Reproductive Biology Of Hetaeria Cristata(Orchidaceae)

Hetalia is a genus of about 20 species in the family of Orchidaceae,There are distributed in tropical Asia,also found in Oceania.There are five species in China,which are produced in the southeast to the southwest.Hetalia.cristata is a perennial native herb of the genus Hetaeria,which is often found on wet roadsides and river ditch stones.this thesis,we studied the reproductive biology of H.cristata in the Jiulianshan National Nature Reserve in Jiangxi Province.observed and measured the flower morphology,pollen flow statistics,breeding system detection,observed the development of embryo sac and embryo and the germination pollen tube.In addition,we conducted the karyotype analysis and determined the ploidy level.Moreover,we tested the seed vigor of plants with 7 different pollination treatments,and non-symbiotic aseptic germination aspect The main conclusions of this study are as follows.1.Through field observation and measurement,we found that the flower color of H.cristata are white to reddish brown.The flower opening is very small under natural conditions,The pollinia length（0.53±0.039 mm,n=40）and width（0.35±0.031mm,n=40）are larger than the flower opening length（0.36±0.034 mm,n=40）and width（0.17±0.016 mm,n=40）.That’s means pollinia are difficult to enter the flower,even with the external force,The elongation of the stigma was not found after flowering,and the distance from the flower opening to the stigma was 1.57±0.10mm（n=40）.That is,even if the pollen block was moved to the flower opening under the action of external force,the pollen block could not contact with the column.Meanwhile the pollen flow and peeling of the cap were unobserved in the 8population surveys.Therefore,It suggest that foreign genes could not be involved in the reproductive process of H.cristata.In the continuous three-year breeding system experiment,the seed setting rate of emasculating and decapitating stigmas were both close to 100%,and the seed setting rates of 7 different pollination treatments showed no significant difference there was no significant difference（F=1.556,df=6,P=0.175）.It indicates that the reproductive mode of H.cristata is apomixis and it has a very high incidence of apomixis.2.By the observation from confocal laser scanning microscopy,we found that the volume of ovule primordial on the placenta was began to increase three days before flowering,and then differentiated into sporogenous cells;One day before flowering,the size of the sporozoites increased and the cytoplasm was concentrated,and then developed into megaspore mother cells;One day after flowering,the volume of megaspore mother cells increased rapidly,and two divisions were made to form a linear quadrant;After 3 days,the megaspore developed into a mononuclear embryo sac;After 7 days,the mononuclear embryo sac was formed an 8-nuclear mature embryo sac through three successive splits.The development of the embryo was carried out after the flower was withered.Two days after the ascension,the two helper cells near the end of the bead gradually degraded,and the egg cells gradually expanded;After 5 days,the two polar nuclei disappeared,and the egg cells continued to expand,and the nuclei were thick and strong;After 10 days,the three antipodal cells degenerated and disappeared,and the egg cells began to divide and develop to form the original embryo.At 15-35 days,the blast cells continued to divide and the number of cells increased,and the embryo sac was filled with blast cells;After 45days,the outer seed coat cells died,and the embryos were fully mature and long oval.The results indicate that the embryo is derived from the egg cell through parthenogenesis.3.After 8 hours of cross-pollination,it was observed under fluorescence microscope that the pollen of H.cristata began to germinate on the stigma and grow rapidly in the column.However,after entering the ovary,the pollen tube grew slowly,and even stopped growing;Between 24 and 96 h,bending occurred at the end of the pollen tube,and no pollen tube continued to grow and enter the ovule.According to karyotype analysis and ploidy detection,the results of the number of chromosomes,type,location of the centromere,and ploidy between H.cristata and 7 different pollination methods of F₁ generations are same and they are both diplont.We speculate that the megaspore mother cells form a tetrad through secondary continuous division instead of meiosis,and the embryo sac develops into a butterfly-type.So the egg cells are diploid,which is a diploid spore apomixis.4.The seed vigor of 7 different pollination treatments was above 91%and there was no obvious difference（F=0.347,d.f.=6,P=0.909）;The seed germination rate was also above 90%and there was no difference（F=0.480,d.f.=6,P=0.821）;After 240 d of sterile culture,the plant height of F₁ seedlings（F=26.955,df=6,P=2.246）,number of leaves（F=0.814,df=6,P=0.563）,leaf length（F=1.718,df=6,P=0.131）,leaf width（F=1.361,df=6,P=0.244）,number of roots（F=1.120,df=6,P=0.361）and root length（F=1.097,df）=6,P=0.374)showed no significant differences between treatments,further indicating that the source of the embryo is so consistent.In summary,the reproductive mode of H.cristata is diploid spore obligate apomixis.