Nghiên cứu sinh học, sinh thái và nhân giống rong bắp sú (kappaphycus striatus) (f schmitz) doty ex p c silva, 1996 TT TIENG ANH

Propagation by the method of embryogenesis through callus undergoes the following stages: preparation of axenic material, callus induction and callus proliferation, somatic embryogenesis

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GRADUATE UNIVERSITY SCIENCE

AND TECHNOLOGY

VU THI MO

STUDIED ON BIOLOGY, ECOLOGY AND

PROPAGATION OF Kappaphycus striatus

(F Schmitz) Doty ex P.C Silva, 1996

SUMMARY OF BIOGICAL DOCTORAL THESIS

KHANH HOA, 2022

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Technology – Vietnam Academy of Science and Technology

Science instructor 1: Prof Dr DUONG TAN NHUT

Science instructor 2: Prof Dr NGUYEN NGOC LAM

The thesis can be found at:

- Library of Graduate university Science and Technology

- Library of Vietnam National

INTRODUCTION

1 The urgency of the thesis

Kappaphycus striatus belongs to the phylum Rhodophyta and is

distributed mainly in tropical waters, in coastal open seas and bays, where there is water exchange, stable high salinity, clear water, and high

light intensity K striatus is not only rich in crude fiber, iron, omega-3

fatty acids, and antioxidants, but also contains biological compounds for the pharmaceutical and biological industries Therefore, the seaweed has economic value and is used as a raw material for K-carrageenan extraction, food, and as agricultural fertilizer

K striatus has been cultivated commercially in Vietnam since 2005,

a transfer of seaweed originally from the Philippines In the early years

of culture, K striatus has a high growth rate and can be grown all year

round in waters with stable high salinity Therefore, seaweed farming contributes to poverty alleviation and has been successful in ensuring livelihoods for many coastal communities However, after nearly

twenty years of culture in the field, the K striatus has not kept the same

biological properties and carrageenan quality as the original

In Vietnam, since the successful migration to the present, propagation by vegetative reproduction is the only method used in the

cultivation of K striatus K striatus has decreased vitality and is very

susceptible to disease outbreaks, leading to a significant reduction in

yield and quality Therefore, K striatus is degraded after a long time

propagated by vegetative reproduction

In vitro culture is a method of propagation that is less dependent on

weather, provides a large number of plants and free, resistant plants that are high-yielding and tolerate high disease levels

disease-more strongly Plants from in vitro culture grow well in nutrient-poor conditions Besides, plants from in vitro culture have a growth rate 1.5

– 1.8 times faster than plants from nature In particular, seaweed has the ability to grow well in high temperature conditions and is resistant to ice-ice disease In addition, the higher carrageenan content, quality, and

nutritional value of plants from in vitro culture are better than those

from nature

However, at present, the studies on the biological as well as

propagation of K striatus, especially in vitro propagation, are still

limited Therefore, in order to study the changes in biological

characteristics of some strains of K striatus after nearly twenty years of culture By selecting a superior strain of K striatus that is highly

adapted to the ecological conditions of Vietnam's waters and carrying

out the propagation based on the advantages of in vitro culture

technology, the PhD student carried out the thesis: "Studied on

biology, ecology, and propagation of Kappaphycus striatus (F

Schmitz) Doty ex P.C Silva, 1996"

2 The research aims of the thesis

Finding strains of K striatus with suitable biological

characteristics as research materials to create high-quality seed

sources by the in vitro culture method

Research to find out suitable in vitro culture conditions for different

morphogenesis processes (callus induction, embryogenesis, and

micropropagule regeneration) of K striatus Evaluation of the quality

of in vitro K striatus plants through field cultivation of

micropropagule regenerated from somatic embryos, content and quality of carrageenan

3 The main research content of the thesis

Biological and ecological characteristics of strains of K striatus Propagation of K striatus by an in vitro culture method

