Subculture Of Callus Research Articles

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 μM picloram as the only PGR. Ten μM picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 μM picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.

IN VITRO NITROGEN METABOLISM AND ORGANOGENESIS OF NICOTIANA TABACUM

The role of N metabolism in organogenesis and growth was studied using tobacco pith callus. Callus was cultured on a solid medium containing 10 μM (1.75 mg/l) IAA and 2 μM (0.43 mg/l) kinetin for 56 days. In the growth experiment, ratios of NH4+-N to NO3--N (0:60, 20:40, 30:30, 40:20 and 60:0 mM) were supplied by (NH4)2 SO4 and KNO3. Callus and media were analyzed for inorganic N. Callus supported by 30:30 and 40:20 media removed the highest amounts of NH4+-N and NO3--N from the media and exhibited organogenesis. Final dry weight was greatest in callus supported by the 30:30 medium. In the organogenesis experiment, the transfer history of the inoculum source affected N uptake, organogenesis and growth. Inorganic N was supplied by NH4NO3 and KNO3-. The net uptake of NH4+-N and NO3--N was lower in shoot-forming than in root-forming and non-organogenic callus subculture from 7-day-old stock cultures. The final pH of the medium supporting shoot-forming callus was lowest. Growth, on a dry weight basis, was lowest in shoot-forming callus. Callus subculture from 60-day-old stock cultures formed no shoots.

IMPROVED PLANT REGENERATION OF SOLANUM AND LYCOPERSICON GENOTYPES FROM LONG-TERM CALLUS CULTURE.

Repeated callus sub-culture reduce the regeneration capacity in many species. Our studies determined the effect of genotype and medium on regeneration of several Solanum and Lycopersicon genotypes from long-term callus cultures. In the first study, 13 genotypes were transferred to regeneration medium, including: Murashige and Skoog plus Gamborg Vitamins (MG); Murashige and Skoog (MS); Gamborg (GM); and white (WM). The greatest shoot regeneration was on the MG medium, containing the highest levels of thiamine. Shoot differentiation was greatest with 0.2 mg/l IAA and 2 mg/l BA. No plants were regenerated on GM or WM medium. In a second study, the effect of thiamine (0 to 200 mg/l) on shoot regeneration of the L. peruvianum genotypes PI199380, PI126945, PI251301, and PI128652, along with Solanum ptycanthum, Solanum nigrum, and L. esculentum `Diego' was evaluated. Shoot regeneration of Solanum ptycanthum, Solanum nigrum, L. peruvianum PI 199380 and PI25301 was best with 20 mg/l of thiamine.

Plant Regeneration from Protoplasts Isolated from Embryogenic Callus of Satsuma

Embryos were obtained from unfertilized and undeveloped seeds of satsuma (Citrus unshiu Marc.) cultured on a modified Murashige and Tucker (MT) medium. Embryogenic callus was induced from the hypocotyl region of the embryos. The callus was successfully maintained through subculturing on MT medium with 185 μm ade-nine, 2.8 μm GA3 and 400 mg malt extract/liter, solidified with Gelrite. Somatic embryogenesis occurred from callus subculture on medium containing 50 g lactose/liter and in the absence of plant growth regulators. Somatic embryos developed into plants on medium with sucrose and GA3. Protoplasts isolated from this callus produced somatic embryos through colony formation: subsequently, normal, entire plants were regenerated.

Temporary chromosome number stabilization in potato cell cultures

Cell cultures of Solanum chacoense (monohaploid) and Solanum tuberosum (tetraploid cultivars and parthenogenetically derived dihaploid clones) were found to be highly mixoploid. Relative stabilization of chromosome number at the ploidy level of the original plant material was achieved in microcalli obtained from single cells or small cell colonies (up to about 5 cells) of stock callus lines. This relative stabilization was maintained over three subcultures, which is sufficient for selection procedures. It has been shown that the stabilization can be maintained during a number of further subcultures. Division centers were repeatedly observed in calli characterized by high mitotic activity. As has been shown for the first time there exist significant differences in the ploidy levels of several division centers within one and the same callus. This is of particular importance to callus subculture.

The histological development of the regenerative tissue derived from cultured immature embryos of wheat ( Triticum aestivum L.)

Histological studies were conducted on the embryogenic tissue of wheat ( Triticum aestivum L.), using primary, subcultured and differentiating callus derived from the scutellum and epiblast of immature embryos. Structures with multiple strata were observed in the late stages of callus induction and on subcultured callus; these were characterized by a middle stratum of ground meristematic tissue with a shoot-forming stratum above and a stratum of large cells below. The shoot-forming protuberances appearing on the differentiating callus were morphologically similar to those observed on subcultured and primary callus and also to the expanding shoot meristem of the cultured embryos. This similarity suggests the continuity of the pre-existing shoot forming tissue during culture in vitro. Typical embryoids have been previously reported on both scutellum and epiblast callus hence this callus could be described as embryogenic. However, organogenesis also occured on the callus as some of the protuberances had a vascular connection to the basal callus. Some of the regenerative outgrowths on the callus were broadly based and were not of single cell origin.

