Callus regeneration from Trifolium subterraneum protoplasts and enhanced protoplast division by low-voltage treatment and nurse cells (original) (raw)
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Elite material of the Latvian red clover variety ‘Agra was included ˙ in the experiment for observation of calli formation and their regeneration. Leaves, stem segments and leaf petiole segments were used as explants. Different frequency of calli formation was observed depending from the explants type. Most frequently calli were formed on leaf segments. The size of calli varied depending fromthe size of explants andwas, in general, independent from the type of explant. The largest calli were developed from the smaller (2 mm) leaf petiole segments. Different types of the calli regeneration were observed. Organogenesis was characteristic feature of regeneration from calli induced on stem segments, rhysogenesis – on leaf segments. Calli induced on leaf petiole segments formed somatic embryos and their posterior regeneration in the whole plant was observed. Embryogenesis was obtained from 12.5% of calli induced on leaf petiole segments.
Euphytica, 1989
Callus cultures were induced from hypocotyl sections of 24 varieties of white clover Trifolium repens L .). The calli did not show any significant difference of growth among the varieties. After the calli has been transferred to three regeneration media, green-spot formation was observed on calli derived from some seedlings. Remarkable intra-and intervarietal variations in the emergence of green spots and some trends between the origin of varieties and the frequency of green spots were observed. In most cases, the green spots turned brown without showing further differentiation, and only two genotypes formed shoots. A callus from a seedling of the Swedish variety 'Undrom' has sustained high levels of plant regeneration throughout 24 months of culture. Protoplasts derived from this selected genotype were divided into cell colonies. 8P Kao, 1977) medium containing 0 .5 mg/1 2,4-D and 0 .5 mg/1 kinetin was the most suitable medium for inducing divisions in protoplasts. When subcultured into solid B5 medium, the colonies produced calli, which when transferred to a regeneration medium, formed shoots. This genotype is expected to a useful subject for genetic engineering of white clover .
In vitro regeneration of commercial cultivars of subterranean clover
Plant Cell, Tissue and Organ Culture, 1993
Regeneration of subterranean clover (Trifolium subterraneum L.) was achieved by both shoot organogenesis and somatic embryogenesis. Shoots derived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was sliced longitudinally into two halves and cultured on shoot induction medium. After 30 days, adventitious shoots were formed from the hypocotyl region while the radicle showed no development. Shoots were then subcultured onto shoot multiplication medium and finally onto a root initiation medium. Histological studies revealed that shoots arose de novo and did not originate from pre-existing meristems. In the second regeneration protocol, shoot apical meristems from young seedlings were induced to form callus. Following four to six weeks culture in the dark, somatic embryos appeared spontaneously on the calli. A majority of embryos had a well-defined root pole, two cotyledonary lobes, and were capable of germination, albeit at a low frequency. Regenerated plants obtained from both protocols appeared phenotypically normal.
Mesophyll protoplasts of white clover: Isolation, culture and organogenesis
Plant Science Letters, 1982
Mesophyll protoplasts of Trifolium repens divided and formed calli with a reproducible plating efficiency of approx. 37% if experimental plants were maintained under a controlled environment and the protoplasts were cultured in a liquid medium containing 2 mg 1-1 each of 2,4~iichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and kinetin, in the dark, at 28°C. Protoplast<lerived callus from one of the three genotypes used differentiated shoot buds. The other genotypes formed only roots. The shoot buds did not develop into complete plants.
Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L
1989
N a n c y L. M a c L e a n and J e r z y N o w a ABSTRACT Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (]979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases~ callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiolederived callus tissuehave so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be nonregenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.
