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:
Our objective is to isolate and characterize papaya sex determination gene(s) that can be used to develop true breeding hermaphrodite lines which will reduce costs of fruit production; this directly contributes to objective 1 of the in-house project. We generated ca. 14,000 chemically induced papaya mutants with the goal of discovering plants with reversed sex. A single male-to-hermaphrodite sex reversal mutant was found. This mutant was propagated via tissue culture and planted in soil where it flowered. Flowers were bagged for self-pollination to produce homozygous lines. Leaves were harvested for genomic DNA sequencing and flower buds were used for gene expression analyses. The expression levels of several potential sex determination genes are being analyzed. A technique called TILLING allowed us to identify 18 mutants for two virus-resistance genes. The DNA base changes in nine of these mutants resulted in amino acid differences that may affect protein function. We are preserving these mutants through hand pollination and creating double mutants for virus resistance testing. Forty-one putative transgenic lines were regenerated from calli bombarded with 11 expression, or silencing, constructs of candidate sex determination genes. All of the first 17 lines tested were verified as carrying the selectable markers gene. Two lines have been transplanted in pots. A new papaya transformation method involving sonication of pollen with DNA constructs is being tested. Approximately 15,000 seeds were collected from 23 fruits pollinated with sonicated pollen. These represent about half of the pollination done so far. Among the first 11,000 seeds planted, 30 seedlings survived kanamycin spraying indicating they may be transformed. The presence of transgenes will be verified molecularly. A field trial permit for this material was granted in April. The first field planting is planned in August after the putative transgenic seedlings from the sonication method are verified and additional transgenic lines are transferred to soil. Suppression of Flowering Suppression of flowering will increase biomass production of sugarcane and other biofuel grasses and can be important for containment of transgenic or weedy germplasm. We HARC Annual Progress Report: 10/1/11 – 9/30/12 July 27, 2012 regenerated 12 independent lines of the model grass Brachypodium transformed for suppression of the flowering time (FT) gene. Four lines were obtained under geneticin selection and eight under hygromycin selection. Proof that these lines are transgenic was verified by amplification of the selectable marker genes. These 12 lines were multiplied in tissue culture and transplanted in soil. Two of the transgenic lines showed delayed flowering. Non-transformed wild type plants flower at three weeks after germination. The transgenic lines have been in tissue culture since September 2011, and were transplanted in April 2012. Only one or two plants from these two transgenic lines had a few flowers by June 2012. Leaf samples were collected from all transgenic and control plants for RNA extraction to see the expression level of FT. Disease Resistance Five sets of potential pathogen-inducible genes and promoters were identified from the papaya genome sequences. These potential pathogen-inducible promoters were subcloned into a binary transformation vector containing the green florescence protein (GFP) as a visual selection marker. Genetic transformations were carried out in Arabidopsis and papaya to verify the functionality of these promoters. Several transformed Arabidopsis lines were obtained. GFP was visualized in different plant organs and was quantified using qRT-PCR. These lines are being selfed to obtain homozygous plants that will be evaluated for disease resistance. Eight molecular markers associated with resistance or tolerance of papaya to Phytophthora palmivora, were obtained by screening the F2 progeny of the tolerant cv. Kamiya crossed to the susceptible cv. SunUp. Seven of the eight makers were sequenced. The sequence data was blasted against the papaya genome database to characterize the potential resistance genes. Four candidate genes that mapped near the two most significant markers were selected for further evaluation. These genes encode a hypersensitive-induced reaction protein and a lipoxygenase involved in signal transduction. These molecular markers have potential for use in marker-assisted-selection to produce tolerant or resistant cultivars. Wildtype Arabidopsis is non-host to the papaya pathogen P. palmivora; however, specific mutants of Arabidopsis are susceptible to this pathogen, indicating the mutated Arabidopsis genes play a role in non-host defense. Arabidopsis mutants for four selected non-host candidate genes were purchased to study their defense functions against Phytophthora. The Arabidopsis mutant plants were selfed and seedlings grown and verified as homozygous. The Arabidopsis knockout mutants had partial loss of resistance to P. palmivora which provides data to enable search for homologs of these genes in papaya. Proteomic profiles of differentially expressed proteins of resistant Kamiya and susceptible SunUp to pathogen inoculation could reveal mechanisms of host interaction with P. palmivora. Twenty-nine differentially expressed proteins were identified and their roles in papaya defense were further evaluated for their RNA expression levels by quantitative RT-PCR. Our results confirmed differential expression in these cultivars. Five thousand genes of cv. SunUp that differentially responded to P. palmivora infection were analyzed by microarrays. Several pathways, such as Jasmonic acid-induction pathway, were identified by advanced genetic software to have significant roles in stress and disease response. Virus and Drought Resistance Transgenic sugarcane plants were produced with resistance to the sugarcane yellow leaf virus (SCYLV). Greenhouse assays of transgenic lines to inoculation with SCYLV were quantified using PCR. Plant growth data and sucrose concentration were also conducted. Transgenic lines maintained a good sugar concentration and had SCYLV resistance comparable to that of the most resistant cultivars. A drought resistant gene, IPT, under a stress-inducible promoter, was cloned into a transformation vector and used to transform sugarcane. Eighty-five transformed lines were obtained. The presence and expression levels of IPT were verified by PCR and gene screen hybridizations. Each independent line was multiplied in tissue culture and some were potted in soil. Plants in tissue culture were used in a salt stress assay while those in soil were used in a water withholding experiment. The pot assays suggest that these transformed plants better withstand drought and salinity. Sugarcane Breeding/Variety Development Eight energycane varieties were transferred from USDA-ARS Houma to HARC. All varieties flowered at the Maunawili Station. Five of the eight varieties were used for biparental and polycrosses with Hawaiian commercial and energycane varieties. High biomass progeny were selected at seven months from plantings on Maui. Thirteen individual F1s selected from crosses involving Ho06-9002 and HoCP72-114 were transferred to Maunawili for making crosses. Two hundred seventy-six S. officinarum accessions were received from USDA-ARS Miami as a backup for the Miami world collection. Two hundred thirty-seven clones came out of quarantine and are being maintained. Coffee Genomics Maintained mapping population of Arabica coffee, Tall Mokka MA2-7 x Catimore 5175-1, at HARC’s Kunia field. The population includes 169 F1 trees and 300 F2 trees derived from 2 F1 trees. Molecular genetics analyses of this population are being conducted by a collaborator. Bioenergy Crops Continue to collect monthly yield data on high yielding selections of jatropha. These selections are producing twice the yield at half the age of unselected trees on an area basis. Some of the higher yield could be due to a wider spacing of the plants allowing sunlight capture but at least part of it is due to increased yield potential. The top performing varieties of the 14 selected lines will be crossed to develop new higher yielding and more drought tolerant varieties. Pruning trials showed a decrease in yield from 2010 to 2011. New trials have been started to determine the cause. This second pruning trial with 20 new plots in the same field will evaluate a fourth pruning on mature trees to see if limited light capture could be the cause. Water use and selection for drought tolerance continues. Selected drought tolerant lines are being used for breeding. A male sterile tree was propagated and is being used as a parent to develop lines with more fruit. Several hybrids including a non-toxic Mexican variety have been planted for field evaluations. Plant uniformity was achieved using seed grown in isolated plots. Plant disease evaluations have begun in an isolation area on trees exhibiting abnormal characteristics including the lack of new growth in over six months, a spotted stem surface, rotting branches, and occasional tree death. Seventy-five trees from Isolation 2 and its offspring are exhibiting the same symptoms which indicates genetic uniformity of the material. The Kumar Oil Expeller has been repaired and will be used to press several hundred pounds of seed. The oil will be converted to biodiesel through a partnership. Top performing high fiber grass types identified in a grow-out at Kunia were transplanted to Ka`u for field trials. Yield data was collected in February and June. The top six varieties in that location are being evaluated in a more highly replicated yield trial. The best performing variety(s) is expected to be planted in a large scale trial.