Combination Of Thidiazuron Research Articles

Thidiazuron‐Induced Tissue Culture Regeneration from Quartered‐Seed Explants of Arachis Paraguariensis

ABSTRACTArachis paraguariensis Chodat & Hassl. is a potential source of novel genes for the genetic improvement of cultivated peanut (Arachis hypogaea L.) because some of its accessions show high levels of resistance to early leaf spot caused by Cercospora arachidicola Hori. In this study, induction of high frequency shoot regeneration from quartered‐seed explants was accomplished for six plant introductions of A. paraguariensis under continuous light on Murashige and Skoog (MS) medium containing 4.4 mg L−1 thidiazuron (TDZ) in combination with 2.2 mg L−1 6‐γ‐γ‐(dimethylallylamino)‐purine (2ip). Recovery of a moderately high number of plantlets per quarter seed cultured was also achieved on medium containing 4.4 mg L−1 thidiazuron in combination with 1.1 to 4.4 mg L−1 6‐benzylaminopurine (BAP) with bud formation occurring as early as 1 wk after culture initiation. There were no differences in seed production or in early leaf spot incidence between plants of two genotypes of A. paraguariensis derived from seeds vs. in vitro tissue culture derived plants; however, cultivated peanut cv. Florunner had a higher incidence of early leaf spot.

Somatic embryogenesis of mangosteen

Young red leaf segments from 1 to 3 year-old seedlings of mangosteen (Garcinia mangostana L.) were incubated on different media, consisting of either B5 or Murashige and Skoog (MS) basal media and supplemented with several plant growth regulator at different levels, including 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin or 2,4-D and 6-benzyladenine (BA) or thidiazuron (TDZ) and BA. The highest percentage of globular structures (36.0 %) was obtained using MS medium with a combination of TDZ and BA, each at a concentration of 0.7 mg l−1. Histological observation of sections of globular structures showed accumulation of dense meristematic cells, a common characteristic of globular formation during somatic embryogenesis. Molecular analysis detected the gene somatic embryogenesis receptor-like kinase 1 (SERK1). This gene sequence showed a high similarity (73.0–87.0 %) to previously reported plant SERK1.

Plant regeneration from mature embryo of commercial Indian bread wheat (Triticum aestivum L.) cultivars.

A simple, efficient, reproducible and comparatively genotype-independent in vitro plant regeneration protocol was developed for ten commercial Indian bread wheat cultivars using mature embryos as the explants. Three different auxins and five different combinations of growth regulators in a modified Murashige and Skoog's basal medium were assessed for their effect on callus induction and plant regeneration, respectively, in a high yielding and widely grown cultivar, PBW-343. The optimized conditions were further evaluated with nine other commercial cultivars. A simple novel approach of physical isolation of regenerable calli from non regenerable structures during the early callus phase was used to improve plant regeneration. Callus induced on 2.0 mg(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) showed a regeneration frequency of 86% with 7.5 shoots per explants on hormone-free medium. A considerable improvement in the regeneration frequency (up to 97%) and the average of shoots (19 shoots per explants) was obtained with a combination of thidiazuron (TDZ) and 2,4-D.

English

In vitro culture response was assessed in tomato ( Lycopersicon esculentum Mill. c. v. Omdurman) for optimum callus induction and plantlet regeneration. Callus induction was achieved within seven to ten days directly on the cut surfaces of both hypocotyls and cotyledon explants cultured on Murashige and Skoog (MS) basal medium supplemented with α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), Thidiazuron (TDZ) and benzyl adenine (BA) alone or in different combinations, but not in hormone free-medium. The highest callusing index (5.3) was obtained on hypocotyls explants cultured on MS medium supplemented with NAA at 0.5 mg/l followed by an index of 5.2 obtained from the same explant by using 0.1 mg/l NAA in combination with BAP at 0.5 mg/l. However, for the cotyledon explants, the highest callusing index (4.7) was obtained on MS medium supplemented with NAA at either 2.0 or 3.0 mg/l. After 8 weeks of culture, organogenesis was observed only on the explants cultured on medium containing different concentrations of TDZ alone or in combination with BAP. The best shoot formation (93%) was obtained for cotyledon explant callus induced on MS medium containing TDZ in combination with BAP both at 0.5 mg/l. The highest number (6) of shoot per explant was obtained when cotyledon explant callus was sub cultured on MS medium supplemented with 3.0 mg/l TDZ. Plain half strength of MS was found to be the best rooting medium, however, addition of IAA at 1.0 mg/l and IBA at 2.0 mg/l were found essential to induce highest number of roots (22.1 ± 0.9) and longer roots (11.0 ± 0.3 cm), respectively. This protocol would be useful to create somaclonal variation and utilize transgenic approaches for varietal improvement of tomato.

