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Improved methods for transformation of Cotton suspension cultures Disclosure Number: IPCOM000033404D
Publication Date: 2004-Dec-09
Document File: 3 page(s) / 121K

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An improved and efficient method is disclosed for transforming and regenerating embryogenic cotton cell cutures.

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This is the abbreviated version, containing approximately 39% of the total text.

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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.

* Agrobacterium-mediated
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

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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...