S. B. Narasimhulu

Species specific shoot regeneration response of cotyledonary explants of Brassicas

A study of shoot regeneration from cotyledons of three basic diploid species of Brassica, B. campestris (AA), B. nigra (BB), B. oleracea (CC) and their amphidiploids B. juncea (AABB), B. napus (AACC) and B. carinata (BBCC) showed species-specific responses for in vitro shoot regeneration. Analysis of the species mean shoot regeneration response over a range of growth regulator combinations revealed that i) B. campestris is the lowest regenerating species, ii) B. nigra and B. oleracea regenerate with high frequencies, iii) In amphidiploids, the presence of B. campestris component brings down shoot regeneration frequency below the value of B. oleracea in B. napus combination and is additive of the combining genomes in B. juncea combination. In B. carinata regeneration frequencies are less than the parental diploid species, iv) Significant intraspecific genotypic differences were observed for B. nigra and B. oleracea among diploids and B. juncea and B. carinata among amphidiploids, when cotyledons of eighteen genotypes were tested in one growth regulator combination.

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa.

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.

Production and characterization of intergeneric somatic hybrids of Trachystoma ballii and Brassica juncea

Intergeneric somatic hybrids, Trachystoma ballii (2n=16)+B. juncea (2n=38), were obtained by fusing mesophyll protoplasts of T. ballii and hypocotyl protoplasts of B. juncea using polyethylene glycol. The heterokaryotic fusion frequency was around 23%. Plants were regenerated from 10 out of 2×104 calli of which four were hybrids. Hybrids were intermediate between the parents in general morphology. However, in some characters one of the parents dominated. All the plants were symmetric in their chromosomal constitution as revealed by the formation of 26 bivalents at metaphase-I of meiosis. Two trivalents and 2 univalents were also observed in some pollen mother cells. Hybrid nature was also confirmed by ‘Southern’ hybridization of DNA of one regenerated plant restricted with Hind III and probed with the nick translated plasmid pTA71 carrying a wheat nuclear r-DNA sequence. Hybrid plant RT 1 showed bands characteristic of both parents. All the plants were absolutely pollen sterile. However, on backcrosses to B. juncea seeds were obtained.

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion.

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.

Comparative shoot regeneration responses of diploid brassicas and their synthetic amphidiploid products.

Comparative shoot regeneration responses of three diploid Brassica species, B. campestris (AA), B. nigra (BB) and B. oleracea (CC) and their synthetic amphidiploid combinations have been investigated. The study indicates that A genome has an inhibitory effect on regeneration as evident from significantly low responses of B. juncea (AABB) and B. napus (AACC) combinations, in comparison with regeneration response of B. nigra and B. oleracea. This inhibition may arise from genomic interactions or from the B. campestris cytoplasm interacting negatively with the alien genome. Significant cytoplasmic influence on regenerability has been observed in B. carinata (BBCC) synthesised from reciprocal crosses of B. nigra and B. oleracea.

Shoot regeneration in stem expiants and its amenability to Agrobacterium tumefaciens mediated gene transfer in Brassica carinata.

Immature stem segments of seven different genotypes of Brassica carinata produced shoots with variable frequencies when cultured in MS medium with BAP and picloram at 0.2 mg/l each. Line 171, which produced shoots with 100% efficiency from both cut ends of the expiant, was selected for testing the amenability of this regeneration protocol for genetic transformation. A non-oncogenic Agrobacterium tumefaciens containing plasmid PCV 730, a binary vector carrying resistance genes for kanamycin and hygromycin, was used. A cocultivation period of 4 d with a bacterial concentration of approximately 2.5×10 cells/ml, followed by a recovery period of 2 d, produced transformed shoots that could be selected and rooted in the presence of kanamycin at 15 mg/l. Transformation was confirmed by neomycin phospho-transferase assay and Southern blot analysis. Seed analysis of transformed plants indicated that kanamycin resistance was inherited in the progeny.

