Somatic embryogenesis and plant regeneration in Heracleum candicans Wall

A protocol has been developed for achieving somatic embryogenesis and plant regeneration from petiole-derived callus of Heracleum candicans Wall. Callus was initiated on MS medium supplemented with 0.5 mg l^–1 2,4-D and 0.5 mg l^–1 BAP and subcultured on a medium containing double strength MS macrosalts, 1 mg l^–12,4-D and 0.25 mg l^–1 Kn. Numerous globular embryos were formed on the surface of the callus upon transfer to auxin-rich MS medium that lacked cytokinins. The globular embryos differentiated into mature embryos only when 2,4-D was removed from the medium. Mature embryo formation was significantly influenced by the pH of the medium and the addition of AgNO₃ and ABA. Eighty-five percent of the somatic embryos were converted into plantlets when transferred to a medium supplemented with 0.01 mg l^–1 BAP and 0.01 mg l^–1 IBA. The regenerated plants have been established in soil and appear to be identical to the parent plants in morphology and chromosome number. Received: 5 November 1997 / Revision received: 9 February 1998 / Accepted: 19 February 1998     

Regeneration of Asparagus officinalis L. through Callus Cultures Derived from Protoplasts

Undifferentiated callus derived from asparagus protoplast cultureshas been used for studies on organogenesis. Root and shoot formationhas been obtained with different hormonal balances. Adeninehas been found to be effective, together with a cytokinin, inpromoting the formation of somatic embryoids in this tissue.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Regeneration from Phylloclade Explants and Callus Cultures of Schlumbergera and Rhipsalidopsis

Phylloclade explants of Schlumbergera and Rhipsalidopsis were cultured in vitro to produce axillary and adventitious shoots. The explants of both species, taken from greenhouse-grown plants, produced only axillary shoots. There was a pronounced improvement in adventitious shoot formation in phylloclade explants of cultivar CB4 of Rhipsalidopsis by increasing numbers of subcultures of axillary shoots used as donor plants. The axillary shoots generated from the explants were either subcultured to produce successive generations of axillary shoot cultures or made into phylloclade explants and tested for adventitious shoot formation at each subculture. The duration of each subculture varied from 6 to 12 weeks. After the first subculture, sporadic adventitious shoot formation began, and after the third subculture 87% explants of cultivar CB4 produced adventitious shoots at a frequency of about 12 shoots per explant. In contrast, there was no improvement in regenerative ability in explants of cultivar Thor-Olga of Schlumbergera up to third subculture. Adventitious shoots could be produced by callus culture too. Cultivar CB4 was highly regenerative, producing as many as 10 adventitious shoots per square cm of callus. In vitro grown plantlets, when transferred to pots continued to show prolific growth.     

Radiation-induced Organogenesis and Isoenzyme Patterns in Long-term Callus Cultures of Datura innoxia

Effect of gamma irradiation on growth, shoot organogenesis andenzyme activities and isoenzyme patterns of -amylase and peroxidaseduring differentiation in long-term calluses of Datura innoxiahave been investigated. Radiation in doses of 0.2 and 1.0 kRstimulated the shoot regeneration frequency as well as the numberof shoots per regenerating callus. The 0.2 kR dose could induceshoot organogenesis even in calluses incubated in the dark oncallusing medium, although with less frequency. Such cultures,however, showed profuse shoot regeneration when sub-culturedonto regeneration medium under light conditions. A higher radiationdose (5.0 kR) was lethal to both growth and shoot differentiation.Prior to shoot regeneration, -amylase and peroxidase specificactivities increased to four- to fivefold and 7–24-fold,respectively. While the amylase isoenzyme pattern remained unchanged,specific changes in the isoperoxidase pattern were observedduring shoot differentiation in callus cultures. The most significantchange was the appearance of fast-moving anodic bands priorto visible shoot differentiation. Thus, such isoperoxidasesprovide useful biochemical markers for shoot differentiation. Datura innoxia, shoot organogenesis, isoenzyme pattern, gamma-radiation, growth regulators     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

Callus, Organogenesis and Plantlet Formation in Tissue Cultures of Clitoria ternatea

