• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Isolation and callus formation of Gracilariopsis bailiniae(Gracilariales, Rhodophyta) protoplasts*

    2018-12-22 07:00:26CHENHaihong陳海紅CHENWeizhou陳偉洲SHIJingyi石經(jīng)儀CHENZepan陳澤攀ZHANGYi張毅
    Journal of Oceanology and Limnology 2018年6期
    關(guān)鍵詞:石經(jīng)海紅張毅

    CHEN Haihong (陳海紅), CHEN Weizhou (陳偉洲), SHI Jingyi (石經(jīng)儀),CHEN Zepan (陳澤攀), ZHANG Yi (張毅)

    Marine Biology Institution, Key Laboratory of Marine Biotechnology of Guangdong Province, Shantou University, Shantou 515063, China

    Abstract This paper reports the first successful isolation of protoplasts from Gracilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol,incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield.The optimized isolation conditions were: 40% base solution 3 (deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2·2H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2%w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5.The highest yield of viable protoplasts, which was obtained in these conditions, was (1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.

    Keyword: Gracilariopsis bailiniae; enzyme; marine bacterium; protoplast isolation; cell division; callus

    1 INTRODUCTION

    Gracilariopsisbailiniaeshows fast growth and heat-resistance (Zhong et al., 2014). It is a food for abalones and a raw material for agar extraction that is farmed in Fujian and Guangdong Provinces, China(Hurtado-Ponce, 1992; Pan and Li, 2010).Agarophytes (Gelidium,GracilariaandGracilariopsis) have considerable industrial importance since they are the principal source of raw material for the agar industry worldwide (Zemke-White and Ohno, 1999; Smit, 2004). In recent years,there has been increasing global demand for agar,which is used widely for microbial culture and in the food, health care products, medical and chemical industries, because of its gel, thickening and stabilization properties (Liu et al., 2013). The supply of agarophytes from wild stocks can no longer meet the demand for agar. Thus, large-scale cultivation of agarophytes is required (Gupta et al., 2013). The continuous supply of seed material is a key step for successful aquaculture (Saminathan et al., 2015).However, the limitations of traditional seed production methods for most agarophytes have restricted exploitation of these seaweeds for industrial use(Mantri, 2009). Thus, development of seed stock methods for agarophytes is crucial to achieve largescale production of seedlings.

    Protoplasts are living plant cells devoid of cell walls which can be applied in somatic hybridization,proteomics, metabolomics, cybridization and protoclonal variation studies (Fujita and Saito, 1990;Davey et al., 2005; Reddy et al., 2008). Theoretically,a protoplast can lead to the regeneration of one or more plants from a single cell due to the totipotence of plant cells (Huddy et al., 2013). Moreover, many protoplasts can be isolated from a small fragment of algal thallus, so protoplasts can be excellent tools for seed stock production and plant breeding (Gupta et al., 2011; Huddy et al., 2013; Wang et al., 2014). Chen(1998) and Chen and Shih (2000) developed methods for producing stocks of seedlings using protoplasts from green algae (UlvafasciataandMonostroma latissimum). Protoplasts fromMonostromaandPorphyrahave also been successfully tested for seeding and regeneration in laboratory conditions(Kito et al., 1998; Dipakkore et al., 2005; Reddy et al., 2006), and protoplasts from some species ofGracilariaandGracilariopsishave successfully been regenerated to whole thalli (Cheney, 1990; Yan and Wang, 1993; Reddy et al., 2008; Yeong et al., 2008;Wang et al., 2014; Huddy et al., 2015). These studies indicated that using protoplasts as seed stocks for cultivation is feasible. However, methods for protoplast isolation and regeneration of agarophyte species have not been sufficiently established (Wang,1994; Baweja et al., 2009). There are very few reports about protoplast isolation fromGracilariaandGracilariopsisspecies (Reddy et al., 2010; Gupta et al., 2011; Huddy et al., 2013; Wang et al., 2014).

    In the present study, we used a marine bacterium,Marinomonassp. YS-70, that can produce agarase.The crude agarase solution from this bacterium mixed with commercial enzymes (cellulase and macerozyme R-10) as a cell wall hydrolase produced good yields of protoplasts fromGracilariopsisbailiniae. This paper reports the optimization of the protoplast isolation conditions and the callus formation of these protoplasts.

    2 MATERIAL AND METHOD

    2.1 Experimental algae

    Thalli ofG.bailiniaewere obtained from the breeding pond of Hainan Ocean and Fisheries Sciences Research Base, Qionghai, Hainan, China. In the laboratory, the seaweed samples were cleaned of mud and epiphytes using a soft brush and filtered seawater. Then, the thalli were maintained in ventilated tanks at 27°C and 40–60 μmol photons/(m2·s) (12 h light:12 h dark) (Zhong et al., 2014).

    2.2 Preparation of agarase

    Marinomonassp. YS-70, which was isolated from red algae, was used for the preparation of agarase.The bacteria were inoculated into 100 mL of 2216E medium in a 500-mL conical flask. The 2216E medium was composed of 5 g tryptone (OXOID), 1 g yeast extract (OXOID), 0.01 g FePO4(Sangon) and 0.2% agar (OXOID), dissolved in 1 000 mL aged seawater (final pH adjusted to 7.3). After incubating at 26°C for 48 h in a reciprocal shaker (120 r/min), the bacterial solution was centrifuged at 10 444 r/min for 30 min at 4°C. The supernatant was collected and stored at -80°C until use in protoplast isolation.

    2.3 Agarase assay

    Agarase assay was carried out by estimating the reducing sugar released using the 3,5-dinitrosalisylic acid (DNS) method (Tang, 2012). The activity of agarase was determined at pH 3.5, 4.5, 5.5, 6.5, 7.5,8.5, 9.5 and 10.5. The reaction mixture was prepared from 0.1 mL crude agarase solution and 0.9 mL buffer(0.05 mol/L) containing 20 g/L agar; the buffers used were citric acid (pH 3.5–5.5), phosphate (pH 6.5–7.5),and glycine-NaOH (pH 8.5–10.5). The reaction mixture was incubated at 40°C for 30 min. Then,1 mL of DNS was mixed with 1 mL of the reaction mixture, heated for 5 min in a boiling water bath, and then cooled. The release of reducing sugar was determined by measuring the absorbance at 540 nm against a standard curve for galactose. One unit of agarase activity was defined as the amount of enzyme(mL) that produced reducing sugar equivalent to 1 μg D-galactose per min in these conditions.

    2.4 Isolation of protoplasts from G. bailiniae

    For the optimization of protoplast isolation, thalli were cleaned with a soft brush and rinsed three times with filtered seawater. After cleaning,G.bailiniaethalli were cut into 2–3 mm long pieces using a sterile scalpel blade in a culture dish and rinsed three times in filtered seawater. Then, approximately 0.5 g of seaweed pieces were incubated in 5 mL of enzyme solution in a 50-mL conical flask placed in the dark on a rotary shaker (90 r/min) for 4 h at pH 6.5. The initial enzyme solution (before various parameters were optimized) contained 1% (w/v) cellulase and 0.8 mol/L sorbitol in 40% base solution (deionized water containing 25 mmol/L 2-(N-morpholino)ethanesulfonic acid-Tris [MES-Tris] and 25 mmol/L CaCl2·2H2O) and 60% crude agarase solution. After incubation, the enzyme mixture was filtered through 45 μm nylon mesh to remove undigested algal pieces,and the remaining filtrate was centrifuged at 1 247 r/min for 8 min at 25°C. After that, 80% of the supernatant was discarded, and the protoplasts were resuspended in 5 mL MES medium containing 0.6 mol/L D-sorbitol and this procedure was repeated twice. Then,protoplast numbers were determined in a blood counting chamber. Each set of conditions was tested with three replicates.

    Fig.1 pH-activity curve of Marinomonas sp. YS-70 agarase

    For protoplast regeneration experiments, the thalli ofG.bailiniaewere prepared more stringently. First,thallus tips (within 3 cm from the apex) were selected,cleaned with a soft brush and rinsed three times with filtered seawater. After that, the thallus tips were treated with an ultrasonic cleaner (KQ-250DB) at 100% power for 3 min before soaking in sterile seawater containing 1.5% KI for 10 min. Finally, the thallus tips were immersed in sterile seawater containing 0.1 g/L ampicillin sulfate, 0.1 g/L kanamycin sulfate, 0.02 g/L neomycin sulfate and 2 mg/L GeO2for 48 h at 25°C.Then, theG.bailiniaethalli were used to isolate protoplasts, as described above.

    2.5 Optimization of protoplast isolation parameters

    For protoplast isolation fromG.bailiniae, first, the base solution was optimized from among seawater(base-solution 1), deionized water (base-solution 2),and deionized water containing 25 mmol/L MES-Tris and 25 mmol/L CaCl2·2H2O (base-solution 3). Then,the concentration of cellulase (0%, 1%, 2%, 3% or 4% w/v), macerozyme R-10 (0%, 0.5%, 1% or 1.5%w/v) and sorbitol (0.2, 0.4, 0.6, 0.8 or 1.0 mol/L), and the incubation time (2, 3, 4, 5 or 6 h), temperature (25,28 or 31°C) and enzyme solution pH (5.9, 6.1, 6.3, 6.5 or 6.7), were also optimized, in order. The optimal conditions identified in the proceeding tests were used in subsequent tests.

    2.6 Protoplast staining

    To confirm the viability of protoplasts, we used 0.5% w/v Evans blue (Biotopped, Beijing, China) to stain protoplasts, which were observed under a light microscope (Zhang et al., 2014). The viable protoplast yields were determined as follows:

    Viable protoplast yield=total number of protoplasts×survival rate.

    Fig.2 Effect of different base solutions on protoplast yield of G. bailiniae thalli

    To confirm true protoplasts lacking a cell wall,0.01% (w/v) Fluorescent Brightener 28 (Sigma) was used to stain protoplasts, and they were observed under a fluorescence microscope (LEICA DMI3000 B) with UV light (Wang et al., 2014).

    2.7 Culture of protoplasts

    Protoplasts ofG.bailiniaewere dispensed into 2 mL MES medium containing 0.6 mol/L sorbitol in 35 mm×10 mm Petri dishes with a protoplast density of 5×104–1×105cells/mL. Then, protoplasts were cultured at 26°C with a 12 h:12 h light:dark cycle(16 μmol/(m2·s)). After culturing for 2 days, the culture medium was replaced with ? MES medium containing 0.6 mol/L sorbitol. Then, the ? culture medium was replaced with MES medium when the protoplasts had been cultured for a total of 4 days.After that, 50% of the culture medium was replaced every 3 days with MES medium.

    2.8 Statistical analysis

    Analysis of variance was used for the comparison of results in different conditions.P<0.05 was considered significant.

    3 RESULT

    3.1 Agarase activity assay

    Marinomonassp. YS-70 agarase activity was high at pH 5.5 to 8.5 (Fig.1) and the highest enzyme activity was observed at pH 5.5. Lower and higher pH values resulted in lower enzyme activity.

    3.2 Effect of the base solution type

    Fig.3 Effect of enzyme constituents and concentrations on protoplast yield from G. bailiniae thalli

    As Fig.2 shows, the protoplast yield was significantly (P<0.05) increased by using basesolution 3 and the highest protoplast yield((12.5±1.3)×105cells/g fresh weight [f. wt]) was obtained with this treatment. There was no significant difference in the protoplast yield between treatment with base-solution 1 and base-solution 2. The lowest protoplast yield ((1.08±0.59)×105cells/g f. wt) was obtained on treatment with base-solution 1.

    3.3 Optimization of cell wall degrading enzyme combination and concentration

    As Fig.3a and b show, the protoplast yields fromG.bailiniaethalli were significantly affected by the concentration of added enzymes (P<0.05). The protoplast yield was highest when the enzyme mixture contained 60% crudeMarinomonassp. YS-70 agarase solution, 2% w/v cellulase and 1% w/v macerozyme R-10. A large quantity of protoplasts could be obtained when the mixture of enzymes consisted of agarase and cellulase only (Fig.3a), i.e., when the concentration of macerozyme R-10 was 0%. However, few protoplasts were released fromG.bailiniaethalli when agarase was mixed with macerozyme R-10 without cellulase (Fig.3b).

    Fig.4 Effects of (a) different incubation pH, (b) sorbitol concentration, (c) incubation temperature, and (d)time on protoplast yield from G. bailiniae thalli

    3.4 Optimization of incubation pH, sorbitol concentration and incubation temperature and time

    pH 6.5 was found to be the optimal pH for protoplast yield ofG.bailiniae, with a significantly increased yield compared with the other pH values assessed (P<0.05) (Fig.4a).

    Fig.5 Protoplasts of G. bailin i ae

    The protoplast yields were not significantly different with sorbitol treatments of 0.2, 0.4 and 0.6 mol/L (Fig.4b); the highest protoplast yield was obtained when the sorbitol concentration was 0.4 mol/L. The yield of protoplast decreased significantly (P<0.05) when the sorbitol concentration was >0.6 mol/L. Based on these results, a sorbitol concentration of 0.4 mol/L was considered optimal for protoplast yield.

    Protoplast yields fromG.bailiniaewere not significantly different between the incubation temperatures of 25 and 28°C, and the optimal yield was obtained at 28°C. The protoplast yield decreased significantly (P<0.05) at 31°C (Fig.4c).

    Protoplasts were released fromG.bailiniaeas early as 2 h after the start of incubation of the thalli in enzyme solution. Protoplast yields increased with incubation time (Fig.4d). However, the survival rate of the protoplasts decreased as the incubation time increased. As Fig.4d shows, the protoplast yield fromG.bailiniaeobtained after 4.0 h was(31.3±4.5)×105cells/g f.wt and the survival rate was 56%, the highest viable yield among the tested incubation times. Therefore, an incubation period of 4 h was considered optimal for viable protoplast yield.

    3.5 Protoplast cell wall regeneration and cell division

    Freshly isolated protoplasts fromG.bailiniaewere spherical with diameter from 7 to 35 μm (Fig.5a).Living protoplasts appeared yellow and dead protoplasts were dark blue when stained with Evans blue (Fig.5b). True protoplasts were red and undigested cell walls were green when they were stained with Fluorescent Brightener 28 and observed under UV light (Fig.6a). Regeneration of the cell wall began from one pole of the protoplast (Fig.6b). After being cultured for 48 h, the surface of most protoplasts was covered with regenerated cell wall and then the cell wall thickened. After 60 h of culture, the cell wall regeneration was almost complete (Fig.6c). We found that the first division of most protoplasts occurred on the third day after protoplasts were isolated. There were two division types for the first division of protoplasts fromG.bailiniae, asymmetric division and symmetric division (Fig.7a). In the former,protoplasts produced a small bud from one side of the cell, and in the latter, protoplasts divided into two cells from the cell middle. After the first division, the divided cells could develop into callus masses over several days of culture (Fig.7b, c).

    4 DISCUSSION

    Common sources of algal cell wall degrading enzymes are digestive gland juices of herbivorous marine invertebrates, culture filtrates of marine bacteria, and commercial preparations (Yan and Wang, 1993; Araki et al., 1998; Mussio and Rusig,2006; Reddy et al., 2008; Yeong et al., 2008; Gupta et al., 2013; Wang et al., 2014; Zhang et al., 2014).However, the enzyme activity of digestive gland juices from marine invertebrates varies with sampling time, sampling species and production batch of enzymes, and this material has highly deleterious effects on protoplast viability (Wang, 1994; Zhao et al., 2005). Commercial cell wall degrading enzyme preparations have sometimes been assessed for protoplast isolation fromGracilariaspecies. Yeong et al. (2008) and Huddy et al. (2013) obtained a large quantity of protoplasts fromGracilariachangiiandGracilariagracilisusing commercial cellulase,macerozyme and agarase. In our tests, protoplasts could not be isolated effectively fromGracilariopsis bailiniaeusing commercial agarase (TAKARA),cellulase and macerozyme R-10 (data not shown), but we obtained many protoplasts fromG.bailiniaewhen the commercial agarase was replaced by crude agarase solution fromMarinomonassp. YS-70. This might be because the commercial agarase is purer and could not completely degrade the complex amorphous matrix including sulfated polygalactans (agarcolloids or agars) in the cell wall ofG.bailiniae, resulting in non-release of protoplasts.

    Fig.6 Cell wall regeneration of protoplasts from G. bailiniae

    Fig.7 Protoplast regeneration of G. bailiniae

    Studies have shown that red algae have a wide variety of matrix polysaccharides in their cell walls,which vary in amount depending on the species(Bellanger et al., 1990; Graham and Wilcox, 2000;Reddy et al., 2008). In our study, cellulase and agarase were necessary for protoplast isolation. We could not obtain a good protoplast yield fromG.bailiniaewith a single cell wall degrading enzyme or enzyme mixtures lacking agarase or cellulase, which suggests the cell wall ofG.bailiniaeis complex. Macerozyme R-10 presumably enhanced the protoplast yield because macerozyme has been shown to digest pectin and polygalactans in many studies of protoplast isolation from agarophyte species (Evans and Bravo,1983; Yeong et al., 2008; Gupta et al., 2011; Huddy et al., 2013). However, the commercial enzyme is partially purified and contains (a) toxic substance(s)(Inoue et al., 2011), so the optimization of cell wall degrading enzyme concentration was necessary. A combination of 2% w/v cellulase, 1% w/v macerozyme and 60% crude agarase prepared fromMarinomonassp. YS-70 resulted in the highest protoplast yield among the combinations and concentrations of enzymes we tested.

    pH is an important parameter for protoplast isolation. In general, the pH used in published reports of algal protoplast isolation ranges between pH 5.8 and 7.0 (Cheney et al., 1986; Bj?rk et al., 1990; Yan and Wang, 1993; Mussio and Rusig, 2006; Reddy et al., 2006; Yeong et al., 2008; Lafontaine et al., 2011).However, Gupta et al. (2011) reported an optimal pH of 7.5 for the isolation of protoplasts fromGracilaria verrucosaandGracilariadura. In our study, the optimal pH was 5.0 for commercial cellulase activity and 5.0 to 6.0 for macerozyme R-10, while the crude agarase fromMarinomonassp. YS-70 had its highest activity at pH 5.5–8.5 (Fig.1). Thus, weakly acidic or neutral solution (pH 5–7) could maintain relatively high activity of all three enzymes. The optimal pH of 6.5 for protoplast yield observed in this study is consistent with this conclusion.

    Protoplast yield is influenced by many physicochemical factors, especially the constituents of the enzyme solution. In some previous studies of protoplast isolation from seaweed, CaCl2·2H2O, MES buffer, or both, were added into the enzyme solution(Dipakkore et al., 2005; Mussio and Rusig, 2006;Yeong et al., 2008; Huddy et al., 2013), but other studies did not use these reagents (Bj?rk et al., 1990;Wang et al., 2014). In the present study, the protoplast yield was significantly enhanced by base-solution 3(deionized water containing 25 mmol/L MES-Tris buffer and 25 mmol/L CaCl2·2H2O). It is possible that the MES-Tris buffer maintained a stable pH for enzyme function and CaCl2·2H2O as a plasma membrane stabilizer protected the plasmalemma of the protoplasts (Wang, 1994). We found that a deionized water-based solution gave a higher protoplast yield than seawater based-solution. Similar results were also reported in studies of protoplast isolation fromUlva,EnteromorphaandMonostroma(Dipakkore et al., 2005). This phenomenon was possibly because ions in the seawater result in reduced activity of cell wall lytic enzymes (Dipakkore et al.,2005).

    In this study, 0.4 mol/L sorbitol was found to be the optimal concentration for protoplast yield. Similarly,sorbitol was used as the osmotic stabilizer by Wang et al. (2014) in protoplast isolation fromGracilariopsis lemaneiformis, but the concentration of sorbitol in their study was 0.8 mol/L. Osmotic conditions help protoplasts to maintain their integrity after the cell walls are removed (Compton et al., 2000). Sugars and sugar alcohols, such as mannitol, sorbitol and glucose,are commonly used as osmotic stabilizers in protoplast isolation (Araki et al., 1998; Lafontaine et al., 2011;Wang et al., 2014; Zhang et al., 2014). Use of the inorganic osmoticum NaCl was also reported in protoplast isolation fromLaminariaspecies (Butler et al., 1989).

    In our experiments, the optimal incubation temperature for protoplast yield fromG.bailiniaewas 28°C, close to the optimal growth temperature ofG.bailiniaein nature (Zhong et al., 2014). An optimal incubation temperature for protoplast isolation close to the optimal growth temperature of the algae was also observed for the tropical speciesGracilaria changiiand the temperate speciesGracilaria Verrucosa(Araki et al., 1998; Yeong et al., 2008;Gupta et al., 2011).

    The protoplast yield increased, but the protoplast survival percentage decreased, when the incubation time increased during isolation. A similar result was found in the protoplast isolation fromKappaphycus alvarezii(Zhang et al., 2014). Optimal incubation times for protoplast isolation from many algae are between 2.5 and 3 h, for example 2.5 h forGracilaria verrucosa(Araki et al., 1998), and 3 h forGracilaria changii(Yeong et al., 2008),Gracilariagracilis(Huddy et al., 2013),MonostromanitidumandPorphyrayezoensis(Kito et al., 1998). Incubation times >6 h cause the cells to be overdigested, resulting in a significant reduction of the protoplast yield(Yeong et al., 2008).

    Cell wall resynthesis of protoplasts was observed to start within 12 h (data not shown) after protoplast isolation fromG.bailiniae. Cell wall regeneration began at one pole of the protoplast. A similar pattern of cell wall deposition, beginning at a single pole of the protoplast, was also noted forGracilariagracilis(Huddy et al., 2013) andK.alvarezii(Zablackis et al.,1993). Two division types for the first division—asymmetric division and symmetric division—were first reported in the protoplast regeneration ofGracilariopsisgenera. After the first division, the divided cells can develop into callus-like masses. In the present study, many callus masses were obtained in the cultivation process, which might have potential for application in production of metabolites ofG.bailiniae. Meanwhile, some seaweeds, such asKappaphycusalvarezii(Reddy et al., 2003), have regenerated whole plants from their calli, so the callus masses obtained in our study might have the potential to produce seed material forG.bailiniae.

    In previous reports, protoplasts ofGracilaria changiiandGracilariagracilisregenerated after 2 months of culture (Yeong et al., 2008; Huddy et al.,2015), and protoplasts ofGracilariopsislemaneiformisregenerated after 90 days of culture (Wang et al.,2014). However, we have been unable to regenerate protoplasts ofG.bailiniaeinto whole plants. The capacity for protoplast regeneration might be related to the growth conditions of the algal thalli. Using algae which are in good condition could improve the yield and the survival rate of protoplasts, and be good for protoplast regeneration (Bj?rk et al., 1990; Wang et al., 2014). The thalli ofG.bailiniaeused in our study had been cultured for 1 month before protoplast isolation, so they might have been in poor condition and unsuitable for subsequent regeneration. In addition, the culture medium used in our study might not be suitable for protoplast regeneration ofG.bailiniae. Huddy et al. (2015) regeneratedGracilaria gracilisprotoplasts into whole plants in Provasoli’s enriched seawater (PES) medium. However, Yeong et al. (2008) obtained wholeGracilariachangiiplants from protoplasts only in MES medium but not PES medium. Moreover, the densities of protoplasts in our culture system were 5×104–1×105cells/mL, which might not be suitable for protoplast regeneration.Thus, further studies, especially to determine the optimum conditions for the growth of protoplasts into wholeG.bailiniaeplants, need to be conducted.

