Cryopreservation of orchid seed or pollen by desiccation prior to LN.
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.
- long‐term conservation
- Bletilla formosana
- genetic resources
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 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 . 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].
The process of cryopreservation preserves structurally intact living cells and tissues by cooling them to very low temperatures . Cryopreservation is one of the most effective methods for the long‐term conservation of plant germplasm at ultra‐low temperatures (–196°C) because through it, the vitality of cells is preserved despite the cessation of almost all of their biological activities [8, 9].During cryopreservation, degradation or somatic mutation phenomenon rarely occurs [4, 8, 9, 18, 19].
The advantages of cryopreservation are as follows:
The ability to preserve the vitality and regenerative potential of cells.
A requirement for minimal tissue to be effective, resulting in minimal space being used for operation.
The prevention of genetic variation and germplasm pollution, and the reduction of somatic cell clone variation rates.
The protection against damage from natural disasters, pests, and diseases by using liquid nitrogen (LN) as the storage material.
The reduction in labor requirements to accomplish the complicated process of subculture.
The possibility of being applied to vegetative propagation plants, nonseed propagation plants, transgenic plants, and gene banks.
3. Cryopreservation of orchid genetic resources: seed and pollen
The main purpose of the long‐term preservation of orchid seeds and pollens is to preserve endangered or economically crucial species. Since orchid seeds and pollens are minute, storing many hundreds of thousands of them in a single small vial is possible, making them ideal materials for long‐term preservation in LN. Furthermore, most species of orchids germinate readily. Thus, for both of these reasons, cryopreservation is economical and convenient . As reported in , maintaining the proper water content (WC) of seeds is critical for successful cryopreservation because excess moisture can result in ‘free’ water in tissues forming damaging ice crystals during freezing. In most species, exposing biological samples to such low temperatures without any WC pretreatment is typically lethal because of intracellular freezing . Therefore, pretreatment technologies, for example the vitrification and desiccation methods, have been developed  to use dehydration for the reduction of the WC of cells and avoid the formation of ice crystals from ultra‐low temperature preservation. Prior to ultra‐low temperature preservation, suitable pretreatment methods are used to increase the survival rate of the materials to be preserved. Three pretreatments, namely desiccation, vitrification, and encapsulation–dehydration, are typically applied for orchids [13, 21
According to the aforementioned reports, three cryopreservation methods are available for orchids.
3.1. Vitrification method
The vitrification technique was introduced by Sakai et al. and is typically used to preserve immature and mature seeds with a higher than average WC for extended periods. Preserved materials are sufficiently dehydrated osmotically by being placed in a high osmolarity vitrification solution (glycerol, dimethyl sulfoxide, and ethylene glycol), which alters their intracellular WC so as to vitrify them through the penetration of cryoprotectants. The chemicals used in this process are toxic. The functions of cryoprotectants are to reduce the amount of freezable water in seed tissue, reduce the freezing temperatures of the intracellular solutes, and inhibit ice nucleation and growth [24
The seeds of some orchids cannot survive, when preserved at cryogenic temperatures even with relatively low WC. For example, mature seeds of
Vitrification has been applied in the cryopreservation of immature and highly hydrated seeds of orchid species from several genera including
3.2. Desiccation method
The desiccation method is more suitable for mature seeds than for immature seeds. Narrow desiccation refers to when seeds are first dehydrated through slow drying with a controlled desiccation rate under a constant relative humidity (RH) or through rapid drying under a laminar flow chamber or with silica gel or a saturated salt solution of CaCl2.6H2O (approximately 33% RH) to reduce seed WC before LN preservation. For example,
|Species||Preserved material||Pretreatment before LN||Storage duration||Germination%||Reference|
|Mature seed||At refrigerator for 7 days-7 years||1 month||55||80|||
|MAPymature seed||Direct LN||1 day||77||2|||
|3MAP mature seed||Air dry|
dry over silica gel for 24 h
|2, 3, 4MAP immature mature seed||Direct LN||30 min||2 MAP:0.2|
|Mature seed||At 8°C for 403 days||1 month||100||100|||
|Mature seed||Dry over silica gel for 24 h||1 week||31||37|||
|Mature seed||Air dry for 1week →20°C||3-24 months||70-83||55-85|||
|Mature seed||Air dry for 1week →20°C||3-24 months||35-80||0-83|||
|Mature seed||Air dry for 1week →20°C||3-24 months||63-95||80-97|||
|Mature seed||At refrigerator for 7 days-7 years||1 month||59||90|||
|Mature seed||At refrigerator for 7 days-7 years||1 month||56||69|||
|Mature seed||At 5-6°C for 2-3 weeks||1 month||33||39|||
|Mature seed||Dry over a saturated salt solution of CaCl26H2O(ca. 33% RH)at 16°C||1-12 months||74||51|||
|Mature seed||At 5-6°C for 2-3 weeks||1 month||36||44|||
|Mature seed||At 5-6°C for 2-3 weeks||1 month||14||27|||
|Mature seed||At 5-6°C for 2-3 weeks||1 month||71||42|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||61||56|||
|Mature seed||Air dry for 1 week →20°C||3-24 months||<20||<20|||
|Mature seed||Air dry for 1 week →20°C||3-24 months||55-85||20-55|||
|Mature seed||Dry over silica gel for 24 h||1 week||37||44|||
|Mature seed||At refrigerator for 7 days-7 years||1 month||100||100|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||52||47|||
|Mature seed||In wit silica gel at 2°C for 8 months||15 min||68||70|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||63||58|||
|Mature seed||In wit silica gel at 2°C for 6-12 weeks||15 min||45||46|||
|Pollen||1.at -1°Cmin-1 by a controlled-rate freezer|
