Cryopreserved Seeds Research Articles

Cryopreservation of Seeds of the Highly Valued Tropical Timber Species Swietenia Mahagoni

BACKGROUND: Swietenia mahagoni wood is one of the most valuable in world trade and, as a result, natural populations have been decimated due to unsustainable harvesting. The decline in natural population levels is being exacerbated by climate change. In order to ensure the preservation of valuable genotypes, there is an urgent need to develop strategies to conserve the genetic diversity present within this species. At present, cryopreservation is the most viable option for the long-term storage of plant germplasm, particularly for long-lived species which are challenging to maintain in the field. OBJECTIVE:To cryopreserve intact seeds of S. mahagoni, with the dual goal of retaining the biosynthetic capacity of plants, which is critical since this species is highly valued for medicinal purposes. MATERIALS AND METHODS: Seeds at a moisture content of 6% were immersed in liquid nitrogen (LN) before warming and recovery. Plantlet establishment and growth were assessed over a period of 70 days and anthraquinone synthesis was determined in roots, stems and leaves. RESULTS: The results showed an initial lag in the germination rate of cryopreserved seeds compared with control seeds; however, this difference disappeared over time. The lag in seedling emergence observed in cryostored seeds was also evident in the plant characteristics measured following 30 days of culture when all plant parameters measured were significantly higher in plants produced from control than cryostored seeds. However, after 70 days of growth, these differences were no longer apparent. Anthraquinone levels were also initially lower (at 30 days) in plants regenerated from cryopreserved seeds than those from control seeds, however, this difference was substantially reduced by 70 days thereby indicating the ability of these plants to accumulate secondary metabolites, albeit at a reduced rate, during the early stages of development. CONCLUSION: In S. mahagoni, the delay in anthraquinone production in plants regenerated from cryostored seeds during the early stages of development may have occurred as a consequence of the preferential allocation of resources towards the initiation of recovery processes in response to the stresses imposed by cryopreservation. Once the stresses were overcome and plant growth resumed, resources could be directed to secondary processes such as anthraquinone synthesis.

Exposure Of Calophyllum Antillanum Seeds To Liquid Nitrogen Delays Seedling Emergence And Decreases Leaf Anthraquinones

BACKGROUND: Trees within the Calophyllum genus are multi-use trees that produce valuable wood, phytochemicals with a range of biological activities, and seed oil as a source of biodiesel. As a consequence of climate change, there is a need to develop strategies to preserve valuable plant genetic resources. Cryopreservation represents the most suitable option for the long-term storage of germplasm with minimal space and maintenance requirements. OBJECTIVE: To determine appropriate methods to cryopreserve seeds of Calophyllum antillanum and maintain secondary compound production. MATERIALS AND METHODS: Seeds at a moisture content of 6% were used to evaluate two treatments: seeds immersed in liquid nitrogen and control seeds. Biosynthetic pathway efficiency was assessed post-cryo by determining anthraquinone contents in roots, stems and leaves following 30 and 75 d of seedling growth. RESULTS: The results indicated that exposure to liquid nitrogen delayed germination and seedling emergence for a period of up to 45 d after seed sowing. By 60 d of cultivation, no significant differences in plant growth were observed for cryostored and control seeds. The levels of anthraquinones, which were also measured in seeds and seedlings, were lower in plants regenerated from cryostored seeds following 30 d of growth, but there were no differences in roots and stems by 75 d of growth. Furthermore, the difference in leaf anthraquinone levels for cryopreserved and control seeds at 75 d was much smaller than at 30 d. CONCLUSION: The low initial anthraquinone levels in emerging seedlings correlated with the initial slow growth of cryopreserved seeds.

Physiological, biochemical, and ultrastructural aspects of Coffea arabica L. seeds under different cryopreservation protocols

ABSTRACT Cryopreservation is a technique that may potentially conserve the germplasm of species of the Coffea genus for an indeterminate time. The aim of this study was to evaluate the physiological, biochemical and ultrastructural characteristics of cryopreserved seeds of Coffea arabica L., cultivar Catucaí amarelo IAC 62, which was subjected to different protocols regarding dehydration, precooling, cooling, rewarming and cathode water use. According to each protocol, the seeds were subjected to fast or slow drying to moisture contents of 17 or 20% (wet basis), cooled in different ways, and then immersed in liquid nitrogen for 24 hours. Different rewarming times in a water bath were also used. Physiological, biochemical and ultrastructural analyses were performed on the seeds after the cryopreservation steps. Moisture content at a 17% wb is the key factor for the cryopreservation of Coffea arabica L. seeds, which have better physiological quality and better preserved cell structures. Precooling of coffee seeds before immersion in liquid nitrogen does not provide advantages compared to direct immersion. The rewarming times tested (2, 4, and 6 minutes) and cathode water use did not cause changes in the physiological and biochemical quality or in the cell structures of Coffea arabica L. cryopreserved seeds. The pattern of cell structure observed in all seeds indicates that the damage from cryopreservation is less drastic in the cells of the embryos than in those of the endosperm, with the latter less tolerant to the stresses of dehydration, precooling, and rewarming.

Influence of dehydration on cryopreservation of Musa spp. germplasm

Cryopreservation is an important technique for the long-term storage of economically important plant germplasm. In this study, an efficient protocol was developed for the long-term conservation of seven economically important Musa taxa: M. acuminata Colla ssp. burmannica N.W. Simmonds, M. acuminata Colla ssp. zebrina (Van Houtte) R.E. Nasution, M. balbisiana Colla, M. basjoo Sieb., M. ornata W. Roxburgh (St. Lavender), M. velutina H. Wendl. et Drude (Velvet Pink Banana), and M. acuminata’ balbisiana. The seeds were dehydrated in a sterile laminar flow cabinet for different exposure times and then they were directly immersed in liquid nitrogen. The critical point was to support the initial germination of cryopreserved seeds and this was achieved by the excision of zygotic embryos after liquid nitrogen treatment that allowed the seed germination. The best moisture content for tolerance to cryopreservation ranged from 15.8% (M. acuminata ssp. zebrina) to 17.1% (M. ornata) and the maximum post-cryopreservation germination rates varied from 86.4% (M. velutina) to 55.0% (M. ornata). All seedlings derived from seeds germinated after cryopreservation were easily rooted and acclimated to greenhouse conditions.

Efficacy of Passion Fruit Cryopreservation Using Cryopotectant Agents

ABSTRACT This study aimed to evaluate the effects of cryoprotectant agents on Passiflora eichleriana, Passiflora crystallina and Passiflora nitida seeds undergoing cryopreservation. The experiment was conducted with non-cryoprotected or non-cryopreserved seeds (control), cryopreserved seeds without cryoprotectant and cryopreserved seeds treated with the following cryoprotectants: 1.73, 2.28, 2.60 or 2.71 mol glycerol; 0.37, 0.46, 0.54 or 0.61 mol sucrose; or 0.37, 0.72, 1.04 or 1.35 mol dimethylsulfoxide (DMSO). After treatment, the seeds were cryopreserved in liquid nitrogen (LN2) for 7 days and then defrosted in 37°C water for 5 min. The seeds were submitted to germination and vigor tests consisting of four replicates of 50 seeds. Percentage of germination (PE) and germination velocity index, percentage of emergence velocity index, shoot and root length, and shoot and root dry mass were evaluated. After cryopreservation, all of germination and vigor values were higher without the use of cryoprotectants. The cryoprotectant glycerol had a deleterious effect on seed viability and vigor for all Passiflora species. Glycerol inhibit seedling emergence and concentrations higher than 2.60 mol seed germination on P. cristalina. Higher concentrations of DMSO decrease seed viability and vigor in P. crystalina and P. nitida.

Effect of desiccation on physiological and biochemical indicators associated with the germination and vigor of cryopreserved seeds of Nicotiana tabacum L. cv. Sancti Spíritus 96

Cryopreservation is a valuable technique for the long-term conservation of plant germplasm and complementary to traditional seed storage methods. However, critical factors such as seed moisture content should be optimized before using this technique as a safe strategy for storing seeds such as those of Nicotiana spp. This study aimed to determine the effect of desiccation on physiological and biochemical indicators associated with germination and vigor in cryopreserved seeds of Nicotiana tabacum cv. Sancti Spiritus 96 (SS-96). The germination and vigor of seeds with a range of moisture content were assessed using electrolyte leakage and accelerated aging tests. In addition, these physiological indicators were related to the oxidative state of the seeds, in terms of the rate of O2 ·− generation and the H2O2 content, and the activity of enzymatic antioxidants superoxide dismutase and catalase. The cryopreserved seeds of N. tabacum SS-96 with a moisture content of 2.1% exhibited higher vigor probably due to the retention of membrane integrity, reflected by lower levels of lipid peroxidation and electrolyte leakage associated with the absence of oxidative stress. The results suggest 2.1% as the optimal moisture content for the storage of seeds of this cultivar, both at cryogenic temperatures and at 5°C.

