V. L. Chopra

Transgenic Indian mustard (Brassica juncea) with resistance to the mustard aphid ( Lipaphis erysimi Kalt.)

Abstract. Wheat germ agglutinin (WGA), the chitin-binding lectin from wheat germ, has been shown to be antimetabolic, antifeedant and insecticidal to the mustard aphid (Lipaphis erysimi. Kalt). A cDNA encoding WGA was transferred to Indian mustard (Brassica juncea cv. RLM-198) through Agrobacterium-mediated transformation. Southern analysis of the transgenics showed the integration of the transgene, while Northern and Western analyses demonstrated that the transgene was expressed in the transgenics. Bioassays using leaf discs showed that feeding on transgenics induced high mortality and significantly reduced fecundity of aphids.

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard.

Cytoplasmic male sterility in alloplasmic Brassica juncea carrying Diplotaxis catholica cytoplasm: molecular characterization and genetics of fertility restoration

Abstract The present study was aimed at characterizing cytoplasmic male sterility (CMS) and identifying the fertility restorer gene for CMS (Diplotaxis catholica) Brassica juncea derived through sexual hybridization. The fertility restorer gene was identified by crossing the CMS line with progeny plants derived from somatic hybrids of B. juncea and D. cathoilca. The CMS line is comparable to the nuclear donor B. juncea in all respects except for flower and silique characteristics. In CMS plants, the flowers have smaller nectaries, and anthers are converted into petals or tubular structures. Gynoecium exhibits a crooked style and trilocular ovary. Seed fertility was reduced in the CMS line. Genetic segregation data indicated that a single, dominant, nuclear gene governs fertility restoration. Restored plants showed a high female fertility and lacked gynoecium abnormalities. In fertility-restored plants, petal development was found to be variable; some flowers had the normal number of four petals, while others had zero to three petals. Interestingly, the trilocular character of the ovary was found to co-segregate with CMS and became bilocular upon male-fertility restoration. Thus, this trait appears to be affected by the interaction of nuclear and mitochondrial (mt) genomes. Restriction fragment length polymorphism analysis indicated that mt-genome of D. catholica is highly divergent from that of B. juncea. However, in Northern analysis, out of eight mt genes studied, an altered transcript pattern was recorded for only atpA. In fertility-restored plants, the atpA transcript became shorter, thereby showing its association with CMS.

Picloram induced somatic embryogenesis in chickpea (Cicer arietinum L.)

Complete plantlets were regenerated via somatic embryogenesis in the widely cultivated chickpea variety BG256. Leaf explants, from 13 day-old axenic seedlings yielded somatic embryos with high efficiency during dark incubation on MS medium supplemented with B 5 vitamins, 0.25 mg/l each of 2,4-D and picloram, and 0.1 mg/l BAP. A modified B 5 medium with 2 mg/l BAP and 0.1 mg/l IAA was found suitable for maturation of the embryos. The well-formed embryos germinated on basal B 5 medium supplemented with 0.25 mg/l BAP and developed into healthy plantlets on basal B 5 medium. Hardened plantlets grew into normal, fertile pod bearing plants upon transfer to soil.

Transgenic expression of hevein, the rubber tree lectin, in Indian mustard confers protection against Alternaria brassicae

The antifungal effect of hevein, the chitin binding lectin from rubber plant (Hevea brasiliensis), has been analyzed in transgenic plants for potential control of Alternaria blight in Indian mustard caused by Alternaria brassicae. A cDNA encoding hevein was transferred into Indian mustard (Brassica juncea cv. RLM-198). Southern analysis of the putative transgenics showed integration of the transgene. Northern and Western analyses proved that the integrated transgene is expressed in the transgenics. In whole plant bioassay under glasshouse conditions, transgenics were found to possess parameters that are associated with resistance such as longer incubation and latent period; smaller necrotic lesion size; lower disease intensity and delayed senescence.

Molecular Cloning and Characterization of FATTY ACID ELONGATION1 (BjFAE1) Gene of Brassica juncea

The Brassica juncea homologue of Arabidopsis thaliana FAE1 gene, which is responsible for elongation of fatty acid chain length from C18 to C20 and C22 was amplified via PCR (Polymerase Chain Reaction) using heterologous primers. The PCR product was cloned into pGEM-T vector, subcloned and sequenced. The BjFAE1 has 1536-nucleotides and shares 93.6% homology with the A. thaliana counterpart. Southern analysis, using the PCR product as probe, indicated that FAE1 gene is of the same size in all the cultivated Brassica species, i.e. B. juncea, B. nigra, B. campestris, B. oleracea, B. napus and B. carinata. It expresses strongly only in the developing seed and podwall.

Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea FAT TY ACID ELONGATION1 (BjFAE1) gene.

In Brassicas, the Fatty Acid Elongation1 (FAE1) gene product, a 3-ketoacyl-CoA synthase, is the first in a 4-enzyme complex involved in the synthesis of erucic acid from oleic acid. The FAE1 homologue from Brassica juncea cv. Pusa Bold was cloned in a binary vector both in sense and antisense orientations under the control of the CaMV35S promoter. The recombinant binary vectors were used to transform B. juncea cv. RLM 198 via Agrobacterium tumefaciens. The presence of the transgene was confirmed by polymerase chain reaction and Southern hybridization. Northern and western analyses showed the expression of the gene and protein, respectively, in the transgenic plants. Analyses of the fatty acid profile of the seed oil from homozygous T4 generation seeds revealed that over-expression of the FAE1 gene caused a 36% increase in the percent of erucic acid (37–49% compared to 36% in untransformed control). The down-regulation of FAE1 caused an 86% decrease in the percent of erucic acid to as low as 5% in the seed oil of transgenic plants. Thus, it is clearly possible to alter erucic acid content of mustard by altering the expression level of the FAE 1 gene.

Genetic and Biotechnological Approaches for Reducing Glucosinolates from Rapeseed-Mustard Meal

The seed meal of oleiferous brassicas is an excellent feed concentrate whose nutritional value is impaired by glucosinolates. Glucosinolates and the products of their hydrolytic cleavage are toxic, goitrogenic and reduce palatability because of their pungency. Efforts to develop low-glucosinolate varieties have not been very successful since the genetics of total glucosinolate biosynthesis is complex and highly regulated. This review summarizes information from genetic and plant breeding studies on identification and mapping of the genetic determinants for glucosinolate biosynthesis in Brassicas. Additional options offered by plant biotechnology using genetic engineering tools such as antisense technology and metabolic pathway engineering that could bring down the seed glucosinolate levels have also been highlighted.

Metabolic Engineering of Plant Lipids

Lipid metabolism in plants provides uncommon opportunities for genetic engineering to produce plant oils suited to a variety of end-uses. These opportunities include improvement of food and nutritional value, creating specialty lipids and feedstocks for high-value products and designing custom-made materials for industry. Genetic engineering intervention for production of novel transgenic plants which elaborate the desired product has graduated from academic exercise to commercial possibilities. It is now realized that transgenic crops can serve as biological factories for upscaling production of premium lipids via molecular farming. This review is an attempt at analyzing the status in this field.

A Simple Protocol for Regenerating Mesophyll Protoplasts of Vegetable Brassicas

We report here a simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli. Protoplasts from in vitro grown leaf material were cultured in Kao’s medium with a supplementation of 2,4-D, NAA, BAP and glucose, initially in dark for 3d and subsequently in light. Dilution of protoplast cultures was effected on the 7th, 10th and 13th day of culture initiation with Kao’s medium supplemented with sucrose, and reduced 2,4-13 content; NAA was omitted. Micro-colonies were plated on a K3 medium having 2,4-D, BAP and sucrose gelled with agarose. Transfer of calli to another K3 medium with zeatin regenerated shoots from cauliflower protoplast derived calli, whereas a medium with kinetin and zeatin supported shoot regeneration in cabbage and broccoli. Shoot regeneration occurred within 6-6 weeks of culture initiation. Shoots were easily rooted on MS medium without growth regulators.