Genetic Analysis And Cytological Observations Of Multi-pistil Mutant (mp1) In Alfalfa

Perennial forage legume alfalfa(Medicago sativa L.)is autotetraploid and cross-pollinating species.The actual seed production of alfalfa is much lower than the potential seed production,and high female infertility is the main factor leading to this phenomenon.Alfalfa female sterile mutants should be important materials for exploring the developmental mechanisms of alfalfa pistil.The researches on cytogenetics of alfalfa sterile mutants will provide the basis for genetic improvement and seed production in alfalfa breeding programs.In this paper,preliminary screened alfalfa natural multi-pistil mutant(mpl)and its fieldwild type 4-23 were cloned and planted in greenhouse and in the field.Using the methods of controlled hybridization and genetic analysis,paraffin section by light and fluorescence microscopic observations,genetic characteristics,we have studied the development of embryo sac and callosity deposition of mpl,the main results were as follows:1)The reciprocal crosses between mutant mpl and wild type 4-23 were conducted under the condition of greenhouse cultivation.The podding rate in direct cross(mpl ?× 4-23?)showed that single-pistil flowers was 28.7%(n=300)and multi-pistil flowers had no pod setting with podding rate as 0%(n= 100).The podding rate in reciprocal cross(mp1? × 4-23?)was 57.5%(n=80).The results suggested that multi-pistil mutant mpl was a partial female sterile mutant with normal male fertility.Furthermore,the inheritance of multi-pistil capacity was weak under greenhouse cultivation.During 2012-2013,the expression stability of multi-pistil trait under greenhouse and field conditions using cloned mpl plants was assessed.The results showed that average multi-pistil rate of mpl plants under greenhouse reduced significantly to 3%(n=225).By contrast,average multi-pistil rate of mpl plants growing in the field maintained to a high level at 60%(n=270).The results above indicated that multi-pistil mutantion of mpl may be regulated by environment factors.The cloned plants before flower bud differentiation of mpl and wild type 4-23 were treated for 30 days in plant growth chamber under low temperature(daytime,15± 1 ?,15h;night,10±1?,9h)and short day(daytime,24±1?,8h;night,18±1?,16h)the result found that flowering time of mpl and 4-23 had no difference under low temperature and short day treatments.However,multi-pistil rate of mpl plants increased with time of low temperature and short day treatments extending.The multi-pistil rate of mpl plants under 30d short daytreatment was 30.53%(n=240),while that under 30d low temperature treatment was 39.0%(n=240).The results preliminary indicated that multi-pistil trait of mpl could be initialted by environmental factors such as photoperiod and temperature.2)Using paraffin section and light microscopic method,comparisons of alfalfa pistil development processing between multi-pistil mutant mpl and wild type 4-23 found that no developmental differences had been observed in sporophyte stage.However,synergid,central cells and antipodal cells in mpl embryo sac had been observed to degredate to a certain exteent during gametophyte stage.The results suggested that female gametophyte development defective of multi-pistil mutant mpl could be involved in abnormal pollen tube guidance and lead to fertilization failure,which may be the main reason for low seed setting of mutant mpl.3)Using paraffin section and fluorescence microscopic method,callose depositon dynamics during embryo sac development processing in alfalfa multi-pistil·mutant mpl and wild type 4-23 showed that mpl nucellus appeared much more callose deposition in sporogonium period.Along with the development process,callose in 4-23 embryo sac was degradation,but still existed in mp1 embryo sac after mitosis and especially accumulated at micropyle end.At the same time,at the beginning of function megaspore formation,double circle of callose appeared in embryo sac of multi-pistil flowers,callose deposition amount was significantly higher than that in wild type 4-23,and callose could not degrade after ovule maturity.All results above in dicated that abnormalities of callose metabolism in mutant mpl may be directly affect its ovule fertility for callose accumulation in ovule micropyle would block pollen tube signal transmission.