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

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY 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 Major: Code: HYDROBIOLOGY 42 01 08 SUMMARY OF BIOGICAL DOCTORAL THESIS KHANH HOA, 2022 i The thesis was completed at: Graduate university Science and Technology – Vietnam Academy of Science and Technology Science instructor 1: Prof Dr DUONG TAN NHUT Science instructor 2: Prof Dr NGUYEN NGOC LAM Reviewer 1: … Reviewer 2: … Reviewer 3: … The thesis will be defended before the Academy level doctoral thesis evaluation committee, meeting at the Graduate university Science and Technology - Vietnam Academy of Science and Technology in … h ’, date … month … year 2022 The thesis can be found at: - Library of Graduate university Science and Technology - Library of Vietnam National ii INTRODUCTION 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 disease-free, diseaseresistant plants that are high-yielding and tolerate high disease levels 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" 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 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 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 MATERIALS, CONTENTS AND RESEARCH METHODS 2.1 Materials The branches of the Payaka brown strain and Sacol brown strain of K striatus were – 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 2.2 Contents research Biological and ecological characteristics of strains belonging to K striatus Propagation of K striatus by an in vitro culture method 2.3 Methods research 2.3.1 The current status of strains of K striatus being cultivated in Van Phong bay and Cam Ranh bay 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 broadspectrum 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 weeks of explant culture, good callus was used to study callus proliferation Somatic embryogenesis induction The 16-week-old callus clumps (2 x mm & 10 mg) were inoculated into the PES medium at different solidities Then, each or combination in NAA (1; 2, mg.L-1) and BAP (1; 2, mg.L-1) were added into PES medium to find out the best conditions for somatic embryogenesis Micropropagules regenerated plants regeneration and field cultivation of 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 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 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 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 cm A: Sacol brown strain B: Payaka brown strain 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 weeks of culture in Dòng nâu Payaka Phong) Payaka brown strain (Vân (Van Phong) Sacolnâu brown strain(Vân (Van Phong) Dòng Sacol Phong) Payaka brown strain (Cam (Cam Ranh) Dòng nâu Payaka Ranh) Sacolnâu brown strain(Cam (Cam Ranh) Ranh) Dòng Sacol 400 Dry weight (g) Fresh weight (g) Van Phong bay (Fig 3.1 and Fig 3.2) 300 200 100 0 Time (week) 10 Payaka brown strain(Vân (Van Phong) Phong) Dòng nâu Payaka Sacol brown strain (Van Phong) Dòng nâu Sacol (Vân Phong) Payaka brown strain (Cam Ranh) Dòng nâu Payaka (Cam Ranh) Sacol brown strain (Cam Ranh) Dòng nâu Sacol (Cam Ranh) 50 40 30 20 10 12 Fig 3.1 Fresh weight of Payaka brown and Sacol brown strain in Van Phong bay and Cam Ranh bay Time (week) 10 12 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) Fresh dried rate (%) Growth rate (%.day-1) after 12 weeks of culture in Cam Ranh bay16(Fig 3.3) Payaka strain (Van Dòng brown nâu Payaka (VânPhong) Phong) Sacol brown Dòng nâu strain Sacol(Van (VânPhong) Phong) Dòng brown nâu Payaka (CamRanh) Ranh) Payaka strain (Cam Dòng nâu strain Sacol(Cam (CamRanh) Ranh) Sacol brown 2 -2 10 12 12 Payaka strain (Van(Vân Phong) Dòng brown nâu Payaka Phong) Sacol brown (Van Phong) Dòng nâustrain Sacol (Vân Phong) Payaka brown strain (Cam Ranh)Ranh) Dòng nâu Payaka (Cam Sacol brown (Cam Ranh)Ranh) Dòng nâustrain Sacol (Cam Time (week) 10 12 Time (week) Fig 3.3 GR of Payaka brown strain and Fig 3.4 Fresh dried rate of Payaka brown strain and Sacol brown Sacol brown strain in Van Phong strain in Van Phong bay and bay and Cam Ranh bay 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 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 11 3.2.2 Callus induction and callus proliferation Callus induced best in PES medium supplemented with 15 g.L-1 agar under light intensity of µmol photons.m-2.s-1 after 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 weeks of culture, n = 30 Media Callus induction rate (%) Survival rate (%) Size (mm) 41,1c ± 2,8* Necrotic Necrotic MS Necrotic Necrotic Necrotic ½ MS Necrotic Necrotic Necrotic MS ½ PES ½ PES PES ½ MPI ½ MPI MPI ½ Necrotic 82,2a ± 0,6 70,0b ± 1,9 72,2b ± 3,4 70,0b ± 3,8 68,9b ± 5,3 67,8b ± 8,6 Necrotic 85,1a ± 2,5 73,1b ± 1,8 75,4b ± 1,3 55,7c ± 3,1 30,7d ± 0,5 28,1d ± 3,3 Control Necrotic 1,83a ± 0,15 1,40b ± 0,10 1,53b ± 0,35 1,30b ± 0,10 1,00c ± 0,10 0,47d ± 0,15 Remark Explants were necrotic after months of culture 80% of explants were necrotic and bleaching after – days of culture 70% of explants were necrotic after – days of culture and bleaching after days of culture Explants were necrotic Big filamentous callus was obsever Small callus was obsever Callus grew slowly Small callus was obsever Small and necrotic callus was obsever “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 weeks of culture Scale bar: 2mm A: Control D: 0.1 mg.L-1 BAP G: 1.0 mg.L-1 NAA + 1.0 mg.L-1 BAP -1 -1 B: 0.1 mg.L NAA E: 1.0 mg.L BAP H: 0.1 mg.L-1 NAA + 1.0 mg.L-1 BAP C: 1.0 mg.L-1 NAA F: 0.1 mg.L-1 NAA + 0.1 mg.L-1 BAP 12 Table 3.6 Effects of light intensity on callus induction of the Sacol brown strain after weeks of culture, n = 30 Callus induction rate (%) Survival (%) Size (mm) Necrotic Necrotic Necrotic 67,0a ± 4,6 77,7a ± 3,5 2,6a ± 0,10 15 56,7b ± 3,3 79,0a ± 4,6 2,5a ± 0,15 35 55,3b ± 1,5 64,7b ± 3,8 1,3b ± 0,20 55 33,1c ± 4,0 40,0c ± 3,5 0,5c ± 0,15 Light intensity (µmol photons.m-2.s-1) Remark Callus induction wasn’t obsever and explants were necrotic Clumps of white to brown filaments big callus were obsever Clumps of white to brown filaments big callus were obsever Clumps of white to brown filaments small callus were obsever Clumps of white to brown filaments small callus and necosis callus were obsever after weeks of culture Image 3.8 Callus morphology of the Sacol brown strain at different agar contents after weeks of culture Scale bar: mm A: g.L-1 B: 10 g.L-1 C: 15 g.L-1 D: 20 g.L-1 For the callus proliferation experiments, 8-week-old filaments callus with a large size (> mm) were obtained in the best treatments Under a stereo microscope, callus induction of the Sacol brown strain was observed during the experiment The results showed that the first white filamentous callus cells were observed from a few cells in the apical region (10%) after – days of culture The callus cluster had a spherical structure consisting of long chains of cells arranged to form fibers after weeks of culture These structures grew rapidly, reaching 2.25 – 2.5 mm in size after weeks of culture The callus growth became prominent and spread all over the cut surface, forming a bright whitish cap–like structure after weeks of culture (Fig 3.9 I, K) 13 Image 3.9 Callus indution of the Sacol brown strain Scale bar: 1mm A: Original explants B: Callus induction in the medullary regions C: Callus induction in the medullary regions and cortical regions D: Callus induction in the cortical regions E, F, G: Callus induction after weeks of culture H: Callus induction after weeks of culture I, K, L, M: Callus induction