The Proteomics Research Of Pollen Abortion And Germplasm Innovation Of Spray Cut Chrysanthemum With Non-dispersing Pollen

Spray cut chrysanthemum is an important cultivars of Chrysanthemum?Chrysanthemum morifolium?,and can be grouped into standard cut chrysanthemum and spray cut chrysanthemum according to its inflorescence type.In recent years,spray cut chrysanthemum has become more and more popular in the world,due to its characteristics such as much branched,color and floral shape enrichment,more flowers and long flowering period.However,in the production of spary cut-flower chrysanthemum often encounter a large amount of pollen dispersal,and affect its ornamental value and flowering time.In addition it is also harmful to people,in particular those with pollen allergies.So the research to develop spray cut chrysanthemum with low or non-dispersed pollen is the most effective means to solve this problem.Aiming at this issue,this study chooses a kind of spray cut chrysanthemum with non-dispersed pollen 'Kingfisher' and a kind of spray cut chrysanthemum with normal pollen as experimental material,using paraffin section method and transmission electron microscopy?TEM?techniques,observes the structure and morphology of anther and pollen cells in its pollen development process,in order to find out the key period of pollen abortion and abnormal tissue.On this basis,using proteomics iTRAQ to analyze differential expression of proteins before and after pollen abortion,and analyze their function,trying to find out the proteins related to pollen abortion.In addition,use "Kingfisher" as female parent,and three different kinds of pollen quantity of male parent 'Noa','Qx-010','14-15' cross-breeding,studying the genetic law of pollen dispersal.This study will not only help elucidate the mechanism of pollen abortion of spray cut chrysanthemum,but will also provide valuable data and intermediate breeding material for development of spray cut chrysanthemum with low or non-dispersed pollen.The main research results and conclusions are as follows:?1?In 'Kingfisher',spore mother cell develop normally until the tetrad phase stage.But after that,microspore can't normal released from tetrad to continue to develop into pollen grains,and gradually degraded in tetrads.At last the anther empty capsule.in addition,by comparing pictures of the 'Kingfisher' and 'Nan Nong Hong Cheng' before and after tetrad stage by paraffin section and transmission electron microscopy?TEM?images,find out the tapetum cell in the anther wall of 'Nan Nong Hong Chen' has a condensation cytoplasm at tetrad stage,then began to degrade after early microspore stage.But in the anther wall of 'Kingfisher',tapetum cell wall showers a thick belt squeezing chamber at tetrad stage,with a dispersive cytoplasm.And we can't see cell wall clearly in the tapetum.The tapetal cell degrading in advance can't provide nutrients to the growth of pollen,and failed to timely secrete callose enzyme to degrade the callose which wrapping tetrad,making microspore couldn't be released timely from tetrad and degrade gradually.These results suggest that the tetrad phase stage is the critical period for pollen abortion of'Kingfisher',and pollen abortion is mainly due to the degradation of tapetal cell in advance.?2?Using proteomics technology iTRAQ,totally got 63 kinds of differential protein from isolated,including 59 kinds of differential protein between tetrad stage and microsporocyte stage?29 kinds of differential protein up-regulated and 30 kinds of differential protein down-regulated?,39 kinds of differential protein between microspore stage and tetrad stage?23 kinds of differential protein up-regulated and 16 kinds of differential protein down-regulated?.We classified the differential protein according to their function.The results showed that the function of most differential proteins mainly concentrated in the synthesis process of energy substances and the metabolic process,with metabolic abnormalities,material synthesis blocked,shortage of energy,leading to the cell components decomposition and pollen abortion.Among these proteins,from microsporocyte stage to tetrad stage,we found 4 kinds of proteins related to apoptosis up-regulated:ubiquitin-conjugating enzyme family protein,14-3-3-like protein D,cysteine protease,26S proteasome regulatory subunit 7.From tetrad stage to microspore stage,we found some proteins reduced which related to the synthesis and metabolism of protein,like S-adenosyl-L-homocysteine hydrolase,40S ribosomal protein S9,Phosphoenolpyruvate carboxylase 1 and so on.These results suggest that these proteins related to apoptosis may result in the degradation of tapetal cell in advance.?3?Among this three male parent,'Noa' has the least pollen dispersal,the average pollen dispersal of their F₁ generation is the least,0-455 grains per anther,the average is 184.3±79.7?47.1%of their male parent?,and we got 'Noa-l' and 'Noa-4' with non-dispersed pollen;'14-15' has the most pollen dispersal,the average pollen dispersal of their F₁ generation is the most,with conglomerate pollen,0-720 grains per anther,the average is 441.0±53.8?71.0%of their male parent?,but we got '14-15-1' with non-dispersed pollen;'Qx-010' has a medium pollen dispersal,the average pollen dispersal of their F₁ generation is moderate,60-455 grains per anther,the average is 328.75 ±92.4?69.2%of their male parent?.These results suggest that the pollen dispersal of F₁ generation and male parent has certain correlation,and‘Noa-1'?'Noa-4' and'14-15-1' will be the intermediate breeding material for development of spray cut chrysanthemum with low or non-dispersed pollen.