Selection and characterization of an improved strain (A-13) of Pyropia yezoensis (Bangiales, Rhodophyta)

Journal Pre-proof Selection and characterization of an improved strain (A-13) of Pyropia yezoensis (Bangiales, Rhodophyta) Hao Jiang, Hongchang Ding, Peng Zhang, Tiegan Wang, Xinghong Yan

PII:

S0304-3770(20)30023-1

DOI:

https://doi.org/10.1016/j.aquabot.2020.103213

Reference:

AQBOT 103213

To appear in:

Aquatic Botany

Received Date:

28 May 2019

Revised Date:

19 November 2019

Accepted Date:

2 February 2020

Please cite this article as: Jiang H, Ding H, Zhang P, Wang T, Yan X, Selection and characterization of an improved strain (A-13) of Pyropia yezoensis (Bangiales, Rhodophyta), Aquatic Botany (2020), doi: https://doi.org/10.1016/j.aquabot.2020.103213

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Selection and characterization of an improved strain (A-13) of Pyropia yezoensis (Bangiales, Rhodophyta) Hao Jiang1,2,3·Hongchang Ding1·Peng Zhang4·Tiegan Wang4·Xinghong Yan1,2,3 1

Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean

University, Ministry of Education, Shanghai 201306, China National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean

University, Shanghai 201306, China

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2

Shanghai Engineering Research Center of Aquaculture, Shanghai 201306, China

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Zhejiang Mariculture Research Institute, Wenzhou 325005, China

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3

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Corresponding author: Xinghong Yan

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E–mail: [email protected]

Address: 999 Huchenghuan Road, Lingang New City, Shanghai, China, 201306

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Phone No: (+86)021–6190-0422

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Fax No: (+86)021–6190-0422

Highlights

We carried out a crossing experiment between the green mutant (C-0, ♀) and red mutant (fre, ♂)

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of Pyropia yezoensis to select an improved strain.

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In this paper, the characteristics of improved strain of Pyropia yezoensis were evaluated from the aspects of yield, quality, stress resistance and released number of conchospores

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An improved A-13 which was fast growth, high-temperature resistance, high quality; large amount of conchospores as well as wild like color was selected.

Abstract A crossing experiment between the green mutant (C-0, ♀) and red mutant (fre, ♂) of Pyropia yezoensis was carried out for breeding an improved strain which was fast growing, high quality and wild type color, finally an improved strain (A-13) was isolated from the F1 gametophytic blades. The mean length and length-width ratio of the 60-day-old blades in A-13 were 73.43 cm and 35.41, which were 1.69 and

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2.92 times higher than those of the wild-type strain (WT) of P. yezoensis, respectively, and the wet weight had no significant difference between the two strains. After being cultured for 60 days, the

contents of chlorophyll a and phycobiliprotein of A-13 were 10.35 and 79.77 mg/g, which were 1.93

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and 1.63 times higher than those of the WT strain, respectively. The thickness of the 60-day-old blades

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of A-13 strain was 19.86 μm, which was 30% thinner than that of WT strain. In addition, the total

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number of conchospores per shell released from A-13 was 1408.83×104, which was 2.45 times higher than that of WT strain. The above results confirmed that A-13 strain was characterized by fast growth,

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high quality; large amount of conchospores as well as wild like color, therefore, A-13 is expected to be

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applied in commercial cultivation.

Keywords Pyropia yezoensis · Gametophytic blade · Growth rate · Improved strain · Intraspecific

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hybridization·Thickness

1.

Introduction

Pyropia yezoensis (Sutherland et al., 2011) is a kind of economical and nutritive marine alga. It is widely cultivated in China, Japan and Korea. In China, it is mainly cultivated in Jiangsu and Shandong

province, and its total industry output has reached about CNY¥ 5 billion (Zhu et al., 2016). Besides that, P. yezoensis also have great ecological benefits, large-scale cultivation of Pyropia can alleviate the ocean eutrophication by removing nutrients like N, C and P (Yang and Fei, 2003). However, because of inbreeding and lack of seed production technology for decades, the yield, seeds quality and production’s quality of Pyropia were reduced. Although there have already had two improved strains of P. yezoensis which were pass the National examination and approval (Zhu et al., 2015; National

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Fisheries Technology Extension Station 2014), these two strains still can’t meet the demand of nori industry.

