1a. Objectives (from AD-416):
Objective 1: Develop and assess transgenic plants to control plant growth and development, disease resistance, and shelf life. [NP 301, C1, PS 1A and NP 301, C3, PS, 3A] Sub-objective 1a: Screen and select transgenic and/or non-transgenic anthuriums for resistance/tolerance to bacterial blight. Sub-objective 1b: Develop and evaluate an improved screening method for determining resistance and tolerance to burrowing nematodes in anthurium. Sub-objective 1c: Develop and screen transgenic anthurium lines with improved gene constructs for resistance/tolerance to bacterial blight and burrowing nematodes. Sub-objective 1d: Develop management options to control flowering of pineapple under Hawaii conditions. Sub-objective 1e: Obtain PRSV-resistant Hawaiian papaya that have reduced amount of fruit blemishes through recurrent selection. Objective 2: Identify genes or genetic elements useful for improving horticulturally and commercially important traits in floral crops. [NP 301, C3, PS 3A] Sub-objective 2a: Identification of pathways and molecular components for floral color improvement of anthurium. Objective 3: Improve horticultural characteristics and cultivation practices of subtropical and tropical crops. [NP 305, C1, PS 1B1] Sub-objective 3a: Develop improved practices for coffee production to mitigate the damage of coffee berry borer (CBB). Sub-objective 3b: Develop improved practices for coffee production that will serve as an industry model. Sub-objective 3c: Develop improved practices for sustainable, tropical/subtropical, diversified crop production that will serve as an industry model for zero waste.
1b. Approach (from AD-416):
1) Use genetic engineering to develop anthurium that are resistant or tolerant to bacterial blight and nematode pests. Vegetatively propagate the best lines and initiate a large screening trial of the few selected bacterial tolerant lines in a cinder bed trial to simulate commercial conditions. Evaluate the impact of plant resistance or tolerance on the nematode (reproduction) and the impact of the nematode on the plant (flower yield, plant growth). Develop new screening methods for long term yield evaluations. Develop transformation protocols in house (PBARC) and use new antimicrobial peptides for bacterial resistance and a combination of cystatin and plant proteinase inhibitors for nematode resistance; optimize for expression in monocots/anthuriums. 2) Use genetic engineering to develop pineapple that are resistant to natural flowering. Develop a robust transformation system and a realistic and effective screening regime for natural flowering. 3) Develop color enhanced cultivars of anthurium through the identification of key pigment pathways and subsequent genetic engineering for transferring the traits. Develop more comprehensive molecular biological and biochemical tools to fill the information gap required for supporting improvement of other commercially important traits and a cooperative resource for cultivar development with the University of Hawaii and industry growers. 4) Mitigate the impact of coffee berry borer through effective use of a biological control agent (Beauveria bassiana). Determine viability and persistence of Beauveria spores on coffee cherries, measure Beauveria infection rates in infested coffee berries and monitor infestation levels from field counts of CBB-infested and uninfested coffee berries. 5) Mitigate the impact of coffee berry borer by using plant hormones to synchronize coffee flowering. Define a method to time flowering and “schedule” harvesting for efficient distribution of labor for hand harvested areas and optimize flowering to one or two major flowering times for mechanically harvested areas. 6) Develop virus resistant Hawaiian solo papaya with blemish-free fruit through recurrent selection. Evaluate fruit and either backcross or self lines to increase freckle free phenotype; select fruits with the lowest amount of freckles, desirable fruit shape, high total soluble sugars and good fruit quality. Determine shipping longevity of freckle free fruit.
