Organogenesis in vitro of Nothofagus alpina (P. et E.) Oerst, Fagaceae

In vitro shoot and root regeneration of 2-year-old Nothofagus alpina plants was achieved from several types of expiants cultured in vitro on a modified Woody Plant Medium formulation. Multiple shoot formation was obtained from leaf expiants using 0.45–2.27 M thidiazuron and 0.0049–0.098 M indolebutyric acid. Excised axillary buds formed shoots and roots in the presence of 0.0049 M benzyladenine and 2.46 M indolebutyric acid, or in the absence of plant growth regulators. Nodal sections rooted when 2.46 M indolebutyric acid at was supplied in the medium. Subcultured shoots originating from nodal sections showed a high regeneration rate through multiple shoot and root formation.Abbreviations BA benzyladenine - GA₃ giberellic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron - WPM McCown's Woody Plant Medium - Z zeatin     

In vitro clonal multiplication of the actinorhizal plant Comptonia peregrina

Micropropagation of the actinorhizal plant Comptonia peregrina of the Myricaceae was achieved successfully by the induction of root buds in excised root culture with cytokinin (1.0 M benzyladenine). Excised root segments with initiated root buds were subcultured in Woody Plant Medium (WPM) lacking growth regulators, developing extensive callus which subsequently gave rise to multiple adventitious buds. Shoot elongation was facilitated by transfer of calluses to more aerated conditions. Root initiation was induced on shoots by brief treatment with auxin (<1 M indolebutyric acid) and transfer to WPM for plantlet development. Controlled light and aeration in liquid medium were critical conditions for successful micropropagation.     

Plant regeneration from nucllar tissues of Aegle marmelos through organogenesis

A protocol for organogenesis from nucellar explants excised from fertilized ovules of immature fruits of Aegle marmelos Corr. was developed. Adventitious buds were initiated on Murashige and Skoog's (MS) medium containing various combinations of 6-benzyladenine (BA), -naphthalene-acetic acid (NAA), 3-indoleacetic acid and gibberellic acid. Medium containing 4.4 m BA and 2.7 M NAA produced the maximum number of adventitious buds per explant. Shoots were elongated by transferring explants with shoot buds to medium with a low concentration of BA (0.44 M). Rooting of in vitro-regenerated shoots was obtained in half-strength MS medium with 4.9 M indole-3-butyric acid. This is the first report of plant regeneration from nucellar explants of A. marmelos.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid     

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.     

Rapid in vitro propagation of Aloe barbadensis Mill

Axillary bud development and adventitious bud formation was obtained with decapitated shoot explants of Aloe barbadensis Mill. Maximal bud growth and rooting of shoots was obtained on a modified medium of Murashige and Skoog supplemented with 5 M IBA. More adventitious and axillary buds developed on nutrient media supplemented with IBA than with NAA. Axillary buds but not adventitious buds developed with IAA in the medium. Morphogenesis was inhibited by 2,4-D. Kinetin, benzyladenine and thidiazuron were toxic to the explants and did not stimulate the development of axillary of adventitious buds. The optimal temperature for bud growth and development was 25°C. Axillary bud growth and the formation of adventitious buds was slowed down at 10°C and totally inhibited by 30°C. The optimal sucrose concentration was 3% with the inhibition of bud growth and development by higher sucrose levels.     

Shoot organogenesis and plant regeneration in mulberry (Morus bombycis Koidz): Factors influencing morphogenetic potential in callus cultures

Regeneration of multiple shoots via callus induction and organogenesis was achieved in mulberry (Morus bombycis). Pre-soaked internodal explants in 4.4–8.9 M benzyladenine (BA) formed callus on Linsmaier and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (9.05 M), -naphthaleneacetic acid (2.85 M) and BA (2.2 M). Explants soaked for 48 to 72 h in low levels of BA produced loose and nodular callus that showed regeneration ability. Calluses developed adventitious shoot buds within 3–4 weeks on medium containing BA (8.9 M). Fifteen-week-old calluses developed fewer shoot buds than five-week-old calluses, indicating a decrease in morphogenetic potential with increasing duration of callus cultures. Semi-thin section microscopy was used to evaluate incapability of sustained regeneration. Development of normal shoot bud primordia, due to sub-surface reorganisation, was high in young calluses. The decline in the frequency of shoot bud primordia formation with callus ageing is due to reduced cell division activity in epidermal as well as sub-epidermal layers.     

Factors affecting in vitro microrhizome production in turmeric

In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium –- MS basal medium + 0.88 M BAP (6-benzylaminopurine) + 0.92 M kinetin + 5% coconut water + 2% sucrose + 0.5% agar –- resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium –- MS basal medium + 2.2 M BAP + 0.92 M kinetin + 5% coconut water + 2% sucrose –- at 25±1°C and 16-h light (at 11.7 mol m^–2 s^–1)/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 25±1°C. Half strength MS basal medium supplemented with 80 g l^–1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2 M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1–2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1–0.4 g), medium (0.41–0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.     

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.     

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro

High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 M indole-3-acetic acid and 50 M benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.     

