Wenxi Guo, Hongchng Ding, Xinghong Yn,＊

aKey Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China

bShanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China

Keywords:

ABSTRACT

1.Introduction

Pyropia

(Sutherland et al., 2011) is a nutrient-rich and high-value seaweed, containing abundant proteins, fats, sugars, inorganic salts,and vitamins necessary to the human body. It also plays an important role in the marine environment and regulating ecological balance (Yang& Fei, 2003). Domestically cultivated

Pyropia

, mainly include

Pyropia yezoensis

and

Pyropia haitanensis

.

P

.

yezoensis

is cold-temperature red algae and is mainly cultivated in the coastal areas of Jiangsu and Shandong, whereas

P. haitanensis

is warm-temperature red algae and is mainly distributed in the coastal areas of Zhejiang, Fujian, and Guangdong; and its production has reached approximately 75% of the total national laver production (Zhu et al., 2016). However, in recent years,seawater temperature has risen due to climate change, leading to serious losses of cultivated

P. haitanensis

(Li, 1992). Monotilla and Notoya(2004) found that the temperature tolerance of

P. suborbiculata,

produced in southern China, is 15–30C and Zhao (2018) confirmed that

P. suborbiculata

is more resistant to higher temperatures than

P. haitanensis

. This finding indicates that

P. suborbiculata

has the potential to be developed into a new cultivar that improves the quality and yield of lavers in China.The life history of

P. suborbiculata

is divided into the large frond and conchocelis stages. Mature blades produce spermatia and carpogonia.The fertilized carpogonia release zygotospores, which germinate into the conchocelis phase (Nelson, Brodie, & Guiry, 1999). In addition, the blades can also produce archeospores through asexual reproduction,which then develop into blades (Aye-mon-sein, Ni-ni-win, San-tha-htun,Soe-htun, & Ohno, 2003). It is important to study the development and differentiation of protoplasts from blades in order to understand blade growth and single-cell differentiation in seaweed. The single cells, or protoplasts, of the blade were isolated using bio-enzymes and cultured under

in vitro

conditions. Next, their morphology, structure, developmental pattern, and final developmental trends were examined. This process has been an effective method for studying the pathways of individual cell development and differentiation of blades (Dai & Bao,1988; Polne, & Gibor, 1984; Tang, 1982; Wang, Zhang, Xun, & Sun,1986). Furthermore, the regenerated plants derived from single cells had a high homozygosity and stable inheritance. Therefore, investigating the development and differentiation of blade cells can also provide an important reference for breeding

P. suborbiculata

(Wang, Kong, Mao, &Yang, 2010).At present, most studies have mainly focused on

P. yezoensis

and

P. haitanensis

. Yan and Wang (1989) have systematically studied the cell development and differentiation of the leafy thalli of

P. haitanensis

and explained that the differentiation process from conchospores to sexual cells can be divided into seven different stages. Lu (1979) reported three developmental pathways of vegetative cells of

P. yezoensis

. Yan, Liu, and Zhang (2004) observed eight regeneration pathways of the cells of the leaf of

P. yezoensis

using an enzymatic hydrolysis method. Yan and Liu(2007) reported on the regeneration and development pathways of individual cells from the male and female fronds of

P. haitanensis.

However, studies on the differentiation and development of the leafy cells of

P. suborbiculata

have not been specifically reported. In the present study, the wild-type strain of

P. suborbiculata

was used to study the development and differentiation of blade cells of

P. suborbiculata

. In addition, our findings provide an experimental basis for the genetic breeding of seaweed at the cellular level.

2.Materials and methods

2.1.Culture of P. suborbiculata blade

We used the wild-type strain (

PS-WT

) of

P. suborbiculata

, where freeliving conchocelis of

PS-WT

were germinated from zygotospores released from a gametophytic blade collected from Yangjiang City,Guangdong Province (N 212845"-224102", E 1111635"-1122151") and then stored in the laboratory (Yan, Li, Cheng, & Aruga,2007). The collection of conchospores and cultivation of

P. suborbiculata

blade was conducted according to previously described methods (Yan &Aruga, 2000). Sterilized seawater, supplemented with MES medium,used as the culture medium in this experiment (Yan et al., 2005).

