Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

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]     

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     

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 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     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 M thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 M -naphthaleneacetic acid or 1.0–2.0 M indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.Abbreviations BAP 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum

Somatic embryogenesis was achieved directly from pseudo-bulblettransverse thin cell layers (tTCL) of Lilium longiflorum.Embryo-like structures (globular embryos) were obtained from different sizeexplants of pseudo-bulblet tTCLs after 45 days culture on Murashige and Skoog,1962 (MS) medium containing 5.4 M naphthalene acetic acid(NAA)and 1.1 M thidiazuron (TDZ). The embryo-like structures werethen isolated and mass proliferated on MS medium, containing 5.4M NAA and 0.4 M TDZ, every 45 days. A0.8–1.0 mm thick explant was shown to be optimal forobtaining the highest number of embryo-like structures. For plant regenerationthese structures were transferred to hormone-free MS medium with 30g/l sucrose. All of these structures formedplantlets after 90 days culture.     

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.     

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.     

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     

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 M thidiazuron (TDZ) or 40 M N⁶-benzylaminopurine. The highest efficiency of induction was achieved with 5 or 10 M 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.Abbreviations VWC medium Vacin and Went medium with 15% (by volume) coconut water - TDZ thidiazuron - BAP N⁶-benzylaminopurine - Plbs protocorm-like bodies     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

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     

Growth and morphogenesis of immature embryos of sweet potato (Ipomoea batatas L.) in vitro

Sweet potato (Ipomoea batatas L.) embryos excised from the fertilized ovules of 6- to 12-day-old capsules were cultured on MS medium supplemented with NAA, BA, GA separately and in combinations. GA was found essential for initial morphogenesis of globular and heart stages. Seedlings were recovered from late globular stage onwards but recovery was best from advanced embryo stages. Differentiated embryos produced multiple shoots on MS medium +1M NAA÷2M BA +0.5M GA.     

Adventitious shoot regeneration from in vitro-cultured leaves of Rubus genotypes

Regeneration of adventitious shoots from leaves of several in vitro-cultured Rubus genotypes was affected by media components and incubation conditions. Leaves cultured at 20°C with a photosynthetic photon flux (PPF) of 40 mol m^-2 s^-1 had a higher regeneration frequency and more shoots per regenerating leaf than ones cultured at 25°C with a PPF of either 40 or 80 mol m^-2 s^-1. Thidiazuron (TDZ) was significantly more effective than benzyladenine. Medium containing 1 M TDZ had the highest percentage regeneration for leaves of Autumn Bliss, Canby, Summit and Sentry red raspberries, whereas leaves of MD-ETCE-1 blackberry hybrid responded more to 10 M TDZ. Higher regeneration frequencies were obtained with 0.5 M indolebutyric acid (IBA) than with 1 M, but no significant difference was observed between 0.5 M and no IBA in other experiments. More shoots per regenerating leaf formed on Murashige and Skoog (MS) and N6 media, than on half-strength MS, Anderson, or Woody Plant media for all genotypes tested. The youngest two expanding leaves near the shoot apex were the most regenerative and yielded the highest number of shoots per regenerating leaf.Abbreviations AND Anderson (1980) - BA benzyladenine - IBA indolebutyric acid - MS Murashige & Skoog (1962) - N6 Chu et al. (1975) - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron (N-phenyl-N'-1, 2, 3-thiadiazol-5-ylurea) - WPM Woody Plant Medium [Lloyd & McCown (1980)]     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron     

Effect of thidiazuron on somatic embryogenesis of Cayratia japonica

Unpollinated ovary explants of Cayratia japonica (Thump.) Gagnep, were cultured on the revised Murashige & Skoog's medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) alone, or in combination with 0.009 M thidiazuron (TDZ) or 0.23 M kinetin for induction of embryogenic callus. The best results were obtained on medium containing 2.3 – 4.6 M 2,4-d and TDZ. When the calluses were subcultured on the basal medium (BM), somatic embryogenesis took place spontaneously at surfaces of the calluses, but only about 5% of the somatic embryos could develop to cotyledonary stage and most of the rest remained at the globular stage of development. If the calluses were transferred onto medium containing TDZ or TDZ combined with 0.27 M -napthaleneacetic acid, the number of cotyledonary somatic embryos increased up to 25%. When the somatic embryos of different stages were transferred onto fresh BM, only the cotyledonary embryos could convert into the plantlets. The results revealed that for the induction of embryogenic callus and somatic embryogenesis of Cayratia japonica, both cytokinin and auxin are required in the medium and the cytokinin activity of TDZ is much stronger than that of kinetin even when the concentration of TDZ used was only 4% of kinetin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea)