Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Initiation and propagation of Glycine max L. Merr.: Plants from tissue-cultured epicotyls

A successful technique for the initiation and proliferation of shoots from epicotyl tissue of soybean, Glycine max, has been developed and is described. Fertile plants were recovered. Seeds were germinated on Murashige and Skoog medium containing 5 M benzyladenine. Explanted epicotyl sections were induced to form callus and shoots on Schenk and Hildebrandt medium containing 5.2 mM monobasic ammonium phosphate, 74 M 3-aminopyridine, and 20 M kinetin for five weeks. Shoot proliferation was maintained on N6 medium containing 1.75 mM ammonium sulfate, 2.1 nM picloram, and 0.1 M benzyladenine. Shoots rooted on Gamborg's B5 medium without growth regulators. Shoot-forming cultures were maintained for 60 months. Although all varieties tested produced shoots, some variation in numbers of shoots obtained was observed.     

Rapid in vitro multiplication of jujube through mature stem explants

Stem explants obtained from a mature tree of Ziziphus mauritiana Lamk were grown on modified Murashige and Skoog medium containing 3800 mg l^-1 potassium nitrate, 2475 mg l^-1 ammonium nitrate, 11 M benzyladenine and 0.5 M indole-3-acetic acid. During successive subcultures 15–20 shoots per inoculum were produced. Rooting was induced by pretreatment with 50 M indolebutyric acid or 1-naphthaleneacetic acid for 24 h followed by transfer to auxin-free White's medium. Plantlets grew well in a soil and vermiculite mixture.Abbreviations IAA Indole-3-acetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

Micropropagation of the Mediterranean species Viburnum tinus

In vitro propagation of the Mediterranean species Viburnum tinus L. was established from an outdoor-grown shrub. Two standard macrosalt formulations (Margara N30K and Murashige and Skoog), a range of benzyladenine and sucrose concentrations were tested for their effect on shoot multiplication. The cytokinin concentration was the most important factor affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-node explants cultured on Murashige and Skoog medium supplemented with 4.4 M benzyladenine. Cytokinin concentration and an interaction of macrosalts and benzyladenine influenced shoot length on the multiplication stage: best shoot growth was observed on MS medium containing 1.1 M benzyladenine. In addition, sucrose concentrations of 87.6–146.0 mM gave the highest multiplication rates and improved shoot growth. Following a shoot ellongation stage, single shoots were rooted on media containing naphtaleneacetic acid (1.3–5.4 M). Although enhanced in vitro rooting was obtained on media containing 5.4 M naphtaleneacetic acid, reducing the auxin concentration to 1.3 M during the in vitro rooting stage improved acclimatisation frequency and further plant growth in a horticultural substrate.     

Micropropagation of common ash (Fraxinus excelsior)

Embryos extracted from dried seeds of common ash (Fraxinus excelsior), were germinated on growth regulator-free culture medium. Cotyledonary nodes from these seedlings were placed onto Murashige and Skoog, Woody Plant or Driver and Kuniyuki culture media with 22.2 or 44.4 M benzyladenine, on which they developed into shoot cultures following the outgrowth of axillary buds. With Murashige and Skoog medium, cultures often died. With Woody Plant Medium, survival of the cultures was considerably improved, but large amounts of callus were produced at the cut ends of the explants, and new axillary shoots had long internodes and small leaves. With Driver and Kuniyuki medium, both survival and callus formation were much improved, and the shoots produced were of high quality. Proliferation of axillary shoots was obtained from both shoot tip and nodal explants placed onto Driver and Kuniyuki medium with 22.2 M benzyladenine. Adventitious root formation was best with shoots inserted into half-strength Woody Plant Medium containing 2.45, 4.9 or 9.8 M indolebutyric acid. All of the rooted plantlets tested have successfully established in soil.     

In vitro axillary shoot proliferation and somatic embryogenesis of yellow pitaya Mediocactus coccineus (Salm-Dyck)

Yellow pitaya (Mediocactus coccineus) seeds were sown on Murashige and Skoog (1962) mineral salt medium. After germination, epicotyls were placed on media enriched with a combination of naphthaleneacetic acid (NAA) (0.05, 0.27 or 0.54 M) and benzyladenine (BA) (2.2 or 4.4 M). The apical tip was excised from half of the shoots and the other half were kept intact. Different values for proliferation rate, shoot length and thickness were observed on each medium. The cotyledons and roots were placed on MS medium supplemented with NAA (2.7 or 5.4 M) and embryogenic calluses were formed. Somatic embryos were induced on these media and then they normally developed on a growth regulator-free medium.Abbreviations BA benzyladenine - MS Murashige and Skoog - NAA -naphthalenacetic acid     

In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.)

