Keywords
Orchidaceae, Epidendrum, Sobralia, seeds, cryoconservation, liquid nitrogen, Tetrazolium.
Orchidaceae, Epidendrum, Sobralia, seeds, cryoconservation, liquid nitrogen, Tetrazolium.
The Republic of Ecuador is located on the South American continent. From north to south the country is crossed by the Andes mountain range and has four climatic regions: Coast, Andes, Amazon and the Insular region1. Its position in the middle of the world, the luminous intensity, the ocean currents and the different altitudes produce 82 types of ecosystems (see Ministry of Environment document on ecosystems in Ecuador) There is a great variety of climatic regions that have an important effect in the diversification of plant formations2. Concerning the Orchidaceae family, in Ecuador as of 2010, 4032 species of orchids have been identified, of which 1714 (42.5%) are endemic3; 4.5% of the orchids of the planet are found in Ecuador. Seed banks allow the conservation of the biodiversity ex situ and prioritize species used for food, medicine and those in danger of extinction. Orchidaceae is a large family with many endangered species and all of them are included in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) I and II4. Cryopreservation is an efficient strategy to safeguard these species, but unfortunately, orchid seeds have short lifetimes5; the longevity depends on the moisture content and storage temperature, so it is necessary to experiment with efficient storage systems for each species5. The advantages of cryopreservation are: storage for an indefinite period, genetic stability of the individuals, reduced infrastructure, can have independent energy and the stored genetic material does not require manipulation6.
Therefore, the objective of this research was to define protocols for cryopreservation of orchid seeds, in order to install a seed bank that promotes the conservation of vulnerable species.
The collection of plant material was made through the authorization of the Ministry of Environment of Ecuador No. 17-2011- Investigación-B- DPMS/MAE,FloraX, N0. 08−2013−0869−I CFAU−F LO−DAPI −UNO−MAE and the Botanical Garden "Orquídeas de Sarina" patent No. 006-2015- FLO-DPAP- MA.
The cryopreservation tests were developed with the seeds of 3 species: Epidendrum quitensium Rchb.f., Sobralia rosea Poepp. & Endl. and Epidendrum anderssonii Hágsater & Dodson (Figure 1). The cryopreservation tests were developed with 3 species:
• 2392 Epidendrum quitensium Rchb.f., (0° 17’52.1"N 78° 22’33.3"W 3200 msnm)
• 2420 Sobralia rosea Poepp.& Endl. (0° 52’11.8"N 78° 26’53.8"W 600 msnm)
• 2706 Epidendrum anderssonii Hágsater&Dodson (0° 50’36.2"N 78° 25’01.5"W 1200 msnm)
A) Epidendrum quitensium, B) Sobralia rosea, C) Epidendrum anderssonii.
The species pertain to three different altitudes and were selected from many sources and have capsules with viable seeds. The seeds collected from the forest were stored in an absorbant paper bag with respective codes for the plant, after they were stored in a Ziplock bag with rice of 12% humidity.
Two types of freezing were tested, suggested according to Mroginski et al7. The sample units had 0.2 g of seeds stored in cryo tubes (091.11.102, ˙ISOLAB, Wertheim, Germany) of 2 ml. Steps of freezing: freezing was carried out in the following sequence, 0°C for 1 hour by placing the samples in an refrigerator (Electrolux, Stockholm, Sweden), -22°C for 1 hour placing the seeds in a freezer (Selecta Templow, Barcelona, Spain), - 60°C for 1 hour inserting the seeds in an ultra low temperature freezer (New Brunswick Scientific, Edision, NJ, USA), then the seeds were held at 196°C by submerging the samples in liquid nitrogen contained in a thermal container. Finally the samples were placed in racks and stored in a thermal tank (STATEBOURNE biorack 5400, Washington, UK). Rapid freezing: the samples were placed directly in liquid nitrogen at 196°C by immersion using a procedure similar to that used in steps of freezing. In addition, four combinations of cryo preservatives were analyzed: 1- DMSO 1M (Fisher Scientific, Hampton, NH, USA); 2- DMSO 1M (Fisher) – glycerol 1M; 3- DMSO 1M (Fisher) – sucrose 1M; 4-DMSO 1M (Fisher) – glycerol 0.5M – sucrose 0.5M (Fisher) (Table 1).
M: molar.
