花生与其近缘野生种间细胞融合及杂种愈伤组织的形成

Callus Formation from Fused Protoplasts Between Peanut and Its Related Species

本研究旨在为体细胞杂交法在花生育种中的应用奠定基础。将花生栽培种Krapts和野生种A.stenosperma幼叶解离的原生质体,用PEG方法融合后,置于添加2mg/L毒莠定(Pic)、0.1mg/L苯基噻二唑基脲(TDZ)、2%的椰乳、5g/L聚乙烯吡咯烷酮(PVP)和0.1%2-吗啉乙磺酸(MES)的改良MSB5(MS无机盐+B5有机成分)液体培养基中进行浅层培养。5周后将形成的小愈伤组织转移到添加3mg/L玉米素(ZT)、0.2mg/L6-苄氨基嘌呤(BAP)、0.1mg/L萘乙酸(NAA)的固体培养基上进行培养,促使愈伤组织增殖。观察发现,融合处理的原生质体在液体培养基上培养4天后开始分裂,2周后形成直径约300μm的细胞团,5周后小愈伤组织直径可达2～3mm。当将小愈伤组织转移到固体培养基上后,愈伤组织迅速增殖,并获得大量愈伤组织。提取愈伤组织DNA进行PCR检测,部分愈伤组织扩增出了双亲特异的DNA条带或双亲都不具有的新条带,说明愈伤组织来自于融合细胞。

The aim was to lay the foundation for the application of somatic hybridization in peanut breeding. The protoplasts isolated from leaves of Krapts and A. stenosperma were fused by PEG method, and then cultured in the modified liquid MSB5 medium （Murashige and Skoog salts plus B5 vitamins） with 2 mg/L picloram （Pic）, 0.1 mg/L thidiazuron （＇H）Z）, 2% coconut cream, 5 g/L polyvinyl pyrrolidone （PVP） and 0.1% 2-（N-morpholino） ethanesulfonic acid （MES）. After 5 weeks culture, the formed small calli were transferred to the solid MSB5 medium supplemented with 3 mg/L zeatin, 0.2 mg/L BAP and 0.1 mg/L NAA for callus proliferation. First cell division was observed within 4 days after culture in the liquid medium. And the formed colonies grew up to 300 μm in diameter after 2 weeks culture and up to 2-3 mm after 5 weeks culture. When the small calli were transferred to the solid MSB5 medium, they grew rapidly. Genomic DNA of calli was extracted and detected by PCR, some of the bands from both parents and the new bands were amplified in some calli. The results indicated those calli were derived from fused cells.