Shoot Morphogenesis Research Articles

Stimulation of development of Cucumis sativus L. plants by low concentration of aluminium in vitro conditions

The culture of cucumber plants <em>Cucumis sativus</em> L. Wisconsin cultivar, from seeds and leaf explants, were carried out on the basic medium ofMurashige and Skoog <em>in vitro</em> conditions. In the culture set from the leaf explants the MS medium was supplemented with IAA (0,5 mg/dm<sup>3</sup>) and BAP (2 mg/dm<sup>3</sup>). Aluminium (as AlCl<sub>3</sub>) was added to the media in concentration Of 1 mg/dm<sup>3</sup>. The media pH was adjusted to 6,2 or 4,2. In seedling culture, aluminium substantially stimulated the growth and development of the root system while a shoot to a small degree only (it advanced leaf formation mainly), causing no morphological and developmental anomalies. In the culture from the leaf explants Al induced the process of rhizogenesis which did not take place on the media without Al. It also stimulated a shoot morphogenesis. After 8 weeks of culture, 32 % leafexplants formed plants with short shoots (2-3,5 cm), long ones (5,5-7 cm) and with long but poorly branched root system„ In the acid conditions (pH 4,2), the effect 0 Al on plant growth was Iower than on the media with pH 6,2. Also a number of regenerated explants with comparable direction of differentiation won a fewer in low pH.

Effect of rhythmic auxin application on shoot morphogenesis in carrot (Daucus carota L.) callus tissue

The possible role of auxin oscillations in plant development has been considered earlier but direct experimental evidence in support of this concept has been limited. In this research, we conducted a series of experiments by employing an instrument of novel design that dispensed auxin droplets on the surface of callus cultures in fixed amounts at controllable time intervals (frequencies). The effects of 60 h exposures of different indole-acetic acid (IAA) concentrations (0.5, 1 and 2 mg l−1) and dispensing time intervals (6, 9, 12, 13.5, 15, 18 and 21 min) were tested on carrot calli and compared with control treatments with steady exposure to IAA and rhythmic application of distilled water. Shoot organogenesis was observed after two weeks only on calli that were exposed to IAA oscillations of 9 and 18 min, while other time intervals and controls, including non-periodical controls and periodical application of distilled water, were ineffective. The results were similar among different IAA concentrations. Rhythmical auxin application is much more efficient compared with steady auxin treatment in terms of efficiency and specificity. This effect is frequency-dependent, i.e. there is a resonant stimulation.

In Vitro Adventitious Shoot Regeneration via Indirect Organogenesis from Inflorescence Explants and Peroxidase Involvement in Morphogenesis of Populus euphratica Olivier

The inflorescences as explants for rapid propagation in vitro remained unknown in Populus euphratica Olivier. Here, we reported that multiple shoots were initiation from calli of both male and female inflorescences. The optimum medium for shoot induction from male inflorescences was lactose sulfite medium containing 1.0mg L(-1) 6-benzylaminopurine (BA) and 0.5mg L(-1) α-naphthalene acetic acid (NAA) or Murashige and Skoog (MS) medium containing 0.5mg L(-1) BA and 0.2mg L(-1) NAA. The optimum medium of shoot induction from female inflorescence calli was the MS medium containing 0.5mg L(-1) BA and 0.2mg L(-1) NAA. Rooting of regenerated shoots was obtained on 1/2 MS medium supplemented with 0.5∼1.0mg L(-1) indole-3-butyric acid (IBA) and the highest frequency rooting was on medium containing 0.5mg L(-1) IBA. No shoots were obtained on medium without BA and NAA. Peroxidase (POD) activity was measured by polyacrylamide gel electrophoresis during shoot induction and differentiation stages. The results showed that two bands of POD (2a and 2b) activity appeared lowest during the early 8days at the dedifferentiation phase of leaves inducing calli, whereas POD 2a, 2b activity appeared to be increasing at the homeochronous dedifferentiation phase of inflorescence. Five most intensive bands, POD 1a, 1b, 1c, 2a, and ab, appeared in 8th and 28th days at the redifferentiation phase during shoot morphogenesis. These results demonstrated that the POD was involved in shoot morphogenesis from both leaf and inflorescence explants of Populus euphratica.

