eChapters

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

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

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

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 [Tr^s] 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

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

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 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

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

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.

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

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.

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.

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).

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.

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.

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

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

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.