Cellular change and callose accumulation in zygotic embryos of Eleutherococcus senticosus caused by plasmolyzing pretreatment result in high frequency of single-cell-derived somatic embryogenesis

Summary. Eleutherococcus senticosus zygotic embryos were pretreated with 1.0 M mannitol or sucrose for 3–24 h. This pretreatment resulted in a high frequency of somatic-embryo formation on hormone-free medium. All the somatic embryos developed directly and independently from single epidermal cells on the surface of zygotic embryos after plasmolyzing pretreatment. Scanning electron microscopic observation revealed that the epidermal cells of hypocotyls rapidly became irregular and showed a random orientation before somatic-embryo development commenced. At the same time, the epidermal cells in the untreated control remained regular. Callose concentration determined by fluorometric analysis increased sharply in E. senticosus zygotic embryos after plasmolyzing pretreatment but remained low in the untreated control. Aniline blue fluorescent staining of callose showed that the plasmolyzing pretreatment of zygotic embryos resulted in heavy accumulation of callose between the plasma membrane and cell walls. On the basis of these results, we suggest that plasmolyzing pretreatment of zygotic embryos induces the accumulation of callose, and the interruption of cell-to-cell communication imposed by this might stimulate the reprogramming of epidermal cells into embryogenically competent cells and finally induce somatic-embryo development from single cells. Correspondence and reprints: Division of Forest Resources, College of Forest Sciences, Kangwon National University, Chunchon 200-701, Republic of Korea.     

Segregation for somatic embryogenesis on stem-internode explants from potato seedlings

Differences in productivity for somatic embryos (SEs) in vitro among 18 potato cultivars and three wild Solanum species in an earlier study led to the hypothesis that regeneration of SEs may be under genetic control. To examine this possibility, three test crosses were initiated; Coastal Russet×AF 186-2; Costal Russet×Lenape; AF 186-2×Lenape. True potato seedlings from these crosses were germinated in vitro. Five stem internode explants from each seedling were excised and cultured on two successive media to promote the formation of SEs. Seedling explants Costal Russet×AF 186-2 cross produced more SE than the other two crosses, and explants from the AF 186-2×Lenape cross generally only produced <10 SEs per explant. SEs were produced on the stem-internode explants from the three crosses at different rates. Data for the number of explants producing SEs and numbers of SEs per explant were highly significant. Regeneration of SEs is probably under nuclear control and the inheritance for regeneration may be quite straightforward. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of plantlets of Eleutherococcus sessiliflorus via somatic embryogenesis and successful transfer to soil

Explants of germinating zygotic embryos of Eleutherococcus sessiliflorus,an important medicinal plant, produced somatic embryos directly on Murashige and Skoog (MS) medium with 4.5 M 2,4-D. In addition, embryogenic callus formed at a low frequency (less than 7%) from hypocotyl segments after prolonged culture. High frequency somatic embryogenesis was obtained through cell suspension culture after the cells were transferred to medium lacking 2,4-D. Maturation and germination of embryos was influenced by the sucrose concentration of the medium. At a low concentration of sucrose (1%), maturation and germination of embryos occurred readily. At over 6% sucrose, somatic embryos did not germinate although this could be overcome by GA₃ treatment. Cold treatment during acclimatization after transfer to soil enhanced survival. Surviving plantlets produced new sprouts after overwintering in the field.     

Glutathione metabolism and antioxidant responses during Eleutherococcus senticosus somatic embryo development in a bioreactor

Compared to non-embryogenic callus, proembryonic mass, globular, and heart-shaped embryos of Eleutherococcus senticosus had higher levels of endogenous reduced glutathione (GSH). GSH content declined during the course of the embryo development (torpedo and cotyledon). Similarly, glutathione reductase that is involved in the recycling of GSH providing a constant intracellular level of GSH was also higher in globular and heart-shaped embryos. The transient increase in GSH contents also correlated with the changes in measured γ-glutamylcysteine synthetase activity over the same period. The endogenous levels of oxidized glutathione showed similar trend during development of the somatic embryos, whereas it declined in maturing somatic embryos. A pronounced increase in glutathione-S-transferase, glutathione peroxidase, catalase, and guaiacol peroxidase activity was observed during somatic embryo maturation. Ascorbate-glutathione cycle enzymes (ascorbate peroxidase; dehydroascorbate reductase and monodehydroascorbate reductase) activities also induced indicated that antioxidant enzymes played an important role during embryo development. These results suggested that the coordinated up-regulations of the antioxidant enzymes and glutathione redox system provide protection during somatic embryo development in E. senticosus. Antioxidant responses through alterations of the glutathione redox systems, have been described in the present studies have a significant role in somatic embryo development.     

