eChapters

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

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

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.

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 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.

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

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 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

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

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

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

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

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

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 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

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

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

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

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.