Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long-term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana ( Musa spp.), Citrus spp., grapevine ( Vitis vinifera ), Prunus spp., raspberry ( Rubus idaeus ), potato ( Solanum tuberosum ) and sweet potato ( Ipomoea batatas ) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing 'citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.     

Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)

Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube.     

Cryopreservation of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)—a review

Grapevine (Vitis genus) is one of the economically most important fruits worldwide. Some species and cultivars are rare and have only a few vines, but represent national heritages with a strong need for preservation. Field collections are labor intensive, and expensive to maintain, and are exposed to natural disasters. In addition, infection with pathogens, especially viruses, is common in grapevine because of vegetative propagation, which is conventionally used for this genus. Cryopreservation provides an alternative and ideal means for the long-term preservation of Vitis germplasm, which can be used as a backup to field collections for important autochthonous cultivars or only as cryo-banks for rare, native cultivars that are worthy of preservation. Cryotherapy, based on cryopreservation protocols, provides an efficient method for the eradication of grapevine viruses. This review provides comprehensive and updated information on cryopreservation for long-term preservation of genetic resources and cryotherapy for virus eradication in Vitis. Additional research in grapevine cryopreservation and cryotherapy is needed.     

茎尖和芽的超低温保存

介绍了植物茎尖和芽超低温保存的意义和现状。影响超低温保存的一些主要因素及其所采取的措施,主要包括预培养、低温锻炼、使用冰冻保护剂以及适当采用不同的降温方法和化冻洗涤方法,并就今后的研究提出了一些看法。     

Cryobiotechnology of forest trees: recent advances and future prospects

Globally, forests are of great economic importance and play a vital role in maintaining friendly ecological environments, sustainability of eco-systems, and biodiversity. Harsh environments, human activities and climate warming have long threatened the diversity of forest genetic resources. Among all conservation strategies, cryopreservation is at present time considered an ideal means for long-term conservation of plant genetic resources. To date, studies on cryopreservation of forest trees have been far behind agricultural and horticultural crops. The present review provides a comprehensive and update information on recent advances in cryopreservation of shoot tips, somatic embryogenic callus and seeds of forest trees. Assessments of genetic stability in the regenerants following cryopreservation were also analyzed and addressed. Further studies on cryopreservation of forest trees are proposed and needed. By doing so, we expect to re-evoke research interests and promote further developments in forest tree cryobiotechnology, thus assisting to ensure maintenance of biodiversity of genetic resources of forest trees.     

Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection

Plant shoot tips do not survive exposure to liquid nitrogen temperatures without cryoprotective treatments. Some cryoprotectant solutions, such as plant vitrification solution 2 (PVS2), dehydrate cells and decrease lethal ice formation, but the extent of dehydration and the effect on water freezing properties are not known. We examined the effect of a PVS2 cryoprotection protocol on the water content and phase behavior of mint and garlic shoot tips using differential scanning calorimetry. The temperature and enthalpy of water melting transitions in unprotected and recovering shoot tips were comparable to dilute aqueous solutions. Exposure to PVS2 changed the behavior of water in shoot tips: enthalpy of melting transitions decreased to about 40 J g H2O(-1) (compared to 333 J g H2O(-1) for pure H2O), amount of unfrozen water increased to approximately 0.7 g H2O g dry mass(-1) (compared to approximately 0.4 g H2Og dry mass(-1) for unprotected shoot tips), and a glass transition (T(g)) at -115 degrees C was apparent. Evaporative drying at room temperature was slower in PVS2-treated shoot tips compared to shoot tips receiving no cryoprotection treatments. We quantified the extent that ethylene glycol and dimethyl sulfoxide components permeate into shoot tips and replace some of the water. Since T(g) in PVS2-treated shoot tips occurs at -115 degrees C, mechanisms other than glass formation prevent freezing at temperatures between 0 and -115 degrees C. Protection is likely a result of controlled dehydration or altered thermal properties of intracellular water. A comparison of thermodynamic measurements for cryoprotection solutions in diverse plant systems will identify efficacy among cryopreservation protocols.     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

Invertebrate pests and diseases of sweetpotato (Ipomoea batatas): a review and identification of research priorities for smallholder production

