Microsoft Word - cet-01.docx CHEMICAL ENGINEERING TRANSACTIONS VOL. 46, 2015 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors: Peiyu Ren, Yancang Li, Huiping Song Copyright © 2015, AIDIC Servizi S.r.l., ISBN 978-88-95608-37-2; ISSN 2283-9216 A Highly Efficient Castor Regeneration System Identified through WUSCHEL Expression Wei Li, Zhenjing Li, Yujia Zhai, Changlu Wang* School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China, clw123@tust.edu.cn The key problems that limit the application of molecular breeding tools in castor are the inconsistency and inefficiency of the existing regeneration response in this plant. In the present study, we have examined and attempted to correlate the WUS gene expression in the castor explants with their shoot bud forming efficacy following the induction of embryogenic cells under various culture conditions using RT-qPCR method. By pre- treating the seedling-derived epicotyls explants with appropriate cytokine, a budding rate of > 80% could be achieved when a treatment of 0.35 mg/l BA for 5 days was employed. The corresponding WUS expression in such explants was found to be concentration and pretreatment duration dependent. The relevance of regeneration system developed in this study is discussed in the light of genetic transformation studies being presently carried out in this plant system 1. Introduction Castor (Ricinus communis) is a very important source for industrial oil. In today’s world, thousands of chemical derivatives are being produced using castor oil as a raw material. Therefore, castor oil is one of the best choices for substituting common energy resources such as petroleum (Moshkin, 1986). The demand for castor oil has surged many attempts have been made to improve the production and quality of castor. Transgenic techniques have been applied to castor breeding in order to improve its efficiency. Researchers have developed several important improvements over the years. Mckeon and Chen (2003) with their patented technique, carried out the first successful castor transformation. Sujatha and Sailaja (2005) successfully obtained the transgenic plant with Agrobacterium-mediated methods but only achieved a 0.08% transformation rate. Sailaja et al. (2008) acquired the transformed plant via the gene gun transformation method using the budded embryo as the explant and obtained a transformation efficiency of 1.4%. In recent years, there have been multiple reports on successful castor transgenic breeding; however, the transformation efficiency is problematic and is very low compared to other agriculture crops (such as rice, soybean, wheat, etc.) (Ahn et al., 2007; Kumari et al., 2008). The low transformation efficiency is due to the difficulty of castor regeneration (Sujatha et al., 2008). A highly efficient regeneration system is the basis for transgenic breeding. Generating a somatic embryo, however, is a very complicated process. Through a series of investigations, many of the key genes involved in somatic embryogenic generation have been discovered. One of which is WUSCHEL (WUS). WUS was originally discovered in Arabidopsis, and it is specifically expressed in the central cells with embryogenic property (School et al., 2000). Through various investigations, the primary function of WUS was determined to be maintaining the embryogenic property as well as the further development and differentiation capabilities of the cells (Ogawa et al., 2008). Therefore, the WUS gene is also used as the marker gene of the embryogenic cells. The relative amount of embryogenic cells can be inferred through detecting the WUS expression under different conditions and then determining the influence of that condition on the late-stage budding rate. Our research compared the WUS gene expression of castor explants under different inducing conditions in order to screen for the optimal condition. Then, the castor regeneration system was further optimized through evaluating the late-stage budding effect to develop regeneration system evaluation methods based on embryogenic cell induction. DOI: 10.3303/CET1546233 Please cite this article as: Li W., Li Z.J., Zhai Y.J., Wang C.L., 2015, A highly efficient castor regeneration system identified through wuschel expression, Chemical Engineering Transactions, 46, 1393-1398 DOI:10.3303/CET1546233 1393 2. Materials and Methods 2.1 Plant material and explant preparation The selected castor seeds were Jiaxiang NO.2 and were provided by the Shandong Academy of Agricultural Sciences, Zibo Branch. After being shelled, the seeds were immerged in a 0.1% KMnO4 solution for 10 min for sterilization before being cleaned 2-3 times with sterile water. Then, the endosperms were carefully extirpated intact embryos, which were transferred into medium MS (Murashige and Skoog, 1962) supplemented with 0.2 mg l-1 6-benzylaminopurine (BA). The embryos were cultured for 5 d in the dark at 26 °C. After the buds grew to around 7-10 mm with a visible embryogenic tip and hypocotyl, we removed the cotyledons and cut the epicotyls as the explant for the embryogenic cell induction. 