Effects of thidiazuron and N6-benzylaminopurine on shoot regeneration of Phalaenopsis

Adventitious bud formation from the vegetative buds of the flower stalks of Phalaenopsis occurred on Vacin and Went medium with 15% coconut water and 5 to 40 μM thidiazuron (TDZ) or 40 μM N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 μM TDZ. Adventitious buds developed into shoots on VWC medium. TDZ was more effective than BAP in stimulating the axillary buds of intact shoots to develop. Regenerated shoots rooted after about two months of culture on VWC medium with 1% sucrose. Shoot tips excised from the regenerated shoots initiated protocorm-like bodies after two months of culture on VWC medium.     

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.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoogs (MS) medium supplemented with 11.25 M of 6-benzylaminopurine (BAP) and 4.5 M kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 M of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.     

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties

Summary Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - TDZ thidiazuron     

A liquid cytokinin pulse induces adventitious shoot formation from Douglas-fir cotyledons

Summary The effects of high-concentration, 2-h liquid pulses of N⁶-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 M BA, and 100 and 200 M TD. Shoots developed best from buds induced with 300, 400, and 800 M BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 M BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.Abbreviations BA N⁶-benzylaminopurine - TD thidiazuron - SH Schenk and Hildebrandt (1972) - NAA 1-naphthaleneacetic acid Paper 2691 of the Forest Research Laboratory, Oregon State University, Corvallis. Mention of trade names or commercial products does not constitute endorsement or recommendation for use by the authors or Oregon State University.     

Somatic embryogenesis and organogenesis in peanut: The role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis

Intact peanut (Arachis hypogaea L.) seeds, incubated on media containing N⁶-benzylaminopurine (BAP) or thidiazuron (TDZ) exhibited de novo regeneration at the hypocotyledonary notch region. Regeneration was observed when seeds were cultured on either TDZ or BAP but the optimal level of media supplementation was 10 mol·L^–1 for TDZ and 50 mol–L^–1 for BAP. Light microscopic observations revealed that the regenerants induced by TDZ were somatic embryos while those induced by BAP were shoots. An alternative approach of exposing the seeds to TDZ was through vacuum infiltration followed by culture on basal media but BAP did not induce regeneration by this method. Although TDZ has often been classified as a synthetic cytokinin, our results clearly demonstrate that seedlings treated with TDZ undergo a different morphological route of development than that induced by purine cytokinins.     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

In vitro propagation of Iphigenia indica,an alternative source of colchicine

The present study involves in vitro propagation of Iphigenia indica (Kunth.) through multiplication of whole corms and corm buds. The whole corms produced very small micro-corms, which developed plants individually whereas corm buds multiplied to produce numerous shoots at variable rates in presence of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). The best response in corm and bud multiplication was obtained in Murashige and Skoog's basal medium (MS) supplemented with 2.69 M NAA and 8.88 M BAP. The shoots regenerated were further cultured on MS medium containing NAA and indole-3-butyric acid (IBA) for initiation of roots. MS medium with 5.38 M NAA and 4.92 M IBA induced highest percentage of roots (81%) within 2 weeks in culture.     

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     

Regeneration in Phaseolus vulgaris L. Promotive role of N6-benzylaminopurine in cultures from juvenile leaves

Plants were regenerated from cultured young leaves of Phaseolus vulgaris L. cv. Kinghorn. For inducing shoot regeneration the expiant had to consist of the petiole and a portion of the lamina, and N⁶-benzylaminopurine (BAP) had to be present in the culture medium. Furthermore, the frequency of shoot regeneration increased more than seven-fold if donor seedlings were raised on a medium containing 5 M BAP, followed by culture of the leaf explants on a medium containing 20 M BAP. Regenerated shoots developed roots on basal (hormone-free) medium and the resulting plantlets could be transplanted to soil.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium This research was supported by operating grants from the Research Board Grants Program of the University of Guelph and the Natural Sciences and Engineering Research Council of Canada to PKS. Technical and photographic assistance from Sangeeta Saxena and Jean Gerrath is gratefully acknowledged.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

In vitro regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. (Rosaceae) from leaf explants

A protocol for shoot regeneration of Hagenia abyssinica (Bruce) J.F. Gmel. has been developed using leaf explants originating from in vitro seedlings and mature material. The explants were cultured on Murashige and Skoog medium containing various concentrations of -naphthaleneacetic acid and thidiazuron (TDZ). Concentrations of TDZ lower than 1.0 M promoted direct shoot regeneration, but higher concentrations promoted callus induction. Around 96–100% regeneration was obtained between 1.0 and 10 M TDZ. The average number of shoots per explant at 1.0 M TDZ was 8.4±4.8. Among the different explants used, the highest percentage of regeneration and shoots per explant was obtained from complete leaf explants. A significant (P0.05) difference in regeneration capacity was observed among the five genotypes examined. The resulting shoots were multiplied on multiplication medium, rooted and acclimatised in a greenhouse.     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 M gibberellic acid (GA₃). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA₃. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.Abbreviations BA 6-benzyladenine - BM basal medium - DPU 1,3-diphenylurea - GA₃ gibberellie acid - 2iP isopentenyladenine - MSM multiple shoot medium - NAA 1-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - Z zeatin     

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.     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid