Stable Transformation in Lotus japonicus

Abstract

[Abstract] This is a protocol to produce stable transgenic plants in Lotus japonicus, which is established based on methods previously reported (Handberg and Stougaard, 1992; Stiller et al., 1997; Thkjaer et al., 1998) with some modifications. In this protocol, hygromycin is used to select transgenic plants. Materials and Reagents 1. Germination plate (1% agar in sterilized water) 2. B5 salt (Wako Pure Chemical Industries, catalog number: 399-00621) 3. Gamborg's vitamin solution 4. BAP (Wako Pure Chemical Industries, catalog number: 020-07621) 5. 1-Naphthaleneacetic acid (NAA) 6. Acetosyringone 7. MES 8. (NH4)2SO4 9. Phytagel (Sigma-Aldrich, catalog number: P8169) 10. Meropen (Dainippon Sumitomo Pharma) 11. Hygromycin B 12. LB medium (Nacalai Tesque, catalog number: 20066-95) 13. Co-cultivation medium (see Recipes) 14. YMB medium (see Recipes) 15. Callus medium (see Recipes) 16. Shoot elongation medium (see Recipes) 17. Root induction medium (see Recipes) 18. Root elongation medium (see Recipes) Equipment 1. Clean bench 2. L. japonicus growth facility (we use several types of Biotrons such as LH-410S purchased