| Ziziphus jujuba Mill.belongs to the family Rhamnaceae and genus Ziziphus.It is a peculiar fruit tree species in China and has high nutritional and economic value.Under the unique geographic and climate conditions of southern China,southern China fresh-eat jujube represented by Ziziphus jujuba Mill.Zhongqiusucui has many advantages,such as high yield,high sugar content,rich flavour,good texture,strong adaptability,and easy storability and transportability,and so on.However,in fruit production,southern China fresh-eat jujube also faces disadvantages,such as severe abscission of flowers and fruits and easy fruit cracking.Thus,cross-breeding is required to acquire the advantages from other jujube cultivars.However,embryo abortion and non-setting fruit of southern China fresh-eat jujube seriously hindered its cross-breeding.Research to identify the causes of embryo abortions is urgently required,because solving this challenge is critical for breeding new cultivars and improving the industry.In this paper,we took Ziziphus jujuba Mill.Zhongqiusucui as experimental material and studied the embryo abortion mechanism of southern fresh-eating jujube mainly from embryology and molecular mechanism.The main results were as follows.1.Observation on embryo abortion in southern fresh-eating jujube.(1)Ziziphus jujuba Mill.Zhongqiusucui is a high embryo abortion variety.(2)There were some differences in embryo abortion of jujube fruits in different batches.There was no significant difference between the first and second batches of jujube fruits.Big jujube fruits had relatively low embryo abortion rate.Small jujube fruits had high embryo abortion rate.Most of the seed kernels contained were single.The third batch of jujube fruits differed greatly from the first and second batches of jujube fruits,with the highest degree of embryo abortion.The fruits with higher embryo abortion rate concentrated on big and small jujube fruits.The seed kernels contained were all single.(3)There were significant differences of embryo abortion rate between two types of jujube bearing branches.The embryo abortion degree of jujube fruits on lignified bearing branches was higher than that on non-lignified bearing branches,and the probability of double-kerneled jujube fruits on non-lignified bearing branches was higher than that on lignified bearing branches.(4)The embryo abortion rate of jujube fruits with smaller fruit shape index and larger fruit shape index was lower,and that of middle type was higher.The embryo abortion rate of round or long jujube fruits was lower,while that of medium-length jujube fruits was higher.(5)The embryo abortion of cracked jujube fruits was 100%.2.Mega and microsporogenesis,the development of female and male gametophyte of fresh-eating jujube in southern China.(1)From the inflorescence development stage to flowering,the grade 0 bud on the inflorescence exhibited an increase in horizontal diameter,longitudinal diameter,peduncle length,and bud weight,but the rates of increase were different.From Day 1 to Day 5 after the inflorescence had developed,floral buds mostly grew horizontally.Day 5 was the floral bud flattening stage.From Day 6 to Day 8 after the inflorescence had developed,floral buds mostly grew longitudinally,and Day 8 was the floral bud enlarging stage.(2)The stamen of Ziziphus jujuba Mill.Zhongqiusucui had 5 anthers,4 locules,and an anther wall consisting of epidermis,endothecium,1-2 layered middle layer,and a secretory-type tapetum;additionally,the development of the anther wall belonged to the basic type.The cytokinesis of the microsporocytes was synchronous;the tetrads mostly arranged as a tetrahedron;and the mature pollen had 3 germ pores,3 grooves,and bicellular pollen.During meiosis,the microsporocytes in each locule were at the same phase and therefore exhibited synchrony.Among the different anthers in the same floral bud,as well as the 4 locules in the same anther,the microsporocytes had asynchronous meiosis.(3)There were a few abnormal pollen sacs during microsporogenesis and male gametophyte development,which did not affect pollen formation as a whole.(4)The pistils in the Ziziphus jujuba Mill.Zhongqiusucui had 2 ovaries;2 anatropous ovules;inner and outer integument,crassinucellar;tetrads formed by the meiosis of megasporocytes aligned linearly along the nucellus;megaspore at the chalazal end that developed into the functional megaspore,which underwent mitotic division three times and developed into the mature embryo sac containing 7 cells and 8 nuclei;and embryo sac development of the polygonum type.(5)The Ziziphus jujuba Mill.Zhongqiusucui has some mature embryo sacs which lack ovum or not fully developed.