Somatic embryogenesis in cell suspension cultures of pigeonpea (Cajanus cajan)

Suspension cultures of calli derived from seedling leaf explants of Cajanus cajan L. var. Vamban-1 produced somatic embryos. The highest embryogenic frequency was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The maximum frequency of somatic embryogenesis was observed when this callus was transferred to MS liquid medium supplemented with 4.52 μM 2,4-D. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent divisions in the proembryo led to globular, heart and torpedo-shaped somatic embryos. The germination of somatic embryos occurred on auxin-free MS basal medium. Effects of various auxins, cytokinins and carbohydrates on induction and frequency of somatic embryogenesis were studied. A medium supplemented with 4.52 μM of 2,4-D and 87.64 mM sucrose was effective in inducing a higher frequency of somatic embryos, whereas cytokinin had no effect and led to recallusing of embryos. About 5–6% of embryos converted into plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration in zygotic embryo cultures of balloon flower

Mature zygotic embryos of balloon flower (Platycodon grandiflorum) formed embryogenic calluses at a frequency of 43% when cultured on Murashige and Skoog medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Cell suspension cultures were established from embryogenic calluses using MS liquid medium with 4.52 μM 2,4-D. Following transfer to solid MS basal medium, cell suspension cultures gave rise to somatic embryos, which then developed into plantlets. Plantlets were transplanted to potting soil and grown to maturity. This revised version was published online in June 2006 with corrections to the Cover Date.     

<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.     

High frequency plant regeneration from immature ovule-derived embryogenic cell suspension cultures of Chelidonium majus var. asiaticum

Culture conditions for high frequency plant regeneration via somatic embryogenesis in cell suspension cultures of Chelidonium majus var. asiaticum are described. Immature ovules formed embryogenic calluses at a frequency of 40% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimum ovule size for embryogenic callus formation ranged from 1 to 1.5 mm in length. Cell suspension cultures were established from embryogenic calluses using MS liquid medium containing 4.52 μM 2,4-D. Upon plating onto MS basal medium, cell aggregates from cell suspension cultures produced somatic embryos which then developed into plantlets. Regenerated plantlets were transplanted to potting soil and grown to maturity in a growth chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

ABA enhances plant regeneration of somatic embryos derived from cell suspension cultures of plantain cv. Spambia (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid MS medium supplemented with 4.5 μM 2,4-D and 1.0 μM zeatin. Upon transfer to semi-solid MS medium of the same composition as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 μM abscisic acid (ABA), maturation of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in the medium. Upon transfer to MS medium supplemented with 1.25 μM 6-benzyladenine (BA), 80% of germinated embryos developed into plantlets.     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

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.     

Development of in vitro plant regeneration of Australian native waxflowers (Chamelaucium spp.) via somatic embryogenesis

Waxflowers (Chamelaucium spp.) are native to Australia and now are grown for the cut flower industry worldwide. As part of an effort to achieve somatic hybridization between the species to improve flower quality, somatic embryogenesis was achieved for Chamelaucium uncinatum and C. repens. Somatic embryos from young leaves of C. uncinatum and C. repens were induced in vitro on Murashige and Skoog (MS) agar medium containing 20 g/l sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D). For C. uncinatum, up to 4% of explants developed somatic embryos at 20 μM 2,4-D and for C. repens, up to 3% developed somatic embryos at 5 μM 2,4-D. Somatic embryos of C. uncinatum were also induced from immature seeds—a maximum of 6% of seed explants producing somatic embryos on MS medium containing 0.05 μM 6-benzyladenine (BA) and 0.5 μM Naphthalene acetic acid (NAA). Somatic embryo cultures maintained on MS medium supplemented with 0.1 μM 2,4-D were induced to develop into plantlets after transfer to a hormone-free medium under light.     

Plant regeneration from embryogenic cells-derived protoplasts of Bupleurum falcatum

Hypocotyl segments of Bupleurum falcatum L. formed embryogenic calluses when cultured on Murashige and Skoog's (MS) medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension cultures were initiated by placing calluses into medium with 0.45 μM 2,4-D. Protoplasts were enzymatically isolated from suspension cultures. They were plated at a density of 5 × 104 protoplasts per ml on MS medium supplemented with 9% mannitol, 9.0 μM 2,4-D, 4.4 μM BA, 4.6 μM kinetin, and 0.6% Seaplaque agarose. After four weeks of culture, microcalluses were formed and subsequently transferred to MS solid medium with 18.1 μM 2,4-D. Upon transfer to MS basal medium, microcalluses gave rise to somatic embryos at a frequency of approximately 10%. They subsequently developed into plantlets. The regenerants were successfully transplanted to potting soil and grown to maturity in a greenhouse. The regenerants had the normal chromosome number of 2n=2x=20 and did not show morphological aberrancy. This revised version was published online in June 2006 with corrections to the Cover Date.     

<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.     

Somatic embryogenesis and plant regeneration of litchi (Litchi chinensis Sonn.) from leaves of mature phase trees

Embryogenic cultures were induced from leaflets from new vegetative flushes of mature ‘Brewster’ litchi trees on B5 medium containing 400 mg l⁻¹ glutamine, 200 mg l⁻¹ casein hydrolysate, 30 g l⁻¹ sucrose, 4.52 μM 2,4-D, 9.30 μM kinetin and 3 g l⁻¹ gellan gum in darkness. Embryogenic cultures consisting of proembryonic cells and masses were maintained either on semi-solid MS medium supplemented with 4.52 μM 2,4-D and 0.91 μM zeatin or as embryogenic suspension cultures in liquid medium of the same composition. Maturation of somatic embryos occurred on semi-solid MS medium with 5–20% (v/v) filter-sterilized coconut water in darkness. Recovery of plants from somatic embryos was improved with 14.4 μM GA₃ on half-strength MS medium with 0.2 g l⁻¹ activated charcoal under a 16 h photoperiod provided by cool white fluorescent lights (60–80 μmol s⁻¹ m⁻²). Plants have been successfully acclimatized in the greenhouse.     

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.     

<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.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

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.     

Repetitive somatic embryogenesis in carnation on picloram supplemented media

An efficient method of repetitive somatic embryogenesis and plant regeneration of two carnation cultivars (Sagres and Impulse) was established using a two-step protocol. In the first step, embryogenic tissue were induced from petal explants on MS culture medium containing 9% sucrose (w/v), 9 μM 2,4-D and 0.8 μM BA. In the second step, embryogenic tissue transferred onto the MS medium containing 3% sucrose supplemented with different concentrations of picloram (0.8, 2, 4, 8 and 16 μM) to produce primary somatic embryos. Precotyledonary clumps and cotyledonary somatic embryos were then isolated and subcultured onto the same media as the second step where they formed secondary somatic embryos in repetitive cycles. Cotyledony somatic embryos were converted into plantlets when they transferred onto the growth regulator-free half-strength MS medium followed by being acclimated to the greenhouse conditions.     

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.     

The embryogenic competency and morphological changes during somatic embryogenesis in Iris pseudacorus

Embryogenic callus was obtained from bulb segments of Iris pseudacorus on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with kinetin. When early globular somatic embryos were subcultured onto MS medium with 4.52 μM 2,4-D, high frequency of somatic embryogenesis was obtained. Deprivation of 2,4-D was required for maturation. Mature somatic embryos had an elongated scutellum with a notch on the base of scutellum. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos from embryo clusters and transfer onto fresh half-strength MS medium with 3% sucrose. After acclimation in artificial soil in greenhouse for 2 months, 96.4% of plantlets survived.     

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%.     

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.