An in vitro propagation technique based on axillary bud proliferation has been developed for matureSapium sebiferum trees. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (1–10 m and -naphthaleneacetic acid (0–0.5 m showed axillary bud proliferation. Shoots proliferated in vitro were multiplied on Murashige and Skoog medium containing 2.5 m benzyl adenine and 0.25 m -naphthaleneacetic acid. Seasonal changes affected the shoot proliferation potential of the initial explant. Shoots were rooted on a half-strength, growth-regulator-free, agar-gelled, MS medium after a 48-h treatment on half-strength MS liquid medium with 10 m indole-3-butyric acid. Rooted plantlets were potted and acclimatized in a growth chamber and then moved to the greenhouse. Four-month-old plants were transplanted to the field.Abbreviations BA Benzyl adenine - IBA Indole-3-butyric acid - 2-ip N⁶-(-dimethylallylamino)purine - MS Murashige and Skoog (1962) medium - NAA -Naphthaleneacetic acid     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Rapid plant regeneration from various explants of Jatropha integerrima

A simple, rapid and reproducible protocol for direct shoot regeneration from different explants of Jatropha integerrima was developed. Prolific adventitious shoot bud initiation was obtained using a combination of 2.2 or 4.4 M benzyladenine and 4.9 M indole-3-butyric acid (IBA). Reduction of IBA concentration (2.5 M) promoted further development of shoots. Regenerated shoots rooted readily on Murashige and Skoog (MS) medium lacking growth regulators. Plantlets were acclimatized and successfully transferred to pots.     

Micropropagation of safed musli (Chlorophytum borivilianum), a rare Indian medicinal herb

In vitro clonal multiplication of safed musli (Chlorophytum borivilianum Sant. et. Fernand.), a rare Indian medicinal herb, has been achieved on Murashige and Skoog's (MS) medium supplemented with 22.2 M benzyladenine using young shoot bases as explants. Shoots multiplied at a rate of four-fold every 3 weeks. All shoots rooted when transferred to MS medium with 3/4-strength inorganic and organic constituents and 9.8 M indolebutyric acid and 67% of the micropropagated plants were successfully established in pots. Such plants produced normal fasciculated storage roots as in wild plants.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Micropropagation of parsley through axillary shoot proliferation

A micropropagation protocol of parsley,Petroselinum crispum (Mill.) Nyman (curled type) has been developed. Surface-sterilized axillary buds cultured on Murashige and Skoog medium supplemented with benzyladenine, kinetin, or thidiazuron developed axillary shoots (rosettes). Kinetin resulted in only a low proliferation rate. The concentrations of thidiazuron or benzyladenine that were optimal for shoot proliferation, resulted in shoots with a low capability to root. During the rooting treatment, these shoots showed wilting signs. Rooting was increased significantly by using a two-week inductive stage with 2.5 M naphthaleneacetic acid directly followed by acclimatization. Two proliferation media (5 M benzyladenine and 0.5 M naphthaleneacetic acid or 5 M kinetin and 2.5 M naphthaleneacetic acid) resulted in moderate proliferation but produced shoots that were easy-to-root. These media have been tested by repeated axillary proliferation on the same medium. The medium with 5 M benzyladenine and 0.5 M naphthaleneacetic acid was optimal.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige & Skoog - NAA -Naphthaleneacetic acid     

Micropropagation procedures for Leontochir ovallei

Leontochir ovallei Phil., an endangered Chilean species in the Alstroemericeae, was micropropagated on Murashige & Skoog medium supplemented with 4 M benzyladenine, 1 M indolebutyric acid and 146 mg l^-1 glutamine. Over 88% of the shoots rooted in vitro when treated with 10 M naphthaleneacetic acid and micropropagated plantlets were successfully transplanted into the greenhouse.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Somatic embryogenesis in callus culture of Mussaenda erythrophylla cvs. Queen Sirikit and Rosea

Somatic embryogenesis was achieved from mid-rib and internodal calluses of Mussaenda erythrophylla L. cvs. Queen Sirikit and Rosea cultured on Murashige and Skoog basal medium containing 8.9 M BA+0.57 M IAA+10 mg l^-1 ascorbic acid. Clumps of somatic embryos were separated and grown into complete plantlets when transferred to 1/2 MS medium+37 M adenine sulphate with 2% (w/v) sucrose.     

Rapid propagation of tuna (Opuntia ficus-indica) and plant establishment in soil

Explants from young joints of mature plants of tuna (Opuntia ficus-indica Mill.) were cultured on Murashige and Skoog (MS) medium containing 8.8 M benzyladenine (BA) and 0.5 M naphthaleneacetic acid (NAA). Shoots produced were utilized as secondary explants. Each shoot was cut longitudinally from apex to base into two explants, and some of these explants were cut transversely into proximal and distal explants. The size and number of shoots produced was affected by size and position of the explant within its source. The shoots were rooted in vitro or ex vitro and plants were successfully established in soil from both rooting methods.Abbreviations AC activated charcoal - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) medium - NAA naphthaleneacetic acid     

Direct somatic embryogenesis and plantlet regeneration from cotyledonary leaves of safflower

Somatic embryos were induced directly on adaxial surface of cotyledonary leaves within 8–10 days of culture on Murashige and Skoog medium containing 5.37 to 10.74 M 1 — napthaleneacetic acid and 2.22 M benzyl adenine. Germinated embryos with shoot axes developed into complete plants after transfer onto half stength Murashige and Skoog medium containing 1.07 M 1 — napthaleneacetic acid. Histological studies suggested direct origin of somatic embryos with broad-base attachment.Abbreviations BA benzyl adenine - MS Murashige and Skoog - NAA 1-napthaleneacetic acid - RH relative humidity     

Somatic embryogenesis from leaf of witloof chicory through suspension culture

A procedure for regeneration of somatic embryogenesis from witloof chicory leaf has been developed. Explants were taken from the distal third part of leaf vein and cultured on Murashige and Skoog medium (MS) containing 100 mg/1 casein hydrolysate 1.3 M 2,4-D, and 1.3 M kinetin. A pale yellowisl nodular callus was formed after 4 weeks which was maintained in the same medium for 8–12 weeks with one change to a fresh medium every 4 weeks. Callus was then suspended in the same medium without agar for 4–6 weeks with one change to a fresh medium every 2 weeks. Embryo-like structures appeared upon transfer to liquid MS containing 1.8 M benzyladenine. Embryo germination was accomplished in half strength MS medium with or without 1 g/l activated charcoal.Abbreviations AC activated charcoal - BA benzyladenine - MS Murashige & Skoog (1962) medium     

Somaclonal variation as a tool to develop pest resistant plants of Torenia fournieri ‘Compacta Blue’

Callus cultures of Torenia fournieri Compacta Blue were initiated on a modified Murashige and Skoog salt medium (MS) with 2.26 M 2,4-dichlorophenoxyacetic acid. Shoots were regenerated from these cultures using MS medium amended with 2.46 M indolebutyric acid and 8.88 M benzyladenine. These shoot cultures were subjected to two-spotted spidermite (Tetranychus urticae Koch.) and the greenhouse whitefly [Trialeurodes vaporariorum (Westwood)]. Pests were allowed to feed until such time that their populations started to decrease due to lack of food. The remaining live tissue of the Torenia was placed on MS medium amended with 2.28 M zeatin to induce new adventitious shoots and plantlets. Newly regenerated plantlets were acclimated to greenhouse conditions and evaluated for resistance to the pest to which they were subjected in vitro. Highly significant differences in pest numbers were found in somaclones for both the two-spotted spidermite and greenhouse whitefly when compared to control plants. A wide range of variability was observed among the somaclonal population. There were significantly fewer mite eggs laid on plants regenerated from in vitro cultures screened with two-spotted spidermites than on seed sown controls. Regenerants from cultures screened with whiteflies in vitro had fewer eggs, immatures and live adults than controls.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog salt medium Storrs Agricultural Research Station Scientific Publication 1641.     

In vitro micropropagation of Piper longum – an important medicinal plant

Efficient and rapid tissue culture systems were developed for Piper longum, an important medicinal plant, through shoot tip multiplication and direct regeneration. Multiple shoots were induced from shoot tips cultured on agar-based Murashige and Skoog (MS) medium containing 4.44–22.19 M benzyladenine (BA) and 4.64–13.9 M kinetin (K). Maximum number of shoots were induced with 8.9 M BA and 4.64 M K. Adventitious shoot regeneration from leaf segments was achieved on MS containing 3.6–22.19 M BA along with 3.31–12.4 M picloram (P). Shoot differentiation occurred directly from the leaf bases without intermediale callus formation. Maximum shoot buds were obtained on MS medium with 17.76 M BA and 8.28 M P. Elongated shoots were separated and rooted in MS supplemented with 2.46 M indole butyric acid (IBA). Plantlets, thus developed were established in soil.