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.     

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.     

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.

Summary Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.     

<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.     

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Japanese honeysuckle

Japanese honeysuckle plant (Lonicera japonica Thunb.) is rich in iridoid secologanin and is a potentially useful model for the study of secologanin biosynthesis. Culture conditions for high frequency plant regeneration via somatic embryogenesis from zygotic embryo cultures and zygotic embryo-derived embryogenic cell suspension cultures of this species are described. Mature zygotic embryos formed embryogenic calluses at a frequency of 46.7% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 M 2,4-dichloro-phenoxyacetic acid (2,4-D). Cell suspension cultures were established with embryogenic calluses using liquid MS medium with 4.52 M 2,4-D. Upon plating onto MS basal medium, embryogenic cell suspension cultures produced numerous somatic embryos, which subsequently developed into plantlets at a frequency of 68%. Regenerated plantlets were transplanted to potting soil and grown to maturity in a greenhouse.     

Cell suspension culture and plant regeneration in the latex-producing plant,Calotropis gigantea (Linn.) R. Br.

A procedure is outlined for the establishment of a proliferating cell suspension culture and subsequent plant regeneration of the latex-producing plant,Calotropis gigantea (Linn.) R. Br. Friable calluses were obtained by culturing hypocotyl explants on modified Murashige and Skoog medium with 2.69 μM α-naphthaleneacetic acid and 4.44 μM 6-benzyl-aminopurine. Friable calluses were transferred to modified Murashige and Skoog liquid medium containing 500 mg l⁻¹ casein hydrolysate, 5% (v/v) coconut water and 5% (w/v) sucrose to initiate suspension cultures. Suspensions were subcultured every 10–12 days and supplemented with 13.56 μM 2,4-dichlorophenoxyacetic acid (2,4-D). After 3–4 subcultures, suspensions consisted of small, highly cytoplasmic cell clumps and large vacuolate cells. Plating the suspension clumps on medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and culturing in darkness produced macroscopic calluses, which subsequently produced a high number of shoots when placed in light and supplemented with 2.22 μM 6-benzyl-aminopurine and 0.45 μM 2,4-dichlorophenoxyacetic acid. Shoots were rooted using Bonner's solution containing 0.49 μM indole-3-butyric acid, and the plants successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration from root segments of Psoralea corylifolia L., an endangered medicinally important plant

Summary An efficient plant regeneration protocol has been developed from root explants of Psoralea corylifolia L., an endangered medicinally important herbaceous plant species belonging to the family Fabaceae. Nodular embryogenic callus was initiated from young root segments cultured on Murashige and Skoog (MS) medium (1962) supplemented with α-naphthaleneacetic acid (NAA; 2.68–13.42 μM) or 2,4-dichlorophenoxyacetic acid (2.4-D; 2.25–11.25 μM) in combination with 6-benzylaminopurine (BA: 2.2. μM). thiamine HCl (2.9 μM), L-glutamine (342.23 μM) and sucrose (3.0% w/v). The highest frequency (95.2%) of embryogenic calluses was obtained on MS medium supplemented with the growth regulators NAA (10.74 μM) and BA (2.2 μM). Development and maturation of somatic embryos was achieved after transfer of embryogenic calluses to MS medium supplemented with 1.34 μM NAA or 1.12 μM 2,4-D and 4.4–13.2 μM BA. The maximum number (13.8±1.34) of cotyledonary stage somatic embryos was obtained on MS medium containing 1.34 μM NAA and 13.2 μM BA. Germination of somatic embryos occurred on MS medium without any growth regulators and also on MS medium enriched with BA (1.1–8.8 μM), although the maximum germination frequency (76.1%) was obtained on 4.4 μM BA plus 1.45 μM gibberellic acid (GA₃). Plant regeneration without complete somatic embryo maturation was also achieved by transferring clumps of nodular embryogenic calluses onto MSO medium or MS medium supplemented with NAA (1.34 μM) and BA (2.2–8.8 μM). The highest frequency of plant regeneration (93.3%) and mean number of plantlets (15.4±0.88) were obtained on MS medium containing 1.34 μM NAA and 4.4 μM BA. Regenerated plants with well-developed root systems were transferred to pots where they grew vigorously, attained maturity and produced fertile seeds.     

Plant Regeneration from Immature Zygotic Embryo-Derived Embryogenic Calluses and Cell Suspension Cultures of Catharanthus roseus

Culture conditions for plant regeneration in immature zygotic embryo-derived embryogenic cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) Little Bright Eye are described. Immature zygotic embryos formed off-white, friable calluses at a frequency of 20% on Murashige and Skoog (MS) medium supplemented with 4.52 µM 2,4-dichlorophenoxyacetic acid (2,4-D) after 8 weeks of culture. After a second subculture using MS basal medium at 4-week intervals, off-white friable calluses formed a small quantity of yellowish, compact embryogenic calluses. Upon transfer to MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Cell suspension cultures were established with embryogenic calluses using liquid MS medium supplemented with 4.52 µM 2,4-D. Embryogenic cell clumps from cell suspension cultures developed into plantlets at a frequency of 56.7% when plated onto MS basal medium. Plantlets were transplanted to potting soil and grown to maturity in a growth chamber.     

