Jon Y. Suzuki

The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus)

Papaya, a fruit crop cultivated in tropical and subtropical regions, is known for its nutritional benefits and medicinal applications. Here we report a 3× draft genome sequence of ‘SunUp’ papaya, the first commercial virus-resistant transgenic fruit tree to be sequenced. The papaya genome is three times the size of the Arabidopsis genome, but contains fewer genes, including significantly fewer disease-resistance gene analogues. Comparison of the five sequenced genomes suggests a minimal angiosperm gene set of 13,311. A lack of recent genome duplication, atypical of other angiosperm genomes sequenced so far, may account for the smaller papaya gene number in most functional groups. Nonetheless, striking amplifications in gene number within particular functional groups suggest roles in the evolution of tree-like habit, deposition and remobilization of starch reserves, attraction of seed dispersal agents, and adaptation to tropical daylengths. Transgenesis at three locations is closely associated with chloroplast insertions into the nuclear genome, and with topoisomerase I recognition sites. Papaya offers numerous advantages as a system for fruit-tree functional genomics, and this draft genome sequence provides the foundation for revealing the basis of Carica’s distinguishing morpho-physiological, medicinal and nutritional properties.

Development of Genetically Engineered Resistant Papaya for papaya ringspot virus in a Timely Manner

Papaya orchards throughout most of the world are severely damaged by the destructive disease caused by the papaya ringspot virus (PRSV). PRSV-resistant papaya expressing the coat protein gene (CP) of PRSV have been used in Hawaii to control PRSV since 1998. This chapter presents the experimental steps involved in the development of transgenic papaya, including transgene construction, transformation, and analysis for virus resistance of the transformed papaya. We also describe the important factors that enabled deregulation, commercialization, and adoption of transgenic papaya to occur in Hawaii in a timely manner. Transfer of this technology to other countries with the similar goal and the development of transgenic papaya in other regions of the world also are described.

Allergenicity assessment of the papaya ringspot virus coat protein expressed in transgenic rainbow papaya.

The virus-resistant, transgenic commercial papaya Rainbow and SunUp (Carica papaya L.) have been consumed locally in Hawaii and elsewhere in the mainland United States and Canada since their release to planters in Hawaii in 1998. These papaya are derived from transgenic papaya line 55-1 and carry the coat protein (CP) gene of papaya ringspot virus (PRSV). The PRSV CP was evaluated for potential allergenicity, an important component in assessing the safety of food derived from transgenic plants. The transgene PRSV CP sequence of Rainbow papaya did not exhibit greater than 35% amino acid sequence homology to known allergens, nor did it have a stretch of eight amino acids found in known allergens which are known common bioinformatic methods used for assessing similarity to allergen proteins. PRSV CP was also tested for stability in simulated gastric fluid and simulated intestinal fluid and under various heat treatments. The results showed that PRSV CP was degraded under conditions for which allergenic proteins relative to nonallergens are purported to be stable. The potential human intake of transgene-derived PRSV CP was assessed by measuring CP levels in Rainbow and SunUp along with estimating the fruit consumption rates and was compared to potential intake estimates of PRSV CP from naturally infected nontransgenic papaya. Following accepted allergenicity assessment criteria, our results show that the transgene-derived PRSV CP does not pose a risk of food allergy.

Genome size in Anthurium evaluated in the context of karyotypes and phenotypes

Little is known about the genome of Anthurium other than chromosome observations, which frequently indicate supernumerary (“B”) chromosomes. New genome size estimates for 34 species and nine cultivars presented here provide insights into genome organization and evolution in this very large genus.

Chemotaxonomy of Hawaiian Anthurium cultivars based on multivariate analysis of phenolic metabolites.

