Agrobacterium, the natures’ genetic engineer, has been used as a vector to create transgenic plants. Agrobacterium-mediated gene transfer in plants is a highly efficient transformation process which is governed by various factors including genotype of the host plant, explant, vector, plasmid, bacterial strain, composition of culture medium, tissue damage, and temperature of co-cultivation. Agrobacterium has been successfully used to transform various economically and horticulturally important monocot and dicot species by standard tissue culture and in planta transformation techniques like floral or seedling infilteration, apical meristem transformation, and the pistil drip methods. Monocots have been comparatively difficult to transform by Agrobacterium. However, successful transformations have been reported in the last few years based on the adjustment of the parameters that govern the responses of monocots to Agrobacterium. A novel Agrobacterium transferred DNA-derived nanocomplex method has been developed which will be highly valuable for plant biology and biotechnology. Agrobacterium-mediated genetic transformation is known to be the preferred method of creating transgenic plants from a commercial and biosafety perspective. Agrobacterium-mediated gene transfer predominantly results in the integration of foreign genes at a single locus in the host plant, without associated vector backbone and is also known to produce marker free plants, which are the prerequisites for commercialization of transgenic crops. Research in Agrobacterium-mediated transformation can provide new and novel insights into the understanding of the regulatory process controlling molecular, cellular, biochemical, physiological, and developmental processes occurring during Agrobacterium-mediated transformation and also into a wide range of aspects on biological safety of transgenic crops to improve crop production to meet the demands of ever-growing world’s population.

Agrobacterium-Mediated Gene Transfer in Plants and Biosafety Considerations

Transgenic technology in Indian mustard has expedited crop improvement programs. Further, there is a need to optimize gene editing protocols and find out the suitable target genes to harvest the benefits of gene editing technology in this important edible oilseed crop. Brassica juncea is an economically and industrially important oilseed crop being grown mainly in India and in some parts of Canada, Russia, China and Australia. Besides being consumed as edible oil, it also has numerous applications in food and paint industry. However, its overall production and productivity are being hampered by a number of biotic and abiotic stress factors. Further, its oil and seedmeal quality needs to be improved for increasing food as well as feed value. However, the lack of resistant crossable germplasm or varieties necessitated the use of genetic engineering interventions in Indian mustard crop improvement. A number of genes conferring resistance to biotic stresses including lectins for aphids’ control, chitinase, glucanase and osmotin for disease control and for abiotic stresses, CODA, LEA and ion antiporter genes have been transferred to Indian mustard. Both antisense and RNAi technologies have been employed for improving oil and seedmeal quality. Efforts have been made to improve the phytoremediation potential of this crop through genetic engineering approach. The deployment of barnase/barstar gene system for developing male sterile and restorer lines has really expedited hybrid development programs in Indian mustard. Further, there is a need to optimize gene editing protocols and to find out suitable target genes for gene editing in this crop. In this review paper, authors have attempted to review various genetic transformation efforts carried out in Indian mustard for its improvement to combat biotic and abiotic stress challenges, quality improvement and hybrid development.

Current achievements and future prospects of genetic engineering in Indian mustard (Brassica juncea L. Czern & Coss.).

Brassica is the most economically important genus in the cruciferae family, remarkable for containing agricultural and horticultural crops than any other genus. It includes over 30 wild species, their cultivars and hybrids including six most important species of Brassicas, Brassica campestris, B. juncea, B. carinata and B. napus which are oil yielding species. Due to their agricultural importance, Brassicas have been the subject of much scientific interest and considerable progress has been made in Brassica biotechnology. The molecular breeding and transformation technology for the introduction of desirable traits are two main strategies for its improvement. Several genes of agronomic importance have been identified in Brassica species and other identified genes have been transferred in Brassica species. Plant regeneration, transformation methods and gene of interest are the main requirements for gene transfer in any crop. Regeneration has been optimized via organogenesis and somatic embryogenesis using various explants. Transformation systems have been developed in almost all the economically important species of Brassica such as B. juncea, B. napus, B. rapa, B. oleracea, B. nigra, and B. carinata. Transformation has improved Brassica species for many traits, such as herbicide resistance, insect resistance, salt tolerance, oil quality improvement, for the production of pharmacological and industrial products, development of male-sterile lines and restoration system. Among different methods of transformation, Agrobacterium mediated genetic transformation is most widely used for Brassica and it is generally quite efficient and practical for most species in the genus. However, there is still a need for development of efficient transformation methods to overcome genotype dependency. In the present paper, the work on regeneration and genetic transformation in the Brassica species will be reviewed.

Genetic transformation in oilseed brassicas - A review

A non-tissue culture approach for the generation of transgenic Indian mustard (Brassica juncea) plants using Agrobacterium tumefaciens was developed. Inflorescences with floral buds were vacuum infiltrated with a suspension of A. tumefaciens strain EHA105 carrying a binary vector with an intron-containing β-glucuronidase (GUS) gene (uidA) as a scorable marker and a neomycin phosphotransferase gene (nptII) as a selectable marker. The seeds of agro-infiltrated plants (T0) were germinated on a medium containing 130 mg l−1 kanamycin, and the seedlings that remained green were considered T1 transgenic plants. Histochemical GUS assays, PCR, Southern analysis, and RT-PCR confirmed that both transgenes were integrated into the genome of T1 plants and were stably transmitted and expressed for over three generations. The transformants were obtained within 3–4 mo at a transformation frequency of 0.8%. This method may facilitate functional genomics and improvement of Brassica with novel desirable traits and with less time and expense.

A non-tissue culture approach for developing transgenic Brassica juncea L. plants with Agrobacterium tumefaciens

The plant family Brassicaceae (Cruciferae) is among the largest angiospermic family belonging to the order Brassicales. It consists of 12–15 tribes with 338–360 genera and about 3709 species distributed all around the globe in all continents. Plants usually grown in fields; their development and growth are mostly affected by environmental stresses, including both biotic and abiotic stresses. From abiotic stresses, mostly plants face drought and salinity stress more than others. Out of these two, agriculture is mostly affected by drought stress. Almost 40% of world’s agricultural land area is affected by drought stress. Drought is one of the major environmental stress factor that alters morphological, biochemical also physiological characteristics of all Brassicaceae plants. Drought also lowers the productivity of various central Brassica spp. via altering normal plant growth as well as their development. In this chapter, we have discussed the physiological and biochemical responses and tolerance of Brassicaceae plants to drought stress. Further, we also describe the most challenging strategies to overcome drought stress in Brassicaceae plants.

Brassicaceae Plants Response and Tolerance to Drought Stress: Physiological and Molecular Interventions

A simple and easy protocol for regeneration and genetic transformation ofBrassica juncea (L.) Czern and Coss cv. Pusa bold has been developed. The optimum regeneration was found on combination of BAP (3.0 mg/l) and IAA (0.2 mg/l). The genetic transformation protocol has been standardized usingAgrobacterium tumefaciens (gv3101). Cotyledonary petioles used as explant were pre-cultured for 48 h and than co-cultivated with Agrobacterium suspension for 2 days. Putative transformants were selected from selection medium containing 20 mg/l Kanamycine and 250 mg/l cefotaxime. Complete plant formed after placing shoots on rooting medium (0.5 mg/l IBA). Confirmation of integration of transgene was done by PCR using nptII gene primer.

	 

	Key words: Indian mustard, Agrobacterium tumefaciens, chickpea lectin gene, cotyledonary petioles, transformation.

Optimization and development of regeneration and transformation protocol in Indian mustard using lectin gene from chickpea (Cicer arietinum (L.))

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Optimization and development of regeneration and transformation protocol in Indian mustard using lectin gene from chickpea (Cicer arietinum (L.))