|Dowd, Michael - Mike|
Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Abstract only
Publication Acceptance Date: 11/10/2004
Publication Date: 1/4/2005
Citation: Moss, S.C., Dowd, M.K., Triplett, B.A. 2005. Initiation and proliferation of gossypol-producing cotton hairy roots. Proc. Beltwide Cotton Conf., New Orleans, LA, p. 2092. http://www.cotton.org/beltwide/proceedings/2005/pdfs/2825.pdf Interpretive Summary:
Technical Abstract: Hairy roots are masses of differentiated, transformed cells resulting from infection with Rhizobium rhizogenes (ATCC 15834) (Agrobacterium rhizogenes). Hairy root cultures derived from many plant species have been valuable research tools for producing secondary plant metabolites and for examining nematode-plant interactions. Gossypol, a di-sesquiterpene, is known to be produced by cotton (Gossypium spp.) leaves, stems, seeds, and roots. Several research groups have reported the inhibitory effects for gossypol and related compounds on in vitro growth of cancer cell lines. Gossypol is also reported to have antiviral activity against enveloped viruses such as HIV and to inhibit the growth of numerous parasitic organisms, fungi, microbes, and insects. This study was initiated to determine whether gossypol and related compounds could be produced in hairy root cultures. Our team is also interested in using hairy root cultures to propagate axenic cultures of nematodes, an economically important pest on cotton. The following variables were investigated to maximize efficiency of transformation: cotton species, plant tissue to inoculate, age of tissue at inoculation, and induction media composition. Over 100 independently transformed lines were established from G. barbadense and G. hirsutum. Stems and cotyledons of the two varieties of cotton seedlings were inoculated with Rhizobium rhizogenes. 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 25-27 ºC 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 25 ºC. 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. Immediately after transfer to liquid culture, hairy root growth was very rapid and gossypol production evident. The production of gossypol, 6-methoxy-gossypol and 6,6’-dimethoxy-gossypol was indicated by the retention times of HPLC peaks relative to standards, and the peak assignments were confirmed from the agreement in the corresponding UV-vis absorbance spectra. We are currently identifying cultures that are producing the highest levels of gossypol and gossypol-related compounds. These cotton hairy root cultures will be valuable for studying the biochemistry of gossypol synthesis and degradation, and for examining plant responses to nematode infection.