Introduction

Somatic embryogenesis has been induced in several conifer species (Haggman et al. 2005); however, this has not yet been reported in L. leptolepis.

Abscisic acid (ABA) regulates many important aspects of plant development, including the synthesis of seed storage proteins and lipids, the promotion of seed desiccation tolerance and dormancy, and the inhibition of the phase transitions from embryonic to germinative growth and from vegetative to reproductive growth (Bellaire et al. 2000). ABA plays an important role in embryo development as well as growth and development throughout the plant life cycle. It has been shown that early stage somatic embryos of most conifers require the addition of 20–50 µM exogenous ABA in order to develop into cotyledonary stage (Stasolla et al. 2002). In several angiosperm and coniferous species, endogenous ABA was found to increase during the formation of mature zygotic embryos (Carrier et al. 1999; Kapik et al. 1995; Kong et al. 1997). In addition, ABA mediates some aspects of physiological responses to tolerance of water, salt, hypoxic and cold stress, and high osmoticum (Shinozaki and Yamaguchi-Shinozaki 2000).

Recently, there have been many experiments carried out on several plant species with the objective of explaining the role of oxidative stress in plant morphogenesis (de Marco and Roubelakis-Angelakis 1996; Cui et al. 1999; Papadakis et al. 2001; Papadakis and Roubelakis-Angelakis 2002). Oxidative stress is inevitably present in somatic embryo development as a particular cell differentiation. Active oxygen species (AOS) could be one possible link between oxidative stress and plant regeneration in tissue culture (Gupta and Datta 2003/2004). H2O2 is a form of AOS. H2O2 is generated via superoxide, presumably in a non-controlled manner, during electron transport processes such as photosynthesis and mitochondrial respiration. H2O2 generation is also induced in plants following exposure to a wide variety of abiotic and biotic stimuli. These include extremes of temperature, UV irradiation, ozone exposure, phytohormones, such as ABA, dehydration, wounding and pathogen challenge (Neill et al. 2002). When plants are exposed to stressful conditions, such as drought, low temperature, high salt, high light intensity or in vitro culture, H2O2 and other AOS can be produced in excess and accumulate in vitro. This phenomenon is commonly known as oxidative stress (Cassells and Curry 2001; Konieczny et al. 2008). Overproduction or accumulation of AOS in cells can disturb the redox system of the cell and influence the main metabolic pathways by direct changes in enzyme activity, membrane properties, lipids, DNA structure and other biomolecules (Cassells and Curry 2001). Plant cells possess well-developed systems of antioxidant enzymes such as SOD, CAT, POD, APX to regulate the level of AOS and the concentration of AOS to effectively reduce oxidative damage (Dat et al. 2000; Mittler 2002). SOD is the first enzyme in the detoxifying process, which converts superoxide anion to H2O2 (Gupta and Datta 2003/2004). The principal H2O2 scavenging enzyme in plants is CAT, which is located in peroxisomes, glyoxysomes and in mitochondria. Differences in the activity of the antioxidant enzymes during organogenesis and somatic embryogenesis in several plant species cultured in vitro were reported (Chen and Murata 2002). APX is a key H2O2 scavenging enzyme in the cytosol, chloroplast, and mitochondria of higher plants (Libik et al. 2005). Additionally, H2O2 is a weak oxidizing agent of relatively long lifetime and has the role of a secondary messenger involved in signal transduction via its influence on the expression of some genes (Neill et al. 2002; Vranov et al. 2002; Libik et al. 2005; Hung et al. 2005). H2O2 can induce the expression of genes encoding APX in germinating rice embryos and in Arabidopsis leaves (Karpinski et al. 1999), and wounding induced the expression of gene encoding a catalase via H2O2 in embryos and leaves of maize (Guan et al. 2000). H2O2 is also involved in the regulation of stomatal closure in response to ABA and elicitors (Pei et al. 2000).

Here, we measured the H2O2 content and compared the transcript level of three antioxidant enzymes genes (SOD, CAT and APX) during somatic embryo development of L. leptolepis cultured on different media. We have also discussed the relationship between the development of somatic embryos of L. leptolepis and the H2O2 content.

Materials and methods

Plant material

Immature seeds were collected from Dagujia Seed Orchard in Liaoning Province, China. These were disinfested with 0.1% HgCl and rinsed three times with distilled water. Seeds were cultured on MS-basal medium containing 30 g l−1 sucrose, 3 g l−1 Phytagel, 1.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l−1 benzyladenine (BA), 2 mg l−1 kinetin (KT), 500 mg l−1 glutamine, and 500 mg l−1 caseine hydrosylate (CH) to induce embryogenic callus. The pH of the medium was adjusted to 5.8.

Embryogenic callus was transferred to somatic embryo maturation medium consisting of MS basal medium, and containing 30 g l−1 sucrose, 3 g l−1 Phytagel, 29 mg l−1 abscisic acid (ABA), 106 g l−1 polyethylene glycol (PEG), 250 mg l−1 glutamine, and 250 mg l−1 CH. The pH of the medium was adjusted to 5.8. Embryo callus induction and propagation of pine were incubated in the darkness at 25°C.

Somatic embryos were collected at days 0, 1, 2, 3, 5, 7, 10, 14, 16, 21, 24, 35, and 45 following transfer to embryo maturation medium (Fig. 1). All samples were frozen in liquid Nitrogen, and stored at −70°C until RNA extraction.

Fig. 1
figure 1

Morphology of somatic embryogenesis of L. leptolepis at 13 time points. a day 1, b day 2, c day 3, d day 5, e day 7, f day 10, g day 14, h day 16, i day 21, j day 24, k day 35, and l day 45

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Measurement of H2O2

H2O2 was measured using the method of Ferguson et al. with slight modifications (Ferguson et al. 1983). Four replicates of somatic embryos at 13 time points (~0.5 g fresh weight) were homogenized in 1.5 ml cold acetone. The extract and washings were centrifuged for 10 min at 3,000 rpm. To 1 ml supernatant, 0.1 ml 5% Ti2SO4 and 0.2 ml NH4OH were added dropwise and mixed thoroughly. The mix was then centrifuged for 10 min at 3,000 rpm, and the precipitates were washed repeatedly with 2 ml acetone until the supernatant was colorless. The precipitates were solubilized in 5 ml 2 N H2SO4, made up to H2SO4 and filtered prior to measurement of A at 415 nm against a blank which had been carried through the same procedure. Standards in the range of 0–100 µmol l−1 H2O2 were also reacted with Ti2SO4 and carried through the procedure.

Quantitative RT–PCR assay of SOD, CAT and PAX transcript levels

Relative quantification of SOD, CAT and PAX expression in somatic embryogenesis was achieved by real-time RT–PCR. Total RNA was extracted from somatic embryo tissue at different developmental stages using the CTAB method. First-strand cDNA was synthesized using MMLV (Toyobo, Japan) according to the manufacturer’s instructions. Real-time RT–PCR was performed using SYBR Green (TOYOBO, Japan). Primers of genes used in real-time RT–PCR were designed according to the sequence of NCBI accessions. Forward (F) and reverse (R) as follows: SODF: 5′-CAGGAGACAACGGTCCCACA-3′, SODR: 5′-AACTCCATCAGAACCCGCAAC-3′, according to the accession number X58579.1, which amplifies a 216 bp fragment; CATF: 5′-GATCGTGCGTTTCTCAACTGT-3′, CATR: 5′-AAGGGATTCCAAGATCATCAA-3′, according to the accession number AJ29945.1, which amplifies a 293 bp fragment; APXF: 5′-GTGAAGAGCAAGACGGGAGG-3′, APXR: 5′-TTAGTGGCATCAGGCAAACG-3′, according to the accession number AF326783, which amplifies a 263 bp fragment. In addition to the primers for the endogenous control, β-actin (forward, 5′-GTTCTCAGTGGTGGTTCTAC-3′, and reverse, 5′-GACCCTGACTCCTCATACTC-3′) reactions were run in duplicate on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) using the following thermal cycling profile: 94°C for 2 min, then at 94°C for 30 s, 55°C 30 s, and 72°C for 30 s for 35 cycles. After 35 cycles, samples were run using the dissociation protocol. This assay was performed twice to minimize variations due to sample handling. In order to compensate for differences in loading and RT efficiency (based on our previous studies, which indicated that the analyzed β-actin mRNA levels are quite constant in tissues, regardless of the developmental or physiological conditions) this β-actin was used as an endogenous control. Thus the reported SOD and CAT transcript levels are normalized relative to β-actin.

Results

Changes in H2O2 content during somatic embryo development

The H2O2 content in somatic embryos during somatic embryo development was measured. The results showed that H2O2 content (~0.1145 µmol/g) was the lowest on day 0. The H2O2 content began to increase dramatically when tissues were transferred on ABA and PEG induction medium, and reached a maximum on day 5 (0.7766 µmol/g). The H2O2 content of tissues in ABA and PEG induction medium increased more than 5.78 times within the first 5 days from day 0. A reduction in H2O2 accumulation was observed from day 7 to day 10, which peaked again on day 21, and then H2O2 content started to decrease again from day 24 to day 35 followed by a dramatic increase from day 45 when somatic embryos have completely matured (Fig. 2).

Fig. 2
figure 2

Changes in H2O2 content during somatic embryo development in callus cultures of L. leptolepis

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Analysis of SOD transcript levels during somatic embryo development by real-time RT–PCR

The mRNAs from 13 samples were detected with the same efficiency. Therefore, the analyzed transcript levels correspond with the sum of the relative quantities. Analysis of the dissociation curves of SOD and β-actin control samples showed a single melting peak (Fig. 3), which indicated a specific signal corresponding to the SOD target sequences and the endogenous control, respectively. The fluorescent signal was not detected in all negative-control samples, proving that the RNA extraction procedure, including the DNase treatment, effectively removed genomic DNA from the RNA samples. No contamination appeared during PCR procedure. A fluorescence threshold value (Ct) was calculated for each sample. For each standard curve (Fig. 4), the correlation coefficients ranged from 0.9978 to 0.9995 indicating a high degree of confidence in the measurement of copy number of molecules in the samples.

Fig. 3
figure 3

Dissociation curves of expression patterns of genes encoding antioxidant enzymes. a β-actin (control), b SOD, c CAT, and d APX

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Fig. 4
figure 4

Standard curves of expression patterns of genes encoding antioxidant enzymes. a β-actin (control), b SOD, c CAT, and d APX. Ct was plotted against the log of the initial level of transcript for each gene to generate standard curves

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The relative abundance of the SOD transcript at 13 developmental stages was demonstrated during somatic embryo development of L. leptolepi by RT–PCR. The transcript levels of the SOD were lowest on day 0 (Fig. 5) tissues cultured on non-ABA and PEG induction medium. But which became high when tissues were transferred to ABA and PEG induction medium, and the expression quantity reached a maximum on d3, and then dropped from day 5 to day 45.

Fig. 5
figure 5

Expression patterns of SOD during somatic embryo development of L. leptolepis

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Analysis of CAT transcript levels during somatic embryo development by real-time RT–PCR

The expression pattern of CAT were analyzed and summarized during somatic embryogenesis of L. leptolepis by real-time RT–PCR. The mRNAs from 13 samples were detected with the same efficiency. Analysis of the dissociation curves of the CAT in 13 samples showed a single melting peak (Fig. 3). The fluorescent signal was detected in the negative-control sample, and the dissociation curves indicate that there was a low melting peak in the negative-control sample (Fig. 3). However, the peak of the negative-control was different from that of the samples, and the negative-control did not show any amplification when the PCR product was detected by electrophoresis on an agarose gel. A fluorescence threshold value (Ct) was calculated for each sample. For the CAT standard curve (Fig. 4) the correlation coefficient was 0.999 which indicates confidence in the measurement of the copy number of molecules in the sample.

The expression pattern of CAT was different to that of SOD. When somatic embryos (day 0) were cultivated on medium without ABA and PEG, expression levels of CAT was also the lowest during the whole somatic embryogenesis. Expression levels of CAT began to increase slightly when somatic embryos were transferred to ABA and PEG induction medium. But expression levels of CAT was lower in early somatic embryogenesis, and there was a very lower expression peak at day 5, and then expression quantity began to drop from day 7 to day 10, followed by increasing from day 14, and reached a maximum at day 21, declined dramatically from day 24 to day 35, and then CAT transcript levels began to increase again when somatic embryos matured at day 45 (Fig. 6).

Fig. 6
figure 6

Expression patterns of CAT during somatic embryo development of L. leptolepis

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Analysis of APX transcript levels during somatic embryogenesis by real-time RT–PCR

The mRNAs from 13 samples were detected with the same efficiency. Therefore, the analyzed transcript levels corresponded with the sum of the relative quantities. Analysis of the dissociation curves of APX from 13 samples showed a single melting peak (Fig. 3), which indicates a specific signal. However, the fluorescent signal was detected in negative-control samples. The dissociation curves show that there was a low melting peak in negative-control samples (Fig. 3), but the location of the peak was different from the samples, and negative-controls did not show any amplification when the PCR product was detected by electrophoresis on an agarose gel. A fluorescence threshold value (Ct) was calculated for each sample. For the standard curve, the correlation coefficient was 0.9983, indicating a high degree of confidence for the measurement of the copy number of molecules in the samples (Fig. 4).

The expression patterns of the CAT were analyzed and summarized during somatic embryo development of L. leptolepis using real-time RT-PCR, which was similar to the expression pattern of CAT. The expression quantity was lowest at day 0 tissue cultivated on the medium without ABA and PEG. Expression of the APX began to increase slowly when somatic embryos were transferred to ABA and PEG induction medium. With a peak at day 3 followed by decreasing APX expression quantity, the expression quantity started to increase significantly from day 14, and reached a maximum at day 21. Expression began to drop at day 24 and day 35, but increased slightly at day 45 when somatic embryos matured (Fig. 7).

Fig. 7
figure 7

Expression patterns of APX during somatic embryo development of L. leptolepis

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Discussion

Changes in H2O2 content during somatic embryo development

H2O2 content was lowest when tissues were cultured in ABA and PEG free medium. As tissues were transferred to somatic embryogenic developmental medium, the H2O2 content began to increase dramatically and reach a maximum at day 5, suggesting that ABA and PEG might induce the production of H2O2. PEG can form non-osmosis water stress in the cell, and cause the water to reduce and enhance the density of cell content, to finally promote the growth of the somatic embryo. The level of ABA also increases in response to stress treatments in various plants, and is thought to act as a signal for the initiation of acclimation to these stresses (Shinozaki and Yamaguchi-Shinozaki 1997; Siddiqui et al. 1998; Hare et al. 1999; Kikuchi et al. 2006). Various stress substances or conditions might stimulate ABA. Different pieces of evidence link ABA to AOS. In guard cells, ABA induces the production of H2O2 and the activation of calcium channels by H2O2 mediates the induction of stomata closure (Staneloni et al. 2008). Another reason might be tissues and cells occupy a stage of rapid fission (Fig. 1, days 1–5). Higher concentration of H2O2 is a signal to promote rapid fission of tissues. A reduction of H2O2 accumulation due to a signal for fission increases development of the cells from day 7 to day 10 Fig. 1, days 7–10), then H2O2 content began to increase slowly and reach a subpeak at day 21, H2O2 content started to reduce again from day 24 to day 35 until the somatic embryo had matured at day 45 (Fig. 1). Sugars, proteins and esters accumulated from day 24 to day 35, and full accumulation of these compounds was essential for somatic embryo maturation and might be a reason for drop of adjustment H2O2 content. H2O2 content also had ascension when somatic embryo completely matures at day 45 (Fig. 1, day 45), this stage was somatic embryo entering the latter stage of ripening. This made the shape and the physiological biochemical characteristics of the somatic embryo more similar with the zygote embryo and normal germination.

The expression of antioxidase genes during somatic embryo development

In the dark, or in nonphotosynthetic tissues, AOS is produced mainly by leakage of the electron transport chain in mitochondria or microsomes (Vanlerberghe and McIntosh 1997) and during fatty acid oxidation (Mittler 2002). Increased production of AOS may lead to oxidative stress and cellular damage resulting in seed deterioration (Bailly 2004; Kranner et al. 2006). To cope with oxidative stress, plants have evolved several enzymatic and non-enzymatic systems. The channels by which H2O2 mediates the induction of stomata closure, is by inducing the expression of antioxidant genes SOD (Sakamoto et al. 1995; Guan and Scandalios 1998a; Bueno et al. 1998), and CAT (Guan and Scandalios 1998b; Guan et al. 2000), and increase the activities of antioxidative enzymes such as SOD, CAT, guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and glutathione reductase (GR) in plant tissues (Prasad et al. 1994; Gong et al. 1998; Bellaire et al. 2000). SOD reduces superoxide radicals (O2 −) to H2O2, CAT reduces H2O2 to water and dioxygen, APX reduces H2O2 to water and monodehydroascorbate, thus preventing the formation of the highly reactive hydroxyl radical (OH) that can cause lipid peroxidation, protein denaturation, and DNA mutations. To date, the antioxidant gene/enzyme systems for CAT, SOD and APX have been well characterized in many plants (Willekens et al. 1995).

In this study, transcript levels of SOD, CAT and APX were the lowest when somatic embryos were cultured on callus-propagation medium without ABA and PEG. When they were transferred on mature somatic embryo induction differentiation medium, the expression patterns of SOD, CAT and APX were different.

The results indicated that the CAT, SOD and APX cooperate to adjust differentiation and development of somatic embryos. H2O2 generated at different times catalyze and scavenge different enzymes, and this mad oxidation system maintain suitable level during somatic embryogenesis. Transcript levels of SOD enhanced gradually at early somatic embryo development. The peak of SOD transcript levels appeared at day 3, when the cell fission formed the multi-cell proembryo. High expression of SOD may promote embryonic cell differentiation as well as the early somatic embryo development. SOD activities were high in the stem and root tip of meristematic tissues (Gupta and Datta 2003/2004). With the multi-cell proembryo further fission and development, the transcript levels dropped gradually, and at day 45 the same level as day 0 was detected. The transcript levels of SOD dropped due to H2O2. Massive accumulation of H2O2 in vivo has suppressed transcription of SOD and destroyed the active oxygen elimination system. Therefore, embryonic cell differentiation might be regulated by the relationships between SOD, O2 with H2O2.

Expression patterns of CAT and APX were different with that of SOD. Expression of CAT and APX began to increase slowly when calli were transferred on ABA and PEG induction medium. The first expression peak of APX appeared at day 3, but there was a very low expression peak of CAT at day 5. And transcript levels of the two genes began to drop gradually until day 14, the expression quantity started to increase significantly, and reached maximum at day 21. Then expression began to drop at day 24 and day 35, but increased slightly at day 35. The expression patterns of CAT and APX was similar to changes of H2O2 content during somatic embryo development. This indicated that H2O2 induced the transcription of CAT and APX during somatic embryogenesis.

Suitable density H2O2 has induction action to somatic embryo development, but mechanism is not actually clear (Cui et al. 1999). In this study, H2O2 obviously induced and promoted the somatic embryo formation of L. leptolepis. It is possible H2O2 influenced gene expression through the cell signal transduction system and thus induced somatic embryo formation. The embryonic cell formation is also a cell differentiation process, and the core of this process is the gene difference expression. Therefore, H2O2 might induce somatic embryogenesis at the molecular level.

In summary, ABA was essential for stimulation of somatic embryogenesis in L. leptolepis. ABA might induce H2O2 and AOS generation, H2O2 caused stresses and induced CAT, SOD, and APX expression during somatic embryogenesis of L. leptolepis. Exogenous H2O2 may improve generation frequency of somatic embryos of L. leptolepis.