    5 CONCLUSION

    In this study, we established a protocol for protoplast isolation fromG.bailiniaeand investigated primary development of the protoplasts. This study thus provides a theoretical basis for seedling production ofG.bailiniae, and a protoplast isolation method for protoplast fusion and hybridization work.Furthermore, a large number of callus masses could be obtained in our study, which might have potential application in production ofG.bailiniaemetabolites.

    6 ACKNOWLEDGEMENT

    The authors thank Professor LIU Tao (Ocean University of China), Professor MEI Zhiping(Shantou University), Dr. WANG Hui (Shantou University) and Ms. WANG Zhongxia (Ocean University of China) for providing much advice and kind assistance.

    猜你喜歡
    石經(jīng)海紅張毅
    生活有錢有閑,妻子為何還想離婚?
    哈代詩歌的民謠藝術(shù)
    石經(jīng)
    寶藏(2022年2期)2022-07-30 07:41:02
    唐石經(jīng)刊刻的善本價值
    鴨綠江(2020年21期)2020-10-14 06:05:46
    《秋水共長天一色》
    青年生活(2019年6期)2019-09-10 17:55:38
    韓國古代石刻文字考釋問題及華嚴石經(jīng)文字的復(fù)原
    海紅姑娘
    黃河之聲(2018年20期)2018-12-14 05:14:38
    Noether Symmetry and Conserved Quantities of Fractional Birkhoffian System in Terms of Herglotz Variational Problem?
    Perturbation to Noether Symmetries and Adiabatic Invariants for Generalized Birkhoff Systems Based on El-Nabulsi Dynamical Model
    Fractional Action-Like Variational Problem and Its Noether Symmetries for a Nonholonomic System
    国产男人的电影天堂91| 国产人妻一区二区三区在| 啦啦啦啦在线视频资源| 国产亚洲精品综合一区在线观看| 永久网站在线| 亚洲av成人av| 1024手机看黄色片| 国产在线精品亚洲第一网站| 成人亚洲欧美一区二区av| 精品久久久久久久久亚洲| АⅤ资源中文在线天堂| 少妇猛男粗大的猛烈进出视频 | 熟女人妻精品中文字幕| 国产欧美日韩精品一区二区| 日韩欧美 国产精品| 国产精品免费一区二区三区在线| 99久国产av精品国产电影| 亚洲国产精品国产精品| 免费无遮挡裸体视频| 亚洲国产精品sss在线观看| 国产精品爽爽va在线观看网站| 别揉我奶头~嗯~啊~动态视频| 国产在线精品亚洲第一网站| 一区二区三区高清视频在线| 国产一区亚洲一区在线观看| av女优亚洲男人天堂| 国产在线精品亚洲第一网站| 在线观看av片永久免费下载| 亚洲内射少妇av| 男人的好看免费观看在线视频| 在线播放国产精品三级| 国产成人精品久久久久久| 色吧在线观看| 久久久国产成人免费| 淫妇啪啪啪对白视频| 亚洲人成网站在线播放欧美日韩| 久久99热这里只有精品18| 一个人观看的视频www高清免费观看| 亚洲欧美精品综合久久99| 亚洲丝袜综合中文字幕| 99国产极品粉嫩在线观看| 欧美性猛交黑人性爽| 国产成人aa在线观看| 中文在线观看免费www的网站| av天堂中文字幕网| 色综合亚洲欧美另类图片| 日日摸夜夜添夜夜爱| 精品日产1卡2卡| 天天一区二区日本电影三级| 国产伦精品一区二区三区视频9| 欧美zozozo另类| 亚洲欧美精品综合久久99| 天天一区二区日本电影三级| 国产成人91sexporn| 亚洲精品影视一区二区三区av| 国产91av在线免费观看| 看十八女毛片水多多多| 午夜福利在线在线| 黄片wwwwww| 亚洲自偷自拍三级| 日本免费a在线| 一本一本综合久久| 午夜精品在线福利| 久久草成人影院| 中文在线观看免费www的网站| 淫妇啪啪啪对白视频| 亚洲av成人精品一区久久| 久久久久久久久中文| 免费无遮挡裸体视频| 91午夜精品亚洲一区二区三区| 婷婷六月久久综合丁香| 亚洲五月天丁香| 亚洲第一电影网av| 一区二区三区高清视频在线| 日韩欧美免费精品| 成年av动漫网址| 看十八女毛片水多多多| 香蕉av资源在线| 久久久久九九精品影院| 久久久久久久亚洲中文字幕| 日韩av不卡免费在线播放| a级毛片免费高清观看在线播放| 美女 人体艺术 gogo| 在线国产一区二区在线| 97人妻精品一区二区三区麻豆| 嫩草影院新地址| 欧美成人精品欧美一级黄| 日韩,欧美,国产一区二区三区 | 国产乱人视频| 国产精品三级大全| 又爽又黄无遮挡网站| 天天一区二区日本电影三级| 丰满人妻一区二区三区视频av| 国产亚洲精品久久久com| 中文字幕久久专区| 欧美日韩在线观看h| 亚洲av第一区精品v没综合| 人妻久久中文字幕网| 亚洲经典国产精华液单| 深夜精品福利| a级毛色黄片| 熟妇人妻久久中文字幕3abv| 免费av毛片视频| 俺也久久电影网| 淫妇啪啪啪对白视频| 22中文网久久字幕| 观看美女的网站| 精品日产1卡2卡| 三级男女做爰猛烈吃奶摸视频| 美女黄网站色视频| 能在线免费观看的黄片| 国产午夜精品论理片| 免费看a级黄色片| 有码 亚洲区| 最近中文字幕高清免费大全6| 美女cb高潮喷水在线观看| 久久99热6这里只有精品| 久久久久久久亚洲中文字幕| 热99re8久久精品国产| 99久久成人亚洲精品观看| 全区人妻精品视频| 中国国产av一级| 亚洲精品456在线播放app| 久久精品国产亚洲av涩爱 | 欧美成人a在线观看| 天堂影院成人在线观看| 成人av一区二区三区在线看| 最近中文字幕高清免费大全6| 别揉我奶头 嗯啊视频| 亚洲中文字幕日韩| 久久综合国产亚洲精品| 久久人妻av系列| 成人毛片a级毛片在线播放| 综合色av麻豆| 欧美另类亚洲清纯唯美| 国语自产精品视频在线第100页| 精品国产三级普通话版| 黄色视频,在线免费观看| 最近的中文字幕免费完整| 国产高清三级在线| 欧美日韩一区二区视频在线观看视频在线 | 黄色日韩在线| 长腿黑丝高跟| 日本撒尿小便嘘嘘汇集6| 久久久久国产精品人妻aⅴ院| 高清日韩中文字幕在线| 麻豆成人午夜福利视频| 又黄又爽又免费观看的视频| 精品久久久久久久久av| 国产精品久久久久久久久免| 国产精品一区二区性色av| 我要看日韩黄色一级片| 亚洲国产精品国产精品| 免费看a级黄色片| 亚洲精品影视一区二区三区av| 大又大粗又爽又黄少妇毛片口| 日产精品乱码卡一卡2卡三| 日日干狠狠操夜夜爽| 国产乱人偷精品视频| ponron亚洲| 91在线精品国自产拍蜜月| 国产aⅴ精品一区二区三区波| 天天躁夜夜躁狠狠久久av| 国产精品女同一区二区软件| 久久人妻av系列| av天堂在线播放| 丝袜美腿在线中文| 亚洲在线观看片| 日日啪夜夜撸| 禁无遮挡网站| 91狼人影院| 搡女人真爽免费视频火全软件 | 女同久久另类99精品国产91| 国产精品爽爽va在线观看网站| 欧美另类亚洲清纯唯美| 男人舔奶头视频| 欧美在线一区亚洲| 久久久欧美国产精品| 日本 av在线| 麻豆国产av国片精品| 成人永久免费在线观看视频| 十八禁网站免费在线| 性欧美人与动物交配| 99视频精品全部免费 在线| 国产一区二区激情短视频| 蜜桃亚洲精品一区二区三区| 国产爱豆传媒在线观看| 男女视频在线观看网站免费| 在线国产一区二区在线| 国产男靠女视频免费网站| 在线免费观看的www视频| 我要搜黄色片| 日韩亚洲欧美综合| 又黄又爽又刺激的免费视频.| 国产黄片美女视频| 草草在线视频免费看| 91久久精品国产一区二区成人| 九九爱精品视频在线观看| 日韩,欧美,国产一区二区三区 | 最新中文字幕久久久久| 最近的中文字幕免费完整| h日本视频在线播放| 男女边吃奶边做爰视频| 国产大屁股一区二区在线视频| 欧美+亚洲+日韩+国产| 免费搜索国产男女视频| 亚洲无线观看免费| 高清日韩中文字幕在线| 亚洲va在线va天堂va国产| 免费看日本二区| 欧美人与善性xxx| 一级a爱片免费观看的视频| 国产精品爽爽va在线观看网站| 在线看三级毛片| 欧美人与善性xxx| 精品熟女少妇av免费看| 麻豆乱淫一区二区| 亚洲成人久久爱视频| 欧美高清性xxxxhd video| 成人欧美大片| 91久久精品电影网| 黑人高潮一二区| 久久午夜亚洲精品久久| 亚洲在线观看片| 亚洲精品亚洲一区二区| 亚洲性夜色夜夜综合| 成人亚洲精品av一区二区| 国产高潮美女av| 狠狠狠狠99中文字幕| 亚洲欧美日韩高清在线视频| 在线观看一区二区三区| 欧美xxxx性猛交bbbb| 久久鲁丝午夜福利片| 国产欧美日韩精品亚洲av| 精品午夜福利视频在线观看一区| 天天躁夜夜躁狠狠久久av| 一区二区三区免费毛片| 97在线视频观看| 免费无遮挡裸体视频| 久久婷婷人人爽人人干人人爱| 亚洲欧美日韩高清专用| 国产高清不卡午夜福利| 日日干狠狠操夜夜爽| 免费一级毛片在线播放高清视频| 日日撸夜夜添| 成人亚洲精品av一区二区| 大型黄色视频在线免费观看| 欧美在线一区亚洲| 国产探花极品一区二区| av在线天堂中文字幕| 精品久久久久久久久久免费视频| 亚洲欧美成人综合另类久久久 | 人人妻,人人澡人人爽秒播| 校园人妻丝袜中文字幕| 尤物成人国产欧美一区二区三区| av福利片在线观看| 亚洲精品成人久久久久久| 亚洲精品影视一区二区三区av| 男女那种视频在线观看| 久久热精品热| 我要搜黄色片| 成年av动漫网址| 日本-黄色视频高清免费观看| 波多野结衣高清作品| 久久99热6这里只有精品| 免费av不卡在线播放| 偷拍熟女少妇极品色| 亚洲三级黄色毛片| 在线免费观看的www视频| 成熟少妇高潮喷水视频| 国产乱人视频| 69av精品久久久久久| 精品人妻一区二区三区麻豆 | 国产一区二区激情短视频| 禁无遮挡网站| 日日摸夜夜添夜夜添av毛片| 国产三级中文精品| 黄色怎么调成土黄色| 成人二区视频| 男女免费视频国产| 18禁在线播放成人免费| 纯流量卡能插随身wifi吗| 精品人妻偷拍中文字幕| 午夜91福利影院| 中文字幕久久专区| 久久久久精品久久久久真实原创| 婷婷色综合www| 久久亚洲国产成人精品v| 欧美bdsm另类| 色婷婷久久久亚洲欧美| 国产精品人妻久久久久久| 免费观看a级毛片全部| av卡一久久| 国产免费又黄又爽又色| 国产午夜精品久久久久久一区二区三区| 天美传媒精品一区二区| 高清毛片免费看| 欧美精品人与动牲交sv欧美| 久久午夜综合久久蜜桃| 晚上一个人看的免费电影| 国产精品麻豆人妻色哟哟久久| 久久久亚洲精品成人影院| 少妇猛男粗大的猛烈进出视频| 国产高清三级在线| 天天操日日干夜夜撸| 欧美国产精品一级二级三级 | 22中文网久久字幕| 国产黄片美女视频| 国产在线免费精品| 亚洲av男天堂| 另类精品久久| 欧美日韩综合久久久久久| 亚洲欧美成人综合另类久久久| 最近的中文字幕免费完整| .国产精品久久| 欧美精品国产亚洲| 三级国产精品片| 五月开心婷婷网| 大码成人一级视频| 日本黄色片子视频| 老司机影院毛片| 亚洲怡红院男人天堂| 嫩草影院新地址| 精品99又大又爽又粗少妇毛片| 99精国产麻豆久久婷婷| 久久久精品94久久精品| 国产爽快片一区二区三区| 国产女主播在线喷水免费视频网站| 丰满乱子伦码专区| 啦啦啦啦在线视频资源| 久久99蜜桃精品久久| 99热6这里只有精品| 久久久久久久久久久久大奶| 下体分泌物呈黄色| 男的添女的下面高潮视频| 日本欧美国产在线视频| 亚洲国产精品一区二区三区在线| 免费观看的影片在线观看| 夫妻性生交免费视频一级片| videossex国产| 女人久久www免费人成看片| 久久精品久久精品一区二区三区| 免费大片黄手机在线观看| 黄色怎么调成土黄色| 97超碰精品成人国产| 欧美少妇被猛烈插入视频| 久久这里有精品视频免费| 又粗又硬又长又爽又黄的视频| 美女视频免费永久观看网站| 大话2 男鬼变身卡| 国产男人的电影天堂91| 美女主播在线视频| 久久国内精品自在自线图片| 免费看日本二区| 欧美激情极品国产一区二区三区 | 久久毛片免费看一区二区三区| 亚洲伊人久久精品综合| 少妇人妻精品综合一区二区| 九九在线视频观看精品| 99热网站在线观看| a 毛片基地| 国产成人精品福利久久| 国产精品欧美亚洲77777| 蜜桃久久精品国产亚洲av| 免费高清在线观看视频在线观看| 午夜老司机福利剧场| 一个人看视频在线观看www免费| 亚洲av男天堂| 亚洲精品第二区| 人妻一区二区av| 国产精品人妻久久久影院| 免费看av在线观看网站| 国产高清不卡午夜福利| 一级毛片电影观看| 老女人水多毛片| 自拍偷自拍亚洲精品老妇| 亚洲婷婷狠狠爱综合网| 亚洲精品一二三| 亚洲综合色惰| 中文乱码字字幕精品一区二区三区| 精品人妻熟女毛片av久久网站| 菩萨蛮人人尽说江南好唐韦庄| 热99国产精品久久久久久7| 国产精品麻豆人妻色哟哟久久| 大陆偷拍与自拍| 欧美精品国产亚洲| 国产69精品久久久久777片| 精品酒店卫生间| 色吧在线观看| 在现免费观看毛片| 亚洲人成网站在线观看播放| 欧美bdsm另类| 在现免费观看毛片| 日韩成人伦理影院| 精品国产一区二区久久| 亚洲av电影在线观看一区二区三区| 简卡轻食公司| 国产精品麻豆人妻色哟哟久久| www.av在线官网国产| 国产一区二区三区av在线| 国产免费视频播放在线视频| 美女中出高潮动态图| 国产极品粉嫩免费观看在线 | 伦理电影免费视频| 国产欧美日韩一区二区三区在线 | 国产精品偷伦视频观看了| videossex国产| 看免费成人av毛片| 少妇丰满av| 亚洲欧美清纯卡通| 日韩,欧美,国产一区二区三区| 亚洲av成人精品一二三区| 色视频www国产| 日韩一本色道免费dvd| 能在线免费看毛片的网站| 在线精品无人区一区二区三| 国产精品欧美亚洲77777| 国产乱人偷精品视频| 日本色播在线视频| 水蜜桃什么品种好| 欧美日韩综合久久久久久| 黄色怎么调成土黄色| 欧美变态另类bdsm刘玥| 亚洲综合色惰| 国产91av在线免费观看| 久久99蜜桃精品久久| 一本一本综合久久| 国产在线视频一区二区| 多毛熟女@视频| 精品久久久噜噜| 少妇人妻久久综合中文| 啦啦啦啦在线视频资源| a级毛片在线看网站| 国产亚洲精品久久久com| 国产成人精品福利久久| 