Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 M 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 M 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l^-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 M abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.     

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 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 zygotic embryo explant cultures of rugosa rose

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (½MS) supplemented with 2.26, 9.05, and 9.05 μM 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ½MS without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.     

Somatic embryogenesis and plant regeneration from embryonic tissues ofCamellia japonica L.

Somatic embryogenesis and plantlet regeneration were achieved from immature and mature zygoticCamellia japonica embryos cultured on Murashige & Skoog's mineral medium without growth regulators or with various combinations of IBA and BAR The dependence of embryogenesis rates on growth regulator levels was not clear, though high concentrations such as 4 mg 1^-1BAP plus 2 mg 1^-1IBA were definitely inhibitory. BAP at 1 or 2 mg 1^-1 did appear to determine the formation of bud-like embryos. By far the most responsive initial explants were immature embryonic axes collected in September, 94% of which produced somatic embryos as against only 20% for embryonic axes from mature seeds collected in October. Cotyledon explants were also embryogenic. Somatic embryos differentiating directly on the hypocotyl of the embryonic axes or the surface of cotyledons passed through typical stages of embryogenesis. Indirect somatic embryogenesis via callus was also evident. Embryogenic potential was maintained by secondary embryogenesis through the successive generations of embryos.     

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 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.     

Somatic embryogenesis and plant regeneration from leaf tissue of jojoba

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength Murashige and Skoog basal medium (MS/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.35–4.52 μM) with 6-benzylaminopurine (1.33–4.43μM) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N′-phenylurea (1.21–4.03μM) or (E)-6-[3-(trifluoromethyl)-but-2-enylamino] purine (1.11–3.71μM) resulted in formation of embryogenic cultures and somatic embryos. After two 30-day subcultures, embryogenic cultures were transferred onto MS/2 medium supplemented with different auxins and cytokinins. Somatic embryo maturation, germination and plantlet formation were achieved using 1-naphthaleneacetic acid (3.75μM) or indole-3-butyric acid (3.44μM) in combination with BA (0.44 or 1.33μM) or F3iP (0.37 or 1.11μM). Histology confirmed each stage of development. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

We established a plant regeneration system for Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis. Embryogenic tissues were successfully induced on three kinds of Smith media from megagametophyte explants containing pre-cotyledonary embryos of C. obtusa plus-trees. Factors affecting somatic embryo maturation were examined. The concentration of polyethylene glycol 4000 in the medium was a critical factor for embryo maturation and its effective concentration was 150 g/l. The addition of 30 g/l maltose to the medium had a positive effect on embryo maturation, but sucrose was ineffective. The mature somatic embryos germinated at a germination frequency of approximately 60%, and the presence of activated charcoal was effective in stimulating plantlet growth. The plantlets acclimatized successfully in a greenhouse. To our knowledge, this is first report describing details of a plant regeneration method for C. obtusa via somatic embryogenesis.Abbreviations ABA Abscisic acid - PEG Polyethylene glycol 4000 - SM1 Smith Standard Embryonic Tissue Capture Medium - SM2 Smith Standard Embryogenesis Medium - SM3 Smith Embryo Develop Medium     

Somatic embryogenesis and plantlet regeneration in semul (<Emphasis Type="Italic">Bombax ceiba</Emphasis>)

Somatic embryogenesis in semul, Bombax ceiba L. (Bombacaceae) was achieved from immature zygotic embryo explants on MS medium. The cytokinin BA induced somatic embryogenesis at higher frequencies than the auxin 2,4-d. The rate of somatic embryogenesis was inversely related to the concentration of BA. A constant supply of 0.44 M BA was necessary for a high multiplication rate. Conversion of somatic embryos into plantlets is reported.     

Somatic embryogenesis and plant regeneration in callus from inflorescences of Hordeum vulgare X Triticum aestivum hybrids

Summary Embryogenic callus cultures were obtained by culturing young inflorescence tissues of Hordeum vulgare cv. PF51811 (2x)XTriticum aestivum cv. Chinese Spring (6x) hybrids on 2,4-D-containing N₆ medium. After subculture for about 10 months the calli retained a high potentiality for somatic embryogenesis and plant regeneration. Of about 300 regenerated plants, approximately 100 were transplanted to potting soil. Eight embryoids and three regenerated plants examined had 28 chromosomes identical to the original hybrid plants, while one regenerated plant was found to be a mixploid composed of cells with 28 and 56 chromosomes. The possibility for obtaining amphiploid hybrids through tissue culture is discussed.     