after weeks of culture N, O: Filamentous and “compact” callus after weeks of culture In the stage of callus proliferation, the 8-week-old callus was incubated in different media (MS, ½ MS, MS ½, PES, ½ PES, PES ½, MPI, ½ MPI, MPI ½) After weeks of culture, the results of callus proliferation in PES medium were better than in other media Then, NAA (1; 2; mg.L-1) or BAP (1; 2; mg.L-1) each or in combination were added to the PES medium After weeks of culture, the results of PES medium supplemented with mg.L-1 NAA + mg.L-1 BAP 14 had the best ability to callus proliferation (215.27 mg) times higher than that of the control (111.33 mg) (Fig 3.10 and Fig 3.11.) Image 3.10 Morphology of callus clumps of the Sacol brown strain in different media after weeks of callus proliferation Scal bar: 1mm A: Solid and necrotic callus after week of culture in MS medium B: Callus continued to grow in PES D: Solid callus in MPI medium C: Callus continued to grow in PES ½ E: Solid callus in MPI ½ medium Image 3.11 The morphology of callus clumps from the Sacol brown strain in PES medium supplemented with NAA and BAP at different concentrations after weeks of culture Scal bar: mm A: Control B: mg.L-1 NAA C: mg.L-1 BAP D: mg.L-1 NAA + mg.L-1 BAP E: mg.L-1 NAA + mg.L-1 BAP F: mg.L-1 NAA + mg.L-1 BAP G: mg.L-1 NAA + mg.L-1 BAP Then, large clumps of 16-week-old callus obtained from the best treatments were used for somatic embryogenesis experiments 15 3.2.3 Induction of somatic embryogenesis from callus Clumps of 16-week-old callus (2 x mm; 10 mg) were inoculated into PES medium at different solidities (solid, semi-solid and liquid) The results showed that the filamentous callus cultured on solid medium was vigorous in growth without the induction of somatic embryos (SEs) Whilst, in semi-solid medium (4 g.L-1 agar), the callus was formed with numerous pigmented filaments after weeks of culture and developed to SEs with 15.67 embryos/explant after weeks of culture In liquid medium, the callus was necrotic after a week of culture (Fig 3.12.) Image 3.12 Dissection morphology of Sacol brown strain callus under different cultural conditions A: Solid medium; B, C: semi-solid medium; D: Liquid medium Then, semi-liquid PES medium (4 g.L-1 agar) was added each or in combination with NAA (1; 2; mg.L-1) or BAP (1; 2; mg.L-1) to study SEs The combination of NAA (1 mg·L-1) and BAP (2 mg·L-1) resulted in the highest SEs induction with 113.33 embryos per explant, 189.67 mg fresh and 18.10 mg dry (Table 3.11) Thus, the callus of the Sacol brown strain is best developed in semiliquid PES medium (4 g.L-1 agar) supplemented with the combination of NAA (1 mg·L-1) and BAP (2 mg·L-1) 16 Table 3.11 Effect of each or in combination with NAA or BAP on somatic embryogenesis from callus of Sacol brown strain after weeks of culture, n = 30 PGRs (mg.L-1) NAA BAP SEs /explant Fresh weight (mg) Dry weight (mg) Remark Formation of pigmented callus after 0 20,00d ± 2,00 63,33e ± 4,93 5,83e ± 0,29 weeks of culture, induction of SEs after 0 0,00f ± 0,00 0,00f ± 0,00 0,00f ± 0,00 69,33e ± 2,08 69,67e ± 2,08 7,67f ± 1,15 6,20e± 0,20 No induction of SEs 6,63de± 0,32 No induction of SEs 0,67f ± 0,21 No induction of SEs 61,67c ± 4,73 84,67d ± 5,51 8,13c ± 0,71 after weeks of culture, induction of SEs 0 0,00f ± 0,00 0,00f ± 0,00 85,67d ± 5,13 5,67f ± 0,58 7,63cd ± 0,15 No induction of SEs 0,53f ± 0,06 No induction of SEs 1 57,33c ± 2,52 126,00c ± 5,20 12,67b ± 2,08 (100 – 200 µm in diameter) after weeks weeks of culture Formation of numerous pigmented callus after weeks of culture 2 2 Formation of numerous pigmented callus after weeks of culture, induction of SEs of culture Formation of numerous pigmented callus after weeks of culture, 113,33a ± 6,66 189,67a ± 3,06 18,10a ± 1,01 induction of SEs (200 – 500 µm in diameter) after weeks of culture 69,00b ± 3,61 125,67c ± 5,51 12,23b ± 0,40 Induction of SEs Formation of numerous pigmented callus after weeks of culture, induction of SEs 73,67b ± 5,03 138,00b ± 4,36 13,33b ± 0,58 (100 - 200 µm in diameter) after weeks of culture The microprogagules' regeneration from callus through the embryo was also monitored and recorded during the experiment with the effect of the PES medium at different solidities The results of anatomical and electron microscopy images of embryos' development showed that the shapes of embryos from all treatments have no morphological differences After weeks of culture, microscopic examination revealed a densely pigmented callus (Fig 3.14.B) Initially, at the wall of two callus cells, forming preembryonic cells These cells are initially very small and brownish in color Each septum usually forms two symmetrical cells on either side, then grows larger, reaching about 50 –100 µm in size These cells had a nucleus, brown color (Fig 3.14.C) 17 and then a cytoplasmic division partition, dividing the old cell into new cells after weeks of culture (Fig 3.14 D) Image 3.14 Somatic embryogenesis (SEs) induction from callus and the regeneration of micropropagules from SE of the Sacol brown strain after weeks of culture Image A was taken with an objective lens x 10, images B–J were taken with an objective lens x 20, and images K and L were taken with a mechanical camera without a microscope A: Embryogenic callus H: The somatic embryo in a crescent B: The pigmented callus shape C: Filamentous type callus with pigments I: Sphere somatic embryo D: Cell was doublicate divided J: Shoes shaped somatic embryo E: Filamentous type callus with cells on K: Image of somatic embryo clump triplicate division L: Micropropagules regeneration in F, G: Callus developed multicellular macroalgae liquid PES medium Next, these cells continued to grow and then divide into cells after weeks of culture, and then into cells after weeks of culture (Fig 3.14 E, F), and multicellular after weeks of culture (Fig 3.14 G) 18 When in multicellular form, it is an embryo The embryo is crescentshaped (Fig 3.14 H), spherical (Fig 3.14 I) or shoe-shaped (Fig 3.14 J) with relatively large sizes (150 – 500 µm) Microscopic observation showed that this cluster of embryonic tissue is brown, and the small embryos are stacked but are essentially separate (Fig 3.14 K) Transfer of such embryogenic callus blocks to liquid cultures on a rotary shaker facilitated morphogenesis of somatic embryos into single SEs at the bud pole of the embryo The first sprout will appear after about weeks of culture (Fig 3.14 L) The single embryos with a size of 0.5 - 0.6 mm obtained from the above experiments were used to study microprogagules regeneration 3.2.4 Micropropagules regeneration and field culture Environmental factors were investigated during the regeneration of micropropagules from SEs After weeks of culture, the results showed that SEs had the best ability to regenerate micropropagules in PES medium with aeration, salinity of 35, and temperature of 25 – 27°C under light intensity of 55 µmol photons.m-2.s-1 (Table 3.12 – Table 3.16) Table 3.12 The effect of different medium compositions on the regeneration of microprogagules in the Sacol brown strain after weeks of culture, n = 30 Medium Survival Propagule No of composition rate (%) branch/plantlet length (mm) Control 16,7cd ± 5,1 1,33c ± 0,58 PES 50,0a ± 5,8 9,67a ± 1,15 ½ PES PES ½ MPI ½ MPI MPI ½ a 46,7 ± 1,9 40,0ab ± 5,8 36,7abc ± 1,9 30,0bcd ± 5,8 26,7bcd ± 5,1 5,67b ± 0,58 5,33b ± 0,58 5,00b ± 1,00 2,33c ± 0,58 1,67c ± 0,58 Remark Low survival rate Light brown and stunt microprogagules formation Vigorous growth of microprogagules, a 22,00 ± 1,00 multibranched with