TDZ：一种有效的植物生长调节剂

人工合成的苯基脲衍生物TDZ（N-苯基-N′-1,2,3-噻二唑-5-脲）是已被广泛用于植 物组织培养形态发生的高效生物调节剂。它能诱导外植体从愈伤组织形成到体细胞胚胎发生 的一系列不同反应,具有生长素和细胞分裂素双重作用的特殊功能。近年来通过研究TDZ启 动的形态发生事件,人们正逐渐揭示出其内在作用机理。许多研究报告指出TDZ通过调节内 源植物生长激素起作用,或者是诱导逆境产生起间接作用。它还能调节细胞膜结构、能量水 平、营养吸收和同化作用。本文将探讨TDZ几种可能的作用机理,并概述近年来有关TDZ诱导的植物离体形态发生效应研究进展。     

Effects of chemical substances on the rapid cultivation of moss crusts in a phytotron from the Loess Plateau,China

In this study, typical moss crusts, which were dominated by the species Didymodon vinealis (Brid.) Zand., were collected from the Loess Plateau and a 65-day cultivation experiment was performed to study the effects of five kinds of nutrient solutions (Knop, Murashige-Skoog (MS), Benecke, Part and Hoagland), two kinds of carbohydrates (glucose and sucrose) and three kinds of plant growth regulators (thidiazuron (TDZ), 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA)) on the coverage, plant density, and plant height of moss crusts. The main conclusions are as follows. (1) All Knop, MS, Benecke, Part and Hoagland nutrient solutions improved the coverage and plant density of moss crusts to different degrees and the promotional effects of the Hoagland nutrient solution were most significant. (2) Glucose and sucrose could promote the formation of moss crusts, but they inhibited the development of moss crusts at concentrations greater than 10?g/L. (3) With an increase in concentration, the effects of TDZ on the development of moss crusts changed from “enhanced” to “inhibited”. Regardless of whether the concentration was high or low, 6BA had no significant effects on the growth of moss crusts, and NAA reduced the development of moss crusts. Results suggest that nutrient solutions (e.g. Hoagland), low concentration carbohydrates solutions, and some plant growth regulators (e.g. 1?mg/L TDZ) enhance the development of moss crusts in Loess Plateau under the appropriate environmental conditions.     

Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants

Summary An efficient regeneration system was developed by culturing immature cotyledons and embryo axes of Arachis hypogaea L. cv. Georgia Green on Murashige and Skoog basal medium (MS) supplemented with various concentrations of thidiazuron (TDZ; 1, 5, 10, and 15 μM). Highly morphogenic callus was produced from 100% of the explants comprising the cotyledon with attached embryo axis when cultured in the dark on 10 μM TDZ. Upon excision and continued culture in the dark on 10 μM TDZ, morphogenic callus grew repetitively during monthly subcultures and retained its regeneration potential. For organogenesis, a gradual reduction in TDZ concentration and exposure to light were necessary before transfer to MS basal medium. Inclusion of indole-3-butyric acid in liquid MS medium favored rooting of recovered shoots. A distinct feature of this investigation is the induction of highly morphogenic callus by TDZ and regeneration of morphologically normal, fertile peanut plants after 8 months of callus subculture.     

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。     

Endogenous Hormonal Levels in <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> Calli Induced by Thidiazuron

Callusing was induced from three kinds of explants of Chinese medical plant Scutellaria baicalensis with thidiazuron (TDZ) as an exogenous growth regulator. Multiple shoots were formed from these calli on hormone-free media. Calli induced by 0.3 mg/l TDZ produced shoots directly on the induction medium after culturing for 30–40 days without any interval, while at a higher concentrations of TDZ (1.0, 2.0, and 5.0 mg/l), regeneration occurred only after transfer to hormone-free medium. Endogenous hormones were detected by reverse-phase HPLC-MS and ELISA. The higher TDZ concentrations decreased endogenous benzyladenine (BA) and IAA levels, while iPA and ZR levels and their ratios to IAA were not correlated with TDZ concentrations, growth of calli, or plant regeneration. BA was the dominant cytokinin during calli growth and bud formation, 10 000-fold more abundant than iPA and ZR. Moreover, low IAA/BA ratio, rather than low IAA or high cytokinin levels alone, is the most important factor for shoot formation by calli. TDZ might act indirectly by changing endogenous hormonal levels, most likely the IAA/BA ratio which controls the calli growth and bud formation.__________From Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 392–398.Original English Text Copyright © 2005 by Zhang, Li, Mao, Zhao, Dong, Guo.This article was submitted by the authors in English.     

