Agrobacterium-Mediated Genetic Transformation of BPT-5204, A Commercially

Karnataka J. Agric. Sci.
, 20(4), (845-847) 2007
Agrobacterium-mediated Genetic Transformation of BPT-5204, a Commercially

Grown Indica Rice Variety*
BPT-5204 (GEB24/TN1//Mashuri), though released Freshly subcultured (for 5 d) embryogenic calli (2-4
during 1986, has spread to almost all the states in India mm in size) were soaked in bacterial suspension for 10 min. Calli
(Anonymous, 2004). It is known for its excellent cooking quality; were then placed on CIM medium with acetosyringone (200
but is susceptible to stem borer and brown plant hopper. Among µM) and co-cultivated for 3 days in the dark at 25°C. Calli were
various approaches used for improvement, genetic then washed thoroughly but gently in sterile water supplemented
transformation with appropriate gene(s) seems to be promising. with 500 mgl-1 cefotaxime, blot dried on filter paper, placed on
In rice, considerable difference exists for response to callus
CIM medium supplemented with 50 mgl-1 kanamycin and 300
induction, plant regeneration and transformation between indica
mgl-1 cefotaxime, and cultured for 3-4 weeks in the dark at 27°C.
and japonica types (Hiei et al., 1997). Tissue culture and genetic
Resistant callus clusters were transferred onto fresh medium
transformation in japonica rices has been well studied compared
(CIM containing 50 mgl-1 kanamycin) and cultured for 2-3 weeks
to indica types (Lin and Qifa, 2005; Yukoh and Toshihiko, 2006;
Yookongkaew et al., 2007). With this background, development (dark, 27°C). Resistant calli were then transferred to regeneration
of a protocol for Agrobacterium-mediated genetic transformation medium in which 2, 4-D was replaced with BAP (3 mgl-1) and
of three different explants viz., mature embryo, scutellum and NAA (1 mgl-1), and kept in the light. Regenerated shoots were
scutellum-derived callus was attempted with cry1Ac gene in transferred to basal MS medium for rooting.
BPT-5204, an indica rice.
The procedure followed for scutellum and mature
Agrobacterium strain LBA4404 harbouring the binary embryo co-cultivation and transformation was essentially same
vector pRD400 (Fig. 1) containing cry1Ac codon optimized gene as that used for scutellum-derived callus except for the starting
in fusion with CaMV35S promoter and nos terminator was plant material. These explants after the co-cultivation were placed
provided by Dr. Altosaar, Canada. This vector carries nptII gene on CIM and MS medium for callus induction and direct
as the selectable marker (Cheng et al., 1998). germination, respectively. Putative transformants (survivors on
Fresh seeds of BPT-5204 were obtained from ARS, kanamycin plates) were tested for the marker gene (nptII) using
Mugad, Dharwad. Three different explants viz., mature embryo, PCR with specific primers (npt_F: 5’ GAGGCTA
scutellum and scutellum-derived callus were used for co- TTCGGCTATGACTG 3’ and npt_R: 5’ ATCGGGAGC
cultivation. Mature embryos were obtained for co-cultivation GGCGATACCGTA 3’) to check for the real transformants.
by first de-husking the seeds, and washing them with 70% Transformation efficiency was calculated as number of PCR
ethanol for 30 sec followed by thorough rinsing with sterile
positive plants regenerated from the initial explants used for co-
distilled water. The seeds were soaked overnight over moistened
cultivation. Regenerants (Fig. 2) from three different explants
sterile filter paper in Petri dishes and then mature embryos were
such as mature embryo, scutellum and scutellum-derived callus,
excised under sterile condition. Scutellum explants were collected
which survived on kanamycin selection, were compared.
by excising from 4-5 days old in vitro grown seedlings. However,
the scutellum-derived calli were obtained by culturing the Considerable difference was noticed for the frequency of
scutellum on callus induction/maintenance (CIM) medium of putative transformants (calculated as the number of plants, which
MS (Murashige and Skoog, 1962) supplemented with 2, 4-D (1 survived kanamycin selection from a co-cultivated explant)
mgl-1) and benzylaminopuirne (BAP, 0.25 mgl-1). One month old among the three different explants used for co-cultivation. Out
scutellum-derived callus was taken for co-cultivation. of 320 mature embryos co-cultivated with Agrobacterium, 220
Table 1. Transformation efficiency with different explants of BPT-5204
Explant No. of Plants Putative

explants co- regenerated on transformants PCR +ve
cultivated selection media (plants survived on selection media) plants
Embryo 320 220 55 1
Scutellum 120 10 6 1
Callus 120 64 19 2
* Part of the M. Sc. (Agri.) thesis submitted by the senior author to the University of Agricultural Sciences, Dharwad-580 005, India.
845
Karnataka Journal of Agricultural Sciences, 20(4), 2007
Figure 1. pRD400 vector with codon optimized cry1Ac
Figure 2. Putative transformants
Figure 3. PCR detection of transformants

(Lane1: 100bp ladder, lane2: negative control, lane3: positive control, and lane4-7: transgenics)
846
Agrobacterium-mediated Genetic......
could produce plants initially, but only 55 (0.17) plants survived repeated PCR check was done to find out how many of the
upon selection with kanamycin. Out of 120 scutellum co- putative transformants were real by using nptII specific primers
cultivated, only 40 could produce callus, which subsequently against the template DNA obtained from all the putative
produced 10 plants and only 6 (0.05) could tolerate kanamycin. transformants. Out of 80 such plants, only 4 were found to
Finally, 120 co-cultivated calli produced 64 plants of which 19 carry nptII (Figure 3). Two transformants originated from
(0.16) survived the kanamycin selection (Table 1). scutellum-derived callus, and one each from mature embryo and
scutellum explant. It is well known that a given transgenic event
Transformation efficiency indicates the number of
need not result in plants with desired level of expression of the
transformants that are PCR positive for the transgene (cry1Ac)
transgene (Butaye et al., 2005). Thus in any crop plant
or marker gene (nptII). In general, scutellum-derived callus
transformation programme, it is necessary to obtain large number
recorded the highest transformation efficiency (1.67%) followed
of independent transgenic events to pick up the best among
by scutellum and mature embryo (Table 1).
them. Hence using more efficient selectable marker genes such
A number of studies have shown that not all plants as hph (for hygromycin) and bar (for basta) may prove useful
regenerated upon kanamycin selection are true transformants as reported in several studies (Cao et al., 1992; Ayres and Park,
(Toriyama et al., 1988; Ayres and Park, 1994). In this study, 1994; Aldemita and Hodges, 1996).
Department of Biotechnology, ANJANA KUMARI

University of Agricultural Sciences, R. S. BHAT
Dharwad-580 005, India. M. S. KURUVINASHETTI
(Received: November, 2006)
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Karnataka J. Agric. Sci.

, 20(4), (845-847) 2007

Agrobacterium-mediated Genetic Transformation of BPT-5204, a Commercially

Table 1. Transformation efficiency with different explants of BPT-5204

Explant No. of Plants Putative

Figure 1. pRD400 vector with codon optimized cry1Ac

Figure 2. Putative transformants

Figure 3. PCR detection of transformants

Department of Biotechnology, ANJANA KUMARI

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