Somatic embryogenesis and plant regeneration from petioles of Parthenocissus tricuspidata planch

Summary A protocol of somatic embryogenesis and plant regeneration from petiole segments of Parthenocissus tricuspidata Planch. has been developed. Embryogenic tissue was induced on B5 (Gamborg) basal medium supplemented with 2.25–9.0 μM 2,4-dichlorophenoxyacetic acid, 500 mg l⁻¹ casein hydrolysate (CH), and 0.1 gl⁻¹ activated charcoal. Somatic embryos were induced on B5 medium containing various concentrations of benzyladenine (BA) (4.44, 6.66, and 8.88 μM) and α-naphthaleneacetic acid (NAA) (0, 0.54, and 1.61 μM) plus 500 mg l⁻¹ CH. Ninety percent of normal somatic embryos were converted into plantlets directly on Murashige and Skoog (MS) medium free of plant growth regulators. Shoots could be induced from abnormal somatic embryos on MS medium containing 4.44 μM BA, 0.05 μM NAA, and 500 mg l⁻¹ CH. Genotypic differences were found in the process of somatic embryogenesis and plant regeneration. Histological analysis confirmed the process of somatic embryogenesis. Regenerated plantlets with well-developed roots were successfully acclimatized in greenhouse and all plants showed normal morphological characteristics.     

Highly efficient embryogenesis and plant regeneration of tall fescue (Festuca arundinacea Schreb.) from mature seed-derived calli

Summary We report a protocol for efficient plant regeneration of four tall fescue (Festuca arundinacea Schreb.) cultivars (‘Surpro’, ‘Coronado’, ‘Summer Lawn’, and ‘Fawn’) via somatic embryogenesis. Calli were initiated from mature seeds grown on modified Murashige and Skoog (MMS) medium supplemented with 7.0mgl⁻¹ (31.7μM) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mgl⁻¹ (0.23 μM) kinetin (Kin). Calli were maintained and proliferated by subculture at monthly intervals on MMS medium containing 4.5 mgl⁻¹ (20.4 μM) 2,4-D and 0.2mgl⁻¹ (0.9 μM) Kin. Somatic embryos (SE) were induced from seed-derived calli on SE-induction medium (MMS supplemented with 2.0 mgl⁻¹ 2,4-D and 0.2mgl⁻¹ Kin). Plantlets were regenerated from somatic embryogenic calli grown on modified SH medium supplemented with 2 mgl⁻¹ Kin. Using this optimized protocol, 78.6–82.3% of mature seeds of all four cultivars produced SE clusters, of which 93.5–95.3% regenerated into plants within 10 wk. The regenerants showed no phenotypic abnormalities.     

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> fruiting and seed set of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. CV. Sweet banana

Summary Plantlets of Capsicum annuum L. ev. Sweet Banana regenerated via somatic embryogenesis from immature zygotic embryos were capable of producing flower, fruit, and seed when cultured in small tissue culture containers. In vitro floral buds were first formed on plantlets that grew on plantlet development medium [agar-gelled Murashige and Skoog (MS) basal medium containing 1 mgl⁻¹ (5.3 μM) α-naphthaleneacetic acid (NAA)] in a growth room at 22°C and continuous illumination. However, floral buds rarely developed further into mature flowers. This problem was overcome using the vented autoclavable plant tissue culture containers. In vitro fruit formation and ripening was observed when liquid half-strength MS basal medium supplemented with 5 μg ml⁻¹ silver thiosulfate, 1 mg l⁻¹ (5.3 μM) NAA, and 3% sucrose was added to the surface of the plantlet development medium. Hand-pollination improved fruit set. Further research in needed to determine why the pepper seeds formed in vitro failed to germinate.     

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.     

Abscisic acid and ancymidol promote conversion of somatic embryos to plantlets and secondary embryogenesis inAsparagus officinalis L.

Summary The effects of abscisic acid (ABA) (0, 0.09 μM, 0.19 μM, 0.28 μM, and 0.38 μM) or ancymidol (0, 0.98 μM, 1.95 μM, 2.93 μM, 3.90 μM) in embryo germination medium on the conversion of primary embryos to plantlets and secondary embryogenesis were evaluated for asparagus. ABA and ancymidol each significantly enhanced both responses. ABA was more effective than ancymidol in promoting the conversion of primary embryos to plantlets, while the converse was true for the production of secondary embryos. The most effective treatments for embryo conversion were 0.19 and 0.28 μM ABA; 75–77% bipolar and 55–57% globular embryos converted to plantlets. For secondary embryogenesis, the most effective treatments were 1.95 and 2.93 μM ancymidol; 99–101 and 84–86 somatic embryos were produced from 10 globular and 10 bipolar embryos, respectively. Bipolar embryos generally converted to plantlets better than globular embryos, but more secondary embryos were produced from globular embryos than from bipolar embryos in all treatments. ABA and ancymidol also affected the morphology of the plantlets produced. The plantlets from the embryos incubated on the medium with ancymidol had strong and thick shoots and roots, while those on the medium with ABA had long, thin shoots and short thin roots.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures

Summary High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mgl⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mgl⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.     