In vitro regeneration of Aristolochia tagala and production of artificial seeds

Protocols for in vitro plant multiplication from somatic tissues and production of artificial seeds through encapsulation of nodes were developed for Aristolochia tagala Cham., a rare and valuable medicinal plant, as a measure of conservation and as a prerequisite for genetic transformation procedure. A maximum number of adventitious shoots were regenerated from leaf-derived callus on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP; 2 μM), α-naphthaleneacetic acid (NAA; 0.5 μM), and phloroglucinol (PG; 10μM). Nodes collected from in vitro established shoot cultures were encapsulated in 3 % (m/v) sodium alginate and 1 % (m/v) calcium chloride. Multiple shoots were successfully regenerated from the encapsulated nodes cultured on MS medium supplemented with 3 μM BAP and 0.5 μM kinetin (KIN). Regenerated shoots from callus and artificial seeds were successfully rooted and acclimated to greenhouse conditions. Since roots of A. tagala are primarily used in traditional medicine, a protocol for regenerating roots directly from the leaf derived callus was also developed. Maximum root length was obtained when the callus was cultured in MS medium supplemented with KIN (1 μM), indole acetic acid (IAA; 0.5 μM), NAA (0.1 μM), and PG (10 μM). Biochemical parameters were studied in calli grown with and without PG in the medium to establish a correlation between these parameters and shoot morphogenesis. An increment of antioxidant enzymes (peroxidase and catalase) and metabolites (sugars and proteins), and a decrease in the amount of polyphenol oxidase was observed in the calli which were grown in the presence of PG.

Methylglyoxal with glycine or succinate enhances differentiation and shoot morphogenesis in Nicotiana tabacum callus

The aim of this study was to evaluate the influence of methylglyoxal (MG) on organogenesis and regeneration of tobacco (Nicotiana tabacum L.) plants from callus in media containing glycine or succinate. The best improvement in shoot proliferation and shoot length was obtained in the medium supplemented with 0.1 mM MG and 0.5 mM glycine or 0.25 mM succinate. The histological studies showed vigorous development of corm like structures and shoot organogenesis from callus tissues cultured in MG supplemented media. Biochemical studies also revealed higher content of δ-aminolaevulinic acid (a precursor of chlorophyll) and of chlorophyll.

Changes in endogenous hormones and oxidative burst as the biochemical basis for enhanced shoot organogenesis in cold-treated Saussurea involucrata explants

A low temperature treatment significantly improved shoot morphogenesis of Saussurea involucrata Kar. et Kir leaf explants. The biochemical mechanisms underlying the cold-induced shoot organogenesis were investigated by measuring endogenous plant growth hormones, peroxide radicals, and superoxide dismutase (SOD) activity in the cold-treated leaf explants compared to controls. The ratio of zeatin (ZT) to indole-3-acetic acid (IAA) significantly increased in leaf explants subjected to a 5-day treatment at 4 °C compared to controls. The accumulation of O2·− also rapidly increased in response to cold treatment, and then decreased as SOD activity catalyzed the dismutation of O2·− to molecular oxygen and H2O2; this resulted in a significant increase in H2O2 concentrations in the cold-treated explants. We propose that a combination of the increased ZT/IAA ratio and H2O2 concentration is the basis for the enhanced shoot morphogenesis in response to cold treatment. These results provide a starting point for an improved understanding of the biochemical mechanisms underlying cold-induced shoot organogenesis of this unique plant species.

Improvement of shoot morphogenesis in vitro and assessment of changes of the activity of antioxidant enzymes during acclimation of micropropagated plants of Desert Teak

