25. Embryogenic culture: a major factor that determines the Agrobacterium-mediated transformation efficiency in Basmati rice

1) Department of Biotechnology and Molecular Biology, DDS Haryana Agricultural University, Hisar 125 004, India
2) Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA

The effect of embryogenic competence of calli/tissues on Agrobacterium-mediated transformation efficiency in two commercial Basmati rice varieties, Taraori Basmati and Pusa Basmati 1 was studied. Mature-seed scutella calli (three-to nine-week-old), immature embryo-derived tissues (after three weeks of culture) and cell suspension cultures of Basmati rice varieties were co-cultivated for 3 days with A. tumefaciens strain LBA4404 (pTOK233) using the Hiei et al. (1994) procedure. Media used for callus initiation, and maintenance of cell suspension cultures and plant regeneration have been described earlier (Jain et al. 1996b). The rice tissues used for co-cultivation, were assessed for their embryogenic nature (by appearance) and the ability to regenerate shoots (% calli or clumps regenerating shoots). The level of GUS expression was examined immediately after co-cultivation and after 3-4 weeks of selection. After co-cultivation, calli were transferred onto selection medium (callus induction medium + 50 mg/l hgromycin + 250 mg/l cefotaxime) for 3-4 weeks. The freshly growing tissues were transferred onto antibiotics-supplemented regeneration media. Both hygromycin and cefotaxime were used in all the media and at all the stages to check the growth of Agrobacterium and reduce the number of escapes. The plants were transferred to soil in pots and grown to maturity in greenhouse. For GUS assay of the plants, root tips (1-2 cm) were used. PCR and Southern blot hybridization analysis were carried out to confirm the presence of transgene in transgenic plants as described earlier (Jain et al. 1996a).

In Basmati rice varieties, frequency of transient GUS expression, as measured immediately after co-cultivation, was about two-fold higher in six-week-old mature-seed scutella (MSS) calli compared to that observed in three- or nine-week-old calli (Table 1). The six-week-old calli also had higher embryogenicity and shoot regeneration potential compared to the three- and nine-week-old calli. Pusa Basmati 1 MSS call compared to that of Taraori Basmati were more embryogenic (by appearance), more responsive to shoot regeneration (Table 1) and showed over three-fold higher frequency of Gus expression than Taraori Basmati. In comparison to the MSS calli, tissues derived from immature embryos were more embryogenic as well as amenable to Agrobaterium-mediated transformation in both the Basmati rice varieties. The differences were markedly higher in the recalcitrant Basmati rice variety, Taraori Basmati. In this variety, frequency of co-cultivated calli showing Gus expression was 76.7% in case of immature-embryo derived tissues compared to 25.9% in mature seed calli. Gus expression data after five weeks of selection showed that 22.8 and 3.6% of the co-cultivated calli (6-week-old MSS calli) produced transgenic GUS-positive calli in Pusa Basmati 1 and Taraori Basmati, respectively (Table 1). Frequency of GUS expressing calli increased in both the Basmati rice varieties when immature-embryo derived tissues were used for transformation; the increase was substantially higher (17.3%) in Taraori Basmati (Table 1). Cell clumps isolated from cell suspensions when directly co-cultivated with Agrobacterium gave low frequencies of transient Gus expression, even though these cells had high meristematic activity and shoot regeneration potential. However, calli obtained after the culture of suspension cell clumps on 1.0% agarose-solidified callusing medium for two weeks, were more responsive to Agrobacterium-mediated transformation and showed higher level of Gus expression. It is not clear why meristematic, embryogenic liquid cell cultures are recalcitrant to agro-infection.

Selected hygromycin-resistant and GUS-positive calli regenerated shoots with frequencies ranging from 11 to 31% depending upon the genotype and age of callus used for

transformation (Table 1). The selected calli, however, displayed lower shoot regeneration frequencies compared to that obtained from the tissues used for transformation. This may be explained as an effect of increased age of the cultures and/or effect of antibiotics used in the media to regenerate shoots from selected calli. In general, shoot regeneration frequencies were higher in Pusa Basmati 1 compared to Taraori Basmati. Transformation efficiency of more than five Gus expressing plants could be obtained by using immature embryo-derived tissues in Taraori Basmati and six-week-old MSS and immature embryo derived tissues in Pusa Basmati 1. The transgenic nature of the plants was confirmed by the PCR and Southern blot analysis (data not shown).

The study shows a distinct positive correlation between the embryogenic potential of Basmati rice calli and their competence to be transformed by A. tumefaciens. Mature seed scutellum (MSS)-derived calli, have been an excellent starting material in our Pusa Basmati 1 transformation experiments, as reported earlier for japonica and other indica rice varieties (Hiei et al. 1994). The failure of Taraori MSS calli to yield transgenic plants can be attributed to its poor embryogenicity that first resulted in lower frequency of Agrobacterium-mediated transformation and later failed to induce regeneration in selected HygR calli. The premium Basmati rice variety Taraori, has been reported to be recalcitrant both for plant regeneration and transformation (Jain et al. 1996b). The results show that even a recalcitrant variety such as Taraori Basmati can be transformed through Agrobacterium using embryogenic tissues derived from the immature embryos.


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