38. Agrobacterium-mediated transformation of Basmati rice to express the barley HVA I gene for enhanced tolerance to abiotic stress
  J.S. ROHILA1, R.K. JAIN2 and R. WU1

1)Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA
2)Department of Biotechnology and Molecular Biology, CCS Haryana Agricultural University, Hisar 125004, India

Drought and salt are major abiotic stresses that cause severe loss of Basmati rice production. During recent years, several genes which are responsible for over-production of osmolytes and stress proteins have been shown to confer increased tolerance to these stresses in transgenic rice plants (Jain and Jain 2000). One particularly promising gene, HVA 1 from barley (Hong et al. 1988), encodes a group 3 late embryogenesis abundant (LEA 3) protein. Previously, we demonstrated a positive correlation between HVA I gene expression and drought and salt-stress tolerance in a japonica variety of transgenic rice (Xu et al. 1996). However, under normal environmental conditions, overproduction of stress proteins using a constitutive promoter, requires extra cellular energy and building blocks, and may hamper the normal growth of rice plants (Su et al. 1998). Therefore for this study, we generated transgenic Basmati rice plants that synthesize a high level of LEA 3 protein only under stress conditions. We previously transformed rice by particle bombardment, in which most plants harbored two to five copies of integrated HVA I gene. Most plants that harbored more than two copies of the transgene showed gene silencing in later generations (Cheng et al. 1998). Consequently, for this investigation we transformed rice through the Agrobacterium mediated system, as this method produces transgenic plants with relatively low copy number of transgene as compared to other transformation methods (Roy et al. 2000).

To investigate the role of LEA 3 accumulation on salt and drought tolerance in Basmati rice, we constructed two plasmids in pCambia 1200, both using the HVA I gene. One of the plasmids, pRKJ 21, contains an ABA inducible promoter, and the other one, pRKJ 6, contains a constitutive promoter. A third plasmid, pRKJ 5, was constructed in pCambia 1201 with the

HVA I gene driven by the rice actin I promoter. Since pCambia vectors lack super-virulent genes and do not give higher transformation frequencies in recalcitrant Basmati rice, we mobilized pCambia vector (harboring the HVA I gene) into Agrobacterium tumefaciens strain LBA4404 containing pSB1 by triparental mating. pSB1 and pCambia plasmids belong to different incompatibility groups.

For this study we used mature seed scutellum-derived embryogenic calli as suggested by Jain et al. 2000. Calli were raised on MS medium containing casein hydrolysate (300 mgL-1), 2, 4-D (2.5 mgL-1 ), proline (560 mgL-1) and maltose (30 gL-1). Calli were infected with Agrobacterium containing either pRKJ 5, pRKJ 6 or pRKJ 21. After co-cultivation (two days) and two or three selection cycles (7-10 days each), hygromycin-resistant calli were transferred onto MS medium plates containing maltose (30 gL-1), kinetin (2 mgL-1), NAA (0.5 mgL-1)and agarose (10 gL-1) to regenerate shoots. Regenerated shoots were then transferred to magenta boxes containing half-strength MS medium plus sucrose (20 gL-1) and NAA (0.25 mgL-1) for efficient rooting of regenerated shoots. Both hygromycin (50 mgL-1) and cefotaxime (250 mgL-1) were used in all the media and at all stages to inhibit the growth of Agrobacterium and reduce the number of escapes. Regenerated plants were then transplanted in sterilized soil and grown in the green-house (30oC day and 24oC night, with a supplemental photoperiod of 10 hours). More than one hundred transgenic Basmati rice plants of various lines have been obtained from these transformation experiments. The presence of the transgene was detected at several developmental stages in R1/R2 generations. Transgenic expression was first detected by GUS activity in calli and then in leaves and roots of transgenic plants (pRKJ5) through histochemical assay with X-gluc (5-bromo-4-chloro-3-indolyl glucoronide) reagent incubated at 37oC overnight. In the segregating population, the leaves of individual plants were cut and dipped into solidified MS medium containing hygromycin (100 mgL-1). One week later, the resistant and sensitive phenotypes were scored. The leaves of resistant rice plants remained green, but leaves from sensitive plants turned yellow and eventually brown (data not shown).

Finally, genomic DNA from hygromycin-resistant transgenic rice plants was analyzed by Southern blot hybridization. For this analysis 8-10 microg of genomic DNA was digested with Sma I restriction enzyme (which cuts once in all three plasmids) and analyzed by Southern blot analysis (Fig. 1) to determine the copy number of the transgene. The results revealed that many hygromycin-resistant plants harbor a single copy of the HVA I gene. The accumulation of barley LEA protein in a number of individual R2 lines was determined by Western Blot analysis using LEA3 polyclonal antibody. The presence of 22-Kd band in SDS-PAGE gel indicated that there was barley LEA 3 protein accumulation in leaves of most of the transgenic plants.

Our results have shown that low copy number of the HVAI gene is integrated, stably inherited and highly expressed in Basmati rice genome. More experiments are underway to test the transgenic Basmati rice plants under different abiotic stress conditions.
This work was generously supported by the Rockefeller Foundation, USA.


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