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Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency... fpls-08-02054 December 2, 2017 Time: 15:57 # 1 ORIGINAL RESEARCH published: 05 December 2017 doi: 10.3389/fpls.2017.02054 Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Edited by: Jose M. Seguí-Simarro, Hook Universitat Politècnica de València, Spain 1,2‡ 3‡ 1,2 1† 1,2 Xiaohong Zhou , Renhua Zheng , Guangxin Liu , Yang Xu , Yanwei Zhou , 4 1,2 5 1,2 1,2 Reviewed by: Thomas Laux , Yan Zhen , Scott A. Harding , Jisen Shi and Jinhui Chen * * Taras P. Pasternak, Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China, Albert Ludwigs University of Freiburg, Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Germany China, The Key Laboratory of Timber Forest Breeding and Cultivation for Mountainous Areas in Southern China, State Thomas Dandekar, Forestry Administration Engineering Research Center of Chinese Fir, Fujian Academy of Forestry, Fuzhou, China, Centre for University of Würzburg, Germany Biological Signaling Studies, Faculty of Biology, Albert Ludwigs University of Freiburg, Freiburg, Germany, School of *Correspondence: Forestry and Natural Resources, University of Georgia, Athens, GA, United States Jinhui Chen [email protected] Jisen Shi Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially [email protected] and ecologically, in southern China. The traditional regenerating methods are based Present address: on organogenesis and cutting propagation. Here, we report the development of a Yang Xu, Research Institute of Subtropical high-frequency somatic embryogenesis (SE) regeneration system synchronized via Forestry, Chinese Academy a liquid culture from immature zygotic embryos. Following synchronization, PEM II of Forestry, Fuyang, China cell aggregates were developmentally equivalent in appearance to cleaved zygotic These authors have contributed embryos. Embryo and suspensor growth and subsequent occurrence of the apical equally to this work. and then the cotyledonary meristems were similar for zygotic and SE embryo Specialty section: development. However, SE proembryos exhibited a more reddish coloration than This article was submitted to Plant Biotechnology, zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature a section of the journal somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, Frontiers in Plant Science low concentrations of endogenous indole-3-acetic acid in initial explants correlated Received: 03 July 2017 with improved proembryogenic mass formation, and high SE competency. Analysis Accepted: 16 November 2017 Published: 05 December 2017 of karyotypes and microsatellites detected no major genetic variation in the plants Citation: regenerated via SE, and suggest a potential in the further development of this system as Zhou X, Zheng R, Liu G, Xu Y, a reliable methodology for true-to-type seedling production. Treatment with polyethylene Zhou Y, Laux T, Zhen Y, Harding SA, Shi J and Chen J (2017) Desiccation glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation Treatment and Endogenous IAA and complemented each other. ABA assisted the growth of embryonal masses, Levels Are Key Factors Influencing whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC High Frequency Somatic Embryogenesis in Cunninghamia EMBRYOGENESIS RECEPTOR KINASE (SERK) and the WUSCHEL homeobox (WOX ) lanceolata (Lamb.) Hook. transcription factor served as molecular markers during early embryogenesis. Our Front. Plant Sci. 8:2054. doi: 10.3389/fpls.2017.02054 results show that ClSERKs are conserved and redundantly expressed during SE. SERK Frontiers in Plant Science | www.frontiersin.org 1 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 2 Zhou et al. Somatic Embryogenesis in Chinese Fir and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses. Keywords: ABA, desiccation, endogenous hormone, PEG, somatic embryo, SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), WUSCHEL homeobox (WOX) et al., 2007a,b; Zhou et al., 2013; Li et al., 2017). Development INTRODUCTION of SE has been handicapped by limited yields and calluses’ Cunninghamia lanceolata (Lamb.) Hook, Chinese fir, is an necrosis (Kang, 2008; Chen et al., 2017). Xi and Shi (2005, important native evergreen tree species in China. This fast- 2006) used explants of cotyledon, hypocotyl and mature zygotic growing conifer has been cultivated for over 3,000 years due embryos for direct SE induction, which is not perspective for to its elite wood attributes and high timber productivity (Yang, mass propagation and further genetic improvements due to low 1998). Chinese fir is now the dominant tree species in southern multiplication rates. Hu et al. (2017) recently established SE China, growing in over 15 southern provinces. The total planted via embryogenic callus using immature zygotic embryos. The 6 2 area of Chinese fir has reached 9  10 hm and accounts for system employed ABA and PEG to successfully produce early 30% of the forested land in China (Lu et al., 2015). Chinese somatic embryos, but normally developed, late stage somatic fir occupies prominent roles in ecological and commercial embryos were not obtained, and there were no reports about the prospects. conversion from somatic embryos to plantlets. Advances in third generation recurrent genetic selection and Members of the SOMATIC EMBRYOGENESIS RECEPTOR hybridization of Chinese fir through conventional breeding KINASE (SERK ) gene family encode leucine-rich repeat- programs have led to great genetic improvements on growth, containing transmembrane proteins that are involved in signal wood qualities and biological or non-biological stress tolerances. transduction and are strongly related to SE (Santos and Aragão, However, conventional breeding improvements are inefficient, 2009). SERK was first discovered as a marker for the somatic costly, and time consuming due to the inherently long life to embryogenic transition in carrot (Schmidt et al., 1997). cycles, unstable maturation, and frequently unavoidable dilution The WUSCHEL (WUS) homeobox (WOX) transcription factor of desirable traits caused by genetic segregation and gene is described as functioning in early embryo patterning and flow (Cairney et al., 1999). As an alternative to conventional lateral organ development in Arabidopsis (Haecker et al., breeding, somatic embryogenesis (SE) is thought to be the 2004). Overexpression of WUS can initiate acquisition of most promising clonal propagation strategy based on recurrent embryogenic competence in Arabidopsis (Zuo et al., 2002) genetic selection for commercial plantations regeneration. The and cotton (Zheng et al., 2014). In Picea abies, PaWOX2 generation circulation time could be reduced, and the extra and PaWOX8/9 were highly expressed in early stage embryo genetic gain of forest trees can be captured through SE with development (Palovaara and Hakman, 2008; Hedman et al., lower risks and costs (Gupta and Grob, 1995). The process 2013), when the primary body axis and radial patterning was can be rigorously controlled, and is theoretically feasible for being established (von Arnold et al., 2016). Therefore, SERK all plant species. A well-established SE line, combined with and WUS prompted SE during early embryogenesis (Guan et al., cryopreservation, would be an excellent platform for long-term 2016). conversation and large-scale planting materials production In this study, we developed an effective synchronized (Park, 2002). SE system from C. lanceolata immature seeds. Cell masses Since embryo-like structures were induced in vitro into were synchronized in liquid suspension before somatic somatic embryos in Pinus banksiana (Durzan and Chalupa, embryo differentiation. This system produces seedlings 1976), much progress has been archived by SE systems in conifers with no cytological variation detectable by karyotype and (Klimaszewska and Cyr, 2002; Cairney and Pullman, 2007; Jain microsatellite analyses. We showed that the competence of et al., 2013). However, most studies have concentrated on pine C. lanceolata seeds to generate somatic embryos correlated with species (Klimaszewska and Cyr, 2002; Sutton, 2002). In vitro developmental stage exhibiting low endogenous indole-3-acetic culture of Chinese fir is more difficult in comparison to the other acid (IAA) hormone levels in the source materials. We further coniferous species. At the same time, a rapid mass propagation demonstrated that morphological differences were caused by system is required to meet the increasing demand for this exposure to polyethylene glycol (PEG) and abscisic acid (ABA) species. To our knowledge, less progress has been achieved for during the early developmental stages of somatic embryos. C. lanceolata SE than for propagation via organogenesis (Zhu In addition, expression profiles of SERK and WUS reveal Frontiers in Plant Science | www.frontiersin.org 2 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 3 Zhou et al. Somatic Embryogenesis in Chinese Fir their applicability as the markers of early embryogenesis of Morphological Analysis Chinese fir. The entire developmental pathway and the embryonic stages were evaluated using a stereoscope (Leica, S8AP0), and micrographs were obtained using an inverted microscope (Leica, MATERIALS AND METHODS DMI4000). Samples were double-stained with acetocarmine and Evan’s blue (Gupta and Holmstrom, 2005). The dense Plant Materials cytoplasmic cells were stained into red, while vacuolated cells Chinese fir cones were collected once a week from late June to late were stained into blue. August 2009 in a National Clonal Seed Orchard at Yangkou and Shaowu Forest Farm, Fujian Province, China. These cones were Karyotype Analysis immediately placed on ice and brought back to the lab within Proembryogenic masses and more than 30 actively growing 24 h. Cones were stored at 4–5 C for no more than 1 week until roots (1.5–2 cm long) of regenerated somatic-embryo derived further used. seedlings were excised. The materials were pretreated in ice- Seeds with immature zygotic embryos of different cold water for 18–24 h and then fixed in Carnoy fixative (95% developmental stages were used as the initial explants to ethanol:acetic acid, 3:1) for 24 h, and then dissociated in 45% induce somatic embryos. The stages of the zygotic embryo (v/v) acetic acid for 2 h. Finally, carbol-fuchsin staining was materials were identified from 5 to 6 randomly selected seeds of used for squash preparations, and then slides were observed and each cone according to Shi et al. (2010). Cones were opened and photographed using the cell workstation software Leica 4000. seeds were collected before sterilization. Seeds were then washed The chromosome number and type were determined for >25 with detergent for 10 min, transferred into running water for cells from squashed Chinese fir root tips. Photos showing well- 30 min, surface-sterilized with 75% (v/v) ethanol for 30 s, treated spread chromosomes were processed further using Photoshop with 0.1% (w/v) HgCl containing a few drops (0.01%) of Tween CS3 and CAD 2010. The chromosome length in five cells was 20 for 8–10 min, and then rinsed three times in sterile distilled averaged. water. Seed coats were removed as previously described (Becwar and Pullman, 1995; Pullman and Johnson, 2002), and the embryo Microsatellite Analysis: Preparation of sacs, including the zygotic embryos, were used for induction of Genomic DNA and PCR Amplification proembryogenic masses (PEMs). More than 30 seeds of the same More than 30 regenerated plants of C. lanceolata and three embryonic stage were used to assess the frequency of outgrowths proliferating PEMs were sampled for DNA isolation. Each sample and the test was repeated three times. was immediately frozen in liquid nitrogen and stored at 80 C until the DNA extraction. Samples were ground to a powder Medium and Culture Conditions in liquid nitrogen, and genomic DNA was extracted using the The embryo sacs were cut near the suspensors and initially DNeasy Plant Mini Kit (Qiagen). cultured for 1 month at 23–25 C in darkness on Gupta and We randomly selected 10 primer pairs from available Durzan medium (DCR) (Gupta and Durzan, 1985), which nuclear microsatellite primers that had already been tested in contained 2.0–6.0 mg L 2,4-dichlorophenoxyacetic acid, 1 1 C. lanceolata to detect genetic stability (see Supplementary 0.5 mg L benzylaminopurine, 500 mg L casein hydrolysate 1 1 Table S1). These nuclear simple sequence repeat (SSR) (CH) (Sigma), 450 mg L L-glutamine, 100 mg L myo- 1 1 microsatellite loci were CFeSSR23, CFeSSR35, CFeSSR63, inositol, 20 g L maltose, and 2.1 g L gellan gum (Sigma). CFeSSR72, CFeSSR98, CFeSSR234, CFeSSR278, CFeSSR284, The medium was adjusted to pH 5.8 using KOH or HCl after CFeSSR312, CFeSSR352, and CFeSSR418. These primers were the addition of all of the ingredients except the gelling agent. The developed from the transcriptomic analysis of genotype 6421 gelling agent was added prior to autoclaving at 121 C for 20 min. (Xu et al., 2016). Maltose was used as a carbon source and autoclaved separately. PCR amplification was performed by adding 2 mL diluted For the synchronization of SE development, approximately genomic DNA to a cocktail, with a final volume of 10 mL 2 g of embryogenic calluses were suspended in 50 mL liquid containing 1 PCR buffer, 25 nmol MgCl , 2 nmol dNTPs, medium consisting of DCR salts, 3 mg L ABA (Sigma), 1 1 1 1 U Taq DNA polymerase (Takara), and 1 nmol forward and 0.5 mg L gibberellic acid (GA ), 500 mg L CH, and 30 g L reverse primers. PCR reactions were performed on a Veriti maltose. ABA was filter-sterilized and added into autoclaved 96-well Thermal Cycler (Applied Biosystems) using the following cooled medium. procedure: 4 min at 94 C, followed by 20 cycles of 45 s at 94 C, After 3 weeks suspension culture, steadily proliferating cell 45 s at the annealing Tm, and 60 s at 72 C, with a final 10 min clumps were dispersed on DCR solid medium containing 1 1 1 extension at 72 C. PCR products were run on 8% (w/v) native 3 mg L ABA, 1 mg L GA , and 120–200 g L PEG polyacrylamide gels. (MW 8000; Amresco). The osmotic potential was measured by a Wescor 5520 vapor pressure osmometer (Wescor, Inc.). Three Analysis of Endogenous Hormone Levels months later, the mature somatic embryos were transferred to basic DCR medium for the regeneration of plantlets, which Fresh seeds at different developmental stages of the genotypes were maintained at 25 C under cool white fluorescent light 4098, 5009, 4009, Y21, and 27 were collected for the (30 mmol m per second, with a 16-h photoperiod). detection of endogenous hormones. Megagametophytes Frontiers in Plant Science | www.frontiersin.org 3 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 4 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 1 | Developmental stages of SE (A–L) and zygotic embryogenesis (M–S) in Cunninghamia lanceolata (Lamb.) Hook. (A,B,D,E) Embryo sacs containing immature embryos for PEM induction: (A,B) induced embryogenic tissues; (D,E) induced non-embryogenic tissues. (E,F–K) Somatic embryo induction at diverse stages in Chinese fir: (E) maintenance of PEMs that were translucent and had pointed surface protrusions; (F) formation of proembryos (arrows); PEMs on a high osmolality medium for 1 week after suspension; end of proembryogeny; (G) shaped early reddish embryos (arrows); end of early embryogeny; (H) reddish embryos with bright yellow tops (arrows); the transition to late embryogeny; (I) early cotyledonary embryos; (J) late cotyledonary embryos; (K,L) seedlings germinated from somatic embryos at 1 week (K) and 1 month (L). (M–S) Zygotic embryo development of Chinese fir based on Pullman and Webb (1994): (M) cleaved polyembryogeny before a dominant embryo forms; (N) proembryo, the beginning of early embryogeny; the dominant embryos has formed; (O) further developed dominant embryo with embryonal mass and suspensor prototypes; ready for late embryogeny; (P) dominant embryo with a more mature embryonal mass and suspensor; (Q) embryonal mass to cotyledon formation; (R,S) maturation of cotyledonary embryo. Bars = 1 mm for (A–J); 500 mm for (M–S); 1 cm for (K,L). (1.0–2.0 g) that contained immature zygotic embryos were and the WOX homologs in P. abies (Palovaara et al., 2010; immediately frozen in liquid nitrogen and stored at80 C until Hedman et al., 2013) and Arabidopsis (Hecht et al., 2001) analysis. (see Supplementary Table S3). The potential genes were The methods for extraction and purification of ABA, IAA, named on the basis of their alignment to Arabidopsis (see GA , and zeatin riboside (ZR) were modified from those Supplementary Table S4). The unrooted neighbor-joining described by Bollmark et al. (1988) and He (1993). ELISA phylogenetic trees were generated from the deduced protein hormonal quantification was following the protocols described by sequences using MEGA5.2 with the help of the Jones- Yang et al. (2001) and Teng et al. (2006). Taylor-Thornton (JTT) model in combination with the gamma-distributed rate model (JTTCG; gamma D 0.97). Quantitative Real-Time PCR (qRT-PCR) Bootstrap values from 1,000 replicates were indicated at each node. To isolate the potential SERK and WOX genes, the transcriptome database of C. lanceolata produced by Huang et al. (2012), Tissues at different developmental stages were collected. Qiu et al. (2013), and Wang et al. (2013) was screened using Total RNA was extracted and purified following the methods the NCBI tblastn algorithm using the SERK homologs in previously described by Lin et al. (2009). RNA (1 mg) was Arabidopsis, Medicago, and rice (see Supplementary Table S2), reverse-transcribed with oligo (dT) and random hexamer primers Frontiers in Plant Science | www.frontiersin.org 4 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 5 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 2 | Microscopic structure of embryogenic and non-embryogenic tissues induced from immature embryos and suspension cultures in Chinese fir. Stage annotation based on Filonova et al. (2000). (A) Embryogenic tissues. Cell aggregates include a clump of rounded, dense cytoplasmic cells surrounded by a set of vacuolated cells that are elongated. (B) Well-organized PEM III structures after a more than half year maintenance period. (C,D) Non-embryogenic tissues also induced from immature embryos of Chinese fir: cell clusters composed of dense cytoplasmic cells but lacking elongated cells (C), or with vacuolated cells in a collapsed cell shape but stuck in dense cytoplasmic cell aggregates (D, arrow represented PEM I structure). (E,F) Suspensions of PEMs stained with acetocarmine and Evan’s blue: (E) PEMs in suspension for 1 week, with more early-stage PEMs dedifferentiated (PEM I) (arrows); (F) PEMs in suspension for 3 weeks, with newly organized PEMs. Bars = 100 mm. using a reverse transcription system (Promega). Verification was zygotic embryos visibly protruded from the megagametophyte performed using qRT-PCR. micropolar end into the medium (Figure 1A). One month qRT-PCR was carried out using the LightCycler 480 System later, outgrowths proliferated to form PEMs (Figure 1B). (Roche Applied Science). Each reaction was performed in a 20- Embryogenic tissues were soggy and recognized as translucent mL final volume containing 10 mL of 2 LightCycler 480 SYBR structures, with pointed protrusions at the surface (Figure 1C). Green I Master, 0.5 mL of each homolog-specific primer pair Microscopy of the cell aggregates revealed clumps of rounded, (see Supplementary Table S5) at 100 nM, 5.0 mL of diluted dense cytoplasmic cells surrounded by a set of vacuolated cDNA template, and 4.0 mL of ddH O. Each reaction was run and elongated cells (Figure 2A), which were referred to as in triplicate with the appropriate negative controls. Amplification PEM II defined by Filonova et al. (2000). After a more than was conducted under the following conditions: activation for half year subculturing period, PEM II in the early stages 5 min at 95 C, followed by 40 cycles of 10 s at 95 C, 15 s at became the assembled and well-organized PEM III (Figure 2B). 60 C, and 15 s at 72 C. Fluorescence detection was performed However, non-embryogenic calluses looked quite different in after the extension step. The melting curve, with a temperature color (Figures 1D,E), and cell clusters were not as well- gradient from 60 to 95 C, was used to further investigate the organized (Figures 2C,D). Some non-embryogenic calluses specificity of each qRT-PCR reaction, and the presence of a single showed a denser lump of cytoplasmic cells but lacked elongated PCR product was also verified by 2% agarose gel electrophoresis. cells (Figure 2C), whereas some contained vacuolated cells The eIF-3 housekeeping gene was selected as the endogenous in collapsed shapes and were stuck in dense cytoplasmic cell reference gene for the relative PCR quantification (Wang et al., aggregates (Figure 2D). 2013). The rate of PEM induction was related to the zygotic embryos’ developmental stage (Figure 3). The frequency was markedly higher for embryo sacs corresponding to early zygotic embryo RESULTS stages (stages A–C, Figure 3), but sharply declined in late- stage embryos (stages D–E, Figure 3). Immature embryos in Generation of Somatic Embryos in proembryogeny and early embryogeny were the best sources of Chinese Fir explants for PEM induction. The development of somatic embryos in Chinese fir is shown Our overarching objective was to develop an efficient system for Chinese fir mass propagation via SE. The appearance of in Figures 1F–L. The maintained PEMs were transferred to liquid PEMs with embryonic suspensor structures occurred on DCR culture to synchronize the development of the cell masses and medium containing a 2.0–6.0 mg L 2,4-dichlorophenoxyacetic to produce vigorously differentiated late stage PEM structures. acid. Outgrowth occurred within 1–4 weeks when one or more With the addition of ABA, PEMs separated into small groups Frontiers in Plant Science | www.frontiersin.org 5 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 6 Zhou et al. Somatic Embryogenesis in Chinese Fir embryo proper became opaque, the apical meristem primordium was clearly visible (Figure 1H). The cotyledon primordia formed accordingly (Figure 1I), but were not as easily distinguishable as in zygotic embryos (Figure 1P). On the whole, somatic embryos were smaller than zygotic embryos after they reached the proembryo stages. PEM Induction Frequency Correlated with Endogenous Auxin Level The induction frequency of the PEMs was related to the developmental stages of the explants, as well as the plant genotypes. We investigated whether these differences correlated with endogenous hormonal levels using ELISA quantification. IAA and ABA levels oscillated during somatic embryo development, whereas ZR and GA levels were low and FIGURE 3 | Box plots for PEM induction frequencies at different steady (Figure 4A). Cleaved embryos had the lowest IAA content developmental zygotic embryo stages in C. lanceolata. More than 30 seeds were analyzed for each embryonic stage. Stage A, cleaved polyembryogeny during proembryogeny of zygotic embryo development (stage stages (Figure 1M); stage B, proembryo, only a dominant embryo 1, Figure 4). The IAA concentration rose during development, (Figure 1M); stage C, further developed dominant embryo (Figure 1O); stage until the dominant embryo formed opaque suspensors at the D, dominant embryo had formed apical meristem primordium (Figure 1P); start of late embryogeny (stages 2–3, Figure 4A), and then a low stage E, more changes occurred in embryonal mass to cotyledon formation level was maintained during late embryogeny until cotyledons (Figure 1Q); stages F and G, maturation of cotyledonary embryo (Figures 1R,S). Minimum and maximum PEM induction frequencies are emerged (stages 4–5, Figure 4A). The endogenous ABA content depicted by short black lines, the box signifies the upper and lower quartiles, increased with the development of a dominant embryo and then and the median and mean are respectively represented by a thicker black line declined during embryo maturation. and a thicker plus sign within the box for each stage. Endogenous IAA levels significantly varied among the genotypes of cleaved embryos (Figure 4B). Notably, genotypes 4098 and 5009 displayed relatively low levels of IAA at the stage of cell masses during the first week. At this point, early-stage of embryo cleavage, whereas IAA levels were higher in genotypes PEMs, with fewer elongated cells, were seen, probably due to 4009, Y21, and 27 (Figure 4B). Not so significant differences in the shearing in the suspension (Figure 2E). Three weeks later, the ABA content were observed among the different genotypes. cell aggregates propagated both cell types and assembled into Genotypes 4098 and 5009 were effective sources for SE using new PEM II structures (Figure 2F). Suspended PEMs were then our method, whereas the genotypes Y21, 4009, and 27 did not dispersed into a high-osmolality environment to complete the yield somatic embryos. Therefore, it appears that SE using this critical transition from PEMs to proembryo, and to facilitate method may benefit from low endogenous IAA levels at the late embryogeny. Proembryos formed, having a dense embryonic proembryogeny stage. head and highly vacuolated long polarized cells, but were still translucent (Figure 1F). In the following days, proembryos then ABA and PEG Are Different Stress turned red (Figure 1G), developed a bright yellowish top, and Signals for Somatic Embryo also increased in size (Figure 1H). A well-developed shoot and Development root pole took shape during this stage. Early cotyledonary-shaped embryos then formed, with the appearance of an indentation Polyethylene glycol (MW 8000) is an important facilitator separating the developing cotyledon structures (Figure 1I). of somatic embryo maturation in conifer (Stasolla et al., Finally, the two cotyledons fully opened, completing maturation 2003), which can mimic the desiccation that occurs during (Figure 1J). Mature cotyledon embryos were generated on zygotic embryo maturation. Different concentrations of hormone and osmoticum (PEG) free medium (Figures 1K,L). PEG (12–20%) were tested for effects on somatic embryo Investigation of the developmental processes of zygotic maturation (Figure 5). The number of somatic embryos embryos indicated that somatic embryos went through increased sharply when PEG concentration increased from analogous developmental phases. Suspension-cultured PEMs 150 to 170 g L (15–17%), followed by a sharp decline at were synchronized and produced active PEM II cell aggregates higher PEG concentrations (Figure 5). Thus, the optimized PEG (Figure 2F), which was the equivalent to cleaved zygotic embryo concentration was 17%. development (Figure 1M). During subsequent maturation, the Abscisic acid, “stress hormone” in plants, is also widely used zygotic embryo proper would then become white and translucent during SE induction (Zavattieri et al., 2010). To further clearly (Figure 1N). Both embryo and suspensor enlarged longitudinally elucidate the effect of high osmolality and exogenous ABA during (Figure 1O). Subsequently, the apical meristem primordium SE, we compared the effects of PEG-, ABA-, and PEG and ABA- became visible (Figure 1P), followed by the appearance of containing maturation media on morphological changes during cotyledon primordium (Figure 1Q). However, SE proembryos embryo development (Figure 6). Differences in morphology