4 New contributions to the thesis

It provides detailed data on the biological and ecological

characteristics of two strains of K striatus after growing in Van Phong

bay and Cam Ranh bay, Khanh Hoa, for nearly twenty years

Using silver nanoparticles (AgNPs) as a new chemical sterilization

for in vitro culture of seaweed

Providing data on the embryogenesis process and building protocol

of K striatus propagation using an in vitro culture method The thesis contributes to training and teaching in the field of in vitro culture

plants in general and seaweeds in particular

CHAPTER 1 OVERVIEW

K striatus is a species of seaweed with economic value, belonging

to the phylum Rhodophyta, widely cultivated in countries such as the Philippines and India In 2005, Vietnam successfully planted three

strains of K striatus: Sacol green, Sacol brown, and Payaka brown

There are many factors affecting the growth and quality of seaweed, such as ecological factors (temperature, light, salinity, etc.) and seed origin In the past few years, a number of studies on the biological and

ecological characteristics of K striatus have been carried out

However, there is still no study to compare the growth as well as the

content and quality of carrageenan of strains of K striatus after nearly

twenty years of being planted in Vietnam

On the other hand, in the world, in vitro propagation has been

carried out on the subjects of K alvarezzi and Eucheuma denticulatum

with direct regeneration and indirect regeneration through somatic

embryogenesis with positive results In vitro culture of seaweed is a

fairly simple but effective method of preserving precious genetic resources for mass production according to traditional methods The process of rapid recovery and multiplication in young seaweed has the effect of "rejuvenating" the seed source, enhancing its resistance to the environment In addition, the combination with genetic techniques

such as gene transfer and cell hybridization in vitro seaweed culture

rapidly increase the quality of the desired seed

In Vietnam, some authors have studied the in vitro propagation of

K alvarezii, but the results are limited and the propagation process has

not been described Therefore, up to now, it has not been applied in

practical production On the K striatus, direct shoot regeneration was

performed However, this method has the disadvantage that the multiplication coefficient is low, which leads to the inability to meet

the demand for seed sources Therefore, research on in vitro

propagation by somatic embryogenesis through callus is necessary The result is a generation of plants with good characteristics, able to tolerate high temperatures and disease resistance, thereby improving the productivity as well as the quality of seaweed Propagation by the method of embryogenesis through callus undergoes the following stages: preparation of axenic material, callus induction and callus proliferation, somatic embryogenesis, micropropagule culture, and field cultivation of regenerated plants from somatic embryos

Therefore, studying in vitro culture conditions suitable for different

morphogenesis processes (callus induction, somatic embryogenesis,

micropropagule culture, etc.) of K striatus to find out the in vitro

propagation process is essential

CHAPTER 2 MATERIALS, CONTENTS AND

RESEARCH METHODS

2.1 Materials

The branches of the Payaka brown strain and Sacol brown strain

of K striatus were 2 – 4 weeks old, healthy, disease-free, light brown

in color, and unscratched (400 g, 30 – 40 cm) They were collected at Van Phong bay and Cam Ranh bay, Khanh Hoa province

Strains of K striatus were collected and classificated Then, the

growth, content, and quality of carrageenan of the strains belonging to

K striatus were tested to find out the strains with the best biological

characteristics to use as in vitro propagation research materials

2.3.2 Propagation of K striatus

Preparation of axenic material

The healthy branches of the good strain that belong to the above results were selected The branches were used to acclimatize to laboratory conditions by growing in different medium compositions for the growth Explants were then treated with AgNPs and broad-spectrum antibiotics to find the best sterilization conditions

Callus induction and callus proliferation

Axenic explants were used for callus induction The investigated culture factors were: Media (MS, ½ MS, MS ½, PES, ½ PES, PES ½, MPI, ½ MPI, MPI ½); adding each or combination of NAA (0,1; 1,0 mg.L-1) and BAP (0,1; 1,0 mg.L-1); agar concentration (5 – 20 mg.L-1) under white fluorescent light at (0 – 55 µmol photons.m-2.s-1) After 8

weeks of explant culture, good callus was used to study callus

proliferation

Somatic embryogenesis induction

The 16-week-old callus clumps (2 x 2 mm & 10 mg) were inoculated into the PES medium at different solidities Then, each or combination in NAA (1; 2, 3 mg.L-1) and BAP (1; 2, 3 mg.L-1) were added into PES medium to find out the best conditions for somatic embryogenesis

Micropropagules regeneration and field cultivation of regenerated plants

Sigle somatics (0.5 – 0.6 mm) were used to regenerate micropropagules The culture factors: Media (MS, ½ MS, MS ½, PES,

½ PES, PES ½, MPI, ½ MPI, MPI ½); disturbance of water (aeration,

50 rpm and 100 rpm); light intensity (0 – 70 µmol photons.m-2.s-1); salinity (20 – 40‰) and incubation temperature (20 – 35°C) were investigated to find the best micropropagules regeneration conditions

Ex vitro culture was performed on micropropagules that were 8 weeks

old The light source and the in vitro micropropagules type were tested The 16-week-old in vitro plants (50 – 70 g) were grown in Van

Phong bay to assess their adaptability in the field

After 10 weeks of growing in the field, seaweed was harvested to

evaluate carrageenan content and quality

2.3.4 Statistical analysis

The experiment was set up in a completely randomized design, with each treatment repeated three times, and the data expressed as mean standard deviation (Mean SD) Data were processed in Microsoft Excel 2010 and compared in SPSS 16.0 using 1-factor ANOVA with Duncan's test (p< 0.05)

CHAPTER 3 RESULTS AND DISCUSSION

3.1 Studied the biological and ecological characteristics of K striatus

3.1.1 The current status of strains of K striatus being cultivated in Van Phong bay and Cam Ranh bay

The results of the investigation showed that there are 2 strains of

K striatus, the Sacol brown (Fig 3.1 A) and the Payaka brown (Fig

3.1 B) that are being cultivated in Van Phong bay and Cam Ranh bay, Khanh Hoa province

Image 3.1 Morphology in two strains of K striatus collected from Van

Phong bay Scale bar 2 cm

C: Change in color on the same plant of the Payaka brown strain

3.1.2 Growth, carrageenan content, and quality of Sacol brown strain and Payaka brown strain grown in Van Phong and Cam Ranh bays

After 12 weeks of cutivation in the field, the results showed that

the fresh and dry weight of the Payaka brown strain (355.33; 42.33 g; respectively) and the Sacol brown strain (343.33; 42.67 g;

respectively) reached maximum biomass after 8 weeks of culture in Van Phong bay (Fig 3.1 and Fig 3.2)

Fig 3.1 Fresh weight of Payaka brown and

Sacol brown strain in Van Phong

bay and Cam Ranh bay

Fig 3.2 Dry weight of Payaka brown

and Sacol brown strain in Van

Phong bay and Cam Ranh

Besides, the cumulative growth rate (GR) of the two strains reared was equal (1.30 %.day-1) in Van Phong bay and higher than the cumulative GR

of the Payaka brown strain (1.26%) and Sacol brown strain (1.05 %.day-1)

after 12 weeks of culture in Cam Ranh bay (Fig 3.3)

Fig 3.3 GR of Payaka brown strain and

Sacol brown strain in Van Phong

bay and Cam Ranh bay

Fig 3.4 Fresh dried rate of Payaka

brown strain and Sacol brown

strain in Van Phong bay and

Cam Ranh bay

In addition, the fresh dried rates of Payaka brown strain (12,45%) and Sacol brown strain (12,09%) were highest when cultivating in Van

Phong Bay after 8 weeks of culture (Fig 3.4)

The fresh weight and the fresh dried rate were used to determine

the time of harvest in two strains The two strains of K striatus were

Payaka brown strain (Van Phong) Sacol brown strain (Van Phong) Payaka brown strain (Cam Ranh) Sacol brown strain (Cam Ranh)

0 10 20 30 40 50

Payaka brown strain (Van Phong) Sacol brown strain (Van Phong) Payaka brown strain (Cam Ranh) Sacol brown strain (Cam Ranh)

0 4 8 12 16

Payaka brown strain (Van Phong) Sacol brown strain (Van Phong) Payaka brown strain (Cam Ranh) Sacol brown strain (Cam Ranh)

Payaka brown strain (Van Phong) Sacol brown strain (Van Phong) Payaka brown strain (Cam Ranh) Sacol brown strain (Cam Ranh)

harvested when cultivating in Van Phong Bay after 8 weeks of culture and Cam Ranh bay after 10 weeks of culture to determine the content and quality of carrageenan The results showed that the carrageenan

content and viscosity of the Sacol brown strain grown in Van Phong

bay were the highest (Table 3.1)

Table 3.1 The carrageenan content and quality of the Payaka brown and

Sacol brown strains were cultivated in Van Phong bay and Cam

Phong

Payaka brown 23,00 b ± 1,00 726,33 a ± 3,51 26,00 b ± 1,00

Sacol brown 24,67 a ± 0,58 741,67 a ± 7,64 29,00 a ± 1,00 Cam

Ranh

Payaka brown 22,33 b ± 0,58 723,33 a ± 5,77 27,10 b ± 1,73

Sacol brown 21,67 b ± 0,76 731,67 a ± 7,64 27,33 ab ± 0,58

In this study, the content and quality of carrageenan in two strains

in Van Phong bay and Cam Ranh bay were lower than in the previous studies (Table 3.2) Thus, after nearly two decades of cultivation in Van Phong Bay and Cam Ranh Bay, the growth rate, concentration, and quality of carrageenan in two strains were lower than before

