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Development of technologies

Shin-ichi Yamamoto, Kuniaki Fukui, Takao Niino Genetic Resources Conservation Research Unit, NIAS

A new cryopreservation method using aluminum cryo-plates was developed for clonally propagated crops. In this method, shoot tips are attached to the cryo-plates and can easily be manipulated for preservation treatments, such as dehydration. The ease of handling, minimal injury to shoot tips, and rapid rates of cooling and warming resulted in high regrowth rates. More than 80% regrowth was obtained for mint and tropical mulberry germplasms. It will be possible to utilize this highly effective method for long term preservation in genebanks. Keywords: vegetatively propagated, long term preservation, vitrification, cryo-plate,

liquid nitrogen, genetic resources Background

Results and Discussion

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To achieve easier manipulation of in–vitro-produced shoot tips, we designed and ordered aluminum plates that fit inside 2-ml cryotubes (Fig. 1). We standardized the storage procedure combined with the vitrification and droplet methods (Fig. 2) and monitored regrowth after liquid nitrogen storage using mint, tropical mulberry, carnation, and Dalmatian chrysanthemum as materials. It was necessary to optimize each treatment, such as pretreatment before excision, osmoprotection, and dehydration to improve regrowth after cryopreservation. We determined the optimal conditions for the crop species listed above (see references). With the V-cryo-plate method, high regrowth rates were observed under the optimal conditions for each crop tested: 88%, 87%, 81%, 95% and 77% for mint, tropical mulberry, strawberry, carnation and Dalmatian chrysanthemum, respectively. The V-cryo-plate method has many advantages as listed below and is suitable for large scale storage in genebanks. a) Shoot tips adhere to the plates, so only the plates need to be moved thus increasing the 　efficiency of treatment. b) The injury and loss of shoot tips are dramatically reduced. c) Regrowth rates are high because of rapid cooling and warming d) Technical staff can implement this method after brief training. Until now, a total of 100 accessions of germplasm such as mint, tropical mulberry, straw-berry, mat rush and others have been successfully cryopreserved (100 buds on 10 cryo-plates for each) in the NIAS Genebank.

　Although the cryopreservation methods such as slow freezing, vitrification, and encapsulation desiccation have been developed extensively, their application has not advanced to the same extent for storage of large scale samples in the genebanks of various countries. One reason for lack of widespread use is that these methods require a high level of technical skill. In this study, we designed a new aluminum plate (cryo-plate) and developed a new cryopreservation method (V-Cryo-plate method) that allows easy manipulation of plant material for improvement of vitrification and droplet vitrification methods. As a result, we have established a cryopreservation procedure suitable for large scale storage in genebanks.

A new cryopreservation method for vegetatively propagated plant genetic resources using aluminum cryo-plates

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Future prospects 1. The cryo-plate method can be expected to facilitate and increase the use of cryopreservation for

stable and cost effective long term storage of vegetatively propagated plant genetic resources. 2. To increase the use of the cryo-plate method, it is necessary to expand it to additional crop

species, to publish a manual with inclusive application examples and to publicize its merits to potential users.

Fig. 1. An aluminium cryo-plate for cryopreservation

References 1. Yamamoto S, Rafique T, Priyantha W.S, Fukui K, Matsumoto T, Niino T (2011) Development

of a cryopreservation procedure using aluminium cryo-plates CryoLetters 32(3):256-265 2. Sekizawa K, Yamamoto S, Rafique T, Fukui K, Niino T (2011) Cryopreservation of in vitro-grown

shoot tips of carnation (Dianthus caryophyllus L.) by vitrification method using aluminium cryo-plates Plant Biotechnology 28(4):401-405

3. Yamamoto S, Fukui K, Rafique T, Khan N.I, Castillo Martinez C.R, Sekizawa K, Matsumoto T, Niino T (2012) Cryopreservation of in vitro-grown shoot tips of strawberry by the vitrification method using aluminium cryo-plates Plant Genetic Resources: Characterization and Utilization 10(01):14-19

Fig. 2. Procedures for cryopreservation of tropical mulberry using the V-crio-plate method