kottayam, Nagercoil (Tamil Nadu), Mananthala (Trivandrum District), Vlathankara (Trivandrum District), Ponmudi (High range, Trivandrum District), Angamaly (Ernakulam District), Punalur (Kollam district), Pattambi (Palakkad district) and Calicut, was conducted. DNA amplification of the accessions was studied using forty decamer primers, involving 43 cycles. Based on the number of bands four primers were selected. The least similarity coefficient of 0.71 was shown by the Pattambi accession, which formed a separate cluster with all other accessions. Nagercoil and Ponmudi accessions showed the highest similarity coefficient of 0.88 with each other.

ABSTRACT Exploitation of genetic resistance available in the local germplasm provides a rational solution to most deadly purple blotch disease problem associated with onion production in India. Keeping this in view, efforts have been made to characterize the identified sources of resistance available in the local germplasm. Three lines namely, MS-65-268, PBR-287 and Arka Kalyan-704 that were identified as resistant after screening, were subjected to Random amplified polymorphic DNA (RAPD) analysis for their precise identification and estimation of genetic relationship among them. Highly susceptible variety Arka Niketan was also included in the study for better interpretation. A total of 160 ten- mer random primers were used in the analysis, out of which, 41 exhibited polymorphism among the accessions. Out of these DNA profile with 5 primers namely, OPB02, OPB13, OPC09, OPC12 and OPF08 distinguished the three resistant accessions clearly from the susceptible one. On an average of 2.65 polymorphic, 11.65 monomorphic and 14.3 total bands were produced per primer. The amplified PCR products were of the range from 300 bp to 2 kbp and were consistent, unambiguous and repeatable. Further genetic dissimilarity analysis based on the Ward’s method using minimum variance algorithm and Principal Component Analysis (PCA) of these genotypes using the data generated from RAPD markers revealed high genetic diversity. Dendrogram clustered these four genotypes into two main clusters containing two each. PCA also confirmed high genetic dissimilarity. The least genetic dissimilarity (40.0%) was between PBR-287 and MS-65-268 where as the maximum (65.0%) was found between genotypes Arka Niketan-709 and MS-65-268.

ABSTRACT In oil palm there are three fruit forms viz. dura, tenera and pisifera. The commercial planting material is tenera produced by crossing dura and pisifera (D X P). Dura has thick shell and less economic part, where as pisifera is an abortic, female sterile type and cannot be reproduced in the normal ways. When tenera is selfed, segregation takes place in the monogenic Mendelian ratio; 1 dura: 2 tenera: 1 pisifera. Since all these palm types are morphologically alike, they can be confirmed only after fruiting, and verifying the internal fruit morphology. Fruiting in pisifera is highly erratic and often prolonged to several years. Proper technique in identifying these palms has been puzzling the breeder all over. Hence, a shell thickness marker is very important for early identification of the oil palm varieties. An attempt has been taken to screen PCR primers and select the probable primers linked to the shell thickness in the present study. ‘Bulk segregation analysis’ approach was adopted for the study and DNA isolation was carried out from 25 dura and 25 pisifera palms. All the palms were developed from same tenera x tenera crosses. DNA from all the 25 duras and 25 pisiferas were pooled separately. Two pooled DNA samples were subjected to RAPD analysis with 50 random primers using the standard RAPD protocol. The amplified samples were electrophoresed and documented. Both the pooled samples showed same banding pattern with all the primers, except 10 primers showing differences. Since the genetic configuration of the pooled dura DNA was likely to be same as that of the pisifera pooled DNA, the primers showing difference in banding pattern were the putative primers linked to shell thickness. All the 10 primers would be used for amplification of individual dura and pisifera DNA for confirmation of shell thickness marker.

ABSTRACT Owing to paucity of available genetic / molecular markers, onion breeding still relies heavily on phenotypic selection methods. DNA markers can provide plant breeders genetic information for indirect selection of plants. All the DNA based procedures require high quality plant genomic DNA. Abundance of polysaccharides and phenolics present in plant tissue often impede PCR amplification. The polysaccharide contaminants can also produce downstream problems by inhibiting many of the enzymes that are employed in subsequent analysis and cloning. RFLP based markers in Allium species pose certain problems due to relatively large size of the genome compared to most other herbaceous crops. As an alternative, RAPD would prove advantageous in genetic analysis of Allium. Here we present our results on optimization of DNA extraction in onion to get amplifiable quality of DNA for further RAPD analysis. Leaf samples were collected from the onion germplasm maintained at Indian Institute of Horticultural Research (IIHR), Bangalore. Dried leaf protocol was suitably modified to obtain fair quality of DNA. After varying each of the ingredients of the extraction buffer we arrived at the optimal extraction buffer, which resulted in good quality DNA. Quality was analyzed by subjecting the DNA to agarose gel electrophoresis and restriction digestion. Extracted genomic DNA was quantified with ‘Hoefer’s Dynaquant’ (Pharmacia, USA). The DNA quantity ranged between 160-2400mg/gm of dried powder. The standardized 25ml PCR mixture contained template DNA (25ng), 5 pmol of primer (Operon Tech. Inc., Alameda), 2mM MgCl2, I unit of Taq polymerase (Bangalore Genei), with 10x buffer along with 200 mM each of dNTPs. Amplifications were performed in thermal cycler (MJ Research, PTC 100) for 45 cycles after an initial denaturation of 95°C for 3 minutes. In each cycle denaturation for one minute at 95°C, annealing for one minute at 35°C and extension for two minute at 72°C was programmed with final extension step at 72°C for 5 minutes. This fine tuned protocol resulted in clear, informative RAPD profile.

ABSTRACT The present investigation was undertaken to standardize the DNA extraction methods, to optimize the PCR conditions and generate RAPD’s to study the genetic variability in chrysanthemum. Total genomic DNA was extracted from freshly emerged leaves of chrysanthemum cultivars (Flirt, Nagpur Red, Snow Ball) using different DNA extraction methods (CTAB method, SDS potassium acetate method, SDS miniprep method and urea phenol method). The highest amount of DNA (349μg/g of leaves) was extracted using CTAB method, while it was lowest (130 μg/g of leaves) in SDS miniprep method. The absorbance ratio was high for DNA extracted by CTAB method (1.82) followed by SDS- potassium acetate (1.65) and SDS miniprep (1.60) methods. For standardization of PCR ampl ification conditions, optimization was carried out to determine the optimal concentrations of template DNA, MgCl2, Taq DNA polymerase and annealing temperature. In general, either no products or products with inconsistent bands were obtained when the concentration of template DNA was 25 ng or MgCl2 was 1.0 mM with high annealing temperature of 45°C.

ABSTRACT Of the several PCR techniques developed during the last two decades, random amplified polymorphic DNA (RAPD) offers a simple and economical means of genotype characterization. The information obtained through RAPD characterization is extensively used for the identification of genotypes, screening of duplicates, assessing genetic diversity and monitoring the genetic stability of conserved germplasm. The present study is a report on the DNA fingerprinting of P. nigrum and P. longum cultivars. Fourteen land races and three advanced cultivars of P. nigrum and eleven land races and one advanced cultivar of P.longum were used in the present study. Forty decamer primers from Operon Tech were screened using two representative genomic DNA samples of black pepper. Of these, 10 primers that yielded clear and dominant band patterns were selected for the final analysis of the 29 accessions. These generated 119 amplification products. The total number of markers per primer varied with respect to both P. nigrum and P. longum. Cultivar specific single bands were obtained for a few land races and accessions of both the Piper species.

ABSTRACT Though the genetic control of sex determination is being explored in several animal systems, no much information is available on the genetic basis for sex specificities in dioecious or sexually polymorphic plant species. Date palm is a unique member of the family Palmaceae which exhibits dioecy. Here we report the sex-specific variation in the genomic DNA of male and female date palms as revealed from an extensive study conducted with palms representing different date palm growing regions of the Kingdom of Saudi Arabia. The genomic DNA content in females was less than 60 per cent compared to those of the male genotypes. Polymerase chain reactions carried out to amplify different segments of the alcohol dehydrogenase (AdhA) gene from the date palm genomic DNA yielded two DNA fragments each in the female genotypes where as the same primers amplified a single fragment in male plants. Genomic Southern analysis of Eco R1 restriction digests showed RFLP between the male and female genotypes with the male plants showing a single hybridization signal of approximately 14 kb size and the females showing six additional bands of varying molecular weights. The results of our investigations reveal the potential of molecular biology in differentiating the sex in date palm at DNA level. Further research is needed to develop DNA markers that will help to unequivocally identify the sex of dioecious plants at the seedling stage.

Introduction Musa species and cultivars are identified mainly on the basis of morphological characters. Somatic mutations and changes brought about by environment are major obstacles to the accurate identification of clones (Kaemmer et al., 1992). Isoenzymatic markers provide an additional tool for the characterization of germplasm and have been successfully employed in banana to provide better understanding of somaclonal variants and to detect genetic differences at clonal level. Being located within the center of diversity of cultivated banana, the South Indian state of Kerala accounts for a large share of variability, especially the AA, AB, AAB and ABB genomic groups.Nendran, the French plantain cultivar indigenous to Kerala (Jacob, 1952) is represented by many ecotypes/clones. Other prominent cultivar groups include Mysore (AAB), Rasthali (AAB), Neypoovan (AB), Kunnan (AB), Monthan (ABB) and Karpooravalli (ABB). The variability has been collected and conserved in the field at the Banana Research Station, Kannara. These have been characterized morphologically employing per INIBAP/IPGRI Musa descriptors, facilitating the identification of distinct cultivars, clones and synonyms.This project was initiated to survey the isozyme polymorphism in the cultivar groups and to select suitable system for clonal identification.Results of the study employing three enzymatic systems -esterase, diaphorase and peroxidase are presented

ABSTRACT Amajor goal of plant biotechnology is to develop effective and lasting means to reduce crop losses due to stress caused by biological organisms. In Indian context, fungal diseases are rated as the most important factor contributing to yield losses. Although, conventional programmes towards development of disease resistant cultivars have been successful , they suf fer from limitations of human health consequences, potential degradation of environment etc. In this scenario, introduction of specific genes encoding for disease resistance through genetic engineering seems to be a viable alternative. Among the various genes that are induced in response to plant defense, chitinases are the most widely studied and used for development of transgenic crops. Keeping this in view, attempt was made to isolate chitinase genes from four fungal species viz., Trichoderma harzianum, Metarhizum anisopliae, Nomuraea rileyi and Alternaria solani by designing primers based on sequence information available in the database. RT-PCR was done using the RNA isolated from the fungal species using the designed primers. Amplified products were eluted, cloned into pTZ57R vector and sequenced. Characterization of sequenced cDNA was done using the BLAST (x) programme of NCBI site. The amplified product (261 bp) from T. harzianum showed 90 per cent homology to endochitinases from T. asperellum and T. viridae. Using this cDNA, full-length chitinase gene of 1556 bp from T. harzianum could be accomplished through RACE (Rapid amplification of cDNA Ends). In M. anisopliae the amplified product of 1500 bp matched the full length gene of 1500 bp and showed 99 per cent homology to M. anisopliae and 96 per cent homology to M. flavoviride. In other two fungal species, viz., Nomuraea rileyi and Alternaria solani, the amplified products did not show any homology to chitinase gene

ABSTRACT Piper colubrinum is a wild relative of the cultivated species of black pepper, Piper nigrum. The enzyme hydroxy methyl glutaryl Co A reductase which catalyses mevalonate synthesis, is the key enzyme in isoprenoid biosynthesis.This is the rate limiting step in the pathway which leads to the formation of sesquiterpene phytoalecins, a group of plant defense metabolites. Piper colubrinum is known to be tolerant to the dreaded disease called foot rot, incited by Phytophthora capsici. The hmgr gene is reported to confer resistance to fungal pathogens. Hence we made an attempt to isolate gene encoding this enzyme. cDNA was synthesised by reverse transcription from total RNA of P.colubrinum using oligo dt primers. Specific degenerate primers designed based on the conserved boxes among various plant species were used for amplifying hmgr gene from the cDNA. The 700 bp amplicon was cloned in pGEMT vector and sequenced with T7 primer. Multiple sequence alignment using clustal W 1.8 revealed homology of the cDNA fragment with hmgr genes in several other plant species. Maximum similarity was observed among P. colubrinum, Solanum xanthocarpum and Nicotiana tabacum. The cDNA library of P. colubrinum constructed in lTriplEx2 when screened with radiolabelled hmgr amplicon, yielded positive signals. Attempts are being made to isolate full length gene from these clones, which could be later used for imparting biotic stress tolerance to cultivated crop species.

ABSTRACT The present study was conducted to screen fungal isolates for chitinolytic activity and to clone full-length endochitinase gene from fungus. Among the ten isolates tested (Trichoderma harzianum 1, 2, 3, Trichoderma koningii, Trichoderma virens, Trichoderma viride, Aspergillus oryzae Metarshizium verrucasia, Bauveria bassiana, Nomuraea releyi), Trichoderma koningii showed higest colloidal chitin hydrolysis when screened in Mandels and Reese (1969) salts supplemented with colloidal chitin as the sole carbon source followed by Aspergillus oryzae. Nomuraea releyi showed least chitinolytic activity. Other isolates were on par with each other. The use of diagnostic primers showed specific amplification of 260 bp in Trichoderma harzianum isolates 1,2 and3, Trichoderma koningii and Trichoderma virens, which confirmed presence of endochitinase gene. Attempts were made to clone full-length endochitinase gene from isolates showing chitinolytic activity. A unique amplicon of 1.7Kb from Trichoderma virens was cloned into linear pTZ57R/T vector with T over hangs. The confirmation of clones was made through blue/white colony assay, plasmid mini-preparation and PCR amplification of these plasmids, sequencing and BLAST search at NCBI. Among the clones confirmed, pSAV804 was sequenced that showed 98% homology with other reported endochitinase genes from Trichoderma spp at DNA and protein level. 85.7% of the protein is conserved. The ORF of the sequence comprises of 822 bases, which deduces 274 amino acids. The position of primer sequence in the insert and BLAST results confirmed the reverse orientation in the vector. The endochitinase gene now needs to clone into the expression and plant expression vectors for further studies.

ABSTRACT There has been conscious effort to discover and apply biological pesticides for the control of insect predation. Insecticidal proteins/ genes from Bacillus thuringiensis have been deployed as sprays and through transgenic route for crop protection against insect attack and have been successful in protecting the crop damage and environment pollution. However, impending fear of development of resistance to Bt proteins by insects has prompted search for new insecticidal proteins. In this context we are exploring and analysing components of insect immunity as targets for restricting insect feeding behaviour. Insects encounter several microbes during different stages of growth and development. Incidental entry of these microbes elicits a host defense response, which mainly consists of a phenoloxidase (PO) pathway. The invasion of the microbe is recognized by a specific protein, which activates the PO pathway consisting of prophenoloxidase (PPO) & prophenoloxidase activating enzyme (PPAE). Understanding the mechanism of insect immunity will allow us to discover potential targets for inactivating the mechanism of immune action in insects and make them vulnerable to natural microbial infection. An agronomically important polyphagous pest, Heliothis armigera (Bollworm) was chosen in the present work for studying the components of insect immunity. H. armigera is the most important insect pest of the developing world accounting for heavy damage on various crops including cotton, vegetables, tobacco, potato, groundnut, chickpea and pigeon pea. We have isolated full-length cDNA encoding prophenoloxidase (PPO) and a partial cDNA encoding PPAE from haemocytes of 5th instar larvae of H. armigera. The open reading frame (ORF) of PPO gene is 2.1 kb, which encodes for a 698 amino acid protein with a molecular mass of 77 kDa. Computer-assisted analysis revealed the presence of 60 amino acid long propeptide at the N terminus of protein and 2 copper binding motifs but no signal peptide was detected. The gene was cloned and expressed in a bacterial expression vector pET43b under the induction of IPTG. The SDS-PAGE analysis showed that the protein is expressing in the supernatant in its native form. The expressed enzyme is catalytically active on 4-Methylcatechol upon activation by cetyl pyridinium chloride (CPC). The regulatory role played enzyme in insect immunity is being examined.

ABSTRACT Production of cardamom in India has been greatly affected by Cardamom mosaic virus (CdMV). The conventional breeding program to develop resistance is a lengthy process. An alternative approach is to develop transgenic cardamom through introduction and expression of viral sequences in the plant. The coat protein (CP) gene of all the CdMV isolates have been cloned in pGEMT vector and sequenced. Sequence comparison studies of the eight isolates of CdMV in their CP and 3’UTR showed that, Kursupara isolate is highly variable from the rest of the isolates (Jacob et al., 2003). Hence the present study was undertaken to introduce the Kursupara CP gene of CdMV into cardamom plants in order to develop pathogen-derived resistance in cardamom. The CP gene of the Kursupara isolate of the virus was amplified with and without the 3’untranslated region (3’UTR) from the viral clone. These PCR products were cloned into the pXcmkn12 vector and sub cloned in to a plant expression vector (pAHC17) at the BamHI site, under maize ubiquitin promoter. Work is in progress towards the biolistic transformation of cardamom with these constructs.

ABSTRACT The cry1A genes of Bacillus thuringiensis (Bt) are widely used to engineer lepidopteran insect resistance in crop plants including horticultural crops. Continuous exposure of a single kind of Bt toxin can lead to resistance development in insect pest. Due to the difference in structure and unique insecticidal mechanism, cry2A genes are promising candidates for management of resistance development. Since India is rich in biodiversity and genetic resources, indigenous Bt strains are valuable tool for identification of novel cry genes. The present study describes the cloning of cry2Ab from a new indigenous strain of Bt, 14RI. The presence of cry2Ab gene in Bt strain 14RI was confirmed by PCR with primers specific to its internal region. Primers corresponding to the flaking region of cry2Ab were used for amplification of cry2Ab orf from the Bt strain 14RI. The amplified cry2Ab orf was first cloned into the T/A cloning vector, and then fused to the downstream of the cry2Aa promoter along with its orf1 and orf2 in the Bt shuttle vector pHT1301. Acrystalliferous Bt strain 4Q7 has been transformed with recombinant pHT1301 to express the cry2Ab of Bt strain 14RI.

ABSTRACT Basic understanding of plant cell proliferation and differentiation is essential for applying modern techniques of genetic transformation. The lack of understanding about the physiological and molecular mechanisms underlying in vitro morphogenesis, together with the non-availability of reliable morphogenetic markers, is one of the most serious constraints for application of gene transfer technology to plant improvement. Plant tissue culture has therefore remained an empirical science. In recent years, concerted efforts are being made to understand the molecular control of cell differentiation. In light of this, present work was carried out to study the role of CKI1 (cytokinin independent 1) gene, one of the genes implicated in shoot regeneration, by transforming the tomato cotyledons through Agrobacterium mediated transformation with two different gene constructs one with CKI 1 gene and another without CKI1 gene. In both the constructs, nptII gene was used as a selectable marker. Transformation of tomato cotyledon explants with CKI 1 gene resulted in higher regeneration of putative transformant s (8.0%) as compared to transformation of explants without CKI1 gene (2.1%). The results show that CKI1 gene has a role in increasing the regeneration frequency in tomato.

ABSTRACT Rapid optimization and improvement of papaya transformation necessacitates, a quick, non-destructive marker, which permits early detection of transformed tissue. We assessed the ability of a modified version of the green fluorescent protein gene (GFP) to act as the reporter. Transgenic papaya (Carica papaya L.) plants were developed using immature zygotic embryo as the expl ants and Agrobacterium tumefaciens as the transformation inducer. GFP gene (pBIN35Smgfp-ER) under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter was used as the reporter gene. Media P (modified MS basal medium with B vitamins supplemented with picloram 1 mg/l) and BN (modified MS basal medium with B vitamins supplemented with BAP: NAA 1:0.1 mg/l) supplemented with kanamycin (50 mg/l) were used for regeneration of transgenic papaya under selection pressure. GFP gene expression was detected at the juvenile stages viz. at early stages of somatic embryogenesis. 35S promoter driven GFP gene activity was also detected in the epidermis and the vascular tissues of the leaf petiole. Visualization under microscope and subsequent DNA analysis (Southern hybridization) showed that kanamycin resistant plants carried and expressed the GFP transgene. Expression of the GFP as green fluorescence served as a useful non-destructive indicator of successful transformation.

ABSTRACT Zingiber officinale is a herbaceous perennial. The absence of seed set in ginger makes conventional breeding methods inapplicable. Genetic modification through biotechnological means is the only way for introducing novel genes into the cultivated types. Transient expression of the gus reporter gene was used in preliminary experiments. Agrobacteruim tumefaciens strains EHA105/p35SGUS Int. effective in expressing b- glucuronidase activity was used for standardizing the optimum conditions for effecting transformation, such as preculture of explants, bacterial dilution, infection time, co-cultivation period etc. In addition, effects of acetosyringone and explant wounding were evaluated using the same strain. Genetic transformation of bud and regenerated plantlets was confirmed by their ability to grow on MS medium containing kanamycin 100 mg l-1 + cefotaxime 300 mg l-1 by histochemical b-glucuronidase assays.

ABSTRACT Role of cytokinins in the regulation of sink development in potato was studied by transgenic approach. Transgenic potato plants over-expressing Agrobacterium native ipt (gene for isopentenyl transferase, rate limiting enzyme in cytokinin biosynthesis) under the transcriptiona l regulation of tuber specific patatin promoter were developed. Transformants obtained showed normal phenotype, while the cytokinin levels showed significant increase over the control plants for the various cytokinins tested, viz., trans zeatin riboside (tZR), dihydrozeatin riboside (DHZR) and isopentenyl adenosine (iPA). The primary transformants were hardened and tested under green house conditions. Green house studies showed a significant increase in the number of tubers in ipt transformants when compared to the wild plant as well as GUS transformant. However, the total weight of tubers did not show appreciable difference between transformants and control plants. Reduction in the weight of individual tubers appears to be due to source limitation.

ABSTRACT Astudy was undertaken with the main objective to standardise the Agrobacterium mediated genetic transformation technique in Dendrobium Sonia 17. Protocorms and protocorm like bodies (PLBs) of Dendrobium orchid were transformed by Agrobacterium tumefaciens strains LBA4404 and EHA105 harbouring the plasmids pCAMBIA2301, pCAMBIA1301 and PBI121. The PLBs were generated in vitro by culturing the shoot apices in half strength MS medium. The protocorms were obtained by culturing the seeds in vitro. The sensitivity of orchid tissues to various antibiotics viz., ampicilin, rifampicin, cefotaxime, carbenicillin, kanamycin and hygromycin was evaluated to use the antibiotics as selection agents. The sensitivity was studied in detail by scoring the tissues, based on the morphological changes as those remaining fully green, partially discoloured, bleached, completely turning brown and dead. The various requirements for transformation viz., the type and size of explant, infection time, wounding, co-cultivation and incubation period, concentration of acetosyringone required, elimination of bacteria, and the selection of transformants were optimised to establish a successful transformation system. The pre- cultured explants were mixed thoroughly by gentle swirling with the bacterial cells obtained by centrifuging the bacterial suspension, and also with the bacterial suspension diluted to 1/5 with fresh medium. Then the explants were cocultivated with the bacteria for 2 days at 28°C. The transformation was performed through two consecutive stages of cultivation, the first stage on half strength MS medium containing cefotaxime and subsequent stage on selection medium containing kanamycin. Initially recovered PLBs following co-cultivation, were allowed for further selection with kanamycin. New PLBs and leaves were formed from the transformed PLBs when they were regenerated under kanamycin selection. Successful transformants were obtained with a maximum transformation efficiency of 3.0 per cent with the addition of acetosyringone 100 mM. Out of the kanamycin resistant PLBs 69.23 per cent were GUS positives. The newly formed PLBs and leaves when subjected to histochemical GUS assay indicated successful transformation and it was detected as distinct blue spots on the PLBs after 24’ hours. The PLBs which were not co-cultivated with Agrobacterium were green in colour. The transformation were further confirmed by PCR analysis.

ABSTRACT Cassava has developed importance mainly as an industrial crop due to its ability to store large quantities of starch (74% to 85% of dry weight) within secondary root structures. Cassava starch is exploited as a source of human food, feed for livestock and as raw material for the food processing and chemical industries. Research accomplishments during the last four decades helped India to have the top position among cassava growing countries with an average yield of 24 t ha-1. However several traits like starch quality, high protein , post harvest deterioration etc can be altered only through the development of transgenics. The present study summarizes the results of the experiments undertaken to optimize the protocol for the development of transgenic cassava through Agrobacterium mediated gene transfer. Friable Embryogenic Callus (FEC) was developed in the cassava var. TMS 60444 us ing Murashige and Skoog (MS) media with picloram (50μM). Experiments were conducted to study the efficiency of different Agrobacterium stains viz, AGL 8, GV3101, C58C1, LBA 4404 and EH105 in transforming FEC. The strains differed in their growth rate. The strains EH105 and AGL were slower in growth as compared to other strains and the protocol was modified accordingly. Among all the strains, EH105 resulted in maximum recovery of transgenic tissues. The paper also summarizes the present status of development of transgenic cassava along with future thrusts.

ABSTRACT Coleus forskohlii Briq. is a threatened medicinal plant , susceptible to fungal disease. The regeneration of this plant has been achieved on MSo medium supplemented with 2 mg/l or 3 mg/l BAP and 0.2 mg/l 2,4-D for callus induction using leaf as explants. Multiple shoot production from the leaf-derived transformed callus of coleus has been standardized using the various concentration of NAA with 2 mg/l BAP. The combination of 2 mg/l BAP and 0.5 mg/l NAA was found to be best for multiple shoot production. Rooting was found to be best on MS medium supplemented with 0.7 mg/l NAA. Coleus forskohlii was transformed with glucanase-chitinase gene along with npt-II gene. The presence of npt-II genes in the leaves of putative transformants were confirmed by PCR analysis. SDS- PAGE analysis and glucanase assay revealed the over expression of glucanase enzyme in the transgenic plants. The transgenic coleus plants survived the infection caused by Fusarium chlamydosporium due to the accumulation of glucanase-chitinase enzyme, where as control plants were susceptible to the disease.

ABSTRACT An efficient in vitro micro propagation protocol was developed for direct shoot produc tion of Contoloupe (Cucumis melo variety (NS Abhijit) using cotyledons as explant. The excised cotyledons were cultured on Murashige and Skoog’s medium (MS) contain- ing plant growth regulator, 6 Benzyl adenine with various concentrations for the study of shoot production. The best condition for the shoot production was with 1.2mg/l Benzyl adenine (BA) in MS medium. The shooting frequency was 65% and three shoots were obtained from each explant after 14 days of culture. The shoots (~1.5 cm length) were rooted most effectively on 0.1mg/l of α Naphthalene acetic acid (NAA) supplemented MS medium. We have also standardized the transformation protocol for GUS gene in the muskmelon variety Eden Gem to see test the feasibility of this variety for Agrobacterium mediated transformation. The transformed explants were confirmed through Indigogenic GUS assay.

ABSTRACT Chilli (Capsicum annuum L.) is a vegetable and spice crop of worldwide importance. Despite the fact that other closely related members of the Solanaceae family are easily transformed by genetic engineering, capsicum still remains recalcitrant to in vitro manipulation, due to lack of proper elongation of multiple shoot initials. An attempt has been made to standardize transformation protocol in two local cultivars of capsicum viz., Byadgi dabbi and Sankeshwar, using shoot tip explants. The Shoot tip explants were co-cultivated (for 2 days) with Agrobacerium strain EHA105 harboring pBinBt3 plasmid having cry1AC gene and nptII marker linked to the CaMV 35S promoter and nos terminator. After co-cultivation shoot tips were transferred to MS medium containing 2mg/l IBA, 100mg/l kanamycin and 300mg/l cefotaxime. Survived plants were checked for the presence of transgene using cry1 AC and npt II specific primers. Expression of cry1AC gene in these plants was checked using DesiGen Xpresstrips.

ABSTRACT Chilli is the universal spice of Kerala, known for its pungency, flavour and colour of fruits. Of the five domesticated species of Capsicum, C. annuum is the most extensively cultivated one. Bell pepper (C. annuum L.) is mainly used as a vegetable, because of its low pungency and good flavour. It is used as raw in salads, and also in cooked form. Application of biotechnological tools in this crop for improving the quality, shelf life and tolerance of the crop to stress conditions has been limited. This is mainly due to lack of an efficient regeneration system. We reported a viable protocol for the regeneration of whole plant from the hypocotyl earlier. We also succeeded in developing a protocol for genetic transformation of bell pepper using Agrobacterium tumefaciens. The strain EHA 105 containing gus intron and npt II was used for co-cultivation. The dilution of bacterial culture and time of infection were standardised. This was followed by killing the bacterial cells using cefotaxim @ 250ppm. Transformants were selected on MS medium supplemented with kanamycin. Staining of the regenerants with X-gluc revealed presence of blue spots, confirming transformation. This protocol could be exploited for improving the commercial traits of bell pepper by incorporating desirable genes through Agrobacterium mediated transformation.

ABSTRACT Holostemma ada-kodien K. Schum, is a laticiferous climber belonging to the family Asclepiadaceae. The root tubers of the plant are medicinally important and are utilized as a major ingredient of the drug, Jivanti. The roots of Holostemma are sweet, ophthalmic, emmolient, alterant, tonic stimulant, aphrodisiac, expectorant and galactagogue and are useful in ophthalmopathy, orchitis, cough, burning sensation, stomachalgia, fever and to cure ‘tridosha’ (Warrier et al., 1995). The sugars and amino acids present in the tuberous roots are responsible for the medicinal properties (Ramiah et al., 1981). There is a huge demand for the root tubers of Holostemma in the South Indian pharmacies and more than 150 metric tons of roots are needed every year (Nair et al., 1992). The destructive and ruthless collection of tuberous roots in recent times has led to acute scarcity of the plant and consequently it has been listed as vulnerable and rare in the FRLHT red list of medicinal plants (FRLHT, 1997). Extending the commercial cultivation of Holostemma is practically impossible due to the increasing land pressure and the perennial nature of the crop. An alternative to meet the increasing demand is to produce the active chemicals in Holostemma through in vitro techniques. In vitro techniques have been extensively studied to produce the different secondary metabolites from various medicinal plants. A strong correlation between secondary metabolite production and morphological differentiation gives more impetus to the application of organized cell culture particularly the root cultures for the large-scale production of phytochemicals in vitro. Transformed root cultures - the so called hairy root cultures obtained after the insertion of T-DNA from the root inducing (Ri) plasmid of the Agrobacterium rhizogenes into the plant genome (Ackermann, 1977) has emerged as an important tool for the production of secondary metabolites in vitro (Rhodes et al., 1987). Hairy roots have advantages of fast growth, organ differentiation and stable secondary metabolite production (Banerjee et al., 1995). We report here the hairy root induction from shoot buds and seedling hypocotyls in infection with A. rhizogenes useful in exploring the possibilities of large-scale in vitro secondary metabolite production in this rare medicinal plant.

ABSTRACT The black pepper, ‘the king of spices’ cultivation fetches foreign exchange worth millions of rupees annually, has been constrained by the quick wilt disease caused by the fungal pathogen Phytophthora capsici. Peptides with systematic variations in cationicity, hydrophobicity and amphipathicity were designed based on Chou-Fasmann parameters and sequential alterations of amino acids were tested for antifungal activity against Phytophthora. The antifungal properties of these biodegradable and ecofriendly peptide peptides were evaluated following in vitro microplate titre assay using Phytophthora zoospores. Two peptides LD1 and LD4 showed very low MIC value as compared to that of well-known antifungal peptides and standard chemical fungicides. Fluorescent staining technique using fluorescent brightener-28 and propidium iodide dyes has revealed that the site of action of the designed peptides is the cell membrane. These peptides showed very low haemolytic potential inspite of their potent antifungal activity. The in vivo evaluation of the peptides on fungi infected detached pepper leaves has demonstrated their high antifungal potential.

ABSTRACT Fruit rot and die back caused by Colletotrichum capsici is the destructive disease in chillies. The potential of Pseudomonas fluorescens for the management of fruit rot of chillies was evaluated under greenhouse and field conditions. Among the various strains of P. fluorescens tested in vitro, the strain Pf1 significantly inhibtied the mycelial growth of C. capsici. Combined application of talc-based powder formulation of P. fluorescens to seed (10 g/kg seed) and foliage (1 kg/ha) effectively controlled the fruit rot disease. Early and high induction of various defense compounds were observed due to P. fluorescens treatment.

ABSTRACT Biofertilizers can supplement the chemical nutrients and helps in reducing the production expenditure in agriculture. Crop biofertilization with microbial consortia is an important strategy to improve the physical, chemical and biological conditions of the soil. The facultative methylotrophs are capable of fixing atmospheric nitrogen, besides it produces phytohormones viz; IAA, GA and cytokinin, vitamin B12, polysaccharides, PHB, and siderophores. The essential nutrients are considered as the most important, among the factors limiting growth and yield of the tomato crop. The application of adequate amount of fertilizers is a pre-requisite for exploiting the genetic potential of tomato variety. Therefore, efficient and economic use of the fertilizer with microbial consortia would help in decreasing the input costs for raising bumper crop. More number of studies carried out on the effect of facultative methylotrophs in different parts of the world and in India. The results confirmed that the facultative methylotrophs have a greater importance in increasing the growth and yield of various crop plants. Keeping this in view an experiment was conducted to find out the interaction effect of facultative methylotrophs with other bioinoculants viz., Azospirillum, Azotobacter, PSB and VAM (by seedling dip method followed by PPFM phyllosphere spray) on yield of tomato var Ruchi. The results showed that the treatment with above microbial consortia (50.71 t/ha) was found to increase the yield of the tomato crop by 62.73 percent over uninoculated control (18.90 t/ha) and individual treatments.

ABSTRACT Phytophthora rot is the most destructive disease of black pepper nursery inflicting heavy crop losses. Considering the seriousness of the disease, the present study was undertaken to isolate and select the efficient antagonists from pepper nurseries raised in Government farms at Chelakkara, Pazhayannur, Mannuthy and Pananchery of Thrissur district. A quantitative estimation of the rhizosphere microflora from different nurseries revealed more abundance of soil bacteria followed by fungi and actinomycetes. Based on cultural characters of the rhizosphere microflora, 22 fungi, 20 bacteria and five actinomycetes were selected for further studies. Antagonistic action of soil microflora against Phytophthora capsici was studied by dual culture method in comparison with that of standard culture of T. harzianum. The study revealed that all fungal isolates and five bacterial isolates were antagonistic towards the pathogen with varying degrees. Among the fungal isolates, 13 isolates including T.harzianum recorded cent per cent inhibition of P.capsici. From among the fungal isolates that showed antagonistic reaction in dual culture, further selection of efficient ones was carried out based on the antagonistic index (AI). The isolate 22 F2 (Trichoderma sp.) showed the maximum AI of 3000 followed by isolate 34 F2 (Trichoderma sp.) and T. harzianum with an AI of 1500 each. The cultural and morphological characters of the efficient native isolates (22 F2 and 34 F) were studied and were identified as Trichoderma longibrachiatum Rifai aggr. and Trichoderma viride Pers. ex S.F. Gray aggr. The three antagonists were found parasitic on P.capsici as evidenced by excessive coiling, penetration and disintegration of the hyphae and thereby these Trichoderma spp. used in the study is found to have effective parasitic effect on the pathogen in addition to some degree of antibiosis and these fungi could be utilised for the management of Phytophthora rot in black pepper nursery.

ABSTRACT Studies on the impact of bio-fertilizers with levels of NPK on bulb yield, dry matter production, nutrient uptake and cost: benefit ratio in onion (cv. Bellary Red) were conducted in medium black soils during kharif and rabi seasons of 1999 and 2000 both under irrigated and rainfed conditions at the Agricultural Research Station, Hiriyur located under Central Dry Zone of Karnataka. The treatments were; T1-Control, T2-Azotobacter, T3-Azotobacter+75% N+PK, T4-Azotobacter +100%N+PK, T5-Azospirillum, T6-Azospirillum+75% N+PK, T7-Azospirillum+100%N+PK, T8-VAM, T9-VAM 75% P+NK, T10- VAM+100%P+NK, T11 ? recommended NPK(125:50:125 kgha-1) and T12-75% recommended NPK. The experiments were laid out in RBD in three replications. The bio-fertilizers Azospirillum brasilence (500g ha-1) and Azotobacter vinelandii(500g ha-1) were applied to seedlings at transplanting separately by dipping the roots in the slurry for 15 minutes. The VAM fungi - Glomus mossae was applied to soil @ 1 kilogram per square meter area of nursery bed. The recommended dose of FYM @ 25 t ha-1 was applied to all the treatments as basal dose. The results of the investigations revealed that the plants provided with Azospirillum + 100% N+PK (T7) produced highest bulb yield (250.29;339.02;232.98 q/ha), highest total dry matter production (7053.96;8992.78;6287.03 kg/ha), maximum uptake of N, P a nd K ( 1 5 7 . 8 8 : 7 3 . 5 6 ; 7 0 . 4 6 N PK k g / ha ) , ( 2 0 6 . 8 6 : 9 6 . 2 5 : 9 1 . 1 0 N PK k g / ha ) , (131.81:50.25:71.24 NPK kg/ha) and also the maximum cost:benefit ratio of 4.00 (1:400), 5.42 (1:5.42), 3.94 (1:3.94) under irrigated condition during kharif and rabi seasons and under rainfed situation during kharif season respectively. Thus, among all the treatments, application of Azospirillum + 100% N + PK (Azospirillum + 125:50:125 NPK kg/ha) was found to be most remunerative considering the nutrient uptake, bulb yield and total dry matter production and cost:benefit ratio, which gave the highest returns. This level can be followed for Bellary Red onion irrespective of seasons both under irrigated and rainfed conditions in the Central Dry Zone of Karnataka.

ABSTRACT Soft rot of ginger caused by the fungus Pythium aphanidermatum is a major disease affecting ginger cultivation. To combat invading pathogen in the event of infection plants are known to produce antimicrobial pathogenesis related proteins (PR proteins) prominent among which are antifungal hydrolases like glucanases and chitinases. Since Pythium lacks chitin in its cell wall, increase in level of glucanase in the plant may enhance the resistance of ginger to soft rot disease. Signal molecule like ethylene plays a prominent role in the defense response of plants. The present study was undertaken to investigate the role of ethylene in enhancing the level of glucanase in the ginger system. Detached leaves of Rio de Janeiro variety of ginger were used in different experiments. Leaves were treated with ethephon at different concentrations and different time intervals. Increase in the glucanase activity by ethephon treatment was quantified by enzyme assay using crude protein extract. Glucanase levels showed an initial increase with increasing concentration of ethephon but decreased at higher concentration (5 mg/ml). A longer exposure of detached leaves to ethephon (48 h) resulted in 3.7 fold increase in glucanase activity compared to control. The level of enhancement of the glucanase transcript also followed a similar trend as revealed by RT PCR analysis. Two acidic isoforms of glucanase were detected using native PAGE and their levels were found to be enhanced by treatment with ethephon. An increase in the levels of glucanase may contribute to the plant’s ability to resist infection by Pythium.

ABSTRACT Chickpea is an important pulse crop of India and most suited to the use of rhizobia for its nitrogen requirement. Mesorhizobium ciceri which interact with the crop are poorly understood. Attempt has been made to select M. ciceri with several beneficial properties. Sixty four isolates of M. ciceri collected from the nodules of several chickpea varieties and mutant lines were screened for their growth properties on nitrogen free medium. Isolates MC59 and MC 18-7 showed normal growth and had ARA values at half level to that of Azotobacter. PCR amplification results suggested the presence of ACC deaminase gene sequence both in Azotobacter and M. ciceri. Invariably all isolates showed the presence of one plasmid in the range of 35 kb to 40 kb size.

ABSTRACT Bacterial wilt caused by Ralstonia solanacearum is the major constraint for cultivation of tomato in Kerala. Biocontrol of plant pathogens is becoming an important component of integrated disease management. Among the various antagonists tested against R. solanacearum of tomato, fungi were more effective than bacterial and actinomycetes organisms. Among them, Trichoderma virens, T. viride (Ozhalapathy and Vellanikkara) and T. pseudokoningii were the effective ones, recording the maximum antagonism index (AI) value of 6000 under in vitro condition. They inhibited the pathogen completely by the mechanism of both lysis and overgrowth. In pot culture study, T. viride and T. pseudokoningii and commercial Pseudomonas fluorescens gave maximum protection against wilt incidence. Under field condition, T. viride (Ozh), T. pseudokoningii and P. aeruginosa showed the lowest wilt incidence of 50.0, 58.3 and 58.3 respectively against cent per cent incidence in the control plot.

ABSTRACT Brinjal is an important solanaceous vegetable, which holds coveted position among the different vegetables. Developing hybrids with high yield and resistance to borer would be highly beneficial. The biochemical parameters are used to study the mechanism of resistance to pests and diseases. With this aim, the biochemical constituents were estimated in five intervarietal hybrids and their parents with susceptible variety. The study on biochemical basis of resistance to shoot and fruit borer showed that among the parents of intervarietal crosses, EP 65 had the highest level of peroxidase, polyphenol oxidase and total phenol content in both shoot and fruit and it also recorded high content of solasodine next to APAU Bagmathi. Among the hybrids, the cross EP 65 x Pusa Uttam had the highest level of peroxidase and polyphenol oxidase activity in both shoot and fruit and high level of total phenols and solasodine content next to EP 104 x APAU Bagmathi . The susceptible check (CO 2) registered the lowest value for all the biochemical constituents .The study revealed that the genotypes with high or moderate level of these biochemical constituents suffered less for shoot and fruit borer infestation. The native gel electrophoretic study showed that for peroxidase enzyme, the intervarietal hybrids EP 65 x Pusa Uttam, EP 12 x MDU 1 and EP 104 x APAU Bagmathi and the susceptible variety CO 2 did not show difference in the induction of isoform (PO-1) and all were single and thick. For polyphenol oxidase (PPO), the intervarietal hybrids and the variety CO 2 showed single and strong band induction The evaluation of genotypes based on biochemical constituents and isozyme studies revealed the superiority of the parents EP 65, APAU Bagmathi and MDU 1and the hybrids EP 65 x Pusa Uttam, EP 12 x MDU 1 and EP 104 x APAU Bagmathi. The identified hybrids with high yield and resistance to shoot and fruit borer could be exploited for commercial cultivation.

ABSTRACT Vesicular arbuscular mycorrhizal (VAM) fungi are known to improve the mineral nutrient and water status of the plants even under various abiotic stresses. During present investigations VAM fungus Glomus fasciculatum was used for developing mycorrhizal association with Sesbania sesban (L.) Merrill. In general mycorrhizal (M) plants exhibited better growth and development and showed significantly higher chlorophyll content, better PS II as well as PS I activities when compared to NM plants. In general, both M and NM plants invariably showed reduced growth and development upon exposure to drought stress. However, the M plants could confront this abiotic stress more effectively than the NM plants. This was vindicated by the faster decline in the chlorophyll content and the photochemical activities in NM plants in comparison to the M plants upon exposure to drought stress. The higher photosynthetic efficiency of the M plants under drought stress indicate the presence of certain adaptive strategies in the form of osmotic adjustment operational in these plants through VAM association. The osmotic potential of the leaf cell sap in M plants was significantly lower than that of NM plants of Sesbania sesban, when exposed to either drought stress. The lower y of M plants as compared to NM plants under drought stress could be due to the accumulation of compatible solutes such as proline and sugars. A higher level of proline accumulation in the leaves of M plants than NM plants exposed to drought stress help them to counteract the adverse effects of stress better than NM plants.

