In vitro propagation, regeneration, attempted tetraploid induction, and agrobacterium-mediated transformation of Euphorbia pulchurrima 'Winter Rose'®

by 1972- Pickens, Kimberly Ann

Poinsettia, Euphorbia pulchurrima, is the number one potted flowering plant in the United States. ‘Winter Rose’™ is a very popular cultivar with more than one million plants sold each year. To further improve this cultivar, particularly for larger flower heads and free branching, this research aimed at establishing some in vitro systems for application of biotechnology to poinsettia genetic improvement. A protocol was established for in vitro axillary bud proliferation using greenhouse grown terminal buds. Buds were placed on Murashige-Skoog (MS) basal medium supplemented with benzlyaminopurine (BA). Explants produced the greatest number of axillary buds on medium containing between 2.2-8.8 µM BA. The number of explants that produced axillary buds increased with increasing BA concentration. An organogenesis system was also established using in vitro grown leaf tissues. The greatest amount of callus and shoots were produced from leaf midvein sections placed on MS medium containing 8.8-13.3 µM benzylaminopurine (BA) and 17.1µM indole-3acetic acid (IAA) for one month and then transferred to medium containing only BA. Adventitious buds were produced only from red-pigmented callus, and explants that produced callus continued to produce adventitious shoots in the presence of IAA. Fivemonth-old shoots derived from shoot culture or organogeneses rooted readily in artificial soil without treatment with IBA or treated with 50 or 100 mg/l IBA and were acclimated in the greenhouse. The effects of colchicine and oryzalin on callus production and adventitious shoot formation and their ability to induce tetraploid formation of Euphorbia pulchurrima iii ‘Winter Rose’™ were evaluated. In vitro grown leaf midvein sections were placed in/on various forms (liquid or solid) of medium supplemented with either colchicine or oryzalin. A range of duration times between 1-4 days was also evaluated. Colchicine was less damaging to leaf tissues at concentrations of 0.25 µM or 250.4 µM. A large amount of callus was produced as well as a few adventitious shoots. Oryzalin inhibited plant regeneration from leaf tissues at all concentrations tested, and caused severe necrosis. Tissues produced callus, but no shoots were formed. A protocol was established for using flow cytometry to determine the ploidy level in poinsettia. Sample calluses and regenerated shoots from colchicine treatments were evaluated using the flow cytometer and were found to be diploid. Callus from explants exposed to oryzalincontaining medium was also tested using the flow cytometer and no tetraploid tissue was found. Since colchicine showed generally less inhibitory effect than oryzalin, colchicine is considered to be a better mitotic inhibitor chemical for tetraploid formation in poinsettia than oryzalin. The factors influencing Agrobacterium-mediated transformation of Euphorbia pulchurrima ‘Winter Rose’™ were also evaluated. Kanamycin at 50 mg/L was sufficient to inhibit poinsettia callus and shoot formation, and appeared to be a suitable selectable antibiotic for selecting transformed cells in poinsettia. Variables evaluated in these studies included plasmid type (pBI121, pMON690), with or without addition of the antibiotics CCK (cefotaxime, carbenicillin, and kanamycin) or acetosyringone. Since all tissues infected with agrobacterium (co-cultivation) died in 1-2 months, it appeared that poinsettia is highly sensitive to agrobacterium infection. Because of this premature death of infected tissues, other variables such as acetosyringone, and CCK could not be iv evaluated effectively as to their effect on the transformation of ‘Winter Rose’™. Further study into antinecrosis chemicals or a change in explant type is needed in order to establish a protocol for Agrobacterium-mediated transformation of E. pulchurrima ‘Winter Rose’™. This research established foundation studies on which to build a biotechnological improvement program for E. pulchurrima ‘Winter Rose’™. v