Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group) (original) (raw)
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Protoplast isolation and culture for banana regeneration via somatic embryogenesis
Fruits, 2009
Protoplast isolation and culture for banana regeneration via somatic embryogenesis. Abstract-Introduction. This protocol describes a method for obtaining protoplasts from banana leaves, calli and cell suspensions, and their sustainable development via somatic embryogenesis from embryogenic cell suspensions. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, and media preparation for protoplast production and culture. Results. The first protoplasts may be seen after 30 min of incubation in enzyme maceration. With protoplasts from embryogenic cell suspension, complete development into a whole plant, through somatic embryogenesis, is observed in 12 weeks. The first cell divisions occur on feeder layers 3-8 days after protoplast plating. Proembryo formation is observed 14-21 days after initiation of protoplast culture. The transfer of derived embryo plantlets, at 8-10 weeks after protoplast plating, onto growth regulator-free medium, leads to plant rooting and elongation. France / Musa sp. / method / culture media / culture techniques / protoplasts / somatic embryogenesis / in vitro regeneration Obtention et culture de protoplastes pour la régénération de bananiers par embryogenèse somatique. Résumé-Introduction. Le protocole décrit une méthode qui permet d'obtenir des protoplastes à partir de feuilles, de cals et de suspensions cellulaires de bananiers, ainsi que le développement, par embryogenèse somatique, des protoplastes issus de suspensions cellulaires embryogènes. Le principe, les principaux avantages de la méthode, le matériel végétal de départ, le temps nécessaire et les résultats attendus sont présentés. Matériel et méthodes. Cette partie décrit le matériel de laboratoire nécessaire, la préparation des milieux pour l'obtention de protoplastes, ainsi que leur culture. Résultats. Dès la première demi-heure d'incubation dans la solution enzymatique, les premiers protoplastes sont libérés. En 12 semaines, les protoplastes, issus de suspensions cellulaires embryogènes, se développent en plantes entières via l'embryogenèse somatique. Sur les couches nourricières, les protoplastes reconstituent leur paroi ; les premières divisions cellulaires s'observent (3 à 8) jours après l'étalement des protoplastes. La formation des proembryons se déroule entre (14 et 21) jours après le début des cultures de protoplastes. Puis, (8 à 10) semaines après l'étalement des protoplastes, leur évolution embryogène conduit à des plantules qui s'enracinent et s'allongent après transfert sur un milieu dépourvu de facteurs de croissance. France / Musa sp. / méthode / milieu de culture / technique de culture / protoplaste / embryogenèse somatique / régénération in vitro
In vitro plant regeneration from embryogenic cultures of a diploid and a triploid, Cavendish banana
Plant Cell Tissue and Organ Culture, 1997
Plant regeneration by somatic embryogenesis was attempted with diploid (Musa acuminata ssp. malaccensis) and triploid ('Grand Nain') bananas. Explants inoculated in vitro were, respectively, immature zygotic embryos and male flower bud primordia. An histological study showed that the embryogenic process involves a sequence of similar events for both species. A yellow-green compact callus was initiated, which consisted of an actively dividing meristematic zone surrounded by several layers of starchy cells. A white and friable callus, characterized by the presence of proembryonic cells, bicellular proembryos and proembryonal masses in its periphery gradually appeared, which finally gave rise to somatic embryos from which plants were recovered. Induction media contained 2,4-D (and also NAA and IAA for the triploid); zeatin and kinetin were necessary for embryo maturation and 6-BA and IAA were used for germination.
