Somatic embryogenesis,organogenesis, and regeneration from leaf callus culture of <Emphasis Type="Italic">Decalepis hamiltonii</Emphasis> Wight &; Arn., an endangered shrub

Summary A novel protocol has been developed for inducing somatic embryogenesis from leaf cultures of Decalepis hamiltonii. Callus was obtained from leaf sections in Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA)+N⁶-benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D)+BA. Nodular embryogenic callus developed from the cut end of explants on media containing 2,4-D and BA, whereas compact callus developed on media containing NAA and BA. Upon subsequent transfer of explants with primary callus onto MS media containing zeatin and/or gibberellic acid (GA₃) and BA, treatment with zeatin (13.68μM) and BA (10.65 μM) resulted in the induction of the highest number of somatic embryos directly from nodular tissue. The maturation of embryos took place along with the induction on the same medium. Embryogenic calluses with somatic embryos were subcultured onto MS basal medium supplemented with 4.56μM zeatin+10.65 μM BA. After 4wk, more extensive differentiation of somatic embryos was observed. The mature embryos developed into complete plantlets on growth regulator-free MS medium. A distinct feature of this study is the induction of somatic embryogenesis from leaf explants of Decalepis hamiltonii, which has not been reported previously. By using this protocol, complete plantlets could be regenerated through indirect somatic embryogenesis or organogenesis from leaf explants in 12–16 wk.     

Somatic embryogenesis in <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Turcz.) Baill.

Summary Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardned, with a survival rate of approximately 67%, and established in the field.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Efficient plant regeneration through direct somatic embryogenesis from leaf explants of <Emphasis Type="Italic">Phalaenopsis</Emphasis> ‘Little Steve’

Summary Leaf segments of the orchid sp. Phalaenopsis ‘Little Steve’ were used as explants testing the effects of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.45, 2.26, 4.52 μM), 6-furfurylaminopurine (kinetin; 2.32, 4.65, 13.95 μM), N⁶-benzyladenine (BA; 2.22, 4.44, 13.32 μM), and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 2.27, 4.54, 13.62μM) on the induction of direct somatic embryogenesis. After 20–30 d of culture in darkness, clusters of somatic embryos formed from leaf surfaces and wounded regions of explants on half-strength Murashige and Skoog medium supplemented with BA and TDZ. However, kinetin had no response on direct embryo induction. In addition, 2,4-D highly retarded the frequency of embryogenesis that was induced by TDZ. Generally, adaxial surfaces near wounded regions had the highest embryogenic competency compared to other regions of explants. Histological sections revealed that somatic embryos mostly arose from epidermal cell layers of the explants. Secondary embryogenesis occurred at basal parts of embryos, and originated from outer cell layers. Following transfer of regenerated embryos onto growth regulator-free medium for 3.5–4 mo., plantlets with three to four leaves and several roots were obtained. This protocol provides a simple way to regenerate plants through direct somatic embryogenesis, and is suitable for further studies on embryo development and genetic transformation of Phalaenopsis.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of cowpea, Vigna unguiculata (L.) Walp.

Summary Embryogenic callus was induced from primary leaves of Vigna unguiculata (L.) Walp. in MS medium (Murashige and Skoog, 1962) containing 2,4-dichlorophenoxyacetic acid (2,4-D). Greenish-white, friable embryogenic calluses were used to establish suspension cultures. A shaking speed of 90 rpm and 0.4 ml packed cell volume per 25 ml medium were found to be optimal for maintaining suspension cultures. Globular, heart-shaped and torpedo-shaped embryos were developed in suspension culture containing 4.52 μM 2,4-D. Maturation of cotyledonary-stage somatic embryos was achieved on 0.05 μM 2,4-D, 5 μM abscisic acid and 3% mannitol. Twenty-two percent of the embryos were converted into plants and survived; survival in the field was 8–10%.     

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak.

Summary Establishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.     

Somatic embryogenesis and plant regeneration in Medicago arborea L.

Summary An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 μM) and kinetin (9 μM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 μM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 μM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18.0±0.70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets (8.0±0.89%) when cultured on basal MS medium with 5 μM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N⁶-benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 μM.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Ontogeny of somatic embryos in cucumber (<Emphasis Type="Italic">cucumis sativus</Emphasis> L.)

Summary Suspension culture of cucumber (Cucumis sativus L.) has been an inefficient method for production of somatic embryos owing to problems with embryo maturation and conversion. Embryogenic callus of cv. Green Long was induced on semisolid Murashige and Skoog (MS) medium containing 6.8 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 μM 6-benzylaminopurine (BA). A large number of globular somatic embryos were obtained on transfer of the callus to MS liquid medium supplemented with 87.6 mM sucrose, 1.1 μM 2,4-D, and improved by the addition of 342.4 μM l-glutamine. MS medium supplemented with 87.6 mM sucrose was more effective in somatic embryo production than other sugars. Subsequent development led to the formation of heart-and torpedo-shaped embryos. Maturation of somatic embryos occurred on plant growth regulator-free MS semi-solid medium containing 175.2 mM sucrose and 0.5 gl⁻¹ activated charcoal. Conversion of embryos into plants was achieved on half-strength MS semi-solid medium containing 87.6 mM sucrose and 1.4 μM gibberellic acid (GA₃) in a 16h photoperiod. Twenty-seven percent of embryos were converted into normal plants.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Plant regeneration by somatic embryogenesis and organogenesis in commercial pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> L.)

Summary Axillary and terminal buds from suckers of Ananas comosus cv. Phuket were established on Murashige and Tucker-based (MT) medium with 2.0 mgl⁻¹ (9.8 μM) indolebutyric acid, 2.0 mgl⁻¹ (10.74 μM) naphthaleneacetic acid, and 2.0 mgl⁻¹ (9.29 μM) kinetin, followed by multiplication on Murashige and Skoog-based (MS) medium containing 2.0 mgl⁻¹ (8.87 μM) benzyladenine (BA) to provide a continuous supply of axenic shoots. Leaves, excised from such cultured shoots, produced adventitious shoots from their bases when these explants were cultured on MS medium containing 0.5 mgl⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mgl⁻¹ (8.87 μM) BA. Embryogenic callus was produced when leaf explants were cultured on MS medium with 3.0 mgl⁻¹ (12.42 μM) 4-amino-3,5,6-trichloropicolinic acid (picloram). Somatic embryos developed into shoots following transfer of embryogenic tissues to MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. Cell suspensions, initiated by transfer of embryogenic callus to liquid MS medium with 1.0 mgl⁻¹ (4.14 μM) picloram or 1.0 mgl⁻¹ (4.52 μM) 2,4-D, also regenerated shoots by somatic embryogenesis, on transfer of cells to semisolid MS medium with 1.0 mgl⁻¹ (4.44 μM) BA. All regenerated shoots rooted on growth regulator-free MS medium, prior to ex vitro acclimation and transfer to the glasshouse. These studies provide a baseline for propagation, conservation, and genetic manipulation of elite pineapple germplasms.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of plants from sugarcane seed-derived callus

Summary Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) seeds were germinated on modified Murashige and Skoog (MS) basal medium alone or supplemented with 2.3, 4.5, 11.3, 22.5, and 45.0 μM thidiazuron (TDZ), or 4.5, 13.6, 22.6, and 45.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 4.1, 12.4, 20.7, and 41.3, μM picloram. Both auxins delayed seed germination by approximately 5 d. Maximum germination was observed on MS medium supplemented with 45.0 μM TDZ. Callus induction occurred for seed germinated on 2,4-D and picloram-containing media, but not on TDZ medium. The greatest amount of callus (554±198mg per seed) was produced on 4.1 μM picloram. For shoot initiation, calluses were transferred to MS medium alone or supplemented with 2.5 μM TDZ. The highest number of shoots was recorded on TDZ medium from callus that had been obtained originally from media containing either 4.1 or 12.4 μM picloram or 13.6 μM 2,4-D (∼500). All shoots developed roots and grew to maturity on medium with 24,6 μM indolebutyric acid.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Multiple shoot formation and plant regeneration from stem nodal explants of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Summary Stem nodal explants of Paphiopedilum philippinense hybrids (hybrid PH59 and PH60) directly formed shoots when cultured on a modified half-strength Murashige and Skoog (1962) basal medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D: 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45 and 4.54 μM) for 6 mo. On hormone-free basal medium, the percentages of explants with shoots were 33.3% and 0% and the shoot numbers per explant were 1 and 0 in hybrid PH59 and hybrid PH60, respectively. In hybrid PH59, 4.52 μM 2,4-D plus 0.45μM TDZ induced a higher percentage of explants with shoots and shoot number per explant than did the hormone-free treatment. In hybrid PH60, although 4.52 μM 2,4-D and 0.45 μM TDZ promoted shoot formation, the highest shoot number was found with 4.52 μM 2,4-D alone. Plantlets, each having several roots, were obtained from regenerated shoots after transferring onto hormone-free basal medium for 3 mo. The plantlets were potted in sphagnum moss, and acclimatized well in a greenhouse.     