2.2.Isolation and regeneration of single cells from blades

The conchospores released from

PS-WT

were cultured and developed into gametophytic blades. Healthy blades were selected for obtaining single cells with the conch enzyme. The thalli were cleaned with fresh seawater three times until no microorganisms or other contaminants were observed on each thallus under the microscope. The surface salts of algae were cleaned with 1 mol Lglucose solution, before algae were cut with a knife and placed into a pre-prepared enzyme solution for enzymatic hydrolysis. The enzyme liquid was composed of 2 mol Lglucose solution and 0.5–1.0% enzyme. The temperature during enzymatic hydrolysis was maintained at 28C for 0.5–1 h. The mixed solution was filtered by a 200-mesh screen and this filtrate was centrifuged at 1,200 r⋅minfor 5 min. The supernatant was removed and the precipitation was retained. High specific gravity seawater (specific gravity=1.040) was added and the sample was centrifuged at 1,200 r⋅minfor 5 min. The supernatant was discarded after three successive centrifugations. Cell precipitate was collected and suspended in MES medium(specific gravity =1.030). Cells were cultured in a 9-cm Petri dish under low light. After 3 days, MES medium with lower proportion was added(specific gravity =1.026) to the Petri dish. The culture conditions of isolated cells were set at 18C and 10 μmol photons⋅m⋅s(10 L: 14 D). After 3 days of cultivation, the light density was increased to 40 μmol photons⋅m⋅s. The culture medium was refreshed once per week.

2.3.Cultivation of cell-derived regenerative plants from blades of different ages

Conchospores attached to nylon were cultured with aeration at 18C and 40 μmol photons⋅m⋅s(10L: 14D). Thalli of different ages were cultured for 20, 30, 40, 50, 60, 70, and 80 days, and then those blades were used to obtain single cells and cultured into a plant using an enzymolysis technique. Cell culture methods and conditions were the same as those described above.

2.4.Cultivation of cell-derived regenerative plants from different parts of the blade

The mother blades used were 60 days of age. The blade of

PS-WT

strain was fan-shaped and algae samples were taken from the outermost layer to the inside in turn, or the apical, middle, and basal blades,respectively. Isolated cells were obtained via enzymatic hydrolysis. Cell isolation and cell culture conditions were the same as those described above.

2.5.Amount of released archeospores

A total of 9 blades of

PS-WT

(30 days old) were collected, with three blades incubated in a plastic cup containing 50 mL of culture medium.The culture medium was then poured into the Petri dish (9 cm in diameter) every 24 h to ensure the presence of released archeospores.The method of releasing archeospores was the same as for conchospores(Ding, Lv, Wu, & Yan, 2018).

3.Results

3.1.Types of in vitro development and differentiation of PS-WT blade cells

The

in vitro

development and differentiation of single

P. suborbiculata

blade cells could be divided into nine types using: the morphology of isolated cells, their rhizoids, cell color, cell size, cell arrangement, and their final developmental results.The first type of cells developed into normal blades (Fig.1a). Under

in vitro

conditions, the cells first divided into two polar cells. One of these cells was small, with a slightly larger vacuole, with one end gradually elongating to form a rhizoid. The other cell underwent a transverse division and formed the single row of a cell blade before developing into a leaf composed of a series of cells after successive longitudinal divisions. The shape of the blades was mostly lanceolate,with smooth edges and closely arranged cells. The size of these cells was uniform and their rhizoids showed a transparent petiole shape and were thick, with strong adhesion. Their development and differentiation were similar to those of conchospores.The second type of cells developed into abnormal blades (Fig.1b).These cells grew into abnormal blades with rhizoids under

in vitro

conditions and their occurrence and developmental pattern were similar to those of conchospores. The shape, cell size, and cell arrangement of the regenerated fronds were similar to those of the normal blades, but their rhizoids were more slender and several times longer than that of the normal rhizoids. Most of them were single roots with weaker adhesion.

The third type of cells also developed into abnormal blades (Fig.1c).The leaves were different from normal leaves, with many different shapes. Most of these shapes were irregular, with cells closely and inconsistently arranged. These leaves had smooth edges and no protrusions with single or multiple rhizoids.

The fourth type of cells also developed into abnormal blades (Fig.1d)without rhizoids under

in vitro

conditions. The base of the blade was smooth and did not protrude. The color of the leaves, cell size, and cell arrangement were nearly the same as those in a normal frond, while the shape of the leaves was irregular and the blades released archeospores easily.The fifth type of cells developed into cell-masses (Fig.1e). Under

in vitro

conditions, the cells formed cell-masses after multiple divisions.The cell size, color, and size of pigment body were similar to those of normal or abnormal fronds. Their cells were arranged in disorderly manner and stacked, they had various shapes, and the edge of their blades was smooth. In the later stages of cultivation, short and transparent rhizoids were sometimes produced, and archeospores were released.