Thirty-six anthurium varieties, sampled from species and commercial cultivars, were extracted and profiled by liquid-chromatography-mass spectrometry (HPLC-MS). Three hundred fifteen compounds, including anthocyanins, flavonoid glycosides, and other phenolics, were detected from these extracts and used in chemotaxonomic analysis of the specimens. Hierarchical cluster analysis (HCA) revealed close chemical similarities between all the commercial standard cultivars, while tulip-shaped cultivars and species displayed much greater chemical variation. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) supported the results from HCA and were used to identify key metabolites characteristic of standard and tulip cultivars and to identify chemical markers indicative of a particular ancestry. Discriminating metabolites included embinin, 4, which was characteristic of standard-shaped spathes and indicated ancestry from Anthurium andraeanum, while isocytisoside 7-glucoside, 7, was found in the majority of tulip-shaped cultivars and suggested that Anthurium amnicola or Anthurium antioquiense had contributed to their pedigree.

Screening promoters for Anthurium transformation using transient expression

Key messageThere are multiple publications onAnthuriumtransformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem.AbstractDifferent promoters and tissue types were evaluated for transient β-glucuronidase (GUS) expression in Anthuriumandraeanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.

Assaying for Pollen Drift from Transgenic ‘Rainbow’ to Nontransgenic ‘Kapoho’ Papaya under Commercial and Experimental Field Conditions in Hawaii

In 1992, papaya ringspot virus (PRSV) was discovered in the Puna district of Hawaii island where 95% of the state of Hawaii’s papaya was being grown. By 1998 production in Puna had decreased 50% from 1992 levels. A PRSV-resistant transgenic papaya ‘Rainbow’ containing the coat protein gene of PRSV was released commercially in Hawaii in 1998, and saved the papaya industry from further devastation. In the ensuing years since the release of the transgenic papaya, a number of farmers grew hermaphrodite nontransgenic ‘Kapoho’ papaya in close proximity to plantings of hermaphrodite transgenic ‘Rainbow’ papaya. These plantings provided a unique opportunity to assay for transgenic-pollen drift under commercial conditions. Between 2004 and 2010, assays for the GUS (beta-glucuronidase) transgene in embryos were done to study transgenic-pollen drift in commercial ‘Kapoho’ plantings and in replicated field plots. Very low pollen drift (0.8%) was detected in fruit of ‘Kapoho’ trees in the border row of one plantation when 90 embryos were assayed per fruit, while no pollen drift was detected in four other commercial plantings in which eight embryos were tested per fruit. Pollen drift averaged 1.3% of tested embryos in field plots where individual hermaphrodite ‘Kapoho’ trees were adjacent to two or four ‘Rainbow’ trees. In contrast, 67.4% of tested embryos were GUS positive in similarly located female ‘Kapoho’ trees. The very low transgene flow to close-by ‘Kapoho’ plantings is likely due to the fact that hermaphrodite trees are used commercially in Hawaii and that these trees are largely self-pollinated before the stigma is exposed to external pollen.

Organ-Specific Transcriptome Profiling of Metabolic and Pigment Biosynthesis Pathways in the Floral Ornamental Progenitor Species Anthurium amnicola Dressler

Anthurium amnicola Dressler possesses a number of desirable and novel ornamental traits such as a purple-colored upright spathe, profuse flowering, and floral scent, some of which have been introgressed into modern Anthurium cultivars. As a first step in identifying genes associated with these traits, the transcriptome from root, leaf, spathe, and spadix from an accession of A. amnicola was assembled, resulting in 28,019 putative transcripts representing 19,458 unigenes. Genes involved in pigmentation, including those for the metabolism of chlorophyll and the biosynthesis of carotenoids, phenylpropanoids, and flavonoids were identified. The expression levels of one MYB transcription factor was highly correlated with naringenin 3-dioxygenase (F3H) and dihydroflavonol-4-reductase (DFR) in leaves, whereas a bHLH transcription factor was highly correlated with flavonoid 3′-monooxygenase (F3′H) and a DFR in spathes, suggesting that these two transcription factors might regulate flavonoid and anthocyanin synthesis in A. amnicola. Gene sequence and expression data from four major organs of A. amnicola provide novel basal information for understanding the genetic bases of ornamental traits and the determinants and evolution of form and function in the Araceae.