Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue

Hosokawa, Munetaka; Matsushita, Yosuke; Uchida, Hiroshi; Yazawa, Susumu

doi:10.1016/j.jviromet.2005.07.001

Cited by 49 publications

(29 citation statements)

References 14 publications

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“…The youngest expanded leaves of each plant, at two and four months after the start of LP-free SAM culture, were used for CSVd assay by micro-tissue (MT) direct RT-PCR (Hosokawa et al, 2006). To evaluate CSVd resistance in the 85 cultivars, we counted the numbers of plants in which CSVd was detected by MT-direct RT-PCR in each of two tests performed two and four months after LP-free SAM culture.…”

Section: Csvd Inoculation By the Lp-free Sam Culture Methodsmentioning

confidence: 99%

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Screening of Chrysanthemum Cultivars with Resistance to Chrysanthemum Stunt Viroid

Nabeshima

Hosokawa

Yano

et al. 2012

J. Japan. Soc. Hort. Sci.

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Cultivars resistant to chrysanthemum stunt viroid (CSVd) are desirable for stable production of chrysanthemum (Chrysanthemum × morifolium Ramat.). We previously reported that CSVd was absent not only from shoot apical meristems (SAMs) but also from leaf primordia (LP) of resistant plants following CSVd inoculation. Using this characteristic as a phenotypic marker, we could identify several resistant cultivars in this study. CSVd was inoculated directly into the SAMs of 85 commercial cultivars by attaching SAMs to infected root tips in vitro, and the cultivars were tested for the presence of CSVd in newly expanded leaves. Of the 85 cultivars, 20 resistant candidates could be identified. These plants were classified into two types according to their CSVd infection characteristics: a CSVd-uninfected or slow titer-increasing type and a CSVd-disappearance type. Scions of four of the 20 candidate cultivars were grafted to CSVd-infected rootstocks for evaluation of the CSVd titer in newly expanded leaves. Although CSVd was detected at high titer in two candidate cultivars over the two-month testing period, 'Sei no Issei' and 'Mari Kazaguruma', other two candidate cultivars, proved to be resistant cultivars. In 'Sei no Issei', CSVd was detected temporarily after grafting, but titers decreased in the newly expanded leaves, while CSVd was not detected in a shoot tip of an infected plant and only at trace levels in a young leaf. 'Mari Kazaguruma' also proved to be a resistant cultivar with a slow titer increase. The different types of CSVd resistance in chrysanthemum cultivars will contribute to CSVd-resistant breeding. With some modifications, the in vitro screening method established here will be available for broadening the diversity of genetic resources resistant to CSVd.

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Section: Csvd Inoculation By the Lp-free Sam Culture Methodsmentioning

confidence: 99%

“…MT direct RT-PCR was performed according to Hosokawa et al (2006). A syringe needle (25G × 1, Terumo, Japan) was used to pierce the youngest expanded leaves once in a random location, and plant sap on the syringe needle dipped into the RT mixture was used for the RT template.…”

Section: Mt Direct Rt-pcrmentioning

confidence: 99%

Screening of Chrysanthemum Cultivars with Resistance to Chrysanthemum Stunt Viroid

Nabeshima

Hosokawa

Yano

et al. 2012

J. Japan. Soc. Hort. Sci.

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“…Detection of begomovirus from tomato plants grown in a greenhouse was conducted using microtissue direct polymerase chain reaction (PCR) according to Hosokawa et al (2006). Briefly, DNA-A of begomovirus was amplified using BM-V and BM-C primers (Briddon and Markham, 1994).…”

Section: Detection Of Begomovirus Using Polymerase Chain Reactionmentioning

confidence: 99%

Production of <i>Tomato Yellow Leaf Curl Virus</i>-free Parthenocarpic Tomato Plants by Leaf Primordia-free Shoot Apical Meristem Culture Combined with <i>in vitro</i> Grafting

et al. 2015

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Tomato yellow leaf curl virus (TYLCV) infections result in decreased tomato growth and reduced yields, and the production is almost entirely lost if plants are infected during early growth. 'Kyo-temari' is the commercial name for the parthenocarpic tomato 'MPK-1', which has been vegetatively propagated and distributed to local farmers in Kyoto City for commercial cultivation. During the winter of 2013, the typical yellow leaf curl symptoms of TYLCV were observed in 10 parthenocarpic tomato cultivars, including 'MPK-1', maintained as mother stock for vegetative propagation at Kyoto University. When microtissue direct polymerase chain reaction was conducted, a begomovirus-specific amplicon was detected in the plants with yellow leaf curl symptoms. Sequencing and phylogenetic analysis clarified that a TYLCV-Mild isolate was infecting the parthenocarpic tomatoes. Because signals of TYLCV were not detected in the shoot apical meristems (SAMs) of TYLCV-infected 'MPK-1' by in situ hybridization, elimination of TYLCV was conducted by regenerating plants from leaf primordia (LP)-free SAMs of parthenocarpic tomato cultivars. By combining the LP-free SAM culture and in vitro grafting, TYLCV-free plants were obtained in approximately three months. The technique developed in this study will contribute to the efficient elimination of TYLCV from vegetatively propagated parthenocarpic tomatoes.

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“…The PCR products were double-digested with XhoI and BamHI and ligated in frame with the coding sequences for the movement and Vp25 capsid proteins flanking the clon- by the amplification of NbPDS-ALSV, could be used as a good indicator to estimate the movement and amplification of wtALSV and ParPDS-ALSV. Successful viral infection was determined by the microtissue direct RT-PCR method 2-3 weeks post inoculation (wpi) according to Hosokawa et al (2006) with slight modifications. PCR amplification was performed with a program of 32 cycles of 95°C for 20 sec, 58°C for 15 sec, and 72°C for 45 sec with an initial heating at 95°C for 3 min using the primer pair ALSV-F and ALSV-R (Table 1) corresponding to nucleotide positions 1396-1421 and 1573-1598 of ALSV RNA2 (AB030941), respectively.…”

Section: Amplification Of Alsv Vectors In N Benthamianamentioning

confidence: 99%

Virus-induced Gene Silencing in Apricot (Prunus armeniaca L.) and Japanese Apricot (P. mume Siebold ^|^amp; Zucc.) with the Apple Latent Spherical Virus Vector System

Kawai

Gonoi

Nitta

et al. 2014

J. Japan. Soc. Hort. Sci.

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Apple latent spherical virus (ALSV) vectors have been shown to effectively induce stable virus-induced gene silencing (VIGS) in a wide range of plant species, including rosaceous fruit tree species such as apple (Malus × domestica Borkh.), pear (Pyrus communis L.), and Japanese pear (P. pyrifolia Nakai). In this study, we attempted to develop a VIGS-based gene evaluation system for two Prunus fruit tree species, apricot and Japanese apricot, using ALSV vectors. A partial sequence of the P. armeniaca PHYTOENE DESATURASE (ParPDS) gene was cloned and ligated into the T-DNA region of a binary vector, pBICAL2, designed based on RNA2 of ALSV. The resultant pBICAL2-ParPDS was introduced into a disarmed Agrobacterium strain, EHA105. pBICAL1, a binary plasmid for the expression of ALSV RNA1 in plants, was also introduced into EHA105. Leaves of Nicotiana benthamiana were infected with pBICAL1/EHA105 and pBICAL2-ParPDS/EHA105 simultaneously to produce and amplify recombinant ALSV particles. The amplified ParPDS-ALSV in N. benthamiana was isolated and infected into the cotyledons of apricot and Japanese apricot seedlings by particle bombardment. Although our attempts to infect wild and recombinant ALSVs into Japanese apricot seedlings were unsuccessful, uniform discoloration of the upper leaves, a typical phenotype of PDS knock down, was observed several weeks after inoculation in apricot seedlings. We discuss the possible use of this VIGS-based gene evaluation system in Prunus.

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Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue

Cited by 49 publications

References 14 publications

Screening of Chrysanthemum Cultivars with Resistance to Chrysanthemum Stunt Viroid

Screening of Chrysanthemum Cultivars with Resistance to Chrysanthemum Stunt Viroid

Production of <i>Tomato Yellow Leaf Curl Virus</i>-free Parthenocarpic Tomato Plants by Leaf Primordia-free Shoot Apical Meristem Culture Combined with <i>in vitro</i> Grafting

Virus-induced Gene Silencing in Apricot (Prunus armeniaca L.) and Japanese Apricot (P. mume Siebold ^|^amp; Zucc.) with the Apple Latent Spherical Virus Vector System

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