Improved methods for transformation of Cotton suspension cultures
Publication Date: 2004-Dec-09
The IP.com Prior Art Database
An improved and efficient method is disclosed for transforming and regenerating embryogenic cotton cell cutures.
Improved methods for transformation of Cotton (Gossypium spp.) using embryogenic cell suspensions.
Agrobacterium-mediated cotton embryogenic suspension culture transformation
1. Collect 10-14 day-old embryogenic suspension cultures for transformation. Centrifuge an overnight liquid culture of an Agrobacterium tumefaciens strain containing a vector of interest and resuspend the cell pellet in liquid MSM (Murashige & Skoog basal salts + vitamins, pH 5.7, D-Maltose 30 g/L) + AS100µM (Acetosyringone 100µM) media at a 1:10 ratio (original culture volume to liquid MSM). Fully submerge approximately 200-300 milligrams of embryogenic culture in 5-mls of the diluted bacterial suspension in a 100 x 15 mm Petri dish for 5 minutes, and then remove the bacterial suspension via pipette aspiration. Using a laboratory spoon, transfer treated tissue to a disposable, sterile Petri dish containing a sterile filter disc (ex. Whatman #1, Cat No. 1001 070) wetted with liquid MSM medium + AS100µM. Disperse the tissue using forceps into small, 2-3 mm mounds of tissue across the filter disc. Seal plates with Parafilm and place in the dark at 25°C for 36-48 hrs.
Particle bombardment-mediated transformation of embryogenic callus
Randomize a collected, 100-milligram pool of 10-14 day-old embryogenic suspension calli onto a liquid MSM-moistened sterile, 5-7 cm filter disc in a sterile dish. Place a surface-sterilized mesh screen (baffle) over the collected tissue. Bombard the tissue with purified plasmid DNA coated onto particles. A typical protocol could use gold microcarriers (0.6 -1.0 um) at 1000-1500 psi with a target distance of 6-9 cm under 28 inches Hg (95 kPa) vacuum.
2. After 36-48 hours of co-cultivation, transfer tissue as 2-3 mm mounds to fresh plates of MSMK (MSM with additional 1.9g/L KNO3, gelling agent 2-10 g/L) + Tim400 (Timentin 400mg/L) + S.A. (Selection Agent such as kanamycin, phosphinothrycin or glyphosate) media. Incubate under a 16 hr light (70-150uE m-2 s-1)/ 8 hr dark photoperiod at 28°C.
* particle bombardment-mediated
Transfer bombarded tissue and filter disc to MSMK media without selection and culture for 2 weeks. Incubate under a 16 hr light (70-150uE m-2 s-1)/ 8 hr dark photoperiod at 28°C. After 2 weeks, transfer tissue (without disc) to MSMK + S.A. media.
Agrobacterium and particle bombardment mediated NOTE: omit Timentin from all subsequent media when using particle bombardment-derived tissue, since there is no Agrobacterium to eliminate.
3. Subculture all tissues to fresh MSMK plates containing Timentin 200 mg/L and Selection Agent every 14-21 days. Proliferation of healthy-appearing tissue upon repeated passages to fresh selection media is indicative of stable transformation
and should be observed in 4-8 weeks. From this point forward, passage only healthy, proliferating tissue masses, while discarding necrotic and browning tissue. Regeneration with associated greening of cotyledonary po...