Regeneration in Phaseolus vulgaris L.: High-frequency induction of direct shoot formation in intact seedlings by N6-benzylaminopurine and thidiazuron

Induction of prolific shoot formation in Phaseolus vulgaris L. cv. Kinghorn Wax was achieved by germinating mature seeds and growing seedlings on a medium supplemented with 10 M thidiazuron (TDZ), a substituted phenylurea, or 80 M N⁶-benzylaminopurine (BAP). Culture for 7 d in the presence of 10 M TDZ was sufficient to induce maximal shoot formation, whereas a continuous presence of BAP was required for the induction and development of shoots. The differentiation of adventitious shoots occurred within four weeks of seed culture, from tissues in the regions of axillary buds on the cotyledonary node and also areas surrounding the shoot apex of the intact seedling. The number of shoots regenerated from intact seedlings was significantly higher than that obtained with expiants. Regenerated shoots developed into flowering plants. Similar results were obtained in six other bean cultivars.Abbreviations BAP N⁶-benzylaminopurine - MS Murashige and Skoog (1962) medium - TDZ N-phenyl-N 1-(1,2,3 thiadiazol-yl)urea (thidiazuron) To whom correspondence should be addressedThis research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada and the University Research Board Grant Programs of the University of Guelph to P.K.S. We thank Drs. Jean Gerrath and R. Rastogi for helpful discussions. Technical assistance from Sangeeta Saxena is gratefully acknowledged.     

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.     

In vitro regeneration of silktree (Albizzia julibrissin) from excised roots

Root segments (1 cm long) were excised from 15–20 day old seedlings of silktree (Albizzia julibrissin) grown on B5 medium. About 50% of the control (no growth regulators added) root explants formed shoot buds within 15 days after placement on the culture medium. After 30 days, there were about 4 shoots per control explant. Addition of low levels of various auxins (0.5 M) did not influence the formation of shoot buds from the explants. Higher concentrations (5M), however, decreased shoot regeneration. Kinetin and 2iP did not influence shoot regeneration at the concentrations tested (1 & 10 M). Addition of benzyladenine, Zeatin, or thidiazuron to the culture medium increased both the percentage of explants that formed shoots and the number of shoots per explant. Thidiazuron was highly effective in stimulating shoot formation at low concentrations (<1 M). At 0.05 M thidiazuron, 95% of the explants produced shoots and about 10 shoots were formed per explant. Compared to TDZ, higher concentrations (10 M) of benzyladenine and Zeatin were required to enhance shoot formation. Upon excision and transfer to B5 medium, regenerated shoots developed into normal rooted plantlets.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - TDZ Thidiazuron - 2ip Isopentenyladenine     

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     

An efficient adventitious shoot regeneration system for Zhanhua winter jujube (Zizyphus jujuba Mill.) using leaf explants

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 M TDZ and 2.85 M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 M benzyladenine and 5.77 M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 M IAA and 2.46 M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube.     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

The in vitro culture of Gerbera aurantiaca

Multiple shoot cultures were obtained from elongating axillary buds of Gerbera aurantiaca Sch. on the medium of Murashige & Skoog (MS) supplemented with 5 M benzyladenine. Shoots rooted in vitro on MS medium supplemented with 5 to 10 M indolebutyric acid or 5 to 10 M -naphthaleneacetic acid. Plantlets were hardened in a mist bed and transferred to soil.     

Micropropagation ofPrunus mume

Shoot proliferation from axillary buds ofPrunus mume Sieb. et Zucc. was obtained on Woody Plant Medium (WPM) supplemented with 1 to 5 M benzyladenine, 3% sorbitol and solidified with 0.5 to 0.7% agar. Effects of different carbon sources on shoot proliferation were examined. Glucose provided better shoot proliferation than sucrose, sorbitol and fructose. In the presence of sucrose, leaf chlorosis occurred and shoots gradually declined. Best rooting percentage was obtained on WPM supplemented with 1 M naphthaleneacetic acid. Rooted plantlets were acclimatized under intermittent mist. However, survival rate was relatively low (20 to 30%).     

Micropropagation of Cunila galioides,a popular medicinal plant of south Brazil

Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.     

Effect of various bud induction treatments on elongation and rooting of adventitious shoots of Canary Island pine (Pinus canariensis)

Three-day-old cotyledonary explants of Pinus canariensis were subjected to 30 induction treatments using half-strength Bornman's medium containing various combinations of N⁶- benzyladenine, zeatin, kinetin and 2-isopentenyl-adenine. The highest numbers of buds were obtained with 10 M 6-benzyladenine, but both kinetin and zeatin influenced shoot elongation. Shoots were maintained on half-strength Schenk and Hildebrandt medium with 2% sucrose and 0.05% activated charcoal. For rooting, shoots were pulsed for 4 h in a 100 M indole-3-butyric acid aqueous solution (pH 4.2–4.5), and planted in peat:vermiculite:perlite (1:1:1). After 8 weeks, the numbers of rooted shoots were similar for most treatments. Therefore, the bud induction treatments did not significantly influence rooting of adventitious shoots of Canary Island pine.     

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) by organogenesis

Clonal propagation of Virginia Pine (Pinus virginiana Mill.) was achieved by organogenesis on cotyledon explants. The influence of several cytokinins and abscisic acid on adventitious shoot production from cotyledon explants was investigated. Benzyladenine was more effective in shoot induction than three other cytokinins tested. Benzyladenine (22.2 M) in combination with naphthaleneacetic acid(0.05 M) in a Gresshoff and Doy (1972) medium was found to increase shoot bud production. Abscisic acid (7.6 M) in combination with benzyladenine and naphthaleneacetic acid enhanced shoot formation by an additional 65%. Root initiation was achieved with 0.5 strength Gresshoff and Doy media amended with naphthaleneacetic acid (1.3 M), indole-3-butyric acid (1.2 M) and benzyladenine (0.4 M). Over 2400 plantlets from 2 families survived and were transferred to a greenhouse in preparation for field planting. After ten months, the maximum number of surviving plantlets/seed explant from these two sources was 57 for family ALPV-38 and 41 for family ALPV-78, respectively.Abbreviations ABA Abscisic acid - BA N⁶-Benzyladenine - GD Gresshoff and Doy (1972) nutrient media - IBA Indole-3-butyric acid - KN Kinetin - NAA -Naphthaleneacetic acid - 2iP 2-isopentenyl adenine - ZN Zeatin