Title: Improved Transformation of Anthurium Authors
|Fitch, M.M.M. -|
|Leong, T.C.W. -|
|He, X. -|
|Mc Cafferty, H.R.K. -|
|Zhu, Y.J. -|
|Moore, P.H. -|
|Aldwinckle, H.S. -|
|Atkinson, H.J. -|
Submitted to: HortScience
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 9, 2010
Publication Date: N/A
Interpretive Summary: Bacterial blight devastated the Hawaiian anthurium industry in the 1980s, and is still a major limiting factor that affects the profitability of the industry. Currently, expensive management practices are implemented to economically grow anthurium in Hawaii. Development of commercial cultivars that are resistant or tolerant to bacterial blight and also to damage by nematodes is a direct way to help the industry become more profitable. In this work, methods were developed to transform two commercial anthurium cultivars, Midori and Marian Seefurth, with genes that could potentially impart resistance or tolerance to these pathogens. Efficient transformation methods were developed and resulted in the establishment of numerous transgenic lines. This work paves the way for the testing and identification of bacterial or nematode resistant or tolerant lines that could increase the profitability of the anthurium industry.
Technical Abstract: Methods to increase transformation efficiency and yields of transgenic Anthurium andraeanum Linden ex. André hybrids were sought while effecting gene transfer for resistance to the two most important pests, bacterial blight (Xanthomonas axonopodis pv. dieffenbachiae) and nematodes (Radopholus similis and Meloidogyne javanica). Differentiated explant tissues, embryogenic calli, and co-mingled mixtures of the two were transformed with binary DNA plasmid constructs that contained a neomycin phosphotransferase II (nptII) selection gene with a nos promoter and terminator. Explants included ~1-cm long laminae, petioles, internodes, nodes, and root sections from light- and dark-grown in vitro plants. Bacterial blight resistance genes were NPR1 from Arabidopsis, attacin from Hyalophora cecropia, and T4 lysozyme from the T4 bacteriophage. For nematode resistance, rice cystatin and cowpea trypsin inhibitor genes were used. Co-cultivation with Agrobacterium tumefaciens strains EHA105, AGLØ, and LBA4404 ranged from 2 to 14 days. Over 700 independent, putatively transformed lines were selected with 5 and 20 mg.L-1 geneticin (G418) for cultivars ‘Midori’ and ‘Marian Seefurth,’ respectively. Putative transgenic lines were selected 1 to 11.5 months, but on average 5.2 to 8.4 months, after co-cultivation depending on the tissue type transformed. Significantly more embryogenic calli (1 line per 5 mg calli) produced transgenic lines than did explants (1 line per 143 mg explants) (P < 0.004) from about 30 mg of tissue. Calli grew selectively from all explant types, but the type of explant from which each selection was made was not recorded since root, internode, and petiole explants were difficult to discern by the time calli developed. Shoots formed three months after calli were transferred to light. Non-transgenic control and transgenic ‘Marian Seefurth’ formed flower buds in the greenhouse about 28 months after co-cultivation. The plants resembled commercially grown plants from a private nursery. No non-transformed escapes were detected among the selections screened for NPTII by ELISA and PCR. The selections were positive for transgenes as assayed by PCR and Southern hybridizations. Southern blots showed single-copy insertions of the NPR1 regulatory gene. The ability to produce large quantities of independent transgenic lines from embryogenic calli in a relatively short time period should enable researchers to evaluate the effectiveness of any transgene by screening numerous anthurium lines for improved performance.