The aim of the study was to develop an improved and rapid regeneration system via axillary shoot proliferation for Tecomella undulata, an important endangered medicinal tree in India. The Murashige and Skoog (MS) medium augmented with different concentrations (0.1, 0.3, 0.5, 0.7, 1.0, 2.5, 5.0, 7.5 and 10.0 μM) of Thidiazuron (TDZ) was tested for the ability to induce axillary shoot development from cotyledonary node explants excised from 7-day-old sterile seedlings. Among the tried concentrations of TDZ, 0.7 μM showed optimum response in inducing maximum number (25.00 ± 2.30) of shoots and shoot length (4.06 ± 0.58 cm) after 3 weeks of incubation. The regenerated shoots when subcultured on MS medium lacking TDZ gave twofold shoot multiplication rate with maximum number (43.00 ± 2.86) of shoots per explant and longer shoot length (7.40 ± 0.34 cm) during the fourth subculture passage. Attempts were also made to study the morphogenetic effect of medium pH and sucrose concentration on axillary shoot induction and proliferation. The highest efficiency of shoot regeneration was recorded in MS medium supplemented with 0.7 μM TDZ and 3% sucrose at pH 5.8 after 3 weeks of culture. Different concentrations of Indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with IBA (200 μM) pulse treatment given to the basal end of the microshoots for 30 min followed by their transfer in plastic cups containing soilrite and eventually established in natural soil with 80% survival rate. During acclimatization of plants, catalase (CAT) activity increased reaching maximum at 28th day after transplantation, while superoxide dismutase (SOD) activity increased reaching a maximum in the 21 days. Likewise, changes in the glutathione reductase (GR) and Ascorbate peroxidase (APX) were also detected. The observed changes reflect the plant’s capacity to develop antioxidant mechanisms during acclimatization which determine the ability to survive in oxidative stress. The standardized protocol for mass propagation of Tecomella undulata should eliminate the dependence on natural stands of plants for pharmaceutical purposes, and will also serve as a means of conservation as the species is highly overexploited.

Genetic regulation of polar auxin transport and its role in control of shoot morphogenesis

The effects of auxin on plant morphogenesis are related to its differential distribution within different plant tissues. Tissue-specific auxin synthesis and its influx into and efflux from cells are shown to be of importance for establishing the auxin gradients. The paper deals with control of these processes. Much attention is given to genetic control of auxin efflux and to the influence of genes regulating this process on plant morphogenesis.

In vitro morphogenic response and metal accumulation in Albizia lebbeck (L.) cultures grown under metal stress

An efficient regeneration protocol for rapid mass propagation and uptake of heavy metals in Albizia lebbeck (L.), a fast growing, medicinally as well as economically important timber yielding tree was developed. Nodal segments derived from a 20-year-old tree were cultured on MS (Murashige and Skoog) medium supplemented with 10 μM 6-Benzyladenine (BA) and 1 μM α-Naphthalene acetic acid (NAA) showed optimum shoot regeneration frequency (76.6%), number of shoots (23.2 ± 0.28) per explant and shoot length (2.86 ± 0.08 cm) after 10 weeks of culture. After standardizing a reliable protocol for micropropagation, effects of ZnSO4 (0.06–0.48 mM), CuSO4 (0.02–0.2 mM) and CdCl2 (0.0001–0.001 mM) on shoot morphogenesis were also assessed. The regenerated shoots maintained on maintenance medium (MS + 10.0 μM BA + 1.0 μM NAA) containing ZnSO4 (0.06 mM) showed maximum response in terms of shoot number (24.5 ± 0.83) and length (5.9 ± 0.05 cm) after 10 weeks of culture. Proline content showed an increasing trend while chlorophyll (a and b) content exhibited decreasing trend with an increased metal concentrations compared to MM cultures, and maximum increase in proline and decrease in chlorophyll content was recorded in cultures grown on Cd-enriched medium. Best rooting was accomplished on half strength MS medium with 2.0 μM IBA and ZnSO4 (0.06 mM). The plantlets thus obtained were successfully hardened and transferred to greenhouse with 75% survival rate and exhibited normal morphological characteristics compared to donor plant.

Auxin stability and accumulation during in vitro shoot morphogenesis influences subsequent root induction and development in Eucalyptus grandis

Recent results showed that after 16 months in the field, micropropagated eucalyptus plants have an inferior root system to cuttings. Such differences may be due to the plant growth regulators supplied during the culture stages of standard protocols, which are targeted at optimising plantlet yields and not root quality. This study investigated such a proposal, focusing on auxins in an easy-to-root clone. Initial results showed that the auxin provided in the standard protocol (NAA for multiplication and IBA for elongation) enabled 100% rooting in auxin-free medium, where rooting was faster than on IBA-rooting media. When auxin supply was omitted from multiplication and restricted to NAA or IAA during elongation, rooting in an auxin-free medium was reduced to 68 and 31%, respectively, reflecting the stabilities of these auxins in plant tissues. Additionally, 15% of shoots from the NAA-medium and 65% from the IAA-medium produced roots with altered graviperception. GC–MS analysis of these shoots revealed a relationship between free IAA-availability and altered graviperception. This was further tested by adding the IAA-specific transport inhibitor 2,3,5-triiodobenzoic acid to rooting media with IBA, IAA or NAA, which resulted in 100, 70.9 and 20.6% rooting, respectively. At least 40% of the sampled root tips had atypical starch grain deposition and abnormal graviperception. It is proposed that, at least in this clone, while IBA and NAA can be used for in vitro root induction, IAA is necessary for development of graviresponse.