2.2 Induction of embryogenic cells We inoculated the isolated epicotyls into different pre-culturing mediums (MS supplemented with 0, 0.2, 0.35, 0.5 mg l-1 BA or 0.05, 0.1, 0.2, 0.4 mg l-1 TDZ) and then cultured under the day/night cycle (16/8 h) for 3, 5, 7 d at constant 26°C. The experiments were performed with 100 to 150 samples per condition, and each treatment consisted of three replicates. We observed the response of the explants under different conditions, obtained some of the explants, washed away any residual medium, and conducted WUS expression analysis. 2.3 Budding and shoot regeneration We inoculated the pre-cultured explant into the shoot induction media (MS supplemented with BA 0.35 mg l-1, IBA 0.25 mg l-1) to induce budding. The explants were cultured in 26 °C with light and the medium was changed every 7 d. When the bud grew to around 2-3 cm, we carefully cut it off from the base and inoculated it into the root induction media (1/2MS supplemented with 0.2 mg l-1 IBA) to induce rooting. Rooted plantlets were transferred into humid soil (Nutrient soil: vermiculite 1:2), and cultured in a greenhouse at 28 °C. 2.4 WUS expression analysis We tested the difference in WUS expression by using explants under different pre-culture conditions. The upper 5mm tissue of the explants (200 mg) was cut to extract the total RNA. (Plant RNA Kit, OMEGA). The RNA integrity and purity were observed through agarose electrophoresis and absorption detection. We then did a reverse-transcription with PrimeScriptTM RT Master Mix (Takara) (used oligodT as a primer), synthesized cDNA, and added 75μL of DNase free water to dilute. The diluted template was used for the WUS semi- quantitative expression analysis. Actin was used as the (GenBank: AY360221.1) internal control and designed the primer A- F： TGATGATGCTCCCAGGGC and A-R： GTGAGAAGCACAGGATGC. The detection primers were designed based on the castor WUS gene sequence (WUS-F： TCACCATCTGGCAACTAT, WUS- R： AATCCATCGCCTGCTCTA). PCR-amplification was conducted in a total volume of 20μL, containing the appropriate diluted cDNA, 0.2μM for each primer, and 10μL of 2×Q-PCRMix (with SYBR Green I; Takara). Negative controls are explants without the hormone stimulation cDNA template. Real-time quantitative PCR (qRT-PCR) was performed in an MxPCR (Agilent Technologies Inc) as follows: 1 min of pre-denaturation at 95°C and then 35 cycles of denaturation at 95°C for 20s, annealing at 56°C for 20s, and fluorescence collection at 72°C for 15s. The results of which were analyzed with MxPro (Agilent Technologies Inc). 2.5 Statistical analysis Statistical analysis was performed using SPSS 17.0 software. Analysis of variance (ANOVA) was followed by Tukey’s pair wise comparison tests, at a level of P＜0.05, in order to determine the significant differences between means. 3. Results 3.1 Hormone response after pre-culture After numerous experiments, we selected epicotyl, which has a relative low differentiation and high mitogen etic capability as the best explant. The pre-culture stage is used to stimulate cell division and inhibit differentiation, which can induce more embryogenic cells. The results showed that there exist substantial differences in the stress responses to various kinds of cytokine in the explants. The post-BA treatment explants showed obviously expanded tissue 3 days after culture with a relative verdant color (Fig. 1 a). With the duration of the treatments, some of the explants also showed a verdant bud-like protrusion at the morphological top (Fig. 1 b). The explants under the TDZ treatment showed expanded tissue as well as color changes due to some red pigments at the early stage of culturing (Fig. 1 d). Furthermore, with the duration of the TDZ treatment, some of the explants also exhibited a lot of budding-like protrusions. However, nearly 50% of the explants had a lot of white, loose calluses growing around them (Fig. 1 e). Once the callus is generated, the late stage budding rate of the explants will be greatly affected. In addition, the results also indicated that the hormone response in this stage is not concentration-correlated. 1394 3.2 Difference in budding induction We summarized the budding 3 weeks after transferring the pre-cultured explants into the budding induction medium (Table 1). The results indicate how the different pre-culture conditions influence the late stage bud induction. The explants with BA treatment showed an “increased then decreased” budding rate with the increased hormone concentration and cultivation duration. Furthermore, the new buds of the BA group are evenly distributed (Fig. 1 c). The maximal budding in the TDZ treatment group is higher than in the BA treatment group. The budding is dense and concentrated with vitrification-like phenomena on some of the buds (Fig. 1 f). While the budding rate was not obviously influenced by the cultivation duration, the budding rate decreased with increasing TDZ concentration. In summary, the budding induction in the BA pretreated group is better, in which the explants after 0.35 mg l-1 pretreatment for 5d have the best budding efficiency with an 80.7% budding rate. Table 1: Effect of pre-treatment of explants on bud formation in castor. Each treatment consisted of three replicates. Means within a row