Cryopreservation And Chrysanthemum Stunt Viroid(CSVd) Eradication In Argyranthemum

Argyranthemum, a member of the family Asteraceae,is a perennial herbaceous plant and widely grown in the world as an ornamental crop. The wild germplasm is mainly composed of white flowers, while the commercially grown cultivars are purple or yellow. Conservation of plant germplasm is a prerequisite for breeding of novel cultivars by both classic breeding and genetic engineering strategies. Cryopreservation has been considered an ideal means for longterm conservation of plant genetic resources. Therefore, it is significant to establish efficient cryogenic prodcedures for Argyranthemum. Chrysanthemum stunt viroid(CSVd)causes severe damage to Argyranthemum production. In addition, CSVd is listed as a quarantine pathogen, thuslimiting exchanges of the plant materials globally. In practice, use of viroid-free plant material is pivotal for control of this pathogen, and for material exchange between countries and regions. The objectives of the present study are, therefore, to:(1) establish an efficient cryopreservation protocol of Argyranthemum shoot tips, assess genetic stability and evaluate field performance in greenhouse-grown plants regenerated from cryopreserved shoot tips;(2) attempt to eradicate CSVd from infected Argyranthemum using various techniques;(3) exploit mechanisms of eradication of CSVd by different techniques; and(4) elucidate the causes responsible for the differences in the ability of CSVd to invade shoot apical meristems of different genotypes of Argyranthemum. The main results are summarized as the followings:In vitro stock shootsof Argyranthemum maderense ‘Yellow Empire’ were established by surface sterilization with 70% ethanol for 1 min, followed by 1% Na OCl for 10 min, and cultured on a medium composed of MS supplemented with 3% sucrose, 0.1mg/L NAA, 1.0mg/L BAP, 0.3mg/L GA3 and 0.6% agar. Shoot tips(about 2.5 mm in length, containing 5-6 leaf primordia) excised from 5-week-old in vitro stock shoots were precultured overnight on MS medium supplemented with 0.5 M sucrose. Precultured shoot tips were treated for 20 min with a loading solution composed of MS medium containing 2 M glycerol and 0.4 M sucrose, followed by dehydration with plant vitrification solution 2(PVS2) for 30 minat 0 °C. Afterwards, each shoot tip was transferred onto 2.5 μl PVS2 droplets carried on a sterile aluminum foil strip(2 cm x 0.8 mm), prior to a direct immerson into liquid nitrogen. Rewarming and unloading were done with a solution composed of 1.2 M sucrose in MS for 20 min at room temperature. Cryopreserved shoot tip were post-cultured on recovery medium containing MS supplemented with 0.05mg/L GA3. Shoot regrowth rate of 70% was obtained after 6 weeks of post-culture. Evaluation of field performance of Argyranthemum plants derived from cryopreserved shoot tipsshowed thatalthough some alternationswere found in root formation and vegetative growth, morphologies of the leaves and flowers, and color, number and size of the flowers remained unchange,compared with the control. Assessments of genetic integrity by inter simple sequence repeat(ISSR) and amplified fragment length polymorphism(AFLP) did not detect any polymorphic bands across the plants tested following cryopreservation. Therefore, the dropletvitrification cryopreservation described in the present study can be considered promising for long-term preservation of Argyranthemum germplasm.A. maderense ‘Yellow Empire’ and A. frutescens ‘Border Dark Red’ were used to observe field behavious of the infected greenhouse-grown plants. The results showed that CSVd infection induced obvious symptoms on greenhouse-grown plants of A.maderense ‘Yellow Empire’ and A. frutescens ‘Border Dark Red’, including stunted growth, irregular shape of plants, flower distortion and color breaking, thus resulting in poor flower quality. Meristem culture(0.2mm, 1-2 leaf primordia), cryotherapy of shoot tips(2.5mm, 5-6 leaf primordia) and chemotherapy using ribavirin,salicylic acid and amantadine, each at 25, 50 and 100 mg/L, cannot eradicate CSVd. Combination of low temperature treatment(5 oC, 2 to 3 months) and meristem culture(0.2mm, 1-2 leaf primordia) can eradicate CSVd, and obtained one viroid-free A. maderense ‘Yellow Empire’ and two viroid-free A. frutescens ‘Border Dark Red’, respectively.In situ hybridization of CSVd revealed that CSVd could infect shoot apical meristems(SAM) and all the leaf primordia of A. maderense ‘Yellow Empire’. Histological studies showed that the cells in SAM including the meristematic cells and first two leaf primordia could survive cryopreservation. Therefore, plants regenerated from cryopreservation are still viroid-infected. Using in situ hybridization of CSVd in shoot tips in the infected plants after low temperature treatment, we found CSVd-free area in the shoot tips of A. frutescens ‘Border Dark Red’ was enlarged, thus explaning viroid-free plants can be obtained by combination of low temperature therapy with meristem culture.In the diseased A. maderense ‘Yellow Empire’ and A. frutescens ‘Butterfly’, CSVd was found in all tissues including the uppermost cell layers in the AD and the youngest leaf primordia 1 and 2. In diseased A. frutescens ‘Border Dark Red’ and ‘Border Pink’, CSVd was detected in the lower part of the AD and elder leaf primordia, leaving the upper part of the AD, and leaf primordia 1 and 2 free of viroid. No differences were found in the ability of CSVd to infect shoots and flower organs between A. maderense ‘Yellow Empire’ and A. frutescens ‘Border Dark Red’. Histological observations and transmission electron microscopy showed similar developmental patterns of vascular tissues and plasmodesmata(PD) in the SAM of A. maderense ‘Yellow Empire’ and A. frutescens ‘Border Dark Red’, while immunolocalization studies revealed a major difference in the number of callose(β-1, 3-glucan) particles deposited at PD in SAM. A lower number of callose particles were found deposited at PD of SAM of A.maderense ‘Yellow Empire’ than A. frutescens ‘Border Dark Red’. This difference is most likely responsible for the differences in ability of CSVd to invade SAM among Argyranthemum cultivars.The present study(1) provided a technical platform for long-term conservation of Argyranthemum germplasm;(2) elucidated why meritem culture and cryotherapy failed toproduce any viroid-free plants, while combination of low temperature and meristem culture canobtain viroid-free plants;(3) revealed the causes responsible for differences in the ability ofCSVd invade shoot apical dome of Argyranthemumgenotypes.