ABSTRACT Anursery experiment was conducted to study the effect of solarization, selected native antagonists viz., T. viride and T. longibrachiatum and the standard culture of T. harzianum for assesseing the growth enhancement and management of Phytophthora rot in black pepper nursery. The experiment was laid out at College of Horticulture with 20 different treatments and replicated thrice. The sprouting percentage of cuttings in different treatments was worked out at different intervals, which showed significant difference. In general, it was observed that the maximum sprouting percentage was noticed in treatments where solarized potting mixture and biocontrol agents were used. The population of soil microflora viz., fungi, bacteria and actinomycetes in different treatments were also estimated. The study indicated a fluctuation in the population of soil microbes in various treatments at different periods of observation. The observations on height and number of leaves of pepper cuttings in different treatments were recorded. It was noticed that cuttings raised in solarized potting mixture incorporated with native antagonists, especially T. viride, exerted a significant effect in increasing the height of cuttings. With regard to total number of leaves, it was also observed that addition of native antagonists in solarized potting mixture had a positive effect in increasing the leaf production. Thus, the effect of solarization enhanced the growth parameters in crop plants by making available the required nutrients. The increased growth response of plants by soil application of Trichoderma spp. may be due to the production of growth promoting substances by the antagonists. Thus, the nursery experiment revealed that, in general, soil solarization and incorporation of antagonists had a favourable effect in promoting the growth of pepper plants besides checking the Phytophthora rot in black pepper nursery.

ABSTRACT Microbial antagonists not only reduce the soil borne disease, but also influence the growth of crop plants. In a study conducted for five years on the effect of microbial antagonists on damping off incidence and biometric characters of solanaceous seedlings, it was observed that seed treatment and soil application of Trichoderma viride, T. harzianum, Pseudomonas fluorescens, Azotobacter chroococcum reduced the damping off incidence considerably in brinjal, chilli and tomato. Application of these microbes also increased the per cent germination and promoted vigour of solanaceous seedlings as compared to control. Among the various antagonists, T. viride was the most effective one reducing the damping off diseases as well as promoting the growth of solanaceous vegetables.

ABSTRACT Banana is one of the most accepted and versatile fruit crops across the globe and in Kerala, it is the most important economically profitable fruit crop grown, mainly by small and marginal farmers, providing a vital source of income. However, the productivity of this crop is very much reduced due to various diseases of which rhizome rot disease is an important one. In recent years, this disease has become a serious problem to the commercial cultivation of banana in southern states of India like Kerala, Karnataka, and Tamil Nadu.In Kerala the disease incidence is found to be nearly 30% and it will soon become cent percent if unnoticed since the disease is more seen in banana cultivated in paddy fields, and the area under the cultivation of banana in paddy fields in Kerala is rapidly increasing. The disease is caused by a gram negative bacterium, Erwinia carotovora. Developing an integrated management package involving biocontrol methods is the only remedy for managing the disease within the financial limits of the marginal farmers. With this intension a pot culture experiment was conducted to study the inhibitory effect of different biocontrol agents and botanicals in the incidence of the disease after testing their efficacy under in vitro cond itions. The results revealed that the biocont rol agent Pseudomonas flourescens (commercial preparation @ 7g /plant) was found to effective in managing the disease. The co- efficient of infection was found to be13.9% as against the control pot where it was 56.24%. With regard to the growth parameters also, the treatment with this biocontrol agent gave superior results. Thus it was proved that Pseudomonas flourescens (commercial preparation @ 7g /plant) could be successfully utilized in the integrated disease management making it cost effective.

ABSTRACT To study the antagonistic potential of rhizobacteria against the soil borne pathogen Rhizoctonia solani, soil bacteria were isolated from the rice fields of Agricultural Research Station, Mannuthy and screened against R. solani. Out of the 22 rhizosphere bacteria, 10 were found to be antagonistic to R. solani. Seven belonged to the genera Bacillus, Pseudomonas, Clostridium and Sporosarcina and the rest three being unidentified. The bacterial isolates viz., 2B, 4B, 19B (Bacillus sp.), 28B and 10B (Sporosarcina sp.) showed increase in inhibition on extending the period of incubation. Maximum inhibition was expressed by 2B on the third day of inoculation followed by 4B, 10B (Sporosarcina sp.), 7B (Bacillus sp.) and Pseudomonas fluorescens. The bacterial isolate 4B and 7B (Bacillus sp.) and 10B (Sporosarcina sp.) completely prevented the sclerotial production of R. solani whereas 1B (Clostridium sp.), 2B, 19B (Bacillus sp.) and P. fluorescens delayed the sclerotial production. Pot culture studies also confirmed the superiority of these rhizobacteria in controlling the infection by R. solani. The potentiality of these rhizobacterial antagonists can be exploited for the management of soil borne pathogens like R. solani.

ABSTRACT Phytophthora pod rot (PPR) incited by Phytophthora palmivora is the most serious disease of cocoa, inflicting heavy crop losses. Since, antagonistic microbes are useful in ecofriendly management of plant diseases, the present study was undertaken to harness the potential of antagonistic epiphytic microflora isolated from cocoa pods for the management of PPR of cocoa. A quantitative estimation of the epiphytic microflora from pods collected from different locations of cocoa gardens of Thrissur district revealed more abundance of bacteria followed by fungi, actinomycetes and fluorescent pseudomonads. The in vitro antagonistic effect of 16 isolates of fungi, 22 isolates of bacteria, five isolates of actinomycetes and two isolates of fluorescent pseudomonads from pod surface against P. palmivora was carried out in comparison with standard cultures of Trichoderma harzianum and Pseudomonas fluorescens. The study revealed that all the epiphytic fungal and bacterial isolates including standard cultures of T. harzianum and P. fluorescens were antagonistic to P. palmivora in varying degrees. Based on the in vitro screening seven promising epiphytic fungal isolates and five bacterial isolates along with standard cultures of T. harzianum and P. fluorescens were evaluated for their efficiency in reducing the infection by P. palmivora on detached pods. The pathogen was inoculated on the pods one hour after application of antagonists. Observations taken 10 days after inoculation of the pathogen revealed that one isolate of T. viride and two isolates of P. fluorescens obtained from pod surface exerted more than 40 per cent efficiency over control in reducing the infection. The standard cultures of fungal and bacterial antagonists recorded 33-35 per cent efficiency. Hence, these efficient epiphytic and standard cultures could be utilized for the management of PPR of cocoa.

ABSTRACT Interaction of cationic dye acridine orange with lipopolysaccharide (LPS) isolated from Rhizobium strains specific (IC59) and non-specific (TAL1000) to chickpea (Cicer arietinum.L L.) has been investigated by spectrophotometric measurements. The strain specific (IC59) lipopolysaccharide induced decrease in absorbance of the α-band of acridine orange at 490 nm and finally upon further addition of LPS /Dye ratio in the range of 30~60 giving rise to α-polymeric metachromatic band at 660 nm. In contrast the LPS from non-specific strain (TAL1000) induces a decrease in absorbance at the α-band till LPS / Dye ratios are 0~50. This suggest that the anionic changes of strain specific LPS is different from non-specific strain enabling formation of large acridine orange aggregate on the surface of specific strain.

ABSTRACT An In vitro study was conducted to evaluate the fungicidal efficiency of four selected fungal antagonists against leaf spot pathogens of ivy gourd by dual culture method. The antagonists evaluated were Trichoderma viride, Trichoderma harzianum, Aspergillus niger and Chaetomium globosum. The major leaf spot pathogens of ivy gourd used for the study were Cercospora cocciniae, Colletotrichum gloeosporioides and Alternaria alternata, causing severe infection on the foliage. Among the four antagonists tested, T. viride and T. harzianum were found to be the most effective recording the highest AI value of 1500 against all the three pathogens followed by A. niger. C. globosum was least inhibitory on C. gloeosporioides and A. alternata, but was effective against C. cocciniae. A complete overgrowth of the antagonists on all these pathogens were observed in the case of T. viride, T. harzianum and A.niger. C. globosum showed aversion to the hyphal growth of C. gloeosporioides and A. alternata, whereas in C. cocciniae, a homogenous growth was observed.

