Wen-Lu Bi

Potential applications of cryogenic technologies to plant genetic improvement and pathogen eradication.

Rapid increases in human populations provide a great challenge to ensure that adequate quantities of food are available. Sustainable development of agricultural production by breeding more productive cultivars and by increasing the productive potential of existing cultivars can help meet this demand. The present paper provides information on the potential uses of cryogenic techniques in ensuring food security, including: (1) long-term conservation of a diverse germplasm and successful establishment of cryo-banks; (2) maintenance of the regenerative ability of embryogenic tissues that are frequently the target for genetic transformation; (3) enhancement of genetic transformation and plant regeneration of transformed cells, and safe, long-term conservation for transgenic materials; (4) production and maintenance of viable protoplasts for transformation and somatic hybridization; and (5) efficient production of pathogen-free plants. These roles demonstrate that cryogenic technologies offer opportunities to ensure food security.

Responses of In vitro-Grown Plantlets (Vitis vinifera) to Grapevine leafroll-Associated Virus-3 and PEG-Induced Drought Stress

Stresses caused by viral diseases and drought have long threatened sustainable production of grapevine. These two stresses frequently occur simultaneously in many of grapevine growing regions of the world. We studied responses of in vitro-grown plantlets (Vitis vinifera) to Grapevine leafroll associated virus-3 (GLRaV-3) and PEG-induced drought stress. Results showed that stress induced by either virus infection or drought had negative effects on vegetative growth, caused significant decreases and increases in total soluble protein and free proline, respectively, induced obvious cell membrane damage and cell death, and markedly increased accumulations of O2·- and H2O2. Co-stress by virus and drought had much severer effects than single stress on the said parameters. Virus infection alone did not cause significant alternations in activities of POD, ROS, and SOD, and contents of MDA, which, however, markedly increased in the plantlets when grown under single drought stress and co-stress by the virus and drought. Levels of ABA increased, while those of IAA decreased in the plantlets stressed by virus infection or drought. Simultaneous stresses by the virus and drought had co-effects on the levels of ABA and IAA. Up-regulation of expressions of ABA biosynthesis genes and down-regulation of expressions of IAA biosynthesis genes were responsible for the alternations of ABA and IAA levels induced by either the virus infection or drought stress and co-stress by them. Experimental strategies established in the present study using in vitro system facilitate investigations on ‘pure’ biotic and abiotic stress on plants. The results obtained here provide new insights into adverse effects of stress induced by virus and drought, in single and particularly their combination, on plants, and allow us to re-orientate agricultural managements toward sustainable development of the agriculture.

Development, histological observations and Grapevine leafroll-associated virus-3 localisation in in vitro grapevine micrografts: Micrografting of virus-infected grapevine

Development, histological process and Grapevine leafroll-associated virus-3 localisation were studied in micrografts of three scion/rootstock combinations: healthy/healthy, healthy/infected and infected/healthy. Earlier bud break and faster growth in scions of micrografts were obtained when the healthy shoot segments were used as scions, while earlier bud break in rootstocks and greater fresh weight of roots in micrografts were produced when the healthy shoot segments were used as rootstocks. All histological processes including callus initiation and formation in micrografting conjunctions, and initiation of new cambial cells followed by vascular bundle development connecting scions and rootstocks were similar in micrografts, regardless of the sanitary status of the scions and rootstocks used for micrografting. Virus infection in micrografting conjunctions and systematic infection in micrografts were much more efficient and faster in micrografting combination of the infected scions/healthy rootstocks than in the healthy scions/infected rootstocks. To the best of our knowledge, this is the first report addressing histological process of micrograft development and virus localisation in micrografts. In vitro culture system established in this study facilitates studies on the ‘pure’ impact of the viral infection on micrografting.

Production of Pathogen-Free Horticultural Crops by Cryotherapy of In Vitro-Grown Shoot Tips

Horticultural crops are economically valuable for sustainable agricultural production. Plant diseases caused by Pathogens including virus, phytoplasma and bacterium have been a great threat to production of horticultural crops. The efficient use of pathogen-free plant materials has overcome the menace of plant diseases and has sustained crop production. Cryotherapy of shoot tips, a novel application of cryopreservation technique, has become a new plant biotechnology tool for plant pathogen eradication. When compared with the traditional methods, cryotherapy of shoot tips produces high frequency of pathogen-free plants, which is independent of shoot tip size and cryogenic methods. Cryotherapy of shoot tips has six major steps to produce pathogen-free plants: (1) introduction of infected plant materials into in vitro cultures; (2) excision of shoot tips; (3) cryotherapy; (4) post-culture for plant regeneration; (5) indexing of pathogens in regenerated plants after cryotherapy; and (6) establishment of pathogen-free nuclear stock plants. The key steps 2, 3, and 4 are similar to cryopreservation, and play a major role in obtaining high pathogen eradication frequency.

Cryopreservation of grapevine (Vitis 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.

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 regeneration via embryo-like structures: histological observations and genetic stability in regenerants of Lilium spp.

