In vitro culture methods in sorghum with shoot tip as the explant material

Twenty-four diverse genotypes of sorghum were evaluated for response to callus induction and plant regeneration with two media viz., MS and NBKNB using shoot tips as the start material to identify a model genotype. None of the genotypes tested showed promising results. Therefore, alternative methods of in vitro pathways using shoot meristem isolated from shoot tips were explored. Shoot apical meristems were isolated and were induced to multiple shoots or multiple shoot buds pathway by manipulation of thidiazuron (TDZ), 6-benzyl adenine (BAP) and 2, 4-dichlorophenoxy acetic acid (2, 4-D). Choice of the pathway whether large-scale multiplication of shoots or production of target tissues for transformation can be exercised based on the needs and applications. A simple procedure, for large scale handling of shoot tips is described in detail. Electron microscopic studies revealed that meristems isolated from 7-day-old seedlings are superior because of possessing greater number of transformation competent cells.     

TDZ高效诱导蝴蝶兰叶片不定芽及植株再生

以蝴蝶兰（Phalaenopsis）无菌幼苗叶片为材料,研究添加TDZ（噻重氮苯基脲）条件下不同基因型、激素组合、叶片大小、暗培养时间对不定芽发生和再生植株的影响。结果表明：在相同培养条件下,不同基因型外植体芽诱导率差异显著,‘红天使’最高,达81.5%,‘汕农姑娘’等2个品种为0,‘满天红’等4个品种为9.2%～34.9%;添加TDZ芽诱导率显著高于6-BA;单独添加TDZ或6-BA芽诱导率显著高于NAA与TDZ或6-BA的组合。叶片越小不定芽诱导率越高;短时间暗处理有利于不定芽的发生。以1～2 cm长叶片为材料、15 d暗处理、在1/2 MS添加3 mg/L TDZ培养基中,‘红天使’的叶片外植体芽诱导率和平均不定芽数分别可达100.0%和18.2个。研究发现,在继代培养中TDZ对芽的伸长有抑制作用。     

TDZ研究进展(综述)

TDZ在组培中应用广泛,植物中许多生理生化现象受TDZ影响。近年来研究初步表明,TDZ是通过调节内源激素起作用,或是诱导胁迫的结果。本文综述TDZ作用机理的研究进展。     

Molecular fate of thidiazuron and its effects on auxin transport in hypocotyls tissues of Pelargonium × hortorum Bailey

Thidiazuron, a synthetic phenylurea-type cytokinin, has previously beenfound to induce somatic embryogenesis and organogenesis in a wide range ofplantspecies and to modulate the metabolism of endogenous auxins and cytokinins. Inspite of these findings, the precise mode of action of TDZ remainsundetermined.The current studies were undertaken to determine the fate of the TDZ moleculeand the effects of TDZ exposure on auxin transport in plants. The fate of tworadiolabelled versions of thidiazuron, [¹⁴C-5-thidiazol]-TDZ and[¹⁴C-U-phenyl]-TDZ, was investigated in sterile hypocotyl culturesofgeranium (Pelargonium×hortorumBailey). Radiolabelled TDZ was recovered from the tissue explants inethanol-insoluble, ethanol-soluble and chloroform fractions as well as inacidic, basic and neutral eluants from Dowex resins. Hypocotyl sections thathadbeen exposed to TDZ were found to accumulate more ¹⁴C-IAA from theculture medium and to translocate the auxin over a greater distance within thetissues. These data provide the first evidence that the TDZ molecule remainsintact in both a free and conjugated form within the plant tissues and providesome indication that TDZ-exposure enhances the accumulation and translocationofauxin within the tissues.     

Node-derived cultures with high-morphogenic competence in barley and wheat

A successful regeneration system is presented for elite cultivars in barley and wheat based on nodes. Nodal explants were excised from in vitro and ex vitro grown plants. A combination of 8.28 μM 4-amino-3,5,6-trichloropicolinic acid and 4.54 μM to 22.71 μm thidiazuron (TDZ) used in MS-based medium containing 60 g l⁻¹ maltose favoured induction of clumps of multiple shoots/buds with or without callus formation in the primary cultures. Within 8–10 weeks upon further subcultures, the proliferation into callus with rapid and continuously forming adventitious buds containing clusters of meristemoids, termed meristematic bulk tissue (MBT) was obtained. Lowering the levels of growth regulators resulted in redifferentiation of shoots, which elongated, rooted, developed into morphologically normal plants and set seeds normally. With a frequency ranging between 37 and 82% the nodes raised from in vitro grown plants were proliferated into MBT independent of TDZ concentration, cultivar and species. The average number of shoots per responding node in different cultivars was 7–15 in barley and 1–6 in wheat after 12–14 weeks. Nodes from greenhouse grown plants mainly responded for callus formation with poor development of MBT.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

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

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

Thidiazuron-induced morphogenetic response in petiole cultures of Pelargonium × hortorum and Pelargonium × domesticum and its histological analysis

The possibility of inducing somatic embryogenesis in petiole cultures of two cultivars of Pelargonium × hortorum and of one cultivar of Pelargonium × domesticum using thidiazuron (TDZ) was investigated. Petioles were cultivated on a modified Murashige and Skoog medium with different concentrations and application periods of TDZ. Regeneration was achieved with all TDZ treatments for all cultivars and was highly variable. Shoots of different shapes and somatic embryo-like structures were observed. Histological examination revealed that no somatic embryos were formed, and regenerants had to be classified as shoots and shoot-like or leaf-like structures. The importance of these results on the classification of regeneration induced by TDZ in these species and on the propagation of these pelargoniums is discussed.     

Direct shoot regeneration from foliar explants of an epiphytic orchid,Acampe praemorsa (Roxb.) Blatter and McCann

An efficient and reproducible procedure is described for the large-scale propagation of an epiphytic orchid,Acampe praemorsa (Roxb.) B latter and McCann using foliar explants. Shoot buds were induced in basal parts of foliar explants on Murashige and Skoog medium supplemented with N⁶-benzyladenine (BA), kinetin (Kn) or thidiazuron (TDZ), the latter being most effective at 1.0 mg/1. Shoots formed to a TDZ-containing medium elongated following transfer to a substrate supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA) and 0.5 mg/1 BA. NAA at lower concentrations had no beneficial effects on shoot regeneration, whether added to the medium along with BA, Kn or TDZ. However, it promoted shoot elongation and leaf expansion. Higher concentrations of NAA suppressed shoot regeneration. The frequency of shoot regeneration was greatly influenced by the developmental stage and orientation of the leaf. Shoots regenerated from the foliar explants were rooted successfully on MS medium containing 1.0 mg/l indole-3-butyric acid. The plantlets were acclimated and eventually transferred to a garden.Abbreviations BA N⁶-Benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid