Somatic embryogenesis and plant regeneration in Quercus acutissima

Immature embryos of Quercus acutissima were collected weekly beginning 5 weeks post-fertilization and cultured on modified MS(Murashige and Skoog) medium containing 1,000 mg/l glutamine and 5 mM proline with different combinations of IBA(0.5–10.0 mg/l) and BA(0 or 1.0 mg/l) in light. The highest percentage of embryogenic cultures occurred on the medium containing 0.5 mg/l IBA or 1.0 mg/l BA and 0.5 mg/l IBA. Four weeks after initiation, the embryogenic cultures were transferred to MS medium without plant growth regulators and cultured for 4 weeks. The somatic embryos were then transferred to germination medium. The best germination results were achieved from WPM(Woody Plant Medium) containing 0.1 mg/l BA. Plantlets from somatic embryos were incubated on WPM supplemented with 0.2 mg/l BA for 4 weeks and plantlets with well developed shoots and roots were transplanted to perlite and peat moss(11, v/v) mixtures and placed in a culture room. After being hardened off for 8 weeks, they were transferred outdoors where they grew.Abbreviation BA N⁶-benzyladenine - IBA indole-3-butyric acid - GA₃ gibberellic acid - ABA abscisic acid - MS Murashige & Skoog Medium - WPM Woody Plant medium     

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μm kinetin or zeatin, but was highest on medium containing 4.5 μm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 μm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 μm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse. Received: 14 January 1997 / Revision received: 17 June 1997 / Accepted: 5 July 1997     

Somatic embryogenesis and plant regeneration from immature embryo cultures of onion (Allium cepa L.)

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies. Received: 16 December 1997 / Revision received: 23 February 1998 / Accepted: 13 March 1998     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

Somatic embryogenesis and plant regeneration in Acanthopanax senticosus

Mature embryos of Acanthopanax senticosus explanted on Murashige and Skoog (MS) medium with 0.5 mg/1 2,4-D developed somatic embryos directly from swollen cotyledon and embryo axes within one to two months. When the somatic embryos were transferred to medium supplemented with 2,4-D (0.5 mg/1) or IAA (1–3 mg/1) or Zeatin (0.5 mg/1) and NAA (0.2 mg/1), additional somatic embryos developed. Most (93%) embryos germinated on the above medium without 2,4-D. Sixty-two percent of the plantlets survived in soil. Histological observations revealed that the somatic embryos originated from cell masses of epidermal and sub-epidermal origin. There was no cytological separation zone between the somatic embryos and cultured expiants. Consequently, embryos were difficult to separate from their expiant tissue.     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Somatic embryogenesis and plant regeneration in Lilium longiflorum Thunb

Friable callus was obtained from styles and flower pedicels of Lilium longiflorum Snow Queen and the Oriental lily hybrid Star Gazer on Murashige and Skoog (MS) media containing either 2 μm dicamba or 2 μm picloram. Cell suspension cultures were established by suspending the callus of L. longiflorum Snow Queen in liquid medium containing 2 μm dicamba. Through a purification process, a fine fast-growing cell suspension was obtained. This suspension was composed of a homogenous population of small dense cells, which tended to organise into embryo like structures (ELS). In liquid culture with the auxin dicamba, the ELS underwent continuous callus formation. When transferred to solidified hormone-free MS medium, the ELS germinated, forming complete plantlets. Histological investigation showed that in the ELS both shoot and root meristems were distinctly evident. It was concluded that the ELS obtained were in fact somatic embryos. Received: 4 April 1997 / Revision received: 13 May 1997 / Accepted: 15 June 1997     

Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Summary Seventeen cultivars of cotton (Gossypium hirsutum L.) were evaluated for callus initiation and maintenance using 3 initiation media and 3 maintenance media. After a series of transfers of a 3% glucose media, calli were placed on a 3% sucrose medium. After several weeks calli were observed for the presence of embryo-like structures. Cultivars Coker 201 and Coker 315 were identified as embryogenic. Embryogenic callus has since been routinely obtained within 6 weeks by initiating callus on glucose media for 3–4 weeks followed by transfer to sucrose media. Histological examination has shown that embryos are derived from isodiametric, densely cytoplasmic cells and follow predictable patterns of development. Upon maturity, transfer to auxin-free media with reduced sucrose levels results in embryo germination. Regenerated plants can be transferred to greenhouse within 90 days of callus initiation.The senior author is presently a Research Geneticist, USDA-ARS, and Assistant Professor Present address     

Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain

Thirty-two barley cultivars grown in Spain, 18 of the two-row type and 14 of the six-row type, were screened for plant regeneration from cultured immature embryos. Although there was much variation in regeneration capacity among the cultivars, plants were obtained from all cultivars except Almunia. No statistical differences were found in the percentage of regeneration between two- and six-row types. The influence of the auxins 2,4-dichlorophenoxyacetic acid, dicamba, and picloram on the induction and maintenance of embryogenesis and regeneration capacity after 3–4 months in culture, were evaluated for cultivars Cobra, Hop and Reinette. Hop had the highest rates of maintenance of embryogenic capacity and plant regeneration. The medium containing dicamba gave the best embryogenic callus induction, maintenance and regeneration. Five regeneration media, differing in growth regulators and micronutrient composition, as well as partial desiccation of the calli before regeneration, were tested. The regeneration medium containing 10 μm copper sulfate gave the best results. Regeneration frequencies after 3–4 months in culture of cultivar Hop were raised from 59.5 to 93.7% in this medium. Silver nitrate and partial desiccation of the calli also enhanced plant regeneration, but the medium containing 10 μm of silver nitrate reduced root formation. Received: 30 October 1997 / Revision received: 3 April 1998 / Accepted: 17 April 1998     

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     

Somatic embryogenesis and plant regeneration in chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura)

 Somatic embryogenesis was observed in ray-floret explants of Dendranthema grandiflorum (Ramat.) Kitamura cv. Aboukyu on Murashige and Skoog medium containing high concentrations of 3-indoleacetic acid (IAA) and kinetin. 1-Naphthaleneacetic acid also induced somatic embryogenesis but indole-3-butyric acid or 2,4-dichlorophenoxy acetic acid did not. Other cytokinins, such as 6-benzylaminopurine (BAP) and thidiazuron, were also not effective. No embryos were seen at lower IAA concentrations with kinetin and various concentrations of BAP, although higher BAP concentrations yielded many adventitious shoots. In contrast, no somatic embryogenesis was observed from leaves using any combination of plant growth regulators. Histologically, primordia showed a typical embryo shape with a well-developed vascular bundle between the shoot and the root primordia. Embryos had both stomata cells and a root system with polarity. Plants were efficiently regenerated from ray floret-derived embryos subcultured in the appropriate medium. Received: 30 April 1999 / Revision received: 7 October 1999 / Accepted: 27 January 2000     

Somatic embryogenesis and plant regeneration in centipedegrass (Eremochloa ophiuroides [Munro] Hack.)

Centipedegrass (Eremochloa ophiuroides [Munro] Hack.) is an important warm-season turfgrass and pasture grass. To explore the potential use of biotechnical tools in breeding of centipedegrass, we established an efficient plant regeneration system for this species. Four basal media and 24 combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP) were examined for their effects on callus induction from mature seed explants. Twenty combinations of naphthaleneacetic acid (NAA) and BAP were tested for their effect on plant regeneration. Results indicated that Murashige and Skoog basal medium supplemented with 4.5 mg l⁻¹ 2,4-D and 1 mg l⁻¹ BAP was the best medium for callus induction, while the combination of 2 mg l⁻¹ BAP and 1 mg l⁻¹ NAA induced the highest rate of regeneration and development of shoots and roots. This work provides a basis for the breeding of centipedegrass through somaclonal variation and genetic transformation.     

High frequency somatic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata

Culture conditions for high frequency somatic embryogenesis and plant regeneration from cotyledonary explants of Codonopsis lanceolata are described. The maximum induction frequency of somatic embryos from cotyledonary explants was 80% on Murashige and Skoog (MS) medium containing 6% sucrose with 1 mg/l 2,4-dichlorophenoxyacetic acid and 10% coconut water. Upon transfer onto MS basal medium containing 3% sucrose, most somatic embryos developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellin a₃ - MS Murashige and Skoog     

Somatic embryogenesis and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.) Marechal]: a recalcitrant grain legume

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with 0.75 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ 6-benzylaminopurine (BA) and with various additives (50 mg l⁻¹ ascorbic acid and 25 mg l⁻¹ each of adenine sulphate, citric acid and l-arginine). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with 0.25 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing 0.2 mg l⁻¹ BA and 2.0 mg l⁻¹ gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.     

