Plant Growth Regulation (2005) 47:59–64 DOI 10.1007/s10725-005-1714-z
Ó Springer 2005
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Integrated application of physiological and molecular methods to forecast determinative morphogenetic events in tissue cultured tobacco (Nicotiana tabacum L. cv Samsun) leaf discs O. Toldi1,*, , K. Ahanen2, G. Kova´cs1, S. Sorvari2, S. To´th1, S. Dulai3 and P. Scott4 1
Agricultural Biotechnology Center, H-2101 Go¨do¨ll} o, P. O. Box 411, Hungary; 2MTT Agrifood Research Finland, Institute of Horticulture, FIN-21500 Piikkio¨, Finland; 3Eszterha´zy Ka´roly College, H-3300 Eger, Eszterha´zy te´r 1, Hungary; 4School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK; *Author for correspondence (e-mail:
[email protected]; phone: +36-28-526100; fax: +36-28-526192)
Received 23 October 2003; accepted in revised form 28 July 2005
Key words: Aerobic respiration, DNA methylation, Metabolism, Nicotiana tabacum L., Photosynthesis, Tissue culture, Tobacco
Abstract The purpose of this study was to search for physiological parameters that provide an early indication of the morphogenetic response of leaf disc explants to different tissue culture-level manipulations in order to design an accelerated optimisation process for this technology. Tobacco (Nicotiana tabacum L. cv. Samsun) was chosen as model in our studies, because this is still one of the most widely used species in experimental plant biology and detailed knowledge is available from its tissue culture system. Large numbers of physiological markers at the whole plant level (rates of photosynthesis and respiration) and cellular level ‘fitness’ (degree of DNA methylation) were measured together with the concentrations of the most abundant metabolites in photosynthetic carbohydrate metabolism, ATP and protein synthesis in leaf discs induced either for callus development or for shoot differentiation. As a result of the above examinations, we are able to show that the efficiency of photosynthesis, the rate of respiration and the degree of DNA methylation can be used as early markers for the changes that precede the appearance of in vitro morphogenesis. By examining these parameters, we were able to predict early ontogenesis (within 48 h) and the optimal concentrations of growth regulators needed to achieve either shoot differentiation or callus development.
Introduction The major obstacle in the genetic manipulation of many plant species continues to be the lack of an effective tissue culture system for the regeneration of whole plants from tissue explants.
Collaborator via a fellowship under the OECD Co-operative Research Programme: Biological Resource Management for Sustainable Agriculture Systems
Despite extensive experimentation work, conventional tissue culture techniques have not resulted in a real breakthrough in solving the unpredictable nature of plant tissues in in vitro culture (Birch 1997). In our opinion there is an opportunity to incorporate molecular and physiological approaches to improve the reliability of tissue culturing to meet future challenges (Toldi et al. 2001). This is particularly apparent when developing new tissue culture systems or when
60 optimising the procedures for a recalcitrant species with a view to reducing the time until the final evaluation. In this study we have investigated how several growth regulators influence metabolism and development in tissue culture, with the aim of developing a methodology for predicting and optimising morphogenesis to accelerate tissue culture development. To achieve this the volume of metabolite flux through photosynthetic carbohydrate metabolism, ATP and protein synthesis were measured in tobacco leaf discs induced either for callus development, or shoot differentiation. To complement metabolite analysis, rates of photosynthesis and respiration were monitored, as measures of physiological condition, and the degree of DNA methylation as a reference refers for cell volume proliferation and transcriptional activity (LoSchiavo et al. 1989). The novelty in our approach is the approach itself, that is, the use of physiological and molecular methods to optimise tissue culture procedures.