SUMMARY Transverse thin-cell layers (tTCLs) excised from bulblets of Lilium longiflorum X Oriental hybrid ‘Triumphator’ were cultured on 1.0X Murashige and Skoog (MS) medium containing 0.1 mg l−1 α-naphthaleneacetic acid (NAA) and 0.1 mg l−1 kinetin (KT) in the dark at 23° ± 2°C. White-yellow, friable, embryo-like structures formed after 6 weeks in culture. The embryo-like structures proliferated well when cultured on 1.0X MS medium containing 1.0 mg l−1 NAA and 0.2 mg l−1 thidiazuron (TDZ), and converted into whole plantlets on 0.5X MS medium without any added plant growth regulator. Histological studies identified the origin of the first cell divisions from single sub-epidermal cells, from which somatic embryos developed. Using this procedure, embryo-like structures (ELS) were obtained at frequencies of 85.5%, 76.8%, 68.4%, 28.5% and 25.6% for L. longiflorum, L. longiflorum X Oriental ‘Triumphator’, Lilium Oriental hybrid ‘Siberia’, Lilium Asiatic hybrid ‘Elite’, and L. davidii var. unicolor, respectively. Embryo-like structures could be proliferated and developed into plantlets, with five-to-ten plantlets per embryo-like structure in all five Lilium species and hybrids tested. No polymorphic bands were detected using inter-simple sequence repeat (ISSR) markers and no change in ploidy level was found by flow cytometry (FCM) among the regenerants of all five Lilium species and hybrids. These five Lilium species and hybrids represented a diverse genetic resource of the genus Lilium. This protocol may be widely applicable for somatic embryogenesis and has potential applications in micropropagation and genetic transformation in Lilium spp.

Shoot tip cryotherapy for efficient eradication of grapevine leafroll-associated virus-3 from diseased grapevine in vitro plants

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, China College of Horticulture, Qingdao Agriculture University, Qingdao, China New Zealand Institute for Plant & Food Research Ltd., Palmerston North, New Zealand USDA-ARS National Laboratory for Genetic Resources Preservation, Fort Collins, Colorado Correspondence Qiao-Chun Wang, State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China. Email: qiaochunwang@nwsuaf.edu.cn We describe a droplet-vitrification cryotherapy method for the eradication of grapevine leafrollassociated virus-3 (GLRaV-3) from diseased in vitro shoots of Vitis plants. The procedure involved pre-culture of 1.0-mm shoot tips containing five to six leaf primordia (LPs) for 3 days with a pre-culture medium containing 0.3 M sucrose, 0.16 mM glutathione and 0.14 mM ascorbic acid, treatment of the pre-cultured shoot tips for 20 min at room temperature with a loading solution composed of 2 M glycerol and 0.4 M sucrose and exposure to plant vitrification solution 2 (PVS2), prior to freezing in liquid nitrogen of dehydrated shoot tips contained in 2.5-μL PVS2 droplets on aluminium foil strips. Virus localisation showed GLRaV-3 was not present in apical dome (AD) and LPs 1–4, but it was detected in the basal shoot tip region, approximately 0.5 mm from the AD, as well as in LP 5 and more mature tissues. Histological observations identify that only freezing in liquid nitrogen results in the death of all cells in areas of shoot tips harbouring virus, whereas PVS2 treatment does not. Thus, freezing in liquid nitrogen is a necessary step that eradicates GLRaV-3. This cryotherapy procedure produced shoot regrowth levels that ranged from 43% to 59%, and all plants recovered after cryotherapy were free of GLRaV-3 in two wine, one table and one rootstock cultivars. Thus, this procedure can be considered to be efficient and wildly applicable for eradication of GLRaV-3 from Vitis spp.

Greenhouse performance, genetic stability and biochemical compounds in Chrysanthemum morifolium ‘Hangju’ plants regenerated from cryopreserved shoot tips

Chrysanthemum morifolium ‘Hangju’ is a valuable medicinal plant. We previously reported cryopreservation of shoot tips of C. morifolium ‘Hangju’. The present study further evaluated greenhouse performance, assessed genetic stability and analyzed biochemical compounds in the greenhouse-grown plants regenerated from cryopreservation. The results showed that although some minor alternations were detected in early vegetative growth, there were no differences in major parameters of vegetative growth and flower production between the plants regenerated from cryopreservation and in vitro shoots (the control). Morphologies of leaves and flowers were identical between the two types of the plants. No polymorphic bands revealed by ISSR and RAPD, and no alternations at ploidy levels analyzed by FCM were found in the cryo-derived plants. The types and number of biochemical compounds analyzed by UPLC-MS/MS were identical between the two types of the plants. Quantitative analyses by HPLC showed no differences in the contents of the five selected biochemical compounds produced between the plants regenerated from cryopreservation and in vitro shoots. Therefore, cryopreservation would provide a technical platform for establishment of cryo-banking of Chrysanthemum germplasm with medicinal values.