| Haploid breeding of wheat anther culture is one of important technologies in wheat breeding, Dwarf male- sterile wheat is an important wheat breeding material which was created by Chinese Academy of Agricultural and was application in many breeding units.We investigate the affecting factors of hybrid progeny anther culture and improve the efficiency of anther culture of hybrids, which have important theoretical and practical significance on wheat breeding with dwarf male-sterile wheat as recurrent parent.Biolistic bombardment is an important method in current wheat transgene,However,the low regeneration of wheat immature embryocallus, mature embryo callus after biolistic bombardment has become an important restriction factor in genetic transformation of wheat.Contrast with wheat immature embryo,mature embryo callus,wheat anther callus have stronger regeneration features and haploid features,it is a potential,excellent wheat biolistic receptor,But the methord has not break through in using wheat anther callus as biolistic receptor.D32 is a transcription factor gene related to the stress resistance characteristics which was isolated from soybean,its products transcription factor can interact with the DRE,and regulate the expression of the downstream range gene which is related to the resistance of drought,low temperature and high salt stress,then enhance the stress resistance characteristics of plants. In this study,we use dwarf-male-sterile wheat F1 anthers of different genotypes as materials,and we find out their culture characteristics,summering characteristics and the method of improving the efficiency of anther culture;Using dwarf-male-sterile wheat F1 anthers callus as receptors,we studied the techniques and methods of using biolistic bombardment transform D32 gene into common wheat anther callus,and obtained the following results:1.Under certain concentrations,hydrolisis casein and L-glutamine on culturing the dwarf-male-sterile wheat F1 anthers all have certain positive effects on promoting F1 anther callus.in their optimum concentrations, The callus induction rate of the former(6.28%)was increased by 47.07%,while the rate of the latter(5.23%)increased by 22.48%,it indicated that hydrolisis casein is to meet the tissue's needs of absorption and utilization on the nitrogen more than L-glutamine.The promoter action of both hydrolisis casein and L-glutamine to F1 fertile anther materials of dwarf-male-sterile wheat have thresholds.beyond the threshold,the effect will be decreased.It indicated that excessive nitrogen will affect the cultured materials absorption and utilization to the other elements,and can lead to the its metabolic disorders,and affects its differentiation efficiency.2.Biotin is one of vitamin B complexes,its role is mainly promoting carbon fixation and carboxyl transfer reaction caused by biotin enzyme and promote the absorption and use of carbon in plants.Appending biotin 2.0,3.0 mg/L in induction medium can increase the Callus induction rate of F1 fertile anther materials of dwarf-male-sterile wheat by 55.27% and 54.33%.3.Genotype is on of the mainly factors in tissue differentiation and green plant re-differentiation.Callus induction rate and differentiation plant frequence have no necessary connection in materials of different genotypes.In other words,a material which has high callus induction is not necessarily have high differentiation plant frequence.So,we should consider both of them and select two are good when selecting appropriate material.4.With 3.0,4.0 mg/L mass concentrations PP333 putting into the media and undergoing continuous subculturing,the cultured green wheat anther plantlets have passed the summer safely.Under the culture condition of high temperature(25±1℃),adding PP333 3.0mg/L into root media,the anther plants shown growth rapid,and in late stage,they overgrowed.However, adding PP333 4.0 mg/L into root media can showed a better effect of slowing the growth,and phytotoxicity phenomenon did not appear.5.The basic procedure of the transformation of wheat pollen callus by biolistic bombardment is as follows:select the anther callus with the mille size and transfer them to hypertonic medium for 4-6 h.The parameters of bombardment:the diametre of gold powder is 1.0μm,the distance between the barrier network and bombarding material is 9.0 cm,the pressure of split film can bear is 1100 Pa,the amount of each gun powder/DNA was 1μg/60μg,bombard once for per culture dish material.After transformation,we continue to culture callus in hypertonic medium for8-9 h,then transfer them to differentiation medium without osmotic agent for 5-7 d. The last step is transferring the callus to screening medium on which they can cultured under light ,then they can redifferentiation.6.The anther callus were bombarded by biolistic bombardment and screened by 45 mg/L kanamycin and regenerated,and the number of transgenic plants we obtained through PCR detection is 0(Dwarf×Xiaoyan 22),2(Dwarf×101),1(Dwarf×Xinong 213)respectively. |
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