Tobacco Tissue Cultures Research Articles

Analysis of formin functions during cytokinesis using specific inhibitor SMIFH2.

The phragmoplast separates daughter cells during cytokinesis by constructing the cell plate, which depends on interaction between cytoskeleton and membrane compartments. Proteins responsible for these interactions remain unknown, but formins can link cytoskeleton with membranes and several members of formin protein family localize to the cell plate. Progress in functional characterization of formins in cytokinesis is hindered by functional redundancies within the large formin gene family. We addressed this limitation by employing Small Molecular Inhibitor of Formin Homology 2 (SMIFH2), a small-molecule inhibitor of formins. Treatment of tobacco (Nicotiana tabacum) tissue culture cells with SMIFH2 perturbed localization of actin at the cell plate; slowed down both microtubule polymerization and phragmoplast expansion; diminished association of dynamin-related proteins with the cell plate independently of actin and microtubules; and caused cell plate swelling. Another impact of SMIFH2 was shortening of the END BINDING1b (EB1b) and EB1c comets on the growing microtubule plus ends in N. tabacum tissue culture cells and Arabidopsis thaliana cotyledon epidermis cells. The shape of the EB1 comets in the SMIFH2-treated cells resembled that of the knockdown mutant of plant Xenopus Microtubule-Associated protein of 215 kDa (XMAP215) homolog MICROTUBULE ORGANIZATION 1/GEMINI 1 (MOR1/GEM1). This outcome suggests that formins promote elongation of tubulin flares on the growing plus ends. Formins AtFH1 (A. thaliana Formin Homology 1) and AtFH8 can also interact with EB1. Besides cytokinesis, formins function in the mitotic spindle assembly and metaphase to anaphase transition. Our data suggest that during cytokinesis formins function in: (1) promoting microtubule polymerization; (2) nucleating F-actin at the cell plate; (3) retaining dynamin-related proteins at the cell plate; and (4) remodeling of the cell plate membrane.

Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a.

Summary Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site‐specific double‐strand breaks to enhance homologous recombination‐mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas‐based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature‐insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium‐mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T‐DNA integration. Thus, we were not only able to obtain CRISPR/Cas‐based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.

Producing of potato varieties resistant to fusarial wilt by cell selection

The experiment has determined the characteristic features of callusogenesis, regeneration as well as of receiving resistant to fusariosis of potatoes cell lines in simulated conditions of biotic stress. The technologic elements of potato cell selection towards its resistance to Fusarium oxysporum and Fusarium sabucinum were presented. We succeeded in separating the lines which during a 3-year period of research were characterised by a higher resistance point as compared to the original varieties Gurman and Tyras. The plants-regenerants received on the selective medium were characterised by higher resistance (by 10–35%) to the pathogens action as compared to the regenerants of a control variant. The received lines were estimated on their yield capacity and starch content. The lines deviations on economically valuable indices either increased or decreased as to characteristic value. Herewith, the deviation upward is 25–75% depending on characteristics. The combination of maximal characteristic values was found in lines 12 and 28 of Gurman variety. The received lines were given to the selection-breeding plot for further testing. Key words: Potatoes; Culture in vitro; Cell selection; Selective medium; Phytotoxic metabolites; Plants-regenerants. References Behnke, �?. (1980). General resistance to late blight of Solanum tuberosum plants regenerated from callus resistant to culture filtrates of Phytophthora infestans. Theor. and Appl. Genet., 9, 151–152. Bolic, M., Foroughi-Wehr, B., & Kohler, F. (1986). In vitro selection for disease resistance in potato and barley. Nuclear Technigues and in vitro Culture for Plant Improvement, 12, 275–285. Kalashnikova, E.A. (2003). Cell selection of plants on their resistance to fungus diseases. Thesis of Doctoral Dissertation. Moscow (in Russian). Murashige, T. & Scoog, A. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15, 473–497. Podgayetskiy, A.A. & Gorbas, S.B. (2013). The feasibility of getting high-starch forms from among the progeny from crossing of interspecific hybrids. Bulletin of Sumy National Agrarian University, 3(25), 224–228 Polozhenets, V.M., Tymoschuk, O.A. & Zhuravska, I.A. (2006). Fusarium dry rot of potatoe. Quarantine and plants protection, 8, 13–15. Zaharchuk, N.A. (2013). The effectiveness of potatoe selection in vitro on its resistance to Fusarium oxysporum and Fusarium sambucinum. Bulletin of Sumy National Agrarian University, 11 (26), 188–228.