for 48 h at 65 °C. At the same time, the optimal concentrations of BAP and NAA for shoot regeneration were selected on the basis of the efficiency of shoot differentiation also after 3 weeks-long incubation. Samples for metabolite assays and physiological measurements were taken initially after the 3 h of incubation, then in every 24 h at 12:00 over the subsequent 3 weeks. Photosynthetic and respiration rates were measured in normal air (345 ppm CO2, 21% O2, in N2) with an infrared gas analyser (Ciras-1, PP Systems, Unit 2, Herts, UK) in an open gas-exchange system. The white light for excitation of photosynthesis was provided by a Schott KL-1500 light source through a fiberopticable. Measurements were replicated three times under different illumination intensities (12.5, 25.0, 50.0, 75.0, 100.0 lmols/m2/s1). The rates of net CO2 fixation were calculated by using the equations of von Caemmerer and Farquhar (1981). The mean maximum values are shown in the Figure 1a,b. c
Materials and methods Tobacco plants (Nicotiana tabacum L. cv Samsun) were grown aseptically on growth regulator-free MS medium (Murashige and Skoog 1962) that contained MS salts and vitamins, 25 g/l sucrose and was solidified with 8 g/l agar. The pH of the micropropagation media was adjusted to 5.8 by 1 M KOH prior to autoclaving. Plantlets were cultured on the above media in VitroVent plastic containers (Duchefa Biochemie bv., Harleem, The Netherlands) under a 16 h light (approximately 69.5 lmols/m2/s1)/8 h darkness photoperiod at 24 ± 2 °C. Leaf discs (1 cm in diameter) from immature leaves and mature leaves were excised from in vitro grown plants and were transferred into different callus induction media that contained MS salts, vitamins and a gradient of 2,4-D concentrations (2.5, 5.0, 7.5, 10.0, 12.5 lM) or into shoot regeneration medium containing MS salts, vitamins, different concentrations of BAP and NAA (2.5, 5.0, 7.5, 10.0, 12.5 lM) alone or in combination. Optimal concentration of 2,4-D for callus induction was defined on the basis of the increase of dry matter content in leaf disc explants. To determine dry matter content, calluses were excised from leaf discs and were dried in an owen
Figure 1. (a) Effects of different growth regulator treatments on the rate of respiration in tobacco leaf discs isolated from immature leaves (juvenile sink leaves) and from mature leaves (fully expanded source leaves) of in vitro grown plants after 3 and 48 h incubation. Statistical analysis as in Tables 1 and 2. Columns with asterics were significantly different from other sample groups according to the LSD test (p = 0.05) following ANOVA. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BAP – 6-benzylaminopurine; NAA – 1-naphthaleneacetic acid; m – leaf discs isolated from mature leaves; m + 2,4-D – mature leaf discs incubated on 5 lM 2,4-D containing MS media; m + BAP, NAA – mature leaf discs incubated on 5 lM BAP and 2.5 lM NAA containing MS media. (b) Effects of different growth regulator treatments on the rate of photosynthesis in tobacco leaf discs isolated from immature leaves of in vitro grown plants after 48 h incubation. Abbreviations and statistical analysis as in Figure 1a, Tables 1 and 2. Symbols indicate significant decrease (*) or increase ( ) compared to growth regulator-free controls according to the LSD test (p = 0.05) following ANOVA. (c) Optimisation of 2,4-D concentrations in tobacco leaf discs isolated from immature and mature leaves during callus induction by measuring 5-methyl cytosine to total cytosine % after the first 48-h of incubation. Abbreviations and statistical analysis as in Figure 1a and Table 1. Columns with asterics were significantly different from other sample groups according to the LSD test (p = 0.05) following ANOVA. (d) Combined optimisation of BAP and NAA concentrations in immature leaf discs during shoot differentiation by measuring 5-methyl cytosine to total cytosine % after the first 48-h of incubation. Abbreviations and statistical analysis as in Figure 1a and Table 2. Columns with asterics were significantly different from other sample groups according to the LSD test (p = 0.05) following ANOVA.
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62 For DNA purification, freeze-dried leaf tissues (1 g FW/sample) were ground with quartz sand, treated with pronase and extracted with chloroform:octanol according to Citti et al., (1983). The methylcytosine/cytosine ratio was measured for DNA hydrolysed with formic acid in sealed glass vials and subsequently run on a C8 reverse-phase column in HPLC and the ratio was determined as described by Citti et al., (1983). Results for continuous variables are expressed as means ± SEM. Significance of differences between treatment groups were determined using Student’s t-test for unpaired samples, analysis of variance (ANOVA), and Fisher’s LSD test as appropriate. If differences were considered significant for p < 0.05, means were separated by LSD at p = 0.05.
