Chinese Journal of Oceanology and Limnology Vol. 28 No. 4, P. 738-748, 2010 DOI: 10.

1007/s00343-010-9908-2

Optimization of conditions for tetraspore release and assessment of photosynthetic activities for different generation branches of Gracilaria lemaneiformis Bory*
WANG Zhiyuan () , WANG Guangce () ,**, NIU Jianfeng () , WANG Wenjun () , PENG Guang ()

College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

Received Jun. 23, 2009; revision accepted Aug. 17, 2009 Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2010

Abstract Gracilaria lemaneiformis Bory is an economically important alga that is primarily used for agar production. Although tetraspores are ideal seeds for the cultivation of G. lemaneiformis, the most popular culture method is currently based on vegetative fragments, which is labor-intensive and time-consuming. In this study, we optimized the conditions for tetraspore release and evaluated the photosynthetic activities of different colonies formed from the branches of G. lemaneiformis using a PAM (pulse-amplitude-modulated) measuring system. The results showed that variations in temperature and salinityhad significant effects on tetraspore yield. However, variations in the photon flux density (from 15 mol m-2 s-1 to 480 mol m-2 s-1) had no apparent effect on tetraspore yield. Moreover, the PAM-parameters Y(I), Y(II), ETR(I), ETR(II) and Fv/Fm of colonies formed from different branches showed the same trend: parameter values of first generation branches>second generation branches>third generation branches. These results suggest that the photosynthetic activities of different colonies of branches changed with the same trend. Furthermore, photosynthesis in G. lemaneiformis was found to be involved in vegetative reproduction and tetraspore formation. Finally, the first generation branches grew slowly, but accumulated organic compounds to form large numbers of tetraspores. Taken together, these results showed that the first generation branches are ideal materials for the release of tetraspores. Keyword: Gracilaria lemaneiformis Bory; tetraspore; pulse-amplitude-modulated; photosynthesis Abbreviation: ETR (I): Relative rates of photosynthetic electron transport of PSI ETR(II): Relative rates of photosynthetic electron transport of PSII F0: Minimum fluorescence yield Fm: Maximum fluorescence yield Fm': Maximum fluorescence yield in illuminated samples Fv/Fm: Optimum quantum yield OD: Optical density PE: Phycoerythrobilin P700: The reaction center of PSI PAR: Photosynthetic active radiation PSI: Photosystem I PSII: Photosystem II Y(I): Effective PSI quantum yield Y(II): Effective PSII quantum yield Y(NA): Nonphotochemical quantum yield of PSI caused by acceptor side limitation Y(ND): Nonphotochemical quantum yield of PSI caused by donor side limitation

1 INTRODUCTION
The genus Gracilaria is found worldwide and has been reported in temperate, tropical and arctic regions. The genus Gracilaria, was first described by Greville in 1830. Originally, the genus only included four species; however, Agardh reexamined the genus in 1852 and the number of species increased to 23.

Currently, the genus Gracilaria contains more than 100 species that covered worldwide (Bird et al.,
Supported by the National Natural Science Foundation of China (No. 30830015), the National Key Technology Research and Development Program of the 11th Five-Year Plan (No. 2006BAD09A04), and the National High Technology Research and Development Program of China (863 Program, Nos. 2006AA10A402, 2007AA09Z406, 2006AA05Z112, 2006AA10A413) Corresponding author: gcwang@ms.qdio.ac.cn

