Plant Growth Regulation 28: 41–47, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Role of endogenous purine metabolism in thidiazuron-induced somatic embryogenesis of peanut (Arachis hypogaea L.) J.M.R. Victor, B.N.S. Murthy, S.J. Murch, S. KrishnaRaj & P.K. Saxena∗ Department of Plant Agriculture, Biotechnology Division, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 (∗ author for correspondence) Received 4 December 1998; accepted in revised form 25 February 1999

Key words: cytokinin, diaminopurine, somatic embryogenesis, thidiazuron

Abstract Somatic embryogenesis was induced at the hypocotyledonary notch region of intact peanut (Arachis hypogaea L.) seedings cultured on a medium containing 10 µmol·L−1 thidiazuron (TDZ). Inclusion of the purine analogs 2,6-diaminopurine (DAP), azaadenine or azaguanine to the thidiazuron amended medium inhibited the embryogenic response of the seedlings. DAP-mediated inhibition was not overcome by the addition of adenine sulphate. Inhibition of the embryogenic response by DAP provides evidence that the TDZ-induced accumulation of purine cytokinins is an essential component of TDZ-induced somatic embryogenesis process. Analyses of the endogenous level of purine metabolites indicated that supplementation of the media with TDZ resulted in an overall increase in the endogenous cytokinins while DAP inhibited the purine recycling resulting in decreased levels of endogenous adenine and zeatin.

1. Introduction Somatic embryogenesis, the ability of individual plant cells to produce embryos in response to appropriate stimuli, is a poorly understood physiological process. In most culture systems, somatic embryogenesis is induced by modulation of the ratio of auxin to cytokinin [2, 11, 21]. The role of auxin in the induction of somatic embryogenesis has been well documented [12] and specifically in peanut, the induction of somatic embryogenesis is dependent on the auxin content of the medium [3, 7, 20]. However, the role of cytokinins in embryo induction and development is poorly understood due, in part, to a lack of cytokinindependent experimental systems and effective inhibitors of cytokinin biosynthesis and metabolism [10]. Thidiazuron [TDZ: N-phenyl-N0-(1,2,3-thidiazol5-yl)urea] a synthetic cytokinin effectively induced somatic embryogenesis in peanut and other legumes [19]. This process of TDZ-induced somatic embryogenesis offers several unique opportunities to investigate the role of plant growth regulators in somatic embryogenesis as in this system: (a) the embryo-

genic response is induced in the absence of any other growth regulating compounds, (b) intact seedlings are cultured allowing for the investigation of the physiological responses and interactions of different plant tissues, and (c) both endogenous auxins and cytokinins accumulate in peanut seedlings exposed to TDZ [17]. The objective of the current research was to investigate the role of endogenous cytokinin metabolism during the process of somatic embryogenesis. Several inhibitors of purine biosynthesis which mediate TDZ-induced somatic embryogenesis have previously been identified [9]. Diaminopurine (DAP) is a purine analogue which inhibits cell growth and multiplication through inhibition of the purine salvage pathway enzyme adenine phosphoribosyltransferase (APRT) [8, 13]. Two other inhibitors of purine biosynthesis, azaadenine and azaguanine, have also been identified as potential inhibitors of TDZ-induced somatic embryogenesis [9]. Therefore, the specific objectives of the current research were: (a) to characterize the inhibition of TDZ-induced somatic embryogenesis by inhibitors of purine biosynthesis, (b) to determine the role of endogenous cytokinins in peanut somatic

42 embryogenesis, and (c) to quantify differences in the levels of endogenous purines in response to purine synthesis inhibitors.

2. Materials and methods 2.1 Plant material Mature seeds of peanut (Arachis hypogea L. cv. Tango) obtained from Dr T.E Michaels, Crop Science Department, University of Guelph, were used throughout the study. Seeds were carefully selected for uniformity and surface sterilized by immersing in 70% ethanol for 3 min. followed by immersion in 30% solution of commercial bleach (sodium hypochlorite), containing 1 drop of Tween 20 for every 100 ml of the solution, for 20 min. The seeds were continuously stirred using a magnetic stirrer throughout the sterilization process. The sterilized seeds were then washed 5 times in sterile distilled water. The seed coats from the sterilized seeds were aseptically removed and 4 seeds were cultured in each Magenta box (300 ml plastic culture vessels; Magenta Corp., Chicago, IL, USA) containing 60 ml of culture medium. The basic culture medium contained Murashige and Skoog salts [14], 3% sucrose, and B5 vitamins [5]. For experiments with the purine inhibitors, azaadenine (AA), azaguanine (AG) and 6-diaminopurine (DAP), (0, 1, 5, 10, 25, 50 and 100 µmol·L−1 ) were added to MS basal medium or to MS basal medium supplemented with 10 µmol·L−1 thidiazuron (TDZ). The potential reversal of the DAP inhibition was examined by incorporating adenine sulphate (AS: 0, 1, 5, 10, 25, 50 and 100 µmol·L−1) into the MS basal media or MS basal medium supplemented with 10 µmol·L−1 TDZ and 10 µmol·L−1 DAP. The pH of all media was adjusted to 5.7 and 3 g·L−1 Gelrite was added to the medium (Schweizerhall, South Plainfield, NJ, USA) prior to autoclaving. The cultures were incubated in the dark at 24 ◦ C for the first seven days followed by incubation in the light (30–50 µE·m−2 ·s−1 ) with a 16h photoperiod at 24 ◦ C. The number of somatic embryos induced were counted on day 35 of the incubation period for each of sixteen replicate seedlings per treatment and the experiments were repeated twice. Data were analysed by Duncan’s multiple range test (P < 0.05).

Figure 1. Effect of TDZ and DAP on the morphology of peanut seedlings. (A) Peanut seedling cultured on media containing 10 µmol·L−1 TDZ (bar = 0.45 cm); (B) Peanut seedling cultured with 10 µmol·L−1 TDZ and 10 µmol·L−1 DAP (left) or 10 µmol·L−1 DAP (right) (bar = 0.66 cm).

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Figure 2. Inhibition of TDZ (10 µmol·L−1 ) induced somatic embryogenesis in peanut by various concentrations of Azaadenine, Azaguanine and DAP. Bars denoted by the same letters are not significantly different at P = 0.05.

2.2 Endogenous cytokinin analysis

3. Results

Four replicate peanut seedlings, germinated on media supplemented with 10 µmol·L−1 TDZ, with or without 10 µmol·L−1 DAP, were harvested from different Magenta boxes on days 0, 1, 3, 5 and 7 of the culture period. The endogenous cytokinin content was analysed as described previously [17]. Briefly, cytokinins were extracted in a 80% methanol solution, dried under vacuum (SpeedVac model SVC200 and Refrigerated Condensation Trap, Savant Instruments, Farmingdale, N.Y.; Vacuum pump model DD-20, Precision Scientific Inc. Chicago, IL) and resuspended in a sample diluent buffer (0.5 mmol·L−1phosphate buffer, pH 7.4 with 5% acetonitrile). An aliquot of 20 µl of each sample was injected into the HPLC system for quantification (LC Module 1 complete system, Millipore/Waters Inc. Mississauga, ON, Canada). Samples were separated on a CSC Hypersil ODS 5 µm column (25 × 0.46 cm: Scientific Products and Equipment, Concord, ON, Canada) and were eluted from the column with the buffers and gradient described previously [17]. The identification and quantification of individual components was confirmed by GC-MS as described earlier [17]. The experiment was repeated twice and data was analysed by the GLM procedure of SAS [18].

3.1 Morphology of peanut seedlings Seeds cultured on all of the treatments germinated within 3 days of culture initiation. There was no difference in either the rate or frequency of germination as a result of supplementation of the media with either TDZ or the cytokinin analogues. Seedlings grown on media containing TDZ exhibited the characteristic stunted growth habit with dark green swollen cotyledons, shortened, thickened primary roots with few or no secondary root growth and the formation of somatic embryos at the hypocotyledonary notch region (Figure 1). Addition of DAP, AA or AG to the TDZ containing medium did not significantly alter the seedling morphology and none of the cytokinin analogue treatments induced somatic embryogenesis in the absence of TDZ. However, unique responses were observed in seedlings grown on media containing DAP, AA or AG in the absence of TDZ. Seedlings grown on media containing 50 or 100 µmol·L−1 AA were pale with a slight reduction in shoot and secondary root development. Similarly, the seedlings cultured on higher concentrations of AG (50 and 100 µmol·L−1 ) demonstrated unusual shoot and root morphologies with the tips of the roots forming callus to produce a ball of

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Figure 3. Partial reversal effect of embryogenic response in peanut by adenine sulphate (AS) in TDZ (10 µmol·L−1 ) and DAP (10 µmol·L−1 ) treated seedlings. Bars denoted by the same letters are not significantly different at P = 0.05.

compact tissue. Supplementation of the medium with DAP induced early flowering in the cultures, stems appeared thicker and the formation of secondary roots was significantly reduced. 3.2 Culture response to inhibitors of cytokinin 3.2.1 Response to azaadenine Addition of azaadenine (AA) to 10 µmol·L−1 TDZ supplemented MS medium resulted in a significant reduction in embryogenic response compared to the TDZ-supplemented control seedlings (10 µmol·L−1 TDZ, Figure 2). Inhibition of the embryogenic response was evident even at the lowest AA (1 µmol·L−1 ) concentration. A maximum of 17 embryos per seedling was produced in seedlings treated with a combination of TDZ (10 µmol·L−1 ) and 1 µmol·L−1 AA, compared to 46 embryos produced in the absence of AA. The rate of inhibition of embryogenesis remained more or less constant in seedlings treated with 10 to 100 µmol·L−1 AA. Morphologically, these seedlings resembled those treated with TDZ (10 µmol·L−1 ). The embryos produced on medium containing 1, 5, 10 µm AA in combination with 10 µmol·L−1 TDZ appeared larger and healthier than the embryos formed on TDZ grown seedlings and the embryos were well isolated from each other and evenly distributed in the hypocotyledonary region.

3.2.2 Response to azaguanine The seedlings cultured on MS media supplemented with 10 µmol·L−1 TDZ and AG (1–100 µmol·L−1 ) exhibited a reduction in embryogenic potential similar to AA and TDZ treated seedlings. The number of somatic embryos induced per seedling decreased from approximately 27 to 8 as the concentration of AG increased in the culture medium from 1 to 100 µmol·L−1 . The embryos initiated on AG+TDZ containing media were morphologically smaller than those produced on seedlings grown on medium containing TDZ (10 µmol·L−1 ) alone.

3.2.3 Response to diaminopurine DAP also inhibited the embryogenic potential of the seedlings significantly (Figure 1). The seedlings grown on media supplemented with TDZ (10 µmol·L−1 ) and DAP (1–100 µmol·L−1), showed a significant reduction in embryogenic response even at lower concentrations. Seedlings grown on DAP concentrations greater than 50 µmol·L−1 produced only a relatively few, odd, embryos distributed in the hypocotyledonary notch region. Inclusion of adenine sulphate (AS) to the medium containing both TDZ and DAP (10 µmol·L−1 ) did not consistently reverse the inhibitory effects of DAP (Figure 3).

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Figure 4. Effect of TDZ (10 µmol·L−1 ) and DAP (10 µmol·L−1 ) on endogenous cytokinins and related metabolites. (A) adenine, (B) adenosine, (C) dihydrozeatin, (D) zeatin, (E) 2-isopentenyladenosine.

3.3 Changes in endogenous purine metabolites in response to DAP and TDZ The concentrations of endogenous purine metabolites were quantified in seedlings germinated on media containing TDZ and DAP. TDZ induced significant increases in the concentrations of adenine (Ade), adenosine (Ado), dihydrozeatin (DHZ) and zeatin (Zea) in the first seven days of culture (Figure 4). Seedlings cultured on media containing only DAP had significantly lower concentrations of endogenous Ade and Ado but had significantly more 2-isopentenyladenosine (2iP) and DHZ than controls. Addition of DAP to the TDZ containing media significantly decreased the levels of Ade, DHZ and Zea

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Figure 5. Proposed sites of inhibition of purine metabolism for TDZ and DAP.

compared to seedlings cultured on media containing only TDZ (Figure 4). In contrast, the TDZ-induced decrease in 2iP concentration was partially reversed by supplementing DAP to the TDZ-containing culture medium (Figure 4).