日韩成人av中文字幕在线观看| av视频免费观看在线观看| 亚洲熟女精品中文字幕| 狂野欧美白嫩少妇大欣赏| 午夜福利网站1000一区二区三区| 少妇猛男粗大的猛烈进出视频| 国产探花极品一区二区| 噜噜噜噜噜久久久久久91| 蜜桃久久精品国产亚洲av| av在线观看视频网站免费| 我要看黄色一级片免费的| av有码第一页| 18禁在线播放成人免费| 久久久精品免费免费高清| 中国三级夫妇交换| 精品99又大又爽又粗少妇毛片| 成人黄色视频免费在线看| 亚洲精品第二区| 中文天堂在线官网| 国内精品宾馆在线| av天堂中文字幕网| 国产淫片久久久久久久久| 男人舔奶头视频| 欧美 日韩 精品 国产| 亚洲av男天堂| 国产午夜精品一二区理论片| 啦啦啦视频在线资源免费观看| 草草在线视频免费看| 日韩 亚洲 欧美在线| 全区人妻精品视频| 国产精品久久久久久av不卡| 男男h啪啪无遮挡| 精品人妻熟女毛片av久久网站| 亚洲国产精品一区三区| 久久精品国产亚洲av涩爱| av卡一久久| 久久国产精品大桥未久av | 国产欧美亚洲国产| 黄色毛片三级朝国网站 | 国产成人免费无遮挡视频| 大香蕉久久网| 国产成人免费无遮挡视频| 伦理电影大哥的女人| 亚洲,一卡二卡三卡| 国产在线男女| 亚洲国产欧美在线一区| 在线观看人妻少妇| 精品久久久久久电影网| 亚洲美女搞黄在线观看| 26uuu在线亚洲综合色| 18禁动态无遮挡网站| 久久精品久久久久久噜噜老黄| 高清毛片免费看| 又大又黄又爽视频免费| 久热久热在线精品观看| 日韩制服骚丝袜av| 久久久精品免费免费高清| 免费观看性生交大片5| 在线天堂最新版资源| 最近2019中文字幕mv第一页| 深夜a级毛片| 老女人水多毛片| 搡老乐熟女国产| 人人妻人人澡人人爽人人夜夜| 日韩亚洲欧美综合| 黄片无遮挡物在线观看| 丝袜喷水一区| 久久综合国产亚洲精品| 你懂的网址亚洲精品在线观看| 国产美女午夜福利| 日韩不卡一区二区三区视频在线| 99久久精品一区二区三区| 一区二区三区四区激情视频| 亚洲第一区二区三区不卡| 久久99热6这里只有精品| 日本vs欧美在线观看视频 | 久久 成人 亚洲| 国产免费一级a男人的天堂| 国产无遮挡羞羞视频在线观看| 我的女老师完整版在线观看| 日韩在线高清观看一区二区三区| 欧美日韩国产mv在线观看视频| 午夜日本视频在线| 高清在线视频一区二区三区| 狂野欧美激情性xxxx在线观看| 久久女婷五月综合色啪小说| 欧美xxxx性猛交bbbb| 免费播放大片免费观看视频在线观看| 日韩一区二区视频免费看| 国产黄片视频在线免费观看| 我的老师免费观看完整版| 免费看日本二区| xxx大片免费视频| 99国产精品免费福利视频| 欧美 亚洲 国产 日韩一| 久久99精品国语久久久| videos熟女内射| 99九九线精品视频在线观看视频| 全区人妻精品视频| 亚洲成人一二三区av| 日韩中文字幕视频在线看片| 精品少妇黑人巨大在线播放| 精品国产国语对白av| 国产极品天堂在线| 中文在线观看免费www的网站| 亚洲av电影在线观看一区二区三区| 精品国产一区二区三区久久久樱花| 国产日韩欧美视频二区| 中国三级夫妇交换| 国产探花极品一区二区| 国产淫片久久久久久久久| 看非洲黑人一级黄片| 日韩精品免费视频一区二区三区 | 亚洲国产av新网站| 久热这里只有精品99| 嫩草影院入口| 亚洲一区二区三区欧美精品| 又爽又黄a免费视频| 在线观看国产h片| 99精国产麻豆久久婷婷| 欧美3d第一页| 国产国拍精品亚洲av在线观看| 成年人免费黄色播放视频 | 99视频精品全部免费 在线| 麻豆乱淫一区二区| 女人精品久久久久毛片| 99re6热这里在线精品视频| 在线 av 中文字幕| 少妇被粗大猛烈的视频| 午夜视频国产福利| 丰满少妇做爰视频| 欧美精品一区二区免费开放| 成人美女网站在线观看视频| 免费观看无遮挡的男女| 亚洲精品亚洲一区二区| 观看免费一级毛片| 日本爱情动作片www.在线观看| 交换朋友夫妻互换小说| 免费黄网站久久成人精品| 多毛熟女@视频| 欧美日韩综合久久久久久| 亚洲成人一二三区av| 少妇的逼好多水| 欧美最新免费一区二区三区| 建设人人有责人人尽责人人享有的| 十分钟在线观看高清视频www | 国产高清国产精品国产三级| 夜夜看夜夜爽夜夜摸| 亚洲国产欧美日韩在线播放 | 国产精品一区二区三区四区免费观看| 51国产日韩欧美| 中国三级夫妇交换| 国产欧美亚洲国产| 99热6这里只有精品| 日韩三级伦理在线观看| 久久鲁丝午夜福利片| 午夜激情福利司机影院| 久久久久视频综合| 久久久久国产网址| 狂野欧美白嫩少妇大欣赏| 日韩大片免费观看网站| 黄色配什么色好看| 欧美xxxx性猛交bbbb| 97超视频在线观看视频| www.av在线官网国产| 免费在线观看成人毛片| 各种免费的搞黄视频| 不卡视频在线观看欧美| 久久久久国产精品人妻一区二区| 97超视频在线观看视频| 女人久久www免费人成看片| a级毛色黄片| 少妇的逼好多水| 熟女电影av网| 午夜精品国产一区二区电影| 国产亚洲最大av| 日本-黄色视频高清免费观看| 18禁动态无遮挡网站| 久久久久人妻精品一区果冻| 国产成人精品久久久久久|