Su Min Jeon

Elimination of Chrysanthemum stunt viroid (CSVd) from meristem tip culture combined with prolonged cold treatment

Chrysanthemum production in Korea has recently been greatly affected by the spread of Chrysanthemum stunt viroid (CSVd) infection, necessitating the use of CSVd-free stocks to ensure successful chrysanthemum cultivation. We investigated the effects of low temperature (4°C), antiviral chemicals (ribavirin and amantadine) and a combination of these treatments on CSVd elimination by meristem tip cultures using plantlets that originated from CSVd-infected chrysanthemum ‘Ency’. Neither antiviral agents led to CSVd elimination, despite the suppression of meristem tip growth in a concentration dependent manner. However, the CSVd elimination rate increased up to 42.8% when meristem tips were excised after storage at 4°C for two months. The most effective results were obtained from a combination of low temperature for three months at 4°C followed by meristem tip culture on media containing 50 and 100 mg·L−1 ribavirin. These results suggest that antiviral agents can also be useful for CSVd elimination if their treatment is combined with prolonged periods of low temperature. This is the first report of eradication of viroids from spray type chrysanthemum bred in Korea.

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum

The objective of this research was to develop an efficient protocol for shoot regeneration from leaf segments of the Chrysanthemum cv. Vivid Scarlet by examining the effects of plant growth regulators, dark incubation period, gelling agents, and silver nitrate. The highest number of shoots per explant (12.3) was regenerated from leaf explants cultured on Murashige and Skoog (MS) medium supplemented with a combination of 1 mgL(-1) of 6-benzyladenine (BA) and 2 mgL(-1) of α-naphthaleneacetic acid (NAA) under light conditions without any initial dark period. Gelrite was the most effective gelling agent for shoot regeneration among those tested, whereas the presence of silver nitrate distinctly inhibited shoot regeneration. Superior plant growth and rooting was observed on a hormone-free MS medium solidified with Gelrite. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions. The established protocol was applicable to shoot regeneration for four out of six cultivars tested. This research will facilitate the genetic transformation and micropropagation of Chrysanthemum cultivars.

An efficient protocol for Agrobacterium-mediated genetic transformation of recalcitrant chrysanthemum cultivar Shinma

Chrysanthemum cultivar Shinma is a standard cultivar and has a large flower size, long vase life, and strong resistance to the white rust disease; thus, it is an important commercial cut flower in the flower markets of Korea. However, its flower color (white) is simple, so variation in flower color by using genetic transformation is necessary to increase its value in flower markets worldwide. Success of genetic transformation in chrysanthemum is dependent on many factors. In this study, factors that affect the efficient genetic transformation of this chrysanthemum were assessed, transgenic plants with the RsMYB1 anthocyanin regulatory gene were produced, and the presence of the transgene and its stable expression were confirmed using PCR and reverse transcription-PCR. Co-cultivation temperature and Agrobacterium strains were observed to be the main factors that affected higher transformation efficiency. However, the protocol developed by a combination of all optimized factors yielded eight- or fourfold higher transformation efficiency than the simple (un-optimized) protocol or individually optimized factors. The herbicide resistance assay revealed that PCR-positive transgenic shoots have stronger resistance to Basta™ than the wild type (WT). We expect that the efficient protocol developed in this study will facilitate the genetic transformation of genes of interest in this cultivar and that the anthocyanin regulatory gene will help in modifying the flower color.

The effect of antifreeze protein on the cryopreservation of chrysanthemums

Abstract To our knowledge, this is the first study to show the application of antifreeze protein (AFP) in the cryopreservation of ornamental plants. We studied the effect of AFP on the cryopreservation of Chrysanthemum grandiflorum (Ramat.) Kitam ‘Borami’, ‘Secret Pink’, and ‘Yellow Cap’. For all cultivars, survival and regrowth rates were higher in shoot tips vitrified with plant vitrification solution 3 (PVS3) containing various concentrations of AFP than in those vitrified with PVS3 alone. The optimal AFP concentration was genotype-dependent. Scanning electron microscopy revealed that shoot tips vitrified with PVS3 containing AFP had less freezing injuries than those treated with PVS3 alone. Analysis of the enthalpy in each sample by differential scanning calorimetry supported the finding that AFP is a potent cryoprotectant that can reduce the freezing point of water in plant tissues. We expect that our results will facilitate the successful application of AFPs in the cryopreservation of rare and commercially important plant germplasm.