4. Discussion TDZ effectively induced somatic embryogenesis in intact peanut seedlings and the embryogenic response was inhibited by the purine analogs AA, AG or DAP. Previous studies have demonstrated an overall acceleration in purine metabolism and accumulation of adenylate compounds in response to TDZ [9, 15, 17]. Incorporation of TDZ in the medium resulted in accumulation of endogenous adenine, adenosine, zeatin and dihydrozeatin and a decrease in the endogenous level of 2iP. This varied modulation of endogenous metabolites substantiates the inhibitory role of TDZ on cytokinin oxidase (Figure 5) [6]. It is therefore likely that TDZ-treatment modifies the metabolism of adenylate compounds by redirection of the cytokinin synthetic route through isopentenyl-AMP and zeatinriboside to zeatin. Also, the TDZ mediated alteration in the cytokinin biosynthetic pathway would therefore be responsible for the depletion of the endogenous 2iP pool and the elevated concentrations of the other purine metabolites. Supplementation of the TDZ medium with DAP inhibited the embryogenic response and also inhibited the TDZ-induced accumulation of adenine, dihydrozeatin and zeatin. In addition, DAP increased the concentration of 2iP over the level observed in TDZ-treated seedlings. Although, adenine sulphate completely reversed the inhibition of somatic embryogenesis in geraniums [9], it did not reverse the inhibition of regeneration in peanut. This may reflect

either an interaction of the various tissues within the seedlings or a unique TDZ-mediated physiological response in peanuts. Also in contrast to the present study, there was no reduction in levels of endogenous growth substances as a result of supplementing DAP in geranium hypocotyl cultures. Instead, DAP caused a further increase in the levels of cytokinins over that observed in response to TDZ treatment [9]. Moffat and Sommerville [13] have suggested that DAP inhibits the enzyme adenine phosphoribosyltransferase (APRT) in the purine salvage pathway (figure 5). The result of this inhibition would be an overall decrease in all of the purine metabolites, similar to that observed in the present investigation. In addition, DAP may interfere with the de novo synthesis of purine nucleic acids resulting in an reduction of the level of available metabolic energy as ATP, NADH and NADPH [1, 22]. Earlier, we found that TDZ induced an accumulation in metabolic energy concurrent with the application of the chemical to intact plants [15]. One potential mode of action of TDZ is the induction of a physiological stress as a result of the applied chemical stress and a chemically-induced mineral stress [15, 16]. We previously hypothesized that the formation of regenerants is an adaptive mechanism for the plant survival of the chemical stress [15]. In this scenario, the inhibition of the embryogenic response in peanut seedlings reflects a lack of available ATP, ADP, AMP, NADPH and NADH for the morphogenic processes necessary for the plant to survive TDZ-induced physiological stress. Our result have clearly demonstrated that the TDZinduced accumulation of purines, either as cytokinins or as sources of metabolic energy, is an essential component of the TDZ-induced somatic embryogenesis process. During somatic embryo development rapid cell division and protein synthesis take place and a continuous supply of purine and pyrimidine bases is necessary to maintain physiological processes and allow for the rapid cell growth and division required for regeneration [4]. TDZ increases the available supply of purines for cellular development and when this accumulation of purine metabolites was decreased by addition of inhibitors, the number of resultant somatic embryos formed was reduced. It is likely that the supplementation of the culture medium with adenine sulphate was insufficient to overcome the DAPmediated inhibition of purine metabolism or to provide the specific purine metabolites required for regeneration. Although the exact mechanism by which this occurs remains unclear, our results indicate that the

47 inhibition of these biosynthetic processes has a consequential effect on the morphogenetic development.

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