Histological evidence of morphogenesis from various explants of Jatropha curcas L.

Jatropha curcas, a non-domesticated energy plant, has emerged as a source of biodiesel as it does not compete with the edible oil supplies. Realizing its importance, in vitro regeneration methods have been established to meet the demand of large scale supply of superior clones. However, no precise histological analysis has been conducted to document the in vitro morphogenesis in J. curcas. Here, we present a detailed histological description of the initiation of growth and morphogenesis from callus induced from immature cotyledon and embryonal axis of J. curcas. Microscopic observations revealed that the cells of sub-epidermis in immature cotyledon became meristematic and divided extensively to result in the formation of meristemoids within 3–4 weeks of culture. The meristemoid cells are compact, small, and exhibited densely stained cytoplasm compared to the loosely packed large non-meristematic cells. Presence of meristemoids in different developmental stages above the cambial zone, and the absence of vascular connection to the cotyledon explant led us to conclude that the origin of the meristemoids was adventitious in nature and regeneration is through organogenesis of morphogenic callus. On the other hand, callus derived from immature embryonal axis followed two types of regeneration—one type of shoot regeneration was via organogenesis while the second type was through multiplication of the pre-existing meristems. It was noticed that under similar experimental conditions, the process of organogenesis varied from explant to explant. The present study contributes to adequate knowledge and understanding the process of in vitro shoot morphogenesis in J. curcas.

Pengaruh Media Induksi terhadap Multiplikasi Tunas dan Pertumbuhan Planlet Pisang Rajabulu (AAB) dan Pisang Tanduk (AAB) pada Berbagai Media Multiplikasi

The aim of this research was to study the effects of explant from various induction media on multiplication and growth of cv Rajabulu (AAB) and Tanduk (AAB) plantain. This research was arranged in factorial complete randomized design with two factors. The fi rst factor was two kinds of explant which came from induction media I1 (MS + BA 2 mg L-1 + IAA 3 mg L-1), and from induction media I2 (MS + BA 2 mg L-1 + IAA 3 mg L-1 + TDZ 0.09 mg L-1); the second factor was 4 kinds of multiplication media, i.e. MS0 (control/without PGR), MS + BA 0.20 mg L-1 + IAA 0.01 mg L-1 (M1), MS + BA 1 mg L -1 + IAA 0.25 mg L-1 (M2), MS + BA 2 mg L-1 + IAA 0.5 mg L-1 (M3). The experiment results were the use of TDZ in the induction medium reduced the use of high cytokinin in the multiplication level. The use of Rajabulu explant that came from media I2 produced more shoots (4.3 shoots per explan) compared to explant from media I1 (3.2 shoots per explan). The use of multiplication media M3 and M2 produced the highest shoot number. The best shoot morphogenesis produced when the shoots after subculture in media with PGR (M3 or M2) to media MS0 (big shoot 3.1 and medium shoot 3.5). Tanduk plantain’s shoot was responsive to cytokinin. The best treatment is I1-M3 with the highest number of shoots and the highest percentage of big and medium shoot is (33%) compared to other treatments (<17 %). In the second subculture, the highest shoot number were obtained from multiplication media M3 and M2. The morphogenesis of Tanduk plantain produced the highest big shoot if subcultured to media M3-M0 and M2-M0, followed to M0-M0. Keywords: TDZ, cytokinin, auxin, morphogenesis, big shoot, medium shoot

High frequency in vitro propagation of Trichopus zeylanicus subsp. travancoricus using branch–petiole explants

Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5 μM 2,4-dichlorophenoxyacetic acid upon subculture to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot morphogenesis. Medium with 13.3 μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower concentrations of BA (4.4, 6.6 and 8.8 μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3 μM BA containing medium, but with reduced number of shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation as well as growth of shoots. The shoot regeneration potential showed no decline up to 5 years. Culture of the in vitro-derived whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6 μM IBA, the whole branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with BA exhibited high frequency shoot formation. The transfer of shoots after 10–15 days culture on half-strength MS medium containing 2.7 μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix: a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants regenerated from calli after 4 years showed no polymorphism. The established plantlets with morpho-floral features similar to that of the source plants flowered normally and set fruits.

STUDY ON SHOOT MORPHOGENESIS IN-VITRO OF MORINDA CITRIFOLIA L.

Morinda citrifolia L. is a valuable medicinal plant, used to treat many diseases, such as sleeplessness, backache, high blood pressure... To study the shoot morphogenesis in Morinda citrifolia L. for propagation in the future, we examined the effects of BA, Zeatin and NAA on adventitious shoot formation of hypocotyl. The results showed that the regeneration of adventitious shoot comprised three steps of morphogenesis. In this process, Zeatin 1mg/l stimulated the formation of shoot primordia in dark (after a week), earlier than BA at the same concentration. Shoot generation was slow (after five weeks) on MS medium supplemented with 0,1mg/l and Zeatin 1mg/l. Roles of respiration rate and endogenous hormones were discussed to understand the physiological changes in the adventitious root formation.

Development of a phosphomannose isomerase-based Agrobacterium-mediated transformation system for chickpea (Cicer arietinum L.)

To develop an alternative genetic transformation system that is not dependent on an antibiotic selection strategy, the phosphomannose isomerase gene (pmi) system was evaluated for producing transgenic plants of chickpea (Cicer arietinum L.). A shoot morphogenesis protocol based on the thidiazuron (TDZ)-induced shoot morphogenesis system was combined with Agrobacterium-mediated transformation of the pmi gene and selection of transgenic plants on mannose. Embryo axis explants of chickpea cv. C-235 were grown on a TDZ-supplemented medium for shoot proliferation. Embryo axis explants from which the first and second flush of shoots were removed were transformed using Agrobacterium carrying the pmi gene, and emerging shoots were allowed to regenerate on a zeatin-supplemented medium with an initial selection pressure of 20 g l(-1) mannose. Rooting was induced in the selected shoots on an indole-3-butyric acid (IBA)-supplemented medium with a selection pressure of 15 g l(-1) mannose. PCR with marker gene-specific primers and chlorophenol red (CPR) assay of the shoots indicated that shoots had been transformed. RT-PCR and Southern analysis of selected regenerated plants further confirmed integration of the transgene into the chickpea genome. These positive results suggest that the pmi/mannose selection system can be used to produce transgenic plants of chickpea that are free from antibiotic resistance marker genes.

Effects of β-lactam antibiotics, auxins, and cytokinins on shoot regeneration from callus cultures of two hybrid aspens, Populus tremuloides × P. tremula and P. x canescens × P. gradidentata

The effects of β-lactam antibiotics (penicillin, carbenicillin and cefotaxime), cytokinins, and auxins including phenylacetic acid, a β-lactam breakdown product, were evaluated during in vitro shoot morphogenesis in two hybrid aspens; P. tremuloides × P. tremula (XTTa) and P. x canescens × P. grandidentata (XCaG). Although different callus and shoot induction media were used for both hybrids, the β-lactams often engendered similar responses. At concentrations of 1,000 mg l−1, carbenicillin adversely impacted shoot elongation and, to a lesser degree, shoot regeneration. Cefotaxime enhanced caulogenesis for all of the concentrations evaluated (125–500 mg l−1) especially when the cytokinin thidiazuron was used for shoot induction. The shoots formed faster and in greater numbers; and the improvements were significant (α = 0.05) for both hybrids. However, hyperhydricity was more problematic when cefotaxime was included in the media. The incidence of shoot hyperhydricity for the XCaG hybrid was more than twice as great for the highest cefotaxime concentration evaluated (500 mg l−1) than for the control (>90% vs. ~40%). Penicillin had an opposite effect. Hyperhydricity frequencies for the XCaG hybrid were lower when the media were supplemented with penicllin and the reductions were statistically significant at concentrations of 500–1,000 mg l−1. The effects of the antibiotics were generally not reproduced by the auxins (0.1–100 μM), including phenylacetic acid, or the other potential β-lactam degradation products evaluated (e.g. phenylmalonic acid, aminopenicillanic acid). The antibiotics may have affected shoot hyperhydicity indirectly via changes in the time course of shoot regeneration.

