Dhumale Dipti Raghunath, Shingote Prashant Raghunath, Dudhare Mahendra Shankarrao, Jadhav Pravin Vishwanath, Kale Prashant Bhaskar
Department of Agricultural Botany, Biotechnology Centre, Dr Panjabrao Deshmukh Agricultural University, Akola, MS, 444104, India.
National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi, 110012, India.
3 Biotech. 2016 Dec;6(2):181. doi: 10.1007/s13205-016-0497-4. Epub 2016 Aug 26.
Shoot regeneration in safflower (Carthamus tinctorius 'AKS 207' and 'PKV Pink') genetically transformed using Agrobacterium was used for assessing various constraints to the efficiency of transformation including infection period, virulence induction medium, co-cultivation period, bacterial titre, selection regime, and the natural phenolic compound acetosyringone. Transformation frequency was promising with 8-10-day-old cotyledonary leaf explants. Therefore, explants of that age cultured on Agrobacterium minimal medium (AB) containing 100 µM acetosyringone were infected with Agrobacterium (cell titre 0.5 OD) for 15 min followed by 48 h of co-cultivation on kanamycin-enriched medium (50 mg/L). Transformation of the shoots was confirmed using β-glucuronidase (GUS) histochemical assay and polymerase chain reaction (PCR). With the transformation protocol thus optimized, the transformation frequency as determined using GUS assays was 54.0 % for AKS 207 and 47.6 % for PKV Pink. The corresponding figures using PCR were 27.0 and 33.3 %. The transformed shoots required 10-14 weeks of culture initiation but produced very few roots.
利用农杆菌介导对红花（“AKS 207”和“PKV Pink”品种）进行遗传转化，通过芽再生来评估转化效率的各种限制因素，包括侵染时间、毒力诱导培养基、共培养时间、细菌浓度、筛选体系以及天然酚类化合物乙酰丁香酮。对于8 - 10日龄的子叶外植体，转化频率很有前景。因此，将该年龄段的外植体接种在含有100 µM乙酰丁香酮的农杆菌基本培养基（AB）上，用农杆菌（细胞浓度0.5 OD）侵染15分钟，然后在添加了卡那霉素（50 mg/L）的培养基上共培养48小时。利用β - 葡萄糖醛酸酶（GUS）组织化学分析和聚合酶链反应（PCR）确认芽的转化情况。通过如此优化的转化方案，使用GUS分析测定的AKS 207的转化频率为54.0%，PKV Pink为47.6%。使用PCR的相应数字分别为27.0%和33.3%。转化后的芽需要10 - 14周的培养起始时间，但生根很少。
