High-frequency somatic embryogenesis from germinated zygotic embryos of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and evaluation of the effects of medium strength,sucrose, GA<Subscript>3</Subscript>, and BA on somatic embryo development

We developed a new protocol for highly efficient somatic embryogenesis and plantlet conversion of Schisandra chinensis. Friable embryogenic callus was induced from cotyledonary leaves and hypocotyls of germinated zygotic embryos on Murashige and Skoog (MS) agar medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Preculture of zygotic embryos on 2,4-D-containing medium increased embryogenic callus induction efficiency. The highest embryogenic callus induction frequency of 56.7% was obtained from shoot apical meristem-containing hypocotyl explants from 1-week-old germinated embryos on MS medium containing 4.0 mg l⁻¹ 2,4-D. Embryogenic callus proliferation, somatic embryo (SE) formation, and subsequent plantlet conversion occurred under optimal culture conditions. The effects of MS medium strength, sucrose, gibberellic acid (GA₃), and 6-benzyladenine (BA) on SE formation and plantlet conversion were evaluated. Low MS medium strength (1/4 to 1/2) was necessary for SE formation, and the optimal sucrose concentration was 2.0%. Supplementing medium with GA₃ negatively impacted SE formation and subsequent development. BA significantly increased the number of SEs and the plantlet conversion capacity. One-third-strength MS medium with 1.0% sucrose and 0.5 mg l⁻¹ BA produced the highest number of SEs (309 embryos from 9 mg embryogenic callus) and the highest frequency of plantlet conversion from germinated SEs (52.6%). When transplanted to soil, 90% of the regenerated plants developed into normal plants.     

Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae)

Pogonatherum paniceum (Poaceae) is a perennial plant with good potential for eco-recovery and ornamental function. This study presents in vitro culture systems of simple hormonal regulation of somatic embryogenesis and shoot organogenesis from mature caryopses. Mature caryopses of P. paniceum were grown on Murashige and Skoog medium with 3% sucrose (w/v) and various concentrations or combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Morphological development was analyzed by light microscope after histological sectioning. Four types of callus were induced by different concentrations of 2,4-D. Type I callus was regenerated via somatic embryogenesis; type II callus failed to produce any regeneration; type III callus had both somatic embryogenesis and shoot organogenesis capacities; and type IV callus only displayed shoot organogenesis capacity. Regarding hormone combinations used in this study, NAA only induced type IV callus and BAP only induced direct multiple shoot formation. The combinations of 2,4-D and NAA induced type III callus. Several of the regeneration pathways were simply controlled by one or two kinds of plant hormones. The established systems will be helpful for further research on the developmental mechanism of switch between somatic embryogenesis and shoot organogenesis.     

Auxin and nutritional stress coupled somatic embryogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L.

Somatic embryos were induced from internodal segment derived callus of Oldenlandia umbellata L., in MS medium supplemented with different concentrations of 2,4-Dichlorophenoxy acetic acid (2,4-D). Initially calli were developed from internodes of microshoots inoculated in 2.5 µM NAA supplemented medium. Then calli were transferred to 2,4-D added medium for somatic embryogenesis. Nutritional stress coupled with higher concentration of 2,4-D triggered somatic embryogenesis. Nutritional stress was induced by culturing callus in a fixed amount of medium for a period up to 20 weeks without any external supply of nutrients. Addition of 2.5 µM 2,4-D gave 100% embryogenesis within 16 weeks of incubation. Callus mass bearing somatic embryos were transferred to germination medium facilitated production of in vitro plantlets. MS medium supplemented with 2.5 µM benzyl adenine and 0.5 µM α-naphthalene acetic acid produced 15.33 plants per culture within 4 weeks of culture. Somatic embryo germinated plants were then hardened and transferred to green house.     