Distinct pattern of zeatin distribution in the shoot apical meristem marks stress conditions in Posidonia oceanica (L.) Delile plants

In the present work, the immunocytochemical detection of zeatin was performed in the shoot and rhizome of Posidonia oceanica (L.) plants, growing in two sites subjected to different types of anthropic pressure. P. oceanica, the predominant seagrass of the Mediterranean basin, plays a pivotal role in marine ecosystems; it is considered an excellent bioindicator of sea disturbance. Due to a wide range of factors, mostly related to human activities, a significant and widespread decline of P. oceanica meadows is currently underway. Hence, there is much interest towards finding novel descriptors of impaired features in this seagrass, which could be used in monitoring projects. Our data resulted in a specific distribution pattern of zeatin in plant shoots in relation to environmental stress factors, while no significant differences were observed in rhizomes. These results are consistent with the role of cytokinins in shoot morphogenesis and growth. On the basis of cytokinin involvement in the light-dependent signaling cascade and stress response, the putative relationship with light availability is also discussed.

Use of cactus flowers as explants for micropropagation

SummaryFlowers and floral parts have seldom been used successfully as explants to initiate tissue culture. Flower buds of Mammillaria albicoma, M. carmenae, and M. schiedeana (Cactaceae) were cultured on solid Murashige and Skoog medium containing 0.1 mg l−1 -naphthaleneacetic acid (NAA) and 5.0 mg l−1 6-benzylaminopurine. The formation of vegetative buds (areoles) and the development of shoots were observed in all three species. Both direct and indirect mechanisms of shoot morphogenesis were observed. Direct shoot morphogenesis from the perianth (especially from the axils of the perianth segments) in M. albicoma and M. carmenae was confirmed by microscopic examination. Shoots that formed on explants were isolated and used to establish proliferating cultures. Finally, proliferated shoots were rooted in vitro on MS medium containing 0.01 mg l−1 NAA and acclimated to ex vitro conditions. M. carmenae shoots were also rooted non-aseptically in horticultural substrate. This is the first report of the complete micropropagation of cacti initiated from floral explants. In this family of leafless stem succulents, harvesting of flowers may be a convenient way to micropropagate valuable genotypes, as it avoids injury to stock specimens.

Effect of cadmium on fatty acid composition of lipids in the shoots of Karelian birch cultured in vitro

The effects of cadmium (10−6, 10−5, 10−4, and 10−3 M) on the growth and development of stem apical meristem and fatty acid (FA) composition of particular lipid fractions (neutral lipids, glyco-, and phospholipids) were investigated in the shoots of Karelian birch (Betula pendula var. carelica (Mercklin) Hamet-Ahti) produced in vitro. Low cadmium concentration (10−6 M) slightly stimulated shoot morphogenesis. However, at the concentration of 10−4 M, shoot growth and development was terminated, and the concentration of 10−3 M turned out to be critical (the meristem died within the first 5 days). The presence of Cd in the medium considerably affected FA composition in the shoots. The most pronounced changes occurred in the fraction of glycolipids, and the extent of adverse influence significantly depended on metal concentration in the medium. At the Cd concentration of 10−4 M, the content of di-and trienoic FAs decreased by 3–4 times, whereas the level of monoenoic FAs rose by 5 times. The revealed differences in the FA composition of lipids in birch were considered as an indicator of adverse effect of cadmium on the structure and functions of chloroplasts and therefore on photosynthesis.

Molecular cloning and expression analysis of a cytokinin oxidase (DhCKX) gene in Dendrobium huoshanense

Cytokinin oxidase plays an important role in the cytokinins regulatory processes. A putative cytokinin oxidase gene, D endrobium h uoshanense cytokinin oxidase (DhCKX), was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) of total RNA isolated from shoot apices of D. huoshanense plantlets in the presence of 20 micromol l(-1) 6-benzylamino-purine (BAP). The full-length cDNA contains an open reading frame (ORF) encoding a putative protein of 537 amino acids with high homology to known CKX genes and with a predicted molecular mass of 60.383 kDa. Compared with the CKX sequences from other species, the DhCKX shows high identity with Dendrobium hybrid "Sonia", 92.2% in nucleotide sequence and 89.9% in amino acid sequence, respectively. Based on the alignment of amino acid sequences, the phylogenetic tree of 15 CKX genes is constructed. Semi-quantitative RT-PCR analysis showed that the expression of the DhCKX gene was induced in roots, shoot apices and leaves, but not in stems, after treatment with 20 micromol l(-1) BAP. The DhCKX gene showed maximum expression in shoot apices at day 12 during continuous treatment with 20 micromol l(-1) BAP. Furthermore, CKX activity appears to play an important role in controlling the cytokinin levels and growth during shoot morphogenesis in D. huoshanense.