followed by the same letters are not significant at P = 0.05 according to DMRT. (‘--’ means not add) Total no. of explants Concentration of growth regulator (mg l- 1) Days of pretreatment Bud frequency (%) Max no. of shoots per explant BA TDZ 231 0 0 3 15.3p 3 242 5 15.1pq 5 216 7 16.6op 5 348 0.2 -- 3 41.3h 5 371 5 71.3b 8 334 7 52.6g 5 453 0.35 -- 3 63.3fg 8 382 5 80.7a 15 314 7 67.3df 12 307 0.5 -- 3 64.0f 11 384 5 70.6c 8 411 7 68.0d 5 364 -- 0.05 3 30.7 12 288 5 23.3l 18 311 7 33.3i 28 317 -- 0.1 3 32.8ij 17 364 5 28.3j 11 245 7 25.6k 21 381 -- 0.2 3 19.3n 23 355 5 17.3o 11 371 7 12.6qr 19 282 -- 0.4 3 20.6m 21 341 5 4.3s 14 328 7 12.8q 19 3.3 The influence of BA on WUS gene expression Explants cultured under different BA concentrations for 3, 5, and 7 d are used to obtain the cDNAs as the template for WUS expression analysis. The results indicated that the WUS expression is low without hormone induction and increases a little with the cultivation duration. After adding the BA stimulation, the WUS expression dramatically increased; the trend experienced an initial increase and then eventual decrease with the duration of the BA treatment (Fig.2, a). A similar trend of an initial increase and then decrease also occurs with increasing BA concentration. In addition, the WUS gene expression is highest in the explants 5 days after 2mg l-1 BA treatment. 1395 Figure 1: a - The explants preprocessed with 0.35 mg l -1 BA for 3d. b - The explants (preprocessed with 0.35mg l-1 BA) after 7 days of bud induction and culture. c - The explants preprocessed with 0.1mg l-1 TDZ for 3d. d - The explants (preprocessed with 0.1mg l-1 TDZ) after 7 days of bud induction and culture, which had a lot of white, loose calluses growing around. Bars=1 cm Figure 2: Expression analysis of WUS after BA and TDZ treatment. C was explants without hormone treatment. B-1, B-2, B-3 were explants treat with 0.2, 0.35, 0.5 mg/L BA. T-1, T-2, T-3, T-4 were explants treat with 0.05, 0.1, 0.2, 0.4mg/L TDZ. Data represent average of 3 experiments; error bars show standard deviations. 3.4 The influence of TDZ on WUS gene expression Again, we used explants cultured under different TDZ concentrations for 3, 5, and 7 d to acquire the cDNA as the template for WUS expression analysis. The results of which showed that the WUS expression in the explants 3-5 days after adding TDZ is not different from that of the control group; however, the WUS gene expression significantly increased after 7 days of culture. Additionally, the explants that were cultured with 0.05 mg l -1 TDZ demonstrated the most obvious increase in WUS expression (Fig. 2, b). With this data, we suspect that TDZ’s effect usually appears after 7 days of treatment, and culture duration has a greater influence than hormone concentration does. 1396 3.5 Castor regeneration period In this research, it was found that, during the castor regeneration, the elongation of new buds is very inefficient. A single explant, regardless of the number of buds it has, normally only 3-4 buds that would grow larger than 2cm. The new castor bud easily roots with a greater than 90% rooting rate under the conditions in our research. The intact explant can grow normally after the hardening treatment and being transferred into the soil. The regeneration period is about 50-60 d. 4. Discussion 4.1 The relationship between WUS gene expression and the budding rate WUS is the key gene of embryogenic cells, and its expression directly represents the number of embryogenic cells in the plant tissue. Furthermore, an embryogenic cell is the foundation of plant regeneration. Therefore, a higher WUS expression should theoretically mean a higher budding rate. Our results also fit this premise. In comparing the budding induction after the BA pretreatment, we found that the changes in WUS completely correlated with the final budding rate. As the BA concentration increased, both the WUS expression and the budding rate showed a trend that initially increased and then decreased, which also demonstrates that the amount of embryogenic cell induction directly influences the budding rate. Also, the expression of WUS again showed a trend that initially increased and then later decreased with the duration of the BA treatment, which could be due to the reduction of the relative amount of the embryogenic cells or due to the CLV3 feedback inhibition of WUS, thereby reducing the expression area [12]. The results also indicate that the budding induction should initiate before the WUS expression reduces in order to achieve a higher budding rate. The WUS expression is not consistent with the final budding rate in the TDZ pre-cultured tissue. In the early TDZ induction stage (3-5 d), the explant shows expanded tissue with loose callus in the surroundings. Furthermore, the WUS gene expression is not changed at this stage, thus indicating that the plant cells are not embryogenic at this stage. This also means that in the early induction stage, TDZ can only promote division and not the generation of embryogenic property. The tissue culture experiments also show that with increased TDZ concentration as well as increased culture duration, the explants more easily