In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets

Freesia hybrida is an important worldwide cut flower, especially in America and Europe. For efficient regeneration of this flower from young inflorescence and rachillae in tetraploid, we developed a simple in vitro micropropagation protocol. Explants of Freesia hybrida can regenerate plantlets through somatic embryogenesis via two kinds of pathways, that is, directly from the epidermal cells or indirectly from an embryonic callus, depending on the exogenous plant growth regulators (PGRs) used in the culture media. In direct embryogenesis, when the explants were cultured on Murashige and Skoog (MS) medium supplemented with 11.43 μM indole acetic acid (IAA) and 4.44 μM 6-benzylaminopurine (6-BA), the induction rate was 84% for young inflorescence and 100% for rachillae. After the multishoots were subcultured on the rooting MS medium containing 1.08 μM α-naphthalene acetic acid (NAA), the rooting rate was close to 100%. In indirect embryogenesis, embryonic calluses were formed when the culture medium contained 22.22 μM 6-BA and 4.52 μM 2,4-dichlorophenoxy acetic acid (2,4-D), and the induction rate was 92.4% for young inflorescence and 100% for rachillae. After the embryonic calluses were transferred to the medium supplemented with 11.43 μM IAA and 13.33 μM 6-BA, they could develop into plantlets with roots. In assessing the two regeneration pathways in terms of genetic and epigenetic fidelity of the regenerants, two kinds of molecular markers [amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP)] were employed. The AFLP analysis used 20 primer pairs that yielded 916 scorable bands among the donor plant and 11 regenerants from direct embryogenesis, of which 8 (0.87%) were polymorphic. The regenerants from indirect embryogenesis had 1075 clear bands of which 3 (0.27%) were polymorphic scorable bands from 18 primer pairs. Moreover, the variant band patterns included two types, that is, loss-of-original and gain-of-novel bands. MSAP analysis revealed that tissue culturing of the flower induced DNA cytosine methylation alterations in both CG and CNG levels and patterns compared with the donor plant. The variation rate was 1.1 and 1.3% for the direct and indirect embryogenesis pathways, respectively. The findings show that tissue culture of flowering plants is a form of stress which can induce some heritable epigenetic variations and should be considered in future long-term genotype preservation programs of Freesia hybrida.     

<Emphasis Type="Italic">Bridelia stipularis</Emphasis>: a new source for anthocyanin production in vitro

The present study prospects Bridelia stipularis (L.) Blume as a new source of anthocyanins through leaf and internode explants-derived callus cultures. Murashige and Skoog (MS) medium fortified with 21.48 μM α-naphthaleneacetic acid was superior for callus growth. Of the different regimes, the anthocyanin production relied on synergic effects of plant growth regulators, pH, light, and carbon source. The calluses incubated in light on MS medium with 4% glucose containing 2.22 μM N⁶-benzyladenine (BA) and 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) at pH 3.5 yielded the highest amount (a mean of 0.42 mg g⁻¹ callus) of anthocyanins. Subsequent cultures of the calluses on the above medium yielded a stable production of anthocyanins. Medium containing glucose was superior to that with sucrose for anthocyanin formation. Kinetin was inhibitory to anthocyanin accumulation. Suspension cultures of MS medium containing 2.26 μM 2,4-D and 2.22 μM BA at pH 5.0 started excretion of anthocyanins into the medium on reaching to pH 4.4–4.6.     

High frequency somatic embryogenesis and plant regeneration in zygotic embryo cultures of Liriope platyphylla Wang et Tang

Mature zygotic embryos of Liriope platyphylla Wang et Tang formed embryogenic calluses at a frequency of 33% when cultured on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos, which then developed into plantlets. Regenerated plantlets were successfully transferred to potting soil, where they were subsequently grown to maturity. This revised version was published online in August 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 through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

High frequency plant regeneration system for <Emphasis Type="Italic">Nymphoides coreana</Emphasis> via somatic embryogenesis from zygotic embryo-derived embryogenic cell suspension cultures

Culture conditions were established for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Nymphoides coreana. Zygotic embryos formed pale-yellow globular structures and calluses at a frequency of 85.6% when cultured on half-strength Murashige and Skoog (MS) medium supplemented with 0.3 mg l⁻¹ of 2,4-D. However, the frequency of pale-yellow globular structures and white callus formation decreased slightly with an increasing concentration of 2,4-D up to 10 mg l⁻¹ with the frequency rate falling to 16.7%. Cell suspension cultures were established from zygotic embryo-derived calluses using half-strength MS medium supplemented with 0.3 mg l⁻¹ of 2,4-D. Upon plating onto half-strength MS basal medium, over 92.3% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted into potting soil and achieved full growth to an adult plant in a growth chamber. The high frequency plant regeneration system for Nymphoides coreana established in this study will be useful for genetic manipulation and cryopreservation of this species.     

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.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

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

Long term maintenance of an anther-derived haploid callus line of the Asiatic hybrid lily ‘Connecticut King’

A haploid callus line from anther cultures of the Asiatic hybrid lily ‘Connecticut King’ was maintained for a long term. The survival and growth of the haploid calluses were affected by auxins of picloram, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) and temperatures of 25, 15 and 7 °C during culture. Picloram was more suitable for maintenance of the haploid calluses, whereas NAA and 2,4-D led to root and shoot formation from the haploid calluses. The best temperature for maintenance was 25 °C. About 90% of cells in calluses were maintained in haploid level during 60 weeks of subculture, and about 80% of cells were haploid in the calluses maintained over 2 years with the MS medium containing 4 μM picloram in the dark at 25 °C. This revised version was published online in June 2006 with corrections to the Cover Date.