**Cryopreservation of Orchid Genetic Resources by Desiccation: A Case Study of** *Bletilla formosana* Cryopreservation of Orchid Genetic Resources by

Desiccation: A Case Study of Bletilla formosana

Rung‐Yi Wu, Shao‐Yu Chang, Ting‐Fang Hsieh, Keng‐Chang Chuang, Ie Ting, Yen‐Hsu Lai and Yu‐Sen Chang Rung-Yi Wu, Shao-Yu Chang, Ting-Fang Hsieh, Keng-Chang Chuang, Le Ting, Yen-Hsu Lai, and Yu-Sen Chang

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http://dx.doi.org/10.5772/65302

#### Abstract

Many native orchid populations declined yearly due to economic development and climate change. This resulted in some wild orchids being threatened. In order to maintain the orchid genetic resources, development of proper methods for the long-term preservation is urgent. Low temperature or dry storage methods for the preservation of orchid genetic resources have been implemented but are not effective in maintaining high viability of certain orchids for long periods. Cryopreservation is one of the most acceptable methods for long-term conservation of plant germplasm. Orchid seeds and pollens are ideal materials for long-term preservation (seed banking) in liquid nitrogen (LN) as the seeds and pollens are minute, enabling the storage of many hundreds of thousands of seeds or pollens in a small vial, and as most species germinate readily, making the technique very economical. This article describes cryopreservation of orchid genetic resources by desiccation and a case study of Bletilla formosana. We hope to provide a more practical potential cryopreservation method for future research needs.

Keywords: long-term conservation, Bletilla formosana, Desiccation, Dry, Orchid, Seed, Pollen, genetic resources

#### 1. Introduction

Germplasm conservation is mostly applied for breeding purpose. Four methods are usually used in orchid preservation. The first method is more easy to preserve whole plant. It preserves the whole plant in the net-house or greenhouse, most orchid breeders follow this method, but

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

the orchid plants are often lost due to natural disasters, pests, diseases, and physiological disorders during cultivation process. The second method is to preserve orchid cells or tissues by tissue culture. Besides much labor requirements, a lot of problems may occur, such as genetic variation, germplasm pollution, and somatic cell clone variation during the continuous subculture process. The third method, dry storage or low temperature method has been carried out for the preservation of orchid genetic resources [1, 2]. In order to achieve a successful hybridization or a special breeding purpose, orchid breeders must preserve pollens from different flowering parents. Moreover, seeds of some important, economic value, particularly endangered species also need to be preserved. Depending on the equipment, cost, and convenience, orchid breeders often preserve pollens or seeds at 4°C in a refrigerator. However, this method does not get an acceptable result in keeping high viability of certain orchids for long period [3–5]. In addition, dry storage and low temperature methods used in case of many orchid seeds are only for short-term preservation for 1–6 months. Viability of most orchid seeds is significantly reduced after less than 1 year for preservation. Furthermore, the seeds of certain orchid species lose their viability quickly upon desiccation [6, 7]. Therefore, the last method, cryopreservation which is a long-term preservation technique has been researched and developed intensively for the need of orchid genetic resources preservation and the orchid industry. Cryopreservation is one of the most reliable methods for long-term conservation of plant genetic resources, because all metabolic processes and physicochemical changes are arrested at the cryogenic temperature (-196°C) [8, 9]. However, it is usually lethal to expose biological specimens to such low temperatures without any pretreatment because of intracellular freezing [4]. Vitrification and desiccation methods have been often used to preserve seeds by removing water from the cells [9–11] because the water content of plant materials may affect cryopreservation success. Orchid PLB (protocorm like body) conservation by combining encapsulation and dehydration has been suggested [12–14].

Bletilla formosana belongs to genus Bletilla in the family Orchidaceae. The species is distributed widely in Taiwan and is renowned for its ornamental value [5, 15, 16]. B. formosana is endangered due to the destruction of its habitat and over collection for ornamental use. Therefore, preservation of B. formosana is urgent to be proceeded. The purpose of this article is to review the cryopreservation of orchid germplasm, describe a practical method of long-term preservation for Bletilla formosana seeds, and to provide potential cryopreservation methods for other orchid species.
