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: Anthurium plants transformed for bacterial blight and nematode resistance were received from our cooperators and assessed for disease resistance in pot assays and field trials. To assess the potential of these transgenic plants to manage disease and help to make Hawaii-grown anthuriums more marketable and competitive in the global market, we optimized and completed cinder bed trials of transformed lines of anthurium that showed tolerance to Xanthomonas axonopodis pv. dieffenbachia. Promising lines from earlier pot trials did not maintain their tolerance under field conditions. A second cinder bed trial which evaluated the resistance of transformed anthurium plants to burrowing nematode was completed. Lines showing tolerance in previous studies did not hold up in heavily infested field conditions. All inoculated plants had high levels of belowground necrosis and root rot. Stem height and flower size of transformed plants showed no significant difference from wild-type controls. Future emphasis will focus on in house transformations with improved anti-bacterial and nematode resistance genes with optimized expression in anthuriums. Anthurium plants transformed with D2A21 and D4E1 (anti-microbial peptides) with a strong monocot promoter have been molecularly confirmed by polymerase chain reaction (PCR). In vitro assays for bacterial blight resistance have identified transformed anthurium plants that suppress bacterial growth after inoculation. Hawaii gold pineapple varieties continue to be transformed for reduced 1-amino-cyclopropane-1-carboxylate synthase synthase expression to control pineapples flowering during the winter season. 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’. Select lines are being propagated by both tissue culture and seeds to develop a stable line for commercial production. Objective 2: To identify genes or genetic elements useful for improving commercially important traits in floral crops, we analyzed gene expression in four horticulturally important organs: the leaf, spathe, spadix and roots of Anthurium amnicola, a parental species of purple Anthurium cultivars. This research was completed through a collaboration with University of Hawaii at Manoa scientists. The sequence information, derived from ribonucleic acid sequencing, includes putative transcription factors and biosynthetic pathway genes of important ornamental traits such as floral pigments and floral volatiles. The method for accurate and sensitive gene expression detection via quantitative polymerase chain reaction (qPCR) was established in the lab, enabling expansion of more accessible PCR based gene expression studies. In collaboration with scientists at Rensselaer Polytechnic Institute, a method to screen genes to determine anthocyanin decorating activity in Escherichia coli was established to support characterization of relevant putative floral pigment biosynthesis genes. Progress has also been made toward optimizing the isolation of inhibition free Anthurium nuclei as a source for large, intact DNA to construct bacterial artificial chromosome, genomic DNA libraries from which candidate promoter DNA regions will be isolated and identified. Organ specific promoters would be valuable for regulating expression of important ornamental traits, for example, in the spathe and spadix, organs specific to Anthurium and other members of the Araceae family. In collaboration with University of Hawaii at Manoa scientists, collection of Anthurium species and heritage cultivars continues as a means to ensure availability of germplasm for future growers, researchers and breeders. 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 at multiple locations to determine the potential for using the commercially available B. bassiana strain GHA as a control method for CBB in Hawaii. 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. Results from these locations have shown that Beauveria spray combined with strip picking (sanitation) provide excellent control of CBB, to levels beneath 5 percent infestation. The monitoring and spray regimes have been adopted by numerous growers. To aid sanitation efforts to reduce CBB populations between coffee seasons, we have developed plant growth regulator applications to modify flowering. Studies conducted over multiple seasons have shown that application of the plant growth regulator treatments are able to reduce the coffee berries left on the trees between seasons. Although large scale application of the plant growth regulator, gibberellic acid, on mechanically harvested fields is able to decrease variation in the coffee berry development, this treatment alone has not been able to compensate for the non-selectivity of the mechanical harvest. Different pruning methods are currently being explored to further optimize the gibberellic acid sprays. In addition to crop management practices, additional revenue streams from culled fruits that would normally be discarded as waste would also help the farmer’s income. A zero-waste approach makes agriculture more profitable and addresses food and energy security issues in Hawaii. Research continued to optimize/increase production scale of Chlorella protothecoides for oil and animal feed production using papaya as a feedstock. A 40-gallon Bioreactor was installed at the ARS facility in Hilo and results from lab scale have been successfully replicated. Demonstration phase production was initiated as a collaboration between ARS scientists, the Hawaii Department of Agriculture Agribusiness Development Corporation and Big Island Biodiesel. The scale-up of the demonstration phase has resulted in multiple successful 150-gallon, 7-day runs of C. protothecoides and papaya in 270-gallon reactors. The cell counts from these experiments were measured and found comparable to small scale experiments done at by ARS scientist in Hilo.
