Cryopreservation of orchid seeds through rapid and step freezing methods

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.

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 region 1 . 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 formations 2 . Concerning the Orchidaceae family, in Ecuador as of 2010, 4032 species of orchids have been identified, of which 1714 (42.5%) are endemic 3 ; 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 II 4 . Cryopreservation is an efficient strategy to safeguard these species, but unfortunately, orchid seeds have short lifetimes 5 ; the longevity depends on the moisture content and storage temperature, so it is necessary to experiment with efficient storage systems for each species 5 . 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 manipulation 6 .
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 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 al 7   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).

Results
The seeds were considered viable when red coloration of the embryo was observed 8 (Figure 2).
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).  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).

Discussion
Currently, cryopreservation is a safe and cost-effective option for the conservation of endangered species 9 . 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 others 10,11 . The use of cryopreservatives is recommended for seeds with a high moisture content, as stated by Reed and others 12-14 . Furthermore, Harding 15 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 cryoconservation 14 . The cryoconservation method that gave the best results was the "Rapid" freezing without the addition of any cryopreservative substance.

Competing interests
No competing interests were disclosed.

Grant information
The author(s) declared that no grants were involved in supporting this work. The authors present step freezing and rapid freezing methods for cryopreservation purposes. There was a significant difference in the results between the species and between the treatments. The cryoconservation method that provided the best results was the "Rapid" freezing, without the addition of any substance.
The introduction is well structured, helping the readers to understand the issue the method tries to solve. Considering that it is a research note the methodology is very detailed.
Nevertheless, in my opinion it is important to highlight the viability of the developed method on a wider spectrum if possible. In the current form of the Discussion, only general statements can be found regarding the viability of the developed method.
A more concrete concluding sentence is missing that could answer the following question: For what kind of other plants, could this method be useful?
If the developed method can be applied "only" for orchid seeds, a concluding sentence should be placed in the Discussion, stating the limiting factors (in terms of species) of the method. Although in this study only 3 species were examined, I believe that its important to inform the readers about the authors' recommendation regarding the potential usefulness in other species.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes 1.

If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 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 different cryoprotectants such as DMSO, glycerol and sucrose. The viability of tested seeds was checked using tetrazolim test. The protocol might have potential for the cryopreservation of these orchids. However, the manuscript cannot be accepted to publish at present and need major revision for the following main reasons:.
Abstract: Authors could not bring the conclusion 'Species of the genus presented a Epidendrum more efficient response in comparison to .', because only one species was be tested in the Sobralia this manuscript.    Figure 1: Epidendrum quitensium, Sobralia rosea, Epidendrum anderssonii should be italic. Figure 2: A) , B) , C) Epidendrum quitensium Sobralia rosea Epidendrum anderssonii A, B, C should be labelled in Photos. Discussion: The discussion should be rewritten. There were few relevant literatures of orchid Cryopreservation were cited in this manuscript. There have lots of related literatures for orchid Cryopreservation at present, which should be discussed and what is the innovation of the manuscript?
All the source data should be executed by statistical analysis.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. 28

