Fertility Identification Of The Stylo Mutant

Discovering and exploiting key germplasm resources is important in plant breeding. Obtaining male sterile line with characteristics of steady and inheritable is crucial to the lynchpin of ameliorating germplasm resources and accelerating crossbreeding for Stylo. Took the fertility variation of stylo mutant as our study material. Used its wild type of S. guianensis cv. Reyan No.5as the control to make comparison with the Stylo mutant through the study of morphologic observation, analytical paraffin section, pollen vitality assay, artificial castration and artificial pollination. Thus, the characters of developmental process (flower bud differentiation, anther development, embryo sacdevelopment) and fertility on pistil and stamen of both normal and Stylo mutant were analysied comprehensively and comparatively. Researched the characters in morphology and embryology of pistil and stamen on its developmental process. All these to provide some reference for studying speciality of male sterility Stylo and crossbreeding. The main results are as followings:There were abnormal morphological characters in stamen of Stylo mutant,such as anther deform and indehiscent with colour turning from yellow to white and no pollen or little with no release. The analysis of the paraffin section of flower bud differentiation and anther development showed that the Stylo mutant was resulted from anther development deform. The ground meristem dysplasia of immature anther which came from the top of stamen primordia caused archesporial cell prosoplasia, then produced no or abnormal sporogenous cell, and failed to differentiate fibrage, middle lamella, tapetum normally. As a result, the deformed anther wall was indehiscent and could not release pollen. Thus some aborted pollens were produced from tapetum development with varied dysplasia in deform anther.Used six methods such as germination in vitro test, inorganic acid test, I2-KI staining, TTC (2,3,5-Triphenyltetrazolium chloride) staining, FDA (fluorescein diacetate) staining, acetocarmine staining for optimal formula to test the viability of pollen of Stylo. By comparing the results of6methods, showed that the germination in vitro method was the best. The best medium components of germination in vitro was:10%sucrose,10%PEG-6000(polyethylene glycol-6000),0.01%H3BO3,0.03%CaCl2-2H2O,0.01%KNO3, pH6.5. Were optimal for pollen germination and tube growth at30℃for10mins, and then pollen germination average rate were84.03%. The staining method maybe simpler and faster, as0.5%I2and1.5%KI components of I2-KI staining with the best result of obvious color difference and the pollen vitality rate high84.59%, and no significant difference with germination in vitro method, which could be the quick vitality testing method. The TTC (2,3,5-triphenyltetrazolium chloride) staining (0.00%), FDA (fluorescein diacetate) staining (80.13%), acetocarmine staining (95.39%), inorganic acid method (93.59%) were unsuitable to assay pollen vitality of Stylo. There was no viability pollen from the indehiscent anther of Stylo mutant, which examined by best medium components of germination in vitro method. The results showed that the Stylo mutant as stamen sterile.There was abnormal morphological character in pistil of Stylo mutant since its style was longer than normal at the same time, which was a characteristic of stigma to expose for pistil after the floret bloom. Analysed the paraffin section of flower bud differentiation and embryo sac development, found that the pistil of Stylo mutant developed normally.We observed the flowering and setting seed process of Stylo, and tried the methods for artificial castration and artificial pollination. The difference of seed setting percentage for artificial pollination were remarkable (P<0.0001) with the floret blooming at7:30and wilting at16:00when the daily high temperature was at29℃in middle November in Hainan. The best time for artificial pollination of Stylo was9:00-11:00am. Comparing the seed setting percentage of artificial pollination in different time with normal Stylo after artificial castration, suggested that the Stylo mutant set seeds normally. There was no significant difference between the results by SAS analysis(F=2.50,Pr>F=0.1320,P>0.05). The results certified that Stylo mutant could produce normal macrospore with fertile pistil.We can conclude that the Stylo mutant possess sterile stamen with fertile pistil, which can be identifed as male sterility Stylo.