These years, researchers have selected a number of improved strains of P. yezoensis by artificial

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mutation technology (Huang et al., 2014; Wang et al., 2012; Liu et al., 2015). In the process of

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selection, red mutants were always selected as improved strains because of its bright color, fast growth

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and high content of pigments. But the result of pilot cultivation showed that the blades were too red which made the color of nori products was not well received (not published). So it is important to select

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and expand an improved strain which is fast growing, high quality and wild type color. Mutation breeding could get lots of pigmentation mutants in a short time, but its direction and

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properties were random, and it was too difficult to achieve targeted regulation (Yan, 1997). While the cross breeding could concentrate good characteristics of the parents into offspring (Lin et al., 2009;

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Ding et al., 2018). Meiosis of P. yezoensis occurs during the first two divisions of the germinating conchospores then form tetrad which continues to undergo mitosis and eventually develops into a thallus containing 2 to 4 colors (Ohme et al., 1986). In addition, Wu (2016) used green mutant and red mutant of P. yezoensis to perform the intraspecific hybridization experiment, found that the proportion of wild like color in F1 color sectored blades was 33%. The aim of this research was to select a strain

with fast growth, high quality and wild like color from the F1 gametophytic blades of heterozygote conchocelis which was produced by cross experiment between green mutant (C-0, ♀) and red mutant (fre, ♂) and cultivate it into an improved strain which suitable to be applied in commercial cultivation.

2.

Material and method

2.1 Materials

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The green mutant (C-0, ♀) and red mutant (fre, ♂) of P. yezoensis were used as the materials of cross

experiment to get the heterozygote conchocelis. The wild type strain (WT) of P. yezoensis was isolated

from curtain in Lvsi marine area, Jiangsu province, China, in 2001, and it was preserved as free-living

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conchocelis in the laboratory. The preserve method was the same as Kato and Aruga (1984).

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2.2 Cross experiment

The method of cross experiment was the same as Yan (1997). The blades of fre strain which appeared

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spermatangia were chosed as male parent; when carpogonium appeared the blades of C-0 strain were chosed as female parent. The blades of these two strains were cocultured in 1:1 ratio with aeration in a

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250 mL flask containing MES culture medium (Wang et al., 1986) until carposporangia appeared. The culture conditions were 19±1 °C, 50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D).

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Then the fertilized blades were transferred to a new flask and cultured under the same conditions until carpospores were released.

2.3 Verification of heterozygous conchocelis Some free-living conchocelis which were developed from single carpospores were incubated at

19±1 °C, 20 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D) for 3 weeks. Then a homogenizer was used to fragment conchocelis and inoculated it to cleaned shells. The inoculated shells were incubated in Petri dishes containing 200 mL MES medium under 19±1 °C, 10 μmol photons m−2 s−1 and 10:14 h light/dark cycle (10L∶14D) . After cultured for 10 days, the shells were washed to clear superfluous conchocelis filaments on the shell surface, the culture medium were refreshed and the photon flux density was increase to 20 μmol photons m−2 s−1. Four weeks later the

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culture temperature was increased to 23±1 °C and transferred to 8:16 h light/dark cycle (8L∶16D) to

induce the formation of conchosporangias. When conchosporangias were formed, conchospores were collected and cultured in natural seawater enriched with MES medium with aeration in a 250 mL flask

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(Fu et al., 2011). The culture conditions were 19±1 °C, 50 μmol photons m−2 s−1 and 10∶14 h

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light/dark cycle (10L∶14D). The culture medium was refreshed every 5 days (Chen et al., 2016). After

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cultured for about 20 days, the heterozygous conchocelis were detected by observing whether there were color sectored blades in F1 gametophytic blades and their proportions in the population. The

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method was the same as Yan (1997).

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2.4 Selection of the improved strains

When conchosporangias were formed, conchospores were collected and cultured in natural seawater

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enriched with MES medium with aeration in a 250 mL flask. The culture conditions were 19±1 °C, 50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D). The culture medium was refreshed every 5 days. After cultured for 45 days, the flask was replaced (1000 mL), and cultured for another 20 days, some wild and wild like color sectors were selected from F1 color sectored blades and cut them off, then puncher (Φ =3 mm) was used to take 15 disks from each of these sectors for culture with

aeration at the same culture condition, the length, width and wet weight were measured every 5 days. After cultured for 30 days, a couple of color sectors with fast growth rate were selected and treated with enzyme which was got from the digestive glands of sea snail to obtain the homozygous free-living form conchocelis (strain) (Wang et al., 2012). Then, compare the growth rate, mature period, pigments content, thickness and amount of releasd conchospores between the selected strains and WT strain, the

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strain with better comprehensive character was selected, namely the improved strain.

2.5 Characteristics of the improved strain 2.5.1 Blade growth

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After cultured for 45 days, 20 blades of each strain were randomly selected and cultured with aeration

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in a 1000 mL flask containing MES culture medium, respectively. The culture conditions were 19±1 °C,

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50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D). The blade length, width and wet weight were measured every 5 days in a 30-day period. The specific and absolute growth rates of the

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blades were calculated as previously reported by Stein (1973).

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2.5.2 Statistical mature period of blades

When blades were cultured for 30 days, 20 blades of each strain were randomly selected and cultured

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with aeration in a 1000 mL flask containing MES culture medium, respectively. The culture conditions were 19±1 °C, 50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D). Microscope was used to observe blades and statistical the number of mature individual.

2.5.3 High-temperature resistance of blades

Ten blades of A-13 and WT strain which were cultured for 40 days at 19±1 °C were randomly selected and cultured with aeration in a 1000 mL flask containing MES culture medium, the culture conditions were 24 °C, 50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D). The culture medium was refreshed, and the lengths of the blades were measured every 5 days.

2.5.4 In vivo absorption spectra and contents of chlorophyll a as well as the contents of

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phycobiliproteins

After being cultured for 60 days, ultraviolet spectrophotometer (UV-2600, Shimadzu) was used to

measure the in vivo absorption spectra, contents of chlorophyll a (Chl. a), phycoerythrin (PE) and

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phycocyanin (PC). The methods of measure the in vivo absorption spectra and content of Chl. a were

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the same as Yan and Aruga (1997), the methods of measure contents of PE and PC were the same as

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Gao (1993) with some modifications. The fresh blades were dried at 80 °C for 6 hours, then weigh 10 mg dried blades and ground after at least three freezing and thawing cycles (between -20 °C and room

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temperature). The extract was centrifuged at 15,000 × g for 20 min, and the supernatant was used to determine the OD568 and OD618. The contents of PE and PC were calculated using the following

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formulae:

PE= OD568/81.5 × V/ W × 104

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PC= OD618/64.5 × V/ W × 104

where V (in mL) is the volume of the extract and W (in mg) is the dry weight of the sample.

2.5.5 Blade thickness After being cultured for 60 days, double-edges blades (Feiying, Shanghai geely) were used to slice the

apical, middle and basal part of each strain by hand. Blade thickness of each part was measured with an optical microscope (Eclipse90i, Nikon) and expressed as the mean thickness of 10 sections of that part.

2.5.6 Amount of released conchospores When a large amount of conchosporangias were formed (Fu et al., 2011), one shell of each strain was placed in a flask which contain 50 mL medium for culture with aeration, the culture conditions were

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19±1 °C, 50 μmol photons m−2 s−1 and 10∶14 h light/dark cycle (10L∶14D). The conchospores

released from each strain were collected at 1 pm every day and inoculated them into Petri dishes (Φ =9 cm). After the conchospores were completely attached to the bottom of the Petri dishes, the amount of

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conchospores was counted under light microscope, the number of conchospores was counted for

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twenty days and the number of conchospores released in twenty days was calculated as the total

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amount. The statistical method of the amount of conchospores was the same as Hou (2012). The

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experiments at each strain were set up three parallel groups.

