Different Concentrations Of Thidiazuron Research Articles

Effects of Thidiazuron, basal medium and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba×P. berolinensis

The effect of Thidiazuron (TDZ), basal media and light quality on adventitious shoot regeneration from in vitro cultured stem of Populus alba×P. berolinensis were determined to establish a high efficiency shoot regeneration system from stem explants of P. alba×P. berolinensis. Stems of Populus alba×P. berolinensis were collected from cultured shoots in vitro derived from dormancy buds of 3-year-old seedlings. The stem explants were cultured on MS medium containing 0.02-mg·L−1 NAA (naphthaleneacetic acid), and 0.1, 0.3, 0.5 and 1.0 mg·L−1 concentrations of TDZ to determine the effect of TDZ on shoot regeneration. Three basal media, i.e. MS, woody plant medium (WPM) and B5, were used to test their influences of different media on adventitious shoot regeneration. Green, red, blue and yellow plastic films in comparison with florescent light as control were used to observe their effects on shoot regeneration. The results showed that different concentrations of TDZ had an evident influence on shoot regeneration. Lower concentration of TDZ (0.1 mg·L−1) resulted in more adventitious shoot regeneration and higher concentration of TDZ (>0.1 mg·L−1) inhibited shoot regeneration. Among different media, MS medium exhibited a high efficiency for shoot regeneration, followed by WPM medium, while B5 medium inhibited shoot regeneration. Normal light and yellow light exhibited better effects on shoot regeneration, compared with other light.

Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees

Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.

In Vitro Regeneration of Red Squill Urginea Maritima (L.) Baker. Using Thidiazuron

ABSTRACTThe study reports an efficient in vitro micropropagation of Red squill (Urginea maritima (L.) Baker), an important medicinal plant of Mediterranean region, from single and double scale explants. The explants were cultured on MS medium containing different concentrations of Thidiazuron with initial culturing in darkfor eight weeks followed by transfer to 16 h light photoperiod or culturing them in 16 h light photoperiod only. The results showed that initial growth of explants in dark for eight weeks was inhibitory and produced negligibly low number of bulbs on both explants. Contrarily, 16 h light photoperiod resulted in varying frequency of bulblet regeneration on single and double scale explants. Double scale explants were more potent for bulblet regeneration compared to single scale explants. Single scales produced only axillary bulblets with a frequency of 0.90–1.33 bulblets per expalnt. Contrarily, both adventitious and axillary bulblets were recorded on double scales. These explants produced maximum number of 1.17 adventitious bulblets and 3.58 axillary bulblets per explant on MS medium containing 0.55 mg/l TDZ. The frequency of axillary bulblets regeneration remained higher compared to the frequency of adventitious bulblet regeneration on all concentrations of TDZ. All axillary bulblets rooted on the mother regeneration media. However, the adventitious bulblets could only be rooted on MS medium supplemented with 1 mg/l IBA. All bulblets established in the growth chamber were transferred to the field for further observations and flowering.

TDZ-induced plant regeneration in Astragalus cicer L.

We developed a regeneration protocol using thidiazuron (TDZ) with a high frequency in vitro root induction in Astragalus cicer. High in vitro germination ratio (75%) for hard-seeds of A. cicer was also achieved. For this, hypocotyl and cotyledon explants were cultured on Murashige and Skoog medium supplemented with different concentrations of TDZ. The highest frequency of shoot regeneration (53.3%) was achieved from hypocotyl segments through an initial callus growth stage on MS medium containing 0.25 mg/l TDZ. The shoots were cultured on the different strength (1/1, 3/4, 1/2 and 1/4) of basal Murashige and Skoog medium containing different concentrations of NAA. High rooting (100%) and survival (100%) were achieved using half strength MS medium supplemented with 0.25 and 0.50 mg/l NAA.

In vitro bud regeneration of Carthamus tinctorius and wild Carthamus species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm-3 KIN or 0.5 mg dm-3 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm-3 each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.

Adventitious Shoot Regeneration from In Vitro Leaves of Formosan Sweetgum (Liquidambar formosana L.)

