Harvest: Using a sterile razor blade, harvest terminal nodes from the parent plant- use small pieces with no more than 2 nascent leaves per cutting, ~.5-1cm. Note- the more tissue you take, the higher the chance of contamination, and the more difficult it is to clear endophytes. NOTE- you can also take lateral stem cuttings, but rooting efficiency is decreased. Harvest into 50ml falcon tube- no more than 25 per tube.
Clear: for each set of 25, pre-fill 50ml falcon tubes with reagents and place in order: Tween, EtoH, NaOCl, H2O:
Tween 1% v/v (4ml in 40ml sterile ddi H2O)
EtoH 70% v/v (28ml in 40ml sterile ddi H2O)
NaOCl 10% v/v (4 in 40 sterile ddi H2O)
In germfree biosafety cabinet:
Pour Tween soln. into falcon tube containing microcuttings, cap, shake gently, and set timer
for 1 min. After 1 min. Pour off tween soln. into waste container. NOTE: pour slowly to prevent
pouring off microcuttings.
Pour EtOH soln. into falcon tube containing microcuttings, cap, shake gently, and set timer
for 1 min. After 1 min. Pour off EtOH soln. into waste container 1.
Pour NaOCl soln. into falcon tube containing microcuttings, cap, shake gently, and set timer
for 10 mins. After 10 mins. Pour off NaOCl soln. into waste container 2. NOTE: USE WASTE
CONTAINER 2 - mixing EtOH and NaOCl will make chloroform.
Pour sterile ddi H2O into falcon tube containing microcuttings, cap, shake gently, and pour
into waste container. Repeat this rinse step 5 times.