turned reddish rather than white (Figure 1G). When the SE between embryos on PEG-free (Figure 6A) and PEG-containing Frontiers in Plant Science | www.frontiersin.org 6 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 7 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 4 | Endogenous hormone content of zygotic embryos (genotype 4098) at different developmental stages (A) and of different genotypes at the cleaved-embryo stage (B). (A) The different developmental stages represented from 1 to 5: stage 1 cleaved polyembryos (Figure 1M); stage 2 proembryo (Figure 1N); stage 3 dominant embryo (Figure 1O); stage 4 columnar embryo (Figure 1P); stage 5 cotyledonary embryo (Figures 1R,S). (B) Genotypes 4098, 5009, 4009, Y21, and 27 were tested, and genotypes 4098 and 5009 were more competent to form somatic embryos. Data are means  SD of three replicates. Karyotype and Microsatellite Analyses Reveal True-to-Type Propagation via SE A karyotype analysis of plants regenerated through SE was performed by assessing >30 actively growing roots from 30 different individuals. All of the regenerated plants were diploids with 22 chromosomes (2nD 2XD 22; see Supplementary Figure S1A). The relative length and the ratio of the long arm to the short arm (see Supplementary Figure S1B and Supplementary Table S6) indicated that the karyotype belonged to 1B and consisted of 10 pairs of metacentric and 1 pair of submetacentric chromosomes. Eleven randomly selected primers developed from a transcriptome analysis of genotype 6421 were used to explore the microsatellite stability of the regenerated plants (see Supplementary Table S1). All primers exhibited monomorphic bands between the 3 tested PEM lines and 30 regenerated plants (see Supplementary Figure S2). FIGURE 5 | Frequency of somatic embryogenesis (SE) at different osmotic potentials. Data are means  SD of three replicates. SERK and WOX May Serve As Markers of Stress-Mediated Cell-Signaling during SE (Figures 6B,C) media were visible after the first 7 days of incubation. Polar PEM III structures were evident when PEG Somatic embryogenesis is also recognized as a reprogrammed was present, whereas embryos on the ABA-only medium did developmental process responding to stress-related signals not differentiate beyond PEM II. After 1 month, variations were (Zavattieri et al., 2010). Several gene regulators of this process more obvious. By that time, PEM III structures with polarity had have been identified. SERK is considered to be the critical formed in the ABA-only media, without clusters of vacuolated switch from somatic to embryogenic development, and WUS cells associated with proembryo formation (Figure 6D). PEG maintains a small group of cells at the dedifferentiation state alone produced more proembryo-like structures; however, the (Smertenko and Bozhkov, 2014). Thus, we examined the suspensor consisted of not only vacuolated cells but also expression patterns of these two widely recognized gene markers dense cytoplasmic cells (Figure 6E). Normal proembryos with of early embryogenesis during SE. embryonal masses from dense cytoplasmic cells and suspensors As there was little gene information for Chinese fir, sequences from vacuolated cells were obtained only on medium containing of SERK and WOX from P. abies, Arabidopsis, maize, rice, and both PEG and ABA (Figure 6F). In total, we found that ABA alfalfa were selected for screening against the transcriptome of contributed the most to embryonal mass development, whereas Chinese fir 6421. We obtained five orthologous contigs each for PEG facilitated suspensor development and orientation. SERK and WOX (see Supplementary Table S4). Neighbor-joining Frontiers in Plant Science | www.frontiersin.org 7 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 8 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 6 | Influence of ABA and PEG on the formation of proembryos. Tissues were stained with acetocarmine and Evan’s blue and photographed under a stereoscope. (A–C) Incubated on maturation medium for 7 days; (D–F) incubated on maturation medium for 1 month; (A,D) incubated on ABA-containing medium; (B,E) incubated on PEG-containing medium; (C,F) incubated on ABA and PEG-containing medium. (A) No obvious changes occurred after 7 days incubation on ABA-containing medium; PEM II structure; (B,C) PEM III structures formed (arrows); (D) PEM III structures without clear bunchy-shaped vacuolated cells (arrows); (E) proembryos shaped with more dense cytoplasmic cells (arrows); (F) normal proembryos [insert, Em of proembryo (arrow)]. Su, suspensor; Em, embryonal mass. Bars = 500 mm. phylogenetic analysis and qRT-PCR were conducted on both contains PaWOX5. Both are divergent from the angiosperm genes. WOX genes. Three different ClWOX13 contigs were included The phylogenetic tree of SERK showed four main branches in the WOX13 clade, the most ancient WOX clade (van der (Figure 7), annotated as SERK1/2, SERK 3/4/5, nuclear shuttle Graaff et al., 2009). ClWOX13-2 and ClWOX13-3 were closely protein interacting kinase (NIK), and Other (Nolan et al., 2011). related to two WOX13-like proteins of the non-vascular moss The SERK1/2 group included two ClSERK1 (39103 and 19371) Physcomitrella patens, whereas ClWOX13-1 was closer to the contigs. MtSERKL1, MtSERKL2, and ClSERK2-1 (20090) were three maize WOX13s. qRT-PCR results (Figure 8B) revealed that in the subclade of the third group and were more similar to ClWOX13-1 and ClWOX13-3 transcript levels were gradually up- NIK in Arabidopsis. The fourth subgroup included ClSERK1- regulated in liquid suspensions containing ABA (from stages 2 1 (18030), ClSERK1-2 (7157), and MtSERKL3. In general, the to 3), and decreased during proembryogeny (stage 4) to early relative expression data showed that the ClSERKs transcript levels embryogeny (stage 5). The expression of ClWOX4 and ClWOX5 were the highest during the development of the proembryos was comparatively high during late embryogeny (stage 6), which and the lowest in developed embryos (Figure 8A). ClSERK1-3 may be due to their functions in the procambia of cotyledon (39103) transcript levels were almost twofold higher at the end embryo and root stem cells, respectively. ClWOX13-2 transcript of proembryogeny (stage 4) compared with embryogenic calluses level was lowest at the end of early embryogeny (stage 5) and had (stage 1), followed by a sharp decrease during late embryogeny a smaller peak during embryo maturation (stage 6). (stages 5 and 6). ClSERK1-4 (19371), although evolutionarily close to ClSERK1-3 (39103), had a different expression pattern. ClSERK1-1 (18030) transcript levels rose significantly when DISCUSSION PEMs were embedded in the suspension containing ABA for differentiation (stage 2), but then declined, reaching an even An Efficient Synchronized SE System of lower level in PEMs when the dominant embryos began to Chinese Fir Was Developed to develop (stage 5). ClSERK2-1 (20090) and ClSERK1-2 (7517) Regenerate True-to-Type exhibited higher expression levels in the PEMs (stage 1) than in the developed embryos (stages 4, 5, and 6). Somatic-Embryo Derived Plants The unrooted tree of the plant WOX proteins (Figure 9) can Somatic embryogenesis of C. lanceolata has the potential to be naturally divided into three clades, referred to as the WUS, produce a large-scale supply of excellent seedlings for plantation intermediate, and ancient clades (van der Graaff et al., 2009). development in commercial timber resources strategic reserve, The ClWOX4 group contains PaWOX4, and the ClWOX5 group and it is an attractive model for studying early embryogenic Frontiers in Plant Science | www.frontiersin.org 8 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 9 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 7 | Neighbor-joining phylogenetic tree of full-length SERK and SERK-like protein sequences generated using the Jones-Taylor-Thornton (JTT) model and gamma-distributed (gamma = 0.97) with 1,000 bootstrap replicates. Four branches, SERK1/2, SERK3/4/5, NIK and Other are marked with different colors. At: Arabidopsis thaliana; Cl: Cunninghamia lanceolata; Mt: Medicago truncatula; Os: Oryza sativa; Zm: Zea mays. Accession numbers are provided in Supplementary Table S2. events due to the inability to access to zygotic embryos. Tissue mature embryos, these embryos were recalcitrant to obtain culture of Chinese fir via adventitious buds multiplication began normal cotyledon embryos and regenerate plantlets. In our in the late 1970s (Que, 1980) and continues to advance (Wang, studies, the yield and improved uniformity of somatic embryos 1999; Zhu et al., 2007a; Zhou et al., 2013). Recently, Li et al. offer a more stable SE system for mass propagation in Chinese (2017) reported on the production of plantlets from leaf cuttings. fir. Moreover, we synchronized PEM development by introducing While, development of an SE system for C. lanceolata has a liquid suspension system before maturation to proembryos also been ongoing for years, much less has been reported on was initiated (Chen and Chen, 2007). From the standpoint of that progress. Three method improvements have recently been operational economics, this system is cost-efficient, significantly published for Chinese fir SE. (1) Explants of cotyledon, hypocotyl shortening the propagation circulation time from 3 to 1 week. and mature embryos (Xi and Shi, 2005, 2006) were used to What’s more, the proembryos developed quite analogously to directly induced somatic embryos, but with a low propagation zygotic embryos, and offer great promise for conifer embryo rate of <10%, which is not suitable for mass propagation; development and transgenic system studies. (2) Hu et al. (2017) induced embryos indirectly from calluses For long-living forest trees, somaclonal variation can initiated from immature dominant zygotic embryos by successive significantly affect the quality of cloned individuals, and culturing on a low auxin/cytokinin concentration medium. This can result in severe economic losses years after planting. It methodology can produce embryos at much higher rate, but frequently occurs due to in vitro culture conditions, such somatic embryos may be contaminated with adventitious buds as high concentrations of growth regulators and long-term via indirect organogenesis that are not readily distinguished. (3) culturing (DeVerno et al., 1999). In our study, high hormone Hu et al. (2017) also used ABA and PEG to induce somatic concentrations during PEM maintenance and proembryo embryos starting with embryogenic calluses from immature induction, high osmolality induced by PEG, and a relatively long cleavage polyembryony-staged embryos. Although they obtained maturation term may contribute to the higher risk of somaclonal Frontiers in Plant Science | www.frontiersin.org 9 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 10 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 8 | qRT-PCR expression studies of putative SERK (A) and WOX (B) genes during the six developmental stages of SE in Chinese fir. The six developmental stages are: (1) PEMs maintained for 20 days, as seen in Figure 1C; (2) PEMs suspended for 2 days; (3) PEMs suspended for 3 weeks, as seen in Figure 2F; (4) proembryos grown on maturation medium for 1 week, as seen in Figure 1F; (5) reddish proembryos, as seen in Figure 1G or 1H; (6) cotyledon embryos, as seen in Figure 1J. Results shown are means  standard errors of three replicates, calibrated to the expression in the starting PEMs (1). variation. Ploidy level of the regenerants via SE was therefore genes. YUCCAs encode key enzymes in auxin biosynthesis which determined by karyotype analysis. In addition all SSR primers increase endogenous IAA levels (Bai et al., 2013), which in turn used showed monomorphic bands, suggesting our “true-to-type” can trigger IAA polar transport (Su et al., 2009). The addition propagation system. of an auxin antagonist PCIB (p-chlorophenoxy isobutyric acid) to maturation medium prevents embryogenic tissue proliferation and prompts the development of mature embryos. Moreover, Endogenous Hormonal Changes the addition of the auxin synergist phloroglucinol increases the Revealed Correlation between Seed proliferation of embryogenic tissues and totally suppresses the Developmental Stages, Genotypes, and maturation. This may indicate that endogenous IAA at high levels Competence Acquisition for SE can improve proliferation but block maturation of embryogenic The induction of PEMs is critical during the whole SE process as it cultures of Nordmann fir (Find et al., 2002). The PCIB treatment was also conducted in Scots pine, which reduced the proliferation may determine the PEM production efficiency and the capability of SE, which probably relies on the seed’s developmental stage of embryogenic tissues and significantly increased the yield of cotyledon embryos (Abrahamsson et al., 2011; Park and Bonga, and genotypic selection. The strategic selection of competent genotypes and explants during the collection period for PEM 2013). There is more detailed understanding of auxin responses induction could significantly reduce production cost and time. In in somatic embryo formation in Norway spruce. Addition of conifers, somatic embryogenic tissues are always induced from the polar auxin transport inhibitor 1-N -naphtylphthalamic acid juvenile tissues. For most pine species, early-stage immature (NPA) (Benková et al., 2003) caused endogenous IAA content embryos embedded in megagametophytes are highly competent to increase in early embryos, but programmed cell death (PCD) for SE (Stasolla and Yeung, 2003; Salajova and Salaj, 2005; decreased and differentiation of the suspensor was abnormal Park et al., 2006). In C. lanceolata, a high efficiency of (Larsson et al., 2008). During