Qiao-Chun Wang

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.

Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration

A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malusxa0×xa0domestica Borkh.). Shoot-tips, of 2.0xa0mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5xa0mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5xa0M sucrose for 7xa0days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20xa0% in 5–7xa0h, followed by a direct immersion in liquid nitrogen for 1xa0h. Frozen shoot-tips were re-warmed in a water bath at 38xa0°C for 2xa0min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75xa0%) and ‘Wangshanhong’ (36xa0%), with a mean shoot regrowth rate of 61xa0% attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.

Cryopreservation of sweetpotato (Ipomoea batatas) and its pathogen eradication by cryotherapy

Sweetpotato (Ipomoea batatas) ranks as the seventh most important staple crop in the world and the fifth in developing countries after rice, wheat, maize and cassava. Sweetpotato is mainly grown in developing countries, which account for more than 95% of total production of the whole world. Genetic resources, including cultivated varieties and wild species, are a prerequisite for novel sweetpotato breeding in both conventional and genetic engineering programs. Various cryopreservation protocols have been developed for shoot tips and embryogenic tissues. The former explants are preferred for long-term conservation of sweetpotato genetic resources, while the latter are valuable for sweetpotato genetic improvement. This review provides update comprehensive information on cryopreservation of sweetpotato shoot tips and embryogenic tissues. Plant pathogens such as viruses and phytoplasma severely hamper high yield and high quality production of sweetpotato. Thus, usage of pathogen-free planting materials is pivotal for sustainable sweetpotato production. Cryotherapy of shoot tips can efficiently eradicate sweetpotato pathogens such as viruses and phytoplasma. The mechanism behind pathogen eradication by cryotherapy of shoot tips has been elucidated. Pathogen eradication by cryotherapy provides an alternative, efficient strategy for production of pathogen-free plants. In addition, cryopreserved tissues may also be considered to be safer for exchange of germplasm between countries and regions.

Three vitrification-based cryopreservation procedures cause different cryo-injuries to potato shoot tips while all maintain genetic integrity in regenerants.

We previously reported successful cryopreservation of shoot tips of potato Zihuabai by three vitrification-based protocols. In the present study, cryo-injury to shoot tips and genetic stability in regenerants recovered from cryopreserved shoot tips by the three vitrification-based protocols were further investigated. The results showed that sucrose preculture caused no obviously different injuries, while dehydration with plant vitrification solution 2 (PVS2) was the step causing major damage to cells of shoot tips, regardless of the cryogenic procedures. Compared with droplet-vitrification and encapsulation-vitrification, vitrification caused the most severe injury to cells of the shoot tips, thus resulting in much longer time duration for shoot recovery and much lower shoot regrowth rate. Cells in apical dome and the youngest leaf primordia were able to survive and subsequently some of them regrew into shoots following all three vitrification-based cryopreservation procedures. Analyses using inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers in shoots regrown from all three vitrification-based protocols did not find any polymorphic bands. The results reported here suggest that vitrification-based cryo-procedures can be considered promising methods for long-term preservation of potato genetic resources.

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 (0.8–1.0xa0cm long and 0.4xa0cm 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 1xa0mm 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 1xa0mg/l naphthaleneacetic acid, 0.5xa0mg/l thidiazuron, 30xa0g/l sucrose, and 7xa0g/l agar (pHu20095.8). The cultures were incubated for 4xa0wk under a 16-h photoperiod at 23u2009±u20092°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.

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 2–3xa0mgxa0L−1 thidiazuron, depending on genotype, and 0.5xa0mgxa0L−1 indole-3-butyric acid. Shoot tips (3xa0mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5xa0M sucrose for 5xa0d, followed by air-drying for 6xa0h 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.

Potato leafroll virus (PLRV) and Potato virus Y (PVY) influence vegetative growth, physiological metabolism, and microtuber production of in vitro-grown shoots of potato (Solanum tuberosum L.)

Effects of Potato leafroll virus (PLRV) and Potato virus Y (PVY) on vegetative growth, physiological metabolism and microtuber production were investigated using in vitro shoot cultures. The results showed that parameters of shoot growth including bud break percentage, shoot length, and node number and length were markedly reduced in the diseased shoots. These negative effects were much more pronounced in shoots co-infected with PLRV and PVY than in those singly infected with either PLRV or PVY. The inhibitive effects on root developments measured by root number and length were observed only in shoots co-infected with PLRV and PVY. Significantly lower contents of chl-a, chl-b and total chl were found in virus infected shoots than in healthy ones. There were striking differences in contents of total soluble protein observed between healthy shoots and PLVR and PVY co-infected ones. The content of total soluble sugar was highest in shoots co-infected with PLRV and PVY, and lowest in healthy shoots. Furthermore, there were no significant differences found in the level of endogenous indole-acetic acid among healthy shoots verses virus infected shoots. However, the level of zeatin-ribosome was much higher in healthy shoots than in virus infected ones. Yet, both healthy and single PLRV infected shoots produced similar levels of gibberillic acid 3, which were much higher than those of PVY single-infected shoots and PLRV and PVY co-infected shoots. Also, there were no significant differences in the number of microtubers among healthy shoots, PLVR single or PVY single infected shoots, but shoots co-infected with PLRV and PVY produced the lowest number of microtubers. Overall, the healthy shoots produced the largest size of microtubers and the highest percentage of microtubers ≥5xa0mm in diameter.

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips.

Culture of shoot tips from adventitious shoots can eradicate Apple stem pitting virus but fails in Apple stem grooving virus

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from ‘Gala’ apple using shoot tips from of adventitious shoots derived from leaf segments. Leaf segments were excised from in vitro 4-week-old stock shoots and cultured on shoot regeneration medium containing 9.1xa0µM thidiazuron and 0.25xa0µM indole-3-butyric acid (IBA) to induce adventitious shoot formation. Shoot tips of different size and different developmental stage were excised from adventitious shoots and cultured on shoot tip culture medium containing 1.1xa0µM 6-benzyladenine and 0.05xa0µM IBA. Results showed size and developmental stage of shoot tips excised from adventitious shoots did not influence survival rate, but significantly affected shoot regrowth rate and ASPV-free frequency. Shoot regrowth rates increased from 10 to 15xa0% in 0.3xa0mm shoot tips containing two leaf primordia (LP) excised after 2–3xa0weeks of shoot regeneration, to 53–55xa0% in those containing three LP excised after 3–4xa0weeks. The highest shoot regrowth rate (82xa0%) was obtained in shoot tips of 0.4xa0mm shoot tips containing four LP excised after 4xa0weeks. ASPV-free frequencies (95–100xa0%) were high in 0.2–0.4xa0mm shoot tips containing two to three LP excised after 2–4xa0weeks, but low (20xa0%) in 0.4xa0mm shoot tips containing four LP excised after 4xa0weeks. None of the shoots regenerated from the shoot tips were ASGV-free, regardless of the size and developmental stage at which shoot tips were excised. Histological studies and virus localization provided explanations to the varying frequencies of the virus eradication using different size of shoot tips that were excised from adventitious shoots at different developmental stages. The protocol described here was characteristic of high frequency ASPV eradication and high efficiency of production of shoot tips that were used for virus eradication, and thus may have potential applications to virus eradication in other plant species.

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.