1. Microbial production of the plant specialty pigment, peonidin. Improving ornamental traits such as flower color requires understanding of complex, multistep, biosynthetic pathways or networks. The decorative anthocyanin known as peonidin imparts a purple hue and is a commercially important flower trait in numerous ornamentals, including tropical floral plants; however, there is still much unknown about the properties of numerous enzymes required for this trait. A collaboration between scientists at Rensselaer Polytechnic Institute, the University of Hawaii at Manoa, and ARS scientists in Hilo, Hawaii, demonstrated for the first time, the production of peonidin from inexpensive precursors and relevant plant pigment pathway genes expressed in the microbe Escherichia coli. Modern gene editing techniques were used to optimize peonidin production by engineering the host E. coli, to produce increased levels of S-adenosyl-L-methionine, a necessary metabolite for the synthesis of peonidin. This study provides a quick and reliable method to study the function of plant genes involved in complex metabolic pathways of important pigments, fragrances and flavors.
2. Determination of expressed genes from a key tropical floral species by ribonucleic acid (RNA) sequencing. Floriculture crops are important in the U.S with a production value of $4.37 billion and retails sales at $31.3 billion for 2015. However, few molecular, genetic and germplasm resources for improvement of these economically important crops exist. A collaboration between ARS scientists in Hilo, Hawaii, and scientists at the University of Hawaii at Manoa, documented for the first time, expression levels of floral pigment biosynthesis pathway genes including anthocyanin, carotenoid, chlorophyll and putative gene regulating transcription factors in four distinct organs of Anthurium amnicola. Results from this study are an important step to identify key components to improve floral pigment and various ornamental traits and also serves as a foundation for understanding differences in physiology, function and formation of different organs in plants within the Aroid family which includes anthurium and food crops such as taro, an important crop for food secutiry in tropical regions.
3. 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. Research conducted by ARS scientists in Hilo, Hawaii, have optimized/increased culture conditions to optimize oil and/or protein production of Chlorella protothecoides using culled papaya as a feedstock. This technology has been transferred to a demonstration project in collaboration with Hawaii Department of Agriculture Agribusiness Development Corporation and Big Island Biodiesel. The scale-up of the demonstration phase has resulted in multiple successful 150-gallon, 7-day runs of C. protothecoides and papaya in 270-gallon reactors with cell counts comparable to small scale experiments conducted by ARS scientists in Hilo. This project validates the next step in the commercialization of a technology that utilizes agricultural waste to develop new products to make farmers more profitable and addresses food and energy security issues, especially important in Hawaii.
4. Rapid Ohia Death is not caused by Ceratocystis from ornamental Syngonium plants. Rapid Ohia Death (ROD) is the cause of widespread mortality of ohia. Ohia is the most ecologically important native tree on Hawaii Island. Greenhouse and growth chamber inoculation experiments on ohia seedlings and saplings have proven that two new species of the Ceratocystis fimbriata complex causes ROD. Several genotypes of C. fimbriata were already present in Hawaii, affecting sweet potato, taro, and Syngonium, a commonly grown ornamental plant. The possibility that C. fimbriata from Syngonium at Hawaii Island nurseries was the culprit of ROD was examined by ARS scientists in Hilo, Hawaii, using pathogenicity tests, cross-host inoculations, and phylogenetic analysis. Results from these studies indicated that no direct link exists between C. fimbriata from syngonium and ohia, thus exonerating the nursery industry from any contribution to the most virulent invasive fungal species in recent years.
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