1a. Objectives (from AD-416):
The objectives of this SCA are: 1) to characterize transgenic line 63-1 for its potential as papaya ringspot virus (PRSV) resistant breeding stock, and 2) to characterize hybrids from transgenic SunUp and a nontransgenic Sunrise selection with excellent appearance and shipping quality.
1b. Approach (from AD-416):
Objective 1. Transgenic line 63-1 is resistant to PRSV and has been deregulated in the US. Unlike line 55-1, which is the parent of the commercial Rainbow papaya, line 63-1 has two coat protein gene inserts and shows broad resistance to other strains of PRSV. Evidence from genetic crosses indicates that the two coat protein transgene inserts segregate independently. Line 63-1 will be characterized for its border sequences in order to clearly distinguish the coat protein gene inserts, and will be used as a crossing parent to broaden the resistance of transgenic lines. Objective 2. The relatively short shelf-life and speckled surface of the transgenic SunUp papaya make it difficult for Hawaii to effectively export papaya to the mainland US east coast especially. A nontransgenic Sunrise papaya selection was identified to have good shelf-life and lacked fruit blemishes. The papaya will be crossed to transgenic SunUp papaya and the progenies evaluated for PRSV resistance, good shelf life, and lack of fruit blemishes. Promising progenies will be further crossed and evaluated.
3. Progress Report:
One objective is the analysis of transgenic papaya line 63-1; this understanding directly contributes to objective 1 of the in-house project. Transgenic line 63-1 is resistant to papaya ringspot virus and was deregulated by APHIS and EPA, but was not further pursued for commercialization because it had large seqments of vector sequences. However, it has two coat protein transgenes and thus is an attractive model for basic studies to explore the impact that these coat protein transgenes would have resistance to papaya ringspot virus. The first step was to determine the host border sequences of the transgenes. Border sequences were obtained from a flanking region of one protein transgene. Analysis of the border sequence showed that the border sequences were of the papaya genomic type DNA. These sequences will help towards locating the coat protein transgene insertion within the papaya genome as our work progresses on deep sequencing of the transgenic -papaya genome. The second objective is to develop transgenic papaya with less fruit blemishes. Fruits of the blemish free papaya (presumably ‘Golden Sunrise’) were obtained from a Trader Joe’s market in California and seeds of the fruit were grown on the Hamakua coast of Hawaii; this line was designated N08-75. Fruit from those plants showed similar characteristics to the commercial variety and indeed were blemish free. The trees were self pollinated and harvested seeds were also grown in the Waiakea region of Hawaii Island that resemble the ‘lava’ type of soil and moisture conditions of Puna, where 90% of Hawaii’s papaya are grown. The resulting fruit of these trees were also free of freckles. To incorporate PRSV-resistance to the line, the freckle free line was crossed with ‘SunUp’, which is a PRSV-resistant transgenic cultivar that was derived by transforming a nontransgenic ‘Sunset’, a sibling line to ‘Sunrise’. Recipricol crosses were made using ‘SunUp’ and N08-75 and seeds of the cross (N08-75 (female) x ‘SunUp’ (male)) have been planted out in Waiakea and also in Puna at a commercial papaya farm. Tracking of the PRSV-resistance of these plants can easily be done by GUS staining since the coat protein transgene segregates with the GUS marker gene. Incorporating PRSV resistance is a prerequisite to practical improvement of papaya in Hawaii since PRSV is the limiting factor for papaya production. Furthermore, a large advantage of the above approach is that the PRSV-resistant ‘SunUp’ and N08-75 share the same lineage. Thus, it is expected that the crosses between ‘SunUp’ and the N08-75 will result in fruits of similar characteristics.