Manchikatla Venkat Rajam

Effect of increased polyamine biosynthesis on stress responses in transgenic tobacco by introduction of human S-adenosylmethionine gene

Abstract Introduction of a human S -adenosylmethionine decarboxylase ( samdc ; EC 4.1.1.50) gene under the control of a constitutive promoter (CaMV35S) in tobacco ( Nicotiana tabacum var. xanthi ) led to increased polyamine biosynthesis. Transgenic lines with increasing spermidine and putrescine titres, especially in the conjugated forms, exhibited tolerance to salinity and drought as well as to fungal wilts caused by Verticillium dahliae and Fusarium oxysporum . This is the first report where tolerance to both abiotic and biotic stresses has been observed in transgenic plants following transfer of a polyamine biosynthesis gene.

Silencing of acetylcholinesterase gene of Helicoverpa armigera by siRNA affects larval growth and its life cycle.

RNA interference is an effective means of regulation of gene expression both in vitro and in vivo. We studied the effect of siRNA on larval development by selective targeting of the acetylcholinesterase (AChE) gene of Helicoverpa armigera. Chemically synthesized siRNA molecules were directly fed to H. armigera larvae along with the artificial diet. The siRNA treatment resulted in specific gene silencing of AChE and consequently brought about mortality, growth inhibition of larvae, reduction in the pupal weight, malformation and drastically reduced fecundity as compared to control larvae. Our studies suggest some novel roles for AChE in growth and development of insect larvae and demonstrate that siRNA can be readily taken up by insect larvae with their diet.

Applications of biotechnology in eggplant

Eggplant (Solanum melongena L.), an economically important vegetable crop in many countries in Asia and Africa, often has insufficient levels of resistance to biotic and abiotic stresses. Genetic resources of eggplant have been assessed for resistance against its most serious diseases and pests (bacterial and fungal wilts, nematodes and shoot and fruit borer). Attempts at crossing eggplant with its wild relatives resulted in limited success due to sexual incompatibilities. However, the ability of eggplant to respond well in tissue culture, notably plant regeneration, has allowed the application of biotechnology, particularly the exploitation of somaclonal variation, haploidisation, somatic hybridisation and genetic transformation for gene transfer. Somaclonal variation has been used to obtain lines with increased resistance to salt and little leaf disease. Traits of resistance against bacterial and fungal wilts have successfully been introduced into the cultivated eggplant through somatic hybridisation. However, most somatic hybrids were sterile when the parental lines were distantly related. In contrast, the use of close relatives as fusion partners or highly asymmetric fusion resulted in the production of fertile hybrids with resistance traits and a morphology close to the cultivated eggplant, thus avoiding the series of backcrosses necessary for introgression of desired traits into eggplant. As far as molecular markers and genetic engineering are concerned, the information available for eggplant is very scanty. Two genetic linkage maps have been established by using RAPD and RFLP markers. In order to analyse the genetic relationships between eggplant and its relatives, some studies based on AFLP and ctDNA analyses have also been conducted. So far only resistance against insects, and parthenocarpic fruit development have successfully been developed in eggplant using Agrobacterium tumefasciens transformation. However, some work on genetic engineering of eggplant for other biotic and abiotic stresses has recently been initiated.

Ornithine decarboxylase transgene in tobacco affects polyamines,in vitro-morphogenesis and response to salt stress

Summary Transgenic tobacco plants expressing the putrescine synthesis gene ornithine decarboxylase from mouse were raised to study the effects of up-regulation of a metabolic pathway as critical as the polyamine biosynthesis on the plant growth and development, in vitro -morphogenesis and their response to salt stress. Further, the response of the alternate pathway (arginine decarboxylase) for putrescine synthesis to the modulation of the ornithine decarboxylase pathway has also been investigated. The over-expression of the odc gene and increased levels of putrescine in tobacco led to a delay in plant regeneration on selection medium which could be overcome by the exogenous application of polyamine biosynthesis inhibitors and spermidine. Further, the lines generated had a variable in vitro morphogenic potential, which could be correlated to the shifts in their polyamine metabolism. These studies have brought forward the critical role played by polyamines in the normal development of plants and also their role in plant regeneration. Since polyamines are known to accumulate in cells under abiotic stress conditions, the tolerance of the transgenics to salt stress was also investigated and the transgenics with their polyamine metabolism up-graded showed increased tolerance to salt stress.