Decoated seeds of Clitoria ternatea L. germinated on Murashigeand Skoog (Physiologia Plantarum 1962, 15, 473–97) basalmedium (BM) and differentiated callus and bipolar embryoids(two-step method) in low frequency. Calluses developed on lateralroots [BM+KN(0.1 mg 1^–1)], on roots and hypocotyls [BM+KN(0.5mg 1^–1)], and on roots [BM+KN+IAA (0.5 mg 1^–1 ofeach)]. On basal medium with KN (0.5 mg 1^–1) and withKN+IAA (0.5 mg1^–1 of each), multiple shoot buds and embryoids(one-step method) were differentiated directly on split hypocotylsand roots. In the former, shoot buds developed even on unsplithypocotyls. Rhizogenesis on isolated shoot buds occurred efficientlyin BM+indole butyric acid (IBA 0.1 mg 1^–1) and BM+IAA(0.1 mg 1^–1 and 0.5 mg 1^–1). Profuse direct embryoidsand shoot buds developing on root systems are interesting morphogeneticphenomena rarely reported. Clitoria ternatea L., callus, embryoids, multiple shoot buds, regeneration     

Efficient Regeneration of Tetraploid Isatis indigotica Plants via Adventitious Organogenesis from Hypocotyl Explants

An efficient in vitro plant regeneration system via hypocotyl segments of tetraploid Isatis indigotica Fort. was established. Murashige and Skoog's (MS) and Gamborg's (GB₅) media were found to be superior to White medium for promoting shoot regeneration. The highest shoot regeneration (92 %) was achieved from hypocotyls cultured on MS medium containing 8.9 M benzyladenine (BA) and 2.7 M naphthaleneacetic acid (NAA), with an average of 4.2 shoots developed per explant. Plant regeneration was also improved when the explants were cultured in MS basal medium containing 3 % (m/v) sucrose and grown under a 12-h photoperiod. The developed shoots were well rooted in a half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA) and were morphologically normal after transfer to soil.     

Chemical Regulation of Callus Growth, Organogenesis, Plant Regeneration, and Somatic Embryogenesis in Antirrhinum majus Tissue and Cell Cultures

Excised stem explants of Antirrhinum majus L. var. ‘Kymosyblanc’ were grown in a denned medium to investigate factorsinfluencing bud and root development, callus induction, andsomatic embryogenesis. Auxins such as indoleacetic acid (IAA)and naphthaleneacetie acid (NAA) caused limited callus developmentand abundant root formation, whereas 2,4-dichlorophenoxyaceticacid (2,4-D) promoted soft friable callus with embryos and occasionaldevelopment of thick abnormal roots. 2-Naphthoxyacetic acid(NOA) and coconut milk (CM) used together induced friable greencallus growth and differentiation of small globular embryoswhich eventually developed into plantlets after transfer toauxin-free agar mineral medium containing sucrose. Cytokininssuch as benzyladenine (BA), zeatin, and kinetin induced compactgreen callus but in the absence of auxin failed to promote organogenesis.The interaction of IAA and kinetin resulted in the regenerationof the whole plant from stem explants. When NAA was used withkinetin, shoot development was totally inhibited and abundantroots were formed. Thus, the alternative morphogenetic eventsprobably reflect the biochemical subtleties occurring withinthe callus as a result of differences of actual endogenous levelsof growth substances in the tissues studied. These experimentshave been performed and interpreted on a histological basis.     

Micropropagation of Heracleum candicans wall: A rare medicinal herb

Summary A protocol was developed for micropropagation of Heracleum candicans Wall. by axillary shoot proliferation. Maximum shoot proliferation was obtained on Murashige and Skoog medium supplemented with 0.5 mg (2.2 μM) 6-benzyladenine per 1 and 0.1 mg (0.4 μM) 1-naphthaleneacetic acid per 1. Regenerated shoots were rooted on MS medium fortified with 1 mg (4.9 μM) indole-3-butyric acid per 1. Complete plants were transferred to soil and all of these plants were morphologically and cytologically identical to the mother plant.     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

Control and Maintenance of Plant Regeneration in Sugarcane Callus Cultures

Chen, W. H., Davey, M. R., Power, J. B. and Cocking, E. C 1988.Control and maintenance of plant regeneration in sugarcane calluscultures.—J. exp. Bot. 39: 251–261. Sugarcane cultures produced three morphologically distinct typesof callus: a white compact callus capable of plant regeneration,a friable non-morphogenic callus and a mucilaginous nodularcallus which could revert to these other two types dependingon the concentration of 2, 4-D in the culture medium. Leaf explantsformed morphogenic callus more readily than excised stem apices,with most prolific callus formation in the dark. Variation wasobserved in the response of leaf explants, from different sugarcanespecies, varieties and commercial cultivars, to callus induction.Visual selection of suitable callus at each subculture combinedwith incubation in the dark and the use of alternating high-low2, 4-D concentrations in the culture medium, were essentialin maintaining the morphogenic callus. Using such a scheme,the capacity for plant regeneration was maintained for over30 months after callus induction. Morphogenic callus youngerthan 12 months from induction formed somatic embryos and/orshoot meristems when transferred to a medium with activatedcharcoal. However, only organogenesis occurred in older cultures.Somatic embryos and/or shoot meristeras produced plants on auxin-freemedium, which rooted on medium with 7% w/v sucrose. Over 90%of potted plants were grown to maturity in the glasshouse. Key words: Saccharum spp. (sugarcane), somatic embryogenesis, organogenesis     

Organogenesis in Callus from Shoot Apices of Pisum sativum

Shoot formation was observed in callus from apical cells of pea (Pisum sativum L. cv. Century). Shoot apices from 4-day-old plants were macerated and the resulting cell masses grown on agar media. The callus formation and shoot production occurred within 4 to 6 weeks in defined media containing 0.2 to 5.0 μM benzyladenine and 1 μM naphthaleneacetic acid. While most callus produced one or more shoots at high frequency, root formation did not occur regularly. Plants obtained by these procedures were grown to maturity producing flowers and pods.     

Changes in Some Enzyme Activities during Growth and Organogenesis in Dark-Grown Tobacco Callus Cultures

Enzymes involved in malate metabolism, viz., glutamic-oxalacetictransaminase, malate dehydrogenase, malic enzyme and phosphoenolpyruvatecarboxylase, had severalfold higher specific activities in organ-formingcallus cultures of tobacco compared to non-organ-forming cultures.These activities increased considerably during the days precedingshoot and root differentiation. While malate accumulated untilday 15 in non-organ-forming callus, it accumulated up to day6 in shoot-and root-forming callus. Total and reducing sugarsaccumulated until day 3 and declined thereafter in all the cultures.Thus, tobacco callus may utilize this pathway for deriving reducingpower which is required for organogenetic processes. (Received April 30, 1987; Accepted December 1, 1987)     

Enzymic Maceration of Citrus Callus and the Regeneration of Plants from Single Cells

The maceration medium comprised a basal nutrient medium (BM)containing an optimum concentration of 3% (w/v) sucrose. Mannitoland sorbitol were inferior osmotica. Addition of potassium dextransulphate adversely affected maceration. ‘Macerozyme’was not as effective as ‘Macerase’ in the productionof single cells. The optimal concentration of ‘Macerase’was found to be 2–3% (w/v). Single cells obtained by filtering the macerate were rinsedwith BM and cultured in, and on, agar media comprising: BM;BM + 500 mg 1^–1 malt extract (ME); and BM + 10% (v/v)coconut milk (CM). No growth or organization was observed incultures where cells were mixed in with warm medium prior togelling. When spread on the surface of gelled media supplementedwith ME and CM, proliferation and organization occurred. Manymicroscopic globular proembryoids developed within 3 weeks onthe supplemented media. Microscopic torpedo-shaped embryoidswere frequently observed on BM + CM, rarely on BM + ME, andnot at all on unsupplemented BM. The high frequency of microscopic globular proembryoids, andlater of macroscopic pseudo-bulbils, formed on BM + ME leadsus to postulate that pseudobulbils are derived from globularproembryoids in which polarity is not established by the 16to 32-cell stage. Microscopic torpedo-shaped embryoids probablygive rise to macroscopic heart-shaped embryoids which developinto plantlets. The technique reported in this article provides an ideal systemfor examining embryogenesis per se and for studying the effectsof various treatments on embryogenesis and organ differentiationin vitro. It also affords excellent opportunities for the breedingof solid mutant plants.