Induction of somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of <Emphasis Type="Italic">eruca sativa</Emphasis> mill

Summary A protocol was developed for high frequency somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of Eruca sativa. Explants grown on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-D formed embryogenic callus after 4 wk of culture. Secondary somatic embryos were also produced from primary somatic embryos on MS medium containing 0.56 μM 2,4-D. Somatic embryos developed into mature embryos on MS medium in the presence of 45 gl⁻¹ polyethylene glycol. After desiccation, somatic embryos developed into plantlets by culturing the mature somatic embryos on 1/2 x MS medium containing 0.24 μM indole-3-butyric acid.     

Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron

Summary A procedure for the regeneration of cacao (Theobroma cacao) plants from staminode explants via somatic embryogenesis was developed. Rapidly growing calli were induced by culturing staminode explants on a DKW salts-based primary callus growth (PCG) medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and thidiazuron (TDZ) at various concentrations. Calli were subcultured onto a WPM salts-based secondary callus growth medium supplemented with 20 g glucose per L, 9 μM 2,4-D, and 1.4 nM kinetin. Somatic embryos were formed from embryogenic calli following transfer to a hormone-free DKW salts-based embryo development medium containing sucrose. The concentration of TDZ used in PCG medium significantly affected the rate of callus growth, the frequency of embryogenesis, and the number of somatic embryos produced from each responsive explant. A TDZ concentration of 22.7 nM was found to be the optimal concentration for effective induction of somatic embryos from various cacao genotypes. Using this procedure, we recovered somatic embryos from all 19 tested cacao genotypes, representing three major genetic group types. However, among these genotypes, a wide range of variation was observed in both the frequency of embryogenesis, which ranged from 1 to 100%, and the average number of somatic embryos produced from each responsive explant, which ranged from 2 to 46. Two types of somatic embryos were identified on the basis of their visual appearance and growth behavior. A large number of cacao plants have been regenerated from somatic embryos and established in soil in a greenhouse. Plants showed morphological and growth characteristics similar to those of seed-derived plants. The described procedure may allow for the practical use of somatic embryogenesis for clonal propagation of elite cacao clones and other applications that require the production of a large number of plants from limited source materials.     

Somatic embryogenesis of <Emphasis Type="Italic">Merwilla plumbea</Emphasis> (Lindl.) Speta

A simple and efficient system was developed for rapid somatic embryogenesis from leaf explants of Merwilla plumbea, a traditional but threatened medicinal plant in South Africa. Friable embryogenic callus (FEC) was obtained from leaf explants on embryogenic callus induction medium containing agar-solidified Murashige and Skoog (MS) salts and vitamins, 8.3 μM picloram, 2.3 μM thidiazuron (TDZ) and 20 μM glutamine. FEC was subsequently incubated in embryogenic callus proliferation medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.1 μM picloram for 7 days before it was transferred to liquid somatic embryo medium (SEM_L) containing MS medium supplemented with 0.4 μM picloram and 0.9 μM TDZ. In SEM_L supplemented with 150 mg L⁻¹ haemoglobin, 5.4–35.6 somatic embryos per settled cell volume of 500 mg FEC were obtained. These embryos were at globular to cotyledonary developmental stages. Embryo maturation, germination and plant formation rate was 94.4% following transfer of SEs to half-strength MS medium supplemented with 1.4 μM gibberellic acid. Plantlets transferred into soil acclimatized in the misthouse and established successfully in the greenhouse (100%). This is the first report on induction of Merwilla plumbea somatic embryogenesis. The protocol developed offers controlled vegetative propagation by alleviating extinction threats, ensures germplasm conservation and provides a system for physiological, biochemical, molecular and cellular studies of embryo development.