Sweetpotato has been the subject of little research worldwide compared with other major crop staples, and this is especially so for less developed countries where sweetpotato is critical for food security. This review synthesises information on plant protection issues that affect smallholder sweetpotato farmers in less developed countries to identify major issues and suggest research priorities. Though the pests and diseases of sweetpotato in less developed countries are largely common to industrialised systems, their relative importance differs and losses tend to be more severe as a result of differing agronomic practices and relative unavailability of management options and technical support that are important in developed countries. Smallholders are heavily reliant on cultural practices such as traditional forms of biological control using ants and livestock, fallowing and composting (sometimes with plant materials having biocidal properties). Crop protection methods that have been developed for use in sweetpotato production in developed countries, such as pathogen‐tested planting material, early maturing varieties, pheromone trapping and pesticides are less accessible to, and relevant for, smallholders. Smallholders also typically harvest a given crop progressively which extends the period over which storage roots are potentially vulnerable to attack but reduces the risk of post‐harvest losses. Human population growth in developing countries is leading to an increase in cropping intensity with shorter fallow periods and more years of continuous crops. This has the dual effect of depleting soil nutrients and increasing the potential for pest and pathogen build‐up. Associated with this, the adoption of strategies to manage crop nutrition, such as not burning crop residues, promote carryover of pests and pathogen inocula. As a consequence of these factors, sweetpotato yield losses from diseases, especially viruses, and pests, particularly weevils, can be high. Climate change is likely to result in more frequent drought and this will increase losses caused by sweetpotato weevils that are favoured by dry conditions. This review of sweetpotato pests and their management options concludes with suggestions for some future research priorities including the combination of traditional practices that have pest management outcomes with relevant practices from industrial production that are able to be transferred or modified for use in smallholder production. Increased technical support for decision making and diagnostics, including molecular approaches that have scope for field use, will be important in reducing the burden imposed by biotic threats to this important global crop.     

RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus

Sweetpotato chlorotic stunt virus (SPCSV; genus Crinivirus , family Closteroviridae) is one of the most important pathogens of sweetpotato ( Ipomoea batatas L.). It can reduce yields by 50% by itself and cause various synergistic disease complexes when co-infecting with other viruses, including sweetpotato feathery mottle virus (SPFMV; genus Potyvirus , family Potyviridae). Because no sources of true resistance to SPCSV are available in sweetpotato germplasm, a pathogen-derived transgenic resistance strategy was tested as an alternative solution in this study. A Peruvian sweetpotato landrace 'Huachano' was transformed with an intron-spliced hairpin construct targeting the replicase encoding sequences of SPCSV and SPFMV using an improved genetic transformation procedure with reproducible efficiency. Twenty-eight independent transgenic events were obtained in three transformation experiments using a highly virulent Agrobacterium tumefaciens strain and regeneration through embryogenesis. Molecular analysis indicated that all regenerants were transgenic, with 1–7 transgene loci. Accumulation of transgene-specific siRNA was detected in most of them. None of the transgenic events was immune to SPCSV, but ten of the 20 tested transgenic events exhibited mild or no symptoms following infection, and accumulation of SPCSV was significantly reduced. There are few previous reports of RNA silencing-mediated transgenic resistance to viruses of Closteroviridae in cultivated plants. However, the high levels of resistance to accumulation of SPCSV could not prevent development of synergistic sweet potato virus disease in those transgenic plants also infected with SPFMV.     

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.

     

Phytoplasma from little leaf disease affected sweetpotato in Western Australia: detection and phylogeny

Symptoms of leaf and stem chlorosis and plant stunting were common in sweetpotato plants (Ipomoea batatas) in farmers’ fields in two widely separated locations, Kununurra and Broome, in the tropical Kimberley region in the state of Western Australia in 2003 and 2004. In the glasshouse, progeny plants developed similar symptoms characteristic of phytoplasma infection, consisting of chlorosis and a stunted, bushy appearance as a result of proliferation of axillary shoots. The same symptoms were reproduced in the African sweetpotato cv. Tanzania grafted with scions from the plant Aus1 with symptoms and in which no viruses were detected. PCR amplification with phytoplasma‐specific primers and sequencing of the 16S‐23S rRNA gene region from two plants with symptoms, Aus1 (Broome) and Aus142A (Kununurra), revealed highly identical sequences. Phylogenetic analysis of the 16S rRNA gene sequences obtained from previously described sweetpotato phytoplasma and inclusion of other selected phytoplasma for comparison indicated that Aus1 and Aus142A belonged to the Candidatus Phytoplasma aurantifolia species (16SrII). The 16S genes of Aus1 and Aus142A were almost identical to those of sweet potato little leaf (SPLL‐V4) phytoplasma from Australia (99.3%–99.4%) but different from those of the sweetpotato phytoplasma from Taiwan (95.5%–95.6%) and Uganda (SPLL‐UG, 90.0%–90.1%). Phylogenetically, Aus1, Aus142A and a phytoplasma previously described from sweetpotato in the Northern Territory of Australia formed a group distinctly different from other isolates within Ca. Phytoplasma aurantifolia species. These findings indicate that novel isolates of the 16SrII‐type phytoplasma pose a potential threat to sustainable sweetpotato production in northern Australia.     

Recovery of cryopreserved silver birch shoot tips is affected by the pre-freezing age of the cultures and ammonium substitution

The aim of the present study was to improve the cryopreservation protocol for silver birch (Betula pendula Roth) in vitro shoot tips. The recovery of shoot tips derived from young cultures (shoot tips excised from less than 20-month-old cultures) was significantly better than that from old cultures (shoot tips excised from cultures at least 55 months old) when the standard protocol, i.e. the slow-cooling procedure with PGD as cryoprotectant, was used. The recovery of shoot tips of both ages was, however, improved by substituting KNO₃ for ammonium during cold hardening and post-thaw cultivation. The substitution of ammonium with KNO₃ improved recovery in most of the genotypes, indicating the possibility of developing a cryopreservation protocol applicable to several genotypes.     

In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

Encapsulation-Dehydration and Encapsulation-Vitrification Based Cryopreservation of in vitro-Grown Shoot-Tips of Medicinal Plant Astragalus membranaceus

Shoot tips of Amembranaceus excised from in vitro grown axillary bud were encapsulated in calcium alginate beads. Subsequently, shoot tips were precultured in liquid MS medium enriched with 075mol·L-1 sucrose for 5d at 25℃ and then desiccated aseptically on dried silica gel for 5h to a water content of 231% (fresh weight basis) prior to immersion in liquid nitrogen (LN) for 1d. After rewarming at a 40℃ water bath for 2-3min and transferred to solid culture medium for shoot tip recovery. About 50% of cryopreserved shoot tips grew into shoots within 2 weeks after plating. Cryopreservation of Astragalus membranaceus (Fisch.) Bge. shoot tips by encapsulation vitrification has also been developed. Excised shoot tips were firstly encapsulated into alginate gel beads and then precultured in liquid MS medium containing 1mg·L-1 6 BA, 005mg·L-1 NAA and 075mol·L-1 sucrose at 25℃ for 3d. After loading for 90min with a mixture of 2mol·L-1 glycerol and 04mol·L-1 sucrose at 25℃, shoot tips were dehydrated with PVS2 for 120min at 0℃ prior to direct immersion in liquid nitrogen for 1d. After rapidly thawing at a 37℃ water bath for 2-3min, shoot tips were washed for 10min with liquid MS medium supplemented with 1mg·L-1 6 BA, 005mg·L-1 NAA and 12mol·L-1 sucrose at 25℃ and then post cultured on solid MS medium supplemented with 2mg·L-1 6 BA, 005mg·L-1 NAA. The regeneration rate of shoot tips amounted to nearly 80%. Both of plantlets regenerated from cryopreserved shoot tips were morphologically uniform, which both showed as that of control plants. Thus, this encapsulation dehydration and encapsulation vitrification technique appears promising as a routine method for the cryopreservation of shoot tips of Amembranaceus.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Shoot regeneration and cryopreservation of shoot tips of apple (Malus) by encapsulation–dehydration

Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation–dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2–3 mg L^?1 thidiazuron, depending on genotype, and 0.5 mg L^?1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by air-drying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of ‘Gala’. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.     

Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH?5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23?±?2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.     

甘薯种质资源遗传稳定性及遗传多样性SSR分析

应用SSR标记检测国家种质徐州甘薯试管苗库中离体保存5年和8年的24份种质资源及其对应的田间圃材料的遗传稳定性,同时对24份甘薯种质的遗传多样性进行分析.20对SSR引物分析表明,24份甘薯材料扩增得到了清晰的DNA条带30条,其中多态性条带2l条,多态性百分率为70%,全部品种在2种保存方式下谱带一致,说明2种保存方式的效果相同.应用NTSYS软件对材料进行遗传相似性和UPGMA聚类分析,24份甘薯种质资源遗传相似系数在0.57～0.93之间,平均为0.74.在0.72的相似系数上24份材料可以聚成三大类,表明我国的甘薯品种种质资源遗传多样性还是比较丰富的.该研究为甘薯种质资源长期离体保存及甘薯杂交育种提供了理论依据.