Electronic Journal of Biotechnology, 2008
Abbreviations: 2.4-D: 2.4-dichlorophenoxiacetic acid BA: benzyladenine CE: conversion of embryos CNEC: compact non-embryogenic calluses ECS: embryogenic cell suspension ES: explants with scalps IAA: indoleacetic acid ME: mature embryos MM: maturation medium MS: Murashige and Skoog medium NECS: non-embryogenic cell suspension NNEC: nodular non-embryogenic calluses NS: number of scalps cm -2 of the multiple meristem mass. PSE: primary somatic embryos SCV: sedimented cell volumen VP: percentages of vitroplants thus obtained. The purposes of this work were to obtain embryogenic cell suspensions (ECS) from scalps and to regenerate plants of the banana CIEN-BTA-03. Shoot apexes were grown in the scalp-induction medium of Murashige and Skoog plus BA and IAA, following four diverse treatments. The first two, ME22 and ME25, were solid media supplemented with (mg L -1 ) 22.7 BA plus 0.192 IAA, and 25 BA plus 0.217 IAA, respectively, all containing 1.8 g L -1 of phytagel, and subcultures were performed monthly and bimonthly over 16 months. The other two treatments, IT22 and IT25, resembled ME22 and ME25 but consisted in temporary immersion for four months without subcultures, followed by two *Corresponding author
Biotecnologia Vegetal, 2012
A method has been developed for plant regeneration via embryogenic cell suspensions from diploid cultivar 'Calcutta 4´. For callus induction with embryogenic structures, different plant tissues such as scalps from cauliflower-like meristems and meristematic domes of axillary sprouted buds in combination with several culture media were evaluated as explants. The best embryogenic response (8%) was noticed with meristematic domes of axillary sprouted buds in culture medium Murashige and Skoog salts at 50%, MS vitamins, 30 g l-1 sucrose, 10 mg l-1 ascorbic acid, 1 mg l-1 2,4-D, 0.22 mg l-1 Zeatine and supplemented with 100 mg l-1 malt extract, 100 mg l-1 glutamine, 1 mg l-1 biotin, 200 mg l-1 casein hydrolysate, 4.0 mg l-1 proline and solidified with 2.0 g l-1 Gelrite. Embryogenic cell suspensions were established and the highest increase of cellular biomass with 0.50 ml settled cell volume (SCV) was obtained 18 days after culture initiation. In a RD1 culture medium, embryogenic masses from 1362 to 2480 embryos were formed. An average of 54.5% germinated embryos in Temporal Immersion System (TIS) was obtained. Results were significantly higher in comparison with the use of semisolid culture media (28%). Regenerated plants are in field conditions to value their genetic stability.
Embryo rescue and plant regeneration in banana
A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5 μM). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15 d followed by cycles of 8 h dark and 16 h light, under white fluorescent lamps with a light intensity of 3,000 lm/m 2 and at temperature of 28±2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9 mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.
Improvement of somatic embryogenesis in highland-papaya cell suspensions
Plant cell, tissue and organ culture, 1996
Axillary buds (2 ram) from 3-year-old Caricapubescens Lenn6 et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in N-N-medium supplemented with different growth regulators (naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2-3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.
2015
Plant regeneration by somatic embryogenesis was attempted with triploid banana Musa cv. (Berangan "AAA"). Cell suspension culture initiated from in vitro proliferating meristems. Embryogenic calli with globular structures developed from three different media. Induction media contained: 2,4 –Dichlorophenxy acetic acid (2,4D); Naphthalene acetic acid (NAA) and Indole -3-acetic acid (IAA) for the triploid ;zeatin and kinetin were necessary for embryo maturity; 6-benzylamino purine (6-BA) and Inidol 3-acetic acid (IAA) were used for germination. Generally, auxin is not required for initiation of the embryo, but it is needed for the growth of callus prior to this stage if embryogenesis cannot be induced directly from the tissue of explants. Somatic embryos (embryoids) from entirely different origins have a striking similar sequence of embryo formation. However, the origin of a somatic embryo may be a single cell or even a protoplast, but it can also develop from multiple cells ...
Biologia, 2019
A simple and efficient protocol developed for plantlet regeneration through in vitro somatic embryogenesis in three ornamental bananas. In vitro propagation, especially somatic embryogenesis has been a method of choice to obtain homogenous planting material in ornamental bananas. In the present investigation, true-to-type plants via somatic embryogenesis were achieved in three ornamental bananas viz. Musa laterita, Musa beccarii and Musa velutina using immature male flower bud as explants. The evaluation was carried out at two stages of somatic embryogenesis, induction, and maturation of somatic embryos. Embryogenic calli desiccated up to 2 h at 25 ± 1°C resulted in higher frequencies of embryo induction and maturation compared to nondesiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using SRAP markers. It was observed that banana plants derived via somatic embryogenesis displayed normal phenotype as that of plants grown from a single elite mother plant. In the present study, the successful establishment of plants from male flower bud was achieved in 6 to 7 months which is a relatively short duration compared to other reports involving different species of banana. The protocol developed could be useful for large-scale micropropagation studies in these commercially important ornamental bananas.
Plant Cell Reports, 2001
Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish subgroup AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3×10 7 protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.