Results and discussion The aim of this study was to investigate how plant growth regulators influence metabolism and development in tissue culture, in order to develop a methodology for forecasting and optimising in vitro morphogenetic processes. More specifically we were looking for physiological parameters that show measurable and specific alterations prior to visible morphogenetic changes, such as callus growth, or shoot differentiation. Data of Table 1 demonstrate that incubation of leaf discs, excised from immature and mature leaves, on 5.0 lM 2,4D containing media resulted in the highest increase of dry matter contents in both types of explants. At the same time, immature leaves responded to a significantly greater extent to 2,4-D treatment than the ones originating from mature leaves. Dry matter increases for different age leaf discs were the lowest in the absence of 2,4-D (control) and at the highest concentration of 2,4-D (12.5 lM). At
the same time, the frequency of shoot differentiation was most efficient when BAP (5 lM) and NAA (2.5 lM) were applied in combination although BAP treatment alone has also resulted in some shoot development (Table 2). These methods were used to model tissue dedifferentiation (callus induction) and tissue differentiation (shoot regeneration) during the following measurements. Concentrations of some abundant metabolites and rates of photosynthesis and respiration were determined as well in relation to the photosynthetic carbohydrate metabolism (3-PGA, Glc, Glc1-P, Glc-6-P, Fru, Fru-6-P, Suc, starch), ATP and protein synthesis (AMP, ATP, free amino acids, glutamine, asparagine, total protein). No significant differences were found between the immature and mature leaf samples, as there were also no differences between the callusing and regenerating tissues in the concentrations of the above metabolites prior to visible morphogenesis (data not shown). At the same time, rates of photosynthesis and respiration, as well as the degree of DNA methylation, changed rapidly and significantly to be useful in predicting future morphogenetic events after 48 h of its hormonal induction. It has been shown that respiration is enhanced and photosynthesis is supressed simultaneously during auxin-induced growth (Halmann 1990). Utilyzing this phenomenon, some synthetic auxins (2,4-D and 2,4,5-T) are used in agricultural practice, because the increased respiration, paralelled by decreased photosynthesis, results in faster fruit ripening and seed maturation in numerous crop plants (Halmann 1990). The data presented here suggests that the application of 2,4-D (5 lM) to immature leaf discs of tobacco also stimulates the rate of respiration in vitro (Figure 1a). This stimulation was twice that observed in immature leaf discs incubated in the absence of 2,4-D. No such
Table 1. The degree of callus development on 2,4-D-treated immature and mature leaf discs was estimated on the basis of dry matter contents of calluses after a 3 weeks-long incubation period (±SEM). Dry matter (% of callus FWs) 2,4-D concentrations: immature leaves mature leaves
0 lM 2.2 ± 0.7 0
2.5 lM 10.9 ± 2.2* 5.6 ± 1.9
5.0 lM 18.2 ± 3.0* 9.1 ± 2.1*
7.5 lM 14.7 ± 2.8* 8.7 ± 2.4*
10.0 lM 12.1 ± 2.0* 7.3 ± 1.6*
12.5 lM 7.9 ± 1.5* 5.4 ± 1.7
Data of two groups of tobacco leaf discs – immature and mature leaf originated – were analysed by ANOVA followed by Fisher’s LSD test. An asterisk indicates where both types of leaf discs show significant difference (p < 0.05) compared to growth regulator-free control, and a dragger indicates where immature leaf-originated leaf discs show significantly increased values compared to mature leaforiginated leaf discs (p < 0.05). Fifty leaf discs were evaluated in each treatment in 3 repetitions.
