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.     

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.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

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.     

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.     

Direct plant regeneration of curry leaf tree (<Emphasis Type="Italic">Murraya koenigii</Emphasis> koenig.), an aromatic plant

Summary An efficient protocol for plant regeneration from stem segments of Murraya koenigii was developed by culturing on Murashige and Skoog (MS) medium supplemented with 2.5 mg l⁻¹ benzyladenine (BA), 25 mgl⁻¹ adenine sulfate, 0.25 mgl⁻¹ indole-3-acetic acid (IAA), and 3% sucrose. The frequency of shoot bud regeneration was higher on similar medium in subsequent subcultures. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.25–0.5 mgl⁻¹ IAA or 1-naphthaleneacetic acid (NAA) within 8–12 d of culture. The maximum percentage of rooting was obtained on MS medium supplemented with IAA and NAA, each at 0.25 mgl⁻¹. During acclimatization, 95% of rooted plantlets survived were grown normally under greenhouse conditions.     

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.     

Effects of type of explant and age,plant growth regulators and medium strength on somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis>

Plant regeneration was achieved through direct and indirect somatic embryogenesis in Eucalyptus camaldulensis. Callus was induced from mature zygotic embryos and from cotyledon explants collected from 10, 15, 25, and 30-day-old seedlings cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of naphthaleneacetic acid (NAA). Maximum callus induction from mature zygotic embryos was obtained on MS basal medium containing 1 mg l⁻¹ NAA. The frequency of callus development varied based on the age of the cotyledon explants 10-day-old explants giving highest percentage on MS basal medium supplemented with 1 mg l⁻¹ NAA. Callus obtained from mature zygotic embryos gave highest frequency of somatic embryogenesis on MS basal medium containing 0.5 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ NAA. Separate age wise culture of the calli, obtained from cotyledons of different ages cultured separately, revealed high somatic embryogenic potential on callus from 10-day-old cotyledons. Direct somatic embryogenesis too was obtained from hypocotyl explants without an intervening callus phase on MS basal medium containing 0.5 mg l⁻¹ BA. The effects of abscisic acid (ABA), sucrose, and different strengths of MS medium on somatic embryo maturation and germination were also investigated. Number of mature somatic embryos increased with lower concentrations (0–1 mg l⁻¹) of ABA while no significant differences were observed at higher concentrations (2–5 mg l⁻¹) of ABA. Compared to basal medium containing lower concentrations of sucrose (1%), the MS medium supplemented with higher levels of sucrose (4%) showed significantly lower frequency of mature somatic embryos. Basal medium without any dilution gave the highest number of immature embryos. However, the number of mature embryos was high at higher medium dilutions.     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Micropropagation of adult cherimoya (<Emphasis Type="Italic">Annona cherimola</Emphasis> Mill.) CV. Fino de Jete

Summary A micropropagation procedure for the adult cherimoya tree (Annona cherimola Mill.) is described. Axillary shoot proliferation was obtained after culturing nodal sections from Annona cherimola cv. ‘Fino de Jete’, on Murashige and Skoog (MS) medium supplemented with 2.28 μM zeatin. Roots were induced after preincubation of shoots for 3d in light on MS basal medium supplemented with lgl⁻¹ activated charcoal, followed by culturing for 10 d (7 d dark and 3 d light) on MS medium with 492 μM indole-3-butyrie acid (IBA), 15 gl⁻¹ sucrose, and 200 mgl⁻¹ citric acid. Sixty-eight percent of induced shoots rooted after transferring to the same medium without auxin and with the macroelements at half strength and the sucrose at 20gl⁻¹. About 65% of rooted shoots survived after acclimatization. The procedures described herein may prove useful for clonal micropropagation of selected genotypes of cherimoya.     

An efficient callus proliferation protocol and rhaponticin accumulation of Rheum franzenbachii Munt., a medicinal plant

An efficient callus proliferation system for Rheum franzenbachii Munt., a rare medicinal plant, has been developed. Callus induced from leaf explants incubated on Murashige and Skoog (MS) medium with appropriate supplements of plant growth regulators. In the 6-benzylaminopurine (6-BAP) in combination with α-naphthalene acetic acid (NAA) treatments, different concentrations of NAA showed different induction effects on explants. When concentration of 6-BAP was as high as 2.0 mgl^?1 in combination with 0.5 mgl^?1 NAA, the callus induction rate reached 58.3%. N-phenyl-N’-1,2,3-thiadiazol-5-ylure (TDZ) in combination with NAA was very suitable for callus proliferation compared to TDZ in combination with 2,4-dicholorophenoxy acetic acid (2,4-D) or TDZ in combination with indole-3-acetic acid (IAA). Fresh and dry weight of callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ in combination with 0.2 mgl^?1 NAA increased 26.3 and 15.0 times within 35 days culture, respectively. Quantitative analysis of rhaponticin by HPLC showed that the phytochemical profile of callus was similar to that of wild plants, and the content of rhaponticin in callus cultured on MS medium supplemented with 0.5 mgl^?1 TDZ and 0.2 mgl^?1 NAA was 16.6 mgg?¹DW compared to that of 4.0 mgg^?1 DW in wild plants.     

Plant regeneration from cotyledon tissues of common buckwheat (Fagopyrum esculentum Moench)

Summary Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l⁻¹) and kinetin (KIN) (0.2 mg l⁻¹) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l⁻¹ KIN, 2.0 mg l⁻¹ BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.     

Induction of somatic embryogenesis in Azadirachta indica

A modified culture protocol has been developed for the induction of somatic embryogenesis in Azadirachta indica (neem). Embryogenic calluses were initiated from cotyledons or hypocotyls using a Murashige and Skoog (MS) agar medium supplemented with 0.5 mg l⁻¹ α-napthaleneacetic acid (NAA), 1 mg l⁻¹ 6-benzylaminopurine (BA), 1 g l⁻¹ casein hydrolysate, and 50 g l⁻¹ sucrose. The calluses, when transferred to a liquid medium similar to the agar medium but with NAA replaced by 0.5 mg l⁻¹ indole-3-acetic acid (IAA), formed globular structures which further developed a rudimentary root, after 4 to 5 weeks incubation. Subsequently, these highly differentiated tissues when transferred into a hormone-free MS medium containing 1 g l⁻¹ casein hydrolysate and 50 g l⁻¹ sucrose, active embryo masses started to appear after 1 to 2 weeks. The embryo production was found to improve more than 2 fold by adding 0.2 mg l⁻¹ zeatin to the medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

Effectiveness of indoleacetic acid,indolebutyric acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus ‘Jork 9’

The production of an antifungal spirostanol saponin designated SC-1 has been detected in cell suspension cultures of the Mexican species Solanum chrysotrichum. Batch cultures of a cell suspension obtained from hypocotyl derived calluses of this species were grown for 25 days in shake flasks containing Murashige & Skoog (MS) medium. Throughout the growth cycle, fresh and dry weight, SC-1 yield, and uptake of sucrose, glucose and fructose were determined. The effects of inoculum size and sucrose concentration on the biomass accumulation and synthesis of the active metabolite, were studied. The maximum SC-1 production, above 14 mg.g⁻¹ (which was fifty times that of field grown plants), was reached after 20 days using a 2% inoculum and complete MS medium supplemented with 2 mgl⁻¹ 2,4-D, 2 mg l⁻¹kinetin, and sucrose between 30 and 45 gl⁻¹. . This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct somatic embryogenesis from cotyledon and cotyledonary node explants in bishop’s weed <Emphasis Type="Italic">Trachyspermum ammi</Emphasis> (L.) sprague

A three-stage procedure for embryogenesis in Trachyspermum ammi was developed from cotyledon and cotyledonary node explants cultured in Murashige and Skoog (MS) liquid medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Globular somatic embryos without intervening callus phase developed in 4 wk. The development of embryos to heart and torpedo stages required second-stage subculture of the explants (along with developing embryos) in liquid medium with lower concentrations of 2,4-D. Further development of embryos required a third-stage subculture in hormone-free liquid medium supplemented with 100 mg l⁻¹ casein hydrolysate. Regeneration of complete plantlets occurred after the fully developed somatic embryos were transferred to solidified half-strength MS medium supplemented with 1 mg l⁻¹ gibberellic acid.     

Regeneration of Syngonium podophyllum ‘Variegatum’ through direct somatic embryogenesis

A simple and effective method of regenerating Syngonium podophyllum ‘Variegatum’ via direct somatic embryogenesis has been established. Leaf and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with either α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos directly formed at one or two sides of petiole explants on MS medium supplemented 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA or 2.0 mg l⁻¹ TDZ with 0.2 mg l⁻¹ NAA or with 0.2 and 0.5 mg l⁻¹ 2,4-D, respectively. The frequency of petiole explants with somatic embryos produced was as high as 86% when cultured on medium containing 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA. Up to 85% of somatic embryos were able to germinate after transferring onto medium containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ NAA. Approximately 50–150 plantlets were regenerated from a single petiole explant. However, there was no somatic embryo formation from leaf explants regardless of growth regulator combinations used. Regenerated plantlets from petiole explants were stable and grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.     

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.