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1984; Liu, 1987; Fei et al., 1998), of which 30 have been recorded in China (Wu, 1998). Gracilaria is characterized by a typical polysiphonia-type life history with an alternation of isomorphic generations. Specifically, mature tetrasporophytes (2n) produce tetraspores (n) through meioses. Some of the tetraspores develop into male gametophytes (n), while others grow to female gametophytes (n), which then form spermatangi and carpogonia, respectively. After fertilization, cystocarps are formed on the female plant, which subsequently release carpospores (2n) that develop into tetrasporophytes (Ogata et al., 1972; de Oliveira et al., 1984; Yamamoto et al., 1988; Kain et al., 1995; Engel et al., 2001). Most species belonging to the Gracilaria genus are economically important algae that are used for agar extraction (Craigie et al., 1984; Glickman, 1987; Santelices et al., 1989; Marinho-Soriano, 2001; Marinho-Soriano et al., 2003; Freile-Pelegrn et al., 2005) or to obtain natural products with important bioactivities, such as phycoerythrin (PE) and hemagglutinin (Mazumder et al., 2002; Melo et al., 2002). Gracilaria also serve as efficient bio-filtration systems (Neori et al., 1996; Troell et al., 1997; Yang et al., 2000; Yang et al., 2005). In the last few decades, most of the Gracilaria biomass has been obtained from wild stocks, while marine aquaculture has only contributed a small amount to the total harvest. However, the need for Gracilaria has increased greatly with the development of the agar industry. Thus, greater attention has been given to the development of artificial Gracilaria cultures in many countries, especially in Southeast Asia (Glenn et al., 1996). By 1991, approximately one-third of the Gracilaria harvested worldwide was obtained from cultured sources (McHugh, 1991). Cultivation of Gracilaria in China began in the 1950s (Tseng, 2001; Zou et al., 2004), and the main species cultivated were Gracilaria asiatica Zhang et Xia, Gracilaria lemaneiformis Bory and Gracilaria tenuistipitata Chang et Xia. Among these species, G. lemaneiformis was regarded as the most important commercial species due to its rapid growth and the high-quality gel produced when compared with other species (Craigie et al., 1984). G. lemaneiformis normally grows in gravel in the lower intertidal zone. G. lemaneiformis growing in these areas are characterized by multiple branches, while the bases of the thallus are covered by sand. The thallus of G. lemaneiformis is reddish purple. The thallus come

out from a large holdfast. The rate of biomass accumulation peaks in spring and autumn (Li et al., 1984). Most propagation methods of G. lemaneiformis are dependent on vegetative fragments rather than tetraspores or carpospores (Hurtado-Ponce, 1990; Santelices et al., 1995; Nelson et al., 2001; Ryder et al., 2004; You et al., 2004). However, it has been found that thallus farming is not economical because it requires large amounts of propagating material to begin a plantation (Glenn et al., 1996). For example, approximately 20%30% of the harvest may be used as seed material in small-scale culture (Hurtado-Ponce et al., 1992). Furthermore, this practice is labor- and time-consuming. Conversely, the spore-cultured method is economical and highly efficient. Thus, development of a method of collecting spores and culturing sporelings in the sea would be very useful and has therefore attracted a great deal of attention (Levy et al., 1990; Glenn et al., 1996; 1998). People rarely use tetraspores to culture G. lemaneiformis because there is still little information available regarding tetraspores (Alberto et al., 1999). Therefore, the development of tetraspore cultivation methods for G. lemaneiformis requires more details regarding the release of tetraspores and the selection of culture conditions. Here, we reported the optimization of tetraspores released from different branchlet colonies and the photosynthetic activities of these different branchlets was studied.

2 MATERIALS AND METHODS

Mature tetrasporohytes of G. lemaneiformis were collected in November 2007 from the intertidal zone of Zhanshan Bay, Qingdao (360N, 12003E), China. After being washed in seawater to remove the epiphytes and sand, rinsed with sterilized seawater twice and soaked in 1% sodium hypochlorite for 2 min, the organisms were acclimated in sterilized seawater (temperature, 20C; photon flux density, 30 mol m-2s-1; salinity, 30) for one week and the algae were then used for the following experiments. 2.1 Branches of G. lemaneiformis G. lemaneiformis has multiple branches that lead to the production of different colonies. Specifically, G. lemaneiformis consist of first, second and third generation branches. First generation branches are caulis that grow directly on the holdfast, while second generation branches are produced by the first

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generation branches and third generation branches are produced by the second generation branches (Fig.1).

Fig.1 Mature Gracilaria lemaneiformis collected in the field

The differences in colony development of the branches are shown by the beeline

2.2 Treatment of thalli used for the release of tetraspores and measurement of the fluorescence and P700 levels The thalli used for the release of tetraspores release were soaked in seawater containing 0.1 g L-1 kanamycin, 0.3 g L-1 penicillin G, 1 g L-1 streptomycin sulphate, 0.02 g L-1 cefotaxine and 0.1 g ml-1 GeO2 for about 6 h. Next, the first, second and third generation branches were detached and cut into small segments (about 5 cm). All of the branches were stored in sterilized seawater prior to use. The first generation branches were prepared for optimization of the release of tetraspores. The fluorescence and P700 levels of the three generation branches were measured using the Dual-PAM-100 system.

2.3 Optimization of tetraspore release conditions Twenty small segments of first generation branches detached from the same individual were placed into individual petri dishes and the weights of the thalli were then recorded. Next, the temperatures of those small segments were measured once a day using a temperature indicator (Midwest Group, M307477, China). The photon flux densities were measured using an illuminometer (Photoelectric Instrument Factory of Beijing Normal University, ST-80C, China). The salinities of the seawater were measured using a hand-held refractometer (ATAGO, S-10E, Japan). The effects of five different temperatures (10C, 15C, 20C, 25C, 30C) were evaluated at a photon flux density of 30 mol m-2 s-1 while the salinity of the seawater was 30. The effects of photon flux was evaluated by culturing sets of thalli at six photon flux densities (15, 30, 60, 120, 240 and 480 mol m-2 s-1) at 20C while the salinity of the seawater was 30. Finally, the effects of five salinities (10, 20, 30, 35, 40) were evaluated at 20C and a photon flux density of 30 mol m-2 s-1. All branchlets were placed in petri dishes (9 cm in diameter) and cultured in PES culture medium in cultivation chambers. Each petri dish contained 2 grams of the first generation branches, and each treatment was replicated in a parallel Petri dish. All materials were incubated under a photoperiod of 12:12 h light:dark scheme and the culture medium was replaced once a week throughout the study period. After the tetraspores were released, the petri dishes were shaken gently so that they would be evenly distributed, after which five fields of view in each petri dish were selected at random and the tetraspore number in each field was counted. The number of released tetraspores in each petri dish was then calculated. 2.4 PSI and PSII measurements The first, second and third generation branches were all detached from the same individual and then cut into small segments. Next, the fluorescence and P700 parameters of the branchlets of different colonies were examined using a Dual-PAM-100 Measuring System (Heinz Walz GmbH, Eichenring 6, 91090 Effeltrich, Germany) that had been optimized for simultaneous assessment of P700 and chlorophyll fluorescence using the pulse-amplitude modulated (PAM) method and the Saturation Pulse (SP) method. The PAM and SP method can be used to obtain P700 and fluorescence signals simultaneously.

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A PAM fluorescence measurement that was nondestructive and could be conducted in-vivo was used in this study. For measurement, the samples (different generation branches) were placed directly between the ends of two 10 10 mm perspex rods and then measured to generate an automated induction and recovery curve as described by the manufacturer. All data were recorded using the Dual-PAM windows software. The Fv/Fm ratio (maximum photochemical quantum yield of PSII) was calculated according to the following equation (van Kooten et al., 1990): F v / F m = ( F m F 0 )/ F m (1)

quantum yields of nonphotochemical dissipation Y(ND) and Y(NA): Y(I) = 1Y(ND) Y(NA)

energy (6)

