In vitro somatic embryogenesis and plant regeneration in Acacia arabica

In vitro somatic embryogenesis and subsequent plant regeneration was achieved in callus cultures derived from immature zygotic embryos of Acacia arabica on semi-solid Murashige and Skoog (MS) basal salts and vitamins supplemented with 8.88 MBA, 6.78 M2,4-D and 30 g l^–1 (w/v) sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to MS medium supplemented with 6.66 M BA, 6.78 M 2,4-D. The maximum number of somatic embryos per callus was 72.6 after 8 weeks of culture on medium containing 6.66 M BA and 6.78 M 2,4-D. The isolated somatic embryos germinated on half-strength basal MS salts and vitamins supplemented with 0.04 M BA, 0.94 M ABA and 2% (w/v) sucrose. The embryo-derived plantlets were acclimatized in the greenhouse and subsequently showed normal growth.     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

High frequency somatic embryogenesis in Coffea canephora

Leaf explants of Coffea canephora (P. ex Fr.) produced a friable yellow callus when they were cultured on a conditioning basal medium with 2.2 M 2,4-D, 2.4 M IBA and 9.8 M 2iP for 4 weeks then on an induction basal medium with 4.4 M 2,4-D and 17.8 M BA for 10 weeks. This calus could be maintained by means of regular subcultures or it could give rise to somatic embryos depending on the culture medium. Cytological studies documented somatic embryogenesis and embryo development.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - 2iP 2-isopentenyladenine - MS Murashige & Skoog medium - NAA -naphthaleneacetic acid - NPR nucleoplasmic ratio - PGR plant growth regulator     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

Somatic embryogenesis from leaf cultures of potato

An efficient procedure has been developed for inducing somatic embryogenesis from leaf cultures of potato cv. Jyothi. Leaf sections were initially cultured on 2,4-dichlorophenoxyacetic acid (2,4-D) + benzyladenine (BA) and -naphthaleneacetic acid (NAA) + BA supplemented Murashige and Skoog (MS) media. Nodular embryogenic callus developed from the cut ends of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. The explants with primary callus were subsequently moved onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA. Treatment with zeatin (22.8 M) and BA (10.0 M) resulted in the induction of the highest number of somatic embryos directly from meristematic centres produced on the nodular tissue. Embryo induction and maturation took place on this medium. The cotyledonary stage embryos developed into complete plantlets on hormone-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis in leaf cultures of potato which has not been reported previously.     

High-frequency plant regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.)

Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 M 2,4-dichlorophenoxyacetic acid (2,4-D), 60 M 4amino-3, 5,6-picolinic acid (picloram), or 30 M 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 M dicamba plus 10 M 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 M dicamba, 20 M BA) resulted in regeneration of shoots from 45% of calli of the cultivar Baron. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyaceticacid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,5,6-picolinic acid - BA 6-benzylaminopurine     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

Stepwise decrease of 2,4-D and addition of BA in subculture medium stimulated shoot regeneration and somatic embryogenesis in buffalograss

Plant regeneration of buffalograss `Texoka' was achieved through both somatic embryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 M 2,4-D combined with either 0.44 M or 1.32 M BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D (4.5 or 9 M) resulted in higher percentages of embryogenic callus while 2,4-D at 2.25 M generated shoot-producing callus but with a lower percentage of embryogenic callus. Transfer of calluses from medium containing 4.5 M 2,4-D and 0.44 M BA to the somatic embryo initiation medium containing 0.9 M 2,4-D gelled with either 7 g 1^–1 agar or 3 g 1^–1 Gelrite led to the formation of somatic embryos. Somatic embryo initiation medium gelled with 3 g 1^–1 Gelrite led to significantly higher frequency of somatic embryo formation than in medium gelled with 7 g 1^–1 agar. Callus of a female genotype `315' generated under similar treatments did not produce shoots or somatic embryos.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Induction of embryogenicTriticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on induction of embryogenic calli from immature embryos of a responsiveTriticum aestivum L. genotype (PCYT 10). Effects were quatified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M signficantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mgl^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D inT. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3359.     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.     

Whole plant regeneration of Cicer arietinum from callus cultures via organogenesis

A protocol for whole plant regeneration of Cicer arietinum L. cv. C-235 via organogenesis from callus has been developed. Callus initiation was best when immature leaflets were cultured on MS medium containing 5 or 25 M 2,4-D or NAA in combination with 10 M BA, or 25 M 2,4-D alone. The callus grew most vigorously on MS medum supplemented with 10 M NAA and 5 MBA. Best shoot differentiation was obtained from calli derived from the basal portion of shoot tips on MS medium supplemented with 10 M BA and 0.1 M IBA. The shoot forming ability of calli was enhanced by adding 5 mM potassium phosphate to the medium. Shoots were rooted on a MS medium containing l M IBA. The regenerated plants were grown to maturity and produced viable seed.Abbreviations 2,4-D 2,4-dichlorophen-oxyacetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - Kn kinetin - 2-ip 6-(,-dimethylallylamino)-purine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium     

Induction of embryogenic Triticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on inducation of embryogenic calli from immature embryos of a responsive Triticum aestivum L. genotype (PCYT 10). Effects were quantified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M significantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mg1^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D in T. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultual Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3358.     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Effects of mefluidide and dicamba on in vitro growth and embryogenesis ofDactylis glomerata (orchard grass)

The effects of N-(2,4-dimethyl-5-(((trifluoromethyl)sulfonyl)amino)phenyl)acetamide (mefluidide) and 3,6-dichloro-o-anisic acid (dicamba) on in vitro growth and somatic embryogenesis ofDactylis glomerata L. (orchard grass) were studied using suspension cultures and explanted leaf bases. All experiments employed modified Schenk and Hildebrandt medium amended with concentrations of dicamba ranging from 15 to 120 M (SH-15 to SH-120) and of mefluidide ranging from 1 to 100 M. SH medium without either growth regulator was used for embryo germination. Embyro production in suspension cultures with SH-30 medium plus 3 g/L casein hydrolysate was significantly reduced by 1 M mefluidide. Only 15% of these embryos germinated and produced plants compared to 84% from controls. Growth, as measured by dry weight, was significantly reduced by 50 or 100 M mefluidide. The number of embryos formed on leaf sections was significantly reduced by 20 or 25 M mefluidide. Embryos that formed with 10 M or more mefluidide were callused on both SH-15 and SH-30 media. Shoot formation was inhibited from individual embryos and embryo/callus masses that developed on either SH-15 or SH-30 medium containing 5 M or more mefluidide. Radicle emergence was significantly reduced with 10 M mefluidide regardless of 15 or 30 M dicamba. Histological examination revealed that mefluidide inhibited both shoot and root meristem development with shoot development being the more sensitive. Inhibition of both was independent of dicamba concentrations. Shoot formation was also reduced from embryos that had developed on SH-30 medium without mefluidide when transferred to medium containing mefluidide without dicamba.     

Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 M NAA and 10 M BAP, and embryo maturation was promoted after transfer to a medium containing 1 M NAA and 10 M BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 M NAA and 1 M BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 M NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.Abbreviations BAP 6-benzylaminopurine - B5 medium of Gamborg et al. 1968 - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium of Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid