eChapters

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

There has been conscious effort to discover and apply biological pesticides for the control of insect predation. Insecticidal proteins/ genes from Bacillus thuringiensis have been deployed as sprays and through transgenic route for crop protection against insect attack and have been successful in protecting the crop damage and environment pollution. However, impending fear of development of resistance to Bt proteins by insects has prompted search for new insecticidal proteins. In this context we are exploring and analysing components of insect immunity as targets for restricting insect feeding behaviour. Insects encounter several microbes during different stages of growth and development. Incidental entry of these microbes elicits a host defense response, which mainly consists of a phenoloxidase (PO) pathway. The invasion of the microbe is recognized by a specific protein, which activates the PO pathway consisting of prophenoloxidase (PPO) & prophenoloxidase activating enzyme (PPAE). Understanding the mechanism of insect immunity will allow us to discover potential targets for inactivating the mechanism of immune action in insects and make them vulnerable to natural microbial infection.

An agronomically important polyphagous pest, Heliothis armigera (Bollworm) was chosen in the present work for studying the components of insect immunity. H. armigera is the most important insect pest of the developing world accounting for heavy damage on various crops including cotton, vegetables, tobacco, potato, groundnut, chickpea and pigeon pea. We have isolated full-length cDNA encoding prophenoloxidase (PPO) and a partial cDNA encoding PPAE from haemocytes of 5th instar larvae of H. armigera. The open reading frame (ORF) of PPO gene is 2.1 kb, which encodes for a 698 amino acid protein with a molecular mass of 77 kDa. Computer-assisted analysis revealed the presence of 60 amino acid long propeptide at the N terminus of protein and 2 copper binding motifs but no signal peptide was detected. The gene was cloned and expressed in a bacterial expression vector pET43b under the induction of IPTG. The SDS-PAGE analysis showed that the protein is expressing in the supernatant in its native form. The expressed enzyme is catalytically active on 4-Methylcatechol upon activation by cetyl pyridinium chloride (CPC). The regulatory role played enzyme in insect immunity is being examined.

ABSTRACT

The present study was conducted to screen fungal isolates for chitinolytic activity and to clone full-length endochitinase gene from fungus. Among the ten isolates tested (Trichoderma harzianum 1, 2, 3, Trichoderma koningii, Trichoderma virens, Trichoderma viride, Aspergillus oryzae Metarshizium verrucasia, Bauveria bassiana, Nomuraea releyi), Trichoderma koningii showed higest colloidal chitin hydrolysis when screened in Mandels and Reese (1969) salts supplemented with colloidal chitin as the sole carbon source followed by Aspergillus oryzae. Nomuraea releyi showed least chitinolytic activity. Other isolates were on par with each other. The use of diagnostic primers showed specific amplification of 260 bp in Trichoderma harzianum isolates 1,2 and3, Trichoderma koningii and Trichoderma virens, which confirmed presence of endochitinase gene.

Attempts were made to clone full-length endochitinase gene from isolates showing chitinolytic activity. A unique amplicon of 1.7Kb from Trichoderma virens was cloned into linear pTZ57R/T vector with T over hangs. The confirmation of clones was made through blue/white colony assay, plasmid mini-preparation and PCR amplification of these plasmids, sequencing and BLAST search at NCBI. Among the clones confirmed, pSAV804 was sequenced that showed 98% homology with other reported endochitinase genes from Trichoderma spp at DNA and protein level. 85.7% of the protein is conserved. The ORF of the sequence comprises of 822 bases, which deduces 274 amino acids. The position of primer sequence in the insert and BLAST results confirmed the reverse orientation in the vector. The endochitinase gene now needs to clone into the expression and plant expression vectors for further studies.

ABSTRACT

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

ABSTRACT

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

Introduction

Musa species and cultivars are identified mainly on the basis of morphological characters. Somatic mutations and changes brought about by environment are major obstacles to the accurate identification of clones (Kaemmer et al., 1992). Isoenzymatic markers provide an additional tool for the characterization of germplasm and have been successfully employed in banana to provide better understanding of somaclonal variants and to detect genetic differences at clonal level.

Being located within the center of diversity of cultivated banana, the South Indian state of Kerala accounts for a large share of variability, especially the AA, AB, AAB and ABB genomic groups.Nendran, the French plantain cultivar indigenous to Kerala (Jacob, 1952) is represented by many ecotypes/clones. Other prominent cultivar groups include Mysore (AAB), Rasthali (AAB), Neypoovan (AB), Kunnan (AB), Monthan (ABB) and Karpooravalli (ABB). The variability has been collected and conserved in the field at the Banana Research Station, Kannara. These have been characterized morphologically employing per INIBAP/IPGRI Musa descriptors, facilitating the identification of distinct cultivars, clones and synonyms.This project was initiated to survey the isozyme polymorphism in the cultivar groups and to select suitable system for clonal identification.Results of the study employing three enzymatic systems -esterase, diaphorase and peroxidase are presented

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

In oil palm there are three fruit forms viz. dura, tenera and pisifera. The commercial planting material is tenera produced by crossing dura and pisifera (D X P). Dura has thick shell and less economic part, where as pisifera is an abortic, female sterile type and cannot be reproduced in the normal ways. When tenera is selfed, segregation takes place in the monogenic Mendelian ratio; 1 dura: 2 tenera: 1 pisifera. Since all these palm types are morphologically alike, they can be confirmed only after fruiting, and verifying the internal fruit morphology. Fruiting in pisifera is highly erratic and often prolonged to several years. Proper technique in identifying these palms has been puzzling the breeder all over. Hence, a shell thickness marker is very important for early identification of the oil palm varieties.

An attempt has been taken to screen PCR primers and select the probable primers linked to the shell thickness in the present study. ‘Bulk segregation analysis’ approach was adopted for the study and DNA isolation was carried out from 25 dura and 25 pisifera palms. All the palms were developed from same tenera x tenera crosses. DNA from all the 25 duras and 25 pisiferas were pooled separately. Two pooled DNA samples were subjected to RAPD analysis with 50 random primers using the standard RAPD protocol. The amplified samples were electrophoresed and documented.

Both the pooled samples showed same banding pattern with all the primers, except 10 primers showing differences. Since the genetic configuration of the pooled dura DNA was likely to be same as that of the pisifera pooled DNA, the primers showing difference in banding pattern were the putative primers linked to shell thickness. All the 10 primers would be used for amplification of individual dura and pisifera DNA for confirmation of shell thickness marker.

ABSTRACT

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.