PROLIFERATION AND PLANT REGENERATION FROM THE NODAL REGION OF ZEA MAYS L. (MAIZE,GRAMINEAE) EMBRYOS

Immature embryos of Zea mays L. (maize), cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 0.25–1.0 mg/l) and sucrose (3, 6 and 12%), produced a narrow ridge of tissue in the nodal region of the embryo axis, opposite the scutellum. Only those embryos which were placed with the scutellum in contact with medium showed such a response. The ridge of tissue further proliferated and formed a compact and opaque callus that appeared similar to the scutellar callus. Morphogenesis took place on media containing low concentrations of both 2,4-D and sucrose. Plant regeneration was achieved and is interpreted to take place by the precocious germination of somatic embryos. It is suggested that the ridge of tissue formed at the node may represent an evolutionarily extinct epiblast of the maize embryo and may be cotyledonary in nature.     

金红花的组织培养快速繁殖研究

金红花顶芽或腋芽培养在ＭＳ基本培养基中。研究植物激素及培养基的物理性质对器官形成的影响。试验结果表明：芽增殖培养基以附加ＢＡ１．０ｍｇ／ｌ和ＮＡＡ０．２ｍｇ／ｌ为好。生根培养基为１／２ＭＳ＋ＮＡＡ０．１ｍｇ／１。糊状培养基有利于苗的生长,试管有根苗和无根苗移栽均获得高的成活率。     

Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.]

Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/l BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/l BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 mg/l IBA. The regenerated plants were successfully transferred to glasshouse conditions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - BAP 6-benzylaminopurine - KN Kinetin     

Somatic embryogenesis and plant regeneration from leaflets of peanut,Arachis hypogaea

Somatic embryos were induced on peanut (Arachis hypogaea) leaflets from aseptically germinated embryo axes. Leaflet size influenced percent somatic embryogenesis; 5–8 mm long cut leaflets were superior to 2–3 mm long uncut leaflets. Maximum embryogenesis of 14.6% was obtained after a 15 d incubation on induction medium (modified MS with B5 vitamins, 30 g/l sucrose, 4 g/l Gel-Gro, 40 mg/l 2,4-D +0.2 mg/l kinetin) followed by transfer to a secondary medium with 5 mg/l 2,4-D+0.2 mg/l kinetin. Primary somatic embryos were fused along the axes with no distinct cotyledons, but secondary embryos had single axes with two cotyledons. Other treatments had lower percent embryogenesis, no secondary embryogenesis, and embryos with single axes with two cotyledons. Some somatic embryos converted into normal plants capable of greenhouse survival.Abbreviations MS Murashige and Skoog (1962) medium - B5 Gamborg et al. (1968) B5 medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6benzylaminopurine - NAA 1-naphthaleneacetic acid     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION IN TISSUE CULTURES OF PANICUM MAXIMUM JACQ.

Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Direct somatic embryogenesis and plantlet regeneration from seedling leaves of winged bean,Psophocarpus tetragonolobus (L.) DC

Excised seedling leaf segments of winged bean [Psophocarpus tetragonolobus (L.) DC.] underwent direct somatic embryogenesis under appropriate incubation conditions. Initiation and development of the somatic embryos occurred using a two-step culture method. The culture procedure involved incubation for 28 days on MS basal medium supplemented with 0.1–0.5 mg/l NAA and 1.0–2.0 mg/l BA (induction medium) before transfer to MS medium supplemented with 0.1 mg/l IAA and 2.0 mg/l BA (embryo development medium). The initial exposure to low levels of NAA coincident with high levels of BA in the induction medium was essential for embryogenic induction. Maximum embryogenesis (43.3%) was obtained with 0.2 mg/l NAA and 2.0 mg/l BA, and at least 14 days on induction medium were required prior to transfer to the embryo development medium. The conversion frequency of cotyledonary embryos was 53.3% upon culture on MS medium containing 0.1 mg/l ABA for 7 days followed by transfer to MS medium supplemented with 0.1 mg/l IBA and 0.2 mg/l BA. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid     

Direct somatic embryogenesis and plant regeneration from leaf, petiole, and stem explants of Golden Pothos

Somatic embryos directly formed at cut edges or on the surface of leaf explants, around cut ends or along side surfaces of petiole and stem explants of Golden Pothos [Epipremnum aureum (Linden & Andre) Bunt.] on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) or N-phenyl-N-1, 2, 3-thiadiazol-5-ylurea (TDZ) with -naphthalene acetic acid (NAA) and a medium called MK containing MS salts with Kaos vitamins, supplemented with 2.0 mg/l TDZ and 0.2 mg/l NAA. Somatic embryos were also produced on MS medium containing 2.0 mg/l kinetin (KN) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) from leaf and petiole explants, MS medium supplemented with 2.0 mg/l CPPU and 0.5 mg/l 2,4-D from petiole and stem explants, and 2.0 mg/l TDZ and 0.2 mg/l or 0.5 mg/l 2,4-D from stem explants. In addition, somatic embryos occurred from stem explants on Chus N₆ medium containing 2.0 mg/l CPPU and 0.2 mg/l NAA. Somatic embryos matured and grew into multiple buds, shoots, or even plantlets after 2–3 months on the initial culture medium. Germination was optimal on MS medium containing either 2 mg/l 6-benzylaminopurine (BA) and 0.2 mg/l NAA or 2 mg/l zeatin and 0.2 mg/l NAA. Shoots elongated better and roots developed well on MS medium with no growth regulators. Approximately 30–100 plantlets were regenerated from each explant. The regenerated plants grew vigorously after transplanting to a soil-less container substrate in a shaded greenhouse.     

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.     

Somatic embryogenesis from integument (perisperm) cultures of coffee

Somatic embryogenesis was induced in integument (perisperm) cultures of C x R hybrid cultivar of coffee, after a culture period of 15 months, using a sequence of 3 modifications of MS medium. Vigorously growing soft, white, watery crystalline calli were obtained on MS + TIBA (1 mg/l) + L-cysteine HCl (50 mg/l) + PVP (100 mg/l). After 45 d, the calli were subcultured to MS + IAA (0.5 mg/l) + 2,4-D (0.05 mg/l) + Kn (8.6 mg/l) and maintained for the next 9 months without any transfer. On this medium, the callus proliferation was initially vigorous which slowed down after 5–6 months, and then the calli turned light brown and somewhat compact. Later, when the calli were transferred to MS + thiamine HCl (10 mg/l) + pyridoxine HCl (3 mg/l) + nicotinic acid (2 mg/l) + 2,4-D (0.2 mg/l) + 2ip (2.5 mg/l) and cultured for 2 months, they turned darker, more compact and the proliferation almost stopped. These calli were subcultured onto fresh medium of the same composition. After another 2 months of culture cream-coloured, highly friable, embryogenic calli appeared, which in turn produced a few clearly identifiable SEs in another 1 month. Further proliferation and maturation of SEs was achieved by culturing the embryogenic calli on MS + ABA (1 mg/l) for 3 months. The SEs were germinated into 2 cm tall plantlets after 2–3 subcultures, each of 2 months duration on 1/2-MS + Kn (0.1 mg/l).Abbreviations MS Murashige and Skoog (1962) basal medium - ABA Abscisic acid - TIBA 2,3,5 -Triiodobenzoic acid - IAA Indole-3-acetic acid - 2,4-D 2,4-dichloro-phenoxyacetic acid - Kn Kinetin - 2ip N⁶-(2-isopentenyl) adenine - PVP Polyvinylpyrrolidone; - SEs Somatic embryos     

Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Plant regeneration from cultured protoplasts of a glutinous rice

Young embryos of rice (Oryza sativa L. subsp, japonicavar. Guo-xiang No. 1) were cultured on MS agar medium(2,4-D 2 mg/l). Calli were formed and subcultured on N6 agar medium (2,4-D 2 mg/l ). After selection, the small, grainy and pale yellowish cell clusters with dense cytoplasm were used in protoplast preparation. Isolated protoplasts were cultured in N6 medium (2,4-D 1 mg/l, 6-BA 0.2 mg/l)~1 with agarose block culture method. The protoplasts grew, divided and formed calli. After inducing differentiation, the regenerated mature plants were obtained.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Initiation of embryogenic callus and suspension cultures of eastern white pine (Pinus strobus L.)