Pre-procambial cells are niches for pluripotent and totipotent stem-like cells for organogenesis and somatic embryogenesis in the peach palm: a histological study

The direct induction of adventitious buds and somatic embryos from explants is a morphogenetic process that is under the influence of exogenous plant growth regulators and its interactions with endogenous phytohormones. We performed an in vitro histological analysis in peach palm (Bactris gasipaes Kunth) shoot apexes and determined that the positioning of competent cells and their interaction with neighboring cells, under the influence of combinations of exogenously applied growth regulators (NAA/BAP and NAA/TDZ), allows the pre-procambial cells (PPCs) to act in different morphogenic pathways to establish niche competent cells. It is likely that there has been a habituation phenomenon during the regeneration and development of the microplants. This includes promoting the tillering of primary or secondary buds due to culturing in the absence of NAA/BAP or NAA/TDZ after a period in the presence of these growth regulators. Histological analyses determined that the adventitious roots were derived from the dedifferentiation of the parenchymal cells located in the basal region of the adventitious buds, with the establishment of rooting pole, due to an auxin gradient. Furthermore, histological and histochemical analyses allowed us to characterize how the PPCs provide niches for multipotent, pluripotent and totipotent stem-like cells for vascular differentiation, organogenesis and somatic embryogenesis in the peach palm. The histological and histochemical analyses also allowed us to detect the unicellular or multicellular origin of somatic embryogenesis. Therefore, our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells. KEY MESSAGE: Our results indicate that the use of growth regulators in microplants can lead to habituation and to different morphogenic pathways leading to potential niche establishment, depending on the positioning of the competent cells and their interaction with neighboring cells.     

The growth responses of coastal dune species are determined by nutrient limitation and sand burial

Past work suggests that burial and low nutrient availability limit the growth and zonal distribution of coastal dune plants. Given the importance of these two factors, there is a surprising lack of field investigations of the interactions between burial and nutrient availability. This study aims to address this issue by measuring the growth responses of four coastal dune plant species to these two factors and their interaction. Species that naturally experience either high or low rates of burial were selected and a factorial burial by nutrient addition experiment was conducted. Growth characteristics were measured in order to determine which characteristics allow a species to respond to burial. Species that naturally experience high rates of burial (Arctotheca populifolia and Scaevola plumieri) displayed increased growth when buried, and this response was nutrient-limited. Stable-dune species had either small (Myrica cordifolia, N-fixer) or negligible responses to burial (Metalasia muricata), and were not nutrient-limited. This interspecific difference in response to burial and/or fertiliser is consistent with the idea that burial maintains the observed zonation of species on coastal dunes. Species that are unable to respond to burial are prevented from occupying the mobile dunes. Species able to cope with high rates of burial had high nitrogen-use efficiencies and low dry mass costs of production, explaining their ability to respond to burial under nutrient limitation. The interaction between burial and nutrient limitation is understudied but vital to understanding the zonation of coastal dune plant species.     

催吐萝芙木离体培养和植株再生体系的建立

为建立催吐萝芙木(Rauvolfia vomitoria Afzel.)的快繁再生体系,以茎段为外植体,比较了植物生长调节剂对其愈伤组织诱导、分化及生根的影响。结果表明,诱导愈伤组织的适宜培养基为MS+2,4-D 1.0 mg L~(–1)+TDZ 0.5 mg L~(–1)或MS+2,4-D2.0 mg L~(–1)+TDZ 0.5 mg L~(–1),出愈率达100%且生长状况良好;诱导丛生芽的最佳培养基为MS+6-BA 3.0 mg L~(–1)+NAA 0.1 mg L~(–1),出芽率为46.6%,平均出芽数为3.04。这为催吐萝芙木的快速繁殖和遗传转化研究奠定了基础。     

