葫芦科植物幼嫩子房壁制片技术的优化及其染色体倍性鉴定

Optimization of Chromosome Preparation Method Using Young Ovary Wall of Cucurbitaceae Plants and Ploidy Identification

该研究利用黄瓜、甜瓜、西瓜和西印度黄瓜这几种葫芦科植物的幼嫩子房壁作为材料进行染色体制片,探索子房材料的样品大小、预处理时间和酶解时间对染色体制片的影响及其优化,并用该制片方法对黄瓜候选单倍体植株的子房壁进行倍性鉴定和荧光原位杂交实验。结果发现:(1)黄瓜、甜瓜、西瓜和西印度黄瓜的幼嫩子房壁最佳预处理时间分别为1 h 30 min、1 h、55 min和45 min,子房长度为0.2~1 cm,子房壁材料切成边长为1~1.5 mm小块,酶解时间为1 h 10 min^1 h 20 min时,用该优化制片方法均可观察到较多的分裂相。(2)利用该方法鉴定结果显示,葫芦科植物黄瓜、甜瓜、西瓜和西印度黄瓜的染色体分别为14、24、22和24条,黄瓜候选单倍体植株的体细胞染色体数为7条。(3)将该制片方法获得的染色体装片用于荧光原位杂交结果显示,在二倍体黄瓜染色体中有3对明亮的45S rDNA杂交信号和1对5S rDNA杂交信号,而单倍体黄瓜中相应信号数量均减半;在甜瓜、西瓜和西印度黄瓜中均有2对45S rDNA杂交信号和1对5S rDNA杂交信号。研究认为,利用葫芦科植物子房壁作为制片材料,不仅可以获得良好的分裂相,还具有易于取材、制片效率高等优点,因此子房壁制片法是研究植物染色体数目和鉴定倍性的有效方法,且该制片方法也适用于进一步的荧光原位杂交分析。

The young ovary wall of Cucurbitaceae plants,including cucumber,melon,watermelon and West Indian cucumber,were used as the material for chromosome preparation.Several main factors including sample size of ovary material,8-hydroxyquinoline pretreatment time and enzymatic hydrolysis time were investigated to analyze their effects to chromosome preparation results,and we also explore how to optimize this method.Using this method,ploidy identification and fluorescence in situ hybridization experiments were carried out on the ovary wall of candidate haploid plants of cucumber.Results show that:(1)the best pretreatment time of young ovary wall is 1 h 30 min for cucumber,1 h for melon,55 min for watermelon,45 min for West India cucumber.To observe clear mitotic metaphase with this optimized method,we should use melon ovary with a length range of 0.2-1 cm,cut the ovary wall material into small pieces with a side length of 1-1.5 mm.And the suitable time for enzyme digestion is 1 h 10 min to 1 h 20 min.(2)The identification results by this method showed that the cucurbitaceae plants cucumber,melon,watermelon and West Indian cucumber had 14,24,22 and 24 chromosomes,respectively.The somatic cell chromosome number in the plant identified as haploid was 7.(3)Fluorescence in situ hybridization showed that there were 3 pairs of bright 45S rDNA hybridization signals and 1 pair of 5S rDNA hybridization signals in cucumber chromosomes,and the number of corresponding signals in haploid cucumbers was 7.There were 2 pairs of 45S rDNA and 1 pair 5S rDNA signals in melon,watermelon and West Indian cucumber.Our studies indicated that the ovary wall chromosome preparation procedure is suitable for all these Cucurbitaceae species.Not only can they obtain good mitotic metaphase,but also have the advantages of easy to get material and high chromosome preparation efficiency.Therefore,when it is difficult to obtain root tips of the rare material,the ovary wall chromosome preparation method is an effective method for studying the chromosome number of plants and identifying ploidy.This method is also suitable for fluorescence in situ hybridization.