3. Progress Report:
Anthuriums suffer from bacterial blight and nematodes. Anthuriums that were transformed to be bacterial blight resistant are being screened and screening methods were improved. Directly inoculating the transgenic anthurium plants on a greenhouse bench rather than incubating them in a humid chamber has allowed us to screen twice as many plants at one time. In addition, a modified bench inoculation method was developed to improve the rate and progression of infection. Within four weeks all test plants become infected and showed systemic infection similar to plants that were incubated for 24 hours in a humid chamber. This inoculation method provided a more efficient way to inoculate a large number of plants and reduced the length of the trials. Anthurium lines that showed increased tolerance to bacterial blight in initial screens were multiplied for greenhouse and field testing. Anthurium cultivars are being transformed with D2A21 and D4E1 genes and promoter combinations are being evaluated for cystatin and combined nematode and bacterial resistance. A containment trial was initiated to evaluate barriers to prevent burrowing nematode movement in anthurium greenhouses. Chemical pesticides were tested against burrowing nematode in anthurium in a potted plant bioassay. In collaboration with researchers at the Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, a quantitative biochemical “fingerprint” of floral pigments (anthocyanins) and related compounds in Anthurium cultivars of different colors and select species was obtained through application of liquid chromatography mass spectrophotometry (LC-MS) analysis. Baseline, reference data and quantitative information on Anthurium floral pigment levels in cultivars as well as parental species are documented to support breeding programs aimed at improving Anthurium floral color traits through a better understanding of the relationship between biochemical pathways, pigment composition, color and inheritance. Polymerase chain reaction (PCR) amplification of specific DNA segments from the Anthurium chloroplast genome was investigated as one approach to develop molecular markers to distinguish different Anthurium species. One chloroplast DNA molecular marker was identified as a useful diagnostic tool for identifying closely related Anthurium species based on PCR product size similarity. Development of diagnostic tools to track molecular or genetic similarities and differences between Anthurium species and their inheritance patterns in hybrids is ongoing to support development of cultivars with superior horticultural or ornamental traits and for identity preservation or verification of accession or cultivar integrity in germplasm collections. Anthurium cultivars in addition to Marion Seefurth and Midori have been placed into culture, disease indexed and multiplied for transformation. Australian pineapples with reduced Aminocyclopropane-1-carboxylic acid (ACC)synthase are being grown in the greenhouse to produce plants for field testing. We are working with the cooperator to obtain permits for field testing. Hawaii gold pineapple varieties are being transformed for reduced ACC synthase expression. Reduced freckles were observed in crosses between SunUp and the freckle free papaya and are currently being tested in a commercial papaya field. Coffee berry borer (CBB) was discovered in the Kona coffee growing region of Hawaii and the industry is currently facing its toughest challenge and is desperately seeking ways to control this problem. Field studies were initiated to determine the persistence and efficacy of the B. bassiana GHA strain and the potential for using the commercially available GHA strain as a control method for CBB in Hawaii. Results indicate that monthly Beauveria sprays contribute to a decrease in infestation rates and reductions in CBB damage. This information provides growers in various locations of Kona with a potential spray strategy for managing CBB on coffee. Plant growth regulator treatments are being tested on a semi-commercial scale to improve harvest and sanitation practices. Coffee berry borer damage to the berry versus the coffee bean is being evaluated to determine the effect of Integrated Pest Management (IPM) in commercial coffee fields. A zero waste approach makes agriculture more profitable and addresses food and energy security issues in Hawaii. Data were collected to evaluate heterotrophic growth and oil yield of select Chlorella protothecoides accessions from the University of Texas Culture Collection of Algae (UTEX) utilizing waste papaya as a feedstock. The four selected strains showed robust growth and accumulated oil to approximately 0.3-1.0 grams per liter of culture volume under the conditions tested.
1. Tropical biogas wastes evaluated for biogas production. Anaerobic digestion (AD) is technology that utilizes locally abundant biomass wastes from agricultural operations or invasive plants into biogas which can be used to produce heat, electricity, and transportation fuels. Tropical biomass wastes, including albizia leaves, albizia chips, taro skin, taro flesh, papaya, and sweet potato, were evaluated by ARS researchers in Hilo, Hawaii, in collaboration with scientists at Ohio State University, as feedstock for methane (energy) potential by anaerobic digestion (AD). In current studies, these tropical biomass wastes were evaluated for biogas production by liquid anaerobic digestion (L-AD) and/or solid-state anaerobic digestion (SS-AD), depending on feedstock characteristics. The AD system showed potential for treating different tropical food wastes, including individual feedstock and their mixtures. Hawaii relies heavily on imported fuel which results in high cost of energy.
Cabos, R.Y.M., K.H. Wang, B.S. Sipes, W.P. Heller, T.K. Matsumoto. 2013. Detection of plant-parasitic nematode DNA in the gut of predatory and omnivorous nematodes. Nematropica. 43:44-48.
Ge, X., T. Matsumoto Brower, L. Keith, Y. Li. 2014. Biogas energy production from tropical biomass wastes by anaerobic digestion. Bioresource Technology. 169:38-44.