Study On Pollination Biology And Hybridization Breeding Of Michelia Crassipes

Michelia rassipes Y.W.Law,a small evergreen shrub to small tree in Magnoliaceae,is a great ornamental plant with fragrant and dark purple flowers.It has become an useful germplasm resource for cross breeding.In order to explain the main reasons for the low natural seed rate of M.crassipes and determine optimum pollinating time for Magnolia cross breeding,we studied the pollination biology of M.crassipes.We also compared the cross-compatibility of Michelia crassipes and that of others three species(M.figo,M.maudiae and M.platypetala),and identified the authenticity of the hybrid F1 generations.The results could lay a foundation for the protection of the resources M.Crassipes and hybridization of Magnolia.In this study,We attempted to study the flowering dynamics and tested the type of breeding system by determining out-crossing index(OCI),the pollen-ovule ratio(P/O),pollen viability and the stigma receptivity,and conducting artificial pollination experiments.We also studied the pollination by observing the visitors and their behaviors.To revealed the cross-compatibility of M.crassipes and that of other three species(M.figo,M.maudiae and M.platypetala),M.crassipes was uesd as female parent and M.figo,M.maudiae and M.platypetala as male parent respectively for artificial hybridization.The pollen viability of the species was determined via culture in vitro,and the pollen tube germination after pollination was observed by fluorescence microscope.The fruit setting rate and seed quality of interspecific hybridization were also investigated.At the same time,IS SR markers combining with morphological characters were used for early identification of offsprings from M.crassipes ×M.figo and M.crassipes ×M.maudiae.The main results are as follows:1.Pollination biology of M.crassipes(1)Flowering phenology:The florescence of the M,crassipes was from mid-April to late May,lasting for approximately 50d with single flower lasting for 4 to 5 days.M.crassipes was monoecious with protogyny and herkogamy,stamen was higher than pistil on spatial location.(2)Breeding system:The OCI and the P/O were respectively 4 and 2671 ± 123.The artificial pollination revealed that spontaneous self-pollination phenomenon was existed and no apomixis phenomenon under field conditions.The fruiting rates and seed quality of artificial cross-pollination were significantly higher than that of natural and self-pollination.The optimal time to pollinate M.Crassipes with M.figo was 36 hours after flowering when the gynoecium began to bend,with the highest successful fruiting rate of 85.7%.(3)Pollinator:The effective pollinators mainly were Apis cerana,Episyrphus balteatus and Allomyrina dichotoma whose frequency of pollination was low and easily affected by weather.Therefore,the breeding system of M.crassipes was partly self-pollination,and mainly cross-pollination which requires pollinators.And its fruit set was limited by pollinators and herkogamy under natural conditions.The success rate of hybridization could be raised by determining the optimum pollination time.2.Hybridization of M.crassipes(1)Determination of pollen viability:The pollen viability of M.crassipes,M.figo and M maudiae were 86.2±6.67%,66.2±0.9%and 69.1 ±3.33%respectively.whereas the pollen viability of M.platypetala was only 29.07± 5.6%.(2)Cross-compatibility:By fluorescence microscope,the pollens grains of M.crassipes,M.figo and M.maudiae were germinated after 2 hours,and pollen tubes grew into a bundle after 12h.Pollen tubes could normally elongate to complete fertilization.When crossed with M.platypetala,the pollen germination rate of M.platypetala on stigma was higher than that of in vitro,but the pollen grains didn’t germinate untill 12-24h,and majority of pollen tubes stopped growing after 3d.Besides,inflated pollen tube,growth stop,and deposition of callose on stigma and stylet were observed.(3)Fruit set:The fruit setting rate and seed germination rate in the cross of M.crassipes ×M.fgo were 91.1±2.33%and 82.5 ± 3.33%respectively,which were significantly higher than that M.crassipes self-breeding.When M.platypetala was male parent,the fruit setting rate and seed germination rate were above 80%,which were also higher than that M.crassipes self-breeding.However,the fruit setting rate of M.crassipes×M.platypetala was only 11.1 ±3.33%,and hybrid embryo was abortive.(4)In conclusion,M.crassipes has the highest affinity with M.figo,slightly inferior with M.maudiae,and the lowest with M.platypetala.The pre-fertilization barriers that caused by the pollen tubes stopping growth could be one of the main barriers of interspecific hybridization between M.crassipes and M.platypetala.3、Identification of F1 hybrids(1)Morphological observation of F1 generation:Sixteen hybrids from M.crassipes×M.figo had the same leaf tip as male parent.Hybrids from M.crassipes×M.maudiae all had the same leaf colour and leaf tip as male parent.Morphological characters showed that all the offsprings were true hybrids.(2)ISSR analysis of F1 generation:Four ISSR primers were respectively selected from 12 ISSR primers,which were used to amplify individuals of two cross combinations.In M.crassipes and M.figo,a total of 43 bands were generated,in which 95.35%of the bands were polymorphic.In M.crassipes and M.maudiae,total 41 bands were amplified,in which 92.68%polymorphic loci were detected.Clustering results divided hybrids into 3 categories:partial male parent,partial female parent and intermediate type.F1 hybrids from two combinations not only had characteristic band of male parent but also generated some new specific loci which were recombinant fragment of parents.Both ISSR markers and morphological characters indicated that 16 F1 progeny from two combinations were all true hybrids.