Plantlets were regenerated from in vitro-grown leaf explants of five genotypes of Liquidambar formosana on WPM basal medium supplemented with different concentrations of TDZ and NAA. With the addition of 0.27 μm NAA, regeneration efficiency was increased by 2- to 4-fold over that with TDZ alone. Lower concentrations of TDZ (0.45–2.27 μm) were beneficial for regenerating shoot clusters. Four genotypes (P2, P6, P9, and P11) showed high regeneration rates (up to 90%), whereas genotype P13 showed a low capability for shoot regeneration on all media tested (<35%). For all five genotypes, the optimum medium for inducing adventitious shoots was WPM supplemented with 1.14 μm TDZ and 0.27 μm NAA, on which regeneration rate ranged from 72.6% to 89.5% and adventitious shoot clusters per regenerating leaf explant ranged from 2.63 to 3.11 in four genotypes (P2, P6, P9, and P11), while for P13, the regeneration rate and number of shoot clusters per regenerating explant were 23% and 1.39, respectively. Transfer of shoot clusters to WPM basal medium containing 0.54 μm NAA, 2.22 μm BA, and 1.44 μm GA3, resulted in shoot elongation. All the elongated shoots were rooted on WPM supplemented with 9.84 μm IBA, and plantlets were transplanted to soil successfully. Chemical names used: 6-benzyladenine (BA), gibberellic acid (GA3), indole-3-butyric acid (IBA), 1-naphthalene acetic acid (NAA), plant growth regulator (PGR), thidiazuron (TDZ), woody plant medium (WPM).

(287) Factors Affecting In Vitro Proliferation and Regeneration of Birch Species

In vitro shoot cultures for two birch species, Asian white birch (Betula platyphylla) and paper birch (Betula papyrifera), were initiated from shoot tips of mature trees and maintained in MS (Murashige and Skoog) medium containing 3% sucrose and 5–10 μM (micromolar) benzyladenine (BA). The effect of such factors as genotype, basal medium, and plant growth regulator (PGR) on proliferation was investigated. Shoots were proliferated in both MS and woody plant medium (WPM) supplemented with different concentrations of thidiazuron (TDZ), BA, and kinetin (Kin). Two birch species responded differently to these factors. In general, more shoots were proliferated in WPM than in MS medium. The maximum proliferation rate of Asian white birch was achieved by being cultured in WPM containing 4–8 μM TDZ, while paper birch gave rise to the maximum proliferation rate in WPM supplemented with 20 μM BA. Interactions between genotype and medium or cytokinin were found. Shoots produced on media with TDZ had thick stems and small, dark green leaves. Microshoots can be rooted both in vitro and ex vitro with or without IBA treatment. Plants were regenerated from leaf tissues of Asian white birch. Adventitious shoots regenerated when in vitro leaves were cultured on WPM supplemented with 10–20 μM BA with 2-week dark treatment. The effect of genotype, PGR, and culture condition on in vitro regeneration of birch species is being tested.

Micropropagation of ‘Amethyst’ Purple Raspberry (Rubus occidentalis L. x R. idaeus L. ‘Amethyst’)

Abstract A micropropagation method was developed for a cold hardy purple raspberry cultivar (Rubus occidentalis × R. idaeus ‘Amethyst’). In vitro shoot cultures were initiated from shoot tips of a 30-year old ‘Amethyst’ plant. The effects of basal medium, plant growth regulator, and temperature on shoot proliferation were investigated. Shoots were produced from explants in both Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) media supplemented with different concentrations of thidiazuron (TDZ) and benzyladenine (BA), solely or combined. One micro molar TDZ gave rise to the maximum proliferation rate. Interactions between BA and medium or TDZ were significant. Shoots produced on media with 1.0 μM TDZ had thick stems and small, dark green leaves whether BA was absent or present. Shoots can be rooted both in vitro and ex vitro with or without IBA at 0 to 1.0 μM. However, combination of rooting and shoot multiplication by adding a low level of TDZ to rooting medium produced multi-cane plants resulting in shortening propagation time, increasing survival rate, and lowering the production cost.