63 Table 2. The frequency of shoot differentiation on immature leaf explants of tobacco after a 3 weeks-long incubation period (±SEM). Number of de novo developed shoots per leaf discs Concentrations of growth regulators: BAP NAA 5lM BAP+NAA
0 lM 0 0 7.2 ± 1.4*
2.5 lM 2.9 ± 0.5 1.1 ± 0.3 16.8 ± 3.0
5.0 lM 7.2 ± 1.4* 0.6 ± 0.2 12.3 ± 3.1
7.5 lM 5.3 ± 1.1 0 6.4 ± 1.7
10.0 lM 6.1 ± 1.3 0 3.9 ± 1.9
12.5 lM 3.9 ± 1.5 0 2.0 ± 1.5
Data of three groups of tobacco leaf discs – BAP-treated, NAA-treated and BAP-NAA-treated – were analysed by ANOVA followed by Fisher’s LSD test. A dagger indicates where the combined use of BAP and NAA resulted in significant increase in the frequency of shoot differentiation (p < 0.05) compared to single BAP or NAA-treatments. Fifty leaf discs were evaluated in each treatment in 3 repetitions. * marks two identical treatments.
effect was noted in tissue cultured, mature leaves of tobacco. This marked increase in respiration rate and immediate inhibition of photosynthesis (Figure 1b) – measured after 3 and 48 h incubation respectively – preceded the formation of callus from the immature leaf discs allowing us to predict future morphogenetic events within the first 48 h induction. It may be that such an increase in respiration rate in tissues exposed to 2,4-D (or other synthetic auxins) is typical of the most responsive tissue explants prior to an efficient callus formation process. Whereas callus forming ability of tobacco leaf discs were closely associated with increased respiration rate and suppressed photosynthesis, on similar explants – incubated on BAP and NAA containing media – the de novo shoot differentiation was preceded by a significant increase in the efficiency of photosynthesis (Figure 1b). It is known that cytokinins stimulate photosynthesis, uptake and import of nutrients in vivo (Halmann 1990). Leaf discs, treated with the optimal concentration of cytokinin (5 lM BAP), also showed a twofold increase in rates of photosynthesis compared to control leaves in vitro. It may be that the elevated uptake of nutrients – maintained by BAP – supported photosynthesis and directed the morphogenesis towards shoot differentiation that requires a much more intense assimilation than the callus growth in tobacco. After determining primary morphogenetic routes, fine-tuning of the concentrations of applied growth regulators was carried out by monitoring of 5-methylcytosine/cytosine ratio as marker of cell cycling activity. The demethylation of cytosine is usually associated with a higher transcriptional activity and a higher rate of cell proliferation (LoSchiavo et al. 1989). In agreement with this, the
rates of cell proliferation in tobacco tissue cultures were highest where the proportion of methylated cytosine was the lowest both in callus induction (Table 1, Figure 1c) and in direct shoot regeneration (Table 2, Figure 1d). On the basis of the more sensitive and significantly stronger response of immature leaf discs to 2,4-D, BAP and NAA (Figure 1a,c), we could verify that immature leaves are more suitable explants for tissue culture level manipulations than the mature leaves. Thus monitoring 5-methylcytosine/cytosine ratio that has been used previously in molecular and cell biology, could be incorporated into tissue culturing providing a new tool for the establishment of novel tissue culture systems. Because of their increased responsiveness to hormonal treatments, leaf discs isolated from immature leaves were used only for further experiments. To make the above measurements complete, not only were the impacts of a single growth regulator on the DNA methylation determined, but also the combined effects of different growth regulators (BAP + NAA) applied simultaneously to induce shoot differentiation (Figure 1d). First, optimal concentrations of the given growth regulators were stated one-by-one, then we checked whether their joint application results in a further significant decrease in the methylation level or not. Data of Figure 1d shows that the joint application of 5 lM BAP and 2.5 lM NAA enhanced their morphogenetic effects synergistically as evident from the frequency of shoot differentiation (Table 2). Taken together, since integrated monitoring of photosynthetic performance, rate of respiration and DNA methylation provides early information about the morphogenetic response of a given plant explant, this method combination can serve as an experimental tool during the optimisation or adaptation of a new tissue culture procedure.
64 Acknowledgement This project was supported by an OECD Cooperative Research Programme entitled as ‘Biological Resource Management for Sustainable Agriculture Systems’.
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