Growth and allocation of the arctic sedges Eriohorum angustifolium and E. vaginatum: effects of variable soil oxygen and nutrient availability

In arctic tundra soil, oxygen depletion associated with soil flooding may control plant growth either directly through anoxia or indirectly through effects on nutrient availability. This study was designed to evaluate whether plant growth and physiology of two arctic sedge species are more strongly controlled by the direct or indirect effects of decreased soil aeration. Eriophorum angustifolium and E. vaginatum, which originate from flooded and well-drained habitats, respectively, were grown in an in situ transplant garden at two levels of soil oxygen, nitrogen, and phosphorus availability over two growing seasons. In both species, N addition had a stronger effect on growth and biomass allocation than P addition or soil oxygen depletion. Net photosynthesis and carbohydrate concentrations were relatively insensitive to changes in these factors. Biomass reallocated from shoots to below-ground parts in response to limited N supply was equally divided between roots (nutrient acquisition) and perennating rhizomes (storage tissue formation) in E. angustifolium. E. Vaginatum only increased its allocation to rhizomes. In the flood-tolerant E. angustifolium, growth was improved by soil anoxia and biomass allocation among plant parts was not significantly affected. Contrary to our initial hypothesis, whole-plant growth in E. vaginatum improved in flooded soils; however, it only did so when N availability was high. Under low N availability growth in flooded soils was reduced by 20% compared to growth in the aerobic environment. Reduced biomass allocation to rhizomes and thus to storage potential under anaerobic conditions may reduce long-term survival of E. vaginatum in flooded habitats.     

Thidiazuron-induced morphogenesis in tamarind seedlings

Summary Germination of tamarind seeds in medium containing thidiazuron (TDZ) resulted in induction of nodular protrusions in and around the cotyledonary node meristem. The structures developed radially in well-defined circles and subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. The structures developed into shoots on transfer to medium devoid of growth regulators. Histological studies revealed that the protrusions initiated from the nodal meristem and extended to the non-meristematic region between the two meristems and also in the proximal part of the hypocotyl in seedlings germinated in 9.08 μM TDZ. Newly formed cell layers and less-differentiated meristematic protrusions were also seen. With the increase in the distance from the meristem, the buds were less differentiated; in the proximal part of the hypocotyl only the multiple layers of meristematic cells were noted. With extension of the period of incubation, the TDZ-induced meristematic activity extended laterally in circles towards the neighboring region. The radial spread of the meristematic activity from the center of the nodal meristem was also evident at 18.16 μM TDZ. From the pattern of the morphogenic development and the histological studies it may be hypothesized that in tamarind, TDZ influences the existing meristems specifically. Subsequently de novo organogenesis is triggered in the neighboring cells.     

Combined effects of enhanced UV-B radiation and additional nutrients on growth of two Mediterranean plant species

Seedlings of two Mediterranean plants, the slow-growing, evergreen sclerophyll Ceratonia siliqua L. and the fast growing drought semi-deciduous Phlomis fruticosa L., were grown for one year in the field at ambient or ambient plus supplemental UV-B radiation (equivalent to a 15% ozone depletion) and two levels of applied fertilizers (NPK). The effects on growth, morphological, anatomical and physiological parameters were measured at final plant harvest. Additional nutrients increased leaf nitrogen, improved growth and reduced the root/shoot ratio in both plants, yet these effects were more pronounced in the fast growing P. fruticosa. A nutrient-induced increase in chlorophyll content was also observed in this plant. The growth responses to UV-B radiation were different for the two species. Growth in C. siliqua was not affected by UV-B radiation at both nutrient levels and the same was true for P. fruticosa at low nutrients. However, at the high nutrient level, supplemental UV-B radiation improved growth in P. fruticosa, indicating a strong interaction between the treatments. Photosystem II (PSII) photochemical efficiency, methanol-extractable UV-B absorbing capacity, total phenolics and tannins were not affected by either treatment in both plants. It is concluded that nutrient levels can strongly modify the UV-B radiation effects on growth of P. fruticosa. We presume that this may be correlated to the fast growing habit of this species.     