The light curves (LCs) of different generation branches were measured. The LC consists of the fluorescence responses to 13 different actinic irradiances (PAR 0, 14, 21, 30, 61, 103, 134, 224, 347, 539, 833, 1 295 and 1 960 mol m-2 s-1). To generate these curves, PAR was applied in increasing order, with duration of 30 s at each level. Ten repeated measurements were conducted for each type of branch. 2.5 Growth measurements Different colonies of branches were placed in petri dishes. Specifically, each petri dish contained three grams of thalli and each branch was replicated in two parallel petri dishes. All branchlets were cultured in PES culture medium in cultivation chambers at 20C, with a salinity of 30 and a temperature of 20C. All thalli were kept under a photoperiod of 12:12 h LD. The culture medium was replaced once a week and all thalli were weighed after 30 days. 2.6 Pigment content The branches of each colony were finely cut and the pigments were then extracted using the following two methods. 2.6.1 Extraction of water-soluble pigment A total of 0.2 g thalli were placed into a mortar, after which 0.5 ml of distilled water was added and the thalli were ground with a pestle. The mortar was then placed at -20C for about ten minutes, after which the samples were ground again. The above steps were repeated four times and the slurry was then centrifuged at 8 800 g for 5 min. Next, the pigment was then scanned using a spectrophotometer (Du650, BECKMEN, USA) at 400 to 800 nm in increments of 2 nm with a 1-cm light path. The phycoerythrin content was calculated according to the following formula (Kursar et al., 1983): PE=155.8OD498.540.0OD61410.5OD651 2.6.2 Extraction of lipid-soluble pigment The pigment was extracted and calculated according to the method described by Jensen (1978), with some modifications. Specifically, 1.0 ml of 80% (7)

where F0 represents the dark fluorescence yield, which was determined after keeping the tissue in darkness for 15 min. Saturating actinic light pulses (SP) were applied to obtain maximum fluorescence (Fm) in the dark-adapted samples. The maximum fluorescence yields in the illuminated samples were recorded as Fm'. The effective PS II quantum yield was calculated using the following formula: Y(II) = (Fm'F)/Fm' (2)

The relative rates of the photosynthetic electron transport of PSII (ETRII) or PSI (ETRI) were calculated as the effective quantum yield (Y) multiplied by the photosynthetic active radiation (PAR) received by PSII or PSI: rETR = YPAR0.5 (3)

The P700 signals ranged from a minimum level (P700 fully reduced) to a maximum level (P700 fully oxidized). The maximum level, which was denoted as Pm, was determined by application of SP in the presence of far-red light (FR). The Pm is analogous to the Fm, Pm was determined after Far-Red preillumination through application of a saturation pulse. Similarly, Pm' was analogous to Fm'. P700 red, which was determined using a saturation pulse, represents the fraction of the overall P700 that is reduced in a given state. The nonphotochemical quantum yields of PSI caused by donor site limitation and acceptor side limitation, Y(ND) and Y(NA), were calculated as follows: Y(ND)=1P700 red Y(NA)=PmPm'/Pm (4) (5)

Y(I) is calculated from the complementary PS I

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acetone was added to a mortar containing 0.5 g of fresh thalli. The G. lemaneiformis tissue was then ground with a pestle until the acetone turned bright, deep green. The liquid was then transferred to a centrifuge tube, after which the mortar and pestle was rinsed with another 1.0 ml acetone. All of the acetone extracts were then combined and filtered into a volumetric flask using a glass funnel. The final volume of the extract was diluted to 25 ml with acetone and the content of the chlorophyll a was calculated using the following formula: C= (D666-D730) V10/890 2.7 Data statistics and analysis The results were analyzed according to one-way ANOVA (analysis of variance) followed by Tukeys-test when appropriate. Statistical analyses were performed using SPSS13.0 with a P<0.05 indicating statistical significance. All data are reported as the meansS.D. (8)

Fig.2 Effect of different temperatures on the tetraspore yield of 1st generation branches of Gracilaria lemaneiformis cultured at a salinity of 30 and photon flux density of 30 mol m-2 s-1 (meansSD; n=5 random microscopic fields) (P<0.05)

