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. Currently, results do not look promising and future emphasis will be placed on transformations with new anti-microbial constructs. Anthurium cultivars transformed with D2A21 and D4E1 (anti-microbial peptides) with a strong monocot promoter have been regenerated on selection media and the presence of the transgene is being confirmed by PCR. To evaluate anthurium plants transformed for resistance to the burrowing nematode, Radopholus similis, two potted plant trials were conducted on lines transformed with cysteine and serine proteinase inhibitors. Anthurium plants were inoculated with burrowing nematodes on benches in a shadehouse. Nematode reproduction, plant growth, and root health were evaluated and compared with wild-type Midori and Marian Seefurth controls. Plants that exhibited resistance or tolerance were propagated by stem cuttings and grown out for additional screening. A cinder bed assay simulating grower conditions to evaluate flowering quality and yield under heavy nematode infestation was established. Stem height and flower size (length x width) are measured and compared to wild-type controls. The second cinder bed assay has also been initiated. Australian pineapples transformed for reduced Aminocyclopropane-1-carboxylic acid (ACC) synthase are being grown in the greenhouse to produce plants for field testing. Plants are being tested by PCR for the presence of the transgene and will be multiplied for slip production for field planting. We are working with the cooperator to establish field plots and applying for permits for field testing. Hawaii gold pineapple varieties continue to be transformed for reduced ACC synthase expression. We are incorporating the blemish or freckle free characteristics from line N08-75 with virus resistance from transgenic ‘SunUp’ to improve the marketability of Hawaii grown papayas. Currently, seeds from the second backcross to the freckle free line N08-75 are being made. We are currently selecting for progeny with freckle free blemishes and fruit taste similar or better than ‘Sun Up’. 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 near completion, this will allow further in house analysis and serves as a baseline for future gene mining experiments. In collaboration with 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 is being 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. The floral parts of the plant including the spadix and spathe in particular are unique to the Aroid family to which Anthurium belongs. 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. A transient method to test expression of putative genes in anthurium spathe tissue has also been developed. 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 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 to determine the potential for using the commercially available B. bassiana GHA strain as a control method for CBB in Hawaii. Initial results have revealed substantial reductions in CBB damage associated with monthly applications of Beauveria. Optimized dose and timing of the biological pesticide provides growers with a potential spray strategy for managing CBB disease on coffee. Modification to flowering to reduce the amount of green coffee cherries at the end of season is being tested by 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. 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. To reduce production costs, alternative carbon feedstocks have been evaluated and show promise as low-cost alternatives. We determined that C. protothecoides isolates are capable of robust cell growth and oil production in growth medium comprised of pH-adjusted puree of culled, waste papaya fruit without any additional growth supplements. Five strains from the UTEX collection were tested for growth on papaya medium. Optimization of culture medium and growth conditions were used for lab-scale strain characterization and demonstration of the potential for scale-up to an industrial bioprocess. An optimized growth medium composition of 35% papaya puree was determined. The most rapid cell doubling time observed was 6.6 hours and the maximum oil production rate observed was 1.9 g/L/day. Oil production of 21 g/kg papaya was achieved. Laboratory experiments utilizing waste glycerol as an additional feedstock commenced with samples received from Big Island Biodiesel.
1. Mystery of `Ohi`a death solved. `Ohi`a is Hawaii’s most common and widespread native tree, occurring from sea level to 2500 m elevation in both dry and wet forests and on substrates ranging from 50 to 4 million years in age. It is the most ecologically important native Hawaiian tree, defining native forest succession and ecosystem function over broad areas, providing critical habitat for rare and endangered native bird and insect species, and exemplifying the strong links between native Hawaiian culture and the islands’ environment. A newly detected disease has been killing large numbers of mature `Ohi`a trees in forests and residential areas of lower Puna and Hilo on Hawaii Island. ARS scientists in Hilo, Hawaii, partnered with the U.S. Forest Service and the University of Hawaii at Manoa, to determine what was killing the trees. Laboratory tests conducted by ARS scientists in Hilo, Hawaii, have identified the causal agent as the vascular wilt fungus Ceratocystis. This pathogen poses a serious threat to Hawaii’s flagship native tree species whose loss would be catastrophic for the diversity, structure, and function of Hawaii’s remaining native forests and the services they provide.
Suzuki, J.Y., Matsumoto Brower, T.K., Keith, L.M., Myers, R.Y. 2014. The chloroplast psbK-psbI intergenic region, a potential genetic marker for broad sectional relationships in Anthurium. HortScience. 49(10):1244-1252.
Ge, X., Matsumoto Brower, T.K., Keith, L.M., Li, Y. 2015. Fungal pretreatment of albizia chips for enhanced biogas production by solid-state anaerobic digestion. Energy and Fuels. 29:200-204.
Wang, K., Myers, R.Y., Srivastava, A., Sipes, B. 2015. Evaluating the predatory potential of carnivorous nematodes against Rotylenchulus reniformis and Meloidogyne incognita. Biological Control. 88:54-60.