Enhancement of centelloside production from cultured plants of Centella asiatica by combination of thidiazuron and methyl jasmonate

In order to produce centellosides from whole plant cultures of Centella asiatica (L.) Urban, we evaluated the synergistic effects of thidiazuron (TDZ) and methyl jasmonate (MJ) on whole plant growth and centelloside production. After 4 weeks of treatment with 0.025 mg/L of TDZ coupled with 0.1 mM MJ, the production of madecassoside and asiaticoside from whole plant cultures was estimated to be 2.40- and 2.44-fold, respectively, above that of MJ elicitation alone. When whole plants were treated with a growth regulator and an elicitor, the growth of whole plants, as compared to the controls, did not differ. Additionally, total phytosyterol content in the leaves of whole plants co-treated with MJ and TDZ was 1.08-fold greater than those of MJ alone. These results demonstrate that combined treatments not only stimulate the accumulation of centellosides in the leaves but also inhibit the reduction of phytosterol levels caused by MJ elicitation.

Effect of hydrogen cyanamide, mineral oil and thidiazuron in combination with tip pruning on bud break, shoot growth and yield in ‘Bourjasotte Noire’, ‘Col de Damme Noire’ and ‘Noire de Caromb’ figs

Commercial fig production is relatively new to the Mediterranean-type climate Western Cape Province of South Africa. A lack of lateral branch development impedes tree structure development and therefore adequate yields of quality fruit. The chemical rest breaking agents, Lift ® (thidiazuron 3 g L −1) at 6%, Dormex ® (hydrogen cyanamide, 520 g L −1) at 4%, mineral oil at 4% and a combination of mineral oil and Dormex ® at 2% each were evaluated in a split plot design in combination with tip-pruning vs. no-pruning to overcome apical dominance and increase complexity (Experiment 1). During the 2008/2009 season, an additional investigation was conducted to evaluate the use of thidiazuron and hydrogen cyanamide for harvest scheduling (Experiment 2). Dormex ® at 3% and Lift ® at 6%, were applied to dormant trees on 30 June 2008, 3 August 2008, 15 August 2008 or 30 August 2008. In general, Lift ® can be used to increase the number of buds breaking in ‘Bourjasotte Noire’ and ‘Col de Damme Noire’, while tip pruning decreased bud break with a resultant increase in shoot length. Dormex ® and oil combined decreased bud break in these two cultivars but was effective on ‘Noire de Caromb’. Where bud break was increased the resultant N + 1 shoot length was decreased. RBAs increased the number of fruit in both the breba and main crop of ‘Noire de Caromb’, but decreased fruit size of the breba crop. Our data are not conclusive as to what the reason for low lateral bud break might be but leans towards strong AD in seasons with 400 Utah CU or more, but might include some delayed foliation response in seasons with less than 200 Utah CH.

In Vitro Shoot Regeneration and Polyploid Induction of Rhododendron ‘Fragrantissimum Improved’

Rhododendron L.‘Fragrantissimum Improved’ is an attractive cultivar with showy, fragrant flowers but has limited potential for breeding because it is a sterile wide hybrid. Protocols for in vitro regeneration and polyploid induction were developed for this cultivar as a means to potentially restore fertility and enhance ornamental traits. Combinations of thidiazuron (TDZ) at 0, 5, 10, 15, or 20 μM and 1-naphthaleneacetic acid (NAA) at 0, 2.5, 5, or 10 μM were used to induce shoot regeneration from leaves. Shoot regeneration was optimized (68% of leaf segments produced shoots) using 8.8 μM TDZ and 10 μM NAA. To induce polyploidy, regenerative callus was treated with 7.5, 15, 30, 60, or 90 μM of the mitotic inhibitor oryzalin for 1, 3, 5, 7, or 14 d in various combinations. Oryzalin significantly affected survival and shoot regenerative capacity. A percentage of homogenous, tetraploid shoots was recovered from treatments of 30 μM oryzalin for 1 (13%) or 3 (13%) days and 7.5 μM oryzalin for 7 (20%) or 14 (7%) days.

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l −1 BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l −1 BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.

In vitro adventitious shoot regeneration of the medicinal plant meadowsweet (Filipendula ulmaria (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA3), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA3. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.

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.

TDZ x IBA induced shoot regeneration from cotyledonary leaves and in vitro multiplication in safflower ( Carthamus tinctorius L.)

