Hairui Cui

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species.

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R5 generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.

Inheritance and expression of the cry1Ab gene in Bt (Bacillus thuringiensis) transgenic rice

Abstract The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt (Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F2 populations. Crosses between japonica intra-subspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indica inter-subspecies led to distorted segregation of the cry1Ab gene in the F2 population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the Cry1Ab protein was kept quantitatively stable up to the R6 generation. The cry1Ab gene, driven by the maize ubiquitin promoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the Cry1Ab protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the Cry1Ab protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the Cry1Ab content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature.

QTL for rice grain quality based on a DH population derived from parents with similar apparent amylose content

A doubled haploid (DH)population consisting of 135 lines, derived from an indica (IR64) and a japonica (Azucena) rice with a similar apparent amylose content (AAC), was used to investigate the genetic factors affecting cooking and eating quality of rice. AAC,gelatinization temperature (GT), gel consistency (GC) and six starch pasting viscosity parameters were measured for quantitative trait loci (QTL) analysis using 193 molecular markers mapped on the DH population. A total of 17 QTLs were detected for the 9 traits, with at least one QTL and as many as 3 QTLs for each individual trait. No QTL for the measured parameters was found at the wx locus,possibly because of the similar AAC between the parents. Several QTLs with important effects on the variations in the measured parameters were detected in the present study which have not been found in earlier reports based on populations derived from parents with different AAC and wxgene alleles. Two interesting loci could be deduced from the present study according to the marker order compared with other genetic linkage maps. A QTL flanked by Amy2A and RG433 on the end of the long arm of chromosome 6, identified for GT, set back and consistency viscosity, might cover the gene encoding starch branching enzyme I. Similarly, a QTL flanked by RG139 and RZ58on chromosome 2, detected for hot paste viscosity and breakdown viscosity, might cover the gene encoding starch branching enzyme III. Generally, traits significantly correlated with each other shared identical QTL, but it was not true in some cases. The fine molecular mechanisms underlying these traits await further elucidation for the improvement of eating and cooking quality of rice.

Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants

The agronomic and morphological characteristics of Agrobacterium-transformed rice plants carrying the synthetic cry1Ab or cry1Ac gene were investigated. Tremendous variations in plant height, seed fertility, grain size and other traits were seen in 80 T1 lines, derived from 80 T0 plants of 9 rice varieties. On average, about 33% T1 lines had either morphological or agronomic variant plants. Most of the variations in T1 plants had no significant correlation with transgene insertion and were proved heritable to their progenies. Genetic analysis in T3 or T4 generations showed some simple mutations such as chlorophyll deficiency and stunted plants were independent of transgene insertion and seemed to be controlled by a pair of single genes. However, in two independent transgenic progenies of Xiushui 11, all plants homozygous for transgenes showed dwarfism while all hemizygous and null segregants had normal plant heights. Two advanced homozygous Bt lines, KMD1 and KMD2, were developed from these two progenies. Comparison of the agronomic traits of KMD1 and KMD2 with their parent displayed marked differences among them in terms of seedling growth, tillering ability, yield components and yield potential. The genetic variation observed was generally not linked to the transgene locus and was ascribed to somaclonal variation, but other causes might also exist in particular cases. The results are discussed in the context of choosing appropriate transformation methodology for rice breeding programs.

Genetic Analysis of Resistance of Bt Rice to Stripe Stem Borer (Chilo suppressalis)

Two transgenic Bt rice lines, KMD1 and KMD2, both containing a synthetic cry1Ab gene from Bt, were crossed with conventional rice varieties. The inheritance of resistance to SSB of KMD1 and KMD2was investigated through LSB and field examination of their progenies, e.g. F1, BC1 and F2 populations. In LSBs, 100.0% of newly hatched SSB larvae died on the second day after feeding on leaf tissues of F1 and GUS positive BC1 plants, of which the area of leaf tissues consumed by SSB is also similar to that of transgenic parents. These results imply that the resistance of Bt rice to SSB is dominantly controlled and could be easily exploited in hybrid rice production. Field evaluation showed that segregation ratios for SSB resistance to susceptibility in BC1 populations fit the ratio of 1:1, which was also confirmed by LSBs. However, in F2 populations, the ratio was significantly smaller than 3:1 for resistant to susceptible plants in all 6 indica × japonica (KMD1 and KMD2) crosses, though it fitted 3:1 in all 4 japonica × japonica crosses. The results implied that the resistance of Bt rice to SSB was controlled by a dominant gene which was present in a homozygous condition in both KMD1 and KMD2, but the inheritance could be affected by other factors. Assays for Cry1Ab protein showed that, in most crosses, the content of Cry1Ab is significantly higher in leaves of GUS positive F1, BC1 and F2 plants than that in transgenic Bt parent plants, which accounts for the high resistance observed in these plants to SSB.