A cultivar comparison of plant regeneration from suspension cells,callus, and cormel slices ofGladiolus

Callus was initiated from either cormel slices or in vitro-grown plants of sixGladiolus cultivars cultured on Murashige and Skoog’s basal salts medium supplemented with either 10 mg/liter (53.8 µM) 1-naphthaleneacetic acid, 2 mg/liter (9.3 µM) dicamba, or 0.5 mg/liter (2.2 µM) 2,4-dichlorophenoxyacetic acid. More plants were regenerated from callus of the cultivar “Peter Pears” as compared to “Jenny Lee,” “Florida Flame,” or “Golden Year.” No plants were regenerated from callus of “Rosa Supreme” or “Purity White.” Plants were regenerated from 2 and 6-mo.-old suspension cells of “Jenny Lee” and “Peter Pears” but not from “Florida Flame.” Cormel slices cultured on Murashige and Skoog’s basal salts medium supplemented with 1 mg/liter (4.4 µM) 6-benzylaminopurine regenerated plants from all six cultivars indicating a cultivar-independent system of plant regeneration.     

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.     

Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

Plant regeneration through somatic embryogenesis from zygotic embryo-derived callus of <Emphasis Type="Italic">Areca catechu</Emphasis> L. (Arecaceae)

Summary An in vitro culture procedure was established for somatic embryogenesis and plant regeneration from callus cultures of the important palm ‘betel nut’ (Areca catechu L.). Segments of zygotic embryos of Areca catechu L. were cultured on Murashige and Skoog basal medium supplemented with dicamba (9.05, 18.1, 27.15, and 36.2 μM). After 7–8 wk in darkness, wounded regions of explants formed callus with yellow, soft, glutinous structures. Proliferation and maintenance of callus was on the same dicamba-containing medium. With regular subculture every 8 wk, the callus showed pale yellow, compact and nodular structures. During subculture, somatic embryos were formed spontaneously from nodular callus tissues within 2–4 mo. The embryos developed into plantlets after 10 wk of culture on basal medium free of plant growth regulators. After subculturing every month for 3 mo., the plantlets were transferred to containers for acclimatization in the greenhouse. The survival rate was 24%.     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

Assessing competence for adventitious shoot formation in hypocotyl explant cultures from<Emphasis Type="Italic">Catharanthus roseus</Emphasis> cultivars

Hypocotyl expiants from 22 cultivars ofCatharanthus roseus were cultured on various shoot-inducing media to assess their competence for adventitious shoot formation. The Murashige and Skoog (MS) media had been supplemented with 14 μM zeatin and 2.5 μM indole-3-butyric acid (IBA), 4.5 μM BA and 0.5 μM α-naphthaleneacetic acid (NAA), or 14 μM thidiazuron and 2.5 μM IBA. After eight weeks, the expiants from ‘Cooler Raspberry Red’ showed the greatest frequency of adventitious shoot formation, followed by ‘Cooler Orchid’ and ‘Cooler Treated’. The highest frequency (86.7%) for ‘Cooler Raspberry Red’ was attained on the medium enhanced with 14 μM zeatin and 2.5 μM NAA. Excised adventitious shoots were then readily rooted on a half-strength MS basal medium. Afterward, the regenerated plantlets were transferred to potting soil and grown to maturity in a greenhouse.     

Somatic embryogenesis and plant regeneration of Gladiolus (Gladiolus hort.)

Summary Friable embryogenic callus and somatic embryos of 4 Gladiolus cultivars were obtained on Murashige and Skoog (MS) medium with various concentration of auxins from the following explants: corm slices, young leaf bases and whole, intact plantlets. Somatic embryos transferred on MS hormone-free medium regenerated into plantlets. All plantlets obtained through embryogenesis did not differ phenotypically from the parental clones. The embryogenic friable callus has been maintained for over 2 years in culture and has retained a very high regeneration capacity.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KIN kinetin - NAA naphthaleneacetic acid - MS Murashige and Skoog Medium (1962) - E embryogenic callus - NE non-embryogenic callus     

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.     

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.     