Resynthesis of Brassica carinata by protoplast fusion and recovery of a novel cytoplasmic hybrid

Brassica carinata (2n=34, BBCC), was synthesized by fusing dark grown etiolated hypocotyl protoplasts of B. nigra (2n=16, BB) with green mesophyll protoplasts of B. oleracea (2n=18,CC) using polyethylene glycol. Heterokaryons could be microscopically distinguished from the parental types by their dark green chloroplasts in the colourless hypocotyl protoplast background. The mean heterokaryotic fusion frequency estimated on the basis of this morphological distinction was about 16%. A total of 626 calli were obtained, of which 92 regenerated shoots after transfer to zeatin (2 mg/l) supplemented MS medium. Of these, 81 calli differentiated into plants morphologically similar to naturally occurring B. carinata and 11 calli yielded plants resembling parental types. Meiosis in seven hybrid plants showed the chromosome number to be 2n=34 the sum of B. nigra and B. oleracea chromosomes. Molecular confirmation of the amphidiploid nature of hybrids was obtained by probing with a B. juncea derived genomic clone. The use of chloroplast and mitochondrial specific gene probes, revealed that one plant was a cytoplasmic hybrid having cp DNA sequences of both B. oleracea and B. nigra and mt DNA sequences of B. nigra.

Rapid and efficient plant regeneration from hypocotyl protoplasts of Brassica carinata.

Protoplasts were isolated from hypocotyls of 7-d-old seedlings of three genotypes of Brassica carinata after enzymatic digestion in cellulase R-10 (0.5%) and pectolyase Y-23 (0.025%). The protoplasts were stabilized with 0.4 M mannitol used as osmoticum, and were cultured in darkness in Kaos liquid medium containing 0.4 M glucose and the growth regulators 2,4-D (1.0 mg/l), NAA (0.1 mg/l) and zeatin riboside (0.5 mg/l). Protoplasts were transferred to 16 h photoperiod conditions after 3 d of dark culture, and the medium was diluted to reduce the osmoticum on the seventh and tenth days of culture. Microcolonies were thus obtained which, upon transfer to MS agarose medium with 2,4-D (0.1 mg/l), BAP (1 mg/l) and 0.1 M sucrose, proliferated further to produce callus clumps. The plating efficiency of the three genotypes varied from 1 to 2%. Calli 2–3 mm in diameter were transferred to MS agarose plates with zeatin (2 mg/l) where they produced shoot buds and shoots with frequencies ranging from 22.5 to 74.2% for the three genotypes. The shoots were rooted in medium with IBA (1 mg/l) and were then established in soil. The time required for protoplast to plant development was 8 to 10 weeks.

The influence of cytoplasmic differences on shoot morphogenesis inBrassica carinata A . Br.

SummaryThe influence of cytoplasmic differences on shoot regeneration response has been investigated inB. carinata (BBCC) synthesised from reciprocal crosses betweenB. nigra (BB) andB. oleracea (CC). Mean shoot regeneration response from cotyledons of C cytoplasmic origin was twice as high as that from cotyledons with B cytoplasm. Statistical analysis of data indicated significant differences between cytoplasms, among growth regulator combinations and between cytoplasms and growth regulator interactions

Rapid and high frequency shoot regeneration from hypocotyl protoplasts of Brassica nigra

Protoplasts, isolated from etiolated hypocotyls of seven day old seedlings of Brassica nigra, were cultured in Kaos liquid medium containing 7.2% glucose, 2,4-d (1 mg 1-1), NAA (0.1 mg 1-1) and zeatin riboside (0.5 mg 1-1). After initial incubation for 3 days in dark at 25±1°C, cultures were transferred to a photoperiod cycle of 16/8 h and diluted on seventh and tenth day with MS medium containing 3.4% sucrose, 2,4-d (0.1 mg 1-1) and BAP (1 mg 1-1). About 62% of the cells divided at least once and 46% of them reached 8–16 cell stage in one week. The dividing cell clusters could be plated on agarose medium on the fifteenth day to obtain proliferating minicalli with a plating efficiency of 1.8%. 56.8% of minicalli, regenerated shoots on a regeneration medium containing 2 IP and IAA at 1 and 0.2 mg 1-1 respectively. The in vitro produced shoots were rooted in MS medium containing 1 mg 1-1 IBA and established in soil without difficulty. The time taken for protoplasts to develop into plants varied from 9 to 10 weeks.