M. L. Lodha

Expression of Chromosome-integrated Hydrogen-uptake Genes in Cicer-Rhizobium

Integration of H2-uptake (hup) gene cosmid pHU52 into the chromosomal DNA, conferred H2- uptake activity on the Hup- Cicer-Rhizobium strain G36–84 in the free-living state and in nodules. In five transconjugants (G36–84:: Tn5:: pHU52) derepressed for hup gene expression, the specific Hup activity ranged from 158 to 256 nmole H2 hr-1 mg-1 protein which was 42 to 64% lower than the activity obtained in transconjugant with pHU52 as an episome. Integration of the cosmid significantly improved the relative efficiency of symbiotic N2-fixation by imparting H2-recycling capability to Hup-Cicer-Rhizobium. Demonstration of Hup activity in the nodules of field grown chickpea plants suggests that the integrated hup genes are stably maintained in natural environment

In Vitro Regeneration from Internodal Explants and Somaclonal Variation in Chickpea (Cicer arietinum L)

Protocols have been developed for the in vitro regeneration of plants from calli derived from internode explants of chickpea (Cicer arietinum L) cv Pusa-372. Callusing was induced on both B5 and MS media supplemented with different combinations and concentrations of auxins and cytokinins, but shoot regeneration was achieved only in B5 medium supplemented with 4.0 mg l−1 IAA and 0.5 mg l−1 BAP after serial subculture of callus on media with increasing concentration of IAA and constant concentration of BAP. Rooting could not be achieved in in vitro regenerated shoots on any one of the media tried. Complete plantlets were, therefore, developed through grafting of the in vitro regenerated shoot on established root stock. The grafting methodology was found to be highly efficient and reproducible. The somaclones developed produced viable seeds which showed variability in terms of seed colour and seed weight. Thus, the protocols developed in this study remove one important bottleneck in the development of transgenic chickpea.

Cloning of a Gene Sequence for co-3 Desaturase from Brassica juncea

AbstractcDNA was synthesized from RNA isolated from developing seeds of Brassica juncea cv Pusa Jai Kisan at 25–45 days after flowering and partial cDNA sequences encoding ω-3 desaturase gene were PCR amplified using specifically designed primers. The amplified products were cloned, and three of the clones subjected to partial sequence analysis. The clones showed high homology to ω-3 desaturase sequence from other sources. A genomic clone corresponding to ω-3 desaturase was isolated from the genomic library of B. juncea constructed in λEMBL-3 vector using one of the PCR clones.

Sequence Analysis of a Plastidial Omega-3 Desaturase Gene from Brassica juncea

A genomic clone (λg2) encoding a plastidial omega-3 desaturase has been isolated from a genomic library of Brassica juncea cv Pusa Bold and sequenced after subcloning. A continuous stretch of 3866 by of the clone sequenced showed homology with omega-3 desaturase from other plant sources but maximum homology was with that of a plastidial omega-3 fad 7 gene. This sequence designated as Bjgfad7 includes 1381 by 5’ untranslated region, 273 by 3’ untranslated region along with an open reading frame of 1290 by interrupted by 7 introns. The 5’ untranslated region contains putative cis elements responsible for seed specificity, light responsive and stress-inducible expression besides minimal promoter elements.

Energy-dependence of the Assimilatory Nitrate Uptake in Azorhizobium caulinodans Strain IRBG 46

Nitrate assimilation by suspensions of Azorhizobium caulinodans strain IRBG 46, as determined by disappearance of nitrate ions from the external medium, displayed the requirement of readily utilizable carbon source. Nitrate uptake was blocked by the uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone and by an inhibitor of ATPase, N, N — dicyclohexyl carbodiimide. The inhibition of nitrate assimilation in the absence of appropriate carbon source was not overcome by the non-physiological terminal electron donor ascorbate plus N-methyl phenazinium methyl sulphate, a substrate combination that allows electron transfer to O2 without the synthesis of ATP. These data suggest that transport of nitrate into the cell is directly dependent on ATP.

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd301. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.

Construction of a Gene Bank of Azorhizobium IRBG-46: Isolation of hup Genes

Genomic DNA from an efficient Hup+Sesbania-Azorhizobium strain IRBG-46 was isolated, partially digested with EcoRI and fractionated on a 10–40% sucrose density gradient to obtain DNA fragments in the size range of 15–23 kb. In order to isolate hup genes from this strain, a gene bank was constructed in Escherichia coil HB101 using a mobilizable plasmid vector pRK290 having a EcoRI cloning site. Approximately 2x104 Tc-resistant transformants were pooled to constitute the gene bank. Using 12.9 kb EcoRI fragment of cosmid pHU52 as a heterologous hup probe, a total of 2,000 clones were screened by colony hybridization. Five positive clones confirmed by secondary screening and ex planta uptake hydrogenase activity were identified. An insert size in the range of 15–22 kb was revealed by restriction analysis with EcoRI. These five recombinant plasmids containing Hup-determlnants of Azorhizobium IRBG-46 have been designated as pSRH1, pSRH2, pSRH3, pSRH4 and pSRH5. These plasm ids were transferred into Hup-Cicer-Rhizobium strain Rcd 301 to check the expression of hup genes in the new genetic background. In the transconjugants so obtained, the hup genes were found to express under ex planta conditions, and uptake hydrogenase activity ranged from 134 to 392 nmol H2 taken up per h per mg protein.

Isolation and Characterization of an Oxygen Sensitive Mutant of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 was isolated by NTG mutagenesis. It was defective in N2 fixation under 3% O2 level, while under 1% O2 it was almost as active as the parent strain IRBG 46. The mutant was also found to be a slow grower with reduced respiratory activity, low azide tolerance and no catalase activity. However, it did not differ from its parent strain with respect to nitrate respiration. Under symbiotic condition the mutant formed smaller, light green nodules as compared to bigger, dark green nodules formed by the wild type strain. The mutant was also defective in N2 fixation under symbiotic condition. Complementation analysis showed that the mutation might be in either fixL or fixJ gene which are involved in O2 regulation of nif/fix gene expression. A possible role of all these factors in conferring a highly O2 tolerant nitrogen fixing system in the organism, has been discussed.

Expression of hup Genes in Azorhizobium caulinodans and Their Coregulation With RuBP Carboxylase

In Azorhizobium caulinodans strain IRBG 46, H2 evolved by nitrogenase Induced uptake hydrogenase ex-planta. The strain expressed an efficient H2 recycling system under both symbiotic and ex-planta conditions. For the first time, a comparable relative efficiency of electron transfer to N2 via nitrogenase under symbiotic as well as ex-planta conditions for both Hup− strain B11 as well as Hup+ strain IRBG 46, has been reported. The study also suggested a coordinate regulatory relationship between rubisco and hup genes.