Elimination of chrysanthemum stunt viroid and chrysanthemum chlorotic mottle viroid from infected chrysanthemum by cryopreservation

Chrysanthemum morifolium ‘Borami’ and ‘Secret Pink’ showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and ‘Yellow Cap’ showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected ‘Borami’ collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in ‘Borami’ collected from Chilgok and Gyeongsan and in ‘Secret Pink’ from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the ‘Yellow Cap’ collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

Combined effects of supplementary light and CO2 on rose growth and the production of good quality cut flowers

Abstract: The effects of supplementary lighting with high-pressure sodium (HSP) lamps alone or in combination with carbon dioxide (CO2) on the growth, yield, and flower stem quality of two rose cultivars (“Loving Heart” and “Top Grace”) were studied. Compared to natural lighting (control), supplementary lighting alone was beneficial for plant growth, and it increased plant height, stem diameter, and the number of axillary shoots. Furthermore, increases in flower stem yield (>70 cm), flower stem diameter, fresh weight, and the number of petals per flower were also observed. The combination of supplementary lighting and CO2 significantly enhanced all of the studied parameters compared to supplementary lighting alone. Moreover, stomatal density and chlorophyll fluorescence were seemingly affected by either supplementary lighting alone or in combination with CO2. This is the first study to examine the beneficial effects of combined supplementary lighting and CO2 conditions, and the resulting information is essential to rose growers and commercial production.

Sodium nitroprusside stimulates growth and shoot regeneration in chrysanthemum

In this study, we demonstrate that the nitric oxide (NO) donor sodium nitroprusside (SNP) improves plant regeneration in chrysanthemums. Internode explants of three different chrysanthemum cultivars, ‘White ND’, ‘White wing × Peach ND’, and ‘Hunt × Lemon ND’, were cultured on shoot induction medium (SIM) containing various concentrations of N6–benzyladenine (2.22 or 4.44 μM BA) and SNP (0.83–6.71 μM) individually and in combination. Most combinations of BA and SNP significantly improved the morphogenetic potential of internode explants and enhanced shoot regeneration in all three chrysanthemum cultivars compared to treatment with BA alone. The cultivar ‘White wing × Peach ND’ displayed the highest regeneration response (98.3%) and shoot regeneration rate (27.3 shoots/explant) in SIM containing optimal BA (4.44 μM) and SNP (0.83 μM) concentrations within 30 days of culture. Individual shoots of ‘White wing × Peach ND’ were transferred to root induction medium (RIM) containing various concentrations of SNP (0.83–6.71 μM) alone. Shoots rooted in the presence of SNP resulted in healthy plantlets within 30 days of culture with improved root (number of roots/shoot, root length, and fresh weight) and shoot (number of leaves, shoot length, and fresh weight) growth characteristics compared to the control. In addition, the regeneration procedure described in this study only requires a short duration (60 days) to obtain rooted plantlets from internode explants of chrysanthemums. Our results suggest that supplementation of chrysanthemum regeneration medium with SNP enhances shoot regeneration and improves plant growth, overcoming problems associated with propagation and genetic transformation.

Absence of AVP1 transcripts in wild type watermelon scions grafted onto transgenic bottle gourd rootstocks

Abstract In this study we confirmed the stable integration of Arabidopsis AVP1 in the genomes of bottle gourd T 3 homozygous lines and its transcription, and additionally evaluated possibility of translocation of the AVP1 mRNA from transgenic bottle gourd rootstocks to wild type watermelon scions. Each AVP1 gene in two bottle gourd T3 lines is abundantly expressed under a field condition. Given the grafting between wild type watermelon scions and AVP1- expressing bottle gourd rootstocks, no translocation of the AVP1 mRNA was detected in leaves, both sexual flowers, and fruits of the scions. Keywords Bottle ground, Gene modified, Graft, RT-PCR, Watermelon Introduction Grafting is now a popular technique for the cultivation of the horticultural crops including cucurbitaceae fruit vegetables, which has been developed not only to control growth and development of the scion but also to enhance tolerance against soil-borne diseases and/or abiotic stresses, such as salinity, low temperature and drought (Jang et al. 2012; Kubota et al. 2008; Lee 1994). In some special regions, where land utility is extremely limited, the allied crops are repeatedly cultivated all the year round (Kubota et al. 2008; Lee 1994), which increases specific pathogens and salinity of the rhizosphere. To overcome the disadvantages of the intensive cultivation, improvement of rootstocks by using genetic engineering is being attempted as a solution (Han et al. 2009; Smolka et al. 2010; Wang et al. 2012). Control of abiotic stresses is an important element to increase total yields in modern agriculture. Plants respond to various abiotic stresses by altering their turgor pressure in vacuoles in order to accomplish selective permeation of solutes through proton pumps (Gaxiola et al. 2001; McNeil et al. 1999). A vacuolar H