late embryo formation, NPA- PEM induction was obtained using proembryo-stage embryos treatment resulted in aberrant apical and basal patterning in mature embryos (Larsson et al., 2008; Palovaara et al., 2010). (Figure 3). However, only two PEM lines induced from cleaved embryos ultimately produced mature somatic embryos, whereas Therefore, maintenance of auxin transport and avoidance of local auxin buildups is important for the transition of embryogenic PEMs of other older stages failed using the same protocol. A large body of experimental observations exists on the central role of tissues to proembryos. In our study, we found that cleaved embryos contained the lowest levels of IAA prior to the endogenous IAA in the regulation of embryo development (Jenik and Barton, 2005). Auxinic herbicide (2,4-D) is widely applied for emergence of the dominant embryos (Figure 4A). Moreover, embryogenic tissue induction, but removal of 2,4-D is critical at genotypes 4098 and 5009, with higher competence levels for certain stages of cultured embryo development. In Arabidopsis SE, exhibited lower cleaved embryo stage IAA levels than the culture, removal of 2,4-D induces the expression of YUCCA recalcitrant genotypes Y21, 4009, and 27 (Figure 4B). The Frontiers in Plant Science | www.frontiersin.org 10 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 11 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 9 | A phylogenetic analysis of full-length WOX protein sequences was performed using the neighbor-joining statistical method and the Jones-Taylor-Thornton (JTT) model, and was gamma-distributed (gamma = 0.97) with 1,000 bootstrap replicates. At: Arabidopsis thaliana; Cl: Cunninghamia lanceolata; Gb: Ginkgo biloba; Mt: Medicago truncatula; Os: Oryza sativa; Pa: Picea abies; Pp: Physcomitrella patens; Vv: Vitis vinifera; Zm: Zea mays. Accession numbers are provided in Supplementary Table S3. same trend between SE competence and genotype was also identifying genotypes that are appropriate for SE. However, more discovered in other conifer. Pre-and post-cleavage stages before experiments need to be conducted for further verification. the appearance of a dominant embryo also showed the strongest ABA and PEG Play Complementary Role responses to somatic embryo initiation in P. strobus (Park, 2002). in Proembryo Formation In Picea morrisonicola Hayata, a less competent line had a 7.5-fold higher endogenous IAA content than a competent line, and a Osmotic changes are critical during SE competence acquisition. reduction in the endogenous IAA content during proliferation In most coniferous species, exogenous ABA is necessary to stop improved the somatic embryo induction competence for the cell proliferation and stimulate somatic embryo development poorly inducing line (Liao et al., 2008). We hypothesize that lower (Zavattieri et al., 2010; Jain et al., 2013). ABA induces cells’ endogenous auxin levels at early somatic embryo developmental desiccation tolerance from a hormonal level (Bozhkov et al., stages can facilitate the acquisition of competence during SE. It 2002), whereas PEG causes osmotic changes that affect cells is widely known that the potential of SE is genotype dependent. turgor from physiological aspect (Kermode, 1990). Most reports Many factors influencing SE competence acquirement has been focused on the quality improvement by ABA especially for investigated during SE development, such as antioxidants, amino promoting late embryogeny (Bozhkov and Von Arnold, 1998; acids, calcium irons, salicylic acid, genetic cues, and so on Bozhkov et al., 2002; Aronen et al., 2009). In our case, exogenous (Teixeira da Silva and Malabadi, 2012). We revealed high ABA triggered PEM differentiation and promoted sustained correlation between endogenous IAA level in juvenile explants vigor in suspension cultures. Moreover, morphological effects and genotype competence. Screening the endogenous IAA level of PEG and ABA on embryogenesis were compared. We in the initial explants had applicability as a useful tool for conducted such an investigation using ABA-, PEG-, and ABA and Frontiers in Plant Science | www.frontiersin.org 11 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 12 Zhou et al. Somatic Embryogenesis in Chinese Fir PEG-containing maturation media. The results demonstrated auxin transport (PAT) regulate embryo patterning during embryo that PEG initiated morphological changes in the first week, development (Palovaara et al., 2010). Expression of WOX2, promoting polarity in PEM III (Figures 6B,C). After a 1-month 8, and 9 orthologs was not apparently linked to Chinese fir incubation, normal proembryos were obtained only on the embryo development, but three ClWOX13 contigs that grouped medium containing ABA and PEG (Figure 6F). The ABA-only into the ancient WOX13 clade (Figure 9). ClWOX13 exhibited medium produced enlarged embryonal masses without clusters significantly higher transcript levels at stage 3, concurrent with suspensors (Figure 6D), whereas proembryo-like structures the proembryo transition (Figure 8B). ClWOX13-1 was more lacking enlarged embryonal masses were produced under PEG- closely related to the three maize WOX13, whereas the other only condition (Figure 6E). Thus, ABA and PEG contribute two, ClWOX13-2 and ClWOX13-3, clustered with two WOX13- in distinct and perhaps in interacting ways to proembryo like genes of the non-vascular moss P. patens (Figure 9). formation. In addition, the early addition of ABA could delay Interestingly, recent studies in P. patens elucidated the function the development of PEM III, which explains why newly formed of two PpWOX13-like genes in the control of cell wall loosening somatic embryos in Norway spruce grow well on plant growth to facilitate stem cell formation, but no regeneration-related regulator–free medium for at least 1 week (von Arnold et al., functions has yet been reported for the AtWOX13 (Sakakibara 2005). et al., 2014). qRT-PCR results revealed that expression of ClWOX13 may have high correlation with stage 3, which is when the critical transition to proembryo occurs. Because new Molecular Evidence Uncovered Early evidence indicates that Cunninghamia is a long and widespread Stress-Related Signaling during SE living fossil that extends back to the mid-Mesozoic period (Shi Development et al., 2014), we postulate that ClWOX13 may still conserve From PEMs to somatic embryos, each transition stage involves some functions of embryogenesis regulation in Chinese fir. Early physiological and metabolic alterations. These changes were WOX transcriptional machinery correlates with the gradient powered by gene regulators. SERK and WUS are two widely endogenous auxin distribution through the expression of the recognized gene markers of early embryogenesis. Recent progress auxin transport-related genes (Breuninger et al., 2008), and SERK in cotton has confirmed the role of SERK in cellular redox related also is auxin-induced (Nolan et al., 2003). From this aspect, it will somatic cell-to-embryo transition (Pandey and Chaudhary, be of particular interest to explore the connection between the 2014). WUS are responsible for keeping cell undifferentiated state WOX and SERK functions and endogenous auxin during early (Laux et al., 1996). embryogenesis in Chinese fir. SERK gene family attributes to the leucine-rich repeat (LRR) subfamily that coded for a transmembrane protein involved in signal transduction that was strongly related to stress responses CONCLUSION of SE (Santos and Aragão, 2009). AtSERK1 from Arabidopsis had direct function on SE and determined the embryogenic In summary, our study provides an efficient synchronized SE competence in culture (Hecht et al., 2001). In Medicago, system via a liquid suspension in Chinese fir that also might lead MtSERK1, orthologous to AtSERK1, was up-regulated by auxin to the improvement of SE in other gymnosperms. Karyotyping and functioned broader than embryogenesis alone (Nolan et al., and microsatellite analysis support preliminary optimism that 2003). In monocots, OsSERK1 can response to defense signaling no major cytological or genetic variation occurred in our plants hormonal activity, such as salicylic acid, jasmonic acid, and regenerated via SE. A hormonal analysis of IAA, ABA, ZR, and ABA (Hu et al., 2005). Phylogenetic results showed that two GA uncovered a potential relationship between endogenous ClSERK1 contigs (39103 and 19371), which are predominantly IAA and genotypic recalcitrance. Lower endogenous IAA of early expressed in PEMs, shared a high similarity with AtSERK1 stage immature embryos tends to higher competence acquisition. and MtSERK1, indicating that ClSERK1-3 and ClSERK1-4 may Morphological studies of PEMs during maturation revealed that function uniformly in SE. MtSERKL1 and MtSERKL2 are more ABA and PEG play complementary roles during proembryo similar to NIK genes in Arabidopsis (Nolan et al., 2011). formation. ABA boosted the growth of embryonal masses, Arabidopsis NIK shares many similarities with SERK; and is the whereas PEG functioned on the organization of proembryo- transducer of a novel layer of plant innate defenses (Santos et al., like structures. Finally, the phylogenetic tree and expression 2010). ClSERK2-1 (20090), grouped together with MtSERKL1 patterns suggested the probable regulation and activities of SERK and MtSERKL2, had higher transcript levels in stages 1 and 3 and WOX during SE, especially in early stages of proembryo (Figure 8), based on which, we postulated that the transition to formation. SERK is conserved and redundantly expressed in SE, late stage PEM may come with their stress related responses. whereas WOX13 is the most promising candidate for exploring WUS is the prototypical member of the WOX family. the function of WOX in Chinese fir embryo development. WUS overexpression increases the acquisition of embryogenic competence in Arabidopsis (Zheng et al., 2014) and cotton (Zuo et al., 2002). In Arabidopsis zygotic embryo development, AUTHOR CONTRIBUTIONS WOX2, 8, and 9 regulate cell fate by determining the early apical and basal patterning events (Haecker et al., 2004). In JC, JS, and TL conceived and designed the experiments, XZ conifer, PaWOX2, WOX8/9, PIN-FORMED (PIN ) and polar performed experiments and analyzed data, RZ prepared all the Frontiers in Plant Science | www.frontiersin.org 12 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 13 Zhou et al. Somatic Embryogenesis in Chinese Fir samples, GL performed the cytological analysis, YX helped in Southern China, Nanjing Forestry University, and Priority to pick up SSR primers and data analysis. YwZ contributed Academic Program Development of Jiangsu Higher Education to plant materials’ subculture, YZ helped with RT-PCR data Institutions. collection, XZ and RZ wrote the manuscript with contributions from all authors, TL and SH contributed to the manuscript revision. ACKNOWLEDGMENTS The authors thank Elizabeth Dell Trippe and Dr. Tim Smalley for the manuscript revision, Services of China Agricultural FUNDING University for endogenous hormones analysis. This work was supported by National High Technology Research and Development Program of China (863 Program, 2013AA102705), Specialized Research Fund for the Doctoral SUPPLEMENTARY MATERIAL Program of Higher Education (SRFDP, 20113204130002), Natural Science Foundation of Jiangsu University grant The Supplementary Material for this article can be found online 13KJA220001, Talent Project by the Ministry of Science and at: https://www.frontiersin.org/articles/10.3389/fpls.2017.02054/ Technology, Co-Innovation Center for Sustainable Forestry full#supplementary-material DeVerno, L., Park, Y., Bonga, J., and Barrett, J. (1999). Somaclonal variation in REFERENCES cryopreserved embryogenic clones of white spruce (Picea glauca (Moench) Abrahamsson, M., Valladares, S., Larsson, E., Clapham, D., and von Arnold, S. Voss.). Plant Cell Rep. 18, 948–953. doi: 10.1007/s002990050689 (2011). Patterning during somatic embryogenesis in Scots pine in relation to Durzan, D., and Chalupa, V. (1976). 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(2005). Organogenesis and somatic embryogenesis from Zuo, J., Niu, Q. W., Frugis, G., and Chua, N. H. (2002). The WUSCHEL gene cotyledons and hypocotyls of Cunninghamia lanceolata hook. Mol. Plant Breed. promotes vegetative-to-embryonic transition in Arabidopsis. Plant J. 30, 349– 6, 94–100. 359. doi: 10.1046/j.1365- 313X.2002.01289.x Xi, M., and Shi, J. (2006). Organogenesis and somatic embryogenesis from mature zygotic embryos of Cunninghamia lanceolata. Sci. Silvae Sin. 42, 29. Conflict of Interest Statement: The authors declare that the research was Xu, Y., Zheng, R., Wang, Z., Wang, Y., Hong, Z., Yang, L., et al. (2016). conducted in the absence of any commercial or financial relationships that could Identification and characterization of genic microsatellites in Cunninghamia be construed as a potential conflict of interest. lanceolata (Lamb.) hook (Taxodiaceae). Arch. Biol. Sci. 68, 417–425. doi: 10. 2298/ABS150608124X The reviewer TP declared a shared affiliation, with no collaboration, with one of Yang, J., Zhang, J., Wang, Z., Zhu, Q., and Wang, W. (2001). Hormonal changes the authors, TL, to the handling Editor. in the grains of rice subjected to water stress during grain filling. Plant Physiol. 127, 315–323. doi: 10.1104/pp.127.1.315 Copyright © 2017 Zhou, Zheng, Liu, Xu, Zhou, Laux, Zhen, Harding, Shi and Chen. Yang, Y. (1998). Studies on the Sustainable Management of Chinese fir Plantations. This is an open-access article distributed under the terms of the Creative Commons Beijing: China Forestry Publishing House. Attribution License (CC BY). The use, distribution or reproduction in other forums Zavattieri, M. A., Frederico, A. M., Lima, M., Sabino, R., and Arnholdt-Schmitt, B. is permitted, provided the original author(s) or licensor are credited and that the (2010). Induction of somatic embryogenesis as an example of stress-related original publication in this journal is cited, in accordance with accepted academic plant reactions. Electr. J. Biotechnol. 13, 12–13 doi: 10.2225/vol13- issue1- practice. No use, distribution or reproduction is permitted which does not comply fulltext- 4 with these terms. Frontiers in Plant Science | www.frontiersin.org 15 December 2017 | Volume 8 | Article 2054 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Plant Science Pubmed Central

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

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fpls-08-02054 December 2, 2017 Time: 15:57 # 1 ORIGINAL RESEARCH published: 05 December 2017 doi: 10.3389/fpls.2017.02054 Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Edited by: Jose M. Seguí-Simarro, Hook Universitat Politècnica de València, Spain 1,2‡ 3‡ 1,2 1† 1,2 Xiaohong Zhou , Renhua Zheng , Guangxin Liu , Yang Xu , Yanwei Zhou , 4 1,2 5 1,2 1,2 Reviewed by: Thomas Laux , Yan Zhen , Scott A. Harding , Jisen Shi and Jinhui Chen * * Taras P. Pasternak, Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, China, Albert Ludwigs University of Freiburg, Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Germany China, The Key Laboratory of Timber Forest Breeding and Cultivation for Mountainous Areas in Southern China, State Thomas Dandekar, Forestry Administration Engineering Research Center of Chinese Fir, Fujian Academy of Forestry, Fuzhou, China, Centre for University of Würzburg, Germany Biological Signaling Studies, Faculty of Biology, Albert Ludwigs University of Freiburg, Freiburg, Germany, School of *Correspondence: Forestry and Natural Resources, University of Georgia, Athens, GA, United States Jinhui Chen [email protected] Jisen Shi Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially [email protected] and ecologically, in southern China. The traditional regenerating methods are based Present address: on organogenesis and cutting propagation. Here, we report the development of a Yang Xu, Research Institute of Subtropical high-frequency somatic embryogenesis (SE) regeneration system synchronized via Forestry, Chinese Academy a liquid culture from immature zygotic embryos. Following synchronization, PEM II of Forestry, Fuyang, China cell aggregates were developmentally equivalent in appearance to cleaved zygotic These authors have contributed embryos. Embryo and suspensor growth and subsequent occurrence of the apical equally to this work. and then the cotyledonary meristems were similar for zygotic and SE embryo Specialty section: development. However, SE proembryos exhibited a more reddish coloration than This article was submitted to Plant Biotechnology, zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature a section of the journal somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, Frontiers in Plant Science low concentrations of endogenous indole-3-acetic acid in initial explants correlated Received: 03 July 2017 with improved proembryogenic mass formation, and high SE competency. Analysis Accepted: 16 November 2017 Published: 05 December 2017 of karyotypes and microsatellites detected no major genetic variation in the plants Citation: regenerated via SE, and suggest a potential in the further development of this system as Zhou X, Zheng R, Liu G, Xu Y, a reliable methodology for true-to-type seedling production. Treatment with polyethylene Zhou Y, Laux T, Zhen Y, Harding SA, Shi J and Chen J (2017) Desiccation glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation Treatment and Endogenous IAA and complemented each other. ABA assisted the growth of embryonal masses, Levels Are Key Factors Influencing whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC High Frequency Somatic Embryogenesis in Cunninghamia EMBRYOGENESIS RECEPTOR KINASE (SERK) and the WUSCHEL homeobox (WOX ) lanceolata (Lamb.) Hook. transcription factor served as molecular markers during early embryogenesis. Our Front. Plant Sci. 8:2054. doi: 10.3389/fpls.2017.02054 results show that ClSERKs are conserved and redundantly expressed during SE. SERK Frontiers in Plant Science | www.frontiersin.org 1 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 2 Zhou et al. Somatic Embryogenesis in Chinese Fir and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses. Keywords: ABA, desiccation, endogenous hormone, PEG, somatic embryo, SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), WUSCHEL homeobox (WOX) et al., 2007a,b; Zhou et al., 2013; Li et al., 2017). Development INTRODUCTION of SE has been handicapped by limited yields and calluses’ Cunninghamia lanceolata (Lamb.) Hook, Chinese fir, is an necrosis (Kang, 2008; Chen et al., 2017). Xi and Shi (2005, important native evergreen tree species in China. This fast- 2006) used explants of cotyledon, hypocotyl and mature zygotic growing conifer has been cultivated for over 3,000 years due embryos for direct SE induction, which is not perspective for to its elite wood attributes and high timber productivity (Yang, mass propagation and further genetic improvements due to low 1998). Chinese fir is now the dominant tree species in southern multiplication rates. Hu et al. (2017) recently established SE China, growing in over 15 southern provinces. The total planted via embryogenic callus using immature zygotic embryos. The 6 2 area of Chinese fir has reached 9  10 hm and accounts for system employed ABA and PEG to successfully produce early 30% of the forested land in China (Lu et al., 2015). Chinese somatic embryos, but normally developed, late stage somatic fir occupies prominent roles in ecological and commercial embryos were not obtained, and there were no reports about the prospects. conversion from somatic embryos to plantlets. Advances in third generation recurrent genetic selection and Members of the SOMATIC EMBRYOGENESIS RECEPTOR hybridization of Chinese fir through conventional breeding KINASE (SERK ) gene family encode leucine-rich repeat- programs have led to great genetic improvements on growth, containing transmembrane proteins that are involved in signal wood qualities and biological or non-biological stress tolerances. transduction and are strongly related to SE (Santos and Aragão, However, conventional breeding improvements are inefficient, 2009). SERK was first discovered as a marker for the somatic costly, and time consuming due to the inherently long life to embryogenic transition in carrot (Schmidt et al., 1997). cycles, unstable maturation, and frequently unavoidable dilution The WUSCHEL (WUS) homeobox (WOX) transcription factor of desirable traits caused by genetic segregation and gene is described as functioning in early embryo patterning and flow (Cairney et al., 1999). As an alternative to conventional lateral organ development in Arabidopsis (Haecker et al., breeding, somatic embryogenesis (SE) is thought to be the 2004). Overexpression of WUS can initiate acquisition of most promising clonal propagation strategy based on recurrent embryogenic competence in Arabidopsis (Zuo et al., 2002) genetic selection for commercial plantations regeneration. The and cotton (Zheng et al., 2014). In Picea abies, PaWOX2 generation circulation time could be reduced, and the extra and PaWOX8/9 were highly expressed in early stage embryo genetic gain of forest trees can be captured through SE with development (Palovaara and Hakman, 2008; Hedman et al., lower risks and costs (Gupta and Grob, 1995). The process 2013), when the primary body axis and radial patterning was can be rigorously controlled, and is theoretically feasible for being established (von Arnold et al., 2016). Therefore, SERK all plant species. A well-established SE line, combined with and WUS prompted SE during early embryogenesis (Guan et al., cryopreservation, would be an excellent platform for long-term 2016). conversation and large-scale planting materials production In this study, we developed an effective synchronized (Park, 2002). SE system from C. lanceolata immature seeds. Cell masses Since embryo-like structures were induced in vitro into were synchronized in liquid suspension before somatic somatic embryos in Pinus banksiana (Durzan and Chalupa, embryo differentiation. This system produces seedlings 1976), much progress has been archived by SE systems in conifers with no cytological variation detectable by karyotype and (Klimaszewska and Cyr, 2002; Cairney and Pullman, 2007; Jain microsatellite analyses. We showed that the competence of et al., 2013). However, most studies have concentrated on pine C. lanceolata seeds to generate somatic embryos correlated with species (Klimaszewska and Cyr, 2002; Sutton, 2002). In vitro developmental stage exhibiting low endogenous indole-3-acetic culture of Chinese fir is more difficult in comparison to the other acid (IAA) hormone levels in the source materials. We further coniferous species. At the same time, a rapid mass propagation demonstrated that morphological differences were caused by system is required to meet the increasing demand for this exposure to polyethylene glycol (PEG) and abscisic acid (ABA) species. To our knowledge, less progress has been achieved for during the early developmental stages of somatic embryos. C. lanceolata SE than for propagation via organogenesis (Zhu In addition, expression profiles of SERK and WUS reveal Frontiers in Plant Science | www.frontiersin.org 2 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 3 Zhou et al. Somatic Embryogenesis in Chinese Fir their applicability as the markers of early embryogenesis of Morphological Analysis Chinese fir. The entire developmental pathway and the embryonic stages were evaluated using a stereoscope (Leica, S8AP0), and micrographs were obtained using an inverted microscope (Leica, MATERIALS AND METHODS DMI4000). Samples were double-stained with acetocarmine and Evan’s blue (Gupta and Holmstrom, 2005). The dense Plant Materials cytoplasmic cells were stained into red, while vacuolated cells Chinese fir cones were collected once a week from late June to late were stained into blue. August 2009 in a National Clonal Seed Orchard at Yangkou and Shaowu Forest Farm, Fujian Province, China. These cones were Karyotype Analysis immediately placed on ice and brought back to the lab within Proembryogenic masses and more than 30 actively growing 24 h. Cones were stored at 4–5 C for no more than 1 week until roots (1.5–2 cm long) of regenerated somatic-embryo derived further used. seedlings were excised. The materials were pretreated in ice- Seeds with immature zygotic embryos of different cold water for 18–24 h and then fixed in Carnoy fixative (95% developmental stages were used as the initial explants to ethanol:acetic acid, 3:1) for 24 h, and then dissociated in 45% induce somatic embryos. The stages of the zygotic embryo (v/v) acetic acid for 2 h. Finally, carbol-fuchsin staining was materials were identified from 5 to 6 randomly selected seeds of used for squash preparations, and then slides were observed and each cone according to Shi et al. (2010). Cones were opened and photographed using the cell workstation software Leica 4000. seeds were collected before sterilization. Seeds were then washed The chromosome number and type were determined for >25 with detergent for 10 min, transferred into running water for cells from squashed Chinese fir root tips. Photos showing well- 30 min, surface-sterilized with 75% (v/v) ethanol for 30 s, treated spread chromosomes were processed further using Photoshop with 0.1% (w/v) HgCl containing a few drops (0.01%) of Tween CS3 and CAD 2010. The chromosome length in five cells was 20 for 8–10 min, and then rinsed three times in sterile distilled averaged. water. Seed coats were removed as previously described (Becwar and Pullman, 1995; Pullman and Johnson, 2002), and the embryo Microsatellite Analysis: Preparation of sacs, including the zygotic embryos, were used for induction of Genomic DNA and PCR Amplification proembryogenic masses (PEMs). More than 30 seeds of the same More than 30 regenerated plants of C. lanceolata and three embryonic stage were used to assess the frequency of outgrowths proliferating PEMs were sampled for DNA isolation. Each sample and the test was repeated three times. was immediately frozen in liquid nitrogen and stored at 80 C until the DNA extraction. Samples were ground to a powder Medium and Culture Conditions in liquid nitrogen, and genomic DNA was extracted using the The embryo sacs were cut near the suspensors and initially DNeasy Plant Mini Kit (Qiagen). cultured for 1 month at 23–25 C in darkness on Gupta and We randomly selected 10 primer pairs from available Durzan medium (DCR) (Gupta and Durzan, 1985), which nuclear microsatellite primers that had already been tested in contained 2.0–6.0 mg L 2,4-dichlorophenoxyacetic acid, 1 1 C. lanceolata to detect genetic stability (see Supplementary 0.5 mg L benzylaminopurine, 500 mg L casein hydrolysate 1 1 Table S1). These nuclear simple sequence repeat (SSR) (CH) (Sigma), 450 mg L L-glutamine, 100 mg L myo- 1 1 microsatellite loci were CFeSSR23, CFeSSR35, CFeSSR63, inositol, 20 g L maltose, and 2.1 g L gellan gum (Sigma). CFeSSR72, CFeSSR98, CFeSSR234, CFeSSR278, CFeSSR284, The medium was adjusted to pH 5.8 using KOH or HCl after CFeSSR312, CFeSSR352, and CFeSSR418. These primers were the addition of all of the ingredients except the gelling agent. The developed from the transcriptomic analysis of genotype 6421 gelling agent was added prior to autoclaving at 121 C for 20 min. (Xu et al., 2016). Maltose was used as a carbon source and autoclaved separately. PCR amplification was performed