以外种皮珠孔组织为外植体的胡椒体细胞胚再生研究

Somatic Cmbryogenesis of Piper nigrum L.Through Micropylar Tissues of Episperm

胡椒是世界知名香料作物,素有“香料之王”的美誉。建立胡椒组培再生技术,对于提升种苗繁育效率,支撑产业快速发展具有重要的应用价值。本研究以我国胡椒主栽品种热引1号(Piper nigrum c.v.Reyin-1)为材料,系统研究外植体灭菌、愈伤诱导、体细胞胚分化等关键环节对体细胞再生的影响,并建立胡椒体细胞再生和组培苗繁育技术。结果表明:以成熟的种子为材料,首先用75%酒精表面灭菌45 s,去除果皮后再用75%酒精对种子灭菌45 s后,0.1%氯化汞浸泡灭菌6~10 min,获得无菌种子。无菌的胡椒种子在黑暗条件下萌发2周后获得脱落的外种皮作为胡椒组培体系的外植体。在黑暗条件下,以胡椒外种皮为外植体诱导愈伤及分化培养,培养2个月后发现珠孔部位可诱导出愈伤组织,培养基MS+1.5%蔗糖+0.80 mg/L 2,4-D+1.200 mg/L KT为最佳的愈伤组织诱导和胚性愈伤分化培养基,愈伤组织诱导率为88.14%,胚性愈伤分化率约为58.18%;将愈伤组织接种在体胚诱导培养基上,培养3个月后发现培养基MS+1.5%蔗糖+0.40 mg/L 2,4-D+0.600 mg/L KT为最佳的体胚诱导培养基,体胚诱导率为13.33%;将幼苗接种在生根培养基上,培养基1/2MS+1.5%蔗糖+0.25%活性炭为最佳生根壮苗培养基,培养2个月后获得胡椒组培苗。综上所述,本研究提出了利用酒精和氯化汞组合的外植体灭菌方法,明确了胡椒的外种皮珠孔组织是高效适宜的组织培养外植体;进一步分析了愈伤诱导、体胚分化、生根培养等体细胞胚胎发生关键过程中的培养基组合及培养条件。研究结果为胡椒高通量组培苗繁育和转基因育种技术研发奠定基础。

Black pepper(Piper nigrum L.) is a world-famous spice crop,known as ‘the king of spice'.The establishment of tissue culture and regeneration technology of black pepper has important application value in improving the efficiency of seedling breeding and industrial development.Here,we investigated the effects of explant sterilization,callus induction,and embryo differentiation in somatic cell regeneration systematically in black pepper,and established the tissue culture technology using the elite cultivar ‘Reyin-1' as the material.The results showed that episperm micropylar tissues could be employed as the explants.The ripe fruits were first sterilized with 75% alcohol for 45 s,and then peeled seeds were sterilized for 45 s in 75% alcohol,followed by 6–10 min of soaking in 0.1% mercuric chloride.The episperm was removed from the plantlet after two weeks culture in darkness,which was used as the explant for somatic embryogenesis.The callus was induced from episperm micropylar tissues within two-month subculture.The optimal culture medium for callus induction and embryogenic callus differentiation was MS+1.5% sucrose+0.80 mg/L 2,4-D+1.200 mg/L KT,and the callus induction rate was 88.14 %,and differentiation rate of embryogenic callus was about 58.18%.The embryo callus were transplanted on somatic embryo induction medium.Somatic embryos were induced on the medium MS+1.5% sucrose+0.40 mg/L 2,4-D+0.600 mg/L KT,with a induction rate of 13.33%.After two months of rooted culture,tissue culture plantlets were obtained,and the optimal medium for rooting culture was 1/2MS+1.5% sucrose+0.25% activated carbon.In conclusion,the study provided a combine method of alcohol and mercuric chloride for explants sterilization,identified that micropylar tissues of the episperm was the best explants for somatic embryogenesis in black pepper.The medium and culture conditions of callus induction,embryo differentiation,rooting culture during somatic embryogenesis were further explored in black pepper.The results would lay a foundation for high-efficiency tissue culture seedling and transgenic breeding in black pepper.