A high frequency adventitious shoot regeneration protocol for safflower (Carthamus tinctorius L.) cv. Dincer with high yield (Turkish cultivar) using cotyledonary leaves of 10 day-old seedlings were optimized by studying the influence of different combinations of thidiazuron (TDZ) and indole-3-butyric acid (IBA). Cotyledonary leaves were cultured on Murashige and Skoog medium (MS) supplemented with different concentrations and combinations of TDZ and IBA. The highest percentage of regenerated shoots (33.33%) and the highest number of shoots per explant (6.5) occurred on a MS medium containing 0.5 mg/l TDZ and 0.25 mg/l IBA. Furthermore, cotyledonary nodes and meristem tips of 14 day-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzylamino-purine (BAP) alone or combination of -naphthalene acetic acid (NAA). Direct multiple shoots from cotyledonary nodes and meristem tips developed within 18 - 21 days in most media tested. 100% shoot multiplication was achieved from cotyledonary node and meristem tip on a range of MS media supplemented with different concentrations of BAP and NAA.

Adventitious shoot regeneration from the leaves of some pear varieties (Pyrus spp.) grown in vitro

The pear (Pyrus spp.) is one of the most important temperate fruit crops. A complete protocol for adventitious shoot regeneration was developed from the leaves of four pear varieties grown in vitro: Abbe Fetel, Yali, Packham’s Triumph and Aikansui, and the Chinese rootstock variety Duli. Shoot explants were collected from the field and cultured in vitro in Murashige and Skoog (MS) media supplemented with 1.0 mg·L−1 6-benzylaminopurine (BA) and 0.1 mg·L−1 indole-3-butyric acid (IBA). After four weeks, leaf explants of all 5 varieties grown in vitro were excised and cultured in MS media supplemented with 0.0 mg·L−1, 0.2mg·L−1, 0.5 mg·L−1, 1.0 mg·L−1 and 2.0 mg·L−1 naphthaleneacetic acid (NAA) and 5.0 mg·L−1 BA or with 1.0 mg·L−1, 2.0 mg·L−1 and 4.0 mg·L−1 thidiazuron (TDZ). The cultures were maintained in darkness for 21 days for shoot induction in the shoot induction medium (IM), then transferred to the shoot expression medium (EM) in 1.0 mg·L−1 TDZ without any auxins and kept in a growth room at (25±2)°C under a 16/8 h light/dark photoperiod regime for 8 weeks. Finally, the shoots were transferred to the MS shoot elongation medium (SEM) supplemented with 0.2 mg·L−1 BA, 0.1 mg·L−1 IBA and 0.2 mg·L−1 gibberellic acid (GA3). A combination of TDZ and NAA had a significant effect on the number of shoot regenerations in all 5 tested varieties. The maximum mean number of shoots and maximum number of shoots per leaf obtained from Yali variety were 11.8 (P⩽0.001) and 22, followed by Aikansui with 6.6 (P⩽0.001) and 4.6, and Duli with 8 (P⩽0.001) and 12, all arising from the combination of 0.2 mg·L−1 NAA with 1.0 mg·L−1 TDZ. For Packham’s Triumph and Abbe Fetel, the maximum mean number of shoots and maximum number of shoots per leaf were 5.6 (P⩽0.001), 4.8 and 8 (P⩽0.001), and 11, respectively, from the combination of NAA (1.0 mg·L−1) and TDZ (2.0 mg·L−1). Abbe Fetel was the only variety which produced significantly higher adventitious shoots from the two different combinations of 1.0 mg·L−1 NAA and 5.0 mg·L−1 BA (P⩽0.05), and 2.0 mg·L−1 NAA and 5.0 mg·L−1 BA (P⩽0.01). Some of the most prominent problems associated with shoot proliferation and regeneration were also observed and discussed in this paper.

Hormone-regulated inflorescence induction and TFL1 expression in Arabidopsis callus in vitro

To study hormone-regulated inflorescence development, we established the in vitro regeneration system of Arabidopsis inflorescences in the presence of cytokinin and auxin. Media containing a combination of thidiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) were used to induce callus formation. Higher frequencies of calli were obtained by using the inflorescence stems as explants. After transferring the calli to media containing a combination of zeatin and indole-3-acetic acid (IAA), the inflorescences were induced from the calli. The morphology of regenerated inflorescences was similar to that of inflorescences in plants; however, flowers of regenerated inflorescences often lacked a few floral organs. Furthermore, TFL1, a gene involved in floral transition in Arabidopsis, was activated during the inflorescence induction. Our results suggest that the TFL1 gene plays an important role in hormone-regulated inflorescence formation.

