Abstract
Apple latent spherical virus (ALSV) vector is a convenient alternative to genetic transformation in horticultural plants, especially in species recalcitrant to genetic transformation. ALSV, an RNA virus, can infect a wide variety of plant species including major horticultural plants without inducing symptoms. Here, methodologies were developed for infection of ALSV vectors to strawberry seedlings and plantlets cultured in vitro. A seed-propagated F1 hybrid strawberry cultivar 'Yotsuboshi' was aseptically grown on half-strength Murashige–Skoog medium for 1 month and true leaves were inoculated with an ALSV RNA preparation by particle bombardment. ALSV vector infection rates varied from 58 to 100% according to the insertion sequences, in ‘Yotsuboshi’ seedlings. Plantlets (‘Dover’) propagated in vitro could also be infected with ALSV vector at a similar infection rate. For virus-induced gene silencing (VIGS), we prepared an ALSV vector carrying a 201 nucleotide segment of the strawberry phytoene desaturase gene. ‘Yotsuboshi’ and ‘Dover’ plants infected by this vector generated completely white leaves at fifth or sixth true leaves and above. For virus-induced flowering (VIF), we used an ALSV vector expressing the Arabidopsis thaliana flowering locus T gene. Strawberry seedlings infected by this vector started to flower from about 2 months post inoculation and bore fruits with viable seeds. The ALSV vector was no longer detected in any of the seedlings from early-flowered strawberries. Thus, the ALSV vector may be beneficial for examination of gene functions by VIGS in strawberry, and VIF using ALSV vector constitutes an effective new plant breeding technique for the promotion of cross-breeding in strawberry.
Highlights
Strawberry (Fragaria × ananassa) is a popular crop worldwide with production exceeding four million tons per year[1]
The amount of RNA per shot (7 μg) in this trial was greater than the standard amount (5 μg) for apple and gentian, and two shots were taken per each leaf, reverse transcription-polymerase chain reaction (RTPCR) analysis performed on upper leaves at 1 month post inoculation revealed that none of the 16 inoculated strawberry plants was infected by wtALSV
In our initial trial of Apple latent spherical virus (ALSV) inoculation to juvenile strawberry seedlings by particle bombardment, wtALSV never infected to soil-cultivated plants, whereas wtALSV infected to 89% of seedlings grown in vitro (Fig. 1; Table 1)
Summary
Strawberry (Fragaria × ananassa) is a popular crop worldwide with production exceeding four million tons per year[1]. Despite the long cultivation history of strawberries from the fiteenth century, strawberry breeding remains a major concern of farmers and researchers with regard to a variety of traits such as fruit quality (taste), fruit colour, fruit firmness, fruit storability, ascorbic acid content, everblooming habit, disease resistance and heat/ cold resistance[2]. Changes in these agronomically important traits are expected to be caused by mutations in genomic DNA. The genetic mapping approach has identified the FaOMT gene mutation as the regulator of variation in the release of mesifurane, one of the volatiles of strawberry fruit, based on complete cosegregation of the identified 30-bp mutation in the FaOMT promoter[4].
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