by adding 2 mL diluted For the synchronization of SE development, approximately genomic DNA to a cocktail, with a final volume of 10 mL 2 g of embryogenic calluses were suspended in 50 mL liquid containing 1 PCR buffer, 25 nmol MgCl , 2 nmol dNTPs, medium consisting of DCR salts, 3 mg L ABA (Sigma), 1 1 1 1 U Taq DNA polymerase (Takara), and 1 nmol forward and 0.5 mg L gibberellic acid (GA ), 500 mg L CH, and 30 g L reverse primers. PCR reactions were performed on a Veriti maltose. ABA was filter-sterilized and added into autoclaved 96-well Thermal Cycler (Applied Biosystems) using the following cooled medium. procedure: 4 min at 94 C, followed by 20 cycles of 45 s at 94 C, After 3 weeks suspension culture, steadily proliferating cell 45 s at the annealing Tm, and 60 s at 72 C, with a final 10 min clumps were dispersed on DCR solid medium containing 1 1 1 extension at 72 C. PCR products were run on 8% (w/v) native 3 mg L ABA, 1 mg L GA , and 120–200 g L PEG polyacrylamide gels. (MW 8000; Amresco). The osmotic potential was measured by a Wescor 5520 vapor pressure osmometer (Wescor, Inc.). Three Analysis of Endogenous Hormone Levels months later, the mature somatic embryos were transferred to basic DCR medium for the regeneration of plantlets, which Fresh seeds at different developmental stages of the genotypes were maintained at 25 C under cool white fluorescent light 4098, 5009, 4009, Y21, and 27 were collected for the (30 mmol m per second, with a 16-h photoperiod). detection of endogenous hormones. Megagametophytes Frontiers in Plant Science | www.frontiersin.org 3 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 4 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 1 | Developmental stages of SE (A–L) and zygotic embryogenesis (M–S) in Cunninghamia lanceolata (Lamb.) Hook. (A,B,D,E) Embryo sacs containing immature embryos for PEM induction: (A,B) induced embryogenic tissues; (D,E) induced non-embryogenic tissues. (E,F–K) Somatic embryo induction at diverse stages in Chinese fir: (E) maintenance of PEMs that were translucent and had pointed surface protrusions; (F) formation of proembryos (arrows); PEMs on a high osmolality medium for 1 week after suspension; end of proembryogeny; (G) shaped early reddish embryos (arrows); end of early embryogeny; (H) reddish embryos with bright yellow tops (arrows); the transition to late embryogeny; (I) early cotyledonary embryos; (J) late cotyledonary embryos; (K,L) seedlings germinated from somatic embryos at 1 week (K) and 1 month (L). (M–S) Zygotic embryo development of Chinese fir based on Pullman and Webb (1994): (M) cleaved polyembryogeny before a dominant embryo forms; (N) proembryo, the beginning of early embryogeny; the dominant embryos has formed; (O) further developed dominant embryo with embryonal mass and suspensor prototypes; ready for late embryogeny; (P) dominant embryo with a more mature embryonal mass and suspensor; (Q) embryonal mass to cotyledon formation; (R,S) maturation of cotyledonary embryo. Bars = 1 mm for (A–J); 500 mm for (M–S); 1 cm for (K,L). (1.0–2.0 g) that contained immature zygotic embryos were and the WOX homologs in P. abies (Palovaara et al., 2010; immediately frozen in liquid nitrogen and stored at80 C until Hedman et al., 2013) and Arabidopsis (Hecht et al., 2001) analysis. (see Supplementary Table S3). The potential genes were The methods for extraction and purification of ABA, IAA, named on the basis of their alignment to Arabidopsis (see GA , and zeatin riboside (ZR) were modified from those Supplementary Table S4). The unrooted neighbor-joining described by Bollmark et al. (1988) and He (1993). ELISA phylogenetic trees were generated from the deduced protein hormonal quantification was following the protocols described by sequences using MEGA5.2 with the help of the Jones- Yang et al. (2001) and Teng et al. (2006). Taylor-Thornton (JTT) model in combination with the gamma-distributed rate model (JTTCG; gamma D 0.97). Quantitative Real-Time PCR (qRT-PCR) Bootstrap values from 1,000 replicates were indicated at each node. To isolate the potential SERK and WOX genes, the transcriptome database of C. lanceolata produced by Huang et al. (2012), Tissues at different developmental stages were collected. Qiu et al. (2013), and Wang et al. (2013) was screened using Total RNA was extracted and purified following the methods the NCBI tblastn algorithm using the SERK homologs in previously described by Lin et al. (2009). RNA (1 mg) was Arabidopsis, Medicago, and rice (see Supplementary Table S2), reverse-transcribed with oligo (dT) and random hexamer primers Frontiers in Plant Science | www.frontiersin.org 4 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 5 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 2 | Microscopic structure of embryogenic and non-embryogenic tissues induced from immature embryos and suspension cultures in Chinese fir. Stage annotation based on Filonova et al. (2000). (A) Embryogenic tissues. Cell aggregates include a clump of rounded, dense cytoplasmic cells surrounded by a set of vacuolated cells that are elongated. (B) Well-organized PEM III structures after a more than half year maintenance period. (C,D) Non-embryogenic tissues also induced from immature embryos of Chinese fir: cell clusters composed of dense cytoplasmic cells but lacking elongated cells (C), or with vacuolated cells in a collapsed cell shape but stuck in dense cytoplasmic cell aggregates (D, arrow represented PEM I structure). (E,F) Suspensions of PEMs stained with acetocarmine and Evan’s blue: (E) PEMs in suspension for 1 week, with more early-stage PEMs dedifferentiated (PEM I) (arrows); (F) PEMs in suspension for 3 weeks, with newly organized PEMs. Bars = 100 mm. using a reverse transcription system (Promega). Verification was zygotic embryos visibly protruded from the megagametophyte performed using qRT-PCR. micropolar end into the medium (Figure 1A). One month qRT-PCR was carried out using the LightCycler 480 System later, outgrowths proliferated to form PEMs (Figure 1B). (Roche Applied Science). Each reaction was performed in a 20- Embryogenic tissues were soggy and recognized as translucent mL final volume containing 10 mL of 2 LightCycler 480 SYBR structures, with pointed protrusions at the surface (Figure 1C). Green I Master, 0.5 mL of each homolog-specific primer pair Microscopy of the cell aggregates revealed clumps of rounded, (see Supplementary Table S5) at 100 nM, 5.0 mL of diluted dense cytoplasmic cells surrounded by a set of vacuolated cDNA template, and 4.0 mL of ddH O. Each reaction was run and elongated cells (Figure 2A), which were referred to as in triplicate with the appropriate negative controls. Amplification PEM II defined by Filonova et al. (2000). After a more than was conducted under the following conditions: activation for half year subculturing period, PEM II in the early stages 5 min at 95 C, followed by 40 cycles of 10 s at 95 C, 15 s at became the assembled and well-organized PEM III (Figure 2B). 60 C, and 15 s at 72 C. Fluorescence detection was performed However, non-embryogenic calluses looked quite different in after the extension step. The melting curve, with a temperature color (Figures 1D,E), and cell clusters were not as well- gradient from 60 to 95 C, was used to further investigate the organized (Figures 2C,D). Some non-embryogenic calluses specificity of each qRT-PCR reaction, and the presence of a single showed a denser lump of cytoplasmic cells but lacked elongated PCR product was also verified by 2% agarose gel electrophoresis. cells (Figure 2C), whereas some contained vacuolated cells The eIF-3 housekeeping gene was selected as the endogenous in collapsed shapes and were stuck in dense cytoplasmic cell reference gene for the relative PCR quantification (Wang et al., aggregates (Figure 2D). 2013). The rate of PEM induction was related to the zygotic embryos’ developmental stage (Figure 3). The frequency was markedly higher for embryo sacs corresponding to early zygotic embryo RESULTS stages (stages A–C, Figure 3), but sharply declined in late- stage embryos (stages D–E, Figure 3). Immature embryos in Generation of Somatic Embryos in proembryogeny and early embryogeny were the best sources of Chinese Fir explants for PEM induction. The development of somatic embryos in Chinese fir is shown Our overarching objective was to develop an efficient system for Chinese fir mass propagation via SE. The appearance of in Figures 1F–L. The maintained PEMs were transferred to liquid PEMs with embryonic suspensor structures occurred on DCR culture to synchronize the development of the cell masses and medium containing a 2.0–6.0 mg L 2,4-dichlorophenoxyacetic to produce vigorously differentiated late stage PEM structures. acid. Outgrowth occurred within 1–4 weeks when one or more With the addition of ABA, PEMs separated into small groups Frontiers in Plant Science | www.frontiersin.org 5 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 6 Zhou et al. Somatic Embryogenesis in Chinese Fir embryo proper became opaque, the apical meristem primordium was clearly visible (Figure 1H). The cotyledon primordia formed accordingly (Figure 1I), but were not as easily distinguishable as in zygotic embryos (Figure 1P). On the whole, somatic embryos were smaller than zygotic embryos after they reached the proembryo stages. PEM Induction Frequency Correlated with Endogenous Auxin Level The induction frequency of the PEMs was related to the developmental stages of the explants, as well as the plant genotypes. We investigated whether these differences correlated with endogenous hormonal levels using ELISA quantification. IAA and ABA levels oscillated during somatic embryo development, whereas ZR and GA levels were low and FIGURE 3 | Box plots for PEM induction frequencies at different steady (Figure 4A). Cleaved embryos had the lowest IAA content developmental zygotic embryo stages in C. lanceolata. More than 30 seeds were analyzed for each embryonic stage. Stage A, cleaved polyembryogeny during proembryogeny of zygotic embryo development (stage stages (Figure 1M); stage B, proembryo, only a dominant embryo 1, Figure 4). The IAA concentration rose during development, (Figure 1M); stage C, further developed dominant embryo (Figure 1O); stage until the dominant embryo formed opaque suspensors at the D, dominant embryo had formed apical meristem primordium (Figure 1P); start of late embryogeny (stages 2–3, Figure 4A), and then a low stage E, more changes occurred in embryonal mass to cotyledon formation level was maintained during late embryogeny until cotyledons (Figure 1Q); stages F and G, maturation of cotyledonary embryo (Figures 1R,S). Minimum and maximum PEM induction frequencies are emerged (stages 4–5, Figure 4A). The endogenous ABA content depicted by short black lines, the box signifies the upper and lower quartiles, increased with the development of a dominant embryo and then and the median and mean are respectively represented by a thicker black line declined during embryo maturation. and a thicker plus sign within the box for each stage. Endogenous IAA levels significantly varied among the genotypes of cleaved embryos (Figure 4B). Notably, genotypes 4098 and 5009 displayed relatively low levels of IAA at the stage of cell masses during the first week. At this point, early-stage of embryo cleavage, whereas IAA levels were higher in genotypes PEMs, with fewer elongated cells, were seen, probably due to 4009, Y21, and 27 (Figure 4B). Not so significant differences in the shearing in the suspension (Figure 2E). Three weeks later, the ABA content were observed among the different genotypes. cell aggregates propagated both cell types and assembled into Genotypes 4098 and 5009 were effective sources for SE using new PEM II structures (Figure 2F). Suspended PEMs were then our method, whereas the genotypes Y21, 4009, and 27 did not dispersed into a high-osmolality environment to complete the yield somatic embryos. Therefore, it appears that SE using this critical transition from PEMs to proembryo, and to facilitate method may benefit from low endogenous IAA levels at the late embryogeny. Proembryos formed, having a dense embryonic proembryogeny stage. head and highly vacuolated long polarized cells, but were still translucent (Figure 1F). In the following days, proembryos then ABA and PEG Are Different Stress turned red (Figure 1G), developed a bright yellowish top, and Signals for Somatic Embryo also increased in size (Figure 1H). A well-developed shoot and Development root pole took shape during this stage. Early cotyledonary-shaped embryos then formed, with the appearance of an indentation Polyethylene glycol (MW 8000) is an important facilitator separating the developing cotyledon structures (Figure 1I). of somatic embryo maturation in conifer (Stasolla et al., Finally, the two cotyledons fully opened, completing maturation 2003), which can mimic the desiccation that occurs during (Figure 1J). Mature cotyledon embryos were generated on zygotic embryo maturation. Different concentrations of hormone and osmoticum (PEG) free medium (Figures 1K,L). PEG (12–20%) were tested for effects on somatic embryo Investigation of the developmental processes of zygotic maturation (Figure 5). The number of somatic embryos embryos indicated that somatic embryos went through increased sharply when PEG concentration increased from analogous developmental phases. Suspension-cultured PEMs 150 to 170 g L (15–17%), followed by a sharp decline at were synchronized and produced active PEM II cell aggregates higher PEG concentrations (Figure 5). Thus, the optimized PEG (Figure 2F), which was the equivalent to cleaved zygotic embryo concentration was 17%. development (Figure 1M). During subsequent maturation, the Abscisic