Exposure of Common Bean Seeds to Liquid Nitrogen Modifies Mineral Composition of Young Plantlet Leaves

Many publications describe cryopreservation techniques but only a few studies have focused on the biochemical and physiological changes occurring in plants regenerated from seeds exposed to liquid nitrogen. This paper aims at describing the effect of common bean seed cryostorage on mineral nutrition of young plantlets. The following elements were measured on leaves of 10-day-old plantlets from non-cryopreserved and cryopreserved seeds: Al, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, Se, Sr and Zn. At 10 days after sowing, both treatments (control and cryopreserved seeds) showed 100% seed germination without any visual phenotypic difference. However, contents of several elements in the leaves were different. Exposure of seeds to liquid nitrogen decreased Cu, Cd and Na uptake and increased absorption of B and Al. Further studies are required to understand the mechanisms underlying the relationship between seed exposure to liquid nitrogen and mineral nutrition during the early stages of plantlet growth.

Conductivity test in seeds of different passion flower species

The objective of this work was to evaluate the use of the conductivity test as a means of predicting seed viability in seven Passiflora species: P. alata, P. cincinnata, P. edulis f. edulis, P. edulis f. flavicarpa, P. morifolia, P. mucronata, and P. nitida. Conductivity of non-desiccated (control), desiccated, and non-desiccated cryopreserved seeds was determined and related to their germination percentage. The obtained results suggest that the electrical conductivity test has potential as a germination predictor for P. edulis f. flavicarpa seed lots, but not for the other tested species.

Cryopreservation the seeds of a Taiwanese terrestrial orchid, Bletilla formosana (Hayata) Schltr. by vitrification.

BackgroundThe cryopreservation of orchid seeds is an important conservation method, studies of the effects of cryopreservation on the seeds of wild orchids are scant. This investigation was to establish a method for the vitrification and cryopreservation of seeds of B. formosana that may be suitable for the long term storage of Taiwan native orchid germplasm for conservation purposes.ResultsThe germination rate and morphological stability of seeds from spontaneous-dehiscent capsules of Bletilla formosana (Hayata) Schltr. were evaluated after cryopreservation by vitrification. The germination rates of cryopreserved seeds varied according to immersion time and the vitrification method used. Seeds that were dehydrated by immersion in loading solution (LS; 2.0 M glycerol, 0.4 M sucrose) for 10 min to 30 min then transferred to plant vitrification solution 2 (PVS2) for 30 min prior to freezing in liquid nitrogen (LN) showed significantly higher germination rates than seeds immersed in PVS2 only. The optimal immersion times were 10 min for LS and 30 min for PVS2, resulting in an in vitro germination rate of 91%. Germination was not observed for cryopreserved seeds that were dehydrated by immersion in LS only. Seed viabilities and germination rates did not vary significantly for cryostorage times from 10 minutes to 1 year.ConclusionsThis study improve, an efficient protocol was established that maintained seed viability and enhanced the germination rates of seeds, compared with previously described cryopreservation methods, and the germinated seeds showed normal morphology of both vegetative and reproductive organs.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-33) contains supplementary material, which is available to authorized users.

Phenotypic and Molecular Characterization of <i>Phaseolus vulgaris</i> Plants from Non-Cryopreserved and Cryopre-served Seeds

The objective of this work was to evaluate if cryostorage of Phaseolus vulgaris L. seeds induced variations in regenerated plants at the phenotypic and molecular levels. A series of agricultural traits was measured on plants grown from control, non-cryopreserved and cryopreserved seeds, and the genetic stability of plants of the second generation was analysed at selected microsatellite loci. The phenotype of the second generation plants was evaluated as well. No statistically significant phenotypic differences were observed for the parameters measured, neither in the first nor in the second generations. Averaging both treatments, about 76% of the seeds had germinated 10 days after sowing. At harvest we recorded plants with about 73 cm in height, 13 stem internodes, 25 fruits, 103 grains and 4 grains per fruit. One hundred seeds weighted about 26 g. The genetic analyses performed on the second generation plants using six nuclear Simple Sequences Repeats (SSR) markers revealed no changes in microsatellite length between control and cryopreserved samples, implying that there was no effect of seed liquid nitrogen exposure on genome integrity. The phenotypic and molecular results reported here confirm that cryostorage is an efficient and reliable technique to conserve P. vulgaris seeds and regenerate true-to-type plants.

