黑鹅绒海芋体细胞胚发生和植株再生

Efficient Plant Regeneration via Somatic Embryogenesis in Alocasia reginula cv.‘Black Velvet’

该研究建立了黑鹅绒海芋(Alocasia reginula)体细胞胚胎发生途径的植株再生体系。通过叶柄诱导获得胚性愈伤组织,以此建立胚性细胞悬浮培养系,并实现了高频率的植株再生。叶柄外植体在添加2.0 mg·L^(-1)2,4-D和1.0 mg·L^(-1)TDZ的MS培养基上诱导胚性愈伤组织的效率最高(达81.3%)。将胚性愈伤组织破碎成细胞团,转移至添加2.0 mg·L^(-1)2,4-D和1.0 mg·L^(-1)TDZ的MS液体培养基中进行悬浮培养,2周继代1次,悬浮培养12周后获得大量细胞团。将悬浮培养28周的细胞团转移至不含植物生长调节剂的1/2MS固体培养基上进行分化培养,平均每个细胞团可再生2.3–2.5棵植株。利用光学显微镜和扫描电镜观察体细胞胚的萌发和形成。再生苗移栽至温室4个月后,成活率为95.3%。通过流式细胞术对随机选取的50株成活植株进行检测,未发现染色体倍性变异,核DNA含量为10.94 pg·(2C)^(-1),基因组大小为5 290.12 Mb·C^(-1)。植株移栽到温室直至自然开花,表型无明显变异。研究结果可为黑鹅绒海芋种苗商业化生产和生物技术育种提供良好的技术支持。

In this study,a plant regeneration system via somatic embryogenesis was established in Alocasia genus.We obtained embryogenic cell suspension cultures of A.reginula through embryogenic calli induced from petioles,and achieved a high frequency of plant regeneration using embryogenic cell aggregates.Efficiency of embryogenic calli induced from petiole explants was highest(81.3%)on a Murashige and Skoog(MS)medium supplemented with 2.0 mg·L^(-1)2,4-dichlorophenoxyacetic acid(2,4-D)and 1.0 mg·L^(-1) thidiazuron(TDZ).The embryogenic calli were crushed into cell aggregates and then transferred to liquid MS media supplemented with 2.0 mg·L^(-1)2,4-D and 1.0 mg·L^(-1) TDZ for suspension cultivation.By subculturing biweekly,lots of cell aggregates were gained from embryogenic suspension cultures after 12 weeks.The cell aggregates within 28 weeks of suspension culture were transferred to solid 1/2MS media without plant growth regulator for differentiation culture,with an average of 2.3–2.5 plantlets regenerated from each cell aggregate.The formation and germination of somatic embryos were observed by optical microscopy and scanning electron microscopy(SEM).After the regenerated plantlets were transplanted to a greenhouse for 4 months,the achieved survival ratio was 95.3%.Flow cytometry(FCM)demonstrated that there was no chromosome ploidy variation in randomly selected 50 surviving plants.In addition,the nuclear DNA content was estimated at 10.94 pg·(2C)^(-1),and the genome size was 5290.12 Mb·C^(-1).There was no significant variation in their phenotypes from the time the plants were transplanted to the greenhouse until they bloomed spontaneously.These results provide good technical support for the commercial production of seedlings and biotechnological breeding of A.reginula.