Additive effects of genotype,nutrient availability and type of tissue damage on the compensatory response of Salix planifolia ssp. planifolia to simulated herbivory

Plant responses to herbivory include tolerance (i.e. compensatory growth) and defense. Several factors influence the tolerance of a plant following herbivory, including plant genetic identity, site nutrient availability, and previous and/or concurrent herbivory. We studied the effects of these factors on the compensatory response of Salix planifolia ssp. planifolia, a shrub species common in the boreal and subarctic regions of North America. We cloned several genets of S. planifolia and submitted them to simulated root and/or leaf herbivory while varying the nutrient availability. Simulated leaf herbivory was more detrimental to the plant than simulated root herbivory, reducing both above- and below-ground tissue production. Leaf demography was unaffected by either simulated herbivory treatment. There was some compensatory growth following simulated leaf and root herbivory, but only the root compartment responded to increased nutrient availability. Simulated leaf herbivory increased leaf transpiration and reduced stomatal resistance, suggesting increased carbon fixation. The unexpected finding of the experiment was the absence of interactions among factors (genotype, nutrient availability and type of tissue damage) on the compensatory response of S. planifolia. These factors thus have additive effects on the species' compensatory ability.     

Development of an efficient micropropagation system for Tecoma stans (L.) Juss. ex Kunth using thidiazuron and effects on phytochemical constitution

The present study was designed to investigate the stimulatory effects of different doses (0.1 to 2.5 μM) of thidiazuron (TDZ) on in vitro shoot induction and proliferation of mature nodal explants of Tecoma stans. Of the tested concentrations, 2.0 μM TDZ proved to be optimal for maximum regeneration (91%) with a mean shoot number of 5.6 ± 0.67, and length of 2.38 ± 0.08 cm, after 4 wk of incubation. To determine the negative effects of prolonged TDZ exposure, after 4 wk of incubation at optimized level of TDZ, the cultures were transferred to a secondary medium either lacking plant growth regulators or supplemented with benzyladenine (BA) alone, or in combination with different auxins (indole-3-acetic acid, indole-3-butyric acid, or α-naphthalene acetic acid; NAA). Among the tested concentrations, 2.5 μM BA in combination with 0.5 μM NAA yielded the maximum mean shoot number (16.60 ± 0.40), and average shoot length (4.76 ± 0.15 cm) after 4 wk of culture. The best rhizogenesis (93%) was achieved on ½ MS medium containing 1.5 μM NAA, with a mean root number of 7.60 ± 0.40 and length of 4.11 ± 0.23 cm, after 4 wk of incubation. The micropropagated plantlets were successfully acclimatized and hardened off in Soilrite™ with a 90% survival rate. The plantlets grew well with normal growth, flowering and showed, by gas chromatography–mass spectroscopy, an increase in the number of bioactive compounds compared with the donor plant. This is the first report on T. stans in vitro regeneration using TDZ.

     

Micropropagation of mature <Emphasis Type="Italic">Pongamia pinnata</Emphasis> Pierre

Murashige and Skoog’s (MS) basal medium with benzylaminopurine (BA), kinetin (KN), zeatin (Z), and thidiazuron (TDZ) were tested for induction of multiple shoots from mature-tree-derived axillary meristems of Pongamia pinnata. Sprouting of buds was 64% on medium devoid of plant growth regulators (PGR). Incorporation of BA, KN, or Z was ineffective in enhancing sprouting frequency or induction of multiple shoots. Sprouting was completely suppressed in the presence of TDZ. Caulogenic buds appeared in nodal meristems of these explants after withdrawal of TDZ. The number of shoot buds was more on explants precultured in higher concentrations. At higher concentrations of this PGR, a swelling developed at the axil. Multiple shoot primordia appeared and differentiated from this swelling after culturing these explants on MS medium for six passages of 2 wk each. Shoots were harvested and cultured on 0.45 μM TDZ for further proliferation. Primary explants after harvesting of shoots were identified as ‘stump’. Reculturing of stumps on 0.45 μM TDZ produced more shoots. This step was followed for six cycles to obtain additional shoots in each cycle. Shoots maintained on 0.45 μM TDZ elongated and rooted (70%) on growth regulator-free medium. Rooted shoots (65%) survived transfer to a sand/soil mixture. This report describes the protocol for micropropagation of P. pinnata using mature-tree-derived nodal meristems. Recycling of mature stock to produce a stream of useable shoots for subculturing and eventual stabilization is of great value and can possibly be generalized as an isolation protocol especially for woody species. Repeated proliferation of caulogenic buds from the same origin may also find application in rescue of endangered germplasm.     

Modulation of TDZ-induced morphogenetic responses by anti-auxin TIBA in bud-bearing foliar explants of Kalanchoe pinnata

This investigation was conducted to study the range of TDZ-induced morphogenetic responses displayed by foliar explants of Kalanchoe pinnata with or without an epiphyllous bud and to assess their possible auxin mediation by using potent anti-auxin TIBA. Each epiphyllous bud developed into a healthy plantlet when cultured in vitro without nutrient/hormonal supplements. The bud on 10⁻⁶ M TDZ-treated leaf disc produced a compact, dwarf and rootless plantlet having achlorophyllous leaves with increased number of marginal notches. TDZ also caused a significant enlargement of the leaf disc as well as induced an organized hypertrophic growth at a specific site, with either effect being more pronounced in discs without an epiphyllous bud. Treatment with 10⁻⁴ M TIBA completely checked the plantlet growth. In combined dispensations, the TDZ-induced responses were modified by the anti-auxin TIBA in more than one way. Whereas it caused a complete reversal of hypertrophic growth on leaf discs and partially restored pigmentation of plantlet leaves, it could not annul the leaf disc enlargement. The diverse morphogenetic modulations were tissue and response specific. A complete reversal of TDZ-induced leaf disc hypertrophy by TIBA indicated an auxin mediation, whereas a complete non-reversal of the induced leaf disc enlargement pointed to the non-involvement of auxin, for the respective responses. The latter response could probably be manifested through cytokinin-activity of TDZ, while still others may not be interpreted exclusively through either one of the two hormonal options.     

Phenolic-rich leaf carbon fractions differentially influence microbial respiration and plant growth

Phenolics can reduce soil nutrient availability, either indirectly by stimulating microbial nitrogen (N) immobilization or directly by enhancing physical protection within soil. Phenolic-rich plants may therefore negatively affect neighboring plant growth by restricting the N supply. We used a slow-growing, phenolic-rich alpine forb, Acomastylis rossii, to test the hypothesis that phenolic-rich carbon (C) fractions stimulate microbial population growth and reduce plant growth. We generated low-molecular-weight (LMW) fractions, tannin fractions, and total soluble C fractions from A. rossii and measured their effects on soil respiration and growth of Deschampsia caespitosa, a fast-growing, co-dominant grass. Fraction effects fell into two distinct categories: (1) fractions did not increase soil respiration and killed D. caespitosa plants, or (2) fractions stimulated soil respiration and reduced plant growth and plant N concentration while simultaneously inhibiting root growth. The LMW phenolic-rich fractions increased soil respiration and reduced plant growth more than tannins. These results suggest that phenolic compounds can inhibit root growth directly as well as indirectly affect growth by reducing pools of plant available N by stimulating soil microbes. Both mechanisms illustrate how below-ground phenolic effects may influence the growth of neighboring plants. We also examined patterns of foliar phenolic concentrations among populations of A. rossii across a natural productivity gradient (productivity was used as a proxy for competition intensity). Concentrations of some LMW phenolics increased significantly in more productive sites where A. rossii is a competitive equal with the faster growing D. caespitosa. Taken together, our results contribute important information to the growing body of evidence indicating that the quality of C moving from plants to soils can have significant effects on neighboring plant performance, potentially associated with phytoxic effects, and indirect effects on soil biogeochemistry.     