Morpho-histological study of direct somatic embryogenesis in endangered species Frittilaria meleagris

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm⁻³ 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

Propagation of <Emphasis Type="Italic">Gentiana macrophylla</Emphasis> (Pall) from hairy root explants via indirect somatic embryogenesis and gentiopicroside content in obtained plants

An effective protocol for plant regeneration from hairy root (HR) via indirect somatic embryogenesis was established in medicinal plant Gentiana macrophylla, a perennial herb in Gentianaceae. On the MS medium containing 0.5–2.5 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) or 2,4-D plus benzylaminopurine (BAP), all the HR explants produced embryogenic calli (ECs). After transfer to plant growth regulator (PGR)-free MS medium, up to 94% of the ECs produced somatic embryos (SEs) of various stages, including cotyledonary SEs. When the calli with cotyledonary SEs were transferred to PGR-free MS medium, the cotyledonary SEs on the calli developed into plantlets (1–12 ones per callus). The cotyledonary SEs showed two types: solitary and fasciculate. The former developed into single plantlets and the latter into fasciculate ones. After transplantation into soil, a half of the plantlets survived, and one of the survivors flowered without fruiting. Morphologically, about 30% plantlets appeared similar to the wild type (WT)-plants, and 70% of them displayed wrinkled dark green leaves with relatively small and dense stomata, long and thick main root with dense lateral roots. The biomass of roots and leaves of the plantlets increased by five- and one-fold, respectively, and the content of gentiopicroside of their roots raised by 72.4%, in comparison with WT-plants. Polymerase chain reaction revealed that the rolC gene integrated into HR genome still existed in the regenerated plants. This study offers us an effective method and material for producing gentiopicroside or other medicinal compounds.     

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.     

Somatic embryogenesis from rhizome explants of <Emphasis Type="Italic">Cymbopogon winterianus</Emphasis>

Plantlet regeneration through indirect somatic embryogenesis was attempted from rhizome derived callus of Cymbopogon winterianus Jowitt (cv. Jorlab2). Optimum callus was induced on Murashige and Skoog (MS) basal medium supplemented with 4 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D). Initially the callus was friable, shiny white and watery in nature. After subculturing on MS medium containing 2,4-D and kinetin (Kn), callus was transferred onto the MS medium supplemented with 2,4 -D, Kn and coconut water to induce somatic embryogenesis. Optimum somatic embryogenesis (78.33 %) was achieved on MS medium containing 3.0 mg dm⁻³ 2,4-D and 0.5 mg dm⁻³ Kn. High frequency (65 %) plantlet conversion from embryos was achieved in MS medium supplemented with 2 mg dm⁻³ N⁶-benzyladenine (BA), 0.5 mg dm⁻³ Kn, 0.2 mg dm⁻³ calcium pantothenate and 0.2 mg dm⁻³ biotin.     

Somatic embryogenesis and plant regeneration from seeds of wild<Emphasis Type="Italic"> Dicentra spectabilis</Emphasis> (L.) LEM

Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro.Abbreviations BAP 6-Benzylaminopurine - 2.4-D 2,4-Dichlorophenoxyacetic acid - KIN Kinetin - SEM Scanning electron microscopyCommunicated by H. Lörz     

Identification of 2,4-D-responsive proteins in embryogenic callus of Valencia sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck) following osmotic stress

The compound 2,4-Dicholorophenoxyacetic acid (2,4-D) is an important growth regulator which is used in the majority of embryogenic cell and tissue culture systems. However, 2,4-D also appears to have a negative effect on growth and development of plant tissues and organs cultured in vitro. For example, 2,4-D exerts inhibition on in vitro somatic embryo initiation and/or development of most citrus species. To understand the molecular mechanism by which 2,4-D inhibits somatic embryogenesis (SE), proteomic changes of Valencia sweet orange (Citrus sinensis) embryogenic callus induced by treatments with a high concentration of 2,4-D (6 mg l⁻¹) was investigated. Nine 2,4-D-responsive proteins were identified, of which eight were up-regulated and one was down-regulated. Interestingly, three of the eight up-regulated proteins were osmotic stress-associated, suggesting that 2,4-D induced osmotic stress in Valencia embryogenic callus. This speculation was supported by results from our physiological studies: 2,4-D treated callus cells exhibited increased cytoplasm concentration with a significant reduction in relative water content (RWC) and an obvious increase in levels of two osmolytes (proline and soluble sugar). Taken together, our results suggested that 2,4-D could inhibit somatic embryo initiation by, at least in part, inducing osmotic stress to citrus callus cells.     

Somatic embryogenesis and plant regeneration from immature zygotic embryo cultures of mountain ash (<Emphasis Type="Italic">Sorbus pohuashanensis</Emphasis>)

Plant regeneration through somatic embryogenesis was achieved using immature zygotic embryos (ZE) of Sorbus pohuashanensis as explants. Over 50% of immature ZEs from immature seed collected at 30 days after pollination produced direct somatic embryos (SEs) on Murashige and Skoog (MS) medium supplemented with 0–0.44 μM 6-benzyladenine (BA) in combination with 5.73 μM naphthaleneacetic acid (NAA) or with 0.91–2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) alone. Fourteen to 23 SEs per explant were regenerated on MS medium supplemented with BA 0.44 μM in combination with NAA 5.73 μM. SE formation decreased when sucrose concentrations were higher than 40 g L⁻¹. Repetitive embryogenesis occurred following culture on solid MS medium containing 12 μM abscisic acid, 75 g L⁻¹ polyethylene glycol, and 20 g L⁻¹ sucrose at 25 ± 1°C under a 16-h photoperiod with a light intensity of 40 μmol m⁻² s⁻¹. Over 40% of the mature SEs germinated on solid MS medium under light condition described previously. Up to 40% of the regenerated plantlets were successfully acclimatized under greenhouse conditions. Plantlets derived from SEs grew vigorously with similar morphology as those germinated from ZEs. Histological studies of explants at various developmental stages of somatic embryogenesis revealed that SEs passed through globular, heart, torpedo, and mature stages. Similar to ZE suspensors, similar structures of SE degenerated in later stages of embryo development. ZE and SE are a effective means of regenerating tissue culture plantlets for S. pohuashanesis.     

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 embryogenesis and plantlet production using rejuvenated tissues from serial grafting of a mature <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis> tree

An effective micropropagation technique via somatic embryogenesis has been developed using tissue from serially grafted shoots generated from a mature Kalopanax septemlobus tree (~40 y old). Callus was induced from leaf segments obtained from the grafts by culturing the explants in Murashige and Skoog (MS) medium supplemented with 2,4-D and 3% w/v sucrose under darkness. The effects of sucrose, coconut water, and polyethylene glycol (PEG-3350) were evaluated as factors to promote development of somatic embryos (SEs) from embryogenic callus. More than 90% of explants formed callus; however, only 2.5%, or 20 leaf segments out of 800 explants, formed embryogenic callus after 8 wk of culture. High sucrose concentrations (3% and 5% w/v) were effective in inducing SEs. Treatment with 2–10% v/v coconut water also had a positive effect on embryo induction. A synergistic effect on SE induction was obtained using sucrose and PEG, with presence of the latter compound resulting in smaller, more uniform SEs. Embryo germination and conversion to plantlets were significantly influenced by the gelling agents. In general, gelrite-gelled medium was superior to agar-gelled medium. In gelrite-gelled medium, gibberillic acid (GA₃) enhanced embryo germination. Converted plantlets in an artificial soil mixture showed a 91% survival rate and displayed no distinct morphological variations. Our results indicate that reliable somatic embryogenesis and plant production can be achieved with rejuvenated tissues after repeated grafting of shoots derived from a mature Kalopanax septemlobus tree.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Plant regeneration through somatic embryogenesis on Holostemma ada-kodien,a rare medicinal plant

Plant regeneration through indirect somatic embryogenesis has been established on Holostemma ada-kodien Schult. Type of auxin significantly influenced somatic embryogenesis. Friable callus, developed from leaf, internode and root explants on Murashige and Skoog (MS) medium supplemented with 2,4-D (1.0 mg l^–1), was most effective for the induction of somatic embryos. Subculture of the friable callus developed on 2,4-D (1.0 mg l^–1) onto solid or liquid 1/2 MS medium with 0.1 or 0.5 mg l 2,4-D turned the callus embryogenic. Suspension cultures were superior to static cultures (solid medium) for the induction of somatic embryos. Transfer of embryogenic callus to liquid 1/2 or 1/4 MS medium with lower levels of 2,4-D (0.05–0.1 mg l^–1) induced the highest number of somatic embryos. An average of 40 embryos were obtained from 10 mg callus. Fifty per cent embryos exhibited maturation and conversion upon transfer to 1/10 MS basal solid medium. Plantlets were established in field conditions and 90 per cent survived.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration through somatic embryogenesis and organogenesis using cotyledons of <Emphasis Type="Italic">Cassia angustifolia</Emphasis> Vahl

