Molecular gene transfer for the generation of salt tolerant rapeseed (Brassica campestris, Brassica napus) varieties in Bangladesh
Department of Genetics & Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh, Fax: +880 91 55810, E-mail: LH@royalten.net
One-third of the population in Bangladesh is located at the southern coastal part of the country. Salinity is a big problem for crop production in these areas. This research aims at production of transgenic Brassica varieties for salt tolerance. In this paper, we describe a protocol for efficient, rapid and stable transformation of B. campestris and B. napus. That may be suitable for genetic modification of Brassica sp for salt tolerance.
Brassica, regeneration, transformation, Agrobacterium, salt tolerance
Rapeseed, Brassica campestris L. and Brassica napus are important oil-yielding crops in Bangladesh. In addition to its cultivation for edible oil, some cultivars of these species are also has industrial applications in plastics, lubricants, lacquers and detergents. Approximately 70% of total Brassica under cultivation is B. campestris. Brassica napus varieties are also getting popularity due to its higher yield. But due to a remarkable crop loss of 30-100% in these species farmers of the coastal areas are reluctant to cultivate these species in Bangladesh. Conventional plant breeding methods alone are insufficient to solve this problem. However, the application of genetic engineering (gene technology) will contribute significantly to combating the situation. Therefore, it is essential to develop a DNA delivery system for salt tolerant characteristics in the local varieties of these cultivated species.
Rapeseed has consistently proven to be one of the most recalcitrant members of the Brassicaceae in tissue culture. In spite of this problem there is great interest in the genetic transformation of this species for the production of transgenic plants. Therefore, efficient gene transfer and plant regeneration systems are necessary for the development of transgenic plants. The aim of this research project has been to establish an efficient method of transformation and regeneration using explants of B. campestris and B. napus via Agrobacterium-mediated transformation.
A protocol was developed for efficient, rapid and stable transformation of the local varieties of B. campestris. Ten local varieties were taken as plant materials. Seeds were submerged in 70% ethanol for 3 minutes and then for 30 minutes in 0.1% mercuric chloride. These seeds were rinsed three times in sterile distilled water and germinated on ½ -strength MS medium without phytohormones for 6 days. After germination the hypocotyls were cut into 1 cm segments, co-cultivated with the bacterial strain along with the plasmids and placed on shoot regeneration medium. MS media were supplemented with ten various combinations of phytohormones for shoot regeneration. Calli were initiated from the hypocotyl segments on MS medium supplemented with phytohormones, solidified with 5 g/l of agar after adjusting the pH to 5.7 under continuous dark at 30ºC.
The following Agrobacterium strains along with different plasmids for transformation were used: Agrobacterium rhizogenes strain LBA 9402 was used for the production of hairy roots (Figure 1). For co-transformation experiments, the strain LBA 9402 was used with the binary vector pBIN19 and p35S GUS INT gene (Vancanneyt et al., 1990). For plant regeneration 0.5 mm sections of the roots were excised and treated with a liquid callus-inducing medium (C23γ) (Guerche et al. 1987) along with a control for three days. After that they were placed on N5 medium with antibiotics (500 mg/l carbenicillin and 200 mg/l claforan). The GUS staining was carried out according to Jefferson et al. (1987) (Figure 2). PCR and southern analysis using the rolC gene as a probe were applied to confirm the presence of the Ri-TL-DNA in transformed plants.
Agrobacterium tumefaciens strains: I) GV3101 with the vir plasmid pMP90 the strain C58C1 ATHV with the vir-plasmid pTiBo542 (=pEHA101; Hood et al. 1986), a strain similar to EHA101 which has been shown to be highly virulent for many of the important leguminous crops (Jin et al. 1997, Hood et al. 1987) was used.
The nptII gene (neomycin phosphotransferase) was used as a selectable marker gene. The β-Glucuronidase-gene (GUS-Gene: Jefferson et al. 1987) under control of the Ubi- and 35S-Promotors, with an Intron (Vancanneyt et al. 1990), were used as the reporter gene.
For the confirmation of transgenic plants, leaf material was taken from the growing plants for DNA isolation. PCR- and Southern analysis was performed to determine the integration and the copy number of the transgene. The GUS-test was performed to demonstrate ß-glucuronidase expression and Northern analysis to test the expression of the inserted genes.
Figure 1. Stem segments with hairy roots
Figure 2. Gus expression in hairy root
An efficient, stable and reproducible Agrobacterium tumefaciens-mediated transformation protocol was developed for local varieties of Brassica campestris. Regeneration itself does not represent any problem. Stem segments proved to be the best explants. Shoot regeneration in Agrobacterium rhizogenes-mediated transformation was not possible till now. In Agrobacterium tumefaciens-mediated transformation successful shoots regeneration (Figure 3) was obtained from the transformed hypocotyls. MS media supplemented with 2 mg/l BAP+ 0.5 mg/l NAA showed the best results in regenerating the transformants. The transformed shoots were kept in a controlled environment for hardening for two weeks. After that they were kept in a net house and grown in plants (Figure 4). Successful flowering occurred and finally the seeds were harvested from the mature plants.
Figure 3. Regenerated shoot on selection media.
Figure 4. Regenerated plants
The transformation protocol will be utilised for the delivery of gene construct with salt tolerance in local varieties of rapeseed. The rapeseed varieties will be used by the farmers of the coastal wetland of Bangladesh that will play important role in poverty alleviation.
The author is grateful to Professor Heiko C. Becker and Dr. Christian Möller of Göttengen University, Germany for providing the bacterial stains and plasmids for this experiment. The work is supported by The United States Department of Agriculture (USDA), The United States of America (USA) under the project No. BG-ARS-113.
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