ABSTRACT The antagonistic organisms are known to bring out action on the pathogen by various mechanisms. Based on in vitro screening of epiphytic microbes from cocoa pod surface against Phytophthora palmivora, the incitant of pod rot of cocoa, a promising isolate of Trichoderma viride and two isolates of Pseudomonas fluorescens were selected. The mechanisms of antagonistic action of these selected epiphytic antagonists against P. palmivora were studied in comparison with the standard cultures of T. harzianum and P. fluorescens. The selected epiphytic fungal antagonist T. viride and standard culture of T. harzianum were found to be efficient parasites of P. palmivora. The hyphae of the pathogen were seen tightly held by coiling with hyphae of the fungal antagonists. The hyphae of the antagonists penetrated the host hyphae at several points and grew to its inner cavity. The fungal antagonists also caused disintegration of the pathogen hyphae, besides free intermingling, which resulted in malformation of host hyphae. The ability of the two epiphytic P. fluorescens isolates and standard culture of P. fluorescens in producing HCN and siderophores was assessed. It was found that these isolates produced HCN and siderophores in appreciable levels thereby suppressing the P. palmivora. Though, more than one mechanism may operate in suppressing the pathogen, the relative importance of a particular mechanism may vary with different conditions, which needed to be further investigated.

ABSTRACT An in vitro evaluation was carried out to find out the efficiency of bacterial and fungal antagonists in checking the growth of pathogens of Kacholam. Out of the two methods tried to find out the effectiveness of Pseudomonas fluorescens against the two isolates of Ralstonia solanacearum, cross streaking produced lysis at the juncture and point inoculation produced clear inhibition zones. Among the two fungal antagonists tested Trichoderma viride was more efficient than Aspergillus niger because of its faster growth over the pathogen. However, both the antagonists showed complete overgrowth and dense sporulation on the area where the pathogen was streaked. To find out the efficacy of Pseudomonas fluorescens against leaf spot pathogens like Colletotrichum gloeosporioides and C. capsici, streaking on both sides yielded more than 50 per cent reduction in growth of both the fungal pathogens. The fungal antagonist T. viride showed a faster growth over pathogens than A. niger eventhough both recorded an antagonism index of 1500. Dense sporulation was seen over the pathogens in case of T. viride. The ability of fungal antagonists to produce volatiles and their effect on fungal pathogens were also assessed. It was noticed that the volatiles produced by T. viride was more effective than that of A. niger. Even though the extent of inhibition was less compared to direct action, this could also be one of the mechanisms of action of both the antagonists. The study revealed that all the three antagonists were effective against both bacterial and fungal pathogens and they could be utilized under field conditions.

ABSTRACT Soil samples from pepper growing tracts of six districts of Kerala viz., Ernakulam, Idukki, Kollam, Kottayam, Thrissur and Pathanamthitta were assessed for total microbial population. Thirty two fungi, 17 bacteria and three actinomycetes were isolated and studied for their antagonistic potential against the foot rot pathogen, Phytophthora capsici. All the 32 fungal isolates were found antagonistic to the pathogen and majority of them belonged to the genera Trichoderma, Aspergillus, Penicillium, Fusarium, Pythium, Cunninghamella and Rhizopus. Among these maximum inhibition was recorded by Trichoderma spp. which included T. pseudokoningii, T. longibrachiatum, T. polysporum and T. harzianum. The laboratory studies were followed by field trials laid out in farmers’ fields (newly planted pepper gardens) in six districts of Kerala. Effect of biocontrol agents in wilt incidence revealed that the two-time treatment of combined application of Trichoderma local isolate and potassium phosphate was superior in checking the foot rot incidence. The treatment, Trichoderma local isolate + Bordeaux mixture applied twice came in the second position. Data on biometric observations revealed a significant increase in the number of laterals in plots treated with Trichoderma local antagonist and potassium phosphate as compared to plots treated with Bordeaux mixture and copper oxychloride. Results of quality analysis of berries revealed that oleoresin content ranged from 6.98 - 11.84 per cent and piperine content from 3.61 - 5.92 per cent and the quality remain unchanged due to various treatments. The study has provided an ‘eco and health friendly’ biological control for the management of Phytophthora foot rot, the most devastating disease of pepper.

ABSTRACT Trichoderma spp. are important group of fungi which are mycoparasitic on several plant pathogenic fungi. Trichoderma spp. are, therefore, used as commercial biofungicides. Genetic manipulation through induced mutagenesis and molecular cloning are two important tools in improving the biocontrol potential of Trichoderma spp. In the present study, attempts were made to induce genetic variability in Trichoderma virens using UV- induced mutagenesis. In the process, a simple protocol was developed for obtaining high frequency mutations in Trichoderma virens. Using a UV-crosslinker, four stable mutants of T. virens have been developed, which are distinctly different from the wild type in appearance and in antagonism against Rhizoctonia solani and Sclerotium rolfsii. One of the mutants, designated as TI is very slow in growth, but sporulates densely, and retained its mycoparasitic potential against R. solani. Though all the mutants were able to antagonize R. solani, all of them were overgrown by the test pathogen S. rolfsii. These mutants could be used to identify genes involved in growth and conidiation in T. virens.

ABSTRACT Black pepper (Piper nigrum L.), known as the ‘King of Spices’ is an important export- oriented spice crop, valued for its medicinal and condimental properties. A perfect protocol for the rapid in vitro multiplication of this plant is available. However, systemic bacterial contamination is the single major constraint in the large scale in vitro production of plantlets. Based on morphological and biochemical characters, this bacterium was identified as Beijerinckia indica It grows profusely on nitrogen-free Jensen’s agar and solubilizes CaCO in the medium by acid production. It is resistant to a range of antibiotics and produces indole acetic acid. The bacterium is slow growing and has pH optima of 5.5 and temperature optima of 27oC. It has both endophytic as well as epiphytic association with the plant, the colonization being more in the stem and petiole. A single plasmid of 4.0 kb size was isolated from the bacterium and characterized. Competent cells of E. coli DH5a, transformed with this plasmid survived on Jensen’s nitrogen free agar, indicating its ability to fix atmospheric nitrogen. We stipulate that the nif genes are located on the plasmid of the bacterium. Since endophytic bacteria are known to stimulate plant growth through antagonistic activity against pathogens, production of growth promoting substances and inducing systemic resistance in several other agricultural crop species, the symbiotic association between Bejerinckia indica and black pepper could be further exploited through integrated nutrient management for improved growth and yield of this spice crop.

ABSTRACT Atotal of 45 Gram positive, spore forming and rod shaped bacteria identified as Bacillus species were isolated from the rhizosphere of Chickpea plants. They were studied for their interaction with root borne fungal pathogens was studied using spot test method. Isolates CBS 29, CBS 113, CBS 123, CBS 132, CBS 153 and CBS 155 inhibited the growth of four or more fungi with inhibition zones varied from 1-5 mm. Two isolates CBS 153 and CBS 155 produced siderophores on CAS supplemented agar medium. Further inoculation of these Bacilli on seeds of Chickpea cultivar C235 and H8618 showed inhibitory, stunting or stimulatory effects on plumule and radicle growth depending upon cultivar, bacterium used and stage of seedling growth. Studies on co-inoculation of these isolates with Mesorhizobium and their plant growth promoting activity are being carried out. These co-inoculation studies are important areas of exploitation to improve crop productivity.

ABSTRACT In Kerala, apart from cultivated sesame species (Sesamum indicum L.) three wild species were identified viz. Sesamum malabaricum, Sesamum mulayanum and Sesamum radiatum. An investigation was carried out to determine the intensity of arbuscular mycorrhizal fungal colonization in cultivated and wild species of sesame. The percentage of arbuscular mycorrhizal fungal infection in roots of different sesame species were found to be variable. The highest percentage of root colonization of arbuscular mycorrhial fungi was found in wild species S. malabaricum, which promotes tolerance to the plant under drought stress conditions.

ABSTRACT Detection of disease tolerance with enhanced defense mechanism is a practical approach in crop variety improvement programme. Toxins being responsible for pathogenicity and virulence of the fungi, regenerating plants from toxin treated calli is an effective and easy approach. The response of tomato calli to the crude toxins of the foliar pathogens viz., Alternaria solani and Septoria lycopersici was studied and the changes in phenol metabolism due to toxin treatment were analysed. Crude toxins of A. solani and S. lycopersici extracted under optimum conditions were serially diluted and tomato calli were treated with different dilutions of the toxins to find out the optimum dilution at which minimum survival percentage was obtained, based on a 1-4 score. Minimum score of 1 was obtained at 1:10 dilution for A. solani and at 1:20 for S. lycopersici. The calli of resistant (LE-625) and susceptible (CO-3) tomato varieties were treated with desired dilutions of the toxins of A. solani and S. lycopersici. Total phenolics and ortho- dihydroxy (OD) phenolics were estimated at 24 h interval to study the extent of necrosis on resistant and susceptible calli due to toxin treatment. The total and OD-phenol contents of calli of susceptible cultivar (CO-3) progressively decreased with increase in time interval consequent to the treatment of toxins of A. solani and S. lycopersici. In the resistant calli of LE-625, an initial reduction of total phenol and OD-phenol content was observed following the application of the toxins of A. solani and S. lycopersici at 24 hours after treatment. Later with lapse of time, the total phenol content and OD-phenol contents progressively increased reaching a maximum at 120 h after treatment. The results clearly indicated the enhancement of biochemical defense in the resistant calli and a fall in defense mechanism in the susceptible calli due to toxin treatment. Such techniques are useful in the preliminary screening of resistant genotypes in crops like tomato.

