Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14–17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.Abbreviation BAP 6-benzylaminopurine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - MS Murashige and Skoog     

Regeneration of fertile plants from embryogenic suspension culture protoplasts of Sorghum vulgare

Protoplasts were isolated from immature inflorescence-derived embryogenic suspension cultures of two cultivars of Sorghum vulgare. The protoplasts were cultured in a modified K8P liquid medium. They started to divide after 4–5 days of culture, and achieved 16.8% division frequency by 10 days. Protocalli proliferated further upon transfer to C₁ solid medium. After that, they were moved to C₁ differentiation medium to induce shoot formation, followed by whole plant regeneration. So far, 60 plants have been obtained, with only two albinos. Some of these have been transplanted to soil in pots and grown to flowering and have set seeds.Abbreviations BA 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - KT kinetin - PVP polyvinylpyrrolidone     

Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.)

Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T₃) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants.Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplastderived calli from Fujiminori, produced whole plants in the regeneration medium.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

High efficiency shoot regeneration from callus of quaking aspen (Populus tremuloides Michx.)

Callus was induced from leaf segments of aspen (Populus tremuloides Michx.) on modified B5 (mB5) medium with 0.1 mg/1 benzyladenine (BA) and 0.5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting callus was either subcultured to solidified Woody Plant Medium (WPM) with 0.5 mg/1 BA directly for shoot regeneration or sieved into liquid mB5 medium for suspension culture. After 3 weeks of suspension culture, when the callus clumps grew to 3–4 mm in diameter, they were transferred onto solidified WPM with 0.5 mg/1 BA for shoot regeneration. Almost 100% of the clumps formed shoots on WPM when subcultured directly from mB5 with an average number of 6 shoots per callus. When transferred from suspension culture in mB5 to WPM, an average of 6 shoots per callus were produced from 51% of calli. These shoots could be easily rooted on either mB5 or WPM with 0.2 mg/1 indole-3-butyric acid (IBA) and transferred to pots. Transplanted plants were kept under intermittent mist for 2–4 weeks before normal growth in the green house.Abbreviations BA 6-Benzyl-adenine - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - mB5 medium modified B5 medium - WPM Woody Plant medium     

Plant regeneration from hordeum spontaneum and hordeum bulbosum immature embryo derived calli

Callus cultures were initiated from isolated immature embryos of Hordeum spontaneum and Hordeum bulbosum on MS or B5 basal medium supplemented with 2 mg/1 2,4-D. Shoot regeneration occurred on transfer of tissue to media containing 1 mg/1 IAA and 1 mg/1 zeatin. The regenerated shoot buds were rooted on basal medium without hormones. The in vitro regenerated plants were transferred to soil and were grown to fertile mature plants. A low percentage of albino plants was observed among the regenerated plants. No major differences were detected between the two species in respect to their potency to form callus or to the regeneration capacity. The regeneration capacity of calli decreased gradually and ended after 6 months in culture.Abbreviations IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - MS Murashige and Skoog medium     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Plant regeneration and initiation of cell suspensions from root-tip derived callus of Oryza sativa L. (rice)

Root-tip derived suspended callus of Oryza sativa cv. Thaipei showed the capacity for plant regeneration via organogenesis. Cell cultures were induced in liquid Murashige-Skoog medium containing 2 mg/l 2.4-dichlorophenoxyacetic acid. Dicamba or Picloram were effective for induction of organogenesis. Shoots and roots differentiated following subculture on medium lacking auxins but containing kinetin. At 1 and 4 mg/l Dicamba and 1 mg/l Picloram normal green plants were regenerated whereas with 7 mg/l Dicamba in the medium only albino plantlets were obtained. Regenerated plantlets were grown to maturity and set seed. Cell suspension cultures, initiated from the root-tip derived calli, provided suitable material for protoplast isolation.Abbreviations BM Basic medium - 2.4 -D 2,4-dichlorophenoxyacetic acid - Dicamba 3,6-dichloro-2-methoxy benzoic acid - Picloram 4-amino-3,5,6-trichloropicolinic acid     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.)

Summary A friable and fast-growing type of callus was isolated from a long term shoot-competent cell culture of wheat. The suspension cultures established from this callus consisted of small, densely cytoplasmic cells which divided more rapidly but with a lower plant regeneration frequency than the original culture. A high yield of protoplasts was released from suspension cells (2 to 3×10⁷ protoplasts per ml packed cell volume) when treated with enzyme mixtures. The isolated protoplasts divided at a relatively high frequency (20% to 50%) in both liquid and agarose-solidified KM8p medium. Up to 0.21% of the dividing protoplasts continued to divide and form micro-calli. Sixty-eight plants were regenerated from micro-calli, and among the 30 plants which were transplanted to the greenhouse, 3 have survived.Abbreviations BAP 6, enzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - FDA Fluorescein diacetate - IAA indole-3-acetic acid - LS Linsmaier and Skoog basal medium (1965) - MES 2, [N-morpholino]-ethanesulfonic acid - MS Murashige and Skoog basal medium (1962) - NAA 1, naphthaleneacetic acid - PCV packed cell volume     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Chromosome stability of cell suspension cultures of Nicotiana spp.

Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Production of emetic alkaloid by in vitro culture of cephaelis ipecacuanha A. Richard

Callus and adventitious roots were induced on leaf segments from shoot culture of Cephaelis ipecacuanha A. Richard on Murashige-Skoog medium containing 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid, 1-naphthaleneacetic acid and kinetin. The contents of emetic alkaloids in calli, roots and root suspension cultures were quantified by HPLC. Roots cultured in solid and liquid Murashige-Skoog media yielded emetine and cephaeline. The amount of the two alkaloids in the root suspension culture was very similar to that of roots from ipecac mother plant grown in a greenhouse. In contrast, calli subcultured on Murashige-Skoog media containing combinations of 2,4-dichlorophenoxyacetic acid and kinetin produced only trace amounts of emetic alkaloids.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA l-naphthaleneacetic acid - Kin kinetin - MS Murashige-Skoog - EM emetine - CP cephaeline - DW dry weight.     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

Embryogenesis in suspension cultures of Datura innoxia Mill.

In vitro plant regeneration via embryogenesis was obtained in suspension cultures of Datura innoxia Mill. Embryogenesis was induced in suspension cultures raised from callus of androgenetic origin, using LS liquid medium supplemented with 0.22 mg/l 2,4-D. The total number of embryos formed was variable over time in culture. Embryos differentiated and matured in the liquid medium itself as also evidenced by histological observation. Embryos germinated to form plantlets on semisolid MS medium without growth substances. The regenerated plants had haploid number of chromosomes.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn Kinetin - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962)