Brittle Stalk Genes Primary Mapping In Mutant Population Of Zea Mays And The Exploring Of Agrobacterium-Mediated Transgenic System In Miscanthus

Genetic identification of brittle stalk mutants is to find out the important genes involved in cell wall formation and biomass production in maize, because the brittle stalk reflects the cell wall composition and related mechanical strength alterations.From a Mutator inserted library constructed by a cross between Zong31 and W22::Mu, a brittle stalk mutant termed Y04 was screened out in this study. The Y04 mutant is a genetic homozygous mutant through several generations of self-crossing and phenotype observation, and its morphological, anatomical, biochemical and genetic characters were compared with wild type. The main results were described below:1) Phenotype observation: At the germination and seedling stages, this mutant did not show any phenotype, but it had brittle leaf vein and stem. 2) The puncture force of the mature stem in mutant is significantly lower than that of wild type.3) Based on free–hand section staining and scanning electronic microscopy observation, the mutant, compared with wild type, showed thinner and looser sclerenchyma with relatively reduced cellulose and increased lignin level. 4) Chemical analysis indicated that the mutant had less cellulose level and lower cellulose crystallinity(Cr I) value than those of wild type, whereas the mutant had significantly higher total soluble sugar amounts. In addition, the mutant exhibited slightly reduced hemicellulose level and increased lignin and pectin contents, consistent with the previous staining observations. 5) By genetic crossing Y04 mutant with Zong31(WT), the segregation ratio between normal and brittle phenotypes remains at 3:1. Map-based cloning suggested the mutation is located on chromosome 9 between SSR markers bnlg1159 and umc1771.Miscanthus sinensis is the most widely distributed Miscanthus species in China. M. sinensis is one of the parental species of M. × giganteus, which is an allotriploid hybrid between the diploid M. sinensis and the tetraploid M. sacchariflorusid. Genetic transformation is one of the most effective approaches for crop engineering, in which Agrobacterium-mediated transformation has been broadly applied for plant genetic engineering due to its cheap and simple operation. In this study, we use immature infloresences induce embryonic callus as receptor and conducted Agrobacterium-mediated transformation by testing various conditions. The main results were described below:1) After 15 days inducing, the culture medium becomes brown, whereas the brown was strongly inhibited while 0.2% activated-carbon(AC) was added. 2) After 80 days inducing, the healing rate was 79%, but it was 39% while 0.2% AC was supplied into the culture. 3) The Agrobacterium concentration(OD600) and infection time affect the transformation efficiency. After the first and second selective cultivation, some callus were survived under the OD600 at 0.7, infection time at 30 min, which should be the optimal conditions. 4) After 0、2、4、8、300、600、1200s ultrasonic treatments with Agrobacterium infection process, almost all callus could be survived from two rounds of selective cultivation. It also indicated that long time ultrasonic treatment could aid Agrobacterium infection for relatively high transformation efficiency.