CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia.
Plant Methods. 2011 May 20;7:12. doi: 10.1186/1746-4811-7-12.
Safflower (Carthamus tinctorius L.) is a difficult crop to genetically transform being susceptible to hyperhydration and poor in vitro root formation. In addition to traditional uses safflower has recently emerged as a broadacre platform for the production of transgenic products including modified oils and pharmaceutically active proteins. Despite commercial activities based on the genetic modification of safflower, there is no method available in the public domain describing the transformation of safflower that generates transformed T1 progeny.
An efficient and reproducible protocol has been developed with a transformation efficiency of 4.8% and 3.1% for S-317 (high oleic acid content) and WT (high linoleic acid content) genotypes respectively. An improved safflower transformation T-DNA vector was developed, including a secreted GFP to allow non-destructive assessment of transgenic shoots. Hyperhydration and necrosis of Agrobacterium-infected cotyledons was effectively controlled by using iota-carrageenan, L-cysteine and ascorbic acid. To overcome poor in vitro root formation for the first time a grafting method was developed for safflower in which ~50% of transgenic shoots develop into mature plants bearing viable transgenic T1 seed. The integration and expression of secreted GFP and hygromycin genes were confirmed by PCR, Southern and Western blot analysis. Southern blot analysis in nine independent lines indicated that 1-7 transgenes were inserted per line and T1 progeny displayed Mendelian inheritance.
This protocol demonstrates significant improvements in both the efficiency and ease of use over existing safflower transformation protocols. This is the first complete method of genetic transformation of safflower that generates stably-transformed plants and progeny, allowing this crop to benefit from modern molecular applications.
红花（Carthamus tinctorius L.）是一种难以进行基因转化的作物，容易出现过度水合和体外生根不良的情况。除了传统用途外，红花最近也成为了生产转基因产品的广泛平台，包括改良油和药用活性蛋白。尽管基于红花的遗传修饰开展了商业活动，但目前还没有公开的方法可以描述转化红花并产生转化 T1 后代的方法。
我们开发了一种高效且可重复的方案，对于 S-317（高油酸含量）和 WT（高亚油酸含量）基因型，转化效率分别为 4.8%和 3.1%。我们还开发了一种改良的红花转化 T-DNA 载体，包括分泌型 GFP，可用于对转基因芽进行非破坏性评估。通过使用iota-卡拉胶、L-半胱氨酸和抗坏血酸，有效地控制了农杆菌感染子叶的过度水合和坏死。为了首次克服体外生根不良的问题，我们为红花开发了一种嫁接方法，其中约 50%的转基因芽发育成成熟植物，并携带有活力的转基因 T1 种子。通过 PCR、Southern 和 Western blot 分析，证实了分泌型 GFP 和潮霉素基因的整合和表达。对九条独立株系的 Southern blot 分析表明，每条株系插入了 1-7 个转基因，T1 后代表现出孟德尔遗传。
与现有的红花转化方案相比，该方案在效率和易用性方面都有显著提高。这是首次完整的红花遗传转化方法，可以生成稳定转化的植物和后代，使红花能够受益于现代分子应用。