Department of Plant Taxonomy and Nature Conservation, University of Gdańsk, Gdańsk, Poland
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 the controlled temperature at 0°C, -22°C, -60°C and -196°C. They also included the application of three different cryoprotectants such as DMSO, glycerol and sucrose. The viability of tested seeds was checked using tetrazolim test. Red coloured embryos were considered as the main indicator of the preserved viability of the tested seeds. I suppose that this work brings a novelty to a cryoscience research, especially due to the geographical origin of the studied material. Neotropical Orchidaceae should be prioritized in various research studies for their ecological vulnerability and/or a shortage of knowledge. Especially, the research achievements from such country as Ecuador (the orchid biodiversity hotspot), are important for science. However, numerous modifications and a deep correction of the text should be accomplished.
English correction is strongly recommended throughout the whole text. There are many errors in English grammar in the current form of the manuscript, which cause huge difficulties in text reading. Abstract: The authors should be focused on the topic of cryopreservation. The first two sentences in the abstract are out of topic. Moreover, the number of the orchid species (provided number 4032) is not correct. The authors should consider to check the work of D. Neill published in 2015 (¿Cuantas especies nativas de plantas vasculares hay en Ecuador?; Revista Amazónica Ciencia y Tecnología) and Cataloque of the Vascular Plants of Ecuador available on , for www.tropicos.org example. Abstract, sentence "In cryopreservation, temperatures reach below -190°C" should be rephrased because the cryostorage is a long-lasting preserving of studied material at temperature of -80°C, -130°C upto -196°C, which is the temperature of the liquid nitrogen. Abstract, the authors should rephrase the sentence "The present research focuses on……" because of repetition of the verb to focus. Instead of "…for a subsequent generation of a seed bank" to use "for future seed collection" for example. Abstract, "the use of cryo-protective substances", better to use the term "cryoprotectants". Introduction; The first half of the abstract is out of the topic of cryopreservation. The authors should be focused on this topic. Moreover, the authors should provide much more citations regarding this topic and discuss shortly the history of cryopreservation of seeds with more interest in orchid seeds. There are many available papers regarding this topic. Methods; The first paragraph should be included in "Acknowledgement" at the end of the paper, not in methods. However, each part of the Methods should be provided with a number, e.g. 1. 1 Collection of the seed material. The sentence "The cryopreservation test were developed with 3 species" is repetition of the first sentence of this paragraph. The authors should use "a.s.l."as the abbreviation for above sea level; the provided abbreviation "msnm" is not understandable. The part of the sentence" The species pertain to three different altitudes….. " is a repetition of the information written before. Authors write "…..were selected from many sources", what does it mean? The authors should provide more detailed information about the sources. Then, the part of the sentence …"and have capsules with viable seeds" does not bring the clear information. Were the capsules opened during collection or not? When did the authors collect the seeds ( In the paragraph "Freezing speed" the authors provide information about placing the tested orchid seeds under the temperature 0°C in a refrigerator. However, this fact is not written in the abstract. The authors should eliminate "…., contained in a thermal container". The sentence "Finally the samples were places in racks and store in thermal tansk……" should be eliminated, because it is not understandable. Freezing speed; In addition,….should be eliminated because the application of cryoprotectants is one of the main objectives of this research paper. It is not something additional. Why did the authors not test application of sucrose and glycerol individually? The authors should clearly demonstrate, which treatment is considered as a control. Table 1 should be deleted, because all the information given in this table is written in the text. Figure 1; Title should be improved, e.g. "Orchid species used in this study". The paragraph of "Seed viability" should be eliminated. Instead just one sentence with a citation of the work, where this method was used as first, should be placed at the end of the previous paragraph. Statistical analysis; The authors should explain better of the meaning "The experimental design 2x5". It is not clear from the written text. Results; The sentence "The seeds were considered viable when red….." is actually the part of Material and methods. Results; this part should be re-written. The information should be provided more clearly. Table 3; What is CI? Better to use term "Gradual" instead of "step". The abbreviations of each treatment are already provided in Material and Methods. Figure 3; Authors should provide information about the meaning of "E" (in e.g. ED, EDG) and "R" (in e.g. RD, RDG). Figure 4; Authors should provide information about the meaning of sp2392, sp2420 and sp2706. It is not clear. Discussion; First, the obtained main result should be provided and discussed with much more available literature. Especially, the authors should compare their findings with achievements in other studies. The provided references are very limited. The works dealing with cryostorage of seeds of other plant families should be included. The given number of cited paper in discussion does not correspond to the number provided in the References.

Are sufficient details of methods and analysis provided to allow replication by others? Partly
If applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
No competing interests were disclosed.

Competing Interests:
Referee Expertise: Isolation, molecular determination and cryopreservation of orchid mycorrhiza fungi and associated bacteria. Experienced with orchid seed banking techniques.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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