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.

‘Fragrant taro’ [Colocasia esculenta (L.) Schott var. antiquorum] micropropagation using thidiazuron and benzylaminopurine

SummaryFragrant taro [Colocasia esculenta (L.) Schott var. antiquorum] is an important regional plant, with a good taste, from Chongming Island in the southeast of China. Its natural vegetative rate of propagation is relatively low and no research on in vitro propagation of ‘fragrant taro’ has been reported.Thus, an in vitro propagation method for ‘fragrant taro’ was developed, and the effects of thidiazuron (TDZ) and benzylaminopurine (BAP) on shoot-tip differentiation and multiplication were analysed. Shoot-tips were first cultured in half-strength Murashige and Skoog (MS) semi-solid medium containing different concentrations of TDZ, BAP and naphthalene acetic acid (NAA) for 30 d.The best result was found with 1.0 mg l–1 TDZ treatment. Plantlets were then sub-cultured in MS liquid medium for another 90 d. After 30 d, the highest multiplication rate (2.5) was observed in medium with 1.0 mg l–1 BAP and 0.5 mg l–1 NAA. The multiplication rate in medium supplemented with 3.0 mg l–1 BAP and 0.1 mg l–1 TDZ was the highest (4.7) after 90 d of culture. Well-developed shoots were rooted in MS medium solidified with 0.6% (w/v) agar, supplemented with 1.0 mg l–1 BAP, 0.5 mg l–1 NAA and 500 mg l–1 active carbon before transplanting. A protocol for propagating ‘fragrant taro’ in vitro was established in which shoots maintained rapid multiplication for a long time. This protocol can also be used for germplasm conservation and exchange of ‘fragrant taro’.

ADVENTITIOUS SHOOT FORMATION ON TEAK (TECTONA GRANDIS L.F.) CALLUS CULTURES DERIVED FROM INTERNODAL SEGMENTS

ADVENTITIOUS SHOOT FORMATION ON TEAK (TECTONA GRANDIS L.F.) CALLUS CULTURES DERIVED FROM INTERNODAL SEGMENTS

(282) In Vitro Indirect Organogenesis of Leucocoryne purpurea, an Ornamental Geophyte Species Endemic to Chile

Callus induction (CI) and later shoot induction (SI) were studied in Leucocoryne purpurea, a native and endemic Chilean geophyte species. Basal leaf portions (BL), bulb basal plate (BP), and root tips (RT) from in vitro plants were used as explants. Treatments for CI included all three explants and media containing different sources and concentrations of auxins and cytokinins as plant growth regulators (PGRs). Plant material was initiated on MS basal medium (Murashige and Skoog, 1962), supplemented with vitamins, 30 g·L-1 sucrose, 6.0 g·L-1 agar and pH adjusted to 5.7 before autoclaving. The experiments were carried on a growth chamber at 24 ± 1.5 °C. CI cultures were maintained in darkness for 16 weeks, and SI for 12 weeks in a 16-hour photoperiod. BL and RT explants did not respond to any of the CI treatments. BP explants cultured on MS basal medium without PGRs also did not produce any callus. The average frequency of callus induction for BP was 78% and the average fresh weight of callus was 10.06 g/explant after 16 weeks of culture. Best treatment for CI was BP cultured on 4.52 μm 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.45 μm 6-benzyladenine (BA), when they were compared to 2,4-D alone or picloram as auxin source. After 16 weeks of culture, calli were transferred to SI medium, supplemented with three different concentrations of thidiazuron (TDZ), either intact or subdivided (150 mg/explant). SI treatments had a greater and significant response when the callus came from a CI medium containing auxin and cytokinin combined, in comparison to those coming from a CI medium containing auxins only.

SOMATIC EMBRYOGENESIS IN RHODODENDRON CATAWBIENSE ‘GRANDIFLORUM’

The genetic stability of regenerants in Rhododendron catawbiense Michx. 'Grandiflorum' and the in vitro regeneration process from leaf explants were determined. Chromosome diploid number (2n=2x=26) in actively growing shoot apices and a high degree of cultivar genetic stability were found. The effect of different concentrations of thidiazuron (TDZ) in the presence of indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D) or 2-isopentenyladenine (2iP) on induction of embryogenic/organogenic mass was compared. Histological evidence demonstrated embryogenic capacity of mucilaginous callus at concentration 11.4-22.7 μM of thidiazuron. Somatic embryo initiation was exclusively induced with TDZ, while IBA, 2,4-D or 2iP alone did not have any effect on formation of the embryogenic structures.

High Frequency Multiple Shoot Regeneration from Decapitated Embryo Axes of Chickpea and Establishment of Plantlets in the Open Environment

Multiple shoot regeneration from the cut plumular ends of embryo axes of chickpea (Cicer arietinum L.) was evaluated on Murashige and Skoog medium having different concentrations of thidiazuron (TDZ) (0.1 to 10.0 mg dm-3) 6-benzylaminopurine (BAP) (0.5 and 1.0 mg dm-3), kinetin (0.5 and 1.0 mg dm-3) or zeatin (2.0 and 4.0 mg dm-3). TDZ (0.2 mg dm-3) was found to be the most effective cytokinin as it produced multiple shoots in 100 % of the explants from genotypes C235, ICC5166, ICC12269, ICC4951, ICC11531, BG256 and a local cultivar. Shoots were elongated on growth regulator-free medium, and rooted on growth regulator-free medium containing 1/4 MS salts + full vitamins + 3 % sucrose. Plantlets formed were acclimatized for 12 - 15 d in MS medium with a gradual reduction in sucrose concentration and transferred into pots filled with soil and kept in the field; this resulted in more than 70 % survival. The plants developed normally and produced fertile flowers and set seeds. Low temperatures, maximum 19.0 °C, and minimum 8.2 °C, during the first 15 d of transfer favoured survival on transfer to pots.

Somatic embryogenesis, secondary somatic embryogenesis, and shoot organogenesis in Rosa

Summary The influence of various 2,4-D concentrations (11.3–181 µmol/L) on induction of callus from leaf tissues of Rosa hybrida cvs. Carefree Beauty and Grand Gala and R. chinensis minima cv. Red Sunblaze was evaluated. Following transfer of callus to a regeneration medium containing different concentrations of thidiazuron (TDZ) (0–90.8 µmol/L), 6-benzyladenine (BA) (0–44.4 µmol/L), or 2.9 µmol/ L gibberellic acid (GA3), alone or in various combinations, the highest frequency of embryogenic (32 %) and organogenic (55.3 %) callus was induced on ‘Carefree Beauty’. Secondary somatic embryos were also induced on somatic embryos of ‘Carefree Beauty’. The effects of different concentrations of TDZ (2.3 µmol/L), BA (2.2 or 4.4 µmol/L), and abscisic acid (ABA) (3.8 or 7.6 µmol/L), alone or in combinations, on proliferation and germination of secondary somatic embryos were also evaluated. ABA was found to be the most effective in promoting proliferation and germination of somatic embryos. The growth rate of secondary embryogenic callus grown on ABA increased by 36fold, while germination rate of somatic embryos was more than five times higher than those derived from embryogenic callus grown on BA and TDZ. For R. chinensis minima cv. Red Sunblaze, only somatic embryogenesis (6.6 %) was observed; while, for R. hybrida cv. Grand Gala, only shoot organogenesis (3.3 %) was observed.