exhibit a vitrification phenomenon, which could explain the mismatch of WUS expression and budding rate. Furthermore, with the increased TDZ culture duration, the explants might actually have a greater number of induced embryogenic cells; however, a large amount of cells appeared at the early stage and lead to the vitrification, which would then affect the differentiation of the embryogenic cells. This will lead to failed budding in addition to a reduced budding rate. The WUS expression increased significantly 7 days after TDZ treatment, which indicates that the culturing duration may be the key factor regulating TDZ-induced budding. Meanwhile, if vitrification can be reduced or even prevented, the budding rate could also possible be improved. As mentioned above, during the ex vivo regeneration, the budding rate of the explant is related to the WUS activity. The higher the WUS activity is, the more embryogenic cells that will be induced, and the higher budding potential the explant will have. 4.2 The stability of budding rate The budding is a very complex process. A normal cell needs to first be induced into an embryogenic state, then form the bud primordium, and finally develop into the visible bud [13]. The current evaluation of a regeneration system usually focuses on the budding efficiency. However, this kind of evaluation has its limitation and may actually be the cause of the low reproducibility and stability for building a plant regeneration system. In this research, we combined the mechanism of plant embryogenesis and began with the induction of embryogenic cells. By examining the WUS expression, we estimated the induction of embryonic cells, determined the optimal induction condition for embryogenic cells, improved the induction rate, and increased the stability of the budding rate. All these ensured the following up regeneration process. As such, our findings can stimulate the initiation of regeneration system construction, which ultimately provides the basis for building a better castor regeneration system. 4.3 Molecular evaluating methods We compared the WUS expression and the budding rate as well as provided the molecular evaluation of a regeneration system, which is beneficial for building the plant regeneration system in the future. Besides WUS, numerous key genes that are involved in plant embryogenesis have been discovered using modern molecular biology: for example, the LCE family that influences embryo formation and the PIN family that influences the plant hormone trafficking (Lotan et al., 1998; Gaj et al., 2005). We can apply these findings to the construction of the plant regeneration system and modify the molecular evaluation of regeneration systems. Furthermore, this will assist in improving the efficiency of regeneration system construction as well as provide a more solid basis for a highly efficient regeneration system. 1397 References Ahn Y.J., Vang L., McKeon T.A., Chen G.Q., 2007, High-frequency plant regeneration through adventitious shoot formation in castor (Ricinus communis L.). In Vitro Cell Dev Biol Plant 43(1): 9-15, DOI: 10.1007/s11627-006-9009-2. Gaj M.D., Zhang S., Harada J.J., 2005, Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis. Plant, 222(6): 977-988, DOI: 10.1007/s00425-005-0041-y. Kumari K.J., Ganesan M., Jayabalan N., 2008, Somatic organogenesis and plant regeneration in Ricinus communis. Biologia plantarum, 52(1): 17-25, DOI: 10.1007/s10535-008-0003-x. Lotan T., Ohto M., Yee K.M., 1998, Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell, 93(7): 1195-1205, DOI: 10.1016/S0092-8674(00)81463-4. Mckeon T.A., Chen G., 2003, Tranformation of Ricinus communis, the castor plant [P]. United States Department of Agriculture. Moshkin, V. A., 1986, Economic importance and regions of cultivation of castor. Castor. Moscow. Kolos: 1-5. Muller, R., Bleckmann, A., Simon, R., 2008, the receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1. The Plant Cell. 20(4): 934-946, DOI: 10.1105/ tpc.107.057547. Murashige T., Skoog F., 1962, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497, DOI: 10.1111/j.1399-3054.1962.tb08052.x. Ogawa M., Shinohara H., Sakagami Y., 2008, Arabidopsis CLV3 peptide directly binds CLV1 ectodomain. Science, 319(5861): 294-294, DOI: 10.1126/science.1150083. Sailaja M., Tarakeswari S.M., 2008, Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds. Plant Cell Rep 27(9):1509-1519, DOI: 10.1007/s00299-008-0580-3. Scheres B., 2001, Plant cell identity: The role of position and lineage. Plant Physiology, 125(1): 112-114, DOI: 10.1104/pp.125.1.112. Schoof H., Lenhard M., Haecker A., 2000, The stem cell population of Arabidopsis shoot meristems is maintained by a regulatory Loop between the CLAVATA and WUSCHEL genes. Cell, 100(6): 635-644, DOI: 10.1016/S0092-8674(00)80700-X. Sujatha M., Reddy T.P., Mahasi M., 2008, Role of biotechnological interventions in the improvement of castor (Ricinus communis L) and Jatropha curcas L. Biotechnology Advances, 6(5): 424-435, DOI: 10.1016/j.biotechadv.2008.05.004. Sujatha M., Sailaja M., 2005, Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds. Plant Cell Rep 23(12): 803- 810, DOI: 10.1007/s00299-004-0898-4. 1398