In vitro regeneration through somatic embryogenesis as well as organogenesis using cotyledon of a woody medicinal legume, Cassia angustifolia is reported. The cotyledons dissected from semi-mature seeds, if inoculated on Murashige and Skoog’s medium (MS) supplemented with auxin alone or in combination with cytokinin, produced direct and indirect somatic embryos. A maximum of 14.36 ± 2.26 somatic embryos per 20 mg of explants including callus were produced in 70% cultures on MS medium with 2.5 μM benzyladenine (BA) + 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Although the percentage of embryogenic cultures was higher (83.33%) at 10 μM 2,4-D + 1 μM BA, the average number of somatic embryos was much less (7.6 ± 0.85) at this level, whereas at 2.5 μM BA and 5 μM 2,4-D, there was a simultaneous formation of both somatic embryos and shoots. The somatic embryos, although started germinating on the same medium, developed into full plantlets only if transferred to MS basal with 2% sucrose. Cytokinins alone did not induce somatic embryogenesis, but formed multiple shoots. Five micromolar BA proved optimum for recurrently inducing shoots in the competent callus with a maximum average of 12.04 ± 2.10 shoots and shoot length of 2.26 ± 0.03 cm. Nearly 91.6% shoots (2–2.5 cm in size) organized an average of 5.12 ± 0.58 roots on half strength MS + 10 μM indole-3-butyric acid. All the plantlets have been transferred successfully to soil. Types of auxin and its interaction with cytokinin significantly influenced somatic embryogenesis.     

High-frequency direct somatic embryogenesis from leaf tissues of <Emphasis Type="Italic">Drimiopsis kirkii</Emphasis> Baker (giant squill)

Whole plants were regenerated from excised leaves of Drimiopsis kirkii Baker (Lily of the Valley) through direct somatic embryogenesis. An initial exposure to a low level of 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 μM) in the medium was essential in inducing the direct formation of somatic embryos. A high concentration of 2,4-D (4.52 μM) in the proliferation medium reduced embryogenesis and enhanced callus formation. The presence of kinetin in the medium enhanced the somatic-embryogenesis-inducing effect of 2,4-D (0.45 μM). The maximum embryogenesis rate (4,026 somatic embryos per gram of leaf) was obtained in explants cultured for 30 d in medium supplemented with 2.33 μM kinetin and 0.45 μM 2,4-D (embryo induction medium). Kinetin (4.65 μM) also enhanced embryo germination (97.6%), but the presence of α-naphthalene acetic acid in the medium drastically reduced embryo germination. Following conversion, the regenerated plantlets were transferred to soil and showed normal morphological characteristics.     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

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.     

Simple one-medium formulation regeneration of fingerroot [<Emphasis Type="Italic">Boesenbergia rotunda</Emphasis> (L.) mansf. Kulturpfl.] via somatic embryogenesis

Summary Most published protocols necessitate different media formulations for multistep somatic embryogenesis. This study aims to establish a simple but effective formulation for the regeneration of plantlets of the pharmaceutically active Boesenbergia rotunda (L.) Mansf. Kulturpfl, formerly Boesenbergia/Kaempferia pandurata (Schult), to ensure a superior and consistent supply of materials for commercialization purposes. In this study, a single-medium formulation of Murashige and Skoog (MS) supplemented with 13.54μM 2,4-dichlorophenoxyacetic acid (2,4-D) was found to be the only medium out of eight formulations to promote the complete somatic embryogenesis process for the culture of B. rotunda (L.). Callus cultures were initiated from a total of 280 explants of rhizome meristem. The percentage of cultures forming embryogenic callus was 23.3 ±4.3% on this MS medium augmented by 13.54μM 2,4-D. The best plantlet regeneration rate was attained from the first subcultured callus with a mean of 6.6±0.1 plantlets per 1 cm diameter aggregate of callus. Somatic embryogenesis characteristic of monocots was evident from histological studies. The regenerated plantlets have been successfully established in soil.