水稻穗期活体植株对潮霉素的敏感性分析及突变体选育

携带潮霉素磷酸转移酶基因(HPT)的转基因作物细胞和组织对抗生素潮霉素具有一定耐性,是作物尤其是单子叶作物遗传转化中常用的选择基因。本研究旨在建立一种活体植株潮霉素抗性快速鉴别技术,用于转基因育种和相关研究。在始穗期前后,用不同浓度潮霉素B溶液(25～100mg/L)喷洒常规及转基因水稻植株,考察叶片和谷粒的中毒症状。发现常规水稻在高于50mg/L的潮霉素溶液处理后,叶片出现褐色斑点,稻穗部分谷粒颖壳呈黄褐色的中毒症状,叶片斑点和黄褐色谷粒的数量均随剂量增大而增加。经100mg/L潮霉素处理后,籼稻品种嘉优99和C10剑叶每平方厘米平均斑点数目为21.1和19.2个,穗上黄褐色谷粒比例分别达27.6%和23.5%;粳稻品种嘉优5号和R5叶片中毒斑点数为11.8和10.7个,黄褐色谷粒比例分别为11.2%和11.9%,表明2份籼稻材料较2份粳稻材料对潮霉素更为敏感。在上述浓度范围内,携带HPT的转Bt基因水稻克螟稻1号均未出现中毒症状。对γ射线诱变处理克暝稻的M2群体(共12万株)穗期喷施100mg/L的潮霉素B溶液,从中筛选到42株潮霉素敏感型突变体,组织化学法分析表明这些植株具有β-葡萄糖苷酸酶GUS活性。对能够取到较嫩叶片的14个单株采用离体叶片法进行抗虫性鉴定,证明这些植株对二化螟仍然表现高抗。上述结果表明,在始穗期喷施100mg/L潮霉素溶液,潮霉素敏感植株可出现明显、直观的中毒症状,可以在活体植株水平上有效鉴别植株是否携带HPT,可用于育种中转基因植株的筛选或转基因安全评估中植株的鉴别。

IN PLANTA ASSAY OF HYGROMYCIN SUSCEPTIBILITY AND MUTANT SCREENING IN RICE AT HEADING STAGE

Cells and tissue of plants carrying the hygromycin phosphotransferase gene(HPT) are tolerant to antibiotic hygromycin and hence become the common selection gene for genetic transformation of crop plants,particularly monocots.The present study aimed at establishing a rapid method for in planta screening of hygromycin tolerant plants in transgenic breeding.At heading stage transgenic and conventional rice plants were sprayed with hygromycin solutions of different concentrations(25 ~ 100mg/L) and toxic symptoms were observed in the following days.It was observed that yellow-brown necrotic spots appeared in the leaves and grains of conventional rice varieties after foliar spraying of 50mg/L hygromycin,and the more spots were occurred with the higher hygromycin solution.There were 21.1 and 19.2 spots per cm2 on flag leaves of indica varieties Jiayou 99 and C10,respectively;while 27.6% and 23.5% grains were yellow-brown in the two varieties,respectively.The corresponding data for two japonica varieties Jiayou 5 and R5 were 11.8 and 10.7 for leave spots and 11.2% and 11.9% for yellow-brown grains,respectively.These results indicated that the two indica varieties are more sensitive to hygromycin than the two japonica varieties.In the treatment of hygromycin with above levels,the transgenic rice line KMD1 with the HPT gene showed no toxic symptoms on leaf or panicles.The γ-rays treated M2 population of KMD1 was sprayed with 100mg/L hygromycin solution at heading stage and 42 plants were screened out with toxic symptoms in leaves and panicles from 120,000 plants.Among the 42 hygromycin susceptible plants,14 plants with leaves available for in vitro feeding assay were subjected to the feeding of rice striped steam borers Chilo supperssalis(Walker)] and all showed high resistance.The above results showed that spraying of 100mg/L hygromycin solution at heading stage would lead to visually apparent toxic symptoms in susceptibility rice plants and hence could be developed as an effective in planta method for distinguishing plants carrying the HPT gene in breeding programs and on-site biosafety monitoring.