Effects of CeCl3 and LaCl3 on callus and root induction and the physical response of tobacco tissue culture

La3+ and Ce3+ have positive effects on plant growth and production. Although it is well known that rare earth elements promote cell growth, the biological effects of La3+ and Ce3+ on callus, shoot and root induction in tobacco are still unclear. The relationships among callus induction, rooting, enzyme activities and stomatal characteristics in tobacco are unknown. The objectives of this study were to identify the relationships between the induction of calluses, shoots, roots, stomata and enzyme activities. The induction percentages of calluses, buds, roots were recorded at 5, 10, 15, 20 and 25 days after La3+ and Ce3+ treatments. Peroxidase isoenzyme activity was determined by electrophoresis. The characteristics of the stomata were observed under an optical microscope. Our results show that low concentrations of Ce3+ (<15 mg/L) result in increases in the induction percentages of calluses, buds and roots, but La3+ (>5 mg/L) inhibits the induction of calluses, buds and roots. There are more peroxidase isoenzyme bands in Ce3+ treatments than in La3+ treatments. This is consistent with the induction percentages of calluses, buds and roots in Ce3+ and La3+ treatments. High enzyme activities may promote the induction of calluses, buds and roots. The stomata area and stomata number of leaves are significantly different between La3+ treatments and Ce3+ treatments. La3+ improves the stomata area and number. Based on these results, we speculate that La3+ may promote the development of the photosynthetic system. Ce3+ may promote tobacco growth and rooting by improving enzyme activities.

Physiological and biochemical effects of morphactin IT 3233 on callus and tumour tissues of Nicotiana tabacum L. cultured in vitro. IV. IAA oxidase activity. Oat coleoptile biotest

IAA oxidase activity in callus and tumour tissue of tobacco subjected to the action of morphactin IT 3233 for shorter and longer periods was determined. Control tumour tissue shows an activity higher by about 40 per cent as compared with that of callus tissue. Morphactin applied for a short time (24-h incubation) does not change the activity of the enzyme. When application is prolonged, a considerable enhancement (up to 140%) of the enzyme activity in callus tissue is observed in dependence on the morphactin concentration. In tumour tissues the activity is stimulated by 45 per cent as compared to control. Oat coleoptile elongation growth induced by IAA is limited to 40 per cent when morphactin is added in the concentrations used for tobacco tissue cultures. The possibility of the morphactin action on tissue growth via IAA metabolism is discussed.

Transcriptional changes related to secondary wall formation in xylem of transgenic lines of tobacco altered for lignin or xylan content which show improved saccharification

In this study, an EST library (EH663598–EH666265) obtained from xylogenic tissue cultures of tobacco that had been previously generated was annotated. The library proved to be enriched in transcripts related to the synthesis and modification of secondary cell walls. The xylem-specific transcripts for most of the genes of the lignification and xylan pathways were identified and several full-length sequences obtained. Gene expression was determined in available tobacco lines down-regulated for enzymes of the phenylpropanoid pathway: CINNAMATE 4-HYDROXYLASE (sc4h), CINNAMOYL-COA REDUCTASE (asccr) and lignification-specific peroxidase (asprx). In addition, lines down-regulated in the nucleotide-sugar pathway to xylan formation through antisense expression of UDP-GLUCURONIC ACID DECARBOXYLASE (asuxs) were also analysed. It is shown herein that most transcripts were down-regulated for both lignin and xylan synthesis pathways in these lines, while CELLULOSE SYNTHASE A3 was up-regulated in lignin-modified lines. The analysis indicates the existence of interdependence between lignin and xylan pathways at the transcriptional level and also shows that levels of cellulose, xylan and lignin are not necessarily directly correlated to differences in transcription of the genes involved upstream, as shown by cell wall fractionation and sugar analysis. It is therefore suggested that cell wall biosynthesis regulation occurs at different levels, and not merely at the transcriptional level. In addition, all lines analyzed showed improved enzymic saccharification of secondary but not primary walls. Nevertheless, this demonstrates potential industrial applicability for the approach undertaken to improve biomass utility.

