| Ginkgo biloba,an ancient relict plant,is a precious economic tree species and a greening tree species.It has a regular tree shape and golden autumn leaves,which has a high ornamental value and has been widely used in urban landscaping.Ginkgo trees have extremely long-life span and there exist a large number of ancient trees in China that have formed characteristic ancient ginkgo community parks in many places.Ginkgo is dioecious and a typical windpollinated species.In recent years,a large number of male ginkgo trees planted in urban greening has led to a dramatic increase in pollen production,which has greatly increased the number of ovules set by female plants.Excessive fruiting not only affects the landscape and may also pollutes the environment,but also excessive consumption of tree nutrition resulting in serious weakness of the tree until death.Previous studies suggested that the ovule development of G.biloba(mainly refers to the endosperm)is not the product of fertilization,but the function of pollination in ovule development is not clear.In this paper,pollinated and non-pollinated ovules were used as experimental materials,and exogenous reagents were sprayed to explore effective treatment methods to regulate the fruiting amount,transcriptome data analysis and structure observation was used to reveal the function of pollination in the development of G.biloba seed.The main results are as follows:(1)G.biloba branches with ovules were clipped before the pollination stage for laboratory hydroponics,and pollination and non-pollinated treatment were performed when pollinating drops appeared at the micropyle.There was no significant difference between pollinated and non-pollinated ovules in the first 7 days.From 7 to 14 days,pollinated ovules showed a slow growth trend,while non-pollinated ovules basically did not develop.From 14 to 28 days,pollinated ovules showed a pattern of rapid development,while non-pollinated ovules showed a trend of decay.The same results were also verified in bagged and non-bagged ovules on the trees,indicating that pollination is crucial to the development of ginkgo ovules and seeds.(2)The pollination process of G.biloba mainly includes the production of pollination drops at the micropylar and the pollen entering the powder storage chamber through the contraction of pollination drops.The structure of pollinated and non-pollinated ovules was observed.Scanning electron microscopy(SEM)showed that pollinated ovules developed normally,the micropylar showed a tight state,and the opening of the powder storage chamber was closed immediately after pollination.However,the micropyle of non-pollinated ovule still showed a funnel-shaped shape of rolling outward,and the opening of the powder chamber was not closed.The results of paraffin section also showed that the micropylar canal of pollinated ovule gradually closed with the development of the integument,and the functional megaspore in the nucellar tissue divided into free nuclei,and the number and volume of free nuclei increased dramatically with the increase in development time.The development of nonpollinated ovule stopped,the micropylar canal could not be closed,free nuclei could still be produced in the nucellus tissue,but the number of free nuclei was small,and the free nuclei stopped dividing and proliferating in the later stage,and the ovule was aborted.The above studies showed that the micropylar canal and the opening of the powder storage chamber of non-pollinated ovules could not be closed,functional megaspores and free nuclei could still differentiate normally,but the proliferation of free nuclei was slow or even stopped,and ovule abortion mainly occurred during the proliferation of free nuclei.(3)To screen the most effective method of preventing pollination and fruit thinning,the exogenous spraying treatments of ginkgo ovules was carried out.It was found that the pollination drop could still be secreted normally after spraying 0.4°Be stone sulfur mixture,and the 500 times diluted anti-transpirant(mainly composed of fulvic acid,ablative acid,and humic acid),and 0.1 g/L polyvinylpyrrolidone(PVP)could effectively control the secretion of pollination drops.After 7 days of spraying treatments,the ovules of the three groups began to wilt and withered to 14 days.However,there were significant differences in the abortion rate between the three treatments.The 500 times diluted anti-transpirant had the best effect(95%abortion rate),followed by the 0.4°Be stone sulfur mixture(88%abortion rate),and 0.1 g/L PVP had a 73%abortion rate.The three reagents had no adverse effects on the growth and development of leaves.(4)Transcriptome sequencing of non-pollinated ovules and ovules pollinated after 14 and 28 days was carried out,which identified 35,788 DEGs(Different Expression Genes).GO analysis showed that DEGs were mainly enriched in defense response,response to biotic stimulus,and inorganic molecular entity transmembrane transporter activity,KEGG analysis showed that the differentially expressed genes were mainly enriched in phenylpropanoid biosynthesis,photosynthesis,plant hormone signal transduction,starch and sucrose metabolism.Sixteen,six,two,and four DEGs were identified in the IAA,CTK,GA,and BRs signal transduction pathways,respectively,and their expression was down-regulated in nonpollinated ovules.Eight,four,four,and six DEGs were identified in the ABA,SA,JA,and ETH signal transduction pathways,and most of the genes in the JA signal transduction pathway were up-regulated in non-pollinated ovules,these imply that pollination influences seed growth by influencing hormone synthesis and distribution within the ovule.(5)A total of 25 differentially expressed GbMADS genes were identified in the transcriptome data of pollinated and non-pollinated ovules.Both transcriptome sequencing data and qRT-PCR results showed that GbMDAS28 and GbMADS33 were significantly decreased in non-pollinated ovules.Further sequence alignment analysis showed that GbMADS28 and GbMADS33 had high homology with GGM13 and AG involved in pistil development in angiosperms,suggested that they may play a crucial role in the development of ginkgo ovules,but their functions and mechanisms still need to be further studied. |
