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J. People Plants Environ > Volume 19(1); 2016 > Article
Choi, Cho, Goo, Lee, Kang, Jeoung, and Chung: Virus Identification Using RT-PCR Method from Meristem Cultured and Ribavirin Treated Plantlet for Producing FreMV-free Freesia ‘Shiny Gold’

ABSTRACT

Freesia is one of the most popular flowers over the world including Korea, due to the fragrance and beauty of the plant flower. The first domestic freesia cultivar ‘Shiny Gold’ was developed by NIHHS, RDA, in 2003, which has yellow double and large petals and strong fragrance. Ten years have passed since ‘Shiny Gold’ was cultivated at floral farms, and the deterioration of cut flower quality and yield are reported from the farms. Virus infection causes a reduction in the quantity and quality of the cut freesia flowers and is one of the most serious problems in Korea. Virus detection was carried by reverse transcription polymer chain reaction (RT-PCR) for FreMV, FreSV, BYMV, CMV, and TRV, as known to infect freesia. FreMV, FreSV, BYMV, and TRV were detected single or multiply, and CMV was not found in the freesia leaves collected from the farms. To produce virus-free freesia, meristem culture of ‘Shiny Gold’ was conducted in MS medium added ribavirin at different concentration. As the increased of ribavirin concentration, the growth of ‘Shiny Gold’ plantlets was inhibited in freesia’Shiny Gold’. The plantlets produced by meristem culture in ‘Shiny Gold’ were virus free at the enzyme-linked immunosorbent assay (ELISA) level.

Introduction

Freesia (Freesia hybrida), one of the bulb flower, is originated from South Africa and approximately 800 cultivars of freesia have been developed since the 19th Century (Wang 2006; Bala and Berecici 2010). Totally 41 freesia cultivars with various colors and shapes were developed in National Institute of Horticultural and Herbal Science (NIHHS), Rural Development Administration (RDA) so far; ‘Shiny Gold’, ‘Bolero’, ‘Shiny Lemon’, ‘Pretty Woman’, ‘Dancing Flame’ and ‘Shiny Bell’ for cut-flower, and ‘Tiny Gold’ and ‘Shy Smile’ for potted flowers (Lim et al., 2004; Cho et al., 2008, 2010, 2011, 2013; Choi et al., 2014). Freesia is recently getting attention as a plant that of heating energy reduction for farmhouses, with the minimum temperature for cultivation at the below 10°C of the winter season in Korea (Cho et al., 2007; MAFRA 2014). Freesia cv. ‘Shiny Gold’, the most popular domestic freesia cultivar, has yellow double petals and strong fragrance (Lim et al., 2004). Ten years have passed since the ‘Shiny Gold’ cultivated in the field, the reduction of the cut flower quality and quantity by virus infection are reported.
Totally 5 viruses reported to infect freesia: Freesia mosaic virus (FreMV), Freesia sneak virus (FreSV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), and Tobacco rattle virus (TRV) (Derks and Vink-van den Abeele, 1987; Brunt, 1995; Vaira et al., 2006; Kumar et al., 2009; Choi et al., 2010; Choi et al., 2013b). FreMV, which belong to potyvirus genus, was first discovered from Freesia refracta and reported in Netherlands, 1954, and it was also reported in Australia, England, India and Korea (Van et al., 1954; Brunt 1995; Kumar et al., 2009; Choi et al., 2010). Moreover, whole genome sequence and structure of strain FreMV-Kr isolated in Korea were reported (Choi et al., 2010). FreSV, transmitted by zoospores of Olpidium brassicae, which is soilborne fungus belong to the family ophioviridae, that was also recently reported in Korea (Yoon et al., 2013). Nucleotide and amino acid sequence of FreSV coat protein was analyzed and registered on the NCBI database, showing 30-70% homology to those of other ophioviruses reported so far (Navarro et al., 2005, Yoon et al., 2013). FreSV-infected freesia plants have shown light yellow mosaic patterns and necrotic purple speckles on leaves. Some plants revealed unclear mosaic patterns, or the leaves are deformed, distorted and bended, poor growth and abnormal flowering (Derks and Vink-van den Abeele, 1987; Choi et al., 2013). To produce high-quality cut freesia, it is required the breeding virus-resistant freesia cultivars and the producing and keeping virus-free plantlets by various virus inhibitor treatment in the meristem culture. In this study, meristem culture with ribavirin treatment as virus inhibitor were performed in order to produce virus-free freesia plant and accessed the virus reduction effect in the cultured freesia plantlets.