acid, “stress hormone” in plants, is also widely used zygotic embryo proper would then become white and translucent during SE induction (Zavattieri et al., 2010). To further clearly (Figure 1N). Both embryo and suspensor enlarged longitudinally elucidate the effect of high osmolality and exogenous ABA during (Figure 1O). Subsequently, the apical meristem primordium SE, we compared the effects of PEG-, ABA-, and PEG and ABA- became visible (Figure 1P), followed by the appearance of containing maturation media on morphological changes during cotyledon primordium (Figure 1Q). However, SE proembryos embryo development (Figure 6). Differences in morphology turned reddish rather than white (Figure 1G). When the SE between embryos on PEG-free (Figure 6A) and PEG-containing Frontiers in Plant Science | www.frontiersin.org 6 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 7 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 4 | Endogenous hormone content of zygotic embryos (genotype 4098) at different developmental stages (A) and of different genotypes at the cleaved-embryo stage (B). (A) The different developmental stages represented from 1 to 5: stage 1 cleaved polyembryos (Figure 1M); stage 2 proembryo (Figure 1N); stage 3 dominant embryo (Figure 1O); stage 4 columnar embryo (Figure 1P); stage 5 cotyledonary embryo (Figures 1R,S). (B) Genotypes 4098, 5009, 4009, Y21, and 27 were tested, and genotypes 4098 and 5009 were more competent to form somatic embryos. Data are means  SD of three replicates. Karyotype and Microsatellite Analyses Reveal True-to-Type Propagation via SE A karyotype analysis of plants regenerated through SE was performed by assessing >30 actively growing roots from 30 different individuals. All of the regenerated plants were diploids with 22 chromosomes (2nD 2XD 22; see Supplementary Figure S1A). The relative length and the ratio of the long arm to the short arm (see Supplementary Figure S1B and Supplementary Table S6) indicated that the karyotype belonged to 1B and consisted of 10 pairs of metacentric and 1 pair of submetacentric chromosomes. Eleven randomly selected primers developed from a transcriptome analysis of genotype 6421 were used to explore the microsatellite stability of the regenerated plants (see Supplementary Table S1). All primers exhibited monomorphic bands between the 3 tested PEM lines and 30 regenerated plants (see Supplementary Figure S2). FIGURE 5 | Frequency of somatic embryogenesis (SE) at different osmotic potentials. Data are means  SD of three replicates. SERK and WOX May Serve As Markers of Stress-Mediated Cell-Signaling during SE (Figures 6B,C) media were visible after the first 7 days of incubation. Polar PEM III structures were evident when PEG Somatic embryogenesis is also recognized as a reprogrammed was present, whereas embryos on the ABA-only medium did developmental process responding to stress-related signals not differentiate beyond PEM II. After 1 month, variations were (Zavattieri et al., 2010). Several gene regulators of this process more obvious. By that time, PEM III structures with polarity had have been identified. SERK is considered to be the critical formed in the ABA-only media, without clusters of vacuolated switch from somatic to embryogenic development, and WUS cells associated with proembryo formation (Figure 6D). PEG maintains a small group of cells at the dedifferentiation state alone produced more proembryo-like structures; however, the (Smertenko and Bozhkov, 2014). Thus, we examined the suspensor consisted of not only vacuolated cells but also expression patterns of these two widely recognized gene markers dense cytoplasmic cells (Figure 6E). Normal proembryos with of early embryogenesis during SE. embryonal masses from dense cytoplasmic cells and suspensors As there was little gene information for Chinese fir, sequences from vacuolated cells were obtained only on medium containing of SERK and WOX from P. abies, Arabidopsis, maize, rice, and both PEG and ABA (Figure 6F). In total, we found that ABA alfalfa were selected for screening against the transcriptome of contributed the most to embryonal mass development, whereas Chinese fir 6421. We obtained five orthologous contigs each for PEG facilitated suspensor development and orientation. SERK and WOX (see Supplementary Table S4). Neighbor-joining Frontiers in Plant Science | www.frontiersin.org 7 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 8 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 6 | Influence of ABA and PEG on the formation of proembryos. Tissues were stained with acetocarmine and Evan’s blue and photographed under a stereoscope. (A–C) Incubated on maturation medium for 7 days; (D–F) incubated on maturation medium for 1 month; (A,D) incubated on ABA-containing medium; (B,E) incubated on PEG-containing medium; (C,F) incubated on ABA and PEG-containing medium. (A) No obvious changes occurred after 7 days incubation on ABA-containing medium; PEM II structure; (B,C) PEM III structures formed (arrows); (D) PEM III structures without clear bunchy-shaped vacuolated cells (arrows); (E) proembryos shaped with more dense cytoplasmic cells (arrows); (F) normal proembryos [insert, Em of proembryo (arrow)]. Su, suspensor; Em, embryonal mass. Bars = 500 mm. phylogenetic analysis and qRT-PCR were conducted on both contains PaWOX5. Both are divergent from the angiosperm genes. WOX genes. Three different ClWOX13 contigs were included The phylogenetic tree of SERK showed four main branches in the WOX13 clade, the most ancient WOX clade (van der (Figure 7), annotated as SERK1/2, SERK 3/4/5, nuclear shuttle Graaff et al., 2009). ClWOX13-2 and ClWOX13-3 were closely protein interacting kinase (NIK), and Other (Nolan et al., 2011). related to two WOX13-like proteins of the non-vascular moss The SERK1/2 group included two ClSERK1 (39103 and 19371) Physcomitrella patens, whereas ClWOX13-1 was closer to the contigs. MtSERKL1, MtSERKL2, and ClSERK2-1 (20090) were three maize WOX13s. qRT-PCR results (Figure 8B) revealed that in the subclade of the third group and were more similar to ClWOX13-1 and ClWOX13-3 transcript levels were gradually up- NIK in Arabidopsis. The fourth subgroup included ClSERK1- regulated in liquid suspensions containing ABA (from stages 2 1 (18030), ClSERK1-2 (7157), and MtSERKL3. In general, the to 3), and decreased during proembryogeny (stage 4) to early relative expression data showed that the ClSERKs transcript levels embryogeny (stage 5). The expression of ClWOX4 and ClWOX5 were the highest during the development of the proembryos was comparatively high during late embryogeny (stage 6), which and the lowest in developed embryos (Figure 8A). ClSERK1-3 may be due to their functions in the procambia of cotyledon (39103) transcript levels were almost twofold higher at the end embryo and root stem cells, respectively. ClWOX13-2 transcript of proembryogeny (stage 4) compared with embryogenic calluses level was lowest at the end of early embryogeny (stage 5) and had (stage 1), followed by a sharp decrease during late embryogeny a smaller peak during embryo maturation (stage 6). (stages 5 and 6). ClSERK1-4 (19371), although evolutionarily close to ClSERK1-3 (39103), had a different expression pattern. ClSERK1-1 (18030) transcript levels rose significantly when DISCUSSION PEMs were embedded in the suspension containing ABA for differentiation (stage 2), but then declined, reaching an even An Efficient Synchronized SE System of lower level in PEMs when the dominant embryos began to Chinese Fir Was Developed to develop (stage 5). ClSERK2-1 (20090) and ClSERK1-2 (7517) Regenerate True-to-Type exhibited higher expression levels in the PEMs (stage 1) than in the developed embryos (stages 4, 5, and 6). Somatic-Embryo Derived Plants The unrooted tree of the plant WOX proteins (Figure 9) can Somatic embryogenesis of C. lanceolata has the potential to be naturally divided into three clades, referred to as the WUS, produce a large-scale supply of excellent seedlings for plantation intermediate, and ancient clades (van der Graaff et al., 2009). development in commercial timber resources strategic reserve, The ClWOX4 group contains PaWOX4, and the ClWOX5 group and it is an attractive model for studying early embryogenic Frontiers in Plant Science | www.frontiersin.org 8 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 9 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 7 | Neighbor-joining phylogenetic tree of full-length SERK and SERK-like protein sequences generated using the Jones-Taylor-Thornton (JTT) model and gamma-distributed (gamma = 0.97) with 1,000 bootstrap replicates. Four branches, SERK1/2, SERK3/4/5, NIK and Other are marked with different colors. At: Arabidopsis thaliana; Cl: Cunninghamia lanceolata; Mt: Medicago truncatula; Os: Oryza sativa; Zm: Zea mays. Accession numbers are provided in Supplementary Table S2. events due to the inability to access to zygotic embryos. Tissue mature embryos, these embryos were recalcitrant to obtain culture of Chinese fir via adventitious buds multiplication began normal cotyledon embryos and regenerate plantlets. In our in the late 1970s (Que, 1980) and continues to advance (Wang, studies, the yield and improved uniformity of somatic embryos 1999; Zhu et al., 2007a; Zhou et al., 2013). Recently, Li et al. offer a more stable SE system for mass propagation in Chinese (2017) reported on the production of plantlets from leaf cuttings. fir. Moreover, we synchronized PEM development by introducing While, development of an SE system for C. lanceolata has a liquid suspension system before maturation to proembryos also been ongoing for years, much less has been reported on was initiated (Chen and Chen, 2007). From the standpoint of that progress. Three method improvements have recently been operational economics, this system is cost-efficient, significantly published for Chinese fir SE. (1) Explants of cotyledon, hypocotyl shortening the propagation circulation time from 3 to 1 week. and mature embryos (Xi and Shi, 2005, 2006) were used to What’s more, the proembryos developed quite analogously to directly induced somatic embryos, but with a low propagation zygotic embryos, and offer great promise for conifer embryo rate of <10%, which is not suitable for mass propagation; development and transgenic system studies. (2) Hu et al. (2017) induced embryos indirectly from calluses For long-living forest trees, somaclonal variation can initiated from immature dominant zygotic embryos by successive significantly affect the quality of cloned individuals, and culturing on a low auxin/cytokinin concentration medium. This can result in severe economic losses years after planting. It methodology can produce embryos at much higher rate, but frequently occurs due to in vitro culture conditions, such somatic embryos may be contaminated with adventitious buds as high concentrations of growth regulators and long-term via indirect organogenesis that are not readily distinguished. (3) culturing (DeVerno et al., 1999). In our study, high hormone Hu et al. (2017) also used ABA and PEG to induce somatic concentrations during PEM maintenance and proembryo embryos starting with embryogenic calluses from immature induction, high osmolality induced by PEG, and a relatively long cleavage polyembryony-staged embryos. Although they obtained maturation term may contribute to the higher risk of somaclonal Frontiers in Plant Science | www.frontiersin.org 9 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 10 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 8 | qRT-PCR expression studies of putative SERK (A) and WOX (B) genes during the six developmental stages of SE in Chinese fir. The six developmental stages are: (1) PEMs maintained for 20 days, as seen in Figure 1C; (2) PEMs suspended for 2 days; (3) PEMs suspended for 3 weeks, as seen in Figure 2F; (4) proembryos grown on maturation medium for 1 week, as seen in Figure 1F; (5) reddish proembryos, as seen in Figure 1G or 1H; (6) cotyledon embryos, as seen in Figure 1J. Results shown are means  standard errors of three replicates, calibrated to the expression in the starting PEMs (1). variation. Ploidy level of the regenerants via SE was therefore genes. YUCCAs encode key enzymes in auxin biosynthesis which determined by karyotype analysis. In addition all SSR primers increase endogenous IAA levels (Bai et al., 2013), which in turn used showed monomorphic bands, suggesting our “true-to-type” can trigger IAA polar transport (Su et al., 2009). The addition propagation system. of an auxin antagonist PCIB (p-chlorophenoxy isobutyric acid) to maturation medium prevents embryogenic tissue proliferation and prompts the development of mature embryos. Moreover, Endogenous Hormonal Changes the addition of the auxin synergist phloroglucinol increases the Revealed Correlation between Seed proliferation of embryogenic tissues and totally suppresses the Developmental Stages, Genotypes, and maturation. This may indicate that endogenous IAA at high levels Competence Acquisition for SE can improve proliferation but block maturation of embryogenic The induction of PEMs is critical during the whole SE process as it cultures of Nordmann fir (Find et al., 2002). The PCIB treatment was also conducted in Scots pine, which reduced the proliferation may determine the PEM production efficiency and the capability of SE, which probably relies on the seed’s developmental stage of embryogenic tissues and significantly increased the yield of cotyledon embryos (Abrahamsson et al., 2011; Park and Bonga, and genotypic selection. The strategic selection of competent genotypes and explants during the collection period for PEM 2013). There is more detailed understanding of auxin responses induction could significantly reduce