High Frequency Plant Regeneration from Astragalus melilotoides hypocotyl and stem explants via somatic embryogenesis and organogenesis

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem segments on MS medium containing 9.05 µM 2,4-D and 2.22–4.44 µM BA. Both somatic embryos and adventitious buds were initiated from hypocotyl-derived calli while only adventitious buds were formed from stem-derived calli in MS medium supplemented with 2.69 µM NAA and 4.44–8.89 µM BA. Somatic embryos or adventitious buds developed into plantlets following being cultured for 3 weeks on MS medium without any growth regulators or with 14.78 µM IBA, respectively. All the regenerated plants were normal with respect to morphology and growth characters, and produced fertile seeds after planting in soil.     

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.     

Plant regeneration via direct embryo axis-like shoot and root formation from excised cotyledon explants of ginseng seedlings

Summary Cotyledon explants of Panax ginseng at various developmental stages were cultured on Murashige and Skoog (MS) medium with 0.5 μM indole butyric acid and 8.8 μM N⁶-benzyladenine. Upon culturing of cotyledon explants from mature zygotic embryos, 34% of the explants formed somatic embryos, and 46% formed adventitious shoots. In the cotyledon explants from 1-wk-old seedlings, embryo axis-like shoots and roots developed at a high frequency (79%) near the excised portion of the cotyledon base. The developmental pattern of embryo axis-like organ formation was structurally different from that of somatic embryos and adventitious shoots but similar to that of parts of the embryo axis of zygotic embryos. In the early stages of embryo axis-like organ formation, epicotyl-like shoot primordia were developed directly from the cotyledon base after 2 wk of culture; subsequently roots developed near the base of the epicotyl-like shoots and eventually regenerated into plantlets with both shoots and roots. The frequency of embryo axis-like organ formation declined as the growth of seedlings proceeded. In addition, the frequency of somatic embryo and adventitious bud formation rapidly declined with the age of the cotyledons. Plant regeneration via embryo axis-like organ formation might be a new pattern of morphogenesis in P. ginseng cotyledon culture.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

High frequency plant regeneration from anther-derived cell suspension cultures via somatic embryogenesis in Catharanthus roseus

Summary A system for high frequency plant regeneration from cell suspension cultures in Catharanthus roseus is described. Calli were obtained from anthers cultured on Murashige and Skoog's medium supplemented with 1 mgl^-1 -naphthaleneacetic acid and 0.1 mgl^-1 kinetin. After the second subculture on solid medium, embryogenic callus was identified and transferred to liquid medium to initiate suspension cultures. Cells dispersed finely in the medium were subcultured at 14-day intervals. Upon plating onto the basal medium, yellowish compact colonies proliferated from the cells and more than 80% of them gave rise to somatic embryos. Subsequently, plantlets developed from the embryos. Both the plantlets and the source plants showed the normal somatic chromosome number of 2n=2x=16.Abbreviations MS Murashige and Skoog - MSNK MS medium + 1 mgl^-1 NAA + 0.1 mgl^-1 kinetin - NAA -naphthaleneacetic acid     

Somatic embryogenesis and plantlet regeneration from cotyledon explants isolated from emblings and seedlings of hybrid firs

Embryogenic tissues have been initiated on cotyledon explants dissected from seedlings or emblings of hybrid firs. Cotyledons of seedling origin (Abies alba x A. cephalonica) gave a relatively low initiation frequency (1.94 percnt;). In embling-derived cotyledons (Abies alba x A. cephalonica, Abies alba x A. numidica), the initiation was cell-line dependent and reached values between 1.25 and 24.28 percnt;. The established embryogenic cell lines are being maintained in long-term cultures.The origin and development of the somatic embryos have been traced histologically. The early stages of somatic embryo development have been characterised by cell division activity (predominantly periclinal) in the epidermal and subepidermal layers of cotyledons and subsequently by development of nodular structures. Further differentiation led to the formation and emergence of somatic embryos on the surface of cotyledons.Somatic embryo development and plantlet regeneration occurred from proliferating tissues initiated from cotyledons of embling as well as seedling origin.     

Plant regeneration via direct somatic embryogenesis in Panax japonicus

Panax japonicus is one of the important medicinal plants. Here, we established the protocol for plant regeneration of P. japonicus via direct somatic embryogenesis. Somatic embryos were directly obtained from the segments of zygotic embryos on MS medium with 4.4 μM 2,4-D. Thereafter, somatic embryos were produced by repetitive secondary somatic embryogenesis. The secondary somatic embryo formation was enhanced by plasmolyzing pretreatment (1.0 M mannitol for 10 h). Frequency of secondary somatic embryo formation from cotyledon segments was lowered by plasmolyzing pretreatment, but the number of somatic embryos per explants was greatly increased. Plasmolyzing pretreatment resulted in retardation of embryo growth and required subculture to fresh medium for further growth of embryos into cotyledonary stage. Without plasmolyzing pretreatment, cotyledonary embryos were obtained after 8 weeks of culture. All the cotyledonary somatic embryos germinated by 5 μM GA₃ treatment, but only 15.3% were germinated on hormone-free medium. After 2 months of culture on 1/2 strength WPM medium, plantlets produced flowers spontaneously. In the anthers of in vitro flowers, microsporogenesis occurred normally with low number of pollen grains.     

TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium

To study the effect of auxin on direct somatic embryogenesis from leaf cultures ofOncidium `Gower Ramsey', 1-cm-long explants have been cultured in vitro testing IAA, 2,4-, quercetin, TIBA and PCIB. On a modified MS medium devoid of plant growth regulators, leaf cells of three regions (leaf tips, adaxial sides and cut ends) formed somatic embryos. After 8 weeks in culture, the frequencies of embryo-forming explants were 55, 52.5 and 30 % on leaf tips, adaxial sides and cut ends, respectively, and the numbers of embryos per dish was 89.3. Except for TIBA, other growth regulators (IAA, 2,4-, quercetin, PCIB) and their combinations tested, all retarded direct embryo formation. In the presence of 0.1 and 0.5 M TIBA, leaf tip, adaxial sides and cuts end of explants gave almost the same embryogenic response as the control. However, 10 and 27.5 % of explants were induced to form embryos from abaxial sides, and these explants did not form embryos on cut ends. In addition, after 8weeks in culture, TIBA at 0.5M highly promoted the mean numbers of embryos per dish to 134.2.     

Somatic embryogenesis and callus production from cotyledon explants of Eastern black walnut

Starting at 8 weeks and continuing until 23 weeks (nut drop) after anthesis,1 m² explants from cotyledons of immature seeds were extracted from Juglans nigra fruits. Explants were placed on Woody Plant Medium with 1 g l^-1 casein hydrolysate and 30 g l^-1 sucrose. The explants remained in light for 4 weeks on primary media containing a 3×3 factorial of 0.05, 0.5, or 5.0 M thidiazuron (TDZ) and 0.1, 1.0, or 10.0 M 2,4-d. Explants were transferred to a secondary medium containing no plant growth regulators and incubated in darkness for 11 weeks. The greatest number of somatic embryos was produced 8, 10, and 12 weeks after anthesis from explants on media with 0.5 or 5.0 M TDZ and 0.1 or 1.0 M 2,4-d. Explants produced the greatest callus volume and dry weight 10, 12, and 14 weeks after anthesis. Throughout the study, callus generally increased with increasing concentrations of both TDZ and 2,4-d.Abbreviations BA 6-benzyladenine - captan 3a,4,7,7a-tetrahydro-2-[(trichloromethyl)thio]-1H-isoindole-1,3(2H)-dione - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - Physan n-alkyl- dimethyl-benzyl ammonium chlorides and n-alkyl-dimethyl-ethylbenzyl ammonium chlorides - TDZ-thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Alfalfa embryo production in airlift vessels via direct somatic embryogenesis

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA abscisic acid - B5 Gamborgs B5 medium (Gamborg et al. 1968) - COT cotyledon embryo state - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - ID internal diameter - MS Murashige and Skoog medium (Murashige & Skoog 1962) - PEG polyethylene glycol - POLY polyembryos - VVM volume of gas/volume of bioreactor     

Continuous long-term somatic embryogenesis in alfalfa

Summary Somatic embryogenic callus was induced on two induction media, B5h and SH4K. Embryos formed on the callus induced on B5h medium when the callus was still on the induction medium. On the other hand, embryos could not form on the callus induced on SH4K medium unless the callus was transferred to a growth regulator-free medium. Callus induced and maintained on B5h medium lost embryogenic capability quickly during the subculture. Callus induced and maintained on SH4K medium, however, consistently remained highly embryogenic. The callus mass showed steady increase during its maintenance on SH4K medium. The embryos induced on SH4K medium showed vigorous germination. Normal and fully fertile plants were recovered from the embryos developed from the callus maintained on SH4K medium.     

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd

Protolasts of Bupleurum scorzonerifolium were prepared from stem node-derived embryogenic calli with an enzyeme mixture, in which snailase was a necessary component. Follolwing cell wall regeneration protoplasts divided and directly formed somatic embryos which developed into plantlets. The conditions favorable to direct embryo formation were investigated, and the nature of the callus used for protoplast preparation was found to be a critical factor. The osmotic concentration and the composition of the culture medium including the phytohormone combinations were also important.     

Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.