Regeneration of almond from immature seed cotyledons

Adventitious shoots were regenerated from immature cotyledons of four almond cultivars (`Ne Plus Ultra', `Nonpareil', `Carmel' and `Parkinson'). Open-pollinated fruit were collected from orchard-grown trees 100–115 days after full bloom. The proximal ends of the cotyledons were excised and the embryonic axes discarded. The effects of different concentrations of thidiazuron (TDZ) and indole-3-butyric acid (IBA) and the presence or absence of light for the first 7 days of culture were determined. Shoot regeneration rates were highest for cotyledons cultured for 8 weeks on Murashige and Skoog (MS) basal medium containing TDZ (10.0 μM), followed by 4 weeks on medium without plant growth regulators. Regeneration levels were further improved if cotyledons were maintained in darkness for the first 7 days. IBA (0.5 μM) significantly reduced the development of adventitious shoots. The frequency of cotyledons that developed adventitious shoots under the optimum tested conditions for `Ne Plus Ultra', `Nonpareil', `Carmel', and `Parkinson' were 80.0%, 73.3%, 100.0% and 86.7%, respectively.

The regeneration and screening of watercress somaclones for resistance to Spongospora subterranea f. sp. nasturtii and measurement of somaclonal variation

A range of watercress (Rorippa nasturtium-aquaticum) explants (stems, hypocotyls, true-leaves, cotyledons and petioles) were tested for their capacity to regenerate adventitious shoots from callus formed using Murashige and Skoog medium containing different concentrations of thidiazuron and 2,4-dichlorophenoxyacetic acid. The highest shoot regeneration rate was a mean of 18 shoots per responding explant from stem callus formed on medium containing 5 μM thidiazuron and 0.05 μM 2,4-dichlorophenoxyacetic acid. A histological study confirmed that shoots originated directly from callus tissue. Twenty five percent of somaclones exhibited somaclonal variation in leaf shape, plant height, axillary branching or ploidy. The variation in 6% of somaclones was heritable to the first selfed generation. A screening protocol was developed to permit the identification of somaclones with increased resistance to the economically damaging watercress root pathogen, Spongospora subterranea f. sp. nasturtii. Although 883 somaclones were screened using this protocol, no significant increase in disease resistance was detected.

Adventitious shoot regeneration from leaf explants of Gypsophila paniculata L.

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering.

Thidiazuron-induced plant regeneration from protoplasts of Vicia faba cv. Mythos

Protoplasts of 10 cultivars of V. faba were isolated from etiolated shoot-tips and tested for their regeneration capacity. After purification, protoplasts were embedded in sodium alginate and cultivated in the medium of Kao and Michayluk (1975) containing 0.5 mg·1(-1) of each 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzylaminopurine. Depending on cultivar, division frequencies of up to 40% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred to Gelrite-solidified media with different combinations of growth regulators. A two step protocol (auxin high/low) was tested for its ability to induce somatic embryogenesis. The formation of globular structures was observed, but no embryo formation could be achieved. In contrast, cultivation of protocalluses on medium supplemented with thidiazuron resulted in shoot development in cultivar Mythos. To generate mature plants, the shoots were grafted onto young seedlings. In order to optimize the in vitro-conditions, different concentrations of thidiazuron alone or in combination with naphthylacetic acid were tested, showing that an increase of thidiazuron and the addition of naphthylacetic acid positively affects both the viability of protocalluses and the regeneration frequency.

MONOTERPENOID CONSTITUENTS OF ROSEMARY SHOOT TIPS CULTURED IN VITRO

Shoot tips (7 to 10 mm long) of rosemary plant (Rosmarinus officinalis L. 'Lockwood de forest') were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) (0, 0.5, 1.0, 1.5, and 2 mg/l) alone or with 3-indole acetic acid (IAA) at 0.5 mg/l. The effect of TDZ and IAA on the proliferation of rosemary shoot tips has been reported in a previous meeting. Here, we report on the effect of TDZ and IAA on the monoterpene constituents identified in the oil of rosemary plants propagated in vitro. The proliferated explants were soaked in hexane as a solvent, then the extractions were used for monoterpene analysis using GC/MS. A significant interaction of TDZ by IAA was found on most of the oil components identified. The highest levels of 1,8-cineole and borneol were obtained at 0.5 mg TDZ/l alone, while the highest level of camphor was obtained at 0.5 mg TDZ/l plus 0.5 mg IAA/l. The highest level of bornyl acetate was at 2 mg TDZ/l.