Department of Life Sciences, South Eastern Kenya University, Kitui, Kenya.
Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture Technology, Nairobi, Kenya.
PLoS One. 2022 Dec 1;17(12):e0278717. doi: 10.1371/journal.pone.0278717. eCollection 2022.
A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.
高产的原生质体和可靠的再生系统是体细胞杂交和基因组编辑研究成功的前提。然而，许多作物包括木薯在内，从原生质体中进行可重复的植物再生仍然是一个瓶颈。我们评估了几种因素，这些因素影响从叶片叶肉中分离出有活力的原生质体、诱导胚性愈伤组织和在三个木薯品种——Muchericheri、TMS60444 和 Karibuni 中再生植物。在含有 5 g/L Macerozyme 和 10 g/L 纤维素酶的酶混合物中，获得了相对较高的原生质体产量。Muchericheri 记录的原生质体产量相对较高，为 20.50±0.50×106，而 TMS60444（10.25±0.25×106）在 10 g/L 纤维素酶和 4 g/L 纤维素酶中的原生质体产量最少。新鲜分离的原生质体细胞接种在含有 MS 基本盐、60 g/L D-葡萄糖、30 g/L 蔗糖、B5 维生素、100 mg/L 肌醇、0.5 mg/L 硫酸铜、100 mg/L 水解酪蛋白、4.55 g/L 甘露醇、0.1 g/L MES、10 mg/L 草甘膦和 3 g/L 琼脂糖的愈伤组织诱导培养基（CIM）固体培养基上，以诱导原生质体的生长和发育。三个品种都形成了菌落，但在这种培养方法中没有观察到进一步的发育。在悬浮培养中，使用不同的细胞密度（1、2 和 3×105 p/mL）进一步评估了原生质体的生长和发育。在细胞密度为 3×105 p/mL 时，观察到具有最多小愈伤组织的发育。获得的小愈伤组织在补充有 10mg/L 草甘膦的 CIM 上培养。在所有细胞密度下，都观察到了愈伤组织的诱导，而在细胞密度为 1×105 p/mL 时，没有观察到体细胞胚胎发生。在 2×105 p/ml 时观察到最高的体细胞胚胎发生，而在细胞密度为 1×105 p/ml 时没有观察到体细胞胚胎发生。体细胞胚胎在补充有 1 mg/L BAP、0.02 mg/L NAA 和 1.5 mg/L GA3 的 EMM 培养基中成熟，然后在无激素培养基中发芽以进行植物再生。该方案使用简单的商业酶混合物，高度可重复，可应用于木薯生物技术研究。
