Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.)

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - 2,4-D dichlorophenoxyacetic acid - IBA indole-3-butyric acid     

Somatic embryogenesis in leaves and leaf-derived protoplasts of Actinidia deliciosa var. deliciosa cv. Hayward (kiwifruit)

Summary The obtention of embryogenic competence in Actinidia deliciosa var. deliciosa cv. Hayward is reported. Axillary buds from shoots submitted to cold (4°C) and starvation for 1.5 months, developed leaves with embryogenic competence. These leaves, cultured in darkness for 1.5 months on a medium containing zeatin as a sole growth regulator, originated compact structures from which embryos developed. The plating orientation and sectioning of leaves strongly affected the expression of the embryogenic potential. A selected fraction of the protoplasts isolated from these leaves was able to develop in an embryogenic way. The germination of the embryos is still only occasional.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 6-dimethylallyl aminopurine - IAA indole-3-acetic acid - NOA naphthoxyacetic acid - SEM Scanning Electron Microscopy     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

High frequency embryoid and plantlet formation from tissue cultures of the Finger millet-Eleusine coracana (L.) Gaertn

Compact nodulated embryogenic callus differentiated from cultured seeds of Eleusine coracana (Finger Millet) on Murashige and Skoog (1962) basal medium with 2,4-dichlorophenoxyacetic acid (1.0, 3.0 mg ^l). This embryogenic callus was maintained on a medium with a lower level of 2,4 — dichlorophenoxyacetic acid. At every subculture the embryogenic callus had some preexisting embryoids in it. With this method of subculture the callus has retained its morphogenic potential for four years. Following transfer to media with different levels of auxins and cytokinins, the callus showed varied patterns of growth and morphogenesis. Embryoids could be germinated in profusion to form plantlets which could be transferred to the field. Shoot buds also differentiated from the whole surface of the embryoid or from the flattened meristemoids.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA napthaleneacetic acid - IBA indolebutyric acid - KN kinetin - MS Murashige and Skoog (1962) - GA₃ Gibberellic acid     

Somatic embryogenesis and subsequent plant regeneration from inflorescence callus of Bambusa beecheyana Munro var. beecheyana

Somatic embryos of bamboo, Bambusa beecheyana Munro var. beecheyana were developed in callus derived from young florets and adventive roots obtained from floret callus. The medium was a modified Murashige and Skoog medium (1962) supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid, 2 mg/l kinetin, a high content of sucrose (6%) and 0.7% agar. The embryoids germinated spontaneously to yield whole plantlets on this medium with or without the hormonal adjuvants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - MS Murashige and Skoog's (1962) medium     

In vitro propagation of Iris pallida

Plantlets were regenerated from callus of Iris pallida, an important perfume plant. Only the leaf base attached to the rhizome had the ability to generate yellow-colored callus on LS medium supplemented with 1 mg/l 2,4-D and 0.1 mg/l KT in the dark. Yellow calli grew with partial differentiation into white tissue, probably embryogenic, during subculture on the same medium with a 16-h photoperiod. Only yellow-colored calli with the white tissue could differentiate into plantlets after transfer to kinetin- or gibberellin- supplemented LS medium. Regenerated plantlets which grew on the medium without growth regulators were transferred to the soil. After 2 years of cultivation in soil, the regenerated plants flowered and formed rhizomes. The components of the essential oil in the rhizome of regenerated plants were essentially the same as those in natural plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - NAA alpha-naphthaleneacetic acid - LS Linsmaier and Skoog (1965) medium     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. II. morphogenetic studies

Plantlets regenerated from shoot apices, cotyledons and callus cultures in Moth bean, Vigna aconitifolia (JACQ) Marechal, a drought resistant legume and pulse crop, were rooted and transferred to soil. Explants for these studies were derived from seedlings pre-conditioned by germination of seeds on B5BA and WMB (control).Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - B5BA B5 basal medium containing BA (2.25 mg/l) - WMB Modified White's medium (Mascarenhas et al 1976) - BA 6-benzyladenine - IAA indole-3-acetic acid - NAA 1-napthaleneaceticacid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP N(^–2 isopentyl) adenine - CM coconut milk NCL Communication No. 3375     

Somaclonal variation inLolium multiflorum L. andL. temulentum L.

Tiller-derived callus ofLolium multiflorum L. gave rise to 65 regenerants displaying variation in height, flowering time, chromosome number and alteration to the banding pattern of the isoenzyme superoxide dismutase. Plants were also regenerated from callus cultures initiated from immature embryos of the related inbreeding speciesLolium temulentum L. Progeny of the regenerated plants from this species displayed variation in height, flowering date, ear length and chlorophyll content.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

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 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     

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 from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

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.     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Embryo culture of Costus speciosus (Koen.) Sm. to regenerate variable diosgenin yielding clones

Mature embryos of Costus speciosus were excised and cultured on Schenk and Hildebrandt's (1972) nutrient medium containing auxins and cytokinins either alone or in combination. Multiple shoots were obtained when kinetin and indole-3-butyric acid were supplemented each at 0.1 mg 1^–1 concentration. Embryo-derived plantlets were multiplied through propagation of rhizomes and the propagules derived from a single embryo were designated as an embryoclone. Twenty such embryo-clones were maintained in the field. Variations in rhizome biomass yield and diosgenin contents of these embryoclones were noted. Thirty-six percent of the embryo-clones studied were high diosgenin yielding types. Diosgenin contents at the intraclonal level were uniform. The in vitro raised plants were morphologically uniform and indistinguishable from their parent.Abbreviations SH Schenk and Hildebrandt (1972) medium - Kn kinetin - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - CA casaminoacids (vitamin free) - TLC thin layer chromatography     

Plant regeneration from callus cultures of Piper longum L. by organogenesis

Plant regeneration from callus cultures of Piper longum was achieved through organogenesis. In vitro grown shoots were used as explants for callus induction. Competent callus was initiated around the nodal ring of tissue using Murashige and Skoog medium supplemented with 1.0 mg.l^–1- naphthaleneacetic acid and 0.2 mg.l^–1 N⁶-benzyladenine. Optimum growth regulator concentrations for shoot induction and shoot elongation were found to be 0.5 mg.l^–1 indole-3-acetic acid with 1.5 mg.l^–1 benzyladenine, and 0.1 mg.l^–1 indole-3-acetic acid with 0.2 mg.l^–1 benzyladenine, respectively. Elongated shoots were rooted on half-strength Murashige and Skoog medium having 0.1 mg.l^–1 indole3-acetic acid. The rooted plants were successfully established in soil.Abbreviations BA, N⁶ Benzyladenine - 2, 4-D 2, 4- dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - 2iP 2-isopentenyladenine - Kn Kinetin - MS Murashige and Skoog (1962) - NAA -Naphthaleneacetic acid