Cryopreservation of Dendrobium hybrid seeds and protocorms as affected by phloroglucinol and Supercool X1000

This study was conducted to investigate two cryopreservation protocols for Dendrobium Swartz. hybrid ‘Dong Yai’. Vitrification was evaluated for cryopreservation of mature seeds, while droplet-vitrification was evaluated for protocorms of the Dendrobium hybrid. The effectiveness of either phloroglucinol or Supercool X1000® as cryoprotectants was evaluated. In vitro germination, protocorm recovery, and seedling development of cryopreserved seeds and protocorms were assessed, respectively. For seed cryopreservation, the use of the PVS2 vitrification solution for 60min at 0°C containing 1% phloroglucinol resulted in 79% seed germination. For protocorm cryopreservation, pretreatment with sucrose 0.3M for 24h followed by exposure to PVS2 solution containing 1% phloroglucinol for 15min at 0°C resulted in 14% protocorm survival after 75 days, compared to 0–6% survival for controls and other treatments. Seeds were germinated and the seedlings produced showed normal growth and development, with no abnormalities. The addition of 1% Supercool X1000® was not effective as a cryoprotectant for seeds. Phloroglucinol at 1% was effective as a cryoprotectant for Dendrobium hybrid seed cryopreservation using the vitrification method. Phloroglucinol provided limited effectiveness for the cryopreservation of Dendrobium hybrid protocorms using droplet-vitrification, but recovery was enhanced as compared to controls and other treatments.

Effect of cryopreservation on seed germination and protocorm development of Vanda tricolor

Germination and subsequent development was assessed after seeds of Vanda tricolor were stored in liquid nitrogen. Mature seeds, harvested 7 months after self-pollination, were directly plunged into liquid nitrogen. Germination of cryopreserved seeds on solid New Dogashima (ND) medium supplemented with 1 mg/l 6-benzyladenine, 0.5 mg/l 1-naphthaleneacetic acid and 2% sucrose was faster than non-cryopreserved seeds (28 days versus 60 days after sowing). Immature seeds, harvested 6 months after self-pollination, were treated with or without loading solution (LS) of 2 M glycerol and 0.4 M sucrose in liquid ND medium, pH 5.4 at 25 °C for 15 min and dehydrated with PVS2 solution for 0-210 min on ice and then cryopreserved by vitrification. The results showed that the germination percentage of cryopreserved seeds treated with LS was higher than without LS. After 90 days of sowing, the highest germination percentage of cryopreserved seeds was 13.6% which was higher than non-cryopreserved seeds (10.5%) when seeds were treated with LS for 15 min and then dehydrated with PVS2 solution for 180 min. After 150 days of sowing, protocorms of non-cryopreserved and cryopreserved seeds were able to form new protocorms (budding protocorms) and developed into shoots after 180 days of sowing. There were no significant differences between growth and development of protocorms derived from non- cryopreserved and cryopreserved seeds.

Effect of cryopreservation, gibberellic acid and mechanical scarification on the seed germination of eight endemic species from the Canary Islands

