Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

Somatic embryogenesis from cultured mature cotyledons of cassava (Manihot esculenta Crantz)

Somatic embryogenesis was obtained from mature cassava cotyledons explants. A two-step medium sequence was developed for efficient embryogenesis. Application of 2,4-D (4 mg l^-1) yielded proembryogenic masses which developed into somatic embryos after transfer to a medium containing NAA (0.01 mg l^-1), BA (0.1 mg l^-1) and GA₃ (0.1 mg l^-1). The 2,4-D concentrations used for embryo initiation strongly influenced embryo development. Among the cultivars tested, TMS 30395 was most responsive. Full strength MS basal medium alone or with 4 x MS micro salts was efficient for the formation of somatic embryos. Casein hydrolysate, adenine sulfate, nicotinic acid, glycine, tryptophan, and serine were ineffective for embryo development. High sucrose concentration (6%, w/v) inhibited the induction of somatic embryos, while 6% sucrose was optimal concentration for the development of somatic embryos after an induction treatment using 2% sucrose. Addition of 0.52 mg l^-1 ABA to the induction media resulted in an increase in somatic embryos production. The ploidy levels of the regenerated plantlets were determined by flow cytometry analysis. Fifty regenerants tested were all tetraploids as the source plants and were morphologically normal. The implications of these results are discussed in relation to genetic transformation using the cotyledons as the explant source.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine - GA₃ gibberellic acid - MCPA methyl- chlorophenoxyacetic acid - NAA naphthalen-acetic acid - PCPA P-chlorophenoxyacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - 2,4,5 T 2,4,5-trichlorophenoxyacetic acid     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)

Hypocotyl segments of 2- to 3-week-old radish (Raphanus sativus L. cv. F₁ Handsome Fall) seedlings produced yellowish compact calli when cultured on Murashige and Skoog's (MS) medium supplemented with 1 mgl^-1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and -naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. When subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced white, compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on medium containing 0.1 to 3 mgl^-1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto medium containing 0.1 mgl^-1 2,4-D and 1 mgl^-1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - GA₃ gibberellin A₃ - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid     

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis

Summary Immature embryos from 5 Cladrastis lutea (Michx.) K. Koch (yellowwood) trees were initially cultured on modified Schenk and Hildebrandt medium (SH) containing either 4.5, 9.0, 13.5 or 23 M 2,4-D. One-third of the explants were transferred to SH medium supplemented with 25.0 M NAA after 2 and 3 weeks respectively. The remaining explants were incubated on the initial 2,4-D containing media for 6 weeks. Groups of somatic embryos formed directly only at the proximal end of cotyledons; only a few formed as single embryos. The greatest numbers were formed from zygotic embryos explanted from 6–8 weeks post-anthesis and initially cultured on medium containing 9 or 13 M 2,4-D. However, all treatments supported somatic embryogenesis. In the second year, explants were initially cultured on SH medium containing either 9.0, 13.5, or 23 M 2,4-D and then transferred to SH medium containing 4.0 M ABA after 2 or 3 weeks. ABA did not affect the development of somatic embryos. Six of 21 somatic embryos germinated on half-strength SH medium without growth regulators. Three entire plantlets were formed, but only one was established in soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - CRAF III chromium trioxide-acetic acidformalin     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Glycerol stimulation of chlorophyll synthesis,embryogenesis, and carboxylation and sucrose metabolism enzymes in nucellar callus of ‘Hamlin’ sweet orange

Anthers of niger (Guizotia abyssinica. Cass) were inoculated onto five different media differing mainly in their inorganic and organic constituents and plant growth regulators to study their influence on callus induction (embryogenic/non-embryogenic) and plant regeneration. LS medium supplemented with 2 mg 1^-1 2,4-d, and 0.3 mg 1^-1 KN favoured the production of EC, whereas 2 mg 1^-1 BAP and 0.5 mg 1^-1 KN promoted the NEC from anthers. Different types of embryos were initiated upon transfer of EC to Chaleff's R-2 medium containing 2 mg 1^-1 NAA and 0.3 mg 1^-1 KN and/or 5 mg 1^-1 ABA. NEC when transferred onto the medium supplemented with 1 mg 1^-1 BAP and 0.1 mg 1^-1 NAA produced on an average 8–12 shoots/callus mass. Embryoids developed from the EC and shoots differentiated from NEC when cultured onto the Chaleff's R-2 and MS media respectively lacking growth regulators, they transformed into whole plantlets. The plantlets thus obtained were successfully hardened and grown to maturity for analysis of various plant characters.Abbreviations EC embryogenic callus - NEC non-embryogenic callus - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - ABA abscisic acid - BAP 6-benzylaminopurine - KN kinetin - MS Murashige and Skoog's medium - LS Linsmaier and Skoog's medium     

