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:
Progress has been made on all three objectives of this project. Objective 1: We developed and assessed transgenic plants to control plant growth and development, disease resistance, and shelf life. Bacterial blight and nematodes are the two most important diseases in anthuriums. Improved screening methods for anthurium bacterial blight resistance have been achieved for bench pots trials and cinder bed trials. Utilizing these improved methods, anthurium lines that showed increased tolerance to bacterial blight in initial screens were multiplied for greenhouse and field testing. To assess the potential for transgenic plants to manage disease and help to make Hawaii-grown anthuriums more marketable and competitive in the global market, we evaluated cinder bed trials of transformed lines of anthurium that showed tolerance to Xanthomonas axonopodis pv. dieffenbachia. Selected lines cloned and propagated were not substantially different than control plants, suggesting that these lines are not bacteria resistant. Current and future emphasis will be placed on transformations with new anti-microbial constructs. Anthurium cultivars transformed in parallel with the ß-Glucuronidase (GUS) reporter gene or D2A21 and D4E1 (anti-microbial peptides) with a strong monocot promoter are being multiplied. Evaluation of the GUS transformants indicates strong expression of the GUS reporter in all tissues tested including leaves, stems and roots. To evaluate anthurium plants transformed for resistance to the burrowing nematode, Radopholus similis, the second cinder bed assay simulating grower conditions was initiated to evaluate flowering quality and yield under heavy nematode infestation. Stem height and flower size (length x width) were measured and compared to wild-type controls. No significant difference in flower size and quality was observed among the lines. Promising lines from earlier bioassays did not maintain their tolerance to R. similis in field conditions with high nematode populations. Australian pineapples transformed for reduced Aminocyclopropane-1-carboxylic acid (ACC) synthase are being grown in the greenhouse to produce plants for field testing. Since the permitting process has been delayed, alternate methods to test flowering within the confines of the greenhouse are being explored. Additional Hawaii gold pineapple varieties continue to be transformed for reduced ACC synthase expression to reduce precocious flowering. To improve the marketability of Hawaii grown papayas we are combining the appearance of the blemish or freckle free characteristics from line N08-75 with the taste and virus resistance from transgenic ‘SunUp’. Seeds from backcrosses between the hybrids and crosses of select line of Kapoho to confer the yellow flesh are being evaluated in cooperator fields. Objective 2: We are identifying genes or genetic elements useful for improving horticulturally and commercially important traits in floral crops. General annotation of the Anthurium amnicola transcriptome is complete and submitted for publication. In collaboration with a scientist at the University of Hawaii at Manoa, the Anthurium transcriptome project is focusing upon anthocyanin and related flavonoid biosynthesis genes that influence floral pigment. To validate the function of a putative, unique anthocyanin biosynthetic enzyme with potential biotechnological applications, a collaborative project has been established with scientists at Rensselaer Polytechnic Institute. We have identified a number of tissue specific transcripts and are currently validating tissue specific expression as well as gene and transcript prevalence in related species and cultivars. Northern analysis has proved difficult for tissue quantitation due to low RNA extraction yields from Anthurium. Methods to measure the relative abundance of the transcripts (qPRC) are being refined to enable quantitative and sensitive gene expression detection and comparison across tissue types. The floral parts of the plant, including the spadix and spathe in particular, are unique to the Aroid family to which Anthurium belongs and no spadix sequence from Anthurium have been published nor spathe or spadix specific gene identified in any aroid. Methodologies for preparation of large MW DNA, which is difficult in Anthurium due to high levels of polyphenolics, are being worked out to enable bacterial artificial chromosome (BAC) library construction for subsequent promoter isolation and analyses. Thus, tissue specific promoters identified from these experiments represent invaluable, previously unavailable biotechnological tools for transgene expression in Anthurium. Constructs that have been successfully used to modify floral color in other species are being transformed into anthuriums. Objective 3: We are improving horticultural characteristics and cultivation practices for coffee and improving sustainable production practices. Coffee berry borer (CBB) was discovered in the Kona coffee growing region of Hawaii and on Oahu in December of 2014. Current recommended control methods include monitoring for CBB through traps or field surveys, pesticide application such as the entomopathogenic strain of Beauveria bassiana, and field sanitation to remove CBB reservoir between seasons. Beauveria persistence and efficacy studies have been conducted at multiple locations to determine the potential for using the commercially available B. bassiana GHA strain as a control method for CBB in Hawaii. Data from these locations over multiple years suggest a strong correlation between environmental conditions and Beauveria persistence. The Beauveria spray regimes and management practices on CBB infestation are being documented on cooperator farms located at different microclimates throughout the Kona coffee region. To aid sanitation efforts to reduce CBB populations between coffee seasons, we have developed plant growth regulator applications to modify flowering. Multiple year studies have shown that applications of the plant growth regulator treatments are able to reduce the amount of green coffee berries left on the trees between seasons. To further this optimization we are looking at pruning methods in addition to plant growth regulator treatments, especially in the Ka`u region of the Big Island where coffee does not have a distinct season. Microalgae derived oils have outstanding potential for use in biodiesel production. Chlorella protothecoides has been shown to accumulate lipids up to 60% of its cellular dry weight with glucose supplementation under heterotrophic growth conditions. Five strains of C. protothecoides from the University of Texas (UTEX) collection were optimized to grow on medium containing 35% papaya puree. In the lab, we observed a rapid cell doubling time of 6.6 hours and maximum oil production rate of 1.9 g/L/day. Oil production of 21 g/kg papaya was achieved in the lab. Currently, these methods are being transferred to a demonstration scale operation (>100 gal). Laboratory experiments utilizing waste glycerol as an additional feedstock commenced with samples received from Big Island Biodiesel.
1. Mini-pilot scale production of biofuel and animal feed from heterotrophic algae. Agricultural waste represents a significant portion of lost revenue for farmers. Microalgae derived oils have outstanding potential for use in biodiesel production and as animal feed. Chlorella protothecoides has been shown to accumulate lipids up to 60% of its cellular dry weight with glucose supplementation under heterotrophic growth conditions. Research conducted by ARS scientists in Hilo, Hawaii, have optimized/increased culture conditions to optimize oil and/or protein production of C. protothecoides using culled papaya as a feedstock. This technology has been transferred by ARS to a demonstration project in collaboration with Hawaii Department of Agriculture Agribusiness Development Corporation and Big Island Biodiesel. This project utilizes agricultural waste to develop new products to make farmers more profitable and addresses food and energy security issues, especially important in Hawaii.
5. Significant Activities that Support Special Target Populations:
Myers, R.Y., Sipes, B.S., Matsumoto Brower, T.K., Mello, C.L., Mello, J.S. 2015. Occurrence and distribution of Heterorhabditid populations in the Hawaiian Islands. Nematropica. 45:198-207.
Matsumoto Brower, T.K., Suzuki, J.Y., Hollingsworth, R.G., Keith, L.M., Tripathi, S. 2016. Protection and coexistence of conventional papaya productions with PRSV resistant transgenic papaya. Acta Horticulturae. 1111:49-54.
Mortenson, L., Hughes, F., Friday, J., Keith, L.M., Barbosa, J., Friday, N., Liu, Z., Sowards, T. 2016. Assessing spatial distribution, stand impacts and rate of Ceratocystis fimbriata induced 'Ohi'a (Metrosideros polymorpha) mortality in a tropical wet forest, Hawai'i Island, USA. Forest Ecology and Management. 377:83-92.