production cost and time. In in somatic embryo formation in Norway spruce. Addition of conifers, somatic embryogenic tissues are always induced from the polar auxin transport inhibitor 1-N -naphtylphthalamic acid juvenile tissues. For most pine species, early-stage immature (NPA) (Benková et al., 2003) caused endogenous IAA content embryos embedded in megagametophytes are highly competent to increase in early embryos, but programmed cell death (PCD) for SE (Stasolla and Yeung, 2003; Salajova and Salaj, 2005; decreased and differentiation of the suspensor was abnormal Park et al., 2006). In C. lanceolata, a high efficiency of (Larsson et al., 2008). During late embryo formation, NPA- PEM induction was obtained using proembryo-stage embryos treatment resulted in aberrant apical and basal patterning in mature embryos (Larsson et al., 2008; Palovaara et al., 2010). (Figure 3). However, only two PEM lines induced from cleaved embryos ultimately produced mature somatic embryos, whereas Therefore, maintenance of auxin transport and avoidance of local auxin buildups is important for the transition of embryogenic PEMs of other older stages failed using the same protocol. A large body of experimental observations exists on the central role of tissues to proembryos. In our study, we found that cleaved embryos contained the lowest levels of IAA prior to the endogenous IAA in the regulation of embryo development (Jenik and Barton, 2005). Auxinic herbicide (2,4-D) is widely applied for emergence of the dominant embryos (Figure 4A). Moreover, embryogenic tissue induction, but removal of 2,4-D is critical at genotypes 4098 and 5009, with higher competence levels for certain stages of cultured embryo development. In Arabidopsis SE, exhibited lower cleaved embryo stage IAA levels than the culture, removal of 2,4-D induces the expression of YUCCA recalcitrant genotypes Y21, 4009, and 27 (Figure 4B). The Frontiers in Plant Science | www.frontiersin.org 10 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 11 Zhou et al. Somatic Embryogenesis in Chinese Fir FIGURE 9 | A phylogenetic analysis of full-length WOX protein sequences was performed using the neighbor-joining statistical method and the Jones-Taylor-Thornton (JTT) model, and was gamma-distributed (gamma = 0.97) with 1,000 bootstrap replicates. At: Arabidopsis thaliana; Cl: Cunninghamia lanceolata; Gb: Ginkgo biloba; Mt: Medicago truncatula; Os: Oryza sativa; Pa: Picea abies; Pp: Physcomitrella patens; Vv: Vitis vinifera; Zm: Zea mays. Accession numbers are provided in Supplementary Table S3. same trend between SE competence and genotype was also identifying genotypes that are appropriate for SE. However, more discovered in other conifer. Pre-and post-cleavage stages before experiments need to be conducted for further verification. the appearance of a dominant embryo also showed the strongest ABA and PEG Play Complementary Role responses to somatic embryo initiation in P. strobus (Park, 2002). in Proembryo Formation In Picea morrisonicola Hayata, a less competent line had a 7.5-fold higher endogenous IAA content than a competent line, and a Osmotic changes are critical during SE competence acquisition. reduction in the endogenous IAA content during proliferation In most coniferous species, exogenous ABA is necessary to stop improved the somatic embryo induction competence for the cell proliferation and stimulate somatic embryo development poorly inducing line (Liao et al., 2008). We hypothesize that lower (Zavattieri et al., 2010; Jain et al., 2013). ABA induces cells’ endogenous auxin levels at early somatic embryo developmental desiccation tolerance from a hormonal level (Bozhkov et al., stages can facilitate the acquisition of competence during SE. It 2002), whereas PEG causes osmotic changes that affect cells is widely known that the potential of SE is genotype dependent. turgor from physiological aspect (Kermode, 1990). Most reports Many factors influencing SE competence acquirement has been focused on the quality improvement by ABA especially for investigated during SE development, such as antioxidants, amino promoting late embryogeny (Bozhkov and Von Arnold, 1998; acids, calcium irons, salicylic acid, genetic cues, and so on Bozhkov et al., 2002; Aronen et al., 2009). In our case, exogenous (Teixeira da Silva and Malabadi, 2012). We revealed high ABA triggered PEM differentiation and promoted sustained correlation between endogenous IAA level in juvenile explants vigor in suspension cultures. Moreover, morphological effects and genotype competence. Screening the endogenous IAA level of PEG and ABA on embryogenesis were compared. We in the initial explants had applicability as a useful tool for conducted such an investigation using ABA-, PEG-, and ABA and Frontiers in Plant Science | www.frontiersin.org 11 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 12 Zhou et al. Somatic Embryogenesis in Chinese Fir PEG-containing maturation media. The results demonstrated auxin transport (PAT) regulate embryo patterning during embryo that PEG initiated morphological changes in the first week, development (Palovaara et al., 2010). Expression of WOX2, promoting polarity in PEM III (Figures 6B,C). After a 1-month 8, and 9 orthologs was not apparently linked to Chinese fir incubation, normal proembryos were obtained only on the embryo development, but three ClWOX13 contigs that grouped medium containing ABA and PEG (Figure 6F). The ABA-only into the ancient WOX13 clade (Figure 9). ClWOX13 exhibited medium produced enlarged embryonal masses without clusters significantly higher transcript levels at stage 3, concurrent with suspensors (Figure 6D), whereas proembryo-like structures the proembryo transition (Figure 8B). ClWOX13-1 was more lacking enlarged embryonal masses were produced under PEG- closely related to the three maize WOX13, whereas the other only condition (Figure 6E). Thus, ABA and PEG contribute two, ClWOX13-2 and ClWOX13-3, clustered with two WOX13- in distinct and perhaps in interacting ways to proembryo like genes of the non-vascular moss P. patens (Figure 9). formation. In addition, the early addition of ABA could delay Interestingly, recent studies in P. patens elucidated the function the development of PEM III, which explains why newly formed of two PpWOX13-like genes in the control of cell wall loosening somatic embryos in Norway spruce grow well on plant growth to facilitate stem cell formation, but no regeneration-related regulator–free medium for at least 1 week (von Arnold et al., functions has yet been reported for the AtWOX13 (Sakakibara 2005). et al., 2014). qRT-PCR results revealed that expression of ClWOX13 may have high correlation with stage 3, which is when the critical transition to proembryo occurs. Because new Molecular Evidence Uncovered Early evidence indicates that Cunninghamia is a long and widespread Stress-Related Signaling during SE living fossil that extends back to the mid-Mesozoic period (Shi Development et al., 2014), we postulate that ClWOX13 may still conserve From PEMs to somatic embryos, each transition stage involves some functions of embryogenesis regulation in Chinese fir. Early physiological and metabolic alterations. These changes were WOX transcriptional machinery correlates with the gradient powered by gene regulators. SERK and WUS are two widely endogenous auxin distribution through the expression of the recognized gene markers of early embryogenesis. Recent progress auxin transport-related genes (Breuninger et al., 2008), and SERK in cotton has confirmed the role of SERK in cellular redox related also is auxin-induced (Nolan et al., 2003). From this aspect, it will somatic cell-to-embryo transition (Pandey and Chaudhary, be of particular interest to explore the connection between the 2014). WUS are responsible for keeping cell undifferentiated state WOX and SERK functions and endogenous auxin during early (Laux et al., 1996). embryogenesis in Chinese fir. SERK gene family attributes to the leucine-rich repeat (LRR) subfamily that coded for a transmembrane protein involved in signal transduction that was strongly related to stress responses CONCLUSION of SE (Santos and Aragão, 2009). AtSERK1 from Arabidopsis had direct function on SE and determined the embryogenic In summary, our study provides an efficient synchronized SE competence in culture (Hecht et al., 2001). In Medicago, system via a liquid suspension in Chinese fir that also might lead MtSERK1, orthologous to AtSERK1, was up-regulated by auxin to the improvement of SE in other gymnosperms. Karyotyping and functioned broader than embryogenesis alone (Nolan et al., and microsatellite analysis support preliminary optimism that 2003). In monocots, OsSERK1 can response to defense signaling no major cytological or genetic variation occurred in our plants hormonal activity, such as salicylic acid, jasmonic acid, and regenerated via SE. A hormonal analysis of IAA, ABA, ZR, and ABA (Hu et al., 2005). Phylogenetic results showed that two GA uncovered a potential relationship between endogenous ClSERK1 contigs (39103 and 19371), which are predominantly IAA and genotypic recalcitrance. Lower endogenous IAA of early expressed in PEMs, shared a high similarity with AtSERK1 stage immature embryos tends to higher competence acquisition. and MtSERK1, indicating that ClSERK1-3 and ClSERK1-4 may Morphological studies of PEMs during maturation revealed that function uniformly in SE. MtSERKL1 and MtSERKL2 are more ABA and PEG play complementary roles during proembryo similar to NIK genes in Arabidopsis (Nolan et al., 2011). formation. ABA boosted the growth of embryonal masses, Arabidopsis NIK shares many similarities with SERK; and is the whereas PEG functioned on the organization of proembryo- transducer of a novel layer of plant innate defenses (Santos et al., like structures. Finally, the phylogenetic tree and expression 2010). ClSERK2-1 (20090), grouped together with MtSERKL1 patterns suggested the probable regulation and activities of SERK and MtSERKL2, had higher transcript levels in stages 1 and 3 and WOX during SE, especially in early stages of proembryo (Figure 8), based on which, we postulated that the transition to formation. SERK is conserved and redundantly expressed in SE, late stage PEM may come with their stress related responses. whereas WOX13 is the most promising candidate for exploring WUS is the prototypical member of the WOX family. the function of WOX in Chinese fir embryo development. WUS overexpression increases the acquisition of embryogenic competence in Arabidopsis (Zheng et al., 2014) and cotton (Zuo et al., 2002). In Arabidopsis zygotic embryo development, AUTHOR CONTRIBUTIONS WOX2, 8, and 9 regulate cell fate by determining the early apical and basal patterning events (Haecker et al., 2004). In JC, JS, and TL conceived and designed the experiments, XZ conifer, PaWOX2, WOX8/9, PIN-FORMED (PIN ) and polar performed experiments and analyzed data, RZ prepared all the Frontiers in Plant Science | www.frontiersin.org 12 December 2017 | Volume 8 | Article 2054 fpls-08-02054 December 2, 2017 Time: 15:57 # 13 Zhou et al. Somatic Embryogenesis in Chinese Fir samples, GL performed the cytological analysis, YX helped in Southern China, Nanjing Forestry University, and Priority to pick up SSR primers and data analysis. YwZ contributed Academic Program Development of Jiangsu Higher Education to plant materials’ subculture, YZ helped with RT-PCR data Institutions. collection, XZ and RZ wrote the manuscript with contributions from all authors, TL and SH contributed to the manuscript revision. ACKNOWLEDGMENTS The authors thank Elizabeth Dell Trippe and Dr. Tim Smalley for the manuscript revision, Services of China Agricultural FUNDING University for endogenous hormones analysis. This work was supported by National High Technology Research and Development Program of China (863 Program, 2013AA102705), Specialized Research Fund for the Doctoral SUPPLEMENTARY MATERIAL Program of Higher Education (SRFDP, 20113204130002), Natural Science Foundation of Jiangsu University grant The Supplementary Material for this article can be found online 13KJA220001, Talent Project by the Ministry of Science and at: https://www.frontiersin.org/articles/10.3389/fpls.2017.02054/ Technology, Co-Innovation Center for Sustainable Forestry full#supplementary-material DeVerno, L., Park, Y., Bonga, J., and Barrett, J. (1999). Somaclonal variation in REFERENCES cryopreserved embryogenic clones of white spruce (Picea glauca (Moench) Abrahamsson, M., Valladares, S., Larsson, E., Clapham, D., and von Arnold, S. Voss.). Plant Cell Rep. 18, 948–953. doi: 10.1007/s002990050689 (2011). Patterning during somatic embryogenesis in Scots pine in relation to Durzan, D., and Chalupa, V. (1976). 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Sci. 68, 417–425. doi: 10. 2298/ABS150608124X The reviewer TP declared a shared affiliation, with no collaboration, with one of Yang, J., Zhang, J., Wang, Z., Zhu, Q., and Wang, W. (2001). Hormonal changes the authors, TL, to the handling Editor. in the grains of rice subjected to water stress during grain filling. Plant Physiol. 127, 315–323. doi: 10.1104/pp.127.1.315 Copyright © 2017 Zhou, Zheng, Liu, Xu, Zhou, Laux, Zhen, Harding, Shi and Chen. Yang, Y. (1998). Studies on the Sustainable Management of Chinese fir Plantations. This is an open-access article distributed under the terms of the Creative Commons Beijing: China Forestry Publishing House. Attribution License (CC BY). The use, distribution or reproduction in other forums Zavattieri, M. A., Frederico, A. M., Lima, M., Sabino, R., and Arnholdt-Schmitt, B. is permitted, provided the original author(s) or licensor are credited and that the (2010). 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