S. L. Mehta

Somaclonal Variation in a Food Legume—Lathyrus sativus

Somaclones of Lathyrus sativus cv P-24 were obtained from leaf, internode and root derived callus cultures. These showed significantly decreased neurotoxin (ODAP) content up to 0.03% compared to that of parent cultivar P-24 (0.3%). The somaclones also showed higher seed yield than parent P24. In addition. somaclone Bio 1–22 showed significantly decreased time for flowering. Other heritable morphological variant features were leaf length, leaf breadth, internode length, flower colour, seed colour, 100 seed weight, neurotoxin content of leaves and red markings on the pods.

β-N-Oxalyl-L-α, β-diaminopropionic Acid in Somaclones Derived from Internode Explants of Lathyrus sativus

Protocols have been developed for in vitro regeneration from internode explants from Lathyrus sativus. Callus raised on B5 medium supplemented with 10.7 μM NAA + 2.2 μM BA permitted shoot regeneration upon transfer to modified MS medium containing 10.7 μM NAA + 2.2 μM BA. Rooting was obtained only on 1/2 MS media containing 0.5 μM IBA. The in vitro regenerated plants, after primary and secondary hardening, were taken to the field. Analysis of ODAP in leaves and seeds was carried out. The low toxin containing progeny of the somaclones were further grown in the field. The toxin contents varied from 0.015% to 0.460% in leaf and 0.030% to 0.539% in seed in R, generation, as compared to 0.258% in leaf and 0.406% in seed for the parent P-24. Statistical analysis showed a positive significant correlation between leaf and seed ODAP contents. Mean seed toxin in R1 generation of some of the somaclones varied from 0.039–0.057% and single plant seed yield varied from 25.8 to 45.0 g. Some plants showed seed toxin content of less than 0.01% from 1–22 progeny. Thus, following in vitro culture of internode explant, toxin content in seeds in R2 generation has been found to be substantially reduced with single plant seed yield either equal to or higher than that of parent cv. P 24.

Cloning and Expression of OX-DAPRO Degrading Genes from Soil Microbe

The strain BYK 1, capable ot utilizing DAP or OX-DAPRO as a sole source ot nitrogen and carbon was Identitied as Pseudomonas stutzeri by various microbial and biochemical tests. Transtormation experiments showed that the OX-DAPRO utilization property Is encoded by the plasmid. Restriction of the plasmid DNA toll owed by cloning ot fragments and screening on OX-DAPRO medium led to Isolation of a clone with DNA Insert of ∼ 2.2 kb (designated as BSKS) which encoded OX-DAPRO degrading gel)e. The growth and OX-DAPRO utilization with this fragment was similar to wild type strain.

Metabolism of the Lathyrus sativus L. Neurotoxin, β-N-Oxalyl-L-α, β-diaminopropionic Acid, by a Pure Culture of a Soil-borne Microbe

Pure cultures of bacteria capable of utilizing the Lathyrus sativus L. neurotoxin, β-N-oxalyl-L-α, β-diaminopropionic acid (ODAP), as their sole carbon and nitrogen source have been isolated from soil-sludge filtrates. Three independent isolates, designated BYA1, BYT1, and BYK1, were selected by repetitive growth on the neurotoxin and purified based upon their antibiotic resistance. Of the three Isolates, strain BYA1 demonstrated the highest capacity for ODAP utilization, degrading greater than 98% of the ODAP present In the culture media within 12 h. Using a variety of morphological and biochemical criteria BYA1 was Identified as an Enterobacter cloacae. The bacterium harbors a single large plasmid (designated pBYA1) approximately 40–50 kb in size that contains the genetic Information for ODAP utilization and antibiotic resistance. Transformation experiments with E. coli recipient strains were used to further define the location of the sequences involved in ODAP metabolism.

Cloning and Characterization of a Gene Responsible for the Lathyrus sativus Neurotoxin Degradation

A plasmid pBYA1, capable of degrading the Lathyrus sativus neurotoxin β-N-oxalyl amino alanine (BOAA), was isolated from a soli microbe and a physical map of the plasmid constructed. A partial Sau3A library of the plasmid was then prepared in pUC18 and a recombinant clone with a 1.8 kb insert capable of growing in M9 medium, with BOAA as sole source of carbon and nitrogen, was isolated. A nested set of deletions of this 1.8 kb fragment was then generated using Bal31 and the shortened fragments subcloned in the vector pUC18. Each of these clones were sequenced by Sanger’s dideoxy method and the sequences overlapped and analysed. The 1.8 kb BOAA degrading fragment was found to contain a 630 nucleotide long coding stretch, coding for 191 amino acids. The initial 56 nucleotides were found to contain sequences resembling the Pribnow-Schaller box of prokaryotes, the “-35” sequences and a sequence resembling the “-43” region of the lac promoter.

Synthesis and Development of Starch Granules in High Lysine Barley Grains

Electron microscopy studies of developing endosperm have shown differences in the synthesis and development of starch granules between high lysine mutant Notch-2 and parent NP 113. The starch granules in Notch-2 were modified and did not develop into characteristic oval granules. Based on iodine absorption and phospholipids analysis, it is suggested that the presence of phospholipids impairs the development of starch granule in Notch-2. This is further confirmed by higher lipid density across starch granule in mutant Notch-2, and its absence in NP 113. Amylopectin from mutant differs from that of NP 113. The results indicate that the lack of geometry and smaller size of starch granules in Notch-2 is ultimately due to specific interaction of lipids with developing starch granules, and this leads to decreased yield.

Protein Bodies at Early Stages of Development in High Lysine Mutant Notch-2 and Parent NP-113 Barley

In barley parent NP-113, endospermic protein bodies originate on rough endoplasmic reticulum, either as electron transluscent vesicles or as very small, spherical, electron dense protein bodies, These are translocated to vacuoles tor enlargement and subsequent storage, Endospermic protein bodies of Notch-2 high lysine mutant are either vacuolar, or confined to distended cisternae of smooth endoplasmic reticulum. Vacuolar protein bodies are of two types one, flocculent, which loosely fill up almost the entire vacuolar space; two, spherical, relatively compact and granular, Protein bodies, confined to smooth endoplasmic reticulum are small, spherical, electron dense or electron transluscent, These protein bodies fuse to form electron dense proteinaceous masses which are deposited in the cytosol due to disruption of the confining smooth endoplasmic reticulum.

Purification of Oxalyl CoA Synthetase Enzyme from Lathyrus sativus and Raising of Antibodies

Oxalyl CoA synthetase, a key enzyme in the biosynthesis of β-oxalyl CoA synthetase, a key enzyme in the biosynthesis of days old seedlings of Lathyrus sativus using affinity chromatography and electroelution. The enzyme existed in three forms. They were designated as OCS-1, OCS-2 and OCS-3 and their molecular weights were found to be 63.1, 39.9 and 17.7 kDa, respectively. The antibodies were raised against all the three enzymes. The monospecificity of the antiserum was checked by immunoblotting. OCS-1 and OCS-2 did not share any common epltopes as no cross-reaction was seen.

Amyloplasts and Starch Characterization in High Lysine Barley Mutant

Starch granules of Notch-2 high lysine barley mutant are small, irregular in shape and show a virtual loss of birefringence and a higher gelatinization temperature, compared to parent NP-113. Starch polymer of Notch-2 is qualitatively different from parent, as evidenced by X-ray diffractograms. There is a higher incidence of V-pattern at early development as also at maturity in mutant as compared to parent. Intensities corresponding to B-pattern are present at early development in both parent and mutant. B-pattern persists at maturity in mutant, while it is replaced by A-pattern in parent. Amyloplasts of mutant are modified in contrast to normal, oblong amyloplasts of parent. Modified amyloplasts show loss of grana, increased granulation and lipid droplets. Extensive lipid complexing with starch polymers in mutant is indicated and possible commercial and nutritional potentials speculated.

Construction and Screening of Barley Genomic Library with a B1-Hordein Gene Probe

Barley genomic library from cv NP113 was made in a replacement vector EMBL-3. The titre was found to be 1.25 x 106 per μg of genomic DNA. The recombinants were screened using a B1-hordein DNA probe. One clone contained a positively hybridizing 4 kb fragment.