3 RESULTS
3.1 The optimal conditions for tetraspore release Tetraspores were released in succession from the cultivated thalli in the lab after 3 weeks. Temperature played an important role in the release of tetraspores (Fig.2). Specifically, the yield of tetraspores increased as the culture temperature increased from 10C to 20C, after which it decreased from 25C to 30C. At below 10C and at 30C, the thalli produced 0.26106 and 0.19106 tetraspores g-1 fresh wt, respectively, which are both insufficient for the release of tetraspores (P<0.05). Additionally, tetraspore yields did not differ significantly among treatments incubated at 15C, 20C and 25C as indicated by mean yields of 2.14106, 2.70106 and 2.66106 g-1 fresh wt, respectively. Thus, 15C, 20C and 25C are suitable temperatures for the release of tetraspores, and the optimal temperature was 20C (P<0.05). The values of the tetraspore yield at photon flux densities ranging from 15480 mol m-2 s-1 did not differ significantly (P>0.05) (Fig.3). Variations in the salinity of the seawater had strong effects on the tetraspore yield. Specifically, salinities of 10 and 40 had adverse effects on the release of tetraspores, as indicated by yields of 0.35106 and 0.24106 g-1 fresh weight, respectively. The yields of tetraspores increased as the salinity increased, with the optimum yield of 2.44106 g-1 fresh wt being observed at 30, above which the

Fig.3 Effect of different photon flux densities on tetraspore yield of 1st generation branches of Gracilaria lemaneiformis cultured at a salinity of 30 and a temperature of 20C (meansSD; n=5 random microscopic fields) (P>0.05)

tetraspore yield decreased (Fig.4). Overall, these findings indicate that a salinity of 3035 was suitable for the release of tetraspores, and the optimum salinity was 30 (P<0.05).

Fig.4 Effect of different salinities on tetraspore yield of 1st generation branches of Gracilaria lemaneiformis cultured under a photon flux density of 30 mol m-2 s-1 and a temperature of 20C (meansSD; n=5 random microscopic fields) (P<0.05)

3.2 Measurement of fluorescence and P700 levels PAM fluorescence parameters Y(II), ETR(II) and Fv/Fm, and the P700 parameters Y(I) and ETR(I) of the different colonies of branches were calculated (Figs.59). The values of the five parameters of different colonies of branches showed the same trend

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overall, with the yields of the first generation branches being the highest and those of the third generation branches being the lowest. As the PAR increased, the parameters changed in different ways. Specifically, the maximum values of Y(I) and Y(II) were observed when the mean photon flux density was 22 mol m-2 s-1 (first generation branches: Y(I) = 0.58, Y(II) = 0.505; second generation branches: Y(I) = 0.48, Y(II) = 0.379; third generation branches Y(I) = 0.4, Y(II) = 0.366), but these values decreased sharply as the photon flux density increased. The minimum values of Y(I) and Y(II) were observed at a photon flux density of 1 362 mol m-2 s-1 (first generation branches: Y(I)=0.23, Y(II)=0.016; second generation branches: Y(I) = 0.02, Y(II)= 0.0127; third generation branches Y(I) = 0.012, Y(II) = 0.01) (Figs.5, 6). However, as the PAR increased, the ETR(I) values increased, with the maximum values being observed when the mean photon flux density was 99 mol m-2 s-1. Specifically, the following values were observed: ETR(I)=14.51; second generation branches: ETR(I) = 10.4; third generation branches ETR(I) = 9.5. These values then decreased as the PAR increased from 99 mol m-2 s-1 (Fig.7). Similarly, as the PAR increased, the ETR(II) values increased until the photon flux density reached 370 mol m-2 s-1 (first generation branches: ETR(II) = 8.99; second generation branches: ETR(II) = 8.32; third generation branches: ETR(II)=5.68), after which they decreased (Fig.8). The Fv/Fm (Maximal PS II quantum yield) did not change as the photon flux density increased, as indicated by mean values of 0.527 4 for first generation branches, 0.420 5 for second generation branches and 0.401 4 for third generation branches (P<0.05) (Fig.9). 3.3 Growth measurements The growth measurement results revealed that the change in the biomass of the different branches differed. Specifically, third generation branches grew the fastest (2.83 g), but first generation branches grew the slowest (1.97 g) (P<0.05) (Fig.10).

