Genetic engineering has the potential to improve disease resistance in taro [Colocasia esculenta (L.) Schott]. To develop a method to produce highly regenerable calluses of taro, more than 40 combinations of Murashige and Skoog (MS) media at full- or half-strength with varying concentrations of auxin [α-naphthaleneacetic acid (NAA) or 2, 4-dichlorophenoxyacetic acid (2, 4-D)], cytokinin [benzyladenine (BA) or kinetin], and taro extract were tested for callus initiation and plant regeneration. The best combination, MS medium with 2 mg·L−1 BA and 1 mg·L−1 NAA (M5 medium), was used to produce regenerable calluses from taro cv. Bun Long initiated from shoot tip explants. After 8 weeks of growth, multiple shoots from these calluses could be induced on MS medium with 4 mg·L−1 BA (M15 medium). The rice chitinase gene (ricchi11) along with the neomycin phosphotransferase (npt II) selectable marker and β-glucuronidase (gus) genes were introduced into these taro calluses through particle bombardment. Transformed calluses were selected on M5 medium containing 50 mg·L−1 geneticin (G418). Histochemical assays for beta-glucuronidase (GUS), polymerase chain reaction (PCR), reverse transcription–PCR, and Southern blot analyses confirmed the presence, integration, and expression of the rice chitinase gene in one transgenic line (efficiency less than 0.1%). Growth and morphology of the transgenic plants appeared normal and similar to non-transformed controls. In pathogenicity tests, the transgenic line exhibited improved resistance to the fungal pathogen, Sclerotium rolfsii, but not to the oomycete pathogen, Phytophthora colocasiae.
Xiaoling He, Susan C. Miyasaka, Yi Zou, Maureen M.M. Fitch and Yun J. Zhu
Xiaoling He, Susan C. Miyasaka, Maureen M.M. Fitch, Sawsan Khuri and Yun J. Zhu
Production of taro [Colocasia esculenta (L.) Schott], a tropical root crop, is declining in many areas of the world as a result of the spread of diseases such as Taro leaf blight (TLB). Taro cv. Bun Long was transformed through Agrobacterium tumefaciens with the oxalate oxidase (OxO) gene gf2.8 from wheat (Triticum aestivum). Insertion of this gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. One independent transformed line contained one gene insertion (g5), whereas a second independent line contained four copies of the gene. Reverse transcriptase PCR (RT-PCR) confirmed the expression of this gene in line g5. Histochemical analysis of the enzyme oxalate oxidase confirmed its activity increased in the leaves of line g5. A bioassay for resistance to TLB used zoospores of Phytophthora colocasiae to inoculate tissue-cultured plantlets. Transgenic line g5 showed the complete arrest of this disease; in contrast, the pathogen killed non-transformed plants by 12 days after inoculation. A second bioassay, in which spores of P. colocasiae were inoculated onto disks of leaves of one-year-old potted plants, confirmed that transgenic line g5 had greatly increased resistance to this pathogen. This is the first report to demonstrate that genetic transformation of a crop species with an OxO gene could confer increased resistance to a pathogen (P. colocasiae) that does not secrete oxalic acid (OA).
Maureen M.M. Fitch, Terryl C.W. Leong, Xiaoling He, Heather R.K. McCafferty, Yun J. Zhu, Paul H. Moore, Dennis Gonsalves, Herb S. Aldwinckle and Howard J. Atkinson
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 comingled 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. Cocultivation 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 cocultivation depending on the tissue type transformed. Significantly more embryogenic calli (one line per 5 mg calli) produced transgenic lines than did explants (one line per 143 mg explants) (P < 0.004) from ≈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 because root, internode, and petiole explants were difficult to discern by the time calli developed. Shoots formed 3 months after calli were transferred to light. Non-transgenic control and transgenic ‘Marian Seefurth’ formed flower buds in the greenhouse ≈28 months after cocultivation. The plants resembled commercially grown plants from a private nursery. No non-transformed escapes were detected among the selections screened for NPTII by enzyme-linked immunosorbent assay and polymerase chain reaction (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.