Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Callus formation and plant regeneration from tubercles of Coryphantha elephantidens (Lem.) Lem.

Summary Callus cultures were established from pith tissue of Coryphantha elephantidens (Lem.) Lem. on Murashige and Skoog (MS) basal medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin. Highest shoot regeneration frequency was observed on a medium containing 6.9 μM kinetin and 2.3 μM 2,4-D under 30 μE m⁻² s⁻¹ light intensity with a 16-h photoperiod. Calluses retained organogenic potential throughout several passages of subculture (18 mo.). Shoots were rooted on MS medium without plant growth regulators. All (100%) plantlets transplanted to soil survived acclimatization. Regenerated plants showed good overall growth and were morphologically similar to the mother plants.     

High frequency plant regeneration of Cymbopogon martinii (Roxb.) Wats by somatic embryogenesis and organogenesis

Embryogenic callus cultures were obtained upon repeated sub-culture of non-embryogenic callus from nodal segments of Cymbopogon martinii (Roxb.) Wats. Murashige and Skoog's medium supplemented with 1mg/l 2,4-dichlorophenoxyacetic acid and 0.5 mg/l kinetin and Linsmaier and Skoog's medium supplemented with 2mg/l 2,4-dichlorophenoxyacetic acid and 0.4 mg/l kinetin were used as maintenance media for non-embryogenic and embryogenic cultures, respectively. Plant regeneration occurred through organogenesis in MS basal media containing 2 mg/l kinetin, 1 mg/l 6-benzylaminopurine, 0.2 mg/l biotin, 0.2 mg/l Ca-pantothonate and 0.1 mg/l napthalene acetic acid. Embryogenesis was induced in LS medium supplemented with 1 mg/l kinetin, 0.5 mg/l 6-benzylaminopurine and 0.1 mg/l 3-indole acetic acid. Plant regeneration at high frequency was recorded both through organogenesis and embryogenesis in different passages of long term callus cultures.Abbreviation MS Murashige and Skoog medium - LS Linsmair and Skoog medium - BAP 6-benzylaminopurine - kin kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - CH Casein hydrolysate - CaP calcium pantothonate - NAA napthalene acetic acid     

Somatic embryogenesis and plant regeneration from callus cultures of Aconitum heterophyllum Wall

Plants were obtained via somatic embryogenesis in callus derived from in vitro raised leaf and petiole explants of Aconitum heterophyllum Wall. Callus was induced on a Murashige-Skoog medium supplemented with either 2,4-dichlorophenoxy acetic acid (2,4-d 1 mg l^-1) and kinetin (KN 0.5 mg l^-1) with coconut water (CW 10% v/v) or naphthalene acetic acid (NAA 5 mg l^-1) and benzylaminopurine (BAP 1 mg l^-1). Somatic embryos appeared after 2–3 months or 2 subculture passages when 2,4-d or NAA induced source of the callus was transferred to a MS medium containing BAP (1 mg l^-1) and NAA (0.1 mg l^-1). For successful plantlet formation, the somatic embryos were transferred to a medium containing 1/4 strength MS nutrient with indole-3-butyric acid (IBA 1 mg l^-1). Alternatively, the somatic embryos were dipped in a concentrated solution of IBA for 5 min and placed on a hormone free medium. Complete plantlets were formed after 4 weeks and were transferred successfully to soil.CIMAP Publication No. 1020.     

Somatic embryogenesis and plant regeneration from leaf-derived cell suspension of a mature tree — Thevetia peruviana L.

Cell suspension cultures, which retained embryogenic potential for almost 2 years, were established from young, expanding, juvenile leaves of a mature Thevetia peruviana L. tree. Calli were obtained by culturing young leaf discs on MS medium supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1 mg/L kinetin. Suspension cultures were initiated by transfer of calli to liquid medium containing 1 mg/L 2,4-D + 0.1 mg/L kinetin, and the cultures were maintained by subculturing to fresh medium at 2 week intervals. Embryogenic frequency of cell aggregates was more than 80% when plated on semi-solid medium containing 0.1 mg/L 2, 4-D and 2 mg/L 2-isopentenyladenine (2-iP). Cell aggregates with developing embryos were transferred to fresh medium lacking growth regulators for embryo maturation. Early embryo development was synchronous and a large number of somatic embryos were produced. These somatic embryos developed into plantlets upon subsequent transfer to modified half-strength MS medium. More than 200 green and rooted plants, at an average of 80 plants per 100 mg of embryogenic callus, were obtained with 60% survival under glass house conditions.Abbreviations 2, 4-D 2 4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine - IAA Indole — 3 — acetic acid - KN Kinetin - MS Murashige and Skoog (1962) basal medium - NAA 1 -Napthalene acetic acid     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghums

Callus and plant regenertion were induced from shoot portions of mature embryos (dry seeds) of five high tannin Sorghum bicolor (L.) Moench cultivars. The explants were cultured on Murashige and Skoog medium with altered concentrations of 5 salts, supplemented with 150 mg/L L-asparagine, 5mg/L 2,4-Dichlorophenoxyacetic acid and 0.05mg/L kinetin. Calli which were yellow and globular were formed with 70–90% frequencies. The subculture medium which gave best results was MS with 2mg/L 2,4-Dichlorophenoxyacetic acid and 0.5mg/L kinetin. Plants were regenerated on MS medium supplemented with 150mg/L L-asparagine and 0.2mg/L kinetin with regeneration frequencies of 11–48%.Abbreviations 2,4-D dichlorophenoxyacetic acid     

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.     

Plant regeneration from stem and petiole protoplasts of sweet potato (Ipomoea batatas) and its wild relative,I. lacunosa

A protoplast-to-plant regeneration system has been established for sweet potato (Ipomoea batatas (L.) Lam.) and its wild relative, I. lacunosa L. Viable protoplasts, isolated from preplasmolyzed stems and petioles of in vitro-grown plants, were cultured on liquid MS (Murashige & Skoog 1962) medium that supported cell division and colony formation. Embryogenic calli of sweet potato were induced on agar-solidified MS medium supplemented with 3% (w/v) sucrose, 50 mg l^-1 casamino acids, 0.2–0.5 mg l^-1 2,4-d, 1.0 mg l^-1 kinetin and 1.0 mg l^-1 ABA. On average, 3 plants were regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 3% (w/v) sucrose, 800 mg l^-1 glutamine, 2.0 mg l^-1 BA or 1.0 mg l^-1 kinetin and 1.0 mg l^-1 GA₃. Embryogenic calli of I. lacunosa L. were initiated on semi-solid MS medium containing 0.2–0.5 mg l^-1 IAA and 1.0–2.0 mg l^-1 BA. An average of 5 plants was regenerated from a single sweet potato callus subcultured on semi-solid MS medium containing 0.5 or 1.0 mg l^-1 GA₃.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole acetic acid - MES 2-(N-morpholino)-ethane sulfonic acid - NAA -naphthaleneacetic acid     

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l⁻¹ kinetin and 1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l⁻¹ kinetin, 1 mg l⁻¹ 2,4-D and 100 mg l⁻¹ ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l⁻¹ kinetin and 0.1 mg l⁻¹ 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l⁻¹ abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l⁻¹ of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l⁻¹ 6-benzyladenine and 1 mg l⁻¹ α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

Secondary metabolites produced by callus cultures of various Ruta species

Callus cultures were established from hypocotyl explants of R. bracteosa, R. chalepensis and R. macrophylla. Calli were maintained for more than three years on MS-medium supplemented with 1 mg l^-1 of each 2,4-D and kinetin. Acridone and furoquinoline alkaloids and coumarins have been isolated from four week old calli grown on a hormone containing and hormone-free medium. A new chlorinated acridone alkaloid has been detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog [6]     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

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.     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

Efficacy of different growth regulators at different stages of somatic embryogenesis <Emphasis Type="Italic">Eryngium foetidum</Emphasis>L.—A rare medicinal plant

Summary High-frequency somatic embryogenesis and plant regeneration were achieved on callus developed from root, stem disc, leaf, and scape explants of Eryngium foetidum L. (Apiaceae). Calluses developed on Murashige and Skoog (MS) medium fortified with 5.37–10.74 μM α-naphthaleneacetic acid (NAA) in combination with 2.32 or 4.65 μM kinetin (Kn) formed somatic embryos after transfer to liquid half-strength MS (1/2MS) mediumwith 2.69 μM NAA and 1.16 μM Kn. Furthermore, promotion of embryo formation was also observed in callus developed on medium with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and Kn after transferral to 1/2MS liquid medium containing 10% coconut water (CW)/coconut toddy (CT). Somatic embryos developed into plantlets at the highest frequency (98%) after transferral to solid 1/2MS medium containing 10% CW/CT. All the plantlets were acclimatized in soil and survived under field conditions, and they were morphologically indistinguishable from the source plant.     

In vitro somatic embryogenesis from callus culture of the timber yielding treeHardwickia binata Roxb.

Somatic embryogenesis was achieved in callus cultures derived from immature cotyledonary explants ofHardwickia binata Roxb., a multipurpose leguminous tree, on semisolid modified Murashige and Skoog's (mMS) medium containing 2900 mg/l potassium nitrate (KNO₃) supplemented with 4.64 µM kinetin (Kn) and 5.37µM a-naphthaleneacetic acid (NAA). Somatic embryos proliferated rapidly after transfer to MS basal medium supplemented with 2052.6 µM L-glutamine and 0.084 µM gibberellic acid (GA₃). Maturation of somatic embryos was achieved on half-strength MS basal medium supplemented with 1.23 µM IBA and 2% (w/v) sucrose. Histological studies confirmed different developmental stages of somatic embryogenesis inHardwickia binata. Abbreviations BA N⁶-benzyladenine - Kn kinetin - NAA a-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) medium - mMS modified Murashige and Skoog (1962) medium     

Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.