WANG Zhongxia, SUI Zhenghong, HU Yiyi ZHANG Si PAN Yulong and JU Hongri

1) Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Kanghua Biotech Co. Ltd., Weifang 261023, P. R. China

A Comparison of Different Gracilariopsis lemaneiformis (Rhodophyta) Parts in Biochemical Characteristics, Protoplast Formation and Regeneration

WANG Zhongxia1),2), SUI Zhenghong1),*, HU Yiyi1), ZHANG Si1), PAN Yulong1), and JU Hongri1)

1) Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, P. R. China

2) Kanghua Biotech Co. Ltd., Weifang 261023, P. R. China

Gracilariopsis lemaneiformisis a commercially exploited alga. Its filaceous thallus can be divided into three parts, holdfast, middle segment and tip. The growth and branch forming trend and agar content of these three parts were analyzed, respectively, in this study. The results showed that the tip had the highest growth rate and branched most, although it was the last part with branch forming ability. The holdfast formed branches earliest but slowly. Holdfast had the highest agar content. We also assessed the difference in protoplast formation and regeneration among three parts. The middle segment displayed the shortest enzymolysis time and the highest protoplast yield; whereas the tip had the strongest vitality of protoplasts formation. Juvenile plants were only obtained from the protoplasts generated from the tip. These results suggested that the differentiation and function ofG. lemaneiformiswas different.

Gracilariopsis lemaneiformis; growth; differentiation; agar; protoplast

1 Introduction

Gracilariopsis lemaneiformisis an economically important seaweed in China; it is one of the major agarophytes (Chenet al., 2009). Agar is widely used in food, pharmaceutical and bioengineering industry, as well as other fields (Armisen, 1995; Liu and Sun, 2007; Marinho-Soriano, 2001). Intensive researches have been carried out forG. lemaneiformis, which included, for example, genetics, hybridization, selective breeding and population structure description (Chenet al., 2011; Panget al., 2010; Patwary and van der Meer, 1992; Zhanget al., 2012). Since early 1970s, researches on the growth characteristics and agar quality isolated fromG. lemaneiformishave also been widely conducted (Liet al., 2010; Peiet al., 2012; Xu and Gao, 2008; Yanget al., 2006). The filaceous thallus ofG. lemaneiformiscan be divided into holdfast which includes both the prostrate disc and the erect part of thallus, middle segment and tip. In the present study, with the aims of elucidating the basic biological property ofG. lemaneiformisand providing theoretical guidance for its breeding, cultivation and efficient economic exploitation, a biochemical analysis was carried out to compare the holdfast, middle segment and tip for their growth and branch forming trend and agar content.

Protoplast technology is a well-known cell engineering approach that provides an effective method for plant breeding. Several reports are available on the successful regeneration in many species ofGracilariaprotoplasts, such asG. verrucosa(Molletet al., 1995) andG. changii(Yeonget al., 2007). However, the success in protoplast regeneration ofG. lemaneiformiswas not met yet. In this study, we analyzed the difference in protoplast formation and regeneration among the three parts of the filaceous thallus, aiming to obtain a high protoplast yield and meet success in protoplast regeneration ofG. lemaneiformis.Our results will be useful for facilitating further application of protoplast technology in the genetic breeding of the alga.

2 Materials and Methods

2.1 Materials

HealthyG. lemaneiformisindividuals were collected in June 2009 from Zhanshan Bay, Qingdao, China. Epiphytes and mud were removed with soft brushes. Youngalga strains, 10 cm in length, were used in the experiment. Segment within 2 cm of apex was defined as the tip. Segment within 2 cm from the base was defined as the holdfast part which includes both the prostrate disc and the erect part of the thallus. Segment within 2 cm from the midpoint of the alga was defined as the middle segment.

2.2 Growth and Branching Measurements

Eight healthy plants were cleaned of attached epiphytes and microorganisms with a soft brush. Segment, 1 cm in length, was cut from the tip, holdfast and middle part of each plant and placed in three sterile Petri dishes (8 segments from each part a Petri dish) with 20 mL Pro medium (Provasoli, 1968), 3 replicates each. The tissues were cultured under an irradiation of 45 μmol m-2s-1with a rhythm of 12 h light and 12 h dark and at 20℃. The culture medium was replaced every three days. The growth and branching status was observed for a period of 16 d and recorded every three days. Visible protuberances on the surface of the filament were recorded as branching events. The growth was recorded as the total length of the main branch plus any additional branches. The recorded data was used to make quantitative growth trend graph and the trend line slope to show the growth trend difference among three parts.

