INDUCTION OF HAIRY ROOT CULTURES FROM GOSSYPIUM HIRSUTUM AND GOSSYPIUM BARDADENSE TO PRODUCE GOSSYPOL AND RELATED COMPOUNDS

Authors: Triplett, Barbara; MOSS, STEPHANIE - UNO DEPT OF BIOLOGICAL SC; Bland, John; Dowd, Michael

Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2008
Publication Date: 7/12/2008
Citation: Triplett, B.A., Moss, S., Bland, J.M., Dowd, M.K. 2008. INDUCTION OF HAIRY ROOT CULTURES FROM GOSSYPIUM HIRSUTUM AND GOSSYPIUM BARDADENSE TO PRODUCE GOSSYPOL AND RELATED COMPOUNDS. In Vitro Cellular and Developmental Biology - Plants. 44:508-517.

Interpretive Summary: Gossypol is a naturally occurring pigment present in cotton seeds, leaves, roots, and other tissues. This compound has not been found in any other plant except cotton and is reported to have pharmacological properties in humans, cattle, and other animals. A number of investigators around the world are examining the efficacy of using gossypol and related compounds as agents against certain types of cancer and viruses. As a natural product in cotton, gossypol serves as a feeding deterrent to insects and herbivores. To expand the use and value of cottonseed as a food, there have been efforts to reduce seed gossypol content while leaving the gossypol glands found in leaves unaltered. The work reported in this manuscript was initiated as an effort to discover how gossypol is produced by the cotton plant, so that strategies for eliminating this compound in seeds can be devised. The objective of this work was to grow cotton tissues that could produce gossypol in sterile culture dishes in the laboratory. Since the growth conditions of cultured plant tissues can be accurately controlled and manipulated, new insights into the biochemistry of gossypol synthesis can be revealed by such an experimental system. Cotton roots were induced to grow after wounding cotton tissues with a microorganism called Rhizobium rhizogenes. Infection with this microbe causes plant tissues to proliferate root masses that can be grown independently from other plant parts. The highly branched root masses are called hairy roots. Several parameters were investigated to optimize cotton hairy root formation including (1) best tissue for infection, (2) best tissue age, (3) two different cotton species, and (4) optimal sugar and plant hormone concentration in culture media. Over 100 independently induced cultures were obtained. Within a few weeks after hairy root formation, the cultured tissues and the culture media were analyzed for gossypol content. Gossypol and two modified derivatives of gossypol were found in the hairy root tissues at roughly the same level found naturally in seeds. Also, a small amount of gossypol was found in the culture medium. The cultures have been propagated in the lab for more than one year. These results confirm that cotton hairy roots from two species of cotton are capable of producing gossypol and related compounds in large quantities under sterile growth conditions. Efforts are being directed at identifying cultures that produce the greatest quantities of gossypol and further defining the optimal growth conditions for these cultures. Since large amounts of sterile, cotton root tissue can be produced by hairy roots, a second use of these cultures is for investigations of nematode-plant interactions. Nematodes are microscopic, soil-dwelling worms that cause significant economic damage on cotton and other crops. Cotton hairy roots represent a well-defined growth substrate for nematode propagation for research purpoes. We hypothesize that by measuring the cotton hairy root response to nematode infection, new strategies for developing resistance to these pests can be found.

Technical Abstract: Hairy root cultures were induced by inoculating cotyledonary leaves and hypocotyl segments from two cotton species, Gossypium hirsutum and Gossypium barbadense, with Rhizobium rhizogenes 15834. Inoculated cotyledonary leaves produced greater numbers of hairy roots than hypocotyl segments. On basal Murashige-Skoog media for hairy root induction, 2.5% of G. barbadense cotyledonary leaf cultures produced hairy roots. Addition of naphthalene acetic (0.1 mg/L) to basal media did not increase the number of induced hairy roots. In contrast, 30 g/L sucrose added to basal induction media increased the frequency of hairy root induction to 12.5% of the cultures. Cotyledonary leaves younger than 14 days after germination produced more hairy roots than older leaf tissue. After transfer to liquid culture, hairy root growth was very rapid. After three weeks in liquid culture, both species of cotton produced gossypol, a di-sesquiterpene secondary metabolite and related compounds. Gossypol was retained within hairy root tissues and also secreted into the culture medium. The high level of gossypol production by these cultures suggests that they will be valuable for characterizing the biochemical and molecular aspects of gossypol biosynthesis, useful for producing radio-labeled forms of gossypol, and capable of generating sufficient quantities of gossypol derivatives and related compounds suitable for discovery of other intriguing biological activities.