Zhenfang Yin

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.

Novel and potential application of cryopreservation to plant genetic transformation.

The world population now is 6.7 billion and is predicted to reach 9 billion by 2050. Such a rapid growing population has tremendously increased the challenge for food security. Obviously, it is impossible for traditional agriculture to ensure the food security, while plant biotechnology offers considerable potential to realize this goal. Over the last 15 years, great benefits have been brought to sustainable agriculture by commercial cultivation of genetically modified (GM) crops. Further development of new GM crops will with no doubt contribute to meeting the requirements for food by the increasing population. The present article provides updated comprehensive information on novel and potential application of cryopreservation to genetic transformation. The major progresses that have been achieved in this subject include (1), long-term storage of a large number of valuable plant genes, which offers a good potential for further development of novel cultivars by genetic transformation; (2), retention of regenerative capacity of embryogenic tissues and protoplasts, which ensures efficient plant regeneration system for genetic transformation; (3), improvement of transformation efficiency and plant regeneration of transformed cells; (4), long-term preservation of transgenic materials with stable expression of transgenes and productive ability of recombinant proteins, which allows transgenic materials to be stored in a safe manner before being analyzed and evaluated, and allows establishment of stable seed stocks for commercial production of homologous proteins. Data provided in this article clearly demonstrate that cryo-technique has an important role to play in the whole chain of genetic transformation. Further studies coupling cryotechnique and genetic transformation are expected to significantly improve development of new GM crops.

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.

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.

Cryopreservation of Embryogenic Cell Suspensions by Encapsulation–Vitrification and Encapsulation–Dehydration

Encapsulation-vitrification and encapsulation-dehydration are two newly developed techniques for cryopreservation of embryogenic cell suspensions. Here, we describe the two protocols using grapevine (Vitis) as a model plant. Cell suspensions at the exponential growth stage cultured in a cell suspension maintenance medium are encapsulated to form beads, each being about 4 mm in diameter and containing 25% cells. In the encapsulation-vitrification procedure, the beads are stepwise precultured in increasing concentrations of sucrose medium up to 0.75 M, with 1 day for each concentration. The precultured beads are treated with a loading solution for 60 min and then dehydrated with plant vitrification solution 2 at 0°C for 270 min before a direct immersion in liquid nitrogen. Following cryostorage, the beads are rapidly rewarmed at 40°C for 3 min and then unloaded with 1 M sucrose solution for 30 min. In the encapsulation-dehydration procedure, the beads are precultured in increasing concentrations of sucrose medium up to 1 M, with 1 day for each concentration, and then maintained on 1 M sucrose medium for 3 days. The precultured beads are dehydrated for 6 h under a sterile air flow, prior to rapid freezing in liquid nitrogen. The freezing and rewarming procedures are the same as used in the encapsulation-vitrification technique. The unloaded beads from encapsulation-vitrification and rewarmed beads from encapsulation-dehydration are postcultured on a recovery medium for 3 days at 25°C in the dark for survival. Surviving cells are transferred to a regrowth medium to induce cell proliferation. Embryogenic cell suspensions are reestablished by suspending the cells in a cell suspension maintenance medium maintained on a gyratory shaker at 25°C in the dark. For plant regeneration, surviving cells are transferred from the recovery medium to an embryo maturation medium and maintained at 25°C under light conditions. Embryos at the torpedo stage are cultured on a rooting medium until whole plantlet regenerates.