Pre-isolation, isolation and regeneration protoplasts from leaf mesophyll of in vivo Malus domestica ‘Anna’ cv.

Abstract To isolate protoplast, a pre-treatment was completed with the order to reduce and identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5% Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation period, sieve pore size, centrifugation period and hormonal balance was estimated using the techniques for isolation. The quantity of cells was computed as the quantity of cells per square on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW medium using a pore size of 25 µm with using incubation period for 20 hours. Moreover, the best protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant recovery using in apple somatic hybridization programs.摘要：为分离原生质体（protoplast），本研究通过预处理实现酚类物质含量的全年降低与鉴定，以促进原生质体分离。以苹果品种‘安娜’的活体叶肉叶片为材料，取15日龄叶片，先在含90 g·L⁻¹甘露醇的培养基中进行质壁分离处理30分钟，随后更换为含130 g·L⁻¹甘露醇的培养基继续处理30分钟，从中分离得到原生质体；分离前需使用包含（1.5%纤维素酶+0.5%果胶酶+1.5% Macrozyme）的酶解混合液。此外，本研究通过相关分离技术，对渗透压、孵育时长、筛孔孔径、离心时长及激素平衡等多种影响因素进行了评估。细胞密度通过血球计数板（haemocytometer）上每方格的细胞数进行计算。在CPW培养基中采用25 µm筛孔、孵育20小时的条件下，原生质体产量显著最高。此外，在添加1.0 mg·L⁻¹萘乙酸（NAA）与0.3 mg·L⁻¹6-苄氨基腺嘌呤（BAP）的MS培养基中，采用2.0×10⁵个原生质体的接种密度时，可获得最佳的原生质体再生效果。本研究建立的分离方案有望为苹果体细胞杂交相关的植株再生工作提供技术支撑。