Spontaneous somatic embryogenesis on in vitro root segment cultures of Rauvolfia micrantha Hook. F.—A rare medicinal plant

Summary Plant regeneration through direct somatic embryogenesis was achieved from root segments derived from in vitro shoots of Rauvolfia micrantha Hook. f. (Apocynaceae) grown for 6 wk in half-strength Murashige and Skoog (MS) medium with 3% sucrose, 100 mgl⁻¹ myo-inositol, and 0.5 mgl⁻¹ α-naphthaleneacetic acid (NAA). The effects of photoperiod and plant growth regulators (PGRs) in half-strength MS medium were studied for the rapid and maximum induction of somatic embryos. The characteristic globular or heart-shaped stages of somatic embryogenesis were not found and cotyledonary stage embryos occasionally appeared without the intervention of callus in total darkness and 16-h photoperiod. Root segments cultured in the medium containing 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ 6-benzyladenine (BA) under 16-h photoperiod showed the maximum frequency (39%) of embryogenesis. The frequency of embryo formation was increased to 63% when they were cultured in medium with 0.1 mgl⁻¹ NAA and 0.2 mgl⁻¹ BA in the dark for 4wk, then grown under the 16-h photoperiod. Explants with developing embryos developed into plants after transfer to half-strength MS medium supplemented with 0.1 mgl⁻¹ BA and 0.05 mgl⁻¹ NAA. The well-developed plants were hardened and most plants (80%) survived and were phenotypically similar to the mother plants.     

Micropropagation of <Emphasis Type="Italic">Kalopanax pictus</Emphasis> tree via somatic embryogenesis

Summary Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacctic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.     

Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis

Summary Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by culturing these explants on callus-inducing solid medium [Murashige and Skoog salts plus vitamins of Gamborg's B₅ medium, 30 g l⁻¹ glucose, 100 mg l⁻¹ myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected over filter mesh 40 subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants.     

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 embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham

Summary A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1⁻¹ (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO₃ on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1⁻¹ (117 mM) sucrose, 0.05 mg 1⁻¹ (0.22 μM) 6-benzylaminopurine, and 10 mg l⁻¹ (58.8 μM) AgNO₃. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO₃ at concentrations below 10 mg l⁻¹ (58.8 μM). A semi-solid medium containing 1.5 g l⁻¹ Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.     

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.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.     

Direct and indirect somatic embryogenesis on cotyledon explants of <Emphasis Type="Italic">Quassia amara</Emphasis> L., an antileukaemic drug plant

Summary In vitro propagation of Quassia amara L. (Simaroubaceae) was attempted using mature and juvenile explants. Attempts to establish in vitro culture using leaf and internode explants from a plant more than 15yr old were unsuccessful due to severe phenolic exudation. Plant regeneration through direct and indirect somatic embryogenesis was established from cotyledon explants. Murashige and Skoog (MS) medium with 8.9 μM N⁶-benzyladenine (BA) and 11.7 μM silver nitrate induced the highest number (mean of 32.4 embryos per cotyledon) of somatic embryos. Direct somatic embryogenesis as well as callus formation was observed on medium with BA (8.9–13.3 μM). Semi-mature pale green cotyledons were superior for the induction of somatic embryos. Embryos developed from the adaxial side as well as from the point of excision of the embryonic axis. More embryos were developed on the proximal end compared to mid and distal regions of the cotyledons. Subculture of callus (developed along with the somatic embryos on medium with BA alone) onto medium containing 8.9 μM BA and 11.7 μM silver nitrate produced a mean of 17.1 somatic embryos. Primary somatic embryos cultured on MS medium with 8.9 μM BA and 11.7μM silver nitrate produced a mean of 9.4 secondary somatic embryos. Most of the embryos developed up to early cotyledonary stage. Reduced concentration of BA (2.2 or 4.4 μM) improved maturation and conversion of embryos to plantlets. Ninety percent of the embryos converted to plantlets. The optimized protocol facilitated recovery of 30 plantlets per cotyledon explant within 80d. Plantlets transferred to small cups were subsequently transferred to field conditions with a survival rate of 90%.     

Plant regeneration through somatic embryogenesis in medicinally important <Emphasis Type="Italic">Centella asiatica</Emphasis> L.

Summary High-frequency somatic embryogenesis and plant regeneration was achieved on callus derived from leaf (petiole and lamina) and internode explants of Centella asiatica L. Growth regulators significantly influenced the frequency of somatic embryogenesis and plant regeneration. Calluses developed on Murashige and Skoog (MS) medium fortified with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 5.37 μM α-naphthaleneacetic acid (NAA), both with 2.32 μM kinetin (Kn), were superior for somatic embryogenesis. Callus developed on NAA and Kn-supplemented medium favored induction and maturation of embryos earlier compared to that on 2,4-D and Kn. Embryogenic callus transferred from NAA and Kn-supplemented medium to suspension cultures of half-strength MS medium with NAA (2.69 μM) and Kn (1.16 μM) developed a mean of 204.3 somatic embryos per 100 mg of callus. Embryogenic callus transferred from 2,4-D and Kn subsequently to suspension cultures of half-strength MS medium with 2,4-D (0.45 μM) and Kn (1.16 μM) developed a mean of 303.1 embryos per 100 mg of callus. Eighty-eight percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS semisolid medium having 0.054 μM NAA with either 0.044 μM BA or 0.046 μM Kn. Embryo-derived plantlets established in field conditions displayed morphological characters identical to those of the parent plant.     

Somatic embryo proliferation, maturation and germination in Catharanthus roseus

In the present study an efficient somatic embryogenesis method has been developed in Catharanthus roseus. Friable embryogenic callus was induced from hypocotyl of in vitro germinated seeds on Murashige and Skoog basal nutrient media supplemented with various auxins particularly 2,4-D (1.0 mg l⁻¹). However, only NAA (1.0 mg l⁻¹) produced somatic embryos in cultures. Embryo proliferation was even high on the same medium added with BAP. Cotyledonary somatic embryo germinated and converted into plantlets in BAP (0.5 mg l⁻¹) added medium following a treatment with gibberellic acid (1.0 mg l⁻¹) for maturation. Carbon sources and concentrations had a marked influence on maturation process. Plantlet conversion was better achieved when embryos were matured on 3% fructose or 3–6% maltose. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as raw material, genetic modification to embryo precursor cell may improve alkaloid yield further.     

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.