Fig.1.Types of in vitro development and differentiation of the single somatic cells isolated from wild-type strain blades of Pyropia suborbiculata. a A normal blade, b An abnormal blade with rhizoid, c An abnormal blade, d An abnormal blade without rhizoid, e A cell-masses of type I, f A cell-masses of type II, g A cell-masses releasing archeospores, h A cell-masses of spermatangia, i A cell-masses of zygotosporangia, j A blade regenerated from a rhizoid cell, k Conchocelis. Scale bar:50 μm.

The sixth type of cells also developed into cell-masses (Fig.1f). The volume of these cell-masses was slightly larger than that of the normal cells. The vacuole of these cells was large and the pigment body was starshaped, while the cells were irregularly arranged. After 3-4 weeks of cultivation, these cell-masses disintegrated.

The seventh type of cells developed into spermatangia (Fig.1h),having differentiated from spermatocytes. Cultivation was carried out for 3–7 days to form spermatangia and release the spermatias.

The eighth type of cells were developmental zygotosporangias(Fig.1i). The cell mass was round, the cell volume was small, and obvious cell separation boundaries were observed. The cell color was dark red, the cell arrangement was irregular, and the spores were easily released and eventually became conchocelis (Fig.1k).

The ninth type of cells developed into a blade that had regenerated from a rhizoid cell (Fig.1j). They were located at the base of the algae.When the cells were separated, they were mostly pear-shaped and had a filament at the thinner end. During cultivation, the speed of cell division was slow and the first polar division occurred after 1 week. It produced two cells, one of which developed into leaves via continuous division,while the other created a rhizoid via split elongation. The rhizoid,containing one shaft wire, was thick and strong. Such cells generally grew to tens of cells before stopping and then gradually became aging cells that were unable to grow into large fronds.

3.2.Relationship between the development of blade cells and the age of algae

Table 1 shows the development of isolated cells from different ageold fronds of

PS-WT

strain. The 20-day-old algae showed only two types of both normal blades and abnormal blades with rhizoids in their regenerated plants. As the age of algae increased, the proportion of normal fronds decreased. Meanwhile, the proportion of abnormal blades with rhizoids increased. When the age of algae was 30–60 days, the proportion of abnormal blades without rhizoid further increased.However, after 60 days of cultivation, the proportion of abnormal blades without rhizoid decreased. When the age of algae was between 30 and 50 days, cell-masses and spermatangias began to appear, respectively,and their proportions increased with algal age. Only a few zygotosporangias appeared when the algae were 80 days old.

3.3.Relationship between the differentiation of blade cells and locations of the cells

Table 2 shows the cells from different parts of the same

PS-WT

frond that were cultured

in vitro

. We found that as cells were taken further from the frond base, the proportion of normal blades and root-filament blades in regenerated plants sharply decreased, while the proportion of abnormal blades and cell-masses gradually increased. In addition, the spermatangia only appeared from the apical part of the algae. Within the leaf, the cells from different parts were in different differentiation states,and the further cells were from the leaf base, the closer the differentiation stage of the cells was to the mother cells.

3.4.Numbers of released archeospores

Fig.2 shows the archeospores released by the wild-type strain during the 20-day cultivation. The many archeospores were released from the

PS-WT

strain. The release time was very early, beginning from the first day and peaking on the 12th day at 10,573 spores.

4.Discussion

4.1.Differentiation pathways of isolated cells in the blades of P.suborbiculata

We considered that the different developmental types could be attributed to the different stages of differentiation of cells. The number of developmental types appearing from the cells of regenerative plants varied from plants of different ages, the different order presented by each developmental type, and the relative changes in the proportion of each developmental type. The development and differentiation of blade cells in

P. suborbiculata,

from the conchospores to the sexual mother cells, can be roughly divided into seven stages.The conchospore had the ability to develop into an intact frond during the primitive stage of developmental differentiation of cell. The germination of the conchospores, in addition to the growth and development of the fronds, increased the number of cells while the differentiation of cells was ongoing. The cells produced by the first few cell divisions of the conchospores were similar to the embryonic cells of the animals. Cell differentiation had not yet started during this first stage and was identified as the first type of cells. After these cells separated,they were able to develop into normal blades like conchospores under

in vitro

conditions. As the fronds grew and developed, most of the cells entered the second stage of differentiation. This second type of cells developed into abnormal blades with rhizoid

in vitro

. The rhizoid was underdeveloped and its adhesion deteriorated. When the algal cells entered the third stage of differentiation, the third type of cells developed into abnormal blades with rhizoid

in vitro

, were characterized by single or multiple rhizoids or like-rhizoids, irregularly-shaped leaves,and regularly-arranged cells. As cells of algae continued to the fourth stage of differentiation, they developed into abnormal blades without rhizoids under

in vitro

conditions and archeospores were released. When the algal cells entered the fifth stage of differentiation, the fifth type of cells could only develop into cell-masses under

in vitro

conditions and the cells were not properly arranged, but the size and color of the cells were similar to those of normal blades. This fifth type of cells might develop into rhizoids in the later stages. When the cells of algae entered the sixth stage of differentiation, the sixth type of cells also developed into cell-masses under the

in vitro

conditions. These cells wereabnormally arranged, the volume of cells became larger, and the cell color became lighter. Such cells also released archeospores. When the cells of algae entered the seventh stage of differentiation, the cells underwent a qualitative change and differentiated into sexual mother cells which became lighter in color. Under the

in vitro

conditions, the cells of the seventh differentiation stage developed into zygotosporangia if they were fertilized female cells. These cells did not divide if they were unfertilized and the volume of cells was larger, the cell color turned red,and the pigment body took a block-like form before disintegrating after a period of cultivation. If it was a male mother cell (the eighth type of cells), it first divided to form a cell-mass and then lightened in cell color.Spermatangia was also formed and released. Rhizoid cells (the ninth cells) were another type of cells that were isolated from the leaves of the algae. They were derived from the original cells produced by the first polar division of the conchospores. Under the

in vitro

conditions,although they could also grow into normal fronds, the growth of the leaves was reduced. After they grew into more than a dozen cells, they stopped developing, and could not grow into large fronds did not release archeospores. The rhizoid was formed, however, indicating that they were highly differentiated and different from other somatic cells.

Table 1 Development types and the percentages of single somatic cells isolated from wild-type strain blades of Pyropia suborbiculata at different ages.

Table 2 Development types and the percentages of the single somatic cells isolated from wild-type strain blades from different parts of Pyropia suborbiculata.

Fig.2.Quantities of the released archeospores of blade of the wild-type strain(PS-WT) of Pyropia suborbiculata.

4.2.Comparison of differentiation pathways of isolated cells from P.yezoensis, P. haitanensis, and P. suborbiculata

The development and differentiation pathways of isolated cells from

P. yezoensis

and

P. haitanensis

have been reported previously (Yan & Liu,2007; Yan et al., 2004). The developmental types of the blade that were regenerated from these three types of seaweed included normal blades,abnormal blades, and cell-masses. Comparing the tendencies of cell development and differentiation, we found that their blade differentiation pathways were generally similar. After comparing the changes in the proportion of their various types of cells from regenerative plants,we found that the situation was similar in

P. yezoensis

and

P. haitanensis

,which rapidly differentiated from normal blades to cell-masses, and formed sexual cells. When the age of frond was 20 days, the proportion of normal and abnormal blades with a rhizoid in the isolated cells of regenerative plant was lower in

P. yezoensis

and

P. haitanensis

. However,in this experiment, we found that when the age of algae was 20–80 days in

P. suborbiculata

(

PS-WT

), the proportion of normal and abnormal blades with a rhizoid in the cell-free regenerative plants was higher. We suspected that this could be attributed to the release of archeospores and that many of the vegetative cells in the algae were in the process of archeospore formation.

4.3.Application prospects of enzymatic seedlings in production

Dai, Bao, Tang, and Liu (1988) and Wang, Sun, Lu, and Wang (1987)have separately attached the single cells of

P. yezoensis

and

P. haitanensis

to nets for cell culture, and while successful results have been achieved,the enzymatic seedlings are still not widely used in practice due to the low adhesion rate. This is probably due to the fact that the proportion of normal blades formed by single cells is too low after the enzymatic hydrolysis of the blades in

P. yezoensis

and

P. haitanensis

(Yan & Liu,2007; Yan et al., 2004). In this experiment, the proportion of normal blades from the isolated cells in

PS-WT

strain was as high as 63.91% in mother blade after 30 days of cultivation, which solved adherence problems and therefore demonstrated promise for seedling applications.In addition, the normal blades obtained by enzymatic separation were homozygous and the filaments were also homozygous, which would convenient for subsequent studies of genetics and breeding of

P. suborbiculata

.

CRediT authorship contribution statement

Wenxia Guo:

Methodology, Formal analysis, Data curation, Writing- original draft.

Hongchang Ding:

Methodology, Supervision, Writing -review & editing.

Xinghong Yan:

Conceptualization, Supervision,Writing - review & editing.

Declaration of competing interest

The authors declare that there is no conflicts of interest.

Acknowledgments

The study was supported in part by 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), Science and Technology Planning Project of Jiangsu Province, China(BE2018335), and Open Program of Key Laboratory of Cultivation and High-value Utilization of Marine Organisms in Fujian Province(2017fjscq02).