Mungbean transformation

Protocol

1. Prepare binary vector ex) pMDC 43
2. Vector are inserted into Agrobacterium competent cell by heat shock method
3. Spreading them on LB agar media with antibiotics for selecting cells (until colonies were visible)
4. Seed planting in vitro, dark condition (during 2 days)
5. Single colony picking into 2ml LB liquid media with antibiotics (over 16h incubate)
6. Prep and confirm inserting DNA using PCR
7. 20ul bacteria suspension + 20ml LB media with antibiotics (over 16h incubate)
8. Harvest cells on 4000 RPM for 10 min
9. Resuspending cells in liquid B₅ plant culture media at a density of 10⁹ cells per ml(OD₅₆₀=1)
10. Immerse plant(Primary leaves with petiole or Cotyledonary nodes with proximal halves of cotyledon or hypocotyl) with suspension and gently shaking
11. Explants were blotted on sterile filter paper and co-cultivated on B₅ basal medium for 3 days under dark conditions at 25±2℃
12. Wash the explants 3~4 times with T.D.W and blot
13. Cultivating on B₅ medium containing 5 * 10^-6 M BAP and 2.5 * 10^-6 M each of 2,4-D and NAA(also antibiotics)
14. The explants along with newly initiated callus were transferred every 2 weeks culture period for 8-10 weeks onto fresh medium