Summary Seed germination of eight endemic species of the Canary Islands (Descurainia burgaeana, Lavatera acerifolia, Lotus mascaensis, Pimpinella cumbrae, Ranunculus cortusifolius, Scilla haemorrhoidalis, Solanum vespertilio and Spartocytisus supranubius) was studied after seed storage in liquid nitrogen (LN, -196 oC), soaking in gibberellic acid (GA3) and mechanical scarification. Lotus mascaensis and Pimpinella cumbrae are classified as “vulnerable” (VU) and Solanum vespertilio as “critically endangered” (CR) according to the IUCN (International Union for the Conservation of Nature) criteria. The germination trials were carried out under controlled conditions of light and temperature. For six of the eight species studied, the final germination percentages of cryopreserved seeds were higher than those of control seeds (non-cryopreserved seeds). Furthermore for three of the species this increase was significant. On the other hand for two of the species, seeds that had been cryopreserved reached a germination percentage lower (Solanum vespertilio) and equal (Spartocytisus supranubius) to the control seeds, however these decreases were not significant. The germination of Solanum vespertilio was significantly enhanced by soaking of the seeds in gibberellic acid (GA3) and the scarified seeds of Lotus mascaensis and Spartocytisus supranubius reached a significantly higher germination percentage than untreated seeds. The germination rate, expressed by the mean germination time (MGT), was significantly lower for two samples of cryopreserved seeds, three samples of seeds pretreated with GA3 and three samples of scarified seeds. Therefore, this study proves seed cryopreservation is a suitable procedure for the long-term seeds conservation of several endemic species from the Canary Islands. Experimental and discussion The eight species chosen in this study, belonging to seven different plant families, they are all endemic species of Canary Islands (Bramwell and Bramwell, 1990). Descurainia burgaeana Webb ex Schulz (Brassicaceae) and Lotus mascaensis Burch. (Fabaceae) are small shrubs endemics of Tenerife island. The shrubby species Spartocytisus supranubius (L.) Webb & Berth. (Fabaceae) and the small chamaephyte Pimpinella

In vitro germination and seedling development of cryopreserved Dendrobium hybrid mature seeds

In vitro germination and seedling development from Dendrobium Swartz. hybrid ‘Sena Red’, ‘Mini WRL’, ‘Jaquelyn Thomas’, and ‘BFC Pink’ seeds cryopreserved through vitrification (PVS2) were evaluated. Germination percentages after cryopreservation (LN) were variable among different controls and treatments, despite the initial high seed viability for all hybrids. Seeds exposed to PVS2 at ice temperature from 1 to 3 h prior to LN exhibited significantly higher germination than seeds exposed to PVS2 at room temperature for the same time periods. No significant differences in germination percentages were observed for time exposure to PVS2 at 1, 2 or 3 h for ‘Sena Red’, ‘Mini WRL’, and ‘BFC Pink’. Seeds of ‘Jaquelyn Thomas’ exposed to 1 h prior to LN showed higher germination percentage than for exposure to 2 or 3 h. The combination of a pre-cooling treatment (ice) with a dehydration treatment (PVS2) for a period of time of 1–3 h was essential to allow proper germination of cryopreserved seeds. Although variability in seed germination among different hybrids and treatments was observed, germination was above 50% of the controls and all germinated seeds developed into normal seedlings with healthy shoot and root formation. No abnormalities, nutritional deficiencies, or diseases were observed in developed seedlings and no significant differences were observed for seedling growth and development from germinated seeds among the different hybrids. Seedlings transplanted to pots acclimatized well and developed into normal plants within 6–8 months in greenhouse. Transplanted seedlings exhibited 100% survival for all hybrids.

Crioconservação em sementes de cebola

O presente trabalho teve como objetivo avaliar os efeitos de diferentes técnicas de crioconservação na qualidade fisiológica, composição química e índice mitótico de sementes de cebola. Sementes de cebola, cultivar Baia Periforme, foram expostas, durante uma hora, a duas técnicas de crioconservação: (a) sem e (b) com crioprotetor (glicerol 50%), seguidas de três métodos de descongelamento: (1) temperatura ambiente de 25°C, durante duas horas; (2) banho-maria a 37°C, durante cinco minutos; (3) microondas em potência de 70W, durante cinco minutos. Antes e após a crioconservação, foram realizadas as seguintes avaliações: teor de água, germinação, vigor (envelhecimento acelerado, primeira contagem de germinação e comprimento de raiz e de parte aérea das plântulas), composição química (teor de açúcar solúvel, proteínas e amido), análise do ciclo celular e índice mitótico. A imersão em nitrogênio líquido, sem o uso de crioprotetor, é uma técnica adequada para a crioconservação de sementes de cebola, não altera a percentagem de germinação e a composição química das sementes, sendo também o tratamento que proporciona maior número de células em divisão celular após o descongelamento das sementes. O tratamento criogênico, com uso de glicerol 50% como crioprotetor, é prejudicial à qualidade fisiológica e altera a composição química de sementes de cebola. O descongelamento das sementes em microondas é mais adequado do que em temperatura ambiente ou banho-maria, porém este último possibilita maior índice mitótico que os demais tratamentos.

The effect of seed cryopreservation on the development of protocorms by the hybrid orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.