Simultaneous exposure to a pulsed and a prolonged anthropogenic stressor can alter consumer multifunctionality

Ecosystems face multiple anthropogenic threats globally, and the effects of these environmental stressors range from individual‐level organismal responses to altered system functioning. Understanding the combined effects of stressors on process rates mediated by individuals in ecosystems would greatly improve our ability to predict organismal multifunctionality (e.g. multiple consumer‐mediated functions). We conducted a laboratory experiment to test direct and indirect, as well as immediate and delayed effects of a heat wave (pulsed stress) and micropollutants (MPs) (prolonged stress) on individual consumers (the great pond snail Lymnaea stagnalis) and their multifunctionality (i.e. consumption of basal resources, growth, reproduction, nutrient excretion and organic‐matter cycling). We found that stressful conditions increased the process rates of multiple functions mediated by individual consumers. Specifically, the artificial heat wave increased process rates in the majority of the quantified functions (either directly or indirectly), whereas exposure to MPs increased consumption of basal resources which led to increases in the release of nutrients and fine particulate organic matter. Moreover, snails exposed to a heat wave showed decreased reproduction and nutrient excretion after the heat‐wave, indicating the potential for ecologically relevant delayed effects. Our study indicates that the immediate and delayed effects of stressors on individual organisms may directly and indirectly impact multiple ecosystem functions. In particular, delayed effects of environmental stress on individual consumers may cumulatively impede recovery due to decreased functioning following a perturbation. Reconciling these results with studies incorporating responses at higher levels of biological complexity will enhance our ability to forecast how individual responses upscale to ecosystem multifunctionality.     

Linking above-ground and below-ground effects in autotrophic microcosms: effects of shading and defoliation on plant and soil properties

Although factors affecting plant growth and plant carbon/nutrient balance – e.g., light availability and defoliation by herbivores – may also propagate changes in below‐ground food webs, few studies have aimed at linking the above‐ground and below‐ground effects. We established a 29‐week laboratory experiment (～one growing season) using autotrophic microcosms to study the effects of light and defoliation on plant growth, plant carbon/nutrient balance, soil inorganic N content, and microbial activity and biomass in soil. Each microcosm contained three substrate layers – mineral soil, humus and plant litter – and one Nothofagus solandri var. cliffortioides seedling. The experiment constituted of the presence or absence of two treatments in a full factorial design: shading (50% decrease in light) and artificial defoliation (approximately 50% decrease in leaf area in the beginning of the growing season). At the end of the experiment a range of above‐ground and below‐ground properties were measured. The shading treatment reduced root and shoot mass, root/shoot ratio and leaf production of the seedlings, while the defoliation treatment significantly decreased leaf mass only. Leaf C and N content were not affected by either treatment. Shading increased NO ₃–N concentration and decreased microbial biomass in humus, while defoliation did not significantly affect inorganic N or microbes in humus. The results show that plant responses to above‐ground treatments have effects which propagate below ground, and that rather straightforward mechanisms may link above‐ground and below‐ground effects. The shading treatment, which reduced overall seedling growth and thus below‐ground N use and C allocation, also led to changes in humus N content and microbial biomass, whereas defoliation, which did not affect overall growth, did not influence these below‐ground properties. The study also shows the carbon/nutrient balance of N. solandri var. cliffortioides seedlings to be highly invariant to both shading and defoliation.     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

In vitro morphogenic response in cotyledon explants of Semecarpus anacardium L.

Three different morphogenic responses??caulogenesis, direct somatic embryogenesis, and callusing??were noted in cotyledon explants of Semecarpus anacardium L. cultured in woody plant medium (WPM) containing thidiazuron (TDZ). Thidiazuron, at all concentrations tested, induced organogenic as well as embryogenic responses. The organogenic buds differentiated to shoots and the embryogenic mass (EM) gave rise to globular embryos which differentiated up to cotyledon-stage embryos on repeated culture in growth regulator (GR)-free WPM medium containing 0.2% activated charcoal after the removal of TDZ. The organogenic and embryogenic responses were optimal in 9.08???M TDZ after the removal of TDZ. Elongated shoots rooted in half-strength liquid WPM medium with 2.46???M indole butyric acid. Plants were successfully acclimatized and transferred to soil. Histological studies confirmed the direct origin of the organogenic buds from the cotyledon explants. The EMs produced somatic embryos on repeated culture in charcoal incorporated GR-free medium. Morphogenic callus formation from the cotyledon explants was also noted. This callus on repeated culture in WPM medium with charcoal differentiated into somatic embryos. Repetitive somatic embryogenesis was evident from direct and indirectly formed primary embryos. The somatic embryos did not convert into plantlets, though sporadic germination of embryos was observed through the emergence of roots.