sharped tip 18,00bc ± 1,00 Vigorous growth of microprogagules 18,67b ± 1,53 Vigorous growth of microprogagules 17,00c ± 1,00 Multibranched with flat and rounded end 7,67d ± 0,58 Multibranched with flat and rounded end 6,67d ± 0,58 micropropagules thin and pale color 1,33e ± 0,58 19 Table 3.13 The effect of culture condition on micropropagules regeneration of the Sacol brown strain after weeks of culture, n = 30 Culture condition Survival rate Propagule No of branch/plantlet length (mm) (%) 100 rpm 40,00ab ± 10,00 3,00b ± 1,00 17,67b ± 1,53 50 rpm 30,00b ± 10,00 2,00b ± 1,00 15,00c ± 1,00 Low growth Aeration 53,33a ± 5,77 9,33a ± 0,58 21,67a ± 0,58 Remark Vigorous growth of light brown microprogagules Vigorous growth of light brown microprogagules Table 3.14 The effect of light intensity on microprogagules regeneration of the Sacol brown strain after weeks of culture, n = 30 Light intensity Survival rate Propagule No of branch/plantlet length (mm) (µmol.m-2.s-1) (%) Necrotic 15 35 55 75 36,67c ± 5,77 40,00bc ± 10,00 53,33a ± 5,77 56,67a ± 5,77 50,00ab ± 10,00 Remark Necrotic of micropropagules complete after a week of culture 3,33d ± 0,58 15,00c ±1,00 Light brown of micropropagules 4,00d ± 1,00 17,33b ±0,58 Light brown of micropropagules 5,67c ± 0,58 20,67a ± 1,53 Light brown of micropropagules 10,00a ± 1,00 21,00a ± 1,00 Vigorous growth, brownish color 8,00b ± 1,00 15,33c ± 0,58 Unhealthy explants Necrotic Necrotic Table 3.15 The effect of salinity on microprogagules regeneration of the Sacol brown strain after weeks of culture, n = 30 Salinity (‰) Survival rate (%) No of branch/plantlet 20 6,7c ± 5,8 2,67c ± 0,58 b c 25 30,0 ± 10,0 3,00 ± 1,00 30 40,0b ± 10,0 7,00b ± 1,00 35 56,7a ± 5,8 9,33a ± 0,58 40 13,3c ± 5,8 3,00c ± 1,00 Propagule length (mm) Remark Necrotic of micropropagules complete after – days of culture 6,00 ± 1,00 Unhealthy explants Vigorous growth, branches with big 17,33b ± 0,58 size and brownish color Vigorous growth, branches with big 22,33a ± 0,58 size and brownish color 5,00cd ± 1,00 Low growth 3,67d ± 0,58 c Table 3.16 The effect of temperature on microprogagules regeneration of the Sacol brown strain after weeks of culture, n = 30 Temperature Survival rate (°C) (%) No of branch/plantlet Propagule length (mm) Remark 20 50,0a ± 10,0 3,33b ± 0,58 17,67b ± 0,58 Low growth 25 60,0a ± 10,0 9,33a ± 1,53 21,00a ± 1,00 27 63,3a ± 5,8 9,67a ± 1,53 30 26,7b ± 11,5 2,67b ± 0,58 35 b 13,3 ± 5,8 b 2,00 ± 1,00 Vigorous growth, branches with big size and brownish color Vigorous growth, branches with 21,67a ± 1,53 big size and brownish color 5,00c ± 1,00 c 4,00 ± 1,00 Low growth, necrotic of tip branches Low growth and necrotic of micropropagules 20 After complete microprogagules regeneration, the microprogagules were transferred to ex vitro culture The 8-week-old in vitro microprogagules were grown in a 100 L glass tank, continuously aerated at 27°C under different light sources Results showed that after weeks of ex vitro culture, the microprogagules that grew well under light source (natural light was reduced ½ by transparent corrugated iron and blue-black net) had the highest survival rate (86.67%), fresh weight (64.7 g) and the highest GR (7.73%/day) (Fig 3.20 D) Image 3.20 The morphology of the Sacol brown strain plants from in vitro microprogagules obtained under different light sources after weeks of ex vitro culture Scale bar: cm A: Light source C: Light source E: Light source B: Light source D: Light source 21 Besides the appropriate light source evaluation results, the type of microprogagules (different in number of branches, size, and weight) was also tested Results showed that the survival rate, fresh weight, and GR of microprogagules type (6.67 branches/plant; 3.01 cm; 1.23 g) were (86.67%; 73.27 g; 7.86%/day