2.3 Measurement of Agar Content

The tissues from three parts ofG. lemaneiformis, 2 g each, were washed with sterile seawater with the agar extracted with autoclave treatment method (Liet al., 2009; Xueet al., 2006). The yield was calculated as the agar produced (g) from every gram of fresh material. The measurement was repeated 4 times.

2.4 Statistical Analysis

The data of growth, branching and agar yield were analyzed using the statistical software SPSS to obtain variance. The significant difference was set at 95%.

2.5 Protoplast Isolation and Regeneration

2.5.1 Preparation of enzyme digestion solution

The combination enzyme solution was as follows: 1% cellulase R-10, 0.8 mol L-1D-sorbitol, 0.75 mol L-1NaCl (Chuet al., 1998) and 2% shellfish enzyme (Liuet al., 2002; Liuet al., 1984; Xuet al., 2007) in distilled water, pH 6.5.

2.5.2 Protoplast isolation and purification

Young, healthy plants were selected and cleaned of other microorganisms with a soft brush. Tip, holdfast and middle segment were cut out and immersed with sterile seawater containing antibiotics for 12 h. The antibiotic mixture consisted of 0.18 g L-1penicillin G sodium and 0.2 g L-1streptomycin sulfate. The segments were then soaked in sterile seawater containing 0.7% KI for 10 min and then rinsed several times with sterile seawater.

The treated alga segments ofG. lemaneiformiswere chopped into smaller explants (1–2 mm2), and then rinsed twice with sterile seawater followed by soaking in 45×10-6MES medium (Wanget al., 1986) for 15 min in the dark to prepare the cells for plasmolysis (Yeonget al., 2007). The treated explants were incubated in protoplast-isolating solution (10 mL g-1) in a 100 mL centrifuge tube placed in the dark and shaked with 100 r min-1at 25℃. The solution containing the protoplasts was then filtered through a 50-μm nylon mesh to remove the undigested tissue and debris. The remaining filtrate containing isolated protoplasts was diluted with 45×10-6MES medium and maintained for 20 min before centrifuged at 500 g for 5 min. Then 90% of the supernatant was discarded and the protoplasts were resuspended with fresh MES medium. This procedure was repeated twice to remove any residual enzyme solution.

The explants were incubated for 2.5, 3, 3.5, 4 and 4.5 h, respectively, in dark with shaking to optimize the enzymolysis time and obtain the highest yield of protoplasts. The final protoplasts were resuspended in 1 mL MES medium and the yield (g) determined as follows:

There were 3 replicates of each treatment.

2.5.3 Protoplast identifying by fluorescent staining

The protoplasts were stained with 0.1% Fluorescent Brightener 28 for 5 min and examined under a UV fluorescence microscope to confirm that they were true protoplasts without cell wall (Zhanget al., 2005a). Fluorescent Brightener 28 is a chitin-binding dye. Cells with cell wall remnants fluoresced blue under fluorescence microscope (Rüchelet al., 2004; Zhaoet al., 2010).

2.5.4 Protoplast vigor identifying by Evans blue staining

The viability of freshly isolated protoplasts was determined by staining with 0.1% Evans blue for 5 min (Yanget al., 1996) and observed under an optical microscope. The viable protoplasts were stained yellow and the dead protoplasts were stained blue.

2.5.5 Protoplast culture and regeneration

Freshly isolated protoplasts were harvested and suspended in high salinity (45) MES medium (Yeonget al., 2007) based on protoplast density, with the final concentration ranging from 1.0×105to 5.0×105cells mL-1. The suspension was dropped onto the cover glass in the Petri dishes, and MES medium was added to a 2/3 volume of the dishes after protoplasts were sunk on the cover glass. From the beginning of the experiment, the dishes were shaded from light with aluminum foil and cultured with a rhythm of 12 h light and 12 h dark at 25℃. The salinity of the MES medium was then gradually reduced to the normal (30) using the following procedure. Half of the culture medium was replaced with fresh medium (30) every2.5 d for the first three times, and then was replaced every 5 d. Meanwhile, the light intensity was gradually increased to 4–5 μmol m-2s-1from day 2 and to 10–12 μmol m-2s-1from day 4.5. The morphology of the protoplasts including their color, size and the point of division, was recorded under an inverted microscope when changing the medium.

