Improved methods for transformation of embryogenic cotton cell cutlures
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 callus.
Agrobacterium-mediated transformation of cotton embryogenic callus
1. Randomize a collected pool of embryogenic cotton calli having a range of developmental characteristics (friable globular embryogenic callus to maturing embryos with identifiable organization or polarity) in a sterile dish. 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) + AS100uM (Acetosyringone 100µM) media at a 1:1 ratio (original culture volume to liquid MSM). Immerse tissue in a small volume of further-diluted (1:5) Agrobacterium suspension. Mix tissue by repeated pipetting, 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) previously infiltrated with liquid MSM + AS100µM. With forceps, distribute the tissue in small, 2-3 mm mounds across the filter disc. Label and seal plates with Parafilm and place in a 25°C dark growth chamber for 36-48 hrs.
Particle bombardment-mediated transformation of embryogenic callus
Randomize a collected, 100-milligram pool of friable globular embryogenic cotton calli onto a 5-7 cm sterile filter disc moistened with liquid MSM media in a Petri 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 a 2-day co-cultivation period, transfer tissue in 2-3 mm mounds to fresh plates of MSMK (MSM with additional 1.9g/L KNO3, gelling agent 2-10 g/L) + Tim500 (Timentin 500mg/L) + S.A. (Selection Agent such as kanamycin, phosphinothrycin or glyphosate) media. Place culture plates in a growth chamber at 28°C with a 16 hr light (70-150uE m-2 s-1, cool white fluorescent lamp)/ 8 hr dark photoperiod. Passage all tissue to fresh selection media every 14-21 days. Proliferation of healthy-looking tissue upon repeated passage to fresh selection media is indicative of stable transformation and should be observed in 6-10 weeks. From this point forward, passage only healthy, proliferating tissue masses, while discarding necrotic and browning tissue. Regeneration with associated greening of cotyledonary portions of the maturing embryo is also indicative of tolerance/resistance to the Selection Agent (i.e., successful transformation). Independent events should be carefully identified and/or isolated during the selection process. Several independent events can be derived from a respective mound of tissue; however, one may conservatively consider each generated...