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×10⁶/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl^–1 GA₃, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.Abbreviations BAP Benzylaminopurine - GA₃ Gibberellic acid - MT Murashige and Tucker medium (1969) - FDA Fluorescein diacetate     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

A new approach to direct somatic embryogenesis in Medicago

A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1–5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35–40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1–5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.Abbreviations ABA Abscisic acid - B5 Medium of Gamborg et al.(1968) - COT Cotyledone stage embryos - 2,4-D 2,4-dichlorphenoxyacetic acid - FW Fresh weight - GA3 Gibberellin A3 - MS Medium of Murashige and Skoog (1962) - PEG Polyethylene Glycol - POLY Polyembryos     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

Somatic embryogenesis in cultured immature kernels of Pistachio,Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl^–1 casein hydrolysate, 114 M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 M benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 M benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine (N⁶-benzyladenine) - EMS embryogenic mass - MS Murashige and Skoog medium (Sigma M-0404) - NAA -naphthalene acetic acid - PGR plant growth regulator - TDZ thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea) - WP McCown's Woody plant medium (Sigma M6774) - ABA abscisic acid     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

Somatic embryogenesis and plant regeneration in cotyledonary explant cultures of Chinese cabbage

Cotyledonary explants of Chinese cabbage were cultured on Murashige and Skoog's medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid. Up to 20% of the cotyledonary explants produced somatic embryos with or without intervening callus production. Explants became more competent as the age of the source seedlings increased up to 8 days, but cotyledonary explants from 10-day-old seedlings were not responsive. Upon transfer to MS basal medium most of the somatic embryos developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity in a phytotron. Among three cultivars used, only cotyledonary explants of Top Salad were capable of producing somatic embryos.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige & Skoog (1962)     

Somatic embryogenesis and plant regeneration in Cichorium intybus L. (Witloof,Compositae)

Somatic embryogenesis of Cichorium intybus L. var. Carolus is induced using cubical pieces of mature tap roots with an intervening callus phase. A Murashige and Skoog's (MS) semi solid basal medium supplemented with 2,4-dichlorophenoxyacetic acid (0.02 or 0.2 mg/l) and benzylaminopurine (0.25 mg/l) and a liquid MS medium devoid of growth regulators are used respectively for induction of callus and somatic embryoids and for further development and germination. Regeneration from the nodular proembryonal stage to the full grown embryoids occurs following different morphological pathways depending on the physical and chemical environment of the culture. Further development of these embryos into plantlets and the possibilities of application of this technique in plantbreeding have been discussed.Abbreviations MS Murashige and Skoog medium - BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid     

Callus formation from leaf mesophyll protoplasts of Malus Xdomestica Borkh. cv. Greensleeves

Large yields (1.85 × 10⁷/g.f.wt.) of viable protoplasts were obtained from leaves of axenic shoot cultures of Malus Xdomestica Borkh. cv. Greensleeves. Protoplasts cultured in liquid or agarose semi-solidified KM8P medium underwent cell wall regeneration and colony formation.Protoplast-derived cell colonies developed to callus on semi-solid KM8 medium. This is the first report of callus formation from mesophyll protoplasts of apple.Abbreviations BAP 6-benzylaminopurine - K kinetin - Z zeatin - GA₃ gibberellic acid - IBA 3-indole butyric acid - NAA 1-naphthalene acetic acid - IAA 3-indole acetic acid - ABA abscisic acid - f.wt. fresh weight - MS Murashige and Skoog (1962)