(6)The external morphology of the Ziziphus jujuba Mill.Zhongqiusucui floral buds correlated with the internal developmental stage of the male and female gametophyte.Therefore,the internal developmental progress of the stamen and pistil can be determined by the external morphological characteristics of the floral buds.3.Stamen morphological development and pollen viability of fresh-eating jujube of southern China.(1)The anthers of Ziziphus jujuba Mill.Zhongqiusucui were basifixed anther,oval,with reticulate ornamentation on the surface,symmetrical pollen sacs and longitudinal dehiscence;the size of the anthers increased first and then decreased on the whole;when the anthers did not dehiscence,they were plump and pollen did not develop;when the pollens matured,the pollens cracked,the pollens dispersed,the pollens became empty gradually,and the pollens became smaller until they withered and withered.The color change of anther was “yellow-green – yellow brown-brown-black ”on the whole;the surface decoration of anthers was a network of irregular circles of different sizes,with the cracking of anthers,the surface decoration deepened into a denser network.(2)The general morphological characteristics of the pollen of Ziziphus jujuba were as follows: the pollen was nearly flat spherical or nearly spherical;the polar view was regular triangle and the equatorial view was elliptic;the germination aperture was three,which were angular pore-shaped.In most periods,there were prominent cytoplasmic sacs at the germination aperture,which were formed at the same time with each germination aperture;the germination ditch was three-cleft,and the ditch scar extends to the poles but was not connected.Pollen grains were reticulate on the outer wall,with shallow meshes,irregular,different sizes and uneven distribution.(3)Pollen belonged to small pollen.(4)Pollen germination rate of Ziziphus jujuba increased first and then decreased from the bud flat stage to the ovary enlargement stage,while that of first blossoming stage,sepal flattening stage and petal anther separation stage were higher.(5)There was a certain relationship between the germination rate of pollen and the maturity of pollen development.That was,with the maturity of pollen,the germination rate of pollen was higher and higher,and the germination rate of pollen decreases with the aging of pollen.(6)The pollen germination rate of Ziziphus jujuba Mill.Zhongqiusucui varies significantly with different storage conditions,different storage time.(7)In terms of storage effect,the vitality of pollen stored at-70 oC and-20 oC were better.The storage life of pollen with high vigour was longer than 25 days under the low temperature drying conditions.4.Stigma shape development and receptivity of fresh-eating jujube in southern China.(1)The stigma was dry stigma and bifid,and there was no mucus secretion on the surface.(2)The mature stigma was covered with a layer of nearly circular papillary cells.(3)The morphological characteristics of the stigma exhibited a specific pattern as the opening of a single flower progressed.From the bud oblate stage to the early flowering stage,two styles were very close to one another,together as a cone shape.The space between the two styles began to increase from the sepal flattening stage and the styles started to grow towards two sides.At the petal flattening stage,the angle between the two styles exhibited a ‘V’ shape,At the stamen flattening stage,the angle between the two styles exhibited a ‘Y’ shape,which lasted until the ovary enlargement stage.(4)The bottom width and length of the style increased as the flowering process progressed.(5)The stigma was flat at the bud oblate stage;from the bud fat stage to the petal flattening stage,the stigma had a pointed,round morphology.Its submicroscopic morphology showed that at that time,the stigmatic surface changed from being wrinkled to full-bodied,then to bulging outwards.At the stamen flattening and petal wilting stages,the stigma was curled outwards,bulging upwards,with a relatively large surface area.At the stamen wilting and ovary enlargement stages,the stigma shrank and dried up.(6)The stigma began to lose its green colour,the stigma receptivity decreased.(7)From the floral bud flattening stage to the ovary enlargement stage,the stigmatic papilla cells on the stigmatic surface at different flowering stages exhibited a changing pattern,first developing to maturity,then reaching the most active stage,followed by shrinking and becoming shrivelled,senesced,and degraded.As the stigmatic papilla cells developed towards maturity and reached physiological senescence,the stigma receptivity exhibited a changing pattern of ‘no receptivity-weak-strong-weak-no receptivity’.(8)Stigma receptivity can be determined based on the developmental level of the stigmatic papillae on the stigmatic surface of the jujube flowers.(9)The optimal stigma receptivity period of Ziziphus jujuba Mill.Zhongqiusucui lasted from the sepal flattening stage to the petal flattening stage,which lasted approximately 6 – 8 h.5.Pollination,fertilization and early embryo development of fresh-eating jujube in southern China.