Table 3.2 Comparison of the growth rate, content and quality of carrageenan

in Payaka brown strain and Sacol brown strain with previous studies

Time

Growth rate (%.day -1 )

Carrageenan content (%/w)

Carrageenan quality

References Strength

(g.cm -2 )

Viscosity (cps)

2005 Van Phong Payaka brown 2,96 – 5,04 25,84 – 28,7 873 –1117 103,32 – 105 (Tran Kha et

al, 2007)

Cam Ranh Sacol brown 5,7 – 5,8 26,2 778 96

2014 Cam Ranh Sacol brown 2,5 – 5,8 25,1 – 28,4 555 – 935 23,8 – 34,6 ( Hung et al,

2019)

2017 Van Phong Payaka brown 1,26 – 1,30 22,33 – 23,00 723,33 –726,33 26 – 27,10 This study

Cam Ranh Sacol brown 1,05 – 1,30 21,67 – 24,67 731,67 – 741,67 27,33 – 29,00

Under the same conditions, the Sacol brown strain had a higher carrageenan content than the Payaka brown strain under the same conditions grown in Van Phong bay Therefore, the Sacol brown strain collected in Van Phong bay was used to conduct in vitro propagation

studies

3.2 Propagation of the Sacol brown strain of K striatus

3.2.1 Preparation of axenic material

After 7 weeks of culture, the branches of the Sacol brown strain were cultured under in vitro conditions The results showed that, the

branches grew best in MPI medium with a brown color, a glossy surface, and no epiphyte on the surface with a weight of 1.85 g The explants were sterilized with a working AgNPs solution (0.02 – 0.1% for 5 – 20 min) and antibiotics (1 – 4% for 24 hours) Treatment

of explants with 0.05% AgNPs for 5 min resulted in 100% of cultured

explants being free of bacteria and a survival rate (80%) after 1 week

of explant culture (Fig 3.4.)

Image 3.4 The morphology of the Sacol brown strain was sterilized with

silver nanoparticles, antibiotics, and different treatment times

after 1 week of culture Scale bar: 2mm

A, B, C, D: Explantswere sterilized with AgNPs for 5; 10; 15; 20 min From left to right: 0,02; 0,03; 0,04; 0,05; 0,1% silver nanoparticles

E: Explantswere sterilized with 1; 2; 3; 4% antibiotics for 24 h

F: Explantswere sterilized with 1% Natri hypochlorite for 15 min

Thus, the demonstration of using AgNPs as a new agent to replace other traditional disinfectants is one of the highlights of the thesis Therefore, the explants treated with 0.05% AgNPs for 5 min were used

in all subsequent experiments

3.2.2 Callus induction and callus proliferation

Callus induced best in PES medium supplemented with 15 g.L-1agar under light intensity of 5 µmol photons.m-2.s-1 after 8 weeks of culture (Tables 3.4, 3.6 and Fig 3.6, 3.8)

Table 3.4 Effects of different media compositions on callus induction of

the Sacol brown strain after 8 weeks of culture, n = 30

rate (%)

Survival rate (%)

Size (mm)

Remark

Control 41,1 c ± 2,8* Necrotic Necrotic Explants were necrotic after 2 months

of culture

MS Necrotic Necrotic Necrotic 80% of explants were necrotic and

bleaching after 2 – 3 days of culture

½ MS Necrotic Necrotic Necrotic

70% of explants were necrotic after 2 – 3 days of culture and bleaching after

7 days of culture

MS ½ Necrotic Necrotic Necrotic Explants were necrotic

½ PES 70,0 b ± 1,9 73,1 b ± 1,8 1,40 b ± 0,10 Small callus was obsever

PES ½ 72,2 b ± 3,4 75,4 b ± 1,3 1,53 b ± 0,35 Callus grew slowly

MPI 70,0 b ± 3,8 55,7 c ± 3,1 1,30 b ± 0,10 Small callus was obsever

½ MPI 68,9 b ± 5,3 30,7 d ± 0,5 1,00 c ± 0,10 Small and necrotic callus was obsever MPI ½ 67,8 b ± 8,6 28,1 d ± 3,3 0,47 d ± 0,15 “Compact” callus was obsever

Image 3.6 Callus morphology of Sacol brown seaweed in PES medium

supplemented with NAA and BAP alone or in combination after

8 weeks of culture Scale bar: 2mm