Differential patterns of dehydroabietic acid biotransformation by Nicotiana tabacum and Catharanthus roseus cells

The aim of this study was to use whole cell catalysts as tools for modification of selected resin acids in order to obtain value-added functional derivatives. The enzymatic bioconversion capacities of two plant species were tested towards dehydroabietic acid. Dehydroabietic acid (DHA) is an abundant resin acid in conifers, representing a natural wood protectant. It is also one of the constituents found in by-products of the kraft chemical pulping industry. DHA was fed to tobacco (Nicotiana tabacum) and Madagascar periwinkle (Catharanthus roseus) plant cell and tissue cultures and bioconversion product formation was monitored using NMR analysis. Both plant species took up DHA from culture medium, and various types of typical detoxification processes occurred in both cultures. In addition, diverse responses to DHA treatment were observed, including differences in uptake kinetics, chemical modification of added substrate and changes in overall metabolism of the cells. Interestingly, Catharanthus roseus, a host species for pharmaceutically valuable terpenoid indole alkaloids, exhibited a very different bioconversion pattern for exogenously applied DHA than tobacco, which does not possess a terpenoid indole pathway. In tobacco, DHA is readily glycosylated in the carbonyl group, whereas in periwinkle it is proposed that a cytochrome P450-catalyzed enzymatic detoxification reaction takes place before the formation of glycosylated product.

Thiopurine Prodrugs for Plant Chemotherapy Purposes

Thiopurine prodrugs are antiviral chemicals used in medical therapy whose mechanisms of action are associated with inhibition of purine biosynthesis. In terms of plant chemotherapy, previous research of 6-mercaptopurine (MP) administration in tobacco tissue culture infected by Tobacco mosaic virus (TMV) showed no inhibition of virus activity. Currently, not enough data exist to confirm thiopurine drug ineffectiveness against viruses in the plant kingdom. This paper presents a screening of MP, 6-methylmercaptopurine riboside (MMPR), 6-thioguanine (6-TG) and 1-amino-6-mercaptopurine (1A-MP) against TMV and Cucumber mosaic virus (CMV) in in vitro tobacco explants and against Grapevine leafroll-associated virus 3 (GLRaV 3) in in vitro grapevine explants. ELISA and RT-PCR were used to evaluate antiviral activity. Higher toxicity levels of MP derivatives, compared to MP, were noted in tobacco and grapevine explants. 1A-MP or 6-TG treatment resulted CMV and GLRaV 3 virus-eradicated explants as obtained with Inosine 5′-monophosphate dehydrogenase inhibitors, whereas TMV was not eradicated by any of the studied drugs.

Effect of NtDCN1 gene activated during embyogenesis on organogenesis in tobacco tissue culture

The role of NtDCN1 gene in organogenesis in the culture of tobacco (Nicotiana tabacum L.) somatic tissues was studied. This gene is specifically expressed in tobacco microspores induced for somatic embryogenesis. This gene knockout resulted in a disturbance of formation and development of embyoids from microspores. In leaf disks and calli derived from tobacco lines with active and inactivated NtDCN1 gene, we studied induction of shoots and roots. A comparative analysis of tobacco line morphogenetic responses in vitro showed that NtDCN1 gene inactivation enhanced shoot formation and suppressed rhizogenesis, whereas this gene reactivation returned organogenesis processes to control level. Difference between lines was manifested only at a definite ratio between exogenous hormones supplied. The involvement of NtDCN1 gene in line responses to exogenous auxin is discussed. The results obtained permit a supposition that the NtDCN1 gene is critical for regulation not only somatic embryogenesis but also organogenesis.

Endogenous and exogenous polyamines in the organogenesis in Curcuma longa L.

