Shoot multiplication in <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10M) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5M TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5M indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Efficient Regeneration of Tetraploid Isatis indigotica Plants via Adventitious Organogenesis from Hypocotyl Explants

An efficient in vitro plant regeneration system via hypocotyl segments of tetraploid Isatis indigotica Fort. was established. Murashige and Skoog's (MS) and Gamborg's (GB₅) media were found to be superior to White medium for promoting shoot regeneration. The highest shoot regeneration (92 %) was achieved from hypocotyls cultured on MS medium containing 8.9 M benzyladenine (BA) and 2.7 M naphthaleneacetic acid (NAA), with an average of 4.2 shoots developed per explant. Plant regeneration was also improved when the explants were cultured in MS basal medium containing 3 % (m/v) sucrose and grown under a 12-h photoperiod. The developed shoots were well rooted in a half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA) and were morphologically normal after transfer to soil.     

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     

Thidiazuron-induced in vitro shoot formation from roots of intact seedlings of Albizzia julibrissin

Seedlings of silktree (Albizzia julibrissin Durrazz.) were grown in vitro on MS-media containing B5 vitamins, 3% sucrose, 0.25% phytagel and various concentrations (0.1–10 M) of thidiazuron (TDZ). Addition of TDZ to the culture medium greatly reduced shoot and root elongation but did not influence shoot production from the cotyledonary node or apex. Within 8–10 days the seedling roots split open, formed large masses of callus, and developed green patches which eventually grew into normal shoots while still within the culture medium containing TDZ at 0.1–1.0 M. Such callus and shoot formation did not occur in control cultures lacking TDZ. At higher TDZ concentrations (2.5–10 M), the green patches formed in the callus did not further develop into shoots. Addition of other cytokinins (kinetin, benzylaminopurine, zeatin) to the culture medium also induced some shoot formation from the roots, but higher concentrations than TDZ were required to induce regeneration. Isopentenyladenine failed to induced shoot formation. Following excision and transfer to MS media with or without 4.9 M IBA, the shoots induced by kinetin or benzylaminopurine rooted 4–7 days earlier than those induced by TDZ, but all excised shoots developed into normal rooted plantlets within 3 weeks.Abbreviations TDZ thidiazuron     

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.     

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     

Shoot regeneration from tissue-cultured leaves of the American cranberry (Vaccinium macrocarpon)

A method for shoot regeneration from leaf explants in two cultivars of cranberry (Vaccinium macrocarpon Ait.) is described. Modified Anderson's medium supplemented with combinations of thidiazuron (TDZ) with or without 1 M NAA (-naphthaleneacetic acid) was used to optimize shoot regeneration. The effect of light or dark incubation was also determined. Maximum regeneration was obtained in the light in the presence of 10 M TDZ and 1 M NAA. While this medium was suitable for leaf explants obtained from shoot cultures, regeneration did not occur from leaves collected from greenhouse-grown plants. Elongation of the regenerated shoot tips did not occur until explants were transferred to growth regulator-free medium at which time only a minority of shoots elongated. Elongated shoots could be dissected away from leaf tissue, rooted easily, and acclimitized to ambient conditions.Abbreviations NAA -naphthaleneacetic acid - TDZ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea     

Thidiazuron-Induced High-Frequency Shoot Regeneration from Root Region of Robinia pseudoacacia L. Seedlings

High-frequency regeneration of shoots was achieved at root region of seedlings of Robinia pseudoacacia L. cultured from seeds on medium supplemented with thidiazuron (TDZ, 1.0 M). The roots of intact seedlings proliferated and formed a compact callus followed by differentiation of numerous shoots. Corresponding cultures on benzylaminopurine-containing medium exhibited much weaker response. Hypocotyl segments also formed shoots at a lower concentration of TDZ (0.1 M). The shoots formed on TDZ-containing medium were well-developed and readily rooted on hormone-free medium. The obtained plants after acclimation in culture room survived after transfer to soil.     

Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 M Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 M IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.     

Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch

Factors were defined for elongation, rooting and acclimatization of micropropagated shoots ofLarix x eurolepis Henry initiated from short shoot buds of plagiotropic stecklings serially propagated for 9 years from an 8-year-old tree. Initiation and multiplication were on Schenk and Hildebrandt (SH) medium supplemented with 5 M 6-benzyladenine (BA) and 1 M indole-butyric acid (IBA). Stem elongation was obtained in 36% of the shoots on SH medium containing 0.5 M BA and 63% of the remaining non-elongated shoots initiated stem elongation after transfer on SH medium devoid of growth regulators. Rooting involved 2 steps: root induction on Campbell and Durzan mineral salts and Murashige and Skoog organic elements, both half-strength (CD-MS/2), supplemented with 1 M of both naphthaleneacetic acid (NAA) and IBA, and root elongation following transfer to CD-MS/2 medium devoid of growth regulators. Repeating this 2-step sequence yielded up to 67% rooted shoots. Acclimatization of plantlets ranged from 83% to 100%. Over 300 plants were transferred to the greenhouse; some showed plagiotropic growth.     

In vitro organogenesis and plant formation in Vigna radiata (L.) Wilczek

In vitro organogenesis was achieved from calluses derived from cotyledon and hypocotyl explants of Vigna radiata on MS medium. Organogenic calluses were induced from both cotyledon and hypocotyl explants excised from 3-day-old seedlings on MS medium containing NAA (1.07 m and BA (2.22 m) and 2,4-D (0.90 m) and BA (2.22 m) combinations respectively. Regeneration of adventitious shoots from cotyledon derived callus was achieved when they were cultured on MS medium supplemented with NAA (1.07 m), BA (8.88 m) and 10% coconut water. Hypocotyl derived calluses produced adventitious shoots when cultured on MS medium fortified with BA (6.66 m), TDZ (2.5 m) and 10% coconut water. Addition of GA at 1.73 m favored maximum 3 elongation of shoots. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 4.90 m IBA. Rooted plantlets, thus developed were hardened and successfully established in field. Among the different carbohydrates and media tested, 87.64 m sucrose and MS+B5 medium proved best for maximum production of shoots. This protocol produced an average of seven plants per hypocotyl derived callus and 15 plants per cotyledon derived callus over a period of 3 months.     

Thidiazuron-induced shoot-bud formation on root segments of Albizzia julibrissin is an apex-controlled, light-independent and calcium-mediated response

Root segments or entire roots of Albizziajulibrissin formed shoot-buds; the former were more responsive thanthe latter. The regeneration capacity of root segments increased with anincreasing distance from the meristem. Shoot regeneration on N₆mineral formulation required either a cytokinin (BAP) or thidiazuron (TDZ); thelatter was more effective than the former, inducing a higher number of shoots ata low concentration (0.1 M) in the light as well as in thedark. The frequency of shoot formation was reduced when the auxin inhibitorsmaleic hydrazide (MH) or triiodobenzoic acid (TIBA) were included, indicating anindirect role of auxin in shoot morphogenesis. Inhibitors of calcium uptake(lanthanum) and calmodulin, trifluoperazine (TFP) or chlorpromazine (CPZ) atvery low levels, resulted in inhibition to reduction in frequency of shootmorphogenesis. This indicates that TDZ-induced shoot formation may be acalcium-mediated response. Increasing the level of calcium in the medium did notpromote shoot formation in the presence of TDZ (0.1 M). At areduced level of calcium, which was ineffective in the presence of low TDZ (0.1M), shoot-buds appeared when the concentration of TDZ wasraised to 1.0 M. This provides indirect evidence that TDZmodulates the tissue level of calcium needed for shoot formation.     

