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25. | Embryogenic culture: a major factor that determines the Agrobacterium-mediated transformation efficiency in Basmati rice |
R.K. JAIN
1, J.S. ROHILLA
2, S. BHUTAI
1, S. JAIN, V.K. CHOWDHURY
1 and R. WU
2 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. |
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