2. air dry 24 h
3. air dry 72 h
|Pollen||1. air dry: 0-30 min|
2. dry over silica gel:120 min
|48 h||1. 65-67|
|Mature seed||Air dry for 1 week→20°C||3-24 months||30-67||5-63|||
|Mature seed||At refrigerator for 7 days-7 years||1 month||100||100|||
|Pollen||AF, AN, PN x||3 days|
|Mature seed||In wit silica gel at 2°C for 6-12 weeks||15 min||43||43|||
|Mature seed||In wit silica gel at 2°C for 6-12 weeks||10 cycle(5 min each)||62||75-82|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||84||79|||
|Mature seed||At 5-6°C for 2-3 weeks||1 month||49||24|||
|Mature seed||Air dry for 1week→20°C||3-24 months||70-85||0-90|||
|Mature seed||Dry over silica gel for 24 h||1 week||49||50|||
|Mature seed||Air dry for 1 week→20°C||3-24 months||85-90||25-75|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||58||55|||
|Mature seed||No wit silica gel at 2°Cfor 6-12 weeks||15 min||62||62|||
|Mature seed||Dry over silica gel for 24 h||1 week||59||67|||
|Mature seed||Air dry for 1week→20°C||3-24 months||25-85||0-80|||
|Mature seed||Air dry for 1week→20°C||3-24 months||20||3-37|||
|Mature seed||At refrigerator for 7 days-7 years||1 month||83||90|||
|Mature seed||Not given||15 min||94||97|||
|Mature seed||Direct LN||1 day||11||1|||
|Immature seed||Direct LN||1 day||26||10|||
A generalized drying method should be included as the storage condition before LN, such as seeds stored in a low RH environment for a period or fresh seeds stored in a refrigerator, then directly immersed in LN. For example, harvested seeds of
The critical factor for successful cryopreservation through desiccation is the existence of the proper WC of tissue, which varies among species . However, because the orchid seed is tiny and light, accurately measuring its moisture context is technically difficult . Therefore, numerous orchid seeds are required to determine their moisture content (MC) prior to desiccation [4, 7, 11]. Moreover, seed viability after cryopreservation by desiccation varies among different species. The seeds of some species lose viability after drying to a specific level of WC. Some seeds remain highly viable after being air‐dried or exposed to a controlled desiccation rate under a constant RH or to rapid desiccation.
The advantages of the desiccation method are simple, practical, nontoxic, and low cost. Also, the method is more suitable for orchid seed with low WC . Therefore, it can be used for long‐term preservation of orchid seed by international plant germplasm centers and private companies.
3.3. Encapsulation‐dehydration method
The third cryopreservation method for orchid materials is encapsulation‐dehydration. This procedure is based on the technology developed for the production of artificial seeds . Preserved tissues encapsulated in alginate beads, pregrown in a liquid medium with enriched sucrose, partially desiccated in the air current of a laminar airflow cabinet or with silica gel to reduce the WC to a suitable level, and then directly immersed in LN .
Among the three aforementioned techniques, the encapsulation‐dehydration method is applied only for particular orchid species. For example, pretreatments prior to LN only improved germination of
4. Factors affecting cryopreservation
For successful cryopreservation, the required WC of tissue is a critical factor, and varies among species [21, 38]. However, studies have revealed that the survival of orchid seeds in storage is also affected by other factors [7, 42]. For example, the WC of preserved tissue is also affected by the desiccation time and the maturity of the preserved material [43, 44]. An inappropriate desiccation time also reduces the viability of the tissue. Furthermore, the WC of seeds gradually decreases after pollination . The impact of these factors is described as follows.
4.1. Water content
For most orchid species, for example,
Pretreatment techniques must be performed before preserving seeds in LN for most orchids. Previous research on plants such as
Some seeds of orchid species have exhibited sensitivity toward extreme desiccation and a reduced viability after short periods of storage under dry conditions (3–5% WC) at high subzero temperatures (e.g., −5°C to −20°C) generally applied in conventional seed banking regimes [4, 24, 36, 45, 48, 49]. The seeds of
Successful cryopreservation is closely related to the WC of plant tissue . Furthermore, the WC of seeds is related to seed maturity. For example, the seeds of
The cryopreservation methods also differ depending on the seed maturity level. The desiccation method is more suitable for mature seeds as a cryopreservation pretreatment. The vitrification method or the encapsulation‐dehydration method may be used for many immature and highly hydrated orchid seeds. The pretreatment in concentrated cryoprotectant solutions before rapid immersion in LN is critical for post‐cryopreservation survival of orchid seeds, for example,
4.3. Other factors
In addition to WC and seed maturity, successful cryopreservation is related to the type of orchid species . Studies have shown that the survival of orchid seeds in storage is affected by factors, such as desiccation time [7, 31, 42]. Desiccation time, by directly affecting the level of seed WC, is crucial for the survival of preserved tissue. Inappropriate desiccation time reduces tissue viability. For example, seed germination of
The values for the pollen germination and WC of the English walnut (
Long‐term preservation of plant genetic resources is not easy. Orchid seeds and pollens are ideal materials suitable for cryopreservation because of the characteristics of tiny volume and low water content. This article describes common pretreatment techniques used in orchid cryopreservation and the factors affecting material viability. Besides, this describes Taiwan's endangered native medicinal and ornamental plants, and the desiccation method applied in a case study of
5. Case study for cryopreservation of
Bletilla formosanaseeds (Orchidaceae) by desiccation
Mature seeds of
The viability and germination rates of fresh seeds of
The authors would like to thank Professor Fure‐Chyi Chen (National Pingtung University of Science and Technology) and Associate Professor Chen Chang (National Chung Hsing University) for assistance and guidance on the experiment.