Li Chunjiang, Yamagishi Noriko, Kasajima Ichiro, Yoshikawa Nobuyuki
1Faculty of Agriculture, Iwate University, Morioka 3-18-8, Iwate, 020-8550 Japan.
2Agri-Innovation Research Center, Iwate University, Morioka 3-18-8, Iwate, 020-8550 Japan.
Hortic Res. 2019 Feb 1;6:18. doi: 10.1038/s41438-018-0106-2. eCollection 2019.
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 F 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 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 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.
苹果潜隐球形病毒（ALSV）载体是园艺植物遗传转化的一种便捷替代方法，尤其是对于那些难以进行遗传转化的物种。ALSV是一种RNA病毒，可感染包括主要园艺植物在内的多种植物物种，且不引发症状。在此，我们开发了将ALSV载体感染草莓幼苗和离体培养苗的方法。以种子繁殖的F1杂交草莓品种‘Yotsuboshi’在半强度Murashige-Skoog培养基上无菌培养1个月，然后通过粒子轰击用ALSV RNA制剂接种真叶。在‘Yotsuboshi’幼苗中，根据插入序列不同，ALSV载体感染率在58%至100%之间。离体繁殖的组培苗（‘Dover’）也能以相似的感染率被ALSV载体感染。对于病毒诱导的基因沉默（VIGS），我们制备了携带草莓基因201个核苷酸片段的ALSV载体。被该载体感染的‘Yotsuboshi’和‘Dover’植株在第五或第六片真叶及以上完全变白。对于病毒诱导的开花（VIF），我们使用了表达该基因的ALSV载体。被该载体感染的草莓幼苗在接种后约2个月开始开花，并结出有活力种子的果实。在早花草莓的任何幼苗中都不再检测到ALSV载体。因此，ALSV载体可能有利于通过VIGS在草莓中检测基因功能，并且使用ALSV载体的VIF构成了一种有效的新植物育种技术，可促进草莓的杂交育种。