(1)There were poor pollination and fertilization and abortion of embryos of Ziziphus jujuba Mill.Zhongqiusucui.(2)Pollen germination and pollen tube elongation were similar on stigma whether inbred or outbred.(3)It takes at least 4 hours for pollen to recognize each other and 6 hours for pollen to germinate on stigma.After 12 hours of pollination,the pollen tube began to extend into the stigma.After 24 hours of pollination,the pollen tube reached 1/4 of the style,and then continued to extend upward to the style tract.After 48 hours of pollination,the pollen tube began to twist.Pollen tube continued to elongate on stigma,twisted and interacted,and expanded globally from 72 to 120 hours after pollination.(4)There were a lot of callose in the style,which obstructed the pollen tube in the style passage.Only a small part of the pollen tube grew to the base of the style and reached the ovary.(5)After 72 hours of pollination,one sperm moved to the vicinity of two polar nuclei and began double fertilization.(6)After 96 hours of pollination,one sperm fused with the secondary nucleus of the embryo sac to form the primary endosperm nucleus,which disintegrated and disappeared after 1 day.After 120 hours of pollination,another sperm fused with egg cells to form zygotes.After 4-5 days of dormancy,the zygotes began to divide into smaller globular embryo,which disintegrated before forming into larger globular embryos.(7)On the 15 th day of fertilization,the embryo degenerated completely and finally formed empty embryo sac.6.Embryological mechanism of embryo abortion of fresh-eating jujube in southern China.(1)Embryo sac development was blocked.In the process of embryo sac development,most of them could not form complete mature embryo sac,and there was phenomenon that single or multiple organelles of antipodal cells,oocytes and helper cells were absent.(2)Pollen tube elongation was blocked.In the process of pollination and fertilization,pollen was fertile and the mature stigma had strong fertility in a certain period of opening.Under the conditions of both of self-pollination and cross-pollination,pollen could germinate on the stigma,but most pollen tubes were blocked in the styloid tract by massive callose.Only a small part of pollen tubes could reach the ovary and completed fertilization.(3)Embryo development was hindered.After the pollen tube released sperm and successfully completed double fertilization,the endosperm nucleus did not divide or only divided for 1-2 times and gradually degenerated and disappeared.Endosperm degeneration will eventually lead to embryo abortion.The zygote divided to form smaller globular embryo,and then smaller globular embryo degenerated before forming into larger globular embryo.Eventually,empty embryo sac was formed,which led to the abortion of embryo.(4)Some ovaries were not fertilized.Pollen germinated on stigma and pollen tube went deep into style.Although fertilization was not completed,auxin synthetase in style or ovary was activated,which promoted ovary to develop into fruit.In the unfertilized embryo sac,the egg cells,polar nuclei,synergids and other cells will disintegrate over time,nucellar tissue will degenerate,mature ovule structure will degenerate,and the whole ovule cavity will be hollow,which will eventually lead to the whole ovule abortion,which is seed abortion in jujube fruit.7.Screening of genes related to embryo abortion of fresh-eating jujube in southern China based on transcriptome sequencing.(1)The ovule abortion of Ziziphus jujuba was one of the reasons for its embryo abortion.(2)By analyzing the transcriptome information,six genes related to ovule development were screened: Zj AGL11,Zj ABCG20,Zj ABCG11,Zj ABCG15,Zj MC1 and Zj MC9.(3)Zj AGL11,Zj ABCG11 and Zj ABCG15 genes may play an important role in regulating ovule development of jujube,and their expression patterns were similar: low expression would lead to abnormal ovule at the critical development stage.(4)Zj ABCG20 gene played an important role in embryo development of jujube,and its low or no expression may lead to ovule abortion.(5)A large number of Zj MC1 genes were expressed during the period of embryo abortion,which led to the changes in downstream gene expression.Furthermore,it was speculated that the great changes of downstream gene expression affected the normal development of ovule,which led to the development of jujube ovule blocked and further abortion.(6)Zj MC9 was abundantly expressed in different degrees of abortive fruit.It was speculated that the high expression of Zj MC9 gene affected the normal development of late ovule,which caused the development of ovule blocked and further abortion. |
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