2.6 Statistic analysis of data

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Excel 2010 was used to process the data, the data were given as mean ± standard deviation (mean ± SD); and analyzed in SPSS 18.0 using paired-sample t-test, P < 0.05 and P < 0.01 were considered as

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significantly different and highly significantly different between the improved strain and WT strain. Origin 9.0 was used to drawn the in vivo absorption spectra.

3.

Result

3.1 Verification of heterozygous conchocelis

1086 blades were chosen from F1 gametophytic blades which were produced by the heterozygous conchocelis, 5 kinds of unsectored blades and 57 kinds of color sectored blades were observed. 94.2 % of them were color sectored blades, two-sectored blade and three-sectored blade accounted for a larger proportion, the proportion were 64.2 % and 28.5 %, respectively, indicating that the conchocelis were heterozygous. In addition, wild like color sectors were in the proportion of 27.1 %.

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3.2 Selection of the improved strains

Conchospores of the heterozygous conchocelis were cultured for 65 days, 25 sectors which were fast

growing and wild like color were selected from the F1 color sectored blades. Then 5 sectors which had

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great advantage in growth were selected. Enzymatic hydrolysis technology was used to get the

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homozygous free-living conchocelis (strain) then compared each strain and the WT strain in color,

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selected and named A-13.

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growth speed and maturation period; finally the strain with better comprehensive characters was

3.3 Blades growth of A-13 strain

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The blades of wild type strain (WT) were wide, thick blade and brown in color (wild type).The blades of the improved strain (A-13) were long and thin, slight blade and reddish brown in color (wild like

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type) (Figure 1).

During 45-75 days old, the mean length of the blades of WT strain was significantly lower than

that of A-13 strain. At 45 days old, the mean length of A-13 strain was 19.91±3.98 cm, 1.29 times longer than that of WT strain. At 75 days old, WT strain was 67.53±14.53 cm, while A-13 strain was as high as 140.68±12.66 cm, 2.08 times longer than WT strain, and the difference was extremely

significant (P<0.01) (Table 1). The ratio of length to width of WT strain was much lower than that of A-13 strain during 45-75 days old, There was a highly significant difference in the ratio (P<0.01). The ratio of length to width of WT strain varied slightly, and its maximum value was 12.38 at the 65 days old. At the age of 70 days old, the length-width ratio of A-13 was 40.96, and then the ratio decreased slightly (Table 1). During age of 45-75 days old, the absolute growth rate of the WT strain blades had a small change

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range, and the maximum value of the absolute growth rate was 1.96 cm/d appearing at the 56-60 days. While the absolute growth rate of A-13 showed a trend of first stability and then decline, and the

maximum value of its absolute growth rate was 4.83 cm/d appearing at the 56-60 days old. In addition,

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specific growth rates of the blades of two strains showed a declining trend, but the maximum value of

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specific growth rates of A-13 was much higher than that of WT strain (Table 2).

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Although the growth rate of its thallus was higher than that of WT strain, there was no significant difference in the mean wet weight of its thallus between 45-75 days old because of relatively thin

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blades of A-13 strain. At the age of 75 days, the mean wet weight of WT and A-13 strain was 1.50±0.55

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and 1.93±0.17 g, respectively (Figure 2).

3.4 Mature period of the F1 blades of A-13 strain

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The blades of WT strain began to mature at the age of 41-45 days, and 25% of individuals matured at 45 days, 80% of individuals matured at 50 days, and all the individuals matured at 55 days. The blades of A-13 strain showed a few individuals (5%) matured at the age of 41-45 days, and then a large proportion of blades began to mature, 60% of individuals matured at 50 days, 90% of individuals matured at 55 days, and all the individuals matured at 60 days old. As a whole, there was no significant

difference in the mature stage of the blades of the two strains (Figure 3).

3.5 High-temperature resistance of the F1 baldes of A-13 strain F1 gametophytic blades of A-13 and WT strain grown at 19 °C until 40-day-old and then cultured for another 30 days at 24 °C. The growth of A-13 blades was better than that of WT strain. The mean length of A-13 was increase to 75.63±5.07 cm when cultured for 30 days at 24 °C, while the mean

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length of WT was 35.00±4.25 cm, and the length of A-13 was 2.16 times longer than that of WT strain (Figure 4).

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3.6 In vivo absorption spectra and main pigment contents of the F1 baldes of A-13 strain

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In vivo absorption spectra of the two strains blades all showed 5 significant peaks (P1-P5) at the wavelength of 350-750 nm, in which P1 was mainly caused by the absorption of Chl. a and β-carotene,

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P2 by PE and β-carotene, P3 by PE, P4 by PC, and P5 by Chl. a. All the 5 peaks of A-13 were

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significantly higher than WT strain (Figure 5).

The contents of Chl. a and total phycobiliprotein in A-13 strain were 10.35±0.30 and 79.77±2.14

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mg/g, respectively, in the 60 days old, which were 1.93 and 1.63 times of those of WT strain,

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respectively. In addition, the ratio of PE to PC was 2.1 in both A-13 and WT strain (Table 3).

3.7 Blades thickness of A-13 strain The mean thickness of basal part of the two strains blades were the highest, followed in turn by middle and apital part, there was no significant difference in the thickness of each part of the thallus (Table 4). The average thickness of A-13 strain was only 19.86±0.53 μm, 30 % lower than that of WT strain, and

the difference was highly significant (P<0.01).

3.8 Number of conchospores released from A-13 strain For 20 consecutive days, the shell conchocelis of the two strains were able to continuously release conchospores, and the two strains had the first release peak in the 3rd -5th day after the beginning of releasing, in which the release amount of A-13 strain was 547.59 ×104 per shell, WT strain was only

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95.77×104 per shell, A-13 was 5.72 times higher than that of WT strain. The total amount of

conchospores released from A-13 and WT strain within 20 days was 1408.83×104 and 575.21 ×104 per

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shell, respectively, and A-13 was 2.45 times than that of WT strain (Figure 6).

Discussions

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The length-width ratio of the blades was one of the most important characters of nori. Generally,

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strains with high length-width ratio grow faster (Tong, 2010). The results of this study showed that, in the early and middle stages of culture, the growth advantage of the improved strain (A-13) thallus was

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obvious, which was positively correlated with its length-width ratio. In the later stage of culture, the growth rate of its thallus slowed down, mainly due to the early maturity of its blades, but the mature

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speed of its thallus was slow and only limited to a small part of the apical, which had little influence on the quality of nori products.

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In production, P. yezoensis can be harvested for several times, and after each harvest, about 7 cm

long of blades will be left to continue its growth (Ecological and Taxonomic Laboratory of Algae, Institute of Oceanology, Chinese Academy of Sciences, 1978). In general, the first harvest period of Pyropia is between 45-55 days old, when the price of blade is the highest. Therefore, the yield of blade harvested at the first time directly affects the income of farmers in that year. In this study, the average

wet weight of A-13 and WT strains was almost the same between 45-55 days old, but the mean length of A-13 strains was about 1.4 times higher than that of WT strain; therefore, under the same cultivation density, the per mu yield of A-13 which harvested at the first time will higher than that of WT strain. In addition, the blades of A-13 strain were slender and long shape. Under the same cultivation area, the seeding density of its conchospores could be appropriately increased, and the yield would be further increased.

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Due to the global warming and rise of ocean temperature, the industry of P. yezoensis suffered a

great loss (Ding et al., 2016). According to the research, long term high temperature destroyed the thylakoid membrane structure of chloroplast, affect enzyme activity and change cytoskeleton system

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(Murata et al., 1997; Wahid et al., 2007), so high temperature resistant becomes an important character

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to P. yezoensis. The results of this experiment confirmed that the blades of A-13 strain were affected by

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high temperature (24 °C), but its growth rate was higher than WT strain, indicating that A-13 strain had stronger resistance to high temperature than WT strain.