Somatic embryogenesis from mesocotyl and leaf-base segments of <Emphasis Type="Italic">Paspalum scrobiculatum</Emphasis> L., a minor millet

Summary Somatic embryos could be induced from embryogenic callus originating from mesocotyl as well as leaf-base segments of Paspalum scrobiculatum on Murashige and Skoog (MS) or Chu et al. (N₆) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9.0, 18.0, and 22.5 μM). N₆ medium was better than MS, for both explants, for high-frequency somatic embryogenesis. Also, mesocotyl tissues were relatively more totipotent than leaf-base segments. The somatic embryos ‘germinated’ and formed plantlets on transfer of embryogenic calluses to hormone-free MS or N₆ regeneration medium. Embryogenic cultures could be maintained on low hormone medium which readily regenerated to form plantlets on hormone-free medium. A higher frequency of plantlet formation occurred on MS than on N₆ medium. In vitro-formed plantlets were gradually acclimatized in the culture room and on transfer to soil flowered and set seed. Somatic embryogenesis and plantlet regeneration from mesocotyl and leaf-base segments are potentially simpler systems than regeneration from ‘embryonic’ explants such as immature embryos and unemerged inflorescences.     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

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.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of <Emphasis Type="Italic">Melia azedarach</Emphasis> (Meliaceae)

Summary A procedure for the regeneration of ‘paradise tree’ (Melia azedarach, Meliaceae) plants from immature zygotic embryos via somatic embryogenesis was developed. Somatic embryos were induced from explants cultured on Murashige and Skoog medium supplemented with 0.45, 4.54, or 13.62 μM thidiazuron. Histological examination revealed that somatic embryos were induced directly from the explants. Further development of somatic embryos was accomplished with Murashige and Skoog medium at quarter-strength with 3% sucrose. A large number of plants were regenerated from somatic embryos and successfully established in soil in a greenhouse. These plants are morphologically similar to those of seed-derived plants. This system may be beneficial for mass propagation as well as for genetic manipulation of the ‘paradise tree’.     

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.     

Regeneration of plantlets from petiole callus of wild viola (Viola patrinii DC.)

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoog's (MS) medium, 5 × 10^–6 M 1-naphthaleneacetic acid and 10^–6 M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.Abbreviations MS Murashige and Skoog - SEM scanning electron microscopy - NAA 1-naphthaleneacetic acid - KIN kinetin     

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 in <Emphasis Type="Italic">Holarrhena antidysenterica</Emphasis> Wall., from shoot segment-derived callus

Summary A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N⁶-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.     

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.     

Efficient plant regeneration of native spearmint (Mentha spicata L.)

Summary Protocols and media constituents for efficient in vitro plant regeneration of Native Spearmint (Mentha spicata L. cultivar ‘Native Spearmint’) have been defined. Adventitious shoots were initiated either directly from morphogenetically competent cells of explants or primary callus. Leaf explants from at least 2-mo.-old in vitro-maintained shoots exhibited the greatest morphogenetic capacity. Explants derived from basal portions of leaves at the bottom of the shoot were most responsive, with up to a 100% regeneration frequency and greater than nine shoots per explant. Highest frequency of meristemoids and morphogenetic callus were initiated from explants cultured onto a basal medium containing Murashige and Skoog (MS) salts, supplemented with 4 mg thidiazuron (TDZ) per L and 25% (vol/vol) coconut water (CW) for 10 to 14 d in darkness. Bud and shoot development required removal of both TDZ and CW from the medium. Shoot propagules were transferred to basal medium supplemented with 0.01 mg α-naphthaleneacetic acid (NAA) per L and grown under low light for about 2 wk to facilitate shoot elongation. Individual shoots about 1 cm tall were dissected and retransferred onto the same medium. Root initiation began within 4 to 6 d and a functional root system developed within 2 to 3 wk. These plantlets were transferred to soil and acclimated successfully for growth and development in a greenhouse. This is the first report of an efficient regeneration system for Native Spearmint based on adventitious organogenesis.     

Regeneration from long-term embryogenic callus of the <Emphasis Type="Italic">Rosa hybrida</Emphasis> cultivar kardinal

Summary Media components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.