Indica rice variety IR64 was transformed with three genes, psy
(phytoene synthase), crtI (phytoene desaturase), and lcy
(lycopene cyclase), that are involved in the biosynthetic pathway of Beta-carotene
in the endosperm (Datta et al. 2003). Homozygous lines were developed
in the T2 generation through transgene-based screening. The
transgenic seeds accumulated total carotenoids in the endosperm of up
to 0.8 micro g/g. Apart from the stability and expression of transgenes,
detailed phenotypic and agronomic characterization of transgenic lines
are indispensable before releasing them to the field. The transgenics
in principle should be at least on a par with the non-transgenic control
for important agronomic characters in addition to having the novel trait
for which they have been transformed. Therefore, in this study, we assessed
the agronomic performance of homozygous transgenic IR64 progenies and
the non-transgenic control under screenhouse conditions at the International
Rice Research Institute, Philippines.
The experimental plot was divided into 10 subplots of equal size (2 m
x 6 m). Eight subplots were assigned to two independent transgenic lines
(four each) and two subplots were assigned to control plants. Each subplot
was transplanted with 100 plants, with a distance of 25 cm between plants.
Homozygosity of the transgenic progenies was confirmed by both PCR (data
not shown) and Southern analysis (Figure 1), which showed a 3.8-kb fragment
corresponding to the crtI expression cassette. Apart from this
expected fragment, other bands were also observed, indicating the integration
of rearranged copies of the crtI gene into the genome, which is
not uncommon in transgenics produced through biolistic transformation.
Agronomic data on nine characters were recorded for ten randomly selected
plants from each subplot, and were used for analysis of variance (ANOVA)
using SPSS software. For all the characters analyzed, the variance between
transgenics (pooled) and the control was statistically nonsignificant
(Table 1), indicating that the transgenics are similar to the control
in terms of yield traits. The overall phenotype and panicle characteristics
of the transgenics were also similar to those of the control (Fig. 2).
Similarly, no intra-transgenic variation was observed in the entire population
of 800 transgenic progenies (data not shown). This substantiated that
manipulation of the Beta-carotene biosynthetic pathway did not cause any
alteration in the agronomic traits of the transgenic plants. Under field
conditions also, transgenic Bt hybrids did not show any phenotypic
trade-off because of the Bt transgene. Rather, they had an approximately
28% yield advantage because of protection against yellow stem borer and
leaffolder (Tu et al. 2000b). Similar work has also been conducted
with a selected event of Xa21-IR72 rice
showing comparable results with the nontransgenic IR72 (Tu
et al. 2000a).
We also compared the transgenic IR64 with the control using 12 Universal
Rice Primers (URPs) (SRILS UniprimerTM Kit, Seoulin Scientific
Co. Ltd., Korea). These primers were shown to result in highly polymorphic
multiple bands among different genotypes of rice (Kang et al. 2000).
In this study, we also included two NHCD Golden rice lines (Datta et
al. 2003) with their control. As expected, polymorphic bands were
observed between IR64 and NHCD (for the transgenic as well as the control).
It is clearly evident that no polymorphism could be detected between the
transgenic and the control of both IR64 and NHCD (Fig. 3). This indicated
that the intact and/or rearranged copies of the transgenes are clustered
in a limited part of the genome, which could not be detected by these
random amplified polymorphic DNA (RAPD) markers derived from the repetitive
sequence of rice.
Our study showed no perceivable difference between IR64 Golden rice and
the IR64 control. However, large-scale multilocational field trials will
provide precise information on yield performance along with the influence
of the environment on Beta-carotene biosynthesis in rice seeds.
We gratefully thank USAID for financial support, Bill Hardy for editorial
assistance, and all involved in the Golden Rice Project.
Datta, K., N. Baisakh, N. Oliva, L. Torrizo, E. Abrigo, J. Tan, M. Rai,
S. Rehana, S. Al-Babili, P. Beyer, I. Potrykus and S.K. Datta, 2003. Bioengineered
'golden' indica rice cultivars with Beta-carotene metabolism in
the endosperm with hygromycin and mannose selection systems. Plant Biotechnol.
J. 1: 81-90.
Kang, H.W., J.G. Seung, J.C. Ryu, K.T. Kim and M.Y. Eun, 2000. Fingerprinting
genomes of various organisms using PCR with URP primers developed from
the repetitive sequence of rice. Plant & Animal Genome VII Conference,
San Diego, CA, USA, Jan. 9-12. http://www.intl-pag.org/8/abstracts/pag8629.html
Tu, J., K. Datta, G.S. Khush, Q. Zhang and S.K. Datta, 2000a. Field performance
of Xa21 transgenic indica rice (Oryza sativa L.), IR72.
Theor. Appl. Genet. 101: 15-20.
Tu, J., G. Zhang, K. Datta, C. Xu, Y. He, Q. Zhang, G.S. Khush and S.K.
Datta, 2000b. Field performance of transgenic elite commercial hybrid
rice expressing Bacillus thuringiensis delta-endotoxin. Nat. Biotechnol.