Somatic embryogenesis and plant regeneration from leaf tissues of Panicum maximum Jacq.

Summary Somatic embryogenesis was induced in proliferating leaf segments of Panicum maximum Jacq., cultured on Murashige and Skoog's nutrient medium containing 2,4-dichlorophenoxyacetic acid and coconut milk. The embryoids gave rise to plants on a medium containing gibberellic acid. The plants were successfully transplanted to soil and grown to maturity. Histological examination of proliferating leaves showed that the embryogenic callus tissue was formed by divisions in cells of the lower epidermis as well as the mesophyll tissue. The regenerated plants showed the normal chromosome number of 2n=4x=32.Florida Agriculture Experiment Station Journal Series No. 2775     

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.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of<Emphasis Type="Italic"> Cryptomeria japonica</Emphasis> D. Don

This report describes the successful plant regeneration via somatic embryogenesis from immature zygotic embryos of Cryptomeria japonica D. Don. For the induction of embryogenic tissue, we determined that the optimal medium contained N⁶-benzyladenine and 2,4-dichlorophenoxyacetic acid. Immature zygotic embryos that were collected at the end of June yielded embryogenic tissue at the highest frequency. Embryogenic tissues that had proliferated in liquid medium included small and loosely packed cells and elongating or elongated cells. We used ten cell lines to determine the optimal medium for the development of somatic embryos. Induced somatic embryos germinated with synchronous sprouting of cotyledons, hypocotyls and roots. Gibberellin A₃ in the germination medium had a positive effect on both the elongation of hypocotyls and the survival of seedlings. The frequencies of induction and germination of somatic embryos differed among the cell lines examined. Most of the seedlings grew normally. This system of somatic embryogenesis required 4–5 months for the regeneration of C. japonica plantlets from immature zygotic embryos.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellin A₃Communicated by F. Sato     

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 in Jatropha curcas Linn., an important biofuel plant

Jatropha curcas L. is one potential source of non-edible biofuel-producing energy crop. Its importance also lies in its medicinal properties. The species is primarily propagated through heterozygous seeds, and thus the seed oil content varies from 4 to 40%. Moreover, due to its perennial nature, seed setting requires 2 to 3 years time. The seed viability and rate of germination are low, and quality seed screening is another laborious task; thus, seed propagation alone cannot provide quality planting material for sustainable use. Somatic embryogenesis, a powerful tool of plant biotechnology for faster and quality plant production has been successfully applied to regenerate plants in Jatropha curcas for the first time. Embryogenic calli were obtained from leaf explants on MS basal medium supplemented with only 9.3 μM Kn. Induction of globular somatic embryos from 58% of the cultures was achieved on MS medium with different concentrations of 2.3–4.6 μM Kn and 0.5–4.9 μM IBA; 2.3 μM Kn and 1.0 μM IBA proved to be the most effective combination for somatic embryo induction in Jatropha curcas. Addition of 13.6 μM adenine sulphate stimulated the process of development of somatic embryos. Mature somatic embryos were converted to plantlets on half strength MS basal medium with 90% survival rate in the field condition. The whole process required 12–16 weeks of culture for completion of all steps of plant regeneration. This protocol of somatic embryogenesis in Jatropha curcas may be an ideal system for future transgenic research.     

Somatic embryogenesis and plantlet regeneration in the genus Secale

Summary A tissue culture of five wild species of the Secale genus, i.e., S. africanum (Stapf.), S. ancestrale (Zhuk.), S. kuprianovii (Grossh), S. segetale (Rosher.), and S. vavilovii (Grossh), from immature embryos of sizes (stages) varying between 1.0 mm to 3.0mm, cultured on MS (1962) mineral nutrient medium supplemented with 0.62 mg/1–5.0 mg/1 of 2,4-D, was established. Initially various types of callus were observed and a correlation between genotype, size of explant and 2,4-D concentration was found. The best embryogenic response was observed when explants were smaller than 1.0 mm. Induction of somatic embryogenesis of 2.0 mm–3.0 mm explants required a higher concentration of 2,4-D. Most embryoids were formed in the presence of 5.0 mg/l of 2,4-D. Secale africanum and S. kuprianovii appeared to have the highest embryogenic capacity among the five investigated species. For embryoids germination to plantlets the MS medium supplemented with GA₃ and cytokinins was used. Ultimately, out of the 932 regenerants obtained 364 originated from somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA3 deGibberellic acid - BAP Benzylaminopurine     

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 different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.