Fig.6 Y(II) values of different colonies of branches cultured under different PAR (photosynthetically active radiation) irradiances (P<0.05)

Fig.7 ETR(I) values of different colonies of branches cultured under different PAR (photosynthetically active radiation) irradiances (P<0.05)

Fig.8 ETR(II) values of different colonies of branches cultured under different PAR (photosynthetically active radiation) irradiances (P<0.05)

Fig.9 Fv/Fm values of different colonies of branches (P<0.05)

3.4 Pigment content

Fig.5 Y(I) values of different colonies of branches cultured under different PAR (photosynthetically active radiation) irradiances (P<0.05)

3.4.1 Water-soluble pigment content The absorption spectra of water-soluble pigments from different branches were measured (Fig.11). According

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Fig.10 Increase in biomass of different colonies of branches after 30 days of culture (P<0.05)

to the formula PE=155.8OD498.540.0OD614 10.5OD651, the crude extract contained 9.119 mg (first generation branches), 7.074 mg (second generation branches) and 6.503 mg (third generation branches) of PE, which corresponded to PE contents of 49.595 mg g-1, 35.37 mg g-1 and 32.515 mg g-1, respectively. These findings clearly demonstrate that the phycoerythrin content of the first generation branches was highest, while that of the third generation branches was lowest. 3.4.2 Lipid-soluble pigment content The absorption spectra of the lipid-soluble pigment of different branches were measured (Fig.12). According to the formula C = (D666D730)V10/890, the crude extract of first, second and third generation branches contained 0.051 mg, 0.044 mg and 0.041 mg chlorophyll a, respectively. Based on these findings, the chlorophyll a levels for first, second and third generation branches were 0.102 mg g-1, 0.088 mg g-1 and 0.082 mg g-1, respectively.

4 DISCUSSION
Natural populations of G. lemaneiformis grow in complex environments that are influenced by a variety of biotic factors including epiphytism (Kuschel et al., 1991; Gonzalez et al., 1993; Troell et al., 1997). Furthermore, it has been reported that many types of bacteria can be found on the thalli of Gracilaria, and that some of these bacteria can inhibit algal spore germination (Egan et al., 2001). Accordingly, the thallus of G. lemaneiformis should be treated with antibiotics prior to experiments conducted to evaluate its release of tetraspores. G. lemaneiformis contains multiple branches that release large numbers of tetraspores upon maturation. Previously, we demonstrated that first generation branches of tetrasporohytes possessed more than 80% of the total tetraspores, while second and third

generation branches comprised only 20% of the total tetraspores. Moreover, the survival rates and germination of tetraspores released from first generation branches have been shown to be higher than those of tetraspores released from other branches (Ye et al., 2006). Based on these findings, first generation branches are ideal materials for the release of tetraspores; therefore, we used these branches of G. lemaneiformis to determine the optimal conditions for the release of tetraspores. Although there is a great deal of information available regarding the life history of Gracilaria, some questions about its heterogenesis still remain. For example, it has been reported that sporophytes and gametophytes occurred synchronously in the thallus of Gracilaria (Liu, 1988), which differs greatly from most organisms. Gracilaria tetraspores are an important stage in life history because they connect sporophytes to gametophytes. The release of tetraspores from G. lemaneiformis is affected by temperature, photon flux densities, gravity of the seawater and weather conditions, which are crucial for the release of tetraspores and their germination. It is not difficult to induce the release and germination of tetraspores on a small-scale in the laboratory (Wang et al., in press). Additionally, small-scale culture studies conducted under natural conditions have demonstrated the feasibility of growing Gracilaria from either carpospores or tetraspores (Levine, 1986; Levy et al., 1990; Prieto et al., 1991; Destombe et al., 1993). However, their large-scale culture has not yet been realized for two reasons, namely, it is difficult to induce the release of substantial numbers of spores and it is difficult to induce the spores to be released synchronously. In fact, it is not easy to control the conditions required for spore release in cultivation chambers. Moreover, the mechanism of spore release is still poorly understood. Thus, it is very important to identify the optimal conditions for the release of tetraspores under large-scale production conditions. During G. lemaneiformis cultivation, we found that first generation branches grew slowly and accumulated a small amount of biomass, while second and third generation branches grew rapidly and accumulated a large amount of biomass. These findings suggested that third generation branches have stronger photosynthetic activity than first generation branches. To validate our viewpoint, we used a Dual-PAM-100 system to measure the photosynthetic activity of different colonies of branches. The photosynthetic activity has increasingly

