The Preliminary Study On Transfer Chromosome-d Of Rape-radish With D Chromosome Addition Line To Brassica Oleracea

Rape-radish with d-chromosome addition line(AACC^+dd,2n=40)is the production:hybrid,obtained from Brassica nupus(AACC,2n=38)×Raphanus,sativus(RR,2n=18),via chromosome doubling and back crossing between Brassica nupus several times.Peterka's study and confirmed that the d chromosome in Raphanus sativus wasnematode resistance gene.In Brassica,plants lack of the kind of gene.The goal of this study is to provide some germplasm materials for breeding a newBrassica nematode resistance variety according to get a hybrid with additionalD-chromosome by sexual cross between Rape-radish with d-chromosome addition lineand cabbage.We selected the addition lines,which had more chromosomes,as the maternalplants,while the cabbage as the father plants,and observed the germination of thefather plants' pollen on the stigma of the addition line under the electron microscope.The papilla cells of addition line secreted a lot of callose after the adhesion ofpollen,even though,part of the pollen can reach the stigma and combine with theovule.In the emasculation pollination of the field hybridization,we found that all theprocess methods of the stigma promoted the hybrid compatibility,no matter deal withGly(100mg/L),GA₃(60mg/L)or repeated pollination.Among these processions,GA3(60mg/L)can increase the seeding rate effectively,but repeated pollination grouphas the highest silique-setting rate.Ovary culture can increase the survival of theembryos.Then we cultured the fertilized ovaries,which were picked from the fieldwithout any special treatment,in different media,and finally harvested 26 seeds.Compared the results among different culture media,we found that the ovaries whichwere cultured in "White(1963)+VB₆(0.5mg/L)+VB₁ (0.5mg/L)+Gly (15mg/L)+Niacin (2.5 mg/L)+IAA(1.5 mg/L)+Sucrose (50g/L),pH=5.6" and "1/2MS+B5 vitamine solution+IAA(1.5 mg/L)+Sucrose (50g/L),pH=5.8" had more seeds.We also found that all the plants resembled their maternal plants morphologically,someplants' morphological marker,eg.the shape of leaves,was between the two.We usedthe known specific molecular markers of radish D-chromosome:OPE07-363 andOPE03-518 to conduct a RAPD test with the parent plants' DNA,and screened 4regenerated plants with d-chromosome marker to make the hybrid identification.Theaccuracy of morphology identification is lower than other methods,so we conduct achromosome count for the 3 surviving regenerated plants.The results showed that thefilial generations including 2 kinds of chromosome number:29 and 48(or 49).Thenwe determined the filial generation as the very hybrid of the addition line and cabbage by PCR amplification of their DNA,which would get 2 kinds of banding patterns thatown the characteristic strips of the parent generation but different from the both at thesame time.The test showed that the pollen viability of filial generation is about 11.32%,the particle of the fertile flower's pollen is full and large,and the shape is between thematernal plant and paternal plant.