Transformation of selenate and selenite to elemental selenium byDesulfovibrio desulfuricans

Summary Desulfovibrio desulfuricans (DSM 1924) can be adapted to grow in the presence of 10 mM selenate or 0.1 mM selenite. This growth occurred in media containing formate as the electron donor and either fumarate or sulfate as the electron acceptor. As determined by electron microscopy with energy-dispersive X-ray analysis, selenate and selenite were reduced to elemental selenium which accumulated inside the cells. Selenium granules resulting from selenite metabolism were cytoplasmic while granules of selenium resulting from selenate reduction appeared to be in the periplasmic region. The accumulation of red elemental selenium in the media following stationary phase resulted from cell lysis with the liberation of selenium granules. Growth did not occur with either selenate or selenite as the electron acceptor and¹³C nuclear magnetic resonance indicated that neither selenium oxyanion interfered with fumarate respiration. At 1 M selenate and 100 M selenite, reduction byD. desulfuricans was 95% and 97%, respectively. The high level of total selenate and selenite reduced indicated the suitability ofD. desulfuricans for selenium detoxification.     

Virulence of Agrobacterium tumefaciens strains with Brassica napus and Brassica juncea

Summary Brassica napus and Brassica juncea were infected with a number of Agrobacterium tumefaciens strains. Tumourigenesis was very rapid and extremely efficient on B. juncea with all but one of the strains. Tumourigenesis on B. napus varied widely. It was very efficient with the nopaline strains, was reduced with the succinamopine strain A281 and was very weak with the octopine strains. The latter observation was confirmed with six different B. napus rapeseed cultivars. The selectivity was due to differences in the virulence of Ti plasmids with B. napus, rather than the tumourigenicity of the T-DNA or virulence of the chromosomal genes associated with the strains. An exception was strain LBA4404. The virulence of the octopine strains was increased by coinfection with more virulent disarmed strains and by induction with acetosyringone.     

Transformation of Brassica juncea (L.) Czern with bacterial codA gene enhances its tolerance to salt stress

The codA gene for biosynthesis of glycinebetaine from Arthrobacter globiformis was used for transforming Brassica juncea cv. Pusa Jaikisan (which lack any means to synthesize glycinebetaine) through Agrobacterium mediated transformation. The stable insertion of the codA gene in the shoots obtained on medium with kanamycin and hygromycin was confirmed by PCR analysis of the nptII gene. Southern hybridization with a codA probe further demonstrated its successful integration. Immunoblot analysis revealed the presence of choline oxidase demonstrating that the bacterial codA gene had been successfully transcribed and translated. The seeds of transgenic lines showed enhanced capacity to germinate under salt stress as compared to that of the wild type. Further, the seedlings of transgenic plants that expressed codA gene showed significantly higher growth than that of the wild type under salt stress conditions. These results demonstrated that the introduction of a biosynthetic pathway for glycinebetaine into Brassica juncea significantly enhanced their salt tolerance. Hence, homozygous genotypes of selected transformed lines can be exploited for improving the salt tolerance of the desirable cultivars of Brassica juncea through breeding programmes.     

Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×10⁴ ml^-1 in Kao's medium supplemented with 1.0 mgl^-12,4-D, 0.1 mgl^-1 NAA and 0.5 mgl^-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 Em^-2s^-1) in a 16/8 h ligø ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl^-1 benzyladenine. On the fifteenth day, microcalli were plated on K₃ medium gelled with 0.5% agarose (Type 1, low EEO, Sigma). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8–10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Genetic and environmental effects on in vitro shoot regeneration from cotyledon explants of Brassica juncea

Twenty-one lines of Brassica juncea were screened for frequency of in vitro shoot formation from cultured cotyledons of 8-day old seedlings. All brown-seeded Indian lines showed good shoot regeneration with 20–50% of cotyledons giving rise to shoots. Very poor shoot regeneration (0–12%) was observed for the predominantly yellow-seeded Chinese/European lines, including two erucic acid-free lines. Germination of seedlings in hydroponic nutrient solution markedly enhanced subsequent shoot regeneration frequency from cotyledons of three Indian lines but had no effect on shoot formation from the recalcitrant Chinese/European lines.     

Plant regeneration from stem cortex expiant and protoplast cultures of Brassica juncea (mustard)

Summary Plant regeneration from stem cortex explants of 13 genotypes of Brassica juncea was assessed. Regeneration was strongly affected by genotype, as up to 50.6 shoots were produced per 100 calli of the most responsive line (Blaze), whereas no shoots were obtained from less responsive lines (Zeml, Vniimk351). Blaze was chosen for B. juncea stem cortex protoplast isolation. After one week of culture, 11–14% of the cells had divided, and about 0.002% produced 1–2 mm colonies within 6 weeks. Up to 7% of these colonies gave rise to shoots upon transfer to plant regeneration medium.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - BAP 6-benzylaminopurine - FDA fluorescein diacetate - IAA indole acetic acid - IBA indole butyric acid - 2ip 2-isopentenyl-adenine - NAA 1-naphthaleneacetic acid - TD thidiazuron     

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.     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

Genotype effects of Brassica napus on its reproductive behavior after pollination with B. juncea

To investigate the cause of variation in the interspecific crossability of Brassica napus, three different genotypes were studied in respect of their reproductive behavior after pollination with B. juncea. There were great differences among maternal genotypes in allowing foreign pollen to germinate on and penetrate into their stigmas, leading to a wide diversity of interspecific fertilization. The division of the hybrid primary endosperm nucleus and zygote appeared normal in all combinations of crosses. While the abundant free nuclei of the endosperm developed properly and never became cellular, the embryos degenerated as early as 10 days after pollination when the cultivar Rucabo, which had the highest fertilization record with species of B. juncea, was involved. When 81007 was used as female parent, the endosperm grew a little but the embryo halted at the heart-torpedo stage. Lack of nourishment might be responsible for the observed embryo abortion. Among the sic hybrid combinations, the cross 84014A x Changyang hunagjie was the only one where endosperm tissue was observable and an abnormal embryo occurred prior to cellular endosperm formation. Apart from the three typical embryological features, significant variation was also demonstrated among each of the cross combinations. Genetic diversity appears to exist not only between varieties, but also within cultivars. In addition, methods for developing interspecific crossable lines are discussed.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.     

Influence of paclobutrazol on growth and development of fruit in Brassica juncea (L.) Czern and Coss

Foliar spraying of paclobutrazol(5, 10 and 20 g mL^–1) on Brassica juncea (cv. RLM 514) plants, at the green floral bud stage of the terminal inflorescence, exerted a profound effect on growth and metabolism of fruits (siliquae). Paclobutrazol caused a decrease in siliqua length but enhanced significantly siliqua width, seed size (diameter) and seed number per siliqua. Paclobutrazol increased dry matter production and also hastened the relative growth rate of the siliqua wall and seed. In paclobutrazol-treated plants the siliquae were darker green, and during active phases of growth both siliqua wall and seed exhibited higher total chlorophyll (Chl) content, decreased chl a/b ratios, greater hill reaction activity of chloroplasts and increased activities of -amylase, -amylase and invertase than controls. Paclobutrazol enhanced the levels of total soluble sugars, starch, protein and free amino acids in mature seeds. The seed oil content increased by 2 to 3 per cent with a treatment of 10 g mL^–1 pacolobutrazol.     

Properties of partially purified leaf ornithine aminotransferase of Brassica juncea under salt stress

Properties of the partially purified L-ornithine: 2-oxoacid aminotransferase (EC 2.6.1.13) of leaves of Brassica juncea salt tolerant somaclone SR3P6-2 and its parent cv. Prakash were studied. The enzyme from the somaclone SR3P6-2 was relatively more efficient in terms of its Km, Vmax, and Ea (activation energy) and required higher levels of chlorides for inhibition as compared to the enzyme from the parent cv. Prakash. These results suggest some salt-stress related changes in the enzyme.     

The genetics of adult-plant blackleg (Leptosphaeria maculans) resistance from Brassica juncea in B. napus

The genetic control of adult-plant blackleg (Leptosphaeria maculans) resistance in a Brassica napus line (579NO48-109-DG-1589), designated R13 possessing Brassica juncea-like resistance (JR), was elucidated by the analysis of segregation ratios in F₂ and F₃ populations from a cross between R13 and the highly blackleg-susceptible B. napus cultivar Tower. The F₂ segregration ratios were bimodal, demonstrating that blackleg resistance in R13 was controlled by major genes. Analysis of the segregation ratios for 13 F₃ families indicated that blackleg resistance in these families was controlled by three nuclear genes, which exhibited a complex interaction. Randomly sampled plants of F₃ progeny all had the normal diploid somatic chromosome number for B. napus. The similarities between the action of the three genes found in this study with those controlling blackleg resistance in B. juncea is discussed.     

