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Brief Report

Cryopreservation of orchid seeds through rapid and step freezing methods

[version 1; peer review: 1 approved, 2 approved with reservations]
PUBLISHED 20 Feb 2018
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Abstract

Ecuador has a great variety of climatic regions that potentiate biodiversity. The family Orchidaceae constitutes one of the most important of the country, having identified about 4032 species with a high degree of endemism, therefore the development and research of alternative methods of storage and conservation of species is a strategy of primary interest for researchers and for society in general. In cryopreservation, temperatures reach below -190°C in order to paralyze the chemical reactions and keep the plant material viable for long periods. The present research focuses on the development of protocols for cryopreservation of seeds, aimed at the preservation of biodiversity, focusing on the family Orchidaceae, for the subsequent generation of a seed bank. The assays were performed on seeds of Epidendrum quitensium, Sobralia rosea, and Epidendrum anderssonii. Two freezing rates were tested: rapid freezing at -196°C; and step freezing at -22°C, -60°C to 196°C, further analyzed four combinations from Dimethylsulfoxide DMSO, glycerol and sucrose (DMSO 1M; DMSO 1M + glycerol 1M; DMSO 1M + sucrose 1M; DMSO 1M + glycerol 0,5M + sucrose 0,5M). The best results were obtained both in rapid and stepped freezing without the use of cryo-protective substances, by introducing the seeds directly into liquid nitrogen. Species of the genus Epidendrum presented a more efficient response in comparison to Sobralia. The viability of the seeds was evaluated by the tetrazolium test.

Keywords

Orchidaceae, Epidendrum, Sobralia, seeds, cryoconservation, liquid nitrogen, Tetrazolium.

Introduction

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.

Methods

Collection of biological material

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. 0820130869−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)

292f551a-5feb-443b-b049-348bcab21278_figure1.gif

Figure 1. Orchids used for cryopreservation tests.

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.

Freezing speed

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).

Table 1. System design and freezing seed symbology used for cryoprotective substances and their concentrations.

M: molar.

TYPE OF FREEZING
GRADUAL (P)Rapid (F)
0° ___ -22° ___ -60° ___ -196-196°
CRYOPRESERVANTESSYMBOLCONCENTRATION
NONEN
DMSOD1M
GLYCEROLG1M0,5M
SUCROSES1M0,5M
COMBINATION OF CRYOPRESERVANTES
NONEN
DMSO
1M
D
DMSO
1M
GLYCEROL
1M
DG
DMSO
1M
SUCROSE
1M
DS
DMSO
1M
GLYCEROL
0,5M
SUCROSE
0,5M
DGS

Seed viability

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.

Statistical analysis

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).

Table 2. Experimental design, testing orchid seeds cryopreservation - design 2x5 with three repetitions, Symbols (N: none, D: DMSO, G: glycerol, S: sucrose, P: Freeze steps, R: Rapid).

STEP (P)FAST (R)
P1P2P3R1R2R3
NPN1PN2PN3RN1RN2RN3
DPD1PD2PD3RD1RD2RD3
DGPDG1PDG2PDG3RDG1RDG2RDG3
DSPDS1PDS2PDS3RDS1RDS2RDS3
DGSPDGS1PDGS2PDGS3RDGS1RDGS2RDGS3

Results

The seeds were considered viable when red coloration of the embryo was observed8 (Figure 2).

292f551a-5feb-443b-b049-348bcab21278_figure2.gif

Figure 2. TTC-stained seeds subjected to the "stepped" cryopreservation process without any cryopreservation substances.

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).

292f551a-5feb-443b-b049-348bcab21278_figure3.gif

Figure 3. Seed cryopreservation: variability by treatment.

Results obtained using the Tukey test.

292f551a-5feb-443b-b049-348bcab21278_figure4.gif

Figure 4. Cryopreservation of seeds: variability by species. Results obtained using the Duncan test, sp2706 (T57.2), sp2392 (T 57.03), sp2420(T39.37).

Table 3. Cryopreservation of orchid seeds.

Values represent percentage of viability assessed by the TTC method, N: cryo preservative; D: DMSO; S: sucrose; G: glycerol.