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.

Radioimmunoassay of Morphinan Alkaloids in Papaver somniferum Hypocotyls, Callus, and Suspension Cultures

Summary Hypocotyls of poppy ( Papaver somniferum L. cv. Marianne) were assayed for morphinan alkaloid content using a commercially available radioimmunoassay (RIA) for morphine (Abuscreen, Roche Diagnostics). The hypocotyls showed a sharp rise in morphinan alkaloid content on day 3 following sowing, reachin a plateau (9 ng·mg- 1 fresh wt) between day 4 and day 5. Hypocotyl explants were cultured for up to 2 months on solid nutrient medium containing α-naphthaleneacetic acid. During this time and while hypocotyls formed callus, the morphinan alkaloid content fell to a «maintenance» level of 0.4 ng·mg −1 fresh weight in culture. Callus masses retained laticifer-like cells. If cell suspensions were plated with medium lacking growth regulators, plantlets were regenerated and the level of alkaloids found for hypocotyls was re-established. If plated cell suspensions were cultured in the presence of growth regulators, organs formed, but plantlets did not regenerate and the alkaloid content remained at or near the maintenance level. Over a period of a year callus subcultures lost their laticifer-like cells, their embryogenic potential, and their ability to maintain low-level alkaloid production.

Embryogenesis and plant regeneration from tissue culture of Canada wildrye, Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.

Shoot-bud regeneration in subcultured callus of engelmann spruce

Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N6-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N6-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures.

Adventitious Shoot Regeneration from in Vitro Subcultured Callus of Rhododendron Exbury Hybrids

Abstract Callus culture establishment and adventitious shoot development from callus of Rhododendron × ‘Gibraltar’ (G) and R. × ‘Old Gold’ (OG) after serial subculture were determined. Callus grew on Anderson's Rhododendron medium containing 2,4- D at 0.0045, 0.009, or 0.018 mm, but was optimum at 0.018 mm, since shoot organogenesis was suppressed at that level. After two callus subcultures, adventitious shoot development from callus on media containing 0, 0.017, 0.034, 0.068, or 0.136 mm zeatin (mixed isomers) was optimum at 0.034 and 0.068 mm zeatin for G and OG, respectively. After 17 weeks (five subcultures), 70- to 75-mg pieces of G and OG callus regenerated ≈20 shoots each. Adventitious shoot regeneration from callus declined with prolonged callus subculture. Regenerated plants are currently being evaluated for somaclonal variation. Chemical names used: (2,4-dichlorophenoxy)acetic acid (2,4-D); (E)-2-methyl-4-(1H-purin-6-ylamino)-2-buren-1-ol (zeatin).

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 10(6) protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10(5) protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

Variation in adventitious regenerants of Begonia rex Putz. ‘Lucille Closon’ as a consequence of cell ontogeny, callus ageing and frequency of callus sub-culture

Plants were regenerated from ground tissues of petioles, from callus aged for 8, 12, 16 or 20 weeks without sub-culture before subsequent transfer to regeneration medium, and from callus sub-cultured at monthly intervals before transfer to regeneration medium. Plants regenerated from leaf epidermal cells served as controls. Karyological studies were carried out on the explants used for callus induction, on the calli and on the regenerants. Callus showed karyological changes on sub-culture and with ageing, with a tendency towards somatic reduction resulting in the production of aneuploid (hypodiploid) cells. Attenuation of variation occurred on regeneration from callus, and regenerants from all but aged calli were phenotypically true-to-type. Polyploid plants were produced from aged callus and petiole ground tissue.

Somatic embryogenesis in Picea glauca (white spruce) and Picea mariana (black spruce)

Embryogenic callus was initiated from immature embryos of Picea glauca (white spruce) and Picea mariana (black spruce) cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (1 × 10−5 M), N6-benzyladenine (5 × 10−6 M), and 1% sucrose. Seeds from cones stored at 4 °C for up to 3 months yielded embryogenic callus. Much higher frequencies of embryogenic callus were obtained from white spruce than from black spruce. Embryogenic callus contained loosely organized cells and somatic embryos of various sizes. The embryos consisted of a cluster of tiny dividing cells (embryonic region) with attached large vacuolated cells (suspensor region). Upon subculture of embrogenic callus to media either lacking growth regulators or with reduced concentrations (5 × 10−7 M, 2,4-dichlorophenoxyacetic acid and 5 × 10−6 M N6-benzyladenine) somatic embryos could be stimulated to develop into plantlets.