3 Results

3.1 Growth and Branching Difference

The growth and branching status of different filaceous parts were observed for 16 d and recorded every three days. The tip grew fastest, which was followed by the middle segment and the holdfast in order. The length growth trend line slope of the tip, middle segment and holdfast was 1.878, 1.426 and 1.057, respectively. The difference in growth rate between the tip and middle segment, middle segment and holdfast, and the tip and holdfast was all significant (P<0.05) (Fig.1).

Fig.1 Length growth trend graph of different parts of the filaceous thallus of G. lemaneiformis. Bar, standard deviation (n=3).

Fig.2 Branching growth trend graph in different parts of the filaceous thallus of G. lemaneiformis. Bar, standard deviation (n=3).

The branching growth trend line slope of tip, middle segment and holdfast was 9.16, 9.10 and 5.00, respectively. The branching growth trend of middle segment was similar to that of tip, but tip produced the most branches. The branching growth trend of holdfast was the slowest but holdfast branched firstly. The difference in branching between tip and holdfast and between middle segment and holdfast was significant (P <0.05); whereas that between tip and middle segment was not (P >0.05) (Fig.2).

3.2 Difference in Agar Content

Holdfast had the highest agar content, up to 16.9% ± 0.16% of dry weight (Fig.3). The difference in agar yield between holdfast and tip and holdfast and middle segment was significant (P<0.05); whereas that between middle segment and tip was not (P>0.05).

Fig.3 Difference in agar content of different parts of the filaceous thallus of G. lemaneiformis. Bar, standard deviation (n=3).

3.3 Difference in Protoplast Isolation and Regeneration

3.3.1 Protoplast identification

Fig.4 Protoplasts of G. lemaneiformis. (a) Freshly isolated protoplasts under light microscope; (b) Freshly isolated protoplasts (marked by white arrows) stained with fluorescent Brightener 28 under UV florescence microscope; (c) Viable protoplasts stained yellow by Evans blue; (d) Three-day-old protoplasts stained with Fluorescent Brightener 28 showing the presence of regenerated cell wall (in blue).

Freshly isolated protoplasts displayed different sizes. The largest ones were approximately 100 μm in diameter with a big vacuole and a dim chromatophore, which were probably produced from medullary cells. Most of theprotoplasts were 7–15 μm and 30–40 μm in diameter and rich in pigment, which were probably produced from epidermal and cortical cells, respectively (Fig.4a). The freshly isolated protoplasts by Fluorescent Brightener 28 staining showed no trace of blue fluorescence when examined under a UV florescence microscope, while true protoplasts without cell walls had been obtained (Fig.4b). Viable protoplasts were stained yellow with Evans blue staining (Fig.4c).

3.3.2 Difference in protoplast yield in different enzymolysis times

The optimal treatment to isolate protoplasts from the algal tips was a 4 h enzyme treatment, resulting in a protoplasts yield of (8.79 ± 0.43)×104g-1fresh material. In contrast, the optimal treatment for middle segments was 3h with a yield of protoplasts up to (12.8 ± 0.45)×104g-1fresh material. For holdfasts, the optimal treatment was 3.5 h enzyme incubation, which produced the best protoplast yield of (7.13 ± 0.40)×103g-1fresh material (Fig.5). Middle segments produced the highest protoplast yield among three algal parts, whereas the protoplast yield of holdfasts was the lowest.

Fig.5 Protoplast yield difference of different parts of the filaceous thallus of G. lemaneiformis in various enzymolysis time. Error bars, standard deviation (n=3).

3.3.3 Protoplast Isolation and Regeneration

The regeneration of protoplasts was significantly different among holdfast, middle segment and tip. After culturing for 3 d, protoplasts from the tip showed the lowest mortality; while those from middle segments showed high mortality and all protoplasts from holdfasts ruptured. Three-day-old protoplasts were stained blue with Fluorescent Brightener 28, indicating that cell walls regenerated (Fig.4d). Preliminary observations showed that cell division only occurred in the protoplasts from tips (Figs.6a, 6b). Cell division continued to develop into macro-colonies and regenerated juvenile plants after 90 d culture (Fig.6c). Further development produced up to 9 plants from the same cell masses after 210 d culture (Fig.6d).

Fig.6 Protoplast regeneration of G. lemaneiformis. (a) Dividing cell of protoplast after 18 d culture; (b) Dividing cell of protoplast after 27 d culture; (c) Plants regenerated from protoplast after 90 d culture; (d) Elongation and increased plants formation from protoplast after 210 d culture.

4 Discussion

4.1 Biochemical Characteristics of the Three Different Parts

Significant difference was found in the growth of holdfasts, middle segments and tips ofG. lemaneiformis: tips had the highest growth rate, which was followed by middle segments and then holdfasts. The reason might be that tips are the growing point of alga with more meristematic cells and higher cell meristematic ability. The difference in branching between tips and holdfasts was also significant. The tips branched the most at the end of cultivation while holdfasts branched earliest, which could meet their function as an attachment to the sand base.

The agar yield in the three parts ofG. lemaneiformiswas also significantly different. The holdfasts had the highest agar content (up to 16.9% of dry weigh), whereas that of the middle segments and tips was lower. The holdfasts had a thick cell wall, which could be the result of its high agar content and its attachment function. The agar content of the middle segments and tips corresponded to their rapid growth.

Our study confirms that the three parts of the filaceous thallus display different biochemical characteristics, suggesting that there is a functional distinction within the tissues ofG. lemaneiformis. In addition, our results also provided useful information for the cultivation and commercialization ofG. lemaneiformis.Tips showed significant superiority in growth and branching, whereas holdfasts had the highest content of agar. Therefore, the tips should be selected as seedlings for the large-scale breeding ofG. lemaneiformis, and the middle segments andholdfasts can be selected for the industrial extraction of agar.

4.2 Protoplast Isolation and Regeneration from the Three Filaceous Thallus Parts

Holdfasts, middle segments and tips had different optimal enzymolysis time, and the optimal protoplast yields. This difference was related to the variation in the cell composition and structures of different parts. Middle segments displayed the shortest enzymolysis time with the highest protoplast yield, which suggests that protoplast isolation is easiest from this part. This is likely to be the result of the larger cross-sectional area of middle segments, which provides a larger area for enzymolysis. A larger proportion of cortical and medullary cells in middle segments can form a loose cell arrangement (Zhanget al., 2005b), which may also increase the enzymolysis efficiency. The protoplast yield of holdfasts was lower than that of tips and middle segments. Considering the long growth time of holdfasts can result in thicker cell walls and higher agar and cellulose contents, it is difficult to isolate protoplasts from this part. Theoretically the protoplast yield could be increased by extending the enzymolysis time. However, the enzymatic solution is toxic to the cells, so this is unlikely to be a viable option (Xiao, 2005). The protoplast yield in holdfasts after 4 h of enzymatic treatment was lower than that in holdfasts after 3.5 h of treatment.

In this study, protoplasts from the three filaceous thallus parts were cultured in MES liquid medium. Protoplast regeneration was only obtained from the protoplasts of tips. This indicated that these protoplasts had the strongest vitality. This is probably because tips are the growing point of alga with high cell meristematic ability and activity.

In summary, protoplast isolation and regeneration in different parts of the filaceous thallus ofG. lemaneiformisshowed obvious difference. Our study provided an important reference not only for the regeneration of protoplasts fromG. lemaneiformis, but also for the application of cell engineering technology, such as protoplast fusion technology, in the genetic breeding ofG. lemaneiformis.

Acknowledgements

This work was supported by the Twelfth Five-Year Plan in National Science and Technology for the Rural Development in China (2012AA10A411) and Public Welfare Project of Ministry of Agriculture of China (200903030).

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(Edited by Qiu Yantao)

(Received November 9, 2012; revised December 20, 2012; accepted December 4, 2013)

? Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

* Corresponding author. Tel: 0086-532-82031128

E-mail: suizhengh@ouc.edu.cn