Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi (original) (raw)
Related papers
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...
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 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...
Cryo letters
The effects of two methods of cryopreservation involving chemical vitrification and air desiccation) were studied on isolated embryonic axes of A. hypogaea. Vitrification with PVS2 and desiccation in a laminar flow cabinet resulted in high levels (70-90%) of whole plant recovery after cryopreservation. A desiccation protocol based on 1h exposure of explants to the air flow was successfully applied to six wild species of section Extranervosae, resulting in recovery levels of 70-90% after liquid nitrogen treatment.
In Vitro Cellular & Developmental Biology - Plant, 2017
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2 3 In Vitro Cellular & Developmental Biology -Plant
Here, we report an efficient and widely applicable method for cryopreservation of Malus shoot tips by encapsulation-dehydration using adventitious shoots. Shoots were induced from leaf segments cultured on a shoot induction medium containing 2-3 mg L −1 thidiazuron, depending on genotype, and 0.5 mg L −1 indole-3-butyric acid. Shoot tips (3 mm in length) containing six leaf primordia excised from 11-wk-old adventitious shoots were encapsulated and precultured with 0.5 M sucrose for 5 d, followed by airdrying for 6 h prior to direct immersion in liquid nitrogen. With our protocol, we obtained a mean organogenesis rate of 100%, a mean of 4.5 adventitious shoots per explant (leaf segment), and a mean shoot recovery of 57.0% from cryopreserved shoot tips in four Malus species. Inter-simple sequence repeat (ISSR) analysis did not reveal any polymorphic bands in regenerants recovered from either leaf segments or cryopreserved shoot tips of 'Gala'. To the best of our knowledge, this is the first report on cryopreservation of Malus shoot tips using adventitious shoots derived from leaf segments and is the most widely applicable protocol so far reported for cryopreservation of Malus. Establishment of this protocol provides an alternative means for cryopreservation of Malus.
Plant Cell Tissue and Organ Culture, 2008
Shoot-tips and somatic embryos are the explants of choice for the in vitro long-term storage of ex situ plant genetic resources in liquid nitrogen. Cryopreservation of organized structures has significantly progressed, especially for species of tropical origin, with the development of several vitrification-based procedures such as encapsulation-dehydration, vitrification and droplet-vitrification approaches. They have allowed improvements in survival and recovery after cryopreservation compared with conventional crystallization-based protocols, proving their effectiveness for large scale application with embryos and shoot-tips of different plants. This review addresses the main physical and technological aspects involved in plant cryopreservation methods, illustrating the development of research with three cases: citrus, cassava and potato. These studies demonstrate how cryopreservation strategies are increasingly applied for their successful employment in the genebanks.
Cryopreservation technology for plant cell cultures
1989
Described in detail is a technique for the cryopreservation, long-term storage, thawing, recovery, and regrowth of embryogenic suspension cultures of maize (Zea mays L. ) With this system we have successfully preserved many different culture lines, and recovered living material after more than 2 yr storage in liquid nitrogen. The recovered tissue is not apparently different from the starting material. With minor variations, the technique has also been used for Dactylis glomerata and other suspension culture cells, as well as callus cultures.
Plant Science, 2002
Embryogenic cell suspensions of grapevine (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration and subsequently regenerated into plants. Embryogenic cell suspensions were first-step precultured for 2 days in MGN liquid medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1 M. Precultured cells were then encapsulated and second-step precultured in MGN liquid medium containing 1 M sucrose for several days. Following preculture, encapsulated cells were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved cells were post-cultured on MGN medium for survival. Surviving cells were then transferred to different media for regrowth and plant regeneration, respectively. An optimal viability of cryopreserved cells was achieved when encapsulated cells were dehydrated to 20.6% water content and subjected to 2 Á/4 days of preculture on 1 M sucrose. MGN solid medium containing 2.5 g/l AC was found to promote viability of cryopreserved cells. Although cryopreserved cells showed a 5-day lag phase in regrowth, and their increase in fresh weight was only half that of control cells, their growth pattern was the same as that of control cells after two subcultures, following re-establishment in cell suspensions. Cryopreservation promoted embryogenesis and subsequent plant germination, compared with both the control and dehydration treatments. Leaf morphologies of plants regenerated from cryopreserved cells appeared to be similar to those of plants from control cells. #