|Dowd, Michael - Mike|
Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Abstract Only
Publication Acceptance Date: 3/22/2004
Publication Date: 5/22/2004
Citation: Moss, S.C., Triplett, B.A., Dowd, M.K. Initiation and proliferation of cotton hairy roots. Congress for In-Vitro Biology. 2004. p. 53A. Interpretive Summary:
Technical Abstract: Hairy roots are masses of differentiated, transformed cells resulting from infection with Rhizobium rhizogenes (also known as Agrobacterium rhizogenes). Soybean hairy root cultures have been an invaluable research tool for examining nematode-plant interactions. Since root-knot and soybean cyst nematodes are also a problem in cotton production, we have investigated the conditions for producing cotton hairy roots. Stems and cotyledons of Gossypium barbadense, St. Vincent Sea Island and Gossypium hirsutum, DeltaPine 90 seedlings were inoculated with the bacterium, Rhizobium rhizogenes (ATCC 15834). The inoculated tissues were placed on four types of Hairy Root Induction Media (HRIM-A: MS salts and vitamins, 7g/L agar, pH 5.8; HRIM-B: MS salts and vitamins, 7g/L agar, 30 g/L of sucrose, pH 5.8.; HRIM-C: MS salts and vitamins, 7g/L agar, 0.1 mg/L NAA, pH 5.8; and HRIM-D: MS salts and vitamins, 7g/L agar, 30 g/L of sucrose, 0.1 mg/L NAA, pH 5.8. Cultures were incubated in a 25-27º C plant growth chamber with 16 hour days and 8 hour nights. Mock-inoculated controls showed that the hairy roots were in fact initiated by the bacterium. Young cotyledons from both G. barbadense and G. hirsutum produced more R. rhizogenes-transformed hairy roots than stem tissues. Inoculated cotyledons on HRIM-B produced more hairy roots than other culture conditions. Over 60 independently transformed lines were established from G. barbadense and over 40 lines were established from G. hirsutum. Once the hairy roots achieved a length of approximately 1.3 cm, they were removed from the cotyledon and transferred to fresh HRIM-B containing 500 mg/L carbenicillin and incubated in the dark at room temperature. The cultures were transferred 3 times at 2 week intervals onto solid HRIM-B plus carbenicillin. Two weeks after the third transfer, 1.3 cm hairy root segments were transferred to liquid HRIM-B minus carbenicillin at 25ºC. At this point, growth was rapid and the amount of root mass increased substantially. Present cultures are growing in 300 ml of liquid HRIM B in 1000 mL Erlenmeyer flasks. This method for producing abundant quantities of hairy roots from cotton tissue is quick and efficient. We have provided our cultures to investigators interested in finding new ways to find resistance to nematodes in cotton germplasm.