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The color of the Pyropia blade was mainly determined by the ratio of PE to PC. Generally, the ratio of PE to PC was greater than or equals to 3, and the blades was reddish; the ratio of PE to PC was

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approximately equal to 2, the color tends to be wild color; the ratio of PE to PC was less than or equal to 1, the color was greenish (Wu et al., 2017; Gu, 2017). In this study, the A-13 strain showed the

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wild-like color with slightly reddish and its ratio of PE to PC value was 2.1, which confirmed the above conclusion. In addition, the color of the hybrid parents in this experiment was red and green, respectively, and the occurrence of wild like type in their offspring was presumed that the genes controlling PE and PC were recombined during meiosis. The contents of phycobiliprotein of Pyropia affect the quality of nori products (Wu et al., 2017).

In this study, the contents of total phycobiliprotein of A-13 strain were 79.77±2.14 mg/g, which were significantly higher than WT strain. The thickness of blade also had a great influence on the quality of nori products. Generally, the blades were relatively thin, and the products made of it was even, while the nori products made of thick blades were uneven and easily appear holes, and the products grade tend to have a large decline (Zhang et al., 2011; Zhao, 2012). The alga thickness of A-13 strain was only 19.86±0.53 μm, reaching the level of great products (Zhang et al., 2011). In addition, with the

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increase of harvesting times, the thickness of blades generally increased gradually (Zhang et al., 2011; Chen et al., 2009). Since the thickness of blades of WT strain reached 28.36±0.13 μm, the harvested

blades can only reach the level of medium products from the second harvest (Wu et al., 2017), while

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the grade of nori products of A-13 strain decreased slowly (Chen et al., 2009).

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In addition, the amount of released conchospores of shell conchocelis directly affected the progress of seeding (Wu et al., 2014). In this study, the total amount of released conchospores of A-13

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strain was 1408.83×104 per shell, 2.45 times higher than those of WT strain, indicating that the amount

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of released conchospores of A-13 strain can fully meet the sedding demand. In conclusion, A-13 strain was characterized by fast growth, high quality, large amount of

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conchospores and the color of blades were similar to the wild type, and it is expected to be cultivated

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into a new strain which is suitable to be applied in commercial cultivation.

Funding information The study was supported in part by the Science and Tecnology Planning Project of Jiangsu Province, China (BE2018335), the National Key Research and Development Program of China

(2018YFD0900606), National Natural Science Foundation of China (31072208), Major Science and Technology Specific Program of Zhejiang Province (2016C02055-6), and Open Program of Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province (2017fjscq02).

Statement of Competing Interest

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The authors declare no conflict of interest that could influence the work reported in this paper.

Acknowledgments

The authors are thankful to Dr. Linbin Huang for his great advices and assistance during the project.

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We also appreciate the invaluable help provided by all experts and researchers of our institute.

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Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Oceanol Limnol Sinica 43(2): 363-369 (in

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Chinese, English abstract)

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Wu HH, 2016. Intraspecific hybridization of red type mutant and green type mutant of Pyropia yezoensis and selection of new recombined strains. Dissertation, Shanghai Ocean University (in

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Chinese, English abstract)

Wu HH, Ding HC, Yan XH, 2017. Selection and characterization of a high-temperature resistant strain

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by hybridization recombination in Pyropia yezoensis. J Fish Chin 41(5): 711-722 (in Chinese, English abstract)

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Wu HX, Yan XH, Song WL, Liu YF, Liu CJ, Wang TG, 2014. Selection and characterization of an improved strain produced by genetic recombinant of inter-specific hybridization between Pyropia haitanensis and Pyropia radi. J Fish Chin 38(8): 1079-1088 (in Chinese, English abstract) Yan XH, 1997. Induction, isolation and characterization of pigmentation mutants in Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Dissertation, Tokyo University of Fisheries

Yan XH, Aruga Y, 1997. Induction of pigmentation mutants by treatment of monospore germlings with NNG in Porphyra yezoensis Ueda (Bangiales, Rhodophyta). Algae 12(1): 39-52 Yang YF, Fei XG, 2003. Prospects for bioremediation of cultivation of large-sized seaweed in eutrophic mariculture areas. J Ocean Univ Qingdao (Natrual Edition) 33(1): 53-57 (in Chinese, English abstract)

