Studies Of Pretreatment Methods On The Anther Culture In Vitro Of Medicago Sativa L.

Alfalfa(Medicago sativa L.)is one kind of leguminous perennial herb,which is widely cultivated over all the world,known as the "queen of forage".Alfalfa is best forage charcteristic of its highly-yield,sufficient-nutrition,ect,which plays a significant role in husbandry economic development.Heterosis is a universal phenomenon in all organisms,which refers to the hybrid offspring in terms of growth and vitality,fertility or seed yield being superior to the parents.Therefore,the utilization of heterosis has been playing a significant role in increasing forage grass yield and improving its quality.The heterosis utilization is an effective method to breed alfalfa varieties,which has a positive effect in promoting the development of husbandry economic.Heterosis utilization is built on the ground of parental with genetic homozygosity and uniformity.Heterosis utilization of alfalfa is well advised to cultivate parent with genetic homozygosity and uniformity.At present,the process of self-crossing is commonly used in the cultivation of parent with genetic homozygosity and uniformity.However,alfalfa is autotetraploid plant that the speed of parents homozygous is slower than the diploid alfalfa featured with self-incompatibility which is difficult to obtain inbred lines.These two reasons make a restriction to apply alfalfa heterosis in production.In order to address the problems in terms of genetic impurity long breeding periods in the ues of alfalfa heterosis,we can take advantage of anther culture that anther and pollen culture can cultivate haploid plants and double chromosomes which could cultivate parent with genetic homozygosity and uniformity.Anther culture become a new approach to solve alfalfa heterosis utilization shortening the breeding periods and breeding varieties to match the breeding object and improving breeding efficiency.In this study,the anthers of the three lines of high-frequency regeneration plant of alfalfa variety named as"Yu Mu NO.1" was used as the materials.Research was made on the effect of temperature pretreatment,centrifugation pretreatment,hypertonic pretreatment and different treatment combinations in alfalfa anther induction,differentiation,plant regeneration and established efficient regeneration system for anther culture.The majoy findings of this experiment were as follows:1.The anthers of three lines were subjected to temperature pretreatment which can significantly improve the rate of callus induction.Callus induction rate of 12 lines was up to 9.33%and the 15th lines was up to 22%,and the 78th lines was up to 9.33%,which was higher than those of the control(75.05%,65.05%and 180.18%,respectively)which reached a significant level.2.After the anthers of three lines were subjected to hypertonic treatment,the effect of different treatment was revealed for the different genotypes.The highest callus induction rate of 12th lines’and 78th lines’ anthers increasing to 9.33%and 5.33%,which was higher than the control(75.05%,and 60.06%,respectively)which reached a significant level.But the callus induction rate of 15th lines’anthers declined.3.Response of the anthers of three lines to combination changed temperature pretreatment and osmotic pressure treatment were different.The anthers callus induction rate of the 12th lines and 78 ’lines was higher than temperature pretreatment alone which did not reach a significant level..As for thel5th lines,the effect of only temperature pretreatment was superior to the effect of combination pretreatment,which callus rate decreased in varied degrees.4.The anthers callus induction rate of three lines were obviously different when they were subjected to the combination in temperature pretreatment and centrifugation pretreatment.Under the treatment 9(temperature pretreatment+2500r/15min),the 12th lines’ callus induction rate reached 15.33%which was higher than the control(187.62%)which reached a significant level.Under the treatment 2(temperature pretreatment+2500r/3min),the 15th lines’ callus induction rate reached 19.33%which did not reach a significant level.Under the treatment 2(temperature pretreatment+1700r/3min),the 78th lines’ callus induction rate reached 18.67%which was higher than the control(460.67%)which reached a significant level.5.On the basis of temperature pretreatment,the orthogonal experiment of centrifugation speed,centrifugation time and osmotic pressure treatment revealed that the main effects that affect anthers callus induction rate are according to different lines.As for the anthers of 12th lines,the influences of factors were significant followed by centrifugation time>hypertonic treatment>centrifugation speed,the optimize combination:3700r/10min+RM-IR.As for the anthers of 15th lines,the influences of factors were significant followed by hypertonic treatment>centrifugation time>centrifugation speed,the optimize combination:1700r/min+3min+SWC.As for the anthers of 78th lines,the influences of factors were significant followed by hypertonic treatment>centrifugation time>centrifugation speed,the optimize combination:2500r/min+15min+RM-IR.6.After induction,calluses were transferred to differentiation culture until plants regeneration.The anthers of the 12th lines were subjected to the osmotic treatment(RMD),the number of regenerated plants was up to 7.The anthers of the 15th lines were subjected to combination treatment(temperature + centrifugation(2500r/10min),the number of regenerated plants was up to 27.The anthers of the 78th lines were subjected to combination treatment(temperature +centrifugation(1700r/3min),the number of regenerated plants was up to 10.