Vegetative propagation of Begonia venosa Skan in vitro from inflorescence explants

Methods are described for the vegetative propagation of Begonia venosa Skan. Young flower buds are capable of producing callus which, contrasting to callus from leaves of adult plants, is very organogenic. For callus induction are required: BA and NAA at a conc. of 0.5 mgl−1, 21 °C and low irradiance. Subculture of organogenic callus is optimal on a medium with 0.1 mgl−1 BA and 2% glucose, whereas NAA is ommitted. Shoot development from preformed adventitious buds is enhanced by lowering the BA and glucose conc. Optimal rooting of excised shoots is obtained on a medium without regulators and a low glucose level. No visible mutations can be detected in the plant material, even not at the flowering stage.

Plant regeneration from embryo-callus culture in barley

Plants were regenerated from callus cultures initiated from immature embryos of barley, Hordeum vulgare L. Immature embryos from seven diverse genotypes were cultured on modified Murashige and Skoog (MS) medium supplemented with 1.5 mg 2,4-D and 6.5 mg IAA/l. Of the 249 embryos cultured, 30% initiated callus within 8 days. Subculture of callus for 80 to 100 days on half-MS medium supplemented with 0.5 mg/l 2,4-D and 1.0 mg/l zeatin resulted in organogenesis. Culture of organogenic calli for 30 days on half-MS medium without growth regulators produced plants which originated mostly via multiple shoot formation. Callusing response of the tested genotypes ranged from zero to 44%; however, only 23% of the calli were regenerative. Regenerated plants included variants for chlorophyll deficiency, plant height, stem thickness, spike shape, pollen fertility, seed set and ploidy.

The ambiguous role of 2,4‐dichlorophenoxyacetic acid in wheat tissue culture

The very basal, highly immature regions of dissected young leaves of Triticum aestivum L. cv. Kite formed adventitious roots on a nutrient medium supplemented with comparatively low concentrations (0.16 to 0.63 μM) of 2,4‐dichlorophenoxyacetic acid (2,4‐D). Higher concentrations (up to 640 μM) had to be applied to stimulate growth from more mature regions higher up the leaf. Yet, already at 2.5 μM roots were less distinct and more callus‐like, and eventually (at 10 to 640 μM) only a subculturable callus of apparently suppressed, slowly proliferating root primordia developed. Furthermore, at the most basal, highly immature regions growth was significantly retarded when the auxin concentration was raised. The leaf culture system appears to reflect the dual action of 2,4‐D known from herbicide research, namely growth stimulation from differentiating (or differentiated) cells, but growth suppression at or in the vicinity of apical meristems. Correspondingly, when the callus of apparently suppressed, slowly proliferating root primordia was transferred to media without 2,4‐D or with low concentrations (0.16–2.5 μM) rapid proliferation commenced, leading to profuse root outgrowth. The system demonstrates the ambiguous role which this auxin appears to have, at least in wheat tissue culture.

Callus induction and plant regeneration in Panicum bisulcatum and Panicum milioides

Surface sterilized seeds and mesocotyls from sterile seedlings from Panicum bisulcatum Thumb., as well as basal parts of leaves and mesocotyls from sterile seedlings, and seeds from Panicum milioides Nees ex. Trin were used as explants to induce callus on a Murashige and Skoog medium supplemented with 2.5 to 10 mg/l of 2,4-D. Subculturing of the white callus from P. milioides and of the brown callus from P. bisulcatum on a medium containing 0.1 mg/l 2,4-D and 10 g/l sucrose led in both species to the appearance of green structures from which plants could be regenerated. Plants were regenerated by an organogenetic process in P. milioides, while P. bisulcatum plants were regenerated both via organogenesis and somatic embryogenesis. 1032 and 94 plants, from P. bisulcatum and P. milioides, respectively, were transferred into soil, and about 90% of them were grown to maturity and set seeds.

Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn

Mature endosperm of Emblica Officinalis (Euphorbiaceae) formed a continously growing callus on MS medium supplemented with an auxin (2,4-D or IAA) and a cytokinin (K or BAP). Subculture of callus on MS with BAP (0.2 mg/l) and IAA (0.1 mg/l) resulted in formation of shoots and embryo-like structures in 50 and 8 per cent cultures, respectively. Regeneration of shoots was more frequent when both BAP (0.2 mg/l) and IAA (0.1 mg/l) were present than on BAP (0.2 mg/l) alone. The embryo-like structures produced plantlets.

Evidence for plantlet regeneration via somatic embryogenesis in the grasses Echinochloa muricata and E. crus-galli var. Oryzicola

Explants for tissue culture were derived from mesocotyl plate tissue of Echinocha crus-galli var. oryzicola and E. muricata seedlings germinated under anaerobic conditions. Callus was initiated in the dark under aerobic conditions on a modified Murashige and Skoog medium plus 10 or 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l of 6-benzyl-aminopurine (BAP). Transfer of this callus tissue into the light on MS medium containing low auxin (≤ 5 mg/l) readily resulted in the formation of green plantlets. Scanning electron microscopic observations revealed that regeneration occured through the formation of somatic embryros. Capacity for regeneration is maintained after repeated callus subculture. This regenerative capacity via somatic embyros provides a valuable research system for continuing the study of the metabolism and developmental physiology of Echinochloa.