eChapters

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

Chilli (Capsicum annuum L.) is a vegetable and spice crop of worldwide importance. Despite the fact that other closely related members of the Solanaceae family are easily transformed by genetic engineering, capsicum still remains recalcitrant to in vitro manipulation, due to lack of proper elongation of multiple shoot initials. An attempt has been made to standardize transformation protocol in two local cultivars of capsicum viz., Byadgi dabbi and Sankeshwar, using shoot tip explants.

The Shoot tip explants were co-cultivated (for 2 days) with Agrobacerium strain EHA105 harboring pBinBt3 plasmid having cry1AC gene and nptII marker linked to the CaMV 35S promoter and nos terminator. After co-cultivation shoot tips were transferred to MS medium containing 2mg/l IBA, 100mg/l kanamycin and 300mg/l cefotaxime. Survived plants were checked for the presence of transgene using cry1 AC and npt II specific primers. Expression of cry1AC gene in these plants was checked using DesiGen Xpresstrips.

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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.