Zelkova sinica Schneid. is a popular landscape plant in China because of its wide adaptation, strong disease resistance, large crown, and beautiful fall color. Immature embryos from Z. sinica seeds were cultured on woody plant medium (WPM) supplemented with 4.5 μM 6-Benzylaminopurine (BA) and 5.4 μM α-naphthaleneacetic acid (NAA) to induce callus, and 60% of immature embryos formed callus. The cream-white, friable, nodular callus with proembryogenic structures was then cultured on WPM containing 5.4 μM NAA in combination with 9.0 or 11.2 μM BA to regenerate shoots; approximately five shoots per explant were induced on 70% callus. Shoots were rooted on WPM containing 0.5 μM indole-3-butyric acid (IBA), on which 62.3% shoots developed roots with an average of 4.2 roots per shoot at 4 weeks. The regenerated plantlets were acclimatized and transplanted into the field. This protocol could be used for mass production for field plantation, genetic improvement, and germplasm exchange of Z. sinica.
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.
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).
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.
In Hawaii, the commercial papaya industry is based on cultivars that segregate as females or hermaphrodites. Multiple seedlings are planted and then thinned at flowering to single hermaphrodites at each site. The aim of this study was to increase propagation efficiency by improving our procedure for micropropagation of hermaphrodite plants only. Initially, shoots were multiplied in vented jars on M2 medium, a Murashige and Skoog formulation containing 0.25 μM 6-benzyladenine (BA) and 0.1 μM α-naphthalene acetic acid (NAA). At weekly intervals, micropropagated shoots were either incubated for 4 to 7 days in IBA2 medium containing 20 μM indole-3-butyric acid (IBA) or were dipped in autoclaved rooting powder containing 0.8% IBA (DIP); then, they were placed in M2 until root initials or small roots were visible. After root induction in both treatments, plants were transferred to an in vitro medium containing ½ MSO and 30 g⋅L−1 sucrose in vermiculite (VER). The IBA2 treatment produced 467 potted plants compared to 475 produced by the DIP treatment; however, the average number of days that each treatment required from root induction to potting of rooted plants was not significantly different (IBA2: 52.42 ± 5.65 days; DIP: 51.94 ± 3.61 days). Plants from both treatments were grown in either wet potting medium (500 mL water/300 g potting medium) or damp potting medium (120 mL water/300 g potting medium) to test the effect of moisture content on plant survival and growth after potting. Use of damp rather than wet potting medium resulted in significantly higher plant survival and growth. These results could facilitate more efficient commercial practice for papaya growers.