Improved cotton somatic embryogenisis and cell line maintenance
Publication Date: 2004-Dec-09
The IP.com Prior Art Database
An improved and efficient method is disclosed for generating and maintaining embryogenic cotton cell cultures.
Improved cotton somatic embryogenesis and cell line maintenance using growth regulator-free media and D-maltose as primary carbon source.
Surface sterilization and seed sowing
1. Fill a disposable, sterile, 50-ml conical centrifuge tube with de-linted cottonseeds
(ex. Coker 312) to the 25-ml graduation mark. Add 70% ethanol to the 45-ml
graduation mark, seal and mix by inversion for 5-10 minutes. Next, perform the
following steps aseptically in a laminar flow hood: a) decant ethanol, using cap to
assist retention of seeds in the tube; b) add 25% household bleach solution with 2
drops of Tween-20 to the 45-ml graduation mark and mix by inversion for 25-35
minutes (ex. use platform tilter apparatus on a slow setting, with 15+/- degrees
inclination); c) decant bleach solution into a waste beaker and rinse seeds 6-8
times with sterile, deionized water (note: seal tube and shake vigorously several
times before decanting each rinse).
2. Decant final rinse and pour seeds into an empty, disposable culture dish to ease subsequent transfer to the next media.
3. Place 15-20 seeds, roughly evenly spaced, on solidified MSM (Murashige & Skoog basal salts + vitamins, pH5.7, D-Maltose 30g/L, gelling agent 8g/L) media. Label, seal with Parafilm and place in a growth chamber under a 16 hr light / 8 hr dark photoperiod at 28°C.
Seed germination and elongation
1. Seeds treated as described above should begin to germinate 2-6 days after sowing. When a primary root has emerged about 2-4 cm from each seed, more importantly, prior to seed coat shedding, transfer 5 germinated seeds each to 20- mls of liquid MSM media (Murashige & Skoog basal salts + vitamins, pH5.7, D- Maltose 30g/L) in 100 x 25 mm deep culture dishes. Place the dishes in a growth chamber (16 hr light/ 8 hr dark) at 28°C to facilitate seed coat shedding, seedling elongation and expansion of cotyledons.
Explant preparation, embryogenesis, embryogenic callus isolation and culture maintenance
1. 3-6 days after transfer to liquid MSM medium, excise fully expanded cotyledons
from a single dish and transfer to a new disposable dish for explant preparation.
Using a fresh scalpel blade, cut 0.5 x 0.75 cm explants from each cotyledon. 20-
30 explants per cotyledon should be generated. Transfer prepared explants to
solidified MSM media.
2. After 8-12 weeks of culture without passage, identify and transfer globular and chunky embryogenic calli to fresh plates of MSMK (MSM wi...