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Agriculture Press, Beijing, p 101 (in Chinese)

Figure captions Figure 1 Morphology of F1 gametophytic blades of the wild-type strain (WT) and improved strain (A-13) in Pyropia yezoensis. a-c. The F1 gametophytic blades of the wild-type strain (WT) after being cultured for 55, 65 and 75 days, respectively; d-f. The F1 gametophytic blades of the improved strain

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(A-13) after being cultured for 55, 65 and 75 days, respectively. Bar: 2 cm

Figure 2 The wet weight per F1 gametophytic blade of the wild-type strain (WT) and improved strain

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(A-13) in Pyropia yezoensis

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Figure 3 Percentage of the mature blades of the wild-type strain (WT) and improved strain (A-13) in

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Pyropia yezoensis

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Figure 4 The mean length of 40-day-old F1 gametophytic blades (at 18 °C) of the wild-type strain (WT)

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and improved strain (A-13) in Pyropia yezoensis after being cultured at 24 °C for another 30 days

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Figure 5 In vivo absorption spectra of F1 gametophytic blades of the wild-type strain (WT) and

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improved strain (A-13) in Pyropia yezoensis after being cultured for 60 days

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Figure 6 Numbers of the released conchospores of the wild-type strain (WT) and improved strain (A-13) in Pyropia yezoensis during a 20- day period

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Tables

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Table 1 Mean length and length-width ratio of the F1 gametophytic blades of the wild-type strain (WT) and improved strain (A-13) in Pyropia yezoensis. Mean length/ cm

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Culture days/ d

A-13

WT

A-13

15.47±4.33

19.91±3.98

11.15±0.84

25.24±5.52**

*

50

24.57±7.72

36.10±4.92

11.13±0.85

29.81±5.13**

55

33.77±9.23

49.26±6.30**

11.61±1.14

31.08±4.83**

60

43.57±10.47

73.43±5.77**

12.12±1.08

35.41±4.80**

65

52.33±11.18

95.63±7.33**

12.38±1.38

37.28±6.61**

70

60.50±13.36

118.75±9.46**

12.27±1.27

40.96±6.56**

67.53±14.53

140.68±12.66**

11.75±0.57

39.19±4.86**

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WT

Length-width ratio

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** Highly significant difference (P < 0.01, t-test), *significant difference (0.01< P < 0.05, t-test)

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Table 2 Growth rates of F1 gametophytic blades of the wild-type strain (WT) and improved strain (A-13) in Pyropia yezoensis Culture days/ d

Absolute growth rate/ cm·d-1

Specific growth rate/ %·d-1

WT

A-13

WT

A-13

46-50

1.82

3.24

9.00

11.91

51-55

1.84

2.63

6.63

6.22

56-60

1.96

4.83

5.25

7.98

61-65

1.75

4.44

3.80

5.28

1.63

4.63

2.86

4.33

71-75

1.41

4.39

2.23

3.39

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66-70

Table 3 Contents of major photosynthetic pigments in F1 gametophytic blades of the wild-type strain (WT) and improved strain (A-13) in Pyropia yezoensis after being cultured for 60 days

Contents（mg/g，dry weight） Strains Chl. a

Phycoerythrin

Phycocyanin

(PE)

(PC)

5.37±0.07

33.18±2.35

15.74±1.88

A-13

10.35±0.30**

54.44±1.41**

25.34±0.75**

48.91±4.23

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WT

PE+PC

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** Highly significant difference (P < 0.01, t-test).

79.77±2.14**

Table 4 Thickness of different parts of F1 gametophytic blades of the wild-type strain (WT) and

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improved strain (A-13) in Pyropia yezoensis after being cultured for 60 days

Apical

A-13

28.22±0.65

19.15±0.81**

Basal

28.33±0.57

28.53±0.54

28.36±0.13

20.42±0.85**

19.86±0.53**

20.02±0.74**

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** Highly significant difference (P < 0.01, t-test).

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Mean thickness/ μm

Middle

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WT

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Mean thickness of different parts of the blades/ μm Strains