Anther culture offers a rapid method of generating homozygous lines for breeding pro- gram and genetic analysis.To produce homozygous transgenic lines of rice(Oryza sativa L.)in one step,we developed an efficient protocol of anther-callus-based transformation mediated by Agro- bacterium after optimizing several factors influencing efficient transformation,including callus induc- tion and Agrobacterium density for co-cultivation.Using this protocol,we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene,Xa21 for resistance to bacterial blight,of which 140 were further confirmed by PCR and Southern hybridization analysis,including haploids(32.1%),diploids(62.1%) and mixoploids(7.5%).Fifteen diploids were found to be doubled haploids,which accounted for 10.7%of the total positive lines.Finally,by including 28 from colchicine induced or spontaneous dip- loidization of haploids later after transformation,a total of 43 doubled haploids(30.7%)of Xa21 transgenic lines were obtained.We also generated two RNAi transgenic haploids of the rice Os- MADS2 gene,a putative redundant gene of OsMADS4 based on their sequence similarity,to inves- tigate its possible roles in rice flower development by this method.Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations,in which lodicules were trans- formed into palea/lemma-like tissues,whereas identities of other floral organs were maintained.The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene.The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene.Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.
The dwarfing gene D-53 Is one of a few dominant genes for dwarfing In rice （Oryza satlva L.）. In the present study, our genetic analysis confirmed that mutant characteristics including dwarfing, profuse tlllerlng, thin stems and small panicles are all controlled by the dominant D-53 gene. We measured the length of each Internode of KL908, a D-53-carrylng line, and classified the dwarfism of KL908 Into the dn-type. In addition, we measured elongation of the second sheath and a-amylase activity In the endosperm, and we characterized KL908 as a dwarf mutant that was neither glbberelllc acid-deficient nor glbberelllc acid-Insensitive. Using a large F2 population obtained by crossing KL908 with a wild-type variety, NJ6, the D-53 gene was mapped to the terminal region of the short arm of chromosome 11, with one simple sequence repeat marker, Ds3, co-segregating, and the other, K81114, located 0.6 cM away.