Biotechnology of eggplant

Eggplant (Solanum melongena L.) is an important vegetable crop grown in various tropical and temperate parts of the world. There is a wide genetic diversity in the cultivated as well as the wild species of eggplant. Cultivated varieties of eggplant are susceptible to a wide array of pests and pathogens as well as to various abiotic stress conditions. In contrast, the majority of wild species are resistant to nearly all known pests and pathogens of eggplant and thereby are a source of desirable traits for crop improvement. Tissue culture protocols for organogenesis, somatic embryogenesis, anther culture and protoplast culture have been well established for the eggplant. Somatic hybridisation has also been attempted for transferring useful genes from wild species to the cultivated plants through protoplast fusion. However, the information on genetic engineering and molecular biology of eggplant is very limited. Transgenic eggplants for insect resistance, for the production of parthenocarpic fruits and abiotic stress tolerance have been accomplished. However, transgenics of eggplant are yet to be developed for improvement of other agronomic traits, including disease and pest resistance, and quality and shelf life of fruits. Molecular markers to complement traditional breeding programs are being developed for genome mapping of agronomic traits. The present review summarises efforts to improve eggplant genetics with an emphasis on the use of biotechnology to introgress genes from wild species into cultivated eggplant.

Polyamines as biomarkers for plant regeneration capacity: improvement of regeneration by modulation of polyamine metabolism in different genotypes of indica rice.

The importance of cellular polyamine (PA) levels and the ratio of putrescine (Put) to spermidine (Spd) for plant regeneration ability via somatic embryogenesis in several commercially grown indica rice varieties is reported here. The genotypes namely NDR-624, IR-20, IR-36, BJ-1 (having Put:Spd ratio approximately 2.3) showed superior plant regeneration while KL, PB-1 and TN-1 (having Put:Spd ratio approximately 3.8) showed moderate plant regeneration ability. The genotypes namely HS, Bindli, DV-85, ACB-72, IR-64 and IR-72 (having Put:Spd ratio approximately 5.0) showed poor plant regeneration ability. In contrast KH-7 (Put:Spd ratio approximately 10.0) showed no response at all. Favorable modification of cellular PA titers and their Put:Spd ratio by the addition of exogenous PAs (Put, Spd) or their biosynthesis inhibitor, difluoromethylarginine (DFMA) led to the induction/promotion of plant regeneration in poorly responding genotypes. These results showed a close relationship between cellular PA levels and their Put:Spd ratio with in vitro morphogenetic capacity in indica rice and suggest that the cellular PAs and Put:Spd ratios are important determinants (biomarkers) of plant regeneration ability in indica rice, and the improvement/induction of plant regeneration in morphogenetically poor and recalcitrant species could be achieved by modulating PA metabolism.

Efficient plant regeneration from long-term callus cultures of rice by spermidine

A significant reduction in regeneration potential with increasing age (upto 12months) in rice (Oryza sativa L. cv.TN-1) embryogenic callus cultures was observed. Spermidine, while having an inhibitory effect on plant regeneration in fresh callus cultures, promoted morphogenesis in long-term callus cultures. A massive accumulation of polyamines, particularly putrescine (5-fold) was observed in 12 month old cultures resulting in a change of putrescine /spermidine ratio, which seems to be important for maintaining the morphogenetic response. Application of exogenous spermidine to 12 month old cultures showed increased levels of polyamines and restored the putrescine/spermidine ratio comparable to that found in freshly induced cultures, concomitantly, promoting the plant regeneration via somatic embryogenesis in long-term rice callus cultures.

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T0 and T1generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.

Thaumatin gene confers resistance to fungal pathogens as well as tolerance to abiotic stresses in transgenic tobacco plants

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.

Effect of genotype, explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 μM indoleacetic acid (IAA)+13.3 μM benzyladenine (BA); 22 μM BA; and 44 μM BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 μM BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 μM IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 μM IAA plus 13.3 μM BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.