Cryopreservation of cultivated and wild Arachis species embryonic axes using desiccation and vitrification methods (original) (raw)
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Cryo letters
A storage protocol at cryogenic temperature was established for shoot apices from in vitro plants of the cultivated groundnut (Arachis hypogaea) and wild Arachis species (A. retusa and A. burchellii) using a basic vitrification protocol with direct immersion in liquid nitrogen (LN). The effect of pre-treatments of donor-plants with ABA as well as of different supplements in the post-thaw culture medium was studied. After rapid warming at 40 C, the explants were cultured on MS medium devoid of growth regulators (MS0) or MS supplemented with 4.4(M benzylaminopurine (BAP) and 0.5(M naphthalene acetic acid (NAA) plus 5(M silver nitrate (AgNO3), 0.25% polyvinylpyrrolidone (PVP) or 0.2% activated charcoal. Non-frozen explants from the three species formed one shoot through meristematic amplification when cultured on MS0 medium. These explants also developed callus on MS supplemented with growth regulators (4.4(M BAP and 0.5(M NAA) alone or plus PVP or AgNO3. Callus formation was suppresse...
Cryo letters
A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to ...
Cryopreservation of Arachis pintoi (leguminosae) somatic embryos
Cryo letters
In this study, we successfully cryopreserved cotyledonary somatic embryos of diploid and triploid Arachis pintoi cytotypes using the encapsulation-dehydration technique. The highest survival rates were obtained when somatic embryos were encapsulated in calcium alginate beads and precultured in agitated (80 rpm) liquid establishment medium (EM) with daily increasing sucrose concentration (0.50, 0.75, and 1.0 M). The encapsulated somatic embryos were then dehydrated with silica gel for 5 h to 20% moisture content (fresh weight basis) and cooled either rapidly (direct immersion in liquid nitrogen, LN) or slowly (1 degree C per min from 25 degree C to -30 degree C followed by immersion in LN). Beads were kept in LN for a minimum of 1 h and then were rapidly rewarmed in a 30 degree C water-bath for 2 min. Finally, encapsulated somatic embryos were post-cultured in agitated (80 rpm) liquid EM with daily decreasing sucrose concentration (0.75 and 0.5 M) and transferred to solidified EM. Us...
Cryopreservation of Malayan kumquat (Fortunella polyandra) embryonic axes by vitrification
2013
Cryopreservation of embryonic axes of Fortunella polyandra was investigated using a vitrification-based technique. Plant vitrification solution 2 (PVS2) was added to cryovials containing embryonic axes (2 mm) and kept for 0, 5, 10, 15, 20, 25 or 30 minutes at 25 degreesC and for 0, 15, 30, 45, 60 or 75 minutes at 0 degreesC before plunging into liquid nitrogen. Another set of embryonic axes with the same treatment but not exposed to liquid nitrogen was used as a control. The highest survival percentage of the embryonic axes was 50.0% with 15 minutes exposure to PVS2 at 25 degreesC and 53.30% with 60 minutes exposure at 0 degreesC. Gradual increase in exposure to PVS2 from 50% to 100% at PVS2 in one to four steps was also studied at both temperatures. The embryonic axis survival rates of 50.0-56.7% obtained for 2- to 4-step PVS2 treatments at both temperatures were significantly better than those of embryonic axes directly exposed to PVS2 (survival of 30%), showing that the period of...
Importance of in vitro culture for developing cryopreservation strategies of woody plants
Acta Horticulturae, 2017
Plant cryopreservation has greatly evolved in recent years, being today a safe cost-effective complementary approach to the traditional ex situ conservation of plant biodiversity. A milestone in the cryopreservation of woody plant material dates back to 1990, when Akira Sakai and co-workers developed the Plant Vitrification Solution n°2 (PVS2) which showed to be very effective for the induction of cell vitrification during ultra-rapid freezing in liquid nitrogen. Since then, the number of PVS2-based protocols, mainly developed for the cryopreservation of shoot tips, increased yearly while, at the same time, new and effective encapsulation-and droplet-based methods were proposed. A range of different cryo-techniques is now available for the cryostorage of woody plant germplasm, allowing the safe long-term conservation in liquid nitrogen of different organs and tissues coming from tissue culture, such as (i) shoot tips, obtained in vitro by axillary or apical buds and used naked or incapsulated in Ca-alginate beads. They are the most used explants with broad-leaf trees, provided that optimized protocols of micropropagation have been achieved; (ii) somatic embryogenic callus, largely used with conifer species for which efficient micropropagation procedures from mature stock plants are rarely available; (iii) seeds and embryonic axes, useful material for the long-term preservation of both seed-propagated, and vegetatively-propagated species, provided that the latter have polyembrionic seeds, such in citrus. Seeds and embryonic axes take advantage from in vitro culture for their development after the recover from liquid nitrogen. The optimization of effective cryo-protocols passes through a correct interpretation of results in post-cryopreservation in terms of "survival" and "regrowth" of explants.
Cryopreservation of Arachis Hypogaea L. Varieties, from the INIAP-Ecuador Germplasm Bank
2021
The peanut (Arachis hypogaea L.) is recognized as one of the most important legume crops globally for its use in human food; it is widely distributed and cultivated in tropical and subtropical regions. The purpose of this study was to evaluate the cryopreservation of five peanut varieties conserved in the INIAP Germplasm Bank, testing cryopreservation methods, evaluating the germination percentage of whole seeds and embryonic shoots. Subsequently, two quantitative variables, shoot length and root, were evaluated. The average germination percentage of varieties and treatments was higher when embryonic axes were isolated with 99.31% than 86.06% seeds. The best germination percentage of the five varieties for seeds and embryonic shoots was obtained by the Peruvian variety with 88.13% and 92.50%. The best treatments by variety for the germination of whole seeds and embryonic axes were obtained by the treatment (desiccation and NL) for whole seeds (GS2) with 95.42% and embryonic axes wit...
Cryopreservation of Arachis Hypogaea L. Varieties, from the INIAP-Ecuador Germplasm Bank
2021
The peanut (Arachis hypogaea L.) is recognized as one of the most important legume crops globally for its use in human food; it is widely distributed and cultivated in tropical and subtropical regions. The purpose of this study was to evaluate the cryopreservation of five peanut varieties conserved in the INIAP Germplasm Bank, testing cryopreservation methods, evaluating the germination percentage of whole seeds and embryonic shoots. Subsequently, two quantitative variables, shoot length and root, were evaluated. The average germination percentage of varieties and treatments was higher when embryonic axes were isolated with 99.31% than 86.06% seeds. The best germination percentage of the five varieties for seeds and embryonic shoots was obtained by the Peruvian variety with 88.13% and 92.50%. The best treatments by variety for the germination of whole seeds and embryonic axes were obtained by the treatment (desiccation and NL) for whole seeds (GS2) with 95.42% and embryonic axes with 92.83%. Ageing and cryopreservation treatments positively affected germination and seedling vigor in whole seeds and embryonic axes. The two quantitative variables, shoot and root length showed variability between the five varieties; significant differences were observed between the four treatments evaluated for whole seeds and embryonic axes. The three treatments for whole seeds (GS1, GS2 GS3) and the non-cryopreserved control treatment (GSC), as well as the treatments for embryonic axes (GEA1, GEA2 GEA3) and the non-cryopreserved control treatment (GEAC), obtained good survival. They germinate whole seeds and embryonic axes with sprout development (aerial part) and root formation. With the most effective treatments for whole seeds (GS2) and embryonic axes (GEA2), the cryopreservation of the national peanut collection of the INIAP Germplasm Bank could be started.
PLoS ONE, 2014
Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time 6 temperature 6 permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80uC variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a , 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0uC and 25uC). VIVcryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.
In Vitro Cellular & Developmental Biology - Plant, 2017
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