Micropropagation with a novel pattern of adventitious rooting in Formosan sweetgum

Cultures of the Formosan sweetgum (Liquidambar formosana Hance) were established from axillary buds of intact plants and from petiole segments of in vitro grown leaves. Petiole segments were generally slower than axillary buds to form multiple shoot cultures. Thidiazuron in combination with 6-benzylaminopurine produced on average up to 9.4 shoots per explant. Shoot elongation was significantly affected by the concentration of 6-benzylaminopurine, regardless of auxin addition to the medium. Adventitious rooting occurred up to 90%. An unexpected pattern of adventitious rhizogenesis was observed from the aerial parts of microshoots not in contact with the rooting medium, including leaf blades and petiole axils. In vitro adventitious root formation from the leaf blade was associated with parenchyma cells surrounding the main vein. The regenerated plants established ex vitro did not show any detectable morphological variation and no aerial roots were formed.

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

Plant Regeneration from Protocorm-derived Callus of Five Paphiopedilum Hybrids

Callus was induced from protocorms of five Paphiopedilum hybrids (Paph. 03-1, Paph. 03-4, Paph. 03-5, Paph. 03-6, and Paph. 03-7) on callus induction medium [MS inorganics (412.5 mg NH4NO3 instead of 1650 mg and 475 mg KNO3 instead of 1900 mg) and vitamins plus (per liter) 0.1 g myo-inositol, 30 g sucrose, and 2.5 g Gelrite; pH 5.5] containing various concentrations and combinations of thidiazuron (TDZ; 4.5 and 45 μm) and 2,4-D (4.5 and 45 μm). Callus formation was greatest for protocorms of Paph. 03-1, Paph. 03-4, Paph. 03-6, and Paph. 03-7. Among the most competent hybrids, callus formation was greatest among protocorms induced in medium containing 4.5 μm 2,4-D and 4.5 to 45 μm TDZ. Induced calli were transferred to 100 × 15 mm petri dishes containing 25 mL of PLB and plant regeneration medium (similar to callus induction medium) containing various concentrations of either benzyladenine (BA; 0.5, 5, or 10 μm), TDZ (0.25, 2.5, or 5 μm) or no growth regulator (control). PLB and plant formation was greatest on medium containing BA.

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. "Schneiders", "Sweetheart", "Starking Hardy Giant", "Kordia" and "Regina" (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.

In vitro Culture and Temporary Immersion Bioreactor Production of Crescentia cujete

Crescentia cujete L. is a widely distributed medicinal tree with a diverse range of phytochemicals used as medicinal compounds. Seedlings of wild-harvested C. cujete were established in vitro and used as the starting material for the establishment of axenic cultures. Shoots were proliferated from nodal segments and were maintained over a period of more than 2 years by sequential subculture on a medium containing 1.0 µmol l−1 kinetin. De novo regeneration was induced on petiole sections cultured onto a medium containing thidiazuron in combination with 2,4-dichlorophenoxyacetic acid. Axenic cultures were also used to test the efficiency of three different cultivation systems for production of biomass of C. cujete. Growth of plantlets in a temporary immersion bioreactor resulted in significant increases in biomass, leaf number, shoot height and transplant efficiency. Plantlets grown in the bioreactors were acclimatized under greenhouse conditions. Together, these experiments have established optimized parameters for propagation and growth of C. cujete plantlets in a sterile controlled environment for biochemical characterization and production of high-quality medicinal products.

Changes in Abscisic Acid During Leaf Yellowing of Cut Stock Flowers

Leaf pigments, such as chlorophyll and carotenoids, are essential plant molecules. They provide carbohydrates and energy during all plant life. Leaf pigments are also important parameters of decorative plants, such as floriculture items, cut foliage and flowers. Leaf yellowing is a form of senescence caused by an internal hormone imbalance, such as a lack of cytokinins. The aim of this study was to investigate the changes in total carotenoids and endogenous ABA in cut flower stock leaves during post-harvest life. The effect of pulse treatment with 5 or 10 μM thidiazuron (TDZ), 150 mg l−1 8-hydroxyquinoline sulphate (8-HQS) and combinations of TDZ with 8-HQS on total carotenoids and ABA concentration was also investigated. Results showed that total carotenoids drastically decreased from 1548 μg cm−2, until reaching 565 μg cm−2 at the end of vase life. Endogenous ABA strongly increased at the same time, going from 167 ng g−1 DW at the beginning of the experiment to 1322 ng g−1 DW at the end of vase life. The TDZ inhibited carotenoid degradation, but did not affect the ABA concentration, while the 8-HQS did not prevent carotenoid degradation and the ABA concentration was only slightly affected. ABA seems to be a secondary senescence bio-product that may have a synergic effect with other senescence inducers dramatically accelerating leaf senescence.

Micropropagation of mature wych elm (Ulmus glabra Huds.).

Explants of mature vigorous donor trees of wych elm ( Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.