火龙果花药培养诱导胚性愈伤组织及遗传稳定性

Embryogenic Callus Induction Via Anther Culture of Purple Pitaya (Hylocereus costaricensis cv. Zihonglong) and their Genetic Stability

成年态火龙果茎段诱导的愈伤组织再分化较困难,进而影响到遗传转化效率。以红皮紫红肉的"紫红龙"火龙果花药为外植体,研究了小孢子不同发育时期、低温预处理时间、不同植物生长调节剂及浓度配比对花药诱导胚性愈伤组织的影响。结果表明,小孢子发育处于单核初期至单核靠边期的花药,其愈伤组织诱导率最高可达23.9%,但未能诱导出胚状体;而4℃低温预处理48 h,愈伤组织的诱导率可达32.8%;最适花药愈伤组织诱导的培养基为MS+2.0 mg/L 6-BA+1.0 mg/L 2,4-D+60 g/L蔗糖,诱导率为37.4%,胚性愈伤组织为黄绿色、致密状,外表有颗粒状突起。愈伤组织增殖培养基以MS+0.4 mg/L TDZ+0.8 mg/L KT效果较好,增殖系数达9.1倍以上。愈伤组织再分化培养基为MS+0.4 mg/L TDZ+0.5 mg/L 2,4-D+0.6 mg/L NAA时,愈伤组织再分化率达36.7%。通过火龙果花药诱导出愈伤组织,增殖获得大量胚性愈伤组织,再分化形成不定芽,经SRAP分子标记检测遗传性一致,能为火龙果基因工程育种和种苗快速繁育提供新的途径。

It is difficult for callus that induced from adult pitaya stem to redifferentiate, which will further affect the efficiency of genetic transformation. In this study, pitaya anthers were used as explants, anthers at different stages of microsporogenesis were cultured, low temperature pretreatment were compared, the induction medium was optimized. As a result, callus was induced, propagated and differentiated, with abundant embryogenic callus generated and adventitious buds formed. Our result indicated that pitaya anthers spanning stages from early-uninucleate to late-uninucleate showed a callus induction rate of up to 23.9%, but no embryoid formed. Low temperature pretreatment of 2 days duration resulted in an induction rate of 32.8%. The optimum medium for callus induction was MS＋2.0 mg/L 6-BA＋1.0 mg/L 2, 4-D＋60 g/L sucrose, with an induction rate of 37.8%. The embryogenic callus was compact in yellowish-green color, with granular bulge on surface. The optimum medium for callus proliferation was MS＋0.4 mg/L, TDZ＋0.8 mg/L, KT, with a multiplication factor over 9.1. Culture medium for callus redifferentiation was MS＋0.4 mg/L TDZ＋0.5 mg/L 2, 4-D＋0.6 mg/L NAA, with a redifferentiation rate of 36.7%. A large number of embryogenic callus were formed through anther callus proliferation, then differentiated to form adventitious shoots, which have the same genetic stability with mother plants by SRAP molecular markers verification. The system we developed herein could be applied in future pitaya rapid micropropagation and genetic engineering.