Embryogenic callus and suspension cultures of eastern white pine (Pinus strobus) have been obtained. The whole female gametophyte was plated on a medium containing 50 mg/l glutamine, 500 mg/l casein hydrolysate, 3% sucrose, 2 mg/1 2,4-D, 1 mg/1 BA and 0.2% Gelrite as a solidifying agent. Embryogenic calli could be seen as early as 5 days following culture. Histological studies indicate proliferation of pre-existing embryogenic tissue in the corrosion cavity followed by extrusion of embryogenic callus through the micropylar end of the gametophyte. Embryogenic suspension cultures were obtained by placing embryogenic callus into liquid medium. Embryogenic suspension cultures were subcultured weekly and proliferated as early-stage embryos with attached suspensors. Embryo development was obtained following transfer of the embryogenic tissue to an auxin-free medium containing 50 mM glutamine, 38 M abscisic acid, and 6% sucrose. Although embryo development could be consistently obtained, whole plants have not yet been recovered from these somatic embryos.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - ABA Abscisic acid - BA 6-Benzyladenine Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC. Journal Article No. 62–89     

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     

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     

Somatic embryogenesis in two Gossypium hirsutum genotypes on semi-solid versus liquid proliferation media

A comparison of semi-solid vs. liquid embryo proliferation media was made using two Gossypium hirsutum L. genotypes (Coker 312 and T25) and two callus initiation media. Sections of petioles from mature, flowering plants were cultured on two modified Murashige and Skoog media. Medium 1 included 4.0 mg l^-1 NAA and 1.0 mg l^-1 kinetin; medium 2 contained 0.1 mg l^-1 2,4-D and 0.1 mg l^-1 kinetin. After six weeks, callus was removed from each explant and divided in half. One callus portion was placed in liquid proliferation medium and the other on semi-solid (0.2% Gelrite) proliferation medium. Composition of proliferation medium was identical to that of initiation medium, except no growth regulators were added. Embryos were counted after eight weeks. The percentage of explants forming callus was influenced by genotype/initiation medium combination. Analysis of variance procedures revealed significant variability for callus initiation media, proliferation media (semi-solid or liquid), and an initiation medium x genotype interaction. Paired t-tests indicated that more embryos were produced in liquid proliferation medium (227.3 embryos/culture) than on semi-solid proliferation medium (134.6 embryos/culture).Abbreviations NAA naphtaleneacetic acid - 2,4-D 2,4-D dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from immature embryo explant of papaya (Carica papaya L. cv. washington and honey dew)

Immature zygotic embryo explants of Carica papaya were cultured on MS medium supplemented with 2,4-D (2.0 mg/l) and formed globular embryos on explants without callus formation in 4-6 weeks. Maturation and conversion of somatic embryos was also achieved on the same medium. Cotyledonary stage embryos germinated to 63.66 and 68.33% in cv. honey dew and washington respectively in MS basal medium supplemented ABA (0.5 microm/l). Robust development and proliferation of plantlet roots in vitro was obtained on MS basal medium. Hardened plantlets have 60% survival rate.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

Effects of ABA on secondary embryogenesis from somatic embryos induced from inflorescence culture ofAralia cordata Thunb

In order to investigate the effect of ABA on secondary embryogenesis from somatic embryos inAralia cordata Thunb., embryogenic callus and somatic embryos were induced from inflorescence on solid MS basal medium supplemented with 1.5 mg/L 2,4-D after eight weeks without subculture. For mass production of somatic embryos, embryogenic cell clumps were maintained in liquid MS medium supplemented with 1.0 mg/L 2,4-D, and then transferred to 2, 4-D-free medium. When developing embryos at various stages were cultured separately in liquid medium with ABA (0 to 2.0 mg/L) for three weeks, and then cultured in ABA-free liquid medium for two weeks, torpedo-shaped embryos exhibited secondary embryogenesis of 65.9% in only 0.2 mg/L ABA pretreatment. Cotyledonary embryos in cultures by 0.2, 0.5 and 1.0 mg/L ABA pretreatment also exhibited secondary embryogenesis (73%, 9.4% and 6.0%, respectively). However, globular and heart-shaped somatic embryos treated with ABA did not form secondary embryos on their hypocotyl surfaces. When cotyledonary embryos were cultured in ABA-free medium or 0.2 mg/L ABA treated medium for three weeks, and then in ABA-free liquid medium for 6 weeks, the germination frequency was lower in medium with 0.2 mg/L ABA (45.9%) than in hormone-free medium (56.8%). This result seems to be related to the high frequency of secondary embryogenesis. It is suggested that secondary embryogenesis by ABA application depends upon the stage of embryo cultured and the ABA concentration.