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).
The disease resistance of a transgenic line expressing the coat protein (CP) gene of the mild strain of the papaya ringspot virus (PRSV) from Hawaii was further analyzed against PRSV isolates from Hawaii and other geographical regions. Line 63-1 originated from the same transformation experiment that resulted in line 55-1 from which the transgenic commercial cultivars, `Rainbow' and `SunUp', were derived. Plants of line 63-1 used in this study consisted of a population from a self pollinated R0 bisexual plant. ELISA and PCR tests provided evidence that there are at least two segregating CP loci. To allow for comparison with reactions of the previously reported line 55-1, virus isolates from Hawaii, Brazil, Thailand, and Jamaica were used to challenge seedlings of 63-1. Unlike line 55-1, a significant percentage of inoculated transgenic plants were susceptible to isolates from Hawaii. However, a proportion of plants were resistant to the non-Hawaiian isolates. In contrast, previous work showed that all plants of the hemizygous line 55-1 were susceptible to PRSV isolates from Brazil, Thailand, and Jamaica. Line 63-1, therefore, presents Hawaii with PRSV-resistant transgenic germplasm that could be used as a source of transgenes for resistance to PRSV isolates within and outside of Hawaii.
Papaya (Carica papaya L.) cultivars and breeding lines were evaluated for resistance to Enterobacter cloacae (Jordan) Hormaeche & Edwards, the bacterial causal agent of internal yellowing disease (IY), using a range of concentrations of the bacterium. Linear regression analysis was performed and IY incidence was positively correlated with increasing inoculum concentrations for susceptible cultivars Kapoho Solo and Laie Gold but not for resistant cultivars or lines. It was determined that the inoculum concentration of 9 to 10 Log10 colony-forming units per milliliter (cfu/mL) was able to reliably differentiate resistant and susceptible papaya germplasm. Red-fleshed cultivars SunUp and Sunrise were the most resistant papaya groups evaluated at this dose concentration. Yellow-fleshed cultivars, Kapoho Solo and Laie Gold, were susceptible to E. cloacae. ‘Rainbow’, an F1 hybrid between ‘SunUp’ and ‘Kapoho Solo’ that is yellow-fleshed, was moderately resistant to E. cloacae, exhibiting limited symptoms of the disease. Yellow-fleshed I-Rb F5/F6, an advanced inbred line derived from ‘Rainbow’, is resistant and offers the potential of improving resistance of yellow-fleshed commercial cultivars. A colorimeter was used to objectively measure internal flesh color and distinguish between infected and non-infected tissue in red- and yellow-fleshed papayas using L*C*H* color space analysis. Symptomatic tissue (72.4 and 79.0°) had higher hue angle means than non-symptomatic tissue (62.8 and 75.0°) for all cultivars or lines in red- and yellow-fleshed papayas, respectively. Yellow (“Y”) hue color also distinguished infected tissue from non-infected tissue. Symptomatic tissue that had Y hue color resulted in 79 to 81° hue angle means among red- or yellow-fleshed papayas. Our results demonstrated the usefulness of colorimetry to help detect infected papaya tissue. In surveys of naturally infected papaya, high populations (8.57 × 107 cfu/g) of E. cloacae were recovered in infected fruit of ‘Kapoho Solo’ and represent a food safety concern for fresh and processed papaya. In isolations from inoculated fruits, we observed decreases of ≈1 to 2 Log10 cfu/g in final bacterial populations when high-dose range inoculum concentrations (9 to 12 Log10 cfu/mL) were used. This dose range may represent a saturation range for E. cloacae inoculation.
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.
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.