The embryogenic potential of rice cell suspensions affects their recovery following cryogenic storage

Summary The rates of recovery from cryogenic storage of suspension cultures of the Japonica rice cultivar Taipei 309, as determined by the reduction of triphenyl tetrazolium chloride and cell regrowth, were significantly influenced by the embryogenic potential of the non-frozen cultures.     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

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. This revised version was published online in August 2006 with corrections to the Cover Date.     

Studies on the distribution of complementary genes causing F1 weakness in common rice and its wild relatives. 2. Distribution of two complementary genes,Hwc-1 and Hwc-2 gene in native cultivars and its wild relatives of tropical Asia

Summary Distribution of two complementary genes Hwc-1 and Hwc-2 causing F₁ weakness was examined using 159 native rice cultivars and 30 strains of wild relatives collected from various sites of tropical Asia including the Himalayan foothills. Hwc-1 is carried by a Peruvian cultivar, Jamaica, a tester cultivar of this experiment. It was not found in the tested cultivars and strains. Hwc-2 was widely distributed in the Japonica types tested. No wild strains had either Hwc-1 or Hwc-2. Hwc-2 was infrequent in the Japonica types collected from Southern Asian countries. Hwc-2 was estimated to be indigenous to the Japonica types, may be linked with certain gene(s) related to adaptation.     

Genetic analysis and fine mapping of LH1 and LH2, a set of complementary genes controlling late heading in rice (Oryza sativa L.)

Heading date in rice (Oryza sativa L.) is a critical agronomic trait with a complex inheritance. To investigate the genetic basis and mechanism of gene interaction in heading date, we conducted genetic analysis on segregation populations derived from crosses among the indica cultivars Bo B, Yuefeng B and Baoxuan 2. A set of dominant complementary genes controlling late heading, designated LH1 and LH2, were detected by molecular marker mapping. Genetic analysis revealed that Baoxuan 2 contains both dominant genes, while Bo B and Yuefeng B each possess either LH1 or LH2. Using larger populations with segregant ratios of 3 : 1, we fine-mapped LH1 to a 63-kb region near the centromere of chromosome 7 flanked by markers RM5436 and RM8034, and LH2 to a 177-kb region on the short arm of chromosome 8 between flanking markers Indel22468-3 and RM25. Some candidate genes were identified through sequencing of Bo B and Yuefeng B in these target regions. Our work provides a solid foundation for further study on gene interaction in heading date and has application in marker-assisted breeding of photosensitive hybrid rice in China.     

The electrofocusing composition of the Large and Small Sub-unit polypeptides of Fraction-1-Protein in rice x wheat and rice x sorghum hybrids

Summary The polypeptide composition of Fraction-1-Protein (F1P) from rice × sorghum, rice × wheat hybrids and their respective parents have been analyzed by a microelectrofocusing method. The large sub-unit (LSU) is composed of three polypeptides and the small sub-unit (SSU) of two polypeptides in rice and sorghum parents and rice × sorghum hybrids. Similarly, LSU is composed of three polypeptides in the rice and wheat parents and rice × wheat hybrids. Two polypeptides occur in the SSU of rice parent and rice × wheat hybrids where as only one polypeptide in the wheat parent. These polypeptides also differ in their isoelectric points. Based on the previous reports of F1P inheritance in hybrids in other crops, F1P analysis of rice × sorghum and rice × wheat hybrids does not seem to be an important marker to identify such intergeneric hybrids. Since this is first such report of F1P inheritance in hybrids between distantly related plants, its implication in different modes of inheritance are discussed.Abbreviations F1P Fraction-1-Protein - IEF Isoelectric focusing - pI Isoelectric points - LSU Large sub-unit - RuBPCase Ribulose 1,5-Bisphosphate Carboxylase-oxygenase - SSU Small sub-unit     

Comparative genetic analysis and molecular mapping of fertility restoration genes for WA,Dissi, and Gambiaca cytoplasmic male sterility systems in rice

The genetic relationship among three cytoplasmic male sterility (CMS) systems, consisting of WA, Dissi, and Gambiaca, was studied. The results showed that the maintainers of one CMS system can also maintain sterility in other cytoplasmic backgrounds. The F₁ plants derived from crosses involving A and R lines of the respective cytoplasm and their cross-combination with other CMS systems showed similar pollen and spikelet fertility values, indicating that similar biological processes govern fertility restoration in these three CMS systems. The results from an inheritance study showed that the pollen fertility restoration in all three CMS systems was governed by two independent and dominant genes with classical duplicate gene action. Three F₂ populations, generated from the crosses between the parents of good-performing rice hybrids, that possess WA, Dissi, and Gambiaca CMS cytoplasm, were used to map the Rf genes. For the WA-CMS system, Rf3 was located at a distance of 2.8 cM from RM490 on chromosome 1 and Rf4 was located at 1.6 cM from RM1108 on chromosome 10. For the Dissi-CMS system, Rf3 was located on chromosome 1 at 1.9 cM from RM7466 and Rf4 on chromosome 10 was located at 2.3 cM from RM6100. The effect of Rf3 on pollen fertility appeared to be stronger than the effect of Rf4. In the Gambiaca-CMS system, only one major locus was mapped on chromosome 1 at 2.1 cM from RM576. These studies have led to the development of marker-assisted selection (MAS) for selecting putative restorer lines, new approaches to alloplasmic line breeding, and the transfer of Rf genes into adapted cultivars through a backcrossing program in an active hybrid rice breeding program.