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Desiccation sensitivity and cryopreservation of excised embryonic axes of Citrus suhuiensis cv. limau madu, Citrumelo [Citrus paradisi macf. × Poncirus trifoliata (l.) raf.] and Fortunella polyandra

Al Zoubi OM, Normah MN

Cryo Letters, 2012 May-Jun;33(3):241-51.
PMID: 22825791

Excised embryonic axes from seeds of three taxa, namely, Citrus suhuiensis cv. limau madu, Citrumelo (Citrus paradisi x Poncirus trifoliate) and Fortunella polyandra, were desiccated in a laminar airflow, over silica gel, and ultra-rapidly. Desiccation sensitivity (WC50) was estimated for each taxon using the quantal response model. High desiccation tolerance (WC50 = 0.11 g water per g dry mass. g/gdw) was observed for limau madu embryonic axes desiccated in a laminar airflow and ultra-rapidly (WC50 =0.10 g/gdw). Desiccation tolerance was substantially lower (WC50 = 0.19 g/gdw) for silica gel dehydration. Similarly, high desiccation tolerance (WC50 = 0.15 g/gdw) was associated with F. polyandra embryonic axes when desiccated in a laminar airflow, while a lower desiccation tolerance (WC50 = 0.17 g/gdw) was observed with silica gel dehydration. Ultra-rapid desiccation led to the highest desiccation tolerance (WC50 = 0.14 g/gdw). The dehydration rate, however, had no influence on desiccation tolerance (WC50 ~ 0.14 g/gdw) for Citrumelo embryonic axes. After each desiccation period, embryonic axes were directly immersed in liquid nitrogen (LN) followed by rapid rewarming. Normal seedling recovery of 80 to 83% for excised embryonic axes of limau madu was observed for laminar airflow and ultra-rapid dehydration, but for silica gel dehydration, 57% recovery was obtained. Similarly, for Citrumelo, high recoveries of 100% and 97% were obtained from axes desiccated in a laminar airflow and using ultra-rapid dehydration, respectively, whereas a lower value was associated with silica gel dehydration (80%). For F. polyandra, 50% recovery was obtained both for laminar airflow and ultra-rapid dehydration, while much lower recovery (43%) was associated with silica gel dehydration. Regardless of the drying method employed, axis survival percentages following exposure to LN were commensurate with the desiccation sensitivity pattern.
Cryopreservation of scalps of Malaysian bananas using a pre-growth method

Sipen P, Anthony P, Davey MR

Cryo Letters, 2011 May-Jun;32(3):197-205.
PMID: 21766149

The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 square cm) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light and 8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40 degree C for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg per liter benzylaminopurine, 0.2 mg per liter indole acetic acid and 10 mg per liter ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48 percent of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10 percent, 13 percent, 42 percent and 48 percent for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.
Cryopreservation of Garcinia cowa shoot tips by vitrification: the effects of sucrose preculture and loading treatment on ultrastructural changes in meristematic cells

Yap LV, Noor NM, Clyde MM, Chin HF

Cryo Letters, 2011 May-Jun;32(3):188-96.
PMID: 21766148

The effects of sucrose preculture duration and loading treatment on tolerance of Garcinia cowa shoot tips to cryopreservation using the PVS2 vitrification solution were investigated. Ultrastructural changes in meristematic cells at the end of the preculture and loading steps were followed in an attempt to understand the effects of these treatments on structural changes in cell membranes and organelles. Increasing preculture duration on 0.3 M sucrose medium from 0 to 3 days enhanced tolerance to PVS2 solution from 5.6 percent (no preculture) to 49.2 percent (3-day preculture). However, no survival was observed after cryopreservation. Examination of meristematic cells by transmission electron microscopy revealed the progressive accumulation of an electron-dense substance in line with increasing exposure durations to 0.3 M sucrose preculture. Treatment with a loading solution (2 M glycerol + 0.4 M sucrose) decreased tolerance of shoot tips to PVS2 vitrification solution and had a deleterious effect on the ultrastructure of G. cowa meristematic cells. This study suggests that G. cowa meristematic cells may lose their structural integrity due to exposure to glycerol present in the loading solution at a 2 M concentration, either due to its high osmotic potential, or due to its cytotoxicity.
Effect of preculture, pvs2 and vitamin C on survival of recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification

Chua SP, Normah MN

Cryo Letters, 2011 Nov-Dec;32(6):506-15.
PMID: 22227711

This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0 degree C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25 degree C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3 percent. It was only 53.3 percent when shoot tips were exposed to PVS2 at 25 degree C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.7 percent) without LN exposure, compared to 73.3 percent for shoot tips not treated with vitamin C. Moreover, 3.3 percent shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.
Cryopreservation of whole seeds and excised embryonic axes of Citrus suhuiensis cv. limau langkat in accordance to their desiccation sensitivity

Makeen MA, Noor NM, Dussert S, Clyde MM

Cryo Letters, 2005 Jul-Aug;26(4):259-68.
PMID: 19827255

Following the investigation of desiccation sensitivity and freezing tolerance of the whole seed of Citrus suhuiensis cv. limau langkat, desiccation sensitivity and cryopreservation of the excised embryonic axes from the seeds of the same species were examined. Three drying conditions were employed: desiccation by equilibrium for the whole seeds and desiccation in laminar airflow and over silica gel for the excised embryonic axes. The relevance of desiccation sensitivity (WC50) to cryopreservation of whole seeds and excised axes was investigated. High desiccation tolerance (WC50 = 0.034 g H2O x g(-1)dw) was acquired for axes desiccated with faster dehydration rate (1.5 g x g(-1) x h(-1)) in laminar airflow compared to substantially lower desiccation tolerance (WC50 = 0.132 and 0.110 g H2O x g(-1)dw) acquired under slower dehydration rates (1.0 and 0.005 g x g(-1) x h(-1)) for axes desiccated over silica gel and whole seeds desiccated by equilibrium respectively. While few whole seeds (8.3%) survived freezing, high recovery percentages of axes (83.3% and 62.2%) after freezing were obtained under laminar airflow and silica gel drying conditions respectively. Irrespective of the drying method employed, axes survival percentages after exposure to LN temperature commensurate with the desiccation sensitivity pattern. For the whole seeds, a factor other than desiccation sensitivity that limits the tolerance to exposure to LN temperature seems to exist and still needs to be defined.
Ultrastructural response of embryonic axes of Fortunella polyandra to dehydration and cryopreservation

Al Zoubi OM, Normah MN

Cryo Letters, 2015 Nov-Dec;36(6):379-91.
PMID: 26963884

To further understand the survival characteristics of desiccation-sensitive excised embryonic axes of Fortunella polyandra to desiccation and cryopreservation it is necessary to study the impact of drying rates on both the ultrastructure and electrolyte leakage.
Cryopreservation of Citrus madurensis embryonic axes by vitrification: importance of loading and treatment with vitrification solution

Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2002 Sep-Oct;23(5):317-24.
PMID: 12447491

This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.
Cryopreservation of Citrus aurantifolia seeds and embryonic axes using a desiccation protocol

Cho EG, Noor NM, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2002 Sep-Oct;23(5):309-16.
PMID: 12447490

The desiccation and freezing tolerance of seeds, with and without testas, and embryonic axes of Citrus aurantifolia were investigated. Seeds were desiccated with silica gel, under the laminar air flow cabinet or by placing them on a laboratory bench. Whatever the desiccation method employed, survival before and after cryopreservation was higher for seeds without testas. When freezing intact seeds, the highest survival percentage (41.3 %) was achieved after desiccation to 7.3 % moisture content (fresh weight basis) on the laboratory bench. Survival of seeds cryopreserved without testas could reach up to 85 % after desiccation under the laminar air flow cabinet or on the laboratory bench, corresponding to moisture contents of 7.1 and 4.5 %, respectively. After desiccation with silica gel, survival reached a maximum of 60.0 %, for a seed moisture content of 3.3 %. Survival of control embryonic axes was high (80-100 %) whatever the sucrose concentration in the preculture medium and the duration of the desiccation period. After cryopreservation, no survival was noted with embryonic axes, which had not been precultured nor desiccated. Survival of non-desiccated embryonic axes after cryopreservation increased progressively in line with increasing sucrose concentrations in the preculture medium, from 7.5 % with 0.1 M sucrose to 77.5 % with 0.7 M sucrose. Survival of desiccated and cryopreserved embryos was always high, whatever the preculture treatment and desiccation period, ranging from 55.8 % to 92.5 %.
Cryopreservation of Citrus madurensis embryonic axes encapsulation-dehydration

Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2002 Sep-Oct;23(5):325-32.
PMID: 12447492

In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.
Cryopreservation of Citrus madurensis zygotic embryonic axes by vitrification: importance of pregrowth and preculture conditions

Cho EG, Hor YL, Kim HH, Rao VR, Engelmann F

Cryo Letters, 2001 Nov-Dec;22(6):391-6.
PMID: 11788881

The role of pregrowth and preculture treatments in terms of both medium composition and exposure duration on survival of embryonic axes of Citrus madurensis after cryopreservation using the vitrification procedure was investigated. The optimal pregrowth treatment for excised embryonic axes was a 3-day treatment with 0.1M sucrose. Preculture was also essential in increasing survival after cryopreservation. Among the various media and treatment durations evaluated, a 24h-preculture of embryonic axes on medium with 0.3M sucrose and 0.5M glycerol was found to be optimal. Using these pregrowth and preculture conditions followed by treatment at 25 degrees C for 20 min each with a loading solution (0.4M sucrose + 2.0M glycerol) and then the PVS2 vitrification solution, direct immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2M sucrose solution for 20 min and transfer of embryonic axes on recovery medium, 82.5% survival and regrowth without intermediary callus formation were obtained with C. madurensis embryonic axes.
Development of a PVS2 droplet-vitrification cryopreservation technique for Aranda Broga Blue orchid protocorm-like bodies (PLBs)

Ping KS, Poobathy RR, Zakaria R, Subramaniam S

Cryo Letters, 2018 5 8;38(4):290-298.
PMID: 29734430

　　BACKGROUND: Conservation of commercially important ornamental plants is important to maintain its unique beauty to cater the market demands.
OBJECTIVE: The main objective is to develop an efficient cryopreservation technique for Aranda Broga Blue orchid PLBs using droplet-vitrification method.
MATERIALS AND METHODS: Several critical factors in cryopreservation were accessed such as preculture concentrations and durations, choice of vitrification solutions, two-step or three-step vitrification, growth recovery medium and PVS2 exposure duration.
RESULTS: The best growth regeneration percentage (5%) was obtained when 3-4mm PLBs were precultured in 0.2M sucrose for 3 days, followed by osmoprotection for 20 minutes, dehydration in PVS2 for 20 minutes at 0 degree C, LN storage, thawed and unloading for 20 minutes, and growth regeneration in VW10 medium. PLBs were found to be very sensitive to osmotic stress imposed by high molecular weight cryoprotectant such as sucrose and glycerol. Osmotic potential of growth recovery medium is one of the main factors that affect growth recovery in cryopreserved PLBs.
CONCLUSION: Current report showed possibilities in cryopreserving Aranda Broga Blue PLBs using droplet-vitrification technique. However, further improvement of growth recovery can be done by focussing on approaches that facilitate sufficient water removal from PLBs without causing severe osmotic injuries to the plant cells.
Improvement of sperm quality of the depik fish, rasbora tawarensis, after cryopreservation using antioxidant

Muchlisin ZA, Afriani D, Eriani K, Hasri I, Nur FM, Maulida S, et al.

Cryo Letters, 2022;44(1):13-19.
PMID: 36625871

BACKGROUND: The cryopreservation of the sperm of the depik fish, Rasbora tawarensis, has previously been developed. However, the quality of the sperm post cryopreservation was not satisfactory and might be improved through the application of antioxidants.
OBJECTIVE: To determine the most suitable antioxidant for the cryopreservation of the depik fish spermatozoa.
MATERIALS AND METHODS: A completely randomized design with a non-factorial experiment was used and the tested antioxidants were glutathione, beta-carotene, ascorbic acid, and butylated hydroxytoluene (BHT) at 6 % concentrations. All treatments had three replications. The sperms were collected from 10 male fishes and diluted with Ringer solution in a ratio of 1: 20 (v/v, sperm: Ringer solution). Then 5% DMSO and 5 % egg yolk were added to the diluted sperms. Furthermore, 6 % of the tested antioxidants were added to the diluents, and then, cryopreservation was carried out in liquid nitrogen for 14 days.
RESULTS: The ANOVA test showed that the application of antioxidants significantly affected the sperm motility, fertility, and hatching rates of the eggs (P < 0.05). Furthermore, the antioxidants also protected the sperm cells during cryopreservation, with glutathione being the best antioxidant.
CONCLUSION: The application of antioxidants during the cryopreservation of depik fish sperm had a significant effect on motility, fertility and hatchability of eggs post-cryo. Furthermore, glutathione was the most suitable antioxidant. doi.org/10.54680/fr23110110312.
Improvement of sperm quality of the depik fish, rasbora tawarensis, after cryopreservation using antioxidant

Muchlisin ZA, Afriani D, Eriani K, Hasri I, Nur FM, Maulida S, et al.

Cryo Letters, 2023;44(1):13-19.
PMID: 36629837

BACKGROUND: The cryopreservation of the sperm of the depik fish, Rasbora tawarensis, has previously been developed. However, the quality of the sperm post cryopreservation was not satisfactory and might be improved through the application of antioxidants.
OBJECTIVE: To determine the most suitable antioxidant for the cryopreservation of the depik fish spermatozoa.
MATERIALS AND METHODS: A completely randomized design with a non-factorial experiment was used and the tested antioxidants were glutathione, beta-carotene, ascorbic acid, and butylated hydroxytoluene (BHT) at 6 % concentrations. All treatments had three replications. The sperms were collected from 10 male fishes and diluted with Ringer solution in a ratio of 1: 20 (v/v, sperm: Ringer solution). Then 5% DMSO and 5 % egg yolk were added to the diluted sperms. Furthermore, 6 % of the tested antioxidants were added to the diluents, and then, cryopreservation was carried out in liquid nitrogen for 14 days.
RESULTS: The ANOVA test showed that the application of antioxidants significantly affected the sperm motility, fertility, and hatching rates of the eggs (P < 0.05). Furthermore, the antioxidants also protected the sperm cells during cryopreservation, with glutathione being the best antioxidant.
CONCLUSION: The application of antioxidants during the cryopreservation of depik fish sperm had a significant effect on motility, fertility and hatchability of eggs post-cryo. Furthermore, glutathione was the most suitable antioxidant. doi.org/10.54680/fr23110110312.
Motility, viability and fertility of goldfish Carassius auratus (Pisces: Cyprinidae) post short-term cryopreservation

Nurlaili N, Eriani K, Salma I, Maulida S, Rahayu SR, Handayani LS, et al.

Cryo Letters, 2023;44(3):169-177.
PMID: 37883170

BACKGRUND: Goldfish Carassius auratus is a popular ornamental fish extensively cultured worldwide. Sperm cryopreservation is a common fish breeding method that ensures sperm availability around the year. Studies on cryopreservation of goldfish sperm, especially on the suitability of cryoprotectant types and pre-freezing time, are scarcely available.
OBJECTIVE: To determine the most suitable type of cryoprotectant and pre-freezing for the successful cryopreservation of goldfish sperm.
MATERIALS AND METHODS: A completely randomized design with two factors was utilized in this study. The first factor is the type of cryoprotectants, which included methanol, ethanol, ethylene glycol, glycerol, and DMSO. The second is pre-freezing times of 10, 20, 30, and 40 min at each of the pre-freezing temperatures of 4 degree C, -10 degree C, and -79 degree C, meaning that the total times for the ramping down of temperature were 30, 60, 90 and 120 min, respectively. The Ringer solution and 10% egg yolk were used as extender and extracellular cryoprotectant. The sperm was stored at -179 degree C for 7 days.
RESULTS: The ANOVA test showed that cryoprotectants and pre-freezing significantly affected the motility, viability, and fertility of goldfish sperm after freezing in liquid nitrogen for 7 days (P<0.05). Furthermore, 10% DMSO combined with 15% egg yolk with an pre-freezing time of 20 min can maintain sperm motility, viability, and fertility higher than other treatments, by 79%, 80%, and 33%, respectively. The agarose gel electrophoresis showed no DNA fragmentation in all samples, including fresh sperm.
CONCLUSION: We conclude that 10% DMSO combined with 15% egg yolk and 20 min pre-freezing is the best treatment for goldfish sperm cryopreservation. DOI: 10.54680/fr23310110412.

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