Studies On Several Aspects On Chinquapin (Castanea Henryi Rehd. & Wils.)'s Biology

Chinquapin(Castanea henryi Rehd.& Wils.)is a special fruit resource in China,with abundant germplasm resources and complex genetic background.The phenomenon of 'empty-seeded ness' is common in chinquapin productions,causing the declines of yield and quality and affecting the efficient application and industry development of the resources.Based on former studies,the experiment was conducted to study the embryology,nutriology, proteomics,molecular biology and palynology of chinquapin,with a view to providing the basis for rational use of chinquapin germplasm rescources and overcoming problems of 'empty-seededness'.The results of major studies were as follows:1.Using 'huangzhen' as the material and taking advantage of paraffin section technology, studies were carried out on the megasporogenesis,female gametophyte formation,the development of embryos and endosperm.The results showed that chinquapin was diclinous with polycarpous compound pistils,axile placentas,commonly 8 to 10 loculament and 2 anatropous ovules in each loculament.An archesporial cell in the nucellus tissue would directly develop into the megaspore mother cell that subsequently formed linear megaspore tetrads.The functional megaspore near the chalazal end developed into mature embryo sac,which was monosporic polygonum-type.After double fertilization,the proembryo would develop into a mature embryo going through globular,heart,torpedo and cotyledon stages.Endosperm is nuclear endosperm and it didn't exist in seeds.The study also found that there were several anomalies in the development of chinquapin embryos.For example,the insides of loculi were fully empty in the early stage when ovules were forming;the loculament would completely shrivel after the fertilization and the embryo sac would wither and have black circular materials around in the later globular stage.The timings when these phenomena happened were also determined.All these may lead to 'empty-seeded hess' phenomenon in chinquapin.2.The fruit growth and development of different varieties of chinquapin were systematically studied.The results indicated that chinquapin fruits could be divided into two parts as the bur and the nut.The burs of most varieties only bore one nut while some few bore one to three nuts. The shapes,sizes and weights varied from varieties to varieties.Most varieties of chinquapin shed flowers between late May and early June and then formed young fruits that would gradually grow and develop.The whole development process could be divided into two stages.In the early stage before early August,the bur rapidly grew while the increase of nuts' weights declined.The anatomies of fruits in different early stages showed that only milky white hairs were seen in 8 to 10 loculi and only an ovule under development was seen on the top of fruits while the others turned brown;during the later stage after middle August,the increase of the volumes of prickly burs slowed down while the weight of single fruit increased rapidly before finally matured.The anatomies showed that ovules quickly developed into seeds with disappearing white hairs.As fruits matured,fleshy cotyledons would occupy the whole fruit space.During this stage,once ovules developed abnormally,the phenomenon of'dmpty-seeded hess' would happen.The single fruit weights of different varieties varied little in the early stage but significantly during the later stage.Other than a few varieties,most varieties had concentrated harvest periods.The transverse and vertical development of Chinquapin fruits were positively related with the changes of single fruit weights.The growth and development of chinquapin fruits featured a S-shape curve representing 'Quick-Slow-Quick' trend.By measuring the changes of the contents of 10 mineral elements in prickly burs during the course of development,the results indicated that as the prickly burs grew larger and nuts gradually developed,the contents of N,P,Cu,Fe,and Zn in prickly burs declined in a whole. Amongst 10 measured mineral elements,only the contents of Zn and Cu were extremely positively related to each other while there were no significant correlations between other mineral elements,demonstrating that there are no significant or extremely significant mutual antagonisms between various mineral elements.Both chinquapin prickly burs and fruits were rich in a variety of mineral elements,most of whose contents varied significantly in the same period.The contents of K,Fe,Mn and Ca in nuts were obviously lower than those in prickly burs,with the contents of the former three minerals in nuts half of those in prickly burs and the content of Ca in nuts one fourth of that in prickly burs.Fruits had a bit higher contents of Mg,B and Cu than prickly burs,especially the content of Zn in nuts 2.2 times than that in prickly burs. The results indicated that as a part of fruit,chinquapin prickly burs at least absorbed half of mineral nutrients,having a significant influence on the development and yield of nuts.3.By using 'chushuhong' under normal development and dmpty-seeded ness 'chushuhong' on August 8 as the samples,a two-dimensional electrophoresis system of high resolution and stability for chinquapin embryonic proteins was established to extract the total protein and conduct two-dimensional electrophoresis.Image Master TM 2D Platinum software was adopted in the analysis.Image Master TM 2D Platinum software was adopted in the analysis.12 differential protein spots were taken out for peptide mass fingerprinting(MALDI-TOF MS) analyses and a complete peptide fingerprinting was obtained.MASCOT was adopted to search the protein database of green plants on NCBInr with 4 proteins successfully identified.These 4 proteins involved in cell wall metabolism,carbohydrate and energy metabolism and cytoskeleton metabolism.The article has reported for the first time the differential proteomic study,providing important information of protein levels for further studies of dmpty-seeded ness phenomena in chinquapin.4.DNA polymorphism analysis was carried out on 22 chinquapin resources in northern Fujian by using ISSR molecular markers.A total of 201 bands were amplified by 16 primers chosen between 100 primers,including 193 polymorphic bands,accounting 96%of the total.On the average,each primer amplified 12 bands.Genetic similarity coefficients of 22 varieties of chinquapin germplasm revealed by ISSR markers ranged from 0.336 to 0.981,with an average of 0.612.These chinquapin germplasm could be classified by polymorphism analysis.The phylogenetic tree showed that chinquapin could be divided into two groups when the genetic distance D equaled 24,with 'huangzhen' as a major class,which was second to none in fruit size. When D equaled 14,the experimental materials could be classified into 6 groups,basically reflecting the similarity relationship between various fruits and pricey burs.The analyses on genetic similarity coefficients of experimental chinquapin showed that there were close relationship between 'wukechangrnang' and 'wenyanghong','hongzizhen' and 'zizhen' as well as 'wuzhen' and 'maisaizi'.5.Naked-eye and microscopic observations were conducted on some properties of male inflorescences and pollens of five major varieties of chinquapin.The results indicated that the lengths of male inflorescences,the sizes and shapes of pollens as well as the lengths of pollen tubes varied in different varieties,while the total numbers of blossom clusters of male inflorescences were close.The longer male inflorescences,the lower densities of clusters,and vise versa.The temperature between 30 to 35℃was most suitable for the germination of chinquapin pollens and the extension of chinquapin pollen tubes while low temperature was favorable for the storage of chinquapin pollens.Different concentrations of gibberellin(GA₃) and boric acid could promote the germination of chinquapin pollens and the extension of chinquapin pollen tubes,with 50 mg/L of gibberellin the most effective and 0.1%to 0.5%of boric acid having better effects.Dry and shading conditions were better for the storage of pollens in arnbient temperatures.The pollens that had been stored for 5 days had the highest germination rate.The results of pollens scanning by electron microscopes further demonstrated that chinquapin pollens were small pollens in prolate shape,with 6-lobate-circular in polar view and oblong-elliptic,broad-elliptic,rhombic shapes in equatorial view.Pollens of different chinquapin varieties were different,principally manifested in the pollen sizes,shapes and ornamentations."huangzhen" pollens were the largest ones with basically the same ridge width and space.There were four groups of pollens with similar sizes.There were no ornamentations exactly the same.However,the properties of different varieties were stable.The reason of 'empty seeded ness' on chinquapin also discussed in this study.The result showed that the 'empty seededness' on chinquapin occurred at the stage of embryo's growth.The factors of variety,nutrition,pollination and impregnation can be regared as some reasons of 'empty seededness'.Variety of 'youzheng' had relative high 'empty seededness' at some producing areas.Varieties with high 'empty seededness' had close relationship by using the technologies of RAPD and ISSR.Mass a blooming,and the development of prickly burs had drastic competition on nutrition,as a result,the storing nutrition of tree reduced.Bad pollination and impregnation may caused embryo's abortion at the forepart,those nonimpregnation had 'empty seededness' in the end.Besides,we also discovered that the disappearing of certain differential protein in 'empty seededness' resulted in several life activities stopped,for example,the matters transportation in cell,cell moving,cell differentiation and growth,cell division and propagating.