The present work evaluated the development of different Curcuma longa L. explants (leaves basis, root tips and ancillary buds from rhizome) stimulated by exogenous polyamines, combined with naphtalen-acetic acid (NAA) or with 6-benzyl-aminopurine (BAP), to produce callus and its subsequent differentiation. The explants, isolated from field plants, were previously subjected to a basic cleaning method and were inoculated onto Murashige and Skoog culture medium (MS) [Murashige, T.S., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497] supplemented with NAA (2.0 mg L −1). Buds were subjected to different treatments, with or without 5.0 and 10.0 mmol L −1 exogenous polyamines (mixture of putrescine:spermine:spermidine, 1:1:1) combined with NAA. The calluses obtained were transferred into the same medium, supplemented with the mixture of polyamines combined with BAP, in order to induce plant differentiation. For C. longa, buds were the most efficient explants for callus induction ( p < 0.05). The application of exogenous polyamines (5.0 and 10.0 mmol L −1) produced the most developed callus, with numerous roots. The medium supplemented with 10 mmol L −1 polyamine mixture, combined with BAP, induced good regeneration, producing vigorous plants and excellent shoot formation.Polyamines addition promoted the formation of callus, roots and leaves, representing an important factor in the determination of indirect organogenesis in C. longa L., and putrescine content may be considered a valuable marker of the differentiation process in this specie, as well as the enzyme peroxidase.

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices ( Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.

Plant regeneration from in vitro cultured leaves of Lanzhou lily ( Lilium davidii var. unicolor)

Lanzhou lily ( Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l −1 TDZ and 1.0 mg l −1 NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l −1 indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.

Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium

Incorporation of a range of higher concentrations of CuSO 4·5H 2O in MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with 240 tobacco tissue cultures. Physiol. Plant 15, 473–497] significantly enhanced direct shoot bud induction and proliferation from cultured leaf and nodal explants taken from mature plants of Stevia rebaudiana. Shoot bud induction medium was supplemented with BAP (2.2 μM) and IAA (2.8 μM). When the concentration of CuSO 4·5H 2O in the induction medium was raised to 0.5 μM (five times the MS level, i.e. 0.1 μM) there was significant increase in percentage response along with increase in shoot bud number per explant. The shoots were healthy, well developed with dark green broader leaves. There was remarkable increase in total biomass and chlorophyll content at increased (0.5 μM) copper level in the medium. During proliferation stage also presence of high copper levels in the medium favoured increase in shoot bud number per explant.

Improved shoot regeneration due to prolonged seed storage

Regeneration in vitro from cotyledon explants of commercial squash ( Cucurbita pepo L.) cultivars is generally efficient on Murashige and Skoog [Murashige, M., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15, 473–497] medium augmented with 4.4 μM benzyladenine. However, cotyledon explants from certain seed batches of cultivars Bareqet and Ma’yan could regenerate only buds, leaf primordia or very small shoots with storage for up to 2 years at 4 °C. Seed storage of cultivars Bareqet and Ma’yan at 4 °C for 6–8 years resulted in significant increases in shoot regeneration, shoot growth and explant growth, returning to the normal range of values for C. pepo. For example, for cv. Bareqet shoots regenerated per explant increased from 0.4 after storage for 2 years to 1.21 after storage for 8 years; shoot length increased from a mean of less than 2 mm after storage for 2 years to 22 mm after storage for 8 years. Additionally, the final explant fresh weight of cv. B increased from 181 mg after storage for 2 years, to 1389 mg after storage for 8 years. Similar responses were observed for seedling-derived explants of cv. Ma’yan following storage’ for 2–7 years. However, total regeneration (number of explants regenerating buds, leaf primordia or shoots) was not affected by prolonged storage for either cultivar. This is the first report of stimulation of in vitro shoot regeneration of a seedling-derived organ caused by prolonged seed storage. Moreover, the great improvement in regeneration due to long-term seed storage provides a new mechanism for the understanding of non-repeatability of plant tissue culture results.

Cadmium accumulation and its effect on the in vitro growth of woody fleabane and mycorrhized white birch

The effect of Cd on woody fleabane ( Dittrichia viscosa (L.) Greuter) and white birch ( Betula celtiberica Rothm. & Vasc.) was examined. Woody fleabane and white birch were grown in vitro in Murashige, T., Skoog, F., [1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–479] (MS) plus Cd (10 mg Cd kg −1) and except for root length in white birch, plant development was inhibited when Cd was added. Cd accumulation in above-ground tissues showed differences among clones, reaching 1300 and 463 mg Cd kg −1 dry wt. in selected clones of woody fleabane and white birch, respectively. Tolerance of Paxillus filamentosus (Scop) Fr. to Cd was also examined before mycorrhization. Plants of mycorrhized white birch grown in the presence of Cd had a better development and accumulated more Cd in their shoots than the non-mycorrhized ones. The use of selected clones of woody fleabane and the mycorrhization of white birch enhance extraction efficiency from contaminated soils in phytoremediation programs.