Diplotaxis catholica + Brassica juncea somatic hybrids: molecular and cytogenetic characterization

Summary Intergeneric somatic hybrids Diplotaxis catholica (2n=18) + Brassica juncea (2n=36) were produced by fusing mesophyll protoplasts of the former and hypocotyl protoplasts of the latter using polyethylene glycol. Out of 52 somatic embryos, 24 produced plants of intermediate morphology. Cytological analysis of 16 plants indicated that 15 were symmetric hybrids carrying 54 chromosomes, the sum of the parental chromosome numbers. One hybrid was asymmetric with 45 chromosomes. Nuclear hybridity of five putative hybrids was confirmed by the Southern hybridization pattern of full length 18s-25s wheat nuclear rDNA probe which revealed the presence of Hind III fragments characteristic of both the parental species. The hybridization pattern of mitochondria specific gene probe cox I indicated that three of the hybrids carried B. juncea mitochondria and one carried mitochondria of D. catholica. Presence of novel 3.5 kb Hind III and 4.8 kb Bgl II fragments suggested the occurrence of mtDNA recombination in one of the hybrids. The hybrids were pollen sterile. However, seeds were obtained from most of the hybrids by back crossing with B. juncea.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

Linked epistasis for six quantitative traits in Indian mustard (Brassica juncea (L.) Czern & Coss)

Summary Gene effects, and interactions, and associations between days-to-flower initiation and maturity, number of secondary branches and siliquae per plant, and 1,000-seed weight and yield per plant were studied in a cross of Indian mustard (Brassica juncea (L.) Czern & Coss) using the parents and F₁, F₂, F₃, B₁, B₂, B₁₁, B₁₂, B₂₁, B₂₂, B_1S, B_2S, B₁F₁, B₂F₁, B_1bip, B_2bip, F₂P₁, F₂F₁, and F_2bip generations. A linked digenic model was adequate for all characters studied. According to this model, the main effects, additive and interactions between linked pairs of genes, were present in varying proportions for days-to-flower initiation and maturity and number of siliquae per plant. The contribution of linked epistatic effects, however, was much greater than that of additive effects. Dominance effects contributed significantly to the inheritance of days-to-flower initiation. Duplicate epistasis was observed for all traits except 1,000-seed weight where epistasis was of the complementary type. A complete association among the genes of similar effect (increasing or decreasing) was observed for number of secondary branches and siliquae, and yield per plant. Coupling phase linkage was observed for days-to-flower initiation whereas repulsion phase linkage was observed for daysto-maturity and 1,000-seed weight.     

Growth of Albugo candida (race unidentified) on Brassica juncea callus cultures

An obligate fungus Albugo candida (Pers. ex Lév.) Ktze. (race unidentified) was successfully grown on host callus tissues of Brassica juncea cv. Varuna. Of the various type of diseased explants used, young (green) hypertrophied inflorescence axis bearing non-erumpent zoosporangial blisters allowed the fungus to multiply asexually over the host calli on modified MS-medium (Murashige and Skoog, 1962). The dual cultures were maintained up to 6–8 subcultures without loss of viability of zoosporangia on MS-medium supplemented with 10.0 mg L^–1 IBA, 0.05 mg L^–1 kinetin, 25.0 mg L^–1 AA, 1.0 mg L^–1 biotin, 1.0 mg L^–1 thiamine-HCl and 1.0 g L^–1 casein hydrolysate. The fungus grew only on the callus cells and not axenically on the medium. Pathogenicity test and histopathology of cultures proved the existence of the viable fungus in vitro.Abbreviations AA ascorbic acid - BAP 6-benzyl aminopurine - CH casein hydrolysate acid hydrolysed - 2,4-D-2,4 dichlorophenoxy acetic acid - FAA formaldehyde acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - HgCl₂ mercuric chloride - Kinetin 6-furfuryl aminopurine - MS Murashige and Skoog (1962) - NAA alpha naphthalene acetic acid - rh relative humidity - sdw sterile distilled water - wt. weights     

Alternaria incidence in some alloplasmic lines of Indian mustard (Brassica juncea (L.) Coss.)

Summary The cytoplasmic substitution lines of Brassica juncea (L.) Coss were evaluated for their field resistance to Alternaria blight (Alternaria brassicae). The euplasmic B. juncea cv. RLM 198 had a mesothetic reaction while alloplasmic B. juncea lines with cytoplasms of B. campestris, B. chinensis, and B. japonica were highly susceptible. B. nigra cytoplasm did not have any effect on the disease reaction of the B. juncea genome. However, the alloplasmic lines with the cytoplasm of B. napus and B. carinata revealed a comparatively higher degree of resistance. The study underlined the utility of cytoplasmic manipulations in modifying the phenotypic expression of nuclear genes.