Oat leaf base: tissue with an efficient regeneration capacity

Summary An efficient short term regeneration system using seedling derived oat (Avena sativa) leaf tissue has been developed. Callus derived from the leaf base showed a higher response of plant regeneration than callus initiated from mesocotyls and more mature parts of the leaves. A correlation between the nuclear DNA content of the donor material, as analysed with flow cytometry, and its ability to form callus was observed. Somatic embryogenesis was histologically recognised from callus derived from tissue close to the apical meristem. Plant regeneration media with various concentrations of auxin were tested. Callus from three different cultivars had a similar regeneration potential with an optimal regeneration frequency of 60%. About 2 months after inoculation regenerated plantlets could be moved to a greenhouse for cultivation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 6-diamidino-2-phenylindole - IAA indole-3-acetic acid - KT kinetin - MS Murashige and Skoog's medium - NAA naphthalene acetic acid     

以根为外植体建立大蒜的组织培养体系

以国内4个大蒜栽培品种为材料,建立了以根为外植体的再生体系。将蒜瓣去皮后灭菌消毒,萌发后选取苗龄为5~7 d的无菌苗的根接种到含不同激素配比的培养基中进行愈伤组织诱导,发现MS+2,4-D 1 mg/L+2ip 0.1 mg/L组合愈伤诱导效率最高,平均为56.06%;愈伤组织经过2~3次继代培养,选取胚性愈伤组织置于不同分化培养基上进行培养,2~3个月后可见小芽产生,分化培养基为MS+KT 1 mg/L时,植株再生效率最高,平均达到35.01 %。本研究建立了一种以根为外植体的高效的大蒜愈伤诱导和再生体系,为大蒜遗传转化体系的建立打下良好基础。     

Study on Tissue and Protoplast Culture of Wild Cotton (Gossypium davidsonii)

This paper deals with the study on the condition of callus formation, embryogenesis, organogenesis, plant regeneration and protoplast culture of wild cotton (G. davidsonii) Callus cultures derived from several organs such as root, stem, leaf, cotyledon and hypocotyl. The results obtained in these cultures showed that the modified MS medium containing 2,4-D 1.0+KT 0.1; 2,4-D 0.1+KT 0.01; NAA (IAA) 2.0+KT 0.1 and NAA (IAA) 1.0+KT 0.1 mg/L were favorable to callus formation. Modified MS medium containing 2,4-D was suitable for initiated callus of G. davidsonii Besides, suspension cultures from callus of G. davidsonii were saccessfully initiated. Optimum concentration of 6BA (or ZT, or 2ip) and NAA (IAA) was for shooting, somatic embryo or leaf formation. Plantlets regenerated from somatic embryo at lower concentration of 6BA, or ZT, or 2ip. As to protoplast culture of this species, the age and physiological condition of callus or suspension cells and concentration of enzymes used for protoplast isolation affected the yield and survival of protoplasts. Protoplast of this species cultured in modified MS medium containing 2,4-D 0.5+NAA 0.5+ZT 0.1–0.2 mg/L. and divied after 3–4 days. The rate of division was 3--4% and cell cluster formed after 14 days, then these cells died.     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/l 2,4-D and 0.2–0.5 mg/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/l 2iPA and 0.2 mg/l NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/l BAP and 0.5 mg/l NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - IAA 3-indoleacetic acid - 2iPA N⁶-(²-isopentenyl) adenosine - BAP 6-benzylaminopurine - KT kinetin - CH casein hydrolysate     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

太白米组织培养的研究

太白米地下鳞茎在MS附加不同浓度KT、BA、IAA、NAA、2,4－D的培养基上,可诱导产生愈伤组织,其中在MS附加NAA 0.5mg/L,KT0.1mg/L的培养基上愈伤组织诱导率最高,可达65％,且生长快;培养在MS附加2,4－D 1.0mg/L,KT 0.1mg/L培养基上的鳞茎可经不定根直接发育成新的鳞茎,由此建立了太白米的鳞茎再生体系,鳞茎再生率达2．17倍。     

CHARACTERIZATION OF AN EMBRYOGENIC CELL SUSPENSION CULTURE DERIVED FROM CULTURED INFLORESCENCES OF PENNISETUM AMERICANUM (PEARL MILLET,GRAMINEAE)

An embryogenic suspension culture was established from cultured inflorescence segments of Pennisetum americanum in Murashige and Skoog's medium supplemented with 2.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) and 5% coconut milk. The suspension was composed of two major cell types: 1) small, richly cytoplasmic and starch-containing cells, generally found in small, compact clumps, here termed embryogenic cells; and 2) elongated, thick-walled cells with large vacuoles. By manipulating the duration of culture and dilution ratios (cell suspension: fresh medium) at the time of subculture, suspensions consisting predominantly of embryogenic cells were obtained. Suspensions grown for 2-3 wks were transferred to agar media with reduced amounts of 2,4-D. This resulted in the production of hundreds of globular and early cotyledonary embryoids. Further development of the embryoids was promoted by their transfer to a medium containing abscisic acid. Many of the embryoids germinated and produced normal green plants. Atypical embryoids, some containing many shoot meristems and a leafy scutellum, were also observed. The relevance of such atypical embryoids in the interpretation of organogenesis and embryogenesis reported in tissue cultures of cereal species is discussed. It is also suggested that somatic embryogenesis occurs in tissue cultures of most, if not all, species of cereals and grasses.     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.     

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 and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

羊草与灰色赖草F1代细胞悬浮系的建立及植株再生

本研究以羊草(L eym us ch inensis)与灰色赖草(L eym us cinereus)杂种F1代幼穗为外植体诱导愈伤组织,在3.0 m g/L 2,4-D M S培养基上继代1次后,转入不同浓度激素(2,4-D、IAA、KT)配比和不同浓度蔗糖的M S液体培养基进行振荡培养,建立杂种F1代细胞悬浮系和植株再生体系.结果表明,细胞悬浮培养时,M S 1.0 m g/L2,4-D 0.1 m g/L KT 4%蔗糖的液体培养基最佳;悬浮细胞分化时,1.0 m g/L 2,4-D 0.1 m g/L KT 4%蔗糖 M S和1.0 m g/L 2,4-D 4%蔗糖 M S培养的悬浮细胞在1.0 m g/L NAA 0.5 m g/L KT M S分化培养基上的绿苗分化率分别达到83%和80%.细胞悬浮系及再生体系的建立为杂种F1代育性恢复的研究奠定了基础.     

Endogenous hormone levels in explants and in embryogenic and non-embryogenic cultures of carrot

Carrot ( Daucus carota L. F1 hybrid Starca) excised hypocotyls were cultured on Murashige and Skoog medium with and without 2,4-dichlorophenoxy acetic acid (2,4-D) to determine the effect of this plant growth regulator on their further development and their endogenous hormone levels. Culture in the absence of 2,4-D stimulated root development at one end of the hypocotyl segments and increased the endogenous levels of free indole-3-acetic acid (IAA), zeatin/zeatin riboside and N ⁶(Δ²-isopentenyl) adenine/ N ⁶(Δ²-isopentenyl) adenosine, as determined by radio-immunoassay. On the other hand, the presence of 2,4-D in the culture medium promoted callus induction and proliferation, together with abscisic acid (ABA) accumulation, in the hypocotyl segments during the first weeks of culture. When the callus segments generated in the hypocotyl sections cultured in the presence of 2,4-D were cultivated further, the development of two callus types was observed, one composed of preglobular and globular embryos and the other translucent, watery and lacking any sign of organisation. The embryos of the first type germinated when callus segments were transferred to regeneration conditions, while no change was observed when the second type was induced to regenerate. Higher levels of free IAA and ABA were obtained in the embryogenic calli when compared to the non-embryogenic, while no differences were observed among callus types in the other hormones evaluated. The possible role of the different plant hormones during induction of somatic embryogenesis is discussed.     

In vitro plant regeneration of Arachis correntina (Leguminosae) through somatic embryogenesis and organogenesis

In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and organogenesis were developed using immature leaves as explants. Morphologically normal somatic embryos were obtained on culture media composed of 20.70 or 41.41 μM picloram (PIC) with the addition of 0.044 μM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively. The source of explants and the developmental stage of the leaves had a marked effect on somatic embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive producing up to 30% embryogenesis in MS+41.41 μM PIC. Embryos converted into plants after transfer to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) with or without 0.044 μM BA, achieving plant regeneration in the induction media. The highest percentage of bud formation was obtained on culture medium composed of␣MS+10.74 μM NAA+0.044 μM BA (12.5%). Roots were formed on all culture media tested. Regenerated plants were transferred to pots and grew well under greenhouse conditions.     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Influence of media and growth regulators on somatic embryogenesis and plant regeneration for production of primary triticales

Basal media and plant growth regulators were tested for the promotion of somatic embryogenesis from immature wheat-rye hybrid embryos. Influence of growth regulators and chilling on plant regeneration were tested on two media. A medium containing four amino acids-glutamine, arginine, glycine and aspartic acid-as the nitrogen source, promoted the production of, on average, twice as much embryogenic callus as the other media, and somatic embryos developed well. The growth regulator dicamba was significantly better than 2,4-dichlorophenoxyacetic acid in promoting somatic embryogenesis and subsequent plant regeneration. Germination of somatic embryos on both regeneration media was enhanced by cold treatment. Supplementing 190-2 plant regeneration medium with a combination of -naphthaleneacetic acid + benzyladenine, indole-3-acetic acid + kinetin or indole-3-acetic acid + zeatin resulted in equally high germination rates.Abbreviations 190-2 Plant regeneration medium of Chuang & Jia - 2,4-d 2,4d Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-o-anisic acid - AA Amino acid medium of Müller & Grafe - IAA Indole-3-acetic acid - BA Benzyladenine - NAA -Naphthaleneacetic acid     

Effect of plant growth regulators on ovary culture of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 μM thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 μM 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y₃ medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA₃) to conversion medium containing 5 μM 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.