In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia)

Shoot apical meristems were used to establish regenerative axillary bud cultures of 9 muscadine grape cultivars. Meristems taken from 10 cm long shoots had less contamination (3%) and a higher survival rate (94%) than those from shorter or longer shoots. Of media tested, MS, 1/2 MS, and C₂D resulted in equivalent shoot proliferation rates, whereas, WPM produced stunted shoots. When pooling results for 3 cultivars, 5, 10 and 20 M BA and 5 M TDZ produced the highest average number of shoots per cultured apex (3.4–3.8). However, shoots produced with TDZ were stunted and did not root well. For rooting of shoots directly in potting mix, a rooting powder pretreatment significantly increased the number of roots per shoot but did not affect percent rooting or root length. For rooting in vitro, 1 M NAA significantly increased all parameters measured. Although more shoots rooted in vitro than in vivo (77% vs. 46%), the latter was judged preferable since acclimatized plants were produced in less time and a major culture step was eliminated. Significant differences among cultivars were noted for measured responses in all experiments.Abbreviations BA benzyladenine - Kin kinetin - MS Murashige & Skoog (medium) - NAA naphthaleneacetic acid - TDZ thidiazuron - WPM woody plant medium     

Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

Plantlet regeneration from cultured leaves of Cydonia oblonga L. (quince)

Summary Adventitious shoots of Cydonia oblonga Quince A were obtained from leaves cultured on MS-N6 medium containing thidiazuron (TDZ) and -naphthaleneacetic acid (NAA). The frequency of regeneration was high (78% of the cultured leaves with 3.2 shoots per regenerating leaf) at 32 M TDZ plus 0.3 M NAA on young leaves obtained from micropropagated shoots. Shoots were rooted by culturing them first on medium containing 5 M NAA for one week and then on auxinfree medium for four weeks. The regeneration protocol may be useful for selection of somaclonal variants with increased tolerance to low Fe and for transformation mediated by Agrobacterium.     

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.     

Adventitious shoot formation from leaf explants of Rhododendron

A protocol for high frequency adventitious shoot regeneration adventitious shoot regeneration from leaf explants of Rhododendron spp. has been developed. The highest percentage of regeneration and the greatest number of shoots were obtained when leaf explants were cultured on Anderson's medium containing 4.9 M IBA and 73.8 M 2iP. Genotypic variation was observed for adventitious shoot regeneration potential among the seven cultivars tested. Regeneration frequencies ranged from 0 to 96%. Lodestar had the highest rate of regeneration after 3 months of culture with 96% shoot regeneration and an average of 14 shoots per explant. Regenerated shoots were rooted in soil in about 2 months. This protocol should be useful in applying gene transfer techniques to Rhododendron improvement.Abbreviations IAA 1-H-indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA 1-H-indole-3-butyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - 2iP N-(3-methyl-2-butenyl)-1-H-purine-6-amine     

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     

Micropropagation for recovery of Cucumis hystrix

A micropropagation protocol that allows for the efficient cloning of C. hystrix was developed using 1 mm shoot-tip explants prepared from plants grown in the greenhouse. Establishment of Stage I cultures was greatest (83%) when shoot tips were cultured in modified MS (Murashige and Skoog, 1962) medium containing (per liter) 30 g sucrose, 0.1 g myo-inositol, and 5 g Agargel plus 1.7 M indole-3-butyric acid (IBA), 0.5 M kinetin and 0.3 M gibberillic acid (GA₃) (IKG). Benzyladenine (BA, 5 M) proved best for Stage II shoot proliferation. Over 35 shoots were obtained per vessel (five shoots per vessel) when explants were cultured in medium containing BA. Less than 10 shoots were obtained per vessel when kinetin (0.5–5 M) and IKG were used. Stage II cultures established from 1 cm shoot tips obtained from Stage I shoots produced more shoots (1.3-fold) than single node explants. Rooting and plantlet acclimatization were best when shoots greater than 1.5 cm were incubated in MS medium containing 0.5 M naphthalene acetic acid (NAA). Higher NAA concentrations stimulated rooting but inhibited shoot elongation and reduced the ability of plantlets to survive acclimatization to ambient conditions. Plantlet height influenced acclimatization. Over 72% of plantlets survived acclimatization if they were at least 4.6 cm when transferred to soilless growing medium.