Seed viability was tested after freezing. Briefly, 5mg of seeds was added to 1.5 ml of 10% sucrose solution and left at 25° C for 24 hours, the seeds were washed with water and 1ml of triphenyl tetrazolium chloride solution (TTC, 1%) (Sigma-Aldrich, St Louis, MI, USA) was added, and then incubated at 40° C for 24 hours. Finally, the seeds were washed with sterilised water and observed under the microscope with a 4x lens (MC100Led, MI-CROS, St. Veit/Glan, Austria). The process for calculating the TTC method was carried out as follows: -Observe the seeds in microscope using lense 4X. -Identify viable seeds and non viable seeds. -Use cross multiplication to determine the average of viability of all seeds.
The experimental design 2x5 with three repetitions was applied to analyse the freezing methods (Table 2). The results were analyzed by unidirectional ANOVA with 95% confidence. To determine the best treatments the Duncan test was used. This analysis was carried out with RStudio 3.1 (package: Agricolae).
STEP (P) | FAST (R) | |||||
---|---|---|---|---|---|---|
P1 | P2 | P3 | R1 | R2 | R3 | |
N | PN1 | PN2 | PN3 | RN1 | RN2 | RN3 |
D | PD1 | PD2 | PD3 | RD1 | RD2 | RD3 |
DG | PDG1 | PDG2 | PDG3 | RDG1 | RDG2 | RDG3 |
DS | PDS1 | PDS2 | PDS3 | RDS1 | RDS2 | RDS3 |
DGS | PDGS1 | PDGS2 | PDGS3 | RDGS1 | RDGS2 | RDGS3 |
The seeds were considered viable when red coloration of the embryo was observed8 (Figure 2).
Viable seeds (dark red embryos) and non-viable (pale embryos). A) Epidendrum quitensium, B) Sobralia rosea, C) Epidendrum anderssonii.
According to the data obtained (Table 3, Figure 3), there is a significant difference in the results when comparing the data between the species and between the treatments. According to the Duncan test, the best treatments were rapid freezing and step freezing without the use of cryopreservatives. The least efficient treatment was step freezing with the use of DMSO as a cryopreservant (Table 4). The species Epidendrum quitensium and Epidendrum anderssonii showed better results (Figure 4).
Values represent percentage of viability assessed by the TTC method, N: cryo preservative; D: DMSO; S: sucrose; G: glycerol.
Results are given as orchid seed viability percentage; a, b, c and d, indicate groups with statistical significance. Classification was made under an alpha of 0.01, and 78 degrees of freedom for error. Symbols (treatment): N: none, D: DMSO, G: glycerol, S: sucrose. Symbols (types of freezing) P: Freeze steps, R: Rapid.
# | Treatment | Mean | |||||
---|---|---|---|---|---|---|---|
1 | RN | 78.00 | a | ||||
2 | PN | 74.66 | a | ||||
3 | RDG | 56.66 | b | ||||
4 | RDS | 56.00 | b | ||||
5 | RDGS | 53.33 | b | c | |||
6 | RD | 46.55 | b | c | |||
7 | PDS | 43.00 | c | d | |||
8 | PDGS | 41.88 | c | d | |||
9 | PDG | 33.33 | d | e | |||
10 | PD | 28.55 | e |
Currently, cryopreservation is a safe and cost-effective option for the conservation of endangered species9. In the present investigation, a protocol was developed for cryopreservation of orchid seeds that provides a high percentage of viability, is easy to apply and economical. The seeds of orchids frozen at -196°C can be kept alive with a moisture content of 12% and do not require cryo-protective substances, confirming what is described by Iriondo et al. and others10,11. The use of cryopreservatives is recommended for seeds with a high moisture content, as stated by Reed and others12–14. Furthermore, Harding15 states that it is necessary to demonstrate the genetic stability of plants regenerated from cryopreserved plant material to approve their release and reintroduction into the environment; but to date, there have been no reports showing changes at the phenotypic, biochemical, chromosomal or molecular levels attributed to storage systems by cryoconservation14. The cryoconservation method that gave the best results was the “Rapid” freezing without the addition of any cryopreservative substance.
Dataset 1: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum quitensium 10.5256/f1000research.13622.d19423315
Dataset 2: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Sobralia rosea 10.5256/f1000research.13622.d19423416
Dataset 3: TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum anderssonii. 10.5256/f1000research.13622.d19423517
Dataset 4: Percentage for seed viability calculations 10.5256/f1000research.13622.d19423618
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Isolation, molecular determination and cryopreservation of orchid mycorrhiza fungi and associated bacteria. Experienced with orchid seed banking techniques.
Alongside their report, reviewers assign a status to the article:
Invited Reviewers | |||
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Version 1 20 Feb 18 |
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