Step
CISpeciesNDDGDSDGS
2392Epidendrum
quitensium
83.2054.8783.2046.3647.01
2420Sobralia rosea55.9312.2910.7219.3418.88
2706Epidendrum
anderssonii
93.5040.8851.8162.8455.94
Rapid
2392Epidendrum
quitensium
74.7948.2962.8752.7951.73
2420Sobralia rosea65.6050.1555.0452.7252.52
2706Epidendrum
anderssonii
84.4918.5741.0763.0860.10

Table 4. Duncan test groups obtained after cryopreservation test.

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.

#TreatmentMean
1RN78.00a
2PN74.66a
3RDG56.66b
4RDS56.00b
5RDGS53.33bc
6RD46.55bc
7PDS43.00cd
8PDGS41.88cd
9PDG33.33de
10PD28.55e
Dataset 1.Dataset 1. TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum quitensium.
http://dx.doi.org/10.5256/f1000research.13622.d194233
Dataset 2.Dataset 2. TTC-stained seeds subjected to the “Rapid” cryopreservation process: Sobralia rosea.
http://dx.doi.org/10.5256/f1000research.13622.d194234
Dataset 3.Dataset 3. TTC-stained seeds subjected to the “Rapid” cryopreservation process: Epidendrum anderssonii.
http://dx.doi.org/10.5256/f1000research.13622.d194235
Dataset 4.Dataset 4. Percentage for seed viability calculations.
http://dx.doi.org/10.5256/f1000research.13622.d194236

Discussion

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 others1214. 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.

Data availability

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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Cerna M, Valdivieso P, Cella R et al. Cryopreservation of orchid seeds through rapid and step freezing methods [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:209 (https://doi.org/10.12688/f1000research.13622.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions
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PUBLISHED 20 Feb 2018
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Reviewer Report 11 Jul 2018
Éva Borbélyné Hunyadi, Research Institute for Organic Agriculture (ÖMKi), Budapest, Hungary 
Approved
VIEWS 9
In the paper entitled "Cryopreservation of orchid seeds through rapid and step freezing methods" written by Marco Cerna et al. two types of freezing technique were tested by the seeds of 3 species:
Epidendrum quitensium Rchb.f., Sobralia rosea Poepp. ... Continue reading
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Hunyadi ÉB. Reviewer Report For: Cryopreservation of orchid seeds through rapid and step freezing methods [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:209 (https://doi.org/10.5256/f1000research.14799.r33998)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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14
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Reviewer Report 09 Apr 2018
Song-Jun Zeng, Key Laboratory of South China Agricultural Plant Molecular Analysis and Gene Improvement & Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China 
Approved with Reservations
VIEWS 14
The authors described a protocol for cryopreservation of seeds of three tropical orchids in liquid nitrogen (LN). They tested rapid and progressive cooling with the controlled temperature at 0°C, -22°C, -60°C and -196°C. They also included the application of three ... Continue reading
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Zeng SJ. Reviewer Report For: Cryopreservation of orchid seeds through rapid and step freezing methods [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:209 (https://doi.org/10.5256/f1000research.14799.r32906)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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Reviewer Report 28 Mar 2018
Alzbeta Novotna, Department of Plant Taxonomy and Nature Conservation, University of Gdańsk, Gdańsk, Poland 
Approved with Reservations
VIEWS 17
The authors describe in the presented manuscript titled “Cryopreservation of orchid seeds through rapid and step freezing methods” a new protocol for cryopreservation of seeds of three tropical orchids in liquid nitrogen (LN). They tested rapid and progressive cooling with ... Continue reading
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CITE
HOW TO CITE THIS REPORT
Novotna A. Reviewer Report For: Cryopreservation of orchid seeds through rapid and step freezing methods [version 1; peer review: 1 approved, 2 approved with reservations]. F1000Research 2018, 7:209 (https://doi.org/10.5256/f1000research.14799.r31828)
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Comments on this article Comments (0)

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Alongside their report, reviewers assign a status to the article:
Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested
Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.
Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions
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