ABSTRACT Apot culture experiment was conducted on tomato with cadmium and lead to identify the selective retention sites of the metals. For this a completely randomized design with five treatments and four replications were laid out at the College of Horticulture, Vellanikkara. The different levels of treatment ranged from 0.5 to 2.0 mg kg -1 soil. Cadmium and lead were directly supplied as a water-soluble source through cadmium chloride and lead nitrate respectively. In tomato the growth and fruiting was permitted only at lower levels of cadmium application. At 1.5 mg kg-1 soil, fruiting failed. While at 2.0 mg kg-1 soil, the very establishment of plant was totally affected. At higher levels of application, cadmium preferred to get accumulated in roots while on the contrary at lower levels, cadmium uptake increased in shoots than in roots with the least content of the metal in fruits. However, in the case of lead, all levels of application in soil permitted growth and fruiting. With increase in rate of lead application, significant selective retention was observed in roots (1.48-6.69 ppm), shoot (0.29-1.62 ppm) and fruits (0.39-1.45 ppm). On comparison of retention sites of lead in tomato, it could be seen that fruits maintained the maximum lead uptake (637.2-2933.0 mg pot-1). Hence, the consumption of such tomato fruits contaminated with heavy metals may result in kidney damage, lack of memory, dissolution of phosphorus in the bones etc.

ABSTRACT The potato is one of the most important food crops both in developed as well as in developing countries. Developing countries today produce 37 per cent of world’s output of potatoes. Potato is an important food crop in India. Both area under potato cultivation and production has increased manifolds during past decades. The efforts are being made to improve potato quality and its post harvest life as well. An experiment was conducted to isolate mitochondria from potato tubers and succinate dehydrogenase (SDH) assay and protein profiling were performed in the tissue. Three varieties namely, Kufri Bahar, Kufri Lauvkar and Kufri Jyoti were taken into consideration for the present investigation. Kufri Bahar showed the minimum values for SDH activity and Kufri Jyoti the maximum SDH activity. On the basis of SDH activity, the mitochondria in Kufri Jyoti were seemed to be more viable, functional and coupled as compared to Kufri Bahar and Kufri Lauvkar. This finding also suggested that the respiratory degradation of starch in Kufri Jyoti was higher as compared to Kufri Bahar and Kufri Lauvkar. Thus these result showed that the shelf life of Kufri Jyoti was less as compared to Kufri Bahar and Kufri Lauvkar. Protein profiling performed by SDS-PAGE was found to be similar in all the potato tubers.

ABSTRACT Biological lipids are chemically diverse group of compounds. Lipids serve two major biological roles namely a structural one as component of membrane, and storage function, particularly in certain seed tissues. Fats and oils are the principal stored form of energy in many organism, and phospholipids and sterols makeup about half of the mass of biological membranes. An experiment was conducted to study phospholipids composition qualitatively as well as quantitatively in the roots of mung bean seedlings. Among the four solvents used for phospholipids isolation, solvent II (methanol) and solvent III (chloroform: methanol: NH3 salt) were proved to be the major phospholipids eluting solvents. The results indicated that Phosphatidyl Choline (PC), Phosphatidyl Inositol (PI), Phosphatidyl Serine (PS), Phosphatidyl Ethanolamine (PE) and Di Phospho Glyceride (DPG) were present in significant amount in the roots of ten days old mung bean seedlings. These phospholipids were also identified by their Rf values using thin layer chromatography. Among all the phospholipids isolated, PI showed the minimum Rf value, whereas PS showed the maximum Rf value. On the basis of column chromatography, four fractions of phospholipids were isolated. The highest amount of phospholipids was found in fraction II and the lowest in fraction I. Fraction II and fraction III contained the maximum phospholipids. In the fraction II PC showed the highest content (μg g-1) and one unidentified phospholipids showed the lowest content (μg g-1), whereas in fraction III, PI showed the maximum content (μg g-1) and PS showed the minimum content (μg g-1).

ABSTRACT Astudy was undertaken at Department of Soil Science & Agrl. Chemistry, College of Horticulture, Vellanikkara to standardize different amelioration techniques to prepare enriched coirpith compost. Twelve treatments were tried using different combinations of coirpith, Pleurotus spp., Schizophyllus spp., urea, rock phosphate, cowdung slurry, mushroom waste, goat manure, glyricidia and water hyacinth. The results of the various enrichment techniques revealed that the temperature in the compost in general, increased to a peak value of 47oC on the 22nd day of composting and later decreased to a stabilized value. The observed temperature of 40-50 °C found during the thermophilic stage is essential for the rapid degradation of lignocellulose, as the thermophilic microfungi and actinomycetes involved in this process thrive at that temperature. Though the pH show a hike at the initial stage of composting, it decreased and stabilized at the later stage of composting which is conducive for growth and proliferation of microbes. The treatment coirpith with Pleurotus spp., glyricidia +20% cowdung slurry and goat manure was found to be ideal for compost preparation.

ABSTRACT The Cry2A proteins are promising candidates for managing the development of Cry1Ac resistance in insects. The Cry2A class of proteins is different from that of Cry1A, and experiments have shown that insects resistant to Cry1Ac are not cross-resistant to Cry2A proteins. However, the Cry2A proteins are less toxic than Cry1Ac to the polyphagous pest Helicoverpa armigera. Structure-based protein engineering of Cry toxins may pave the way for directed evolution of variants with broad spectrum of insect species specificity, enhanced potency, and stability. In the present study homology modeling of Cry2Ac was done using DeepView software. The template for Cry2Ac protein sequence (accession number X57252) was selected using ExPdb template search. The raw sequence of the Cry2Ac was superimposed over the Cry2Aa PDB template (1I5P). The superimposed model was submitted to the SWISS Model server. After receiving the Cry2Ac model, energy minimization was done using Gromos96. The threading energy and force field energy were calculated. The homology model of Cry2Ac made by us along with the Cry2Ac model from the SWISS-MODEL Repository was used for comparative analysis of receptor binding regions of Cry2Aa. Results showed variation between the two highly homologous Cry2A proteins in their receptor binding regions which may account for differences in spectrum of insecticidal activity.

The Distributed Information Sub Center at Kerala Agricultural University, Thrissur was set up in May 1995 as the 23rd center of the BTISNet of DBT, Government of India. The area of specilisation of the Centre is Agricultural Biotech- nology. The Centre passed through several phases involving Internet connectivity through the ordinary C band connectivity, then the Ku band connectivity etc. Now the commu- nication facilities of the Centre have been strengthened by procuring an ISDN Internet connection and a connection through ASIANET. The Center caters to the needs of the students (UG, M Sc & Ph D) and faculty of all departments of the Colleges in the main campus as well as of the other nearby academic and research centers of the University. Over the years, DISC has come to occupy a position in the KAU in the area of Infor- mation Technology , information sources and services based on Internet, CD-ROMs etc. The Centre has procured databases in the field of Biotechnology and developed several databases in the field of Agriculture and related fields. The center regularly conducts training programmes in Bioinformatics. The research activities in the field of Bioinformatics have been enhanced. The Centre has now been upgraded to DIC to facilitate intensive research on Bioinformatics in an effective manner. During the year 2004, the Centre has also started offering a course on Bioinformatics for the M.Sc Biotechnology students of the College of Horticulture. Biotechnology research in KAU spread over various research stations and cam- puses includes Plant Biotechnology, Animal Biotechnology and Fisheries Biotechnol- ogy. Thrust areas for Plant Biotechnology include developing in vitro protocols for mass multiplication of elite types of fruit plants, ornamentals, medicinal plants, spices, plantation crops and forest species. The other thrust areas include checking clonal stability and genetic fidelity of in vitro derived plants using molecular markers, DNA finger printing and characterisation of germplasm with molecular markers, imparting biotic and abiotic stress tolerance in otherwise accepted crop varieties, genetic trans- formation for imparting/ improving expression of desired traits, in vitro germplasm conservation, enhancement of secondary metabolites production etc. The crops identified for biotechnology research at KAU include spice crops like black pepper, ginger, turmeric, vanilla, cardamom; plantation crops like cashew, coco- nut and medicinal & aromatic plants such as Kaempferia, Trichopus, Gymnema, Holostemma and Coscinium. For improvement of such crops through molecular breed- ing, it is highly essential to identify/design specific probes/primers, micro-satellites, etc. By combining the research results generated at the Biotechnology Centre and informa- tion retrieved from the Bioinformatics Centre, suitable probes, primers are being de- signed for characterization and improvement of these unique crops through molecular breeding.