Effect of genotype, explant size, position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l −1 thidiazuron (TDZ), 0.8 mg l −1 adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l −1 indole-3-butyric acid (IBA), 0.5 mg l −1 6-benzyladenine (BA) and 2.0 mg l −1 kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l −1 IBA. All plantlets acclimatized well in the greenhouse.

Effect of sucrose concentrations on somatic embryogenesis in carnation (Dianthus caryophyllus L.)

Abstract The effect of sucrose concentration on callus induction followed by differentiation of embryogenic callus derived from petal explants of four carnation cultivars (Nelson, Sagres, Spirit and Impulse) was investigated. Embryogenic calli were produced on Murashige and Skoog [Murashige, T., Skoog, F.A., 1962. Revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 154, 73–479] basal medium (MS) culture medium containing six concentrations of sucrose (3, 6, 9, 12, 15 and 18%, w/v) all supplemented with 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 μM 6-benzyladenine (BA). Maximum frequency of embryogenic callus was obtained from the media containing 9 and 12% sucrose. Somatic embryos were induced on a hormone-free MS media containing the seven concentrations of sucrose. Development of somatic embryos was enhanced by increasing sucrose concentration from 1.5 to 12%, while it was reduced in higher concentrations of 15 and 18%. However, normal embryos were not developed in the media containing 1.5 and 3% sucrose. Ninety-five percent of somatic embryos were regenerated to form the entire plantlets when they transferred onto the half-strength hormone-free MS culture medium containing 3% sucrose. Plantlets were also continued to grow normally under greenhouse condition.

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.

Development of an embryogenic suspension culture of bitter melon (Momordica charantia L.)

Abstract We have optimized a system for the somatic embryogenesis via embryogenic suspension cultures in bitter melon (Momordica charantia L.). Friable calli could be induced in 30-day-old leaves on semi-solid MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.] medium supplemented with 1.0 mg/l 2,4-D. Large number of globular embryos (24.6%) were noticed when the calli was subcultured in liquid medium containing 1.5 mg/l 2,4-D. The complete removal of 2,4-D in the later stages of culture, stimulated their further development to heart and torpedo stages. Microscopic examination revealed the ontogeny of somatic cell development via the formation of cell clusters, which then enlarged to pro-embryos, and gave rise to heart and torpedo stages within a period of 2 weeks. Somatic embryos successfully germinated on agarified MS medium with no additional growth regulators. An effect of media, other components and stimulating factors such as carbohydrates, amino acids has also been evaluated for somatic embryogenesis. The full strength MS medium containing 50 mg/l PVP and 40 mg/l glutamine was effective to achieve a high frequency of somatic embryo induction, maturation and further development. An average of 6.2% young plants was achieved from friable callus and was phenotypically normal. To our knowledge, there is no published report on somatic embryogenesis of bitter melon (M. charantia L.) via embryogenic callus or cell suspension cultures. These results are likely to facilitate genetic transformation of bitter melon.

Micropropagation of Maytenus senegalensis (Lam.) Excell

A micropropagation protocol for Maytenus senegalensis was established using in vitro germinated 6-week-old seedlings as a source of explants. Shoot proliferation was only achieved using nodal explants. The presence of cytokinins was necessary for shoot induction and growth. While benzyl-6-aminopurine (BA) generally promoted the average number of shoots produced per explant, kinetin promoted the mean length of shoots. Inclusion of auxin (IAA and IBA) in the shoot induction and growth medium did not have significant effects on either the average number of roots produced per shoot or the mean length of shoots. Low levels of BA (0.5 or 1.0 mg l − 1 ) were optimal for shoot induction and growth. As a result, shoot multiplication was done on a medium supplemented with BA (0.5 mg l − 1 ) alone. Rooting was achieved in 2 stages. In the first stage, 8-week-old shoots (2–3 cm long) were pulse-treated in the dark using ½ strength, MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologia Plantarum 15, 473–497] liquid medium containing auxins and then transferred to a solid hormone-free medium at a 12/12 h photoperiod. The shoots pulse-treated with 25 mg l − 1 IBA for 120 h produced the highest number of roots per shoot. IBA concentration and the period of pulse treatment had significant effects on the average number of roots produced per shoot. The rooted plantlets were successfully acclimatized.