Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented wiith 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) - LN Liquid nitrogen     

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented with 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylamino-purine - MS Murashige and Skoog (1962) - LN Liquid nitrogen     

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     

Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson

Prolific shoot bud differentiation was induced in callus and suspension cultures of hypocotyl origin in Brassica campestris cv. Yellow Sarson on MS medium supplemented with K (13.9–23.2 M) or BA (13.3–22.1 M). Plantlets were obtained by rooting the in vitro differentiated shoots. Histological studies revealed a unique mode of meristemoid formation.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA Benzyladenine - IAA Indole-3-acetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthalene acetic acid     

Callus induction and adventitious organogenesis of kenaf (Hibiscus cannabinus L.)

Callus production along with caulogenesis and rhizogenesis were obtained from internodal stem explants of kenaf (Hibiscus cannabinus L.) after 4 weeks in culture. Murashige and Skoog medium was used for two 4×4 matrix experiments designed to determine suitable growth regulator combinations (NAA/BAP or 2,4-D/kinetin) and concentrations (0.1, 0.3, 1.0, 3.0 mg/L). The most abundant callus production was observed at 0.3/3.0 and 1.0/3.0 mg/L 2,4-D/kinetin and at 1.0/1.0 and 3.0/1.0 mg/L NAA/BAP. Rhizogenesis was most extensive with NAA/BAP at concentrations of 0.1/3.0 and 0.3/ 3.0 mg/L. Adventitious shoots developed on both auxin/cytokinin matrixes when each concentration was at 0.3 mg/L or less. These protocols will facilitate the development of in vitro approaches to kenaf improvement and the study of certain host-pathogen interactions.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthyleneacetic acid - SDS sodium dodecyl sulfate     

Plant regeneration from protoplasts of a wild lettuce species (Lactuca saligna L.)

Protoplasts isolated from young, unexpanded leaves of the wild lettuce species, Lactuca saligna, divided to give colonies, when plated at low density in KP8 medium solidified with 1% (w/v) agarose. Sustained colony development was dependent upon the incorporation of the bead culture approach. Plant regeneration, via organogenesis, followed the transfer of colonies firstly to K8 medium and then to M/S medium supplemented with IAA and 6-BAP, both solidified with 1% (w/v) agarose. Excised shoots were rooted on agar-solidified M/S medium lacking phytohormones. The potential of this species as a source of race-non-specific mildew resistance for transfer into lettuce (L. sativa) via somatic hybridisation is discussed.Abbreviations 6-BAP 6-benzylamine purine - IAA indoleacetic acid - M/S Murashige and Skoog (1962) - fwt fresh weight     

Production of somatic hybrid tissues following chemical and electrical fusion of protoplasts from albino cell suspensions ofMedicago sativa andM. borealis

Protoplasts isolated from cell suspensions of albinoMedicago borealis andM. sativa were fused chemically, using two methods, and electrically. Although a small scale method of chemical fusion gave the highest fusion frequency, electrofusion was the superior technique on the basis of throughput of green somatic hybrid cell colonies. Chlorophyll-containing tissues were confirmed as being somatic hybrid by isoenzyme and cytological analyses. This is the first report of the application of albino complementation to produce somatic hybrid cells in forage legumes.Abbreviations AC alternating current - DC direct current - 2,4-D 2,4-dichlorophenoxyacetic acid - f.wt. fresh weight - PEG polyethylene glycol - K8P and K8 Kao (1977) protoplast and cell culture media - MS Murashige and Skoog (1962) medium - UM Uchimiya and Murashige (1974) medium     

Production of aposporous gametophytes from Drymoglossum piloselloides (L.) Presl. frond strips

Strips of Drymoglossum piloselloides fronds in a modified Murashige and Skoog medium in 1% agar produced aposporous gametophytes in about 2 months. Young fronds showed a greater ability to develop aposporous gametophytes than older fronds. The addition of kinetin to the medium improved the ability of older fronds while the presence of 1-naphthaleneacetic acid enhanced the effect of kinetin.Abbreviations Kinetin 6-furfurylaminopurine - NAA naphthaleneacetic acid; Murashige and Skoog (1962) medium     

Regeneration of plants from mesophyll protoplasts of the wild crucifer Eruca sativa Lam.

Protoplasts isolated from mesophyll cells of Eruca sativa Lam., cultured on suitable medium, underwent sustained cell divisions to form calli. The plating efficiency was found to be 0.4%. The protoplast-derived calli subsequently produced plantlets through organogenesis (15.71%) and somatic embryogenesis (11.25%). Regenerated plants exhibited normal appearance. These results indicate potential to introgress desirable traits from this wild crucifer into important oilseed and cole Brassicas by protoplast fusion and hybrid recovery.Abbreviations B5 Gamborg et al., 1968 - K3 Kao and Michayluk, 1974 - MS Murashige and Skoog, 1962 - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - GA₃ Gibberellic acid     

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     

The effect of gelling agents on plaunotol accumulation in callus cultures ofCroton sublyratus Kurz

Callus cultures established from the leaves ofCroton sublyratus Kurz (Euphorbiaceae) contained little or no plaunotol on various media with different hormone combinations. Plaunotol accumulation was observed in calli which were cultured in media with increasing concentrations of gelling agents, especially gellan gum and agarose. The accumulation was observed within three weeks after transfer to the medium and was accompanied by chlorophyll increase, tracheid development and slow growth. Light and increasing concentration of gelling agents were observed to be indispensable for plaunotol accumulation in the callus.Abbreviations MS Murashige and Skoog (1962) basal medium - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine     

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 M 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid     

Plant regeneration from protoplasts of long term haploid suspension cultures of N. plumbaginifolia

A predominantly haploid cell suspension culture of N.plumbaginifolia has been established. The ploidy has remained stable for nearly four years in culture (and is similar in cells recovered after preservation in liquid nitrogen). Protoplasts isolated from these cells regenerate into plants with a high frequency.Abbreviations MS Murashige and Skoog medium (Murashige and Skoog, 1962) - B5 Gamborg's B5 medium (Gamborg, 1970) - 2,4D 2,4 dichlorophenoxyacetic acid - NAA 1 - Naphthylacetic acid - BAP 6 - Benzylaminopurine, IAA=Indole - 3 - acetic acid - MES (2 (m - morpholino)) ethane sulphonic acid - FDA fluorescein diacetate     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

In vitro propagation and low temperature storage ofSaussurea lappa C.B. Clarke — An endangered,medicinal plant

A procedure forin vitro multiplication ofSaussurea lappa (Asteraceae) is described. On Murashige and Skoog's medium (MS) containing benzylaminopurine and gibberellin 3.5-fold shoot multiplication occurred every three weeks. Shoots rooted on MS containing 0.5 M naphthaleneacetic acid with 90% efficiency. The shoot cultures stored at 5°C in the dark for 12 months without an intervening subculture survived with 100% viability. The shoots cold stored for 6 months or more showed higher rates of multiplication under culture room conditions than the untreated shoots.Abbreviations MS Murashige and Skoog 1962 - BAP Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - GA₃ Gibberellin     

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