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

Somatic embryogenesis and plantlet regeneration from cell suspension cultures of Fagus sylvatica L

Summary Embryogenic cell suspension cultures and somatic embryos of five genotypes of beech, were obtained from aged cultures derived from immature zygotic embryos cultured on solid medium containing both 2, 4-dichlorophenoxyacetic acid and N6-benzyladenine. The origin of somatic embryos was traced from single cells. Embryos remained arrested at the globular stage on liquid media, further development was achieved after plating embryogenic aggregates on Murashige and Skoog's medium with half strength major salts supplemented with glutamine and low levels of growth regulators. Cultures of different genotypes showed significant differences in maturation frequency which was not affected by the hormone treatments assayed. The frequency of conversion of embryos into plantlets was low. This frequency increased after cold storage of embryos for up to 7 months.Abbreviations BA N6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EtOH ethanol - GA₃ giberrellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - WPM woody plant medium (Lloyd and McCown, 1980) - Z zeatin     

Meta-topolin enhances regeneration and Agrobacterium-mediated genetic transformation in radish (Raphanus sativus L.)

In Vitro Cellular & Developmental Biology - Plant - Meta-topolin (mT) is a novel aromatic cytokinin that stimulates morphogenesis and is an alternative source of cytokinins frequently employed...     

Somatic embryogenesis and plant regeneration from pistil thin cell layers of Citrus

 Callus induction, somatic embryogenesis and plant regeneration were obtained in six different citrus species [Citrus deliciosa Ten. (cv 'Avana'), C.limon (L.) Burm. (cv 'Berna'), C.madurensis Lour. (cv 'CNR P9'), C.medica L. (cv 'Cedro di Trabia'), C.tardiva Hort. ex Tan. (cv 'CNR P6'), C.sinensis (L.) Osb. (cv 'Ugdulena 7')] from cultures of pistil transverse thin cell layer explants [(t)TCL]. Explants were cultured on three different media: the nutrients and vitamins of Murashige and Skoog medium alone (MS) or MS supplemented with either 500 mg l^–1 malt extract (MS I) or 500 mg l^–1 malt extract and 13.3 μM 6-benzylaminopurine (MS II). Sucrose (146 mM) was used as the carbon source. Somatic embryos were visible 2–5 months after culture initiation. The different genotypes showed a different embryogenic frequency from stigma, style and ovary (t)TCL explants. All of the cultivars regenerated somatic embryos. Percentages of style (t)TCL explants producing somatic embryos ranged from 0% (C.deliciosa, C.madurensis, C.sinensis and C.tardiva on the three different media) to 5.2% (C.limon on MS II). Embryo formation in stigma (t)TCL explants ranged from 0% (C.madurensis on MS and MS I, C.sinensis on MS, C.deliciosa and C.tardiva on the three different media) to 42.4% (C.limon on MS II). Embryo formation in ovary (t)TCL explants ranged from 0% (C.deliciosa on MS, C.limon, C.medica, and C.sinensis on the three different media) to 9.3% (C.tardiva on MS I). After about 12 weeks somatic embryos developed into plantlets at a high frequency. Received: 22 September 1998 / Revision received: 6 November 1998 / Accepted: 23 November 1998     

Somatic embryogenesis and plant regeneration from immature seeds of Magnolia obovata Thunberg

We have tested plantlet formation by somatic embryogenesis using immature seeds of Magnolia obovata. Seed collection date appeared to be critical for embryogenic cell induction. The optimal collection date was 3–4 weeks postanthesis. The embryogenic cells proliferated, formed somatic embryos, and were subsequently converted into normal plantlets under optimized culture conditions. Somatic embryo formation from the embryogenic calli was better on sucrose medium than on glucose medium. The optimum level of sucrose appeared to be 3% with an average of 28 somatic embryos per plate. About 25% of somatic embryos were converted into normal plantlets in 1/2 MS medium containing gibberellic acid (GA₃). During somatic embryo germination, secondary embryogenesis was frequently observed in the lower part of the hypocotyl or radicle ends of germinating somatic embryos. Finally, about 85% of converted plantlets survived in an artificial soil mixture, were transferred to a nursery, and have grown normally.