Research method

1. Plant material

The leaves of freesia ‘Shiny Gold’ showing symptoms of virus infection were collected from Gyeonggi (Suwon, Icheon), Chungnam (Seocheon, Buyeo), and Jeonbuk (Jeonju), in 2013- 2014 and stored at -70°C until use. Freesia seedlings were obtained from those cross-fertilized by the NIHHS, RDA in Suwon, which were sowed in the growth chamber at 25°C and for 12 hours in day length. Plant materials were collected when the normal leaves grew about 10cm, which were stored at -70°C for virus detection. To produce virus free freesia plant, corm less than 1cm harvested by the NIHHS, was used for the meristem culture.

2. Virus detection

Total RNA was extracted using Plant RNeasy® Mini kit (Qiagen, USA), and cDNA was synthesized using PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Japan) according to instructions. Reverse Transcription (RT)-PCR analysis was carried out with EmeraldAmp PCR master Mix (Takara, Japan), 94°C5 min. at, of 94°C, 30 sec, 62°C, 30 sec, 72°C, 1min (40 cycles), 72°C, 5 min (Table 1). The amplified DNA fragments were identified with 0.8% Agarose gel. enzyme-linked immunosorbent assay (ELISA) test of the collected freesia leaves were analyzed using freesia mosaic virus reagent set (LOEWE, Germany) and buffer pack (agdia, USA) according to instructions.
Table 1.
Primers for RT-PCR analysis.
Virusz Primer sequence Length
FreMV F 5’-GCAACCAGCGCACCAGAGCAACT-3’ 23mer
R 5’-CATGTGACGTACACCCAACAGACT-3’ 24mer
FreSV F 5’-ATAGTGAATCCATAAGCTGCTCGA-3’ 24mer
R 5’-TACAAGTCTGGTGATGTAATCGTG-3’ 24mer
BYMV F 5’-TCTGACCAAGAACAACTCAATGCA-3’ 24mer
R 5’-CTAAATACGAACACCAAGCATGGTG-3’ 25mer
CMV F 5’-ATGGACAAATCTGAATCAACCAGTGCT-3’ 27mer
R 5’-TCAGACTGGGAGCACTCCAGATGTGGG-3’ 27mer
TRV F 5’-CGATGAGGAGTTTGATAGCAAAGC-3’ 24mer
R 5’-ACCCACCTAGTGTTGTACGATTTC-3’ 23mer

z Freesia mosaic virus (FreMV), Freesia necrosis virus (FreNV), Bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), Tobacco rattle virus (TRV).

3. Meristem culture

The corms of ‘Shiny Gold’ harvested in NIHHS, RDA was soaked in 70% Et-OH for 30 seconds and washed with sterilized water, after which it was sterilized for 20 minutes in 1% sodium hypochloride solution and washed 3 times with sterilized water. It was planted in Murashige and Skoog (MS) medium, 3% sucrose (w/v), 0.8% agar, pH 5.8, and was incubated at 25°C for 12 hours of day length. Approximately 3 week after, the shoot grew up to be 0.5~1cm from planting, the meristem was extracted at the size of 0.2~0.5mm. The apical meristem cuttings were incubated in MS liquid medium with 3% sucrose (w/v), pH 5.8, BA 1, IAA0.5 ppm and the ribavirin, as a virus inhibitor, was treated 0, 20, 50, 100, and 500μM, respectively. The medium was replaced once a week. The callus and shoot were moved to MS medium contained with 0.8% (w/v) agar and incubated for 4 weeks in the same condition. After that, the length was measured and the virus on leaves was verified by RT-PCR with specific primers for FreMV, FreSV, BYMV and TRV detection and ELISA analysis.

Results and discussions

Total 299 leaves of freesia ‘Shiny Gold’ with virus infected symptoms such as light yellow and brown mosaic patterns, yellowing and necrosis were collected from Gyeonggi (Suwon, Icheon), Chungnam (Seocheon, Buyeo), and Jeonbuk (Jeonju) in 2013-2014. Virus was detected by RT-PCR using virus specific primer set (Fig. 1,Fig2, Table 1). The amplified DNA fragment was 95-97% homology with FreMV, FreSV, BYMV and TRV in the NCBI Blast database (data not shown). The collected freesia leaves either had single or multiple infections of FreSV, BYMV and TRV (Table 2). Out of total 229 freesia ‘Shiny Gold’ plants, 51.5% (118 plants) had single infection by FreMV, followed by 36.2% (83 plants) with multiple infection with FreMV and BYMV, 8.7% (20 plants) with multiple infection by FreMV and FreSV, 3.1% (7 plants) with FreMV, FreSV and BYMV respectively, and only one freesia with multiple infection by FreMV, BYMV, and TRV. However, none of the freesia plantlets was infected by four or more viruses, and CMV was also not detected. As a result of virus identification using RT-PCR analysis, total 96 plantlets of freesia seedlings, 15.6% (15 of them) were infected by FreMV. FreMV could be infected mechanically through wounds using sap, and is reported that it does not infect seeds (Plant Virus Online, 2014).
Fig. 1
Virus-like symptoms on the freesia leaves.
KSPPE-19-1_19_F1.jpg
Fig. 2
Virus detection of FreMV (left) and FreSV (right) using RT-PCR analysis in freesia leaf of (A) shoot from bulb, and (B) seedling.
KSPPE-19-1_19_F2.jpg
Table 2.
Identification of freesia viruses using RT-PCR analysis in freesia cultivars ‘Shiny Gold’.
Virusz Freesia ‘Shiny Gold’
FreMV 118/229y
FreSV 0/229
BYMV 0/229
TRV 0/229
FreMV + FreSV 20/229
FreMV + BYMV 83/229
FreMV + TRV 0/229
FreSV + BYMV 0/229
FreSV + TRV 0/229
BYMV + TRV 0/229
FreMV + FreSV + BYMV 7/229
FreMV + FreSV + TRV 0/229
FreMV + BYMV + TRV 1/229
FreSV + BYMV + TRV 0/229
FreMV + FreSV + BYMV + TRV 0/229

z Freesia mosaic virus (FreMV), Freesia necrosis virus (FreNV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Tobacco rattle virus (TRV).

y Number of plants showing positive reactions/number of plants assayed.

Table 3.
Plant height from meristem cultured plantlets on freesia ‘Shiny Gold’.
Concentration of ribavirin (μM) Plant height of ‘Shiny Gold’ (cm)
0 18.3az
20 15.3b
50 13.1c
100 5.4d
Significance ***

z The data were analyzed with ANOVA and Duncan’s honestly significant difference test at p<0.001.

Fig. 3
Meristem cultured plantlets of freesia ‘Shiny Gold’ (A, ribavirin 0μM ; B, 20μM; C, 50μM; D, 100μM).
KSPPE-19-1_19_F3.jpg
Table 4.
Identification of freesia viruses using ELISA analysis in freesia ‘Shiny Gold’.
Ribavirin concentration (μM) NMCPsz PC NC BC Significance
0 20 50 100
0.0980cdyx 0.0963cd 0.0996cd 0.0686d 0.3232b 0.3782a 0.1364c 0.1084c ***

z NMCPs, non-meristem cultured plantlets; PC, positive control; NC, negative control; BC, blanc

y Concentration of FreMV

x The data were analyzed with ANOVA and Duncan’s honestly significant difference test at p<0.001

After 4 weeks planting ‘Shiny Gold’ corms, the meristem of plant meristem were extracted in the size of 0.2~0.5mm, and then incubated at 25°C shaking incubator with MS liquid medium including ribavirin concentration of 0, 20, 50, 100 μM, respectively. After 4 weeks, the plantlets were sub-cultured in MS solid media with the same conditions. As increasing the ribavirin concentration, the shoot length of meristem cultured ‘Shiny Gold’ plantlets was decreased. The average length of plantlets in MS media contained ribavirin 0, 20, 50, 100μM was 18.9, 15.8, 13.1, and 6.1cm, respectively (Fig. 3, Table 3). ELISA were analyzed for the detection of FreMV, all of the meristem cultured plantlets of ‘Shiny Gold’ showed an average of 0.0963-0.0980 in 0, 20, 50, 100μM ribavirin treatment, which was lower than the negative control (NC) average 0.1364, thereby not showing FreMV at the ELISA level (Table 4). The initial culture without meristem culture showed an average of 0.3232, which was lower than the positive control (PC) average 0.3732 but higher than the NC average, indicating that it is infected by virus and thus the virus concentration decreased by meristem culture. However, FreMV was detected in the RT-PCR testing of meristem culture, indicating that the concentration of FreMV is reduced by meristem culture or ribavirin treatment, but it is not completely removed. This experiment show the reduction effect of FreMV by meristem culture, but not ribavirin, because FreMV was not detected all of the ribavirin concentration (0-100μM) by the ELISA diagnostic tool. For more accurate detection, it is necessary to perform real-time PCR test and have long-term inspection on the reduction of FreMV by increasing the period of cultivation.
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