Intra-varietal variation for in vitro plant regeneration in the genus Trifolium
Euphytica, 1984
Trifolium repens, white clover, Trifolium pratense, red clover, Trifolium arvense, plant regeneration, somatic embryogenesis, genotypic variation . SUMMARY Seedlings of Trifolium repens showed considerable variation with regard to the morphology and growth of their calli, and their ability for in vitro differentiation of shoots . One of the lines selected for regeneration in primary callus cultures also showed shoot formation from protoplasts . Somatic embryogenesis in callus cultures of T. pratense and T. arvense occurred only in selected seedling lines . This paper highlights the importance of screening a large number of plants within a cultivar of outbreeding species to achieve reproducible plant regeneration from tissue culture . ' Present address :
Journal of Plant Physiology, 1996
Plants of napiergrass or elephantgrass (Pennisetum purpureum Schum.) were regenerated from embryogenic calli derived from leaf and inflorescence segments, cell suspensions, protoplasts, and cryopreserved cell suspension cultures. The induction of somatic embryogenesis in segments of young leaves was found to be under developmental control, allowing a zone of cells competent for somatic embryogenesis to be mapped along the length of young leaves. Somatic embryogenesis was also initiated in segments of immature inflorescences. Factors important for the development of embryogenic suspension cultures were investigated. The starting callus tissues and gradual change of liquid medium were found to be critical for establishing embryogenic suspension cultures, which provided a critical source of protoplasts for the regeneration of plants. Plants regenerated from suspensions as well as protoplasts isolated from them grew to maturity in the field with normal morphology and chromosome numbers. Plants were also regenerated from cryopreserved cells after two and a half years of cryogenic storage, and established in soil. a quality forage for ruminants . Previously, plants of napiergrass have been regenerated from cultured leaf and inflorescence explants. Plantlets were also recovered at very low efficiency from protoplasts isolated from suspension cultures, but they neither continued growth nor could be transplanted to soil . In this report, we confirm the utility of leaf and inflorescence segments for the establishment of highly embryogenic callus cultures, which were utilized for the initiation of embryogenic cell suspension cultures. Plants regenerated from the suspensions as well as protoplasts isolated from them were grown to maturity in soil. Plants were also regenerated from cryopreserved cell suspensions after two and a half years of cryogenic storage.
Regeneration Efficiency in Trifolium apertum as Influenced by Explant Media Interaction
CYTOLOGIA, 2005
In vitro response of interaction of various growth regulators and explants was studied in Trifolium apertum (genotype EC 401712), a diploid species domesticated in Russia. The species is considered as a progenitor of T. alexandrinum, a very important fodder legume cultivated in Indian sub-continent. The callus induction was found to be dependent on level of growth regulators, auxin and cytokinin ratio and the explants. Medium 'A' with low level of growth regulators failed to induce callus in 5 out of 6 explants. High hormone medium 'D' showed good callogenesis in hypocotyl, cotyledon and petiole explants. In collar, leaf and root explants the frequency of callus induction was very low. Friable green calli were observed in hypocotyl and petiole explants. The high auxin: cytokinin ratio favoured callus induction. NAA in high concentration was found to be better than Picloram as auxin source. The differences in callus induction frequency were found to be statistically significant for both explant and media as well as their interaction. The calli from petiole explant-'D' medium combination showed very good organogenetic and embryogenetic potential on sub-culture. 50% of sub-cultures showed shoot induction and a large number of them showed root induction in rooting media. A suitable protocol for regeneration and transfer of plantlets in vitro in this genotype of T. apertum has been developed for first time. The cytology of the plants revealed normal meiotic pairing with 8 bivalents.
Plant regeneration from callus and protoplasts in Medicago polymorpha
Plant Cell Tissue and Organ Culture, 1993
Seventeen ecotypes of the wild species Medicago polymorpha adapted to a Sardinian (Italy) environment have been evaluated for their response to tissue culture. The accession Samughero-Albi was the more respondent for callus induction and, together with Usassai, showed the highest regeneration capacity on media containing 1 mg l-1 2iP and 0.1 mg l-1 IAA. The morphogenetic response was also affected by the explant source. The hypocotyl-derived-calli were the best regenerating tissues. Regenerated plantlets were difficult to root and it was possible to obtain plants with a well developed root system only after 5–7 weeks of culture on media containing 2iP and IAA both at 0.2 mg l-1. Mesophyll cells were the best protoplast yielding source but only those isolated from roots were able to divide and to regenerate plants. Results are discussed in relation to the genotype specificity for the morphogenetic response and the feasibility of using M. polymorpha in the somatic hybridization with M. sativa.