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.
Papaya seedlings segregate for sex expression as females or hermaphrodites. Typically only hermaphrodite fruit are marketed in Hawaii. The agronomic practice of growing multiple seedlings that are later thinned to a single hermaphrodite tree is wasteful of seed, labor, and resources, especially when seed is costly. We compared growth of plants propagated by the clonal methods of micropropagation or rooting vegetative cuttings versus plants initiated as seedlings and transplanted. The seedlings were either single-planted hermaphrodites as identified by the polymerase chain reaction (PCR) or multiple-planted, thinned seedlings. The experiments were carried out in three different locations on two islands in Hawaii. Clonally propagated plants were significantly shorter than seedlings and bore flowers earlier and lower on the trunk at all locations. Stem diameter differences were not significant even though plant size was different at planting time. Percentage of trees in bud varied significantly in the third month after transplanting when about 90% of the rooted cuttings and large micropropagated plants had formed flower buds while only one multiple-planted seedling developed a bud. Overall, the clonally propagated plants were more vigorous and earlier bearing than were the seedling plants. There is good potential for adoption of clonal propagation when production becomes efficient enough to compete in price with the current practice of over planting and thinning.
Gynodioecious papaya (Carica papaya L.) seedlings in commercial cropping systems in Hawaii are typically multiple-planted and thinned upon flowering to a single hermaphrodite because seedlings segregate for sex expression. Use of clonally propagated hermaphrodites would eliminate the over-planting practice and may provide other advantages. Yields of clonally propagated hermaphrodites were compared with single- and multiple-planted seedlings in three fields on two islands in Hawaii. Cloned hermaphrodites were either rooted cuttings or in vitro micropropagated plants. Clonally propagated plants bore ripe fruit 1 to 3 months earlier than thinned seedlings and had significantly higher early and cumulative yields. At each site, cumulative yields of thinned seedlings never reached the same level as those of clonally propagated plants. The yield benefit from clonally propagated plants was greatest at Keaau, the lowest sunlight and least productive test site.