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.     

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.     

In vitro breeding of Brassica juncea L. to enhance metal accumulation and extraction properties

In vitro breeding and somaclonal variation were used as tools to improve the potential of Indian mustard (Brassica juncea L.) to extract and accumulate toxic metals. Calli from B. juncea were cultivated on a modified MS medium supplemented with 10–200 μM Cd or Pb. Afterwards, new B. juncea somaclones were regenerated from metal-tolerant callus cells. Three different phenotypes with improved tolerance of Cd, Zn and Pb were observed under hydroponic conditions: enhanced metal accumulation in both shoots and roots; limited metal translocation from roots to shoots; reduced accumulation in shoots and roots. Seven out of thirty individual variants showed a significantly higher metal extraction than the control plants. The improvement of metal shoot accumulation of the best regenerant (3× Cd, 1.6× Zn, 1.8× Pb) and metal extraction (6.2× Cd, 3.2× Zn, 3.8× Pb) indicated a successful breeding and selection of B. juncea, which could be used for phytoremediation purpose.     

Ectopic expression of Atleafy in Brassica juncea cv. Geeta for early flowering

High temperature stress during pod filling severely affects the yield of Brassica juncea. Early flowering can evade the terminal heat stress and result in early maturity of the crop. In this study, a regeneration and transformation protocol has been standardized for B. juncea cv. Geeta. Hypocotyl from 5-day-old seedlings were used as explants. Of the various combinations of auxins and cytokinins tried along with Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium, MS + IAA (0.2 mg/l) + BA (3 mg/l) proved best for shoot regeneration with 89.9 % regeneration efficiency. To induce early flowering Leafy gene from Arabidopsis thaliana was transformed using Agrobacterium mediated transformation method. After 12 weeks transgenic plants showed flowering in vitro whereas their untransformed counterpart did not flower even after 16 weeks. The maximum transformation frequency was 4 %.     

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.     

Mining expressed sequence tags of rapeseed (Brassica napus L.) to predict the drought responsive regulatory network

Physiology and Molecular Biology of Plants - It is of great significance to understand the regulatory mechanisms by which plants deal with drought stress. Two EST libraries derived from rapeseed...     

The role of cotyledonary tissue in the differentiation of shoots and roots from cotyledon explants of Brassica juncea (L.) Czern.

In cotyledon cultures of Brassica juncea, shoots and roots invariable differentiate at the cut end of the petiole. Organogenesis occurred only if the proximal cut end of the petiole was in contact with the medium. In the absence of the petiole, differentiation from the lamina was rare. Hence investigations were carried out to study the influence of the cotyledonary lamina on regeneration of shoots and roots from the petiolar cut end. The lamina tissue was surgically removed from the cotyledon explants at different durations (0–10 days) after culturing them on either root-forming (basal medium) or shoot-forming (basal medium containing 5.0 M N⁶-benzyladenine) media. The differentiation of roots or shoots from the petioles was dependent on the presence of the lamina for at least 7 days of culture. Quantitative removal of the laminar tissue had a corresponding negative effect on shoot bud differentiation from the petiole. The nature of the lamina factor was found to be auxin-like.     

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     

Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288

Background

Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288.

Results

Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line “J163-4” are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity.

Conclusion

The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-322) contains supplementary material, which is available to authorized users.