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Fig.11 Absorption spectra of water-soluble pigments of Gracilaria lemaneiformis

A. 1st generation branches; B. 2nd generation branches; C. 3rd generation branches

been examined using pulse-amplitude-modulated (PAM) fluorescence techniques (Aline et al., 2007). Chlorophyll (Chl) a fluorescence measurements are widely used to assess the physiological state of higher plants and algae due to their being rapid, simple and non-invasive methods (Juneau et al., 2005).

I n f o r ma t i o n r e g a r d i n g t h e p h o t o s y n t h e t i c performance can be obtained with the aid of Light Curves (LC). Specifically, the parameters Y(I), Y(II), ETR(I) and ETR(II) obtained from LC reflected the photosynthetic capacity of different colonies of branches, and the Fv/Fm (Maximal PS II quantum

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Fig.12 Absorption spectra of lipid-soluble pigments of Gracilaria lemaneiformis

A. 1st generation branches; B. 2nd generation branches; C. 3rd generation branches

yield) represents the potential maximum photosynthetic capacity of different colonies of branches. In this study, it was found that different colonies of branches had different parameter values, and that these parameters changed in a similar fashion, namely first generation branches>second

generation branches>third generation branches. These findings indicate that the sequence of photosynthetic activity and the potential maximum photosynthetic capacity was as follows: first generation branches>second generation branches>third generation branches. These findings completely

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contradict our original presumption that the photosynthetic activity of third generation branches is stronger than that of first generation branches. Based on our findings, photosynthesis is involved in two important steps in the cultivation of G. lemaneiformis, vegetative reproduction and the accumulation of organic compounds to form tetraspores. Additionally, we found that first generation branches grew slowly and accumulated large numbers of tetraspores, while third generation branches grew rapidly and accumulated few tetraspores, indicating that first generation branches have a stronger photosynthetic activity than third generation branches. The pigment content of different colonies of branches also reflected this phenomenon, with first generation branches showing the highest content of phycoerythrin and chlorophyll and third generation branches having the lowest levels of these compounds. Normally, when spores are collected from Gracilaria they are collected from third generation branches rather than the first generation branches. However, according to the results of this experiment and those of our previous studies, first generation branches of tetrasporohytes possessed more than 80% of the total tetraspores, while second and third generation branches contained only 20% of the total tetraspores (Ye et al., 2006). Therefore, first generation branches are ideal materials for spore release. Accordingly, future studies to evaluate the cultivation of G. lemaneiformis should focus on first generation branches. G. lemaneiformis is a valuable alga that has not been extensively exploited. With the development of the agar industry, the demand for Gracilaria and its price have increased sharply, which has resulted in serious damage to natural stocks (Hanisak, 1998). Indeed, the price of dried G. lemaneiformis was 1 500 US dollars per ton in China in 2007. Additionally, Gracilaria has become scarce along the coast of China. Thus, artificial cultivation of Gracilaria should be widely developed to complement the available natural resources. In addition, further studies should be conducted to facilitate the cultivation and improve the available strains of Gracilaria.
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