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 Uniprimer^TM 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.

Acknowledgment

We gratefully thank USAID for financial support, Bill Hardy for editorial assistance, and all involved in the Golden Rice Project.

References

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. 18: 1101-1104.