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Plant tissue culture regeneration is chiefly regulated by exogenous phytohormones. To stop regeneration and induce undifferentiated callus growth auxins are used. Unfortunately auxins influence many plant responses, most unrelated to development. Using the thymidine analogue 5-bromodeoxyuridine (BrdU) a phytohormone independent means for differentiation inhibition has been developed. Studies were focused on the target site and mechanism of BrdU action. The BrdU inhibited step in development is indicative of a plant response necessary for normal differentiation. BrdU (5-30 uM) interrupts callus growth in all tested plants. Exogenous cytokinin does not restore growth while thymidine and deoxycytidine rescue plant growth and differentiation in the presence of BrdU. Endogenous cytokinin levels are not greatly affected by subtoxic BrdU levels and indicate that cytokinin and BrdU act upon independent sites. Domestic carrot cells were used in further studies. Normally carrot cells are undifferentiated in medium with the auxin 2,4D. When 2,4D is removed, somatic embryogenesis takes place. By including 5 uM BrdU in the hormoneless medium, the cells fail to differentiate. The growth of carrots in 2,4D is not affected by 5 uM BrdU. Thus, BrdU influences growth during differentiation to a greater extent than the growth of callus cells. BrdU is effective in halting development when applied 0-24 hours after differentiation induction. An event required for differentiation (the first and second replications) must take place at this time. BrdU action begins with DNA incorporation. The consequent cellular replication becomes slowed and DNA repair results. At the same time RNA and protein levels are similar in BrdU treated and untreated cultures. BrdU thymidine substitution into DNA increases from 28% (2 days) to 68% (3 days) after embryogenic induction. A second BrdU effect follows DNA incorporation. Factors (MIFs) in the medium of BrdU treated cells arrest differentiation. After BrdU is repaired from the DNA, the cells are only able to differentiate after a medium change. Understanding MIF production could explain why some plants differentiate more readily than others. BrdU provides the means for further study of MIF's in the auxin-free inhibition of development.

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Gene transfer vectors based on the Agrobacterium tumefaciens Ti plasmid were used to develop a successful disarmed Agrobacterium tumefaciens-mediated transformation method for Lotus comiculatus. A binary vector construct, pJIT73, was used during the development of the Agrobacterium tumefaciens transformation system due to its selectable (Aph IV, nos- neo) and scorable markers. The effects of the antibiotics geneticin (G-418) and hygromycin B were studied. Use of kill curves and selection delay experiments allowed potentially suitable selection pressure parameters to be proposed. Using such selection during transformation experiments led to further optimisation of this stage of transformation. The influence of plant hormones on the regeneration of Lotus comiculatus explants was investigated and a modification of an established protocol using leaf explants was introduced as an attempt to reduce the overall time of regeneration. Various explants were used but leaf pieces were chosen as the most suitable explant on which to focus research. So, through alteration of various stages, including length of cocultivation and subsequent decontamination within the transformation process, a successful method was developed. Experiments indicated the optimum Agrobacterium tumefaciens strain to be used with Lotus comiculatus was the disarmed Ach5 type, LBA4404(pAL4404). Transgenic Lotus comiculatus plants were produced which expressed the scorable marker β-Glucuronidase gene (GUS) and the selectable marker for hygromycin B resistance, AphIV. Gene transfer was confirmed by Southern blotting. The new Agrobacterium tumefaciens-mediated vector system was used to introduce the cowpea trypsin inhibitor gene (CpTi) into Lotus comiculatus. However, although there was evidence for transformed callus development, no shoots were induced. By the use of previously established Agrobacterium rhizogenes-mediated system, an attempt was made to introduce the pea lectin gene (psl) into Lotus corniculatus. Hairy root regenerants were produced but genetic transfer was unconfirmed and attempted investigation of the plant - Rhizobium symbiosis involving Lotus corniculatus was not fulfilled.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990. / Vita. Abstract. Includes bibliographical references. Also available via the Internet.

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