1a. Objectives (from AD-416):
Produce new knowledge of molecular biology, genetics, and crop traits of selected fruit, vegetable, and ornamental crops grown in Hawaii, and preserve selected sugarcane germplasm that is more suitable for growing in Hawaii. Use genetic engineering approaches to enhance the disease resistance of ornamentals such as anthurium and orchids. Develop improved germplasm for the nation’s sugarcane industry through increased biomass and/or sugar, increased resistance to abiotic stress, pathogens, and pests. Evaluate the horticultural characteristics of Jatropha, kukui, and other tropical crops for their potential as biofuel when grown in Hawaii. Assess the potential commercial application and the potential environmental and biosafety risks of transgenic plants that are developed.
1b. Approach (from AD-416):
Continue tissue culture multiplication of transgenic anthurium lines for subsequent screening by PBARC for bacterial and nematode resistance. Use HARC land to grow selected sugarcane lines that cannot be consistently maintained at the sugarcane clonal repository in Miami, Florida. Cross lines of material selected by the national sugarcane centers and distribute seeds from these crosses for their environmental selection for production of sugar and biomass. Cross lines of target crops to produce mapping populations for developing molecular markers associated with important agronomic traits. Use laboratory and field based approaches to evaluate and develop value added products from potential tropical biofuel crops, such as sugarcane, Jatropha, and kukui. Work with PBARC to develop risk mitigating gene constructs that consists of short-linked segments of genes or computer-generated consensus sequences derived from sequences of a family of genes or gene segments, and use them to develop transgenic plants, such as papaya, for resistance to pathogens and enhanced agronomic traits. Documents SCA with Hawaii Agriculture Research Center (HARC); formerly 5320-21000-011-01S (5/18/087).Formerly 5320-21000-011-11S (11/10).
3. Progress Report:
This is the final report for this project. The goal of this collaborative project to develop genetic approaches for improvement of selected crops grown in Hawaii and is directly related to objectives 1 and 2 of the inhouse parent project, "Develop the genomics of papaya for producing new knowledge about the regulation of transgenic disease resistance", and, "Develop methods for improved manipulation and expression of transgenes in key tropical/subtropical ornamental and fruit crop species". Papaya Papaya trees have flowers bearing either male, or female, or both male and female sex organs (hermaphrodite flowers). Hermaphrodite trees are preferred because of their higher productivity and fruit quality. Papaya sex types do not breed true; only 2/3 of the seed from hermaphrodite fruit produces hermaphrodite plants and the other 1/3 produces other sexes that must be chopped from the field to maximize productivity. The practice of planting excess seedlings that are later culled results in inefficient use of time, labor, water, and fertilizer. We identified and cloned papaya’s sex determination genes that will be used to genetically alter superior varieties into true breeding hermaphrodite lines. Towards this goal we genetically mapped the sex determination location, physically mapped and fine sequenced this region, cloned candidate genes, and are in the process of proving the function of these candidate genes. Hawaii Agriculture Research Center (HARC) participated in a multi-institutional collaboration that produced a draft genome sequence of papaya, the first sequenced fruit tree. We produced bacterial artificial chromosome (BAC) libraries that were used to construct a genome physical map to assist whole genome shotgun sequence assembly and integration with the genetic map. These data were used to identify chromosomal regions containing the sex determination genes and to clone the major genes controlling fruit flesh color. After the BAC clones in the male-specific Y chromosome (MSY) and its corresponding female-specific X regions were sequenced, the BAC sequences were joined into two large pseudomolecules for comparing male, female and hermaphrodite sequences. The male sequences had fewer sites of methylation indicating a relatively fast mutation rate compared to its corresponding female X region. More than 14,000 plants of two cultivars of papaya were chemically mutagenized and grown in the field to verify candidate sex determination genes via reverse genetics. One mutant showing partial male-to-hermaphrodite sex reversal phenotype was identified and sequencing of its MSY region is underway. Forty-one transgenic lines were generated to verify candidate sex determination genes identified from the MSY region. The sex types of the transgenic plants will be identified once they flower. Eight molecular markers associated with resistance or tolerance of papaya to its major pathogen, Phytophthora palmivora, were identified by screening F2 progeny of a cross between tolerant ‘Kamiya’ and susceptible ‘SunUp’. Seven of the eight makers were sequenced and the data blasted against the papaya genome database to identify potential resistance genes. Proteomic profiles of ‘Kamiya’ and ‘SunUp’ roots after inoculation with P. palmivora were used to identify proteins associated with papaya response to this rot pathogen and to predict the responsible genes. Twenty-nine differentially expressed proteins were identified and their roles in papaya defense to pathogens are being studied. Over 20 variants of Papaya RingSpot Virus (PRSV) disease reduces papaya production worldwide. Plants transformed for resistance to multiple variants were produced. Seventy-eight lines were confirmed by molecular analysis to contain transgenes. These lines will be transplanted to field trials for evaluation of their performance and reaction to the prevailing strains of PRSV. Sugarcane Polymerase chain reaction (PCR) quantification of sugarcane yellow leaf syndrome virus (SCYLV) indicated that there may be more than one strain in Hawaii with differential capacity to elicit disease symptoms. Sequencing of SCYLV collected from a field at Hawaiian Commercial & Sugarcane Company (HC&S), Maui and from the HARC Substation on Oahu confirmed the variability and differential pathogenicity of SCYLV in Hawaii and suggested how the virus strains may have evolved. Technologies to improve the genetic transformation efficiency and expression of transgenes were developed for sugarcane. These technologies include engineering gene constructs with promoters for higher levels of expression in specific organs and for reducing the silencing of these genes by the transformed plant. The P0 protein of the Sugarcane Yellow Leaf Virus was evaluated for its ability to suppress transgene silencing. Continued breeding to produce sugarcane cultivars for Hawaii and the mainland and selection of progeny for desired agronomic traits across contrasting environmental zones. Produced hybrids from several interspecific crosses to develop molecular marker mapping populations of S. officinarum, S. robustum, and S. spontaneum. Conducted yield trials of extreme segregants of F2 individuals in biomass to develop DNA markers for high biomass for bioenergy. Thirty-one high biomass Saccharum species and their hybrids, and related grasses, e.g. banagrass (Pennisetum) were planted at Kunia for preliminary biomass evaluations while increasing planting materials for bioenergy field trials. Erect clones with high biomass were selected from the preliminary trial and planted for bioenergy feedstock field production trials on the islands of Maui and Hawaii. Results of this trial will be used in future screening to determine the best varieties for Hawaii’s diverse environments. Suppression of flowering will improve biomass production of sugarcane and other biofuel grasses. Flowering suppression is also important for containment of weedy species or transgenic plants. Flowering Locus T (FT) encodes a mobile signal, called florigen, that is produced in leaves and transported to the short apex to induce flowering. Silencing FT expression is expected to suppress flowering of grasses. We developed thirteen lines of transgenic plants, two of which showed reduced flowering. Bioenergy crops Germplasm of potential biofuel perennial crops (Jatropha, Kukui, and Moringa) for initial field evaluations and eventual breeding for agronomic improvement was collected. Kukui, Hawaii’s candlenut tree, was so slow-growing that no data were collected for this species as a potential oil crop. Moringa seed oil content was measured as 29.6% by weight but the oil potential of this crop is problematic because of the difficulty in harvesting seed from such a large tree. Therefore, work focused on Jatropha from six locations; India, Madagascar, Honduras, Mexico, Oahu, and Hawaii. Several high yielding varieties of Jatropha were evaluated in replicated yield trials. Trees from seed obtained in isolation plots showed uniformity of branching and height, both are important for mechanical harvesting of seed. Jatropha flowered year round with heavy bursts in early summer and early fall and produced fruits within 6 months after planting in all test plots. In a test comparing irrigation rates, the highest yielding plots produced enough seed to yield ~56 gallons of oil per acre after 14 months. Selection for superior trees showed accessions from India and Madagascar had highest productivity. The Honduras accessions showed significant genetic variation in leaf size, fruit cluster size and seed size and appears to hold the most potential for long-term crop improvement, especially with the identification of a male-sterile tree. Yield trials showed that early stage aggressive pruning delayed fruit production and did not lead to higher yields. At least seven varieties showed promise for future breeding. Three isolated plots were established including; a supposed ‘non-toxic’ strain of Jatropha from Mexico, a high oil content variety from China, and a male-sterile variety from Honduras. These traits are agronomically important and will be improved by breeding. Anthurium The two most important disease and pest problems restricting the production of anthuriums are a bacterial disease known as blight and susceptibility to root nematodes. These two problems greatly restrict yield of cut flowers and increase the grower costs by reducing the length of a crop cycle. Although there are some cultivar differences in susceptibility to blight and nematodes, the leading varieties have very little tolerance so that increased resistance is needed. The only way to obtain increased resistance is through genetic transformation. We produced more than 800 transformed anthurium lines that were shipped to Hilo for field testing for response to bacteria blight and nematodes. About 200 lines were screened for blight tolerance producing 5 promising lines that will be re-tested and 60 new lines will be tested soon. Twenty-five lines with improved tolerance to blight were selected for further testing. Nematode tolerance testing was conducted by the ARS station Pacific Basin Research Center (PBARC). Lines found to be tolerant to either nematodes or Phytophthora rot will be multiplied and provided to the growers to replace their currently produced susceptible lines. Pineapple F1 populations of pineapple cultivars F153 (Smooth Cayenne) and Hana64 (spiny) were planted for mapping of genes associated with leaf spines. Crosses were made to generate true F2 populations. Approximately 700 F2 plants were plated in the field. Transcriptome sequencing of the two parents was completed. The sequences will be analyzed to develop markers that will be used for mapping.