View source for Mungbean transformation

== Protocol<ref>''Agrobacterium tumefaciens''-mediated genetic transformation of mungbean (''Vigna radiata'' L. wilczek) - a recalcitrant grain legume, Pawan et al, 2001, Plant Science</ref> ==
# Prepare binary vector ex) pMDC 43
# Vector are inserted into Agrobacterium competent cell by [[heat shock]] method
# Spreading them on LB agar media with [[antibiotics]] for selecting cells (until colonies were visible)
# Seed planting in vitro, dark condition (during 2 days)
# Single colony picking into 2ml LB liquid media with [[antibiotics]] (over 16h incubate)
# [[Mini prep|Prep]] and confirm inserting DNA using PCR
# 20ul bacteria suspension + 20ml LB media with [[antibiotics]] (over 16h incubate)
# Harvest cells on 4000 RPM for 10 min
# Resuspending cells in liquid B<sub>5</sub> plant culture media at a density of 10<sup>9</sup> cells per ml(OD<sub>560</sub>=1)
# Immerse plant(Primary leaves with petiole or Cotyledonary nodes with proximal halves of cotyledon or hypocotyl) with suspension and gently shaking
# Explants were blotted on sterile filter paper and co-cultivated on B<sub>5</sub> basal medium for 3 days under dark conditions at 25±2℃
# Wash the explants 3~4 times with T.D.W and blot
# Cultivating on B<sub>5</sub> medium containing 5 * 10<sup>-6</sup> M BAP and 2.5 * 10<sup>-6</sup> M each of 2,4-D and NAA(also [[antibiotics]])
# The explants along with newly initiated callus were transferred every 2 weeks culture period for 8-10 weeks onto fresh medium

==Reference==
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