Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.     

Direct somatic embroygenesis from immature embryos of rosewood (Dalbergia latifolia Roxb.)

Direct regeneration of somatic embryos was obtained from immature zygotic embryos of Dalbergia latifolia. Immature embryos dissected from green pods 90 d after flowering gave the highest frequency of somatic embryo formation. Preculture on high 2,4-D medium for 4 weeks induced direct somatic embryogenesis, which was expressed during the second culture phase in the presence of low 2,4-D along with a high sucrose concentration. Embryos were separated and transferred to the maturation medium containing MS + 0.5–1.0 mg/L BAP, where embryos developed into plantlets. Somatic embryos failed to convert into complete plants without BAP treatment. This method of direct regeneration of somatic embryos without a callus phase has direct application for genetic manipulation studies.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - KIN Kinetin     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

Improvement of somatic embryogenesis and plant recovery in cassava

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BA Benzyl aminopurine - GA Giberellic acid - MS Murashige and Skoog - NAA Naphthalene acetic acid     

Berberine synthesis by callus and cell suspension cultures of Coscinium fenestratum

Callus and cell suspension cultures of Coscinium fenestratum were established from sterile petiole segments on Murashige & Skoog (MS) medium, supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyl amino purine (BAP). The cells in the culture produced berberine as the major compound. NAA stimulated the product synthesis over 2,4-D. Presence of light inhibited the growth and enhanced the berberine synthesis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - HPLC high pressure liquid chromatography - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - TLC thin layer chromatography     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Somatic embryogenesis and plant regeneration in olive (Olea europaea L.)

Leaf discs from olive (Olea europaea L.) grown in vitro and immature zygotic embryos collected at 50, 75, 90 and 105 days after full bloom were tested for their somatic embryogenic capacity. The embryos were grown in half-strength MS medium and half-strength OM medium with BAP combinated with either 2,4-D or NAA. Incubation was either in an initial dark period followed by 16h daylight or in 16h daylight throughout. Somatic embryogenesis, approx. 40%, mostly directly from the embryos, was observed only in 75-day-old embryos in medium containing low cytokinin and auxin concentrations. Differentiation was inhibited by 2,4-D whereas NAA did not. In leaf discs and younger and older zygotic embryos, only callus and root formation was observed. Somatic embryos were germinated and then potted-up to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA naphtaleneacetic acid     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Somatic embryogenesis and plant regeneration in callus culture derived from immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson

Callus was induced on the wounded immature seeds and mature zygotic embryos of Dysosma pleiantha (Hance) Woodson (Berberidaceae) on a medium based on Murashige and Skoog's (1962) formula supplemented with 1 mg/l 2,4-dichlorophenoxy-acetic acid (2,4-D). Spontaneous embryoid formation occurred on the media containing low concentrations of 2,4-D (0.1–0.5 mg/l). These embryoids germinated in either MS or B5 medium containing 1 mg/l N⁶-benzyladenine and 1 mg/l gibberellic acid. The regenerated plantlets were successfully transferred to soil.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GA₃ gibberellic acid - MS medium Murashige and Skoog's (1962) medium     

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.     

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     

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.     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

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     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Sandalwood plantlets from ‘Synthetic seeds’

Somatic embryos of sandalwood (Santalum album) were encapsulated in an alginate matrix to prepare Synthetic seeds. Encapsulated single embryos germinated to form plants with roots and shoots. Embryogenic cell suspensions encapsulated and stored at 4°C for 45 days produced embryos when recultured as suspensions.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indole acetic acid - IBA indole butyric acid - MS Murashige and Skoog (1962) - TW tap water - WM White's medium     

Anther culture in Helianthus annuus L., influence of genotype and culture conditions on embryo induction and plant regeneration

Summary Production of microspore-derived embryos from cultured anthers is now a well established technique for the isolation of homozygous lines in many crop plants. We describe here a culture method for embryo induction and plant regeneration from anthers of four sunflower genotypes. For preliminary experiments, anthers of uninucleate microspores were cultured on four types of basal media viz., Murashige and Skoog's MS, Gamborg's B5, Nitsch and Nitsch, and White's W, supplemented with 1.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 mg/l 6-benzylaminopurine and 40 g/l sucrose. MS basal medium, being more responsive for embryo induction, was used for further experimentation. To optimise the culture requirement MS basal medium was supplemented with 0.2–2.0 mg/l 2,4 dichlorophenoxy acetic acid and 0.5 and 1.0 mg/l 6-benzylaminopurine. The effect of cold pretreatment, hormone regime and sucrose concentration were tested for embryogenic efficiency. Genotype had a significant effect on the capacity of embryo induction. Addition of silver nitrate (2.5 mg/l), an ethylene inhibitor, stimulated embryo germination. Plantlets were obtained (10–15%) from embryos of only one genotype.Abbreviations 2,4-D 2,4 dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - IAA indole-3-aceticacid - BAP 6-benzylaminopurine - KN Kinetin - ABA abscisic acid - GA₃ gibberellic acid     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog