Preliminary Study On The Role Of Lignin Formation In Abortion And In Vitro Culture Of Seedless Grape Ovules

Grape(Vitis spp.)as one of the oldest fruit trees species in the world,has high economic value.Seedless grapes have no seeds,and are conducive to direct processing and eating,so it has long been one of the most concerned traits in the grape industry.But at present,seedless grape varieties cultivation management methods are complex,flower and fruit drop seriously,grape fruit is small.In the actual production and sales process,the market demand cannot be met.It is urgent to cultivate new seedless grape varieties with good quality and convenient cultivation and management.In order to cultivate more new seedless grape varieties,the research of grape seed abortion mechanism and embryo rescue mechanism is the focus of scientific research.This experiment takes ’Lihongbao’,’Wuhecuibao’ and ’Qiuhongbao’ as the research objects,which has the same female parent and the seeds are significantly different.Collected ovules at different developmental stages and cultured ovules in vitro,and studied the morphology,lignin content,PAL(Phenylalanine ammonia-lyase)activity,C4H(Cinnamate-4-hydroxylase)activity,4CL(4-CoumarateCo Aligase)activity,CAD(Cinnamyl alcohol dehydrogenas)activity and transcriptome analysis.We tried to explore the mechanism of seed abortion in seedless grapes from the perspective of lignin metabolism and the reverse regulation process of embryo rescue on seed abortion.It is expected to provide a theoretical basis for the research on the mechanism of grape seed abortion and embryo rescue.The main findings are as follows:(1)The fruit development of ’Qiuhongbao’ grape showed a traditional double ’S’ curve,while’Wuhecuibao’ and ’Lihongbao’ showed a ’S’ curve.It may be due to the existence of seed abortion of seedless grapes,which shortens or prematurely terminates the hard-core period in fruit development.And cause the differences in the fruit development process.(2)There are differences in the size of three grape ovules,according to the order from large to small are ’Qiuhongbao’ > ’Wuhecuibao’ > ’Lihongbao’.But in the same period,the degree of seed difference from large to small was ’Lihongbao’ > ’Wuhecuibao’ > ’Qiuhongbao’,and the variation coefficient of seedless varieties increased with the development of seeds.(3)Seedless grape ovules can be divided into four stages from the perspective of overall development.In the first stage,because the developmental degrees were divided into high developmental ovules and low developmental ovules,and the higher developmental ovules could obtain more nutrients,the lower developmental ovules would be suppressed by high developmental ovules.In the second stage,high developmental ovules began to abort,while low developmental ovules were suppressed and began to develop rapidly.In the third stage,ovules with low degree of development began to abort,and ovules with high degree of development were completely aborted,some of them were shriveled and depressed,and the other part began to appear fleshy growth of ovule seed coat.In the fourth stage,the ovules with low developmental level were also completely aborted.And all ovules either to shrink,shrivel,or to fleshy growth,polarization increased.(4)In the middle period of natural development,seedless grape ovules will appear ovule deformity phenomenon.In addition,concave,browning and tearing wounds will appear at the junction.After artificial embryo rescue,the ovules will also be deformed.In addition to some browning,there are also ovule seed coat expansion and callus formation.(5)The lignin accumulation pattern of seedless grape ovule is different from that of seedless grape.The lignin content of ’Qiuhongbao’ grape ovule was higher than that of ’Lihongbao’ and ’Wuhecuibao’grape ovule.And ’Qiuhongbao’ grape ovule in addition to 62 d~67 d lignin content decreased,the overall trend of continuous accumulation.The lignin accumulation of ’Lihongbao’ continued from 27 d to 42 d after anthesis,which included the time point(36 d)when the ovule of ’Lihongbao’ began to abort,that is,after the abortion of ’Lihongbao’ ovule,the lignin accumulation was affected.The lignin content in the ovule of ’Wuhecuibao’ continued to accumulate within 27 d~37 d,while the lignin content began to decrease after the ovule abortion(36 d).(6)Lignin content was significantly positively correlated with C4 H activity,significantly correlated with PAL and CAD,but not significantly correlated with 4CL activity.Therefore,PAL,C4 H and CAD are highly regulated by embryo abortion in lignin biosynthesis,or have other roles in ovule abortion.(7)A total of 14 differentially expressed PAL genes,3 C4 H genes,2 4CL genes and 4 CAD genes were screened by high-throughput transcriptome sequencing.Genes such as VIT＿11s0052g01090,VIT＿16s0039g02040,VIT＿06s0004g08150,VIT＿08s0040g01710,VIT＿13s0019g04460,VIT＿02s0025g03100 were highly expressed in ovules after embryo salvage,which may be involved in the reverse regulation of ovule abortion in ovule culture.