Morphological Identification And Chromosomal Ploidy Identification Of Some Persimmon Species

Diospyros has a long history of cultivation and numerous germplasm resources,mainly distributed in the tropics and subtropics.There are 58 species and varieties of Diospyros found in China.Among them,10 species have been preserved in the National Persimmon Germplasm Resources Garden,including D.yunnanyemaoensis,D.lotus Linn,D.glaucifolia Metc,D.jinzaoensis,D.oleifera Cheng,D.deyangensis,D.rhombifolia Hemsl,D.cathayensis Steward,D.virginiana dynamic,D.kaki and D.var silvestris Makino.In this research,firstly,the different growth stages and growth states of 10 species were photographed and recorded,based on the BBCH standard;and then,we used the enzymolysis dropping method to optimize the chromosome observation method,and observed the chromosomes of 72 resources.Finally,the ploidy of 112 germplasm resources was identified by flow cytometry.At the same time,the relationship between ploidy and stomata and seeds of persimmon was explored.The results are as follows:1)The phenological periods and characters of 10 species in the National Persimmon Germplasm Resources Garden were recorded and observed.It was found that the germination period of D.cathayensis Steward,Diospyros lotus L.and D.rhombifolia Hemsl was the earliest,in early March,the germination period of D.deyangensis was the latest,in early April;and the bud stage of D.cathayensis Steward is the earliest,in late March,almost synchronous with the leaf stage,D.virginiana and D.deyangensis is relatively late,in late April;D.jinzaoensis fruit mature earliest,about in early September,and D.oleifera Cheng until mid October mature;the shortest dormancy period of D.cathayensis Steward is only about 100 days,while the longest dormancy period of D.deyangensis is about 140 days.Finally,according to the characteristics of 10 species,the identification index table of 10 species in the national persimmon germplasm resources nursery was compiled.2)The chromosome observation system of Diospyros was optimized.The results were as follows: the germinated,expanded and green buds were collected from 8 : 30 to 11 : 30 a.m.every day from the end of March to the middle of April,and the young leaves were peeled off to see the growth point of the bud tip.The 2 – 3 mm growth cone of the bud tip was taken and directly put into the Carnoy’s Fluid,which was temporarily stored in a refrigerator at 4 °C.The buds were removed from the Carnoy’s Fluid before making the film,washed three times with deionized water,and dried with filter paper.The water on the growth point was digested with 2.5 % mixed enzyme for 120 min – 160 min,and then smashed with 70 % ethanol for five times.After centrifugation at 6000 r / min for 4 min,30 μL acetic acid was added into the centrifuge tube,vortexed,and 10 μL was dropped on the glass slide.The difference was observed under a dark for 5 min.The chromosome observation of 72 persimmon plants using the optimized scheme showed that the chromosome number of D.yunnanyemaoensis was 30 diploid,and that of Gushi Niunaishi and Shigang Wuhe(Diospyros lotus L.)was 45 triploid,which were first reported in China.Among the identified materials,D.glaucifolia Metc,D.jinzaoensis,D.oleifera Cheng and Diospyros lotus L.were diploid,D.deyangensis and D.rhombifolia Hemsl were tetraploid,Diospyros kaki Thunb.,D.cathayensis Steward and D.virginiana were hexaploid.The chromosome numbers of the three mutant varieties of Zhoushan Changshi,Jiandinghuoguan and bianyuanhuoguan in the resource nursery were observed,and it was found that the chromosome numbers of the mutant varieties were consistent with those of the original varieties.3)Morphological identification results showed that stomatal density,seed area,perimeter,length and width were positively correlated with ploidy but could not be used as the basis for ploidy identification.The determination of chromosome ploidy by flow cytometry is suitable for the preliminary screening of a large number of ploidy identification materials.For the more important materials,the enzymatic drop method is still needed to observe,and the results are more accurate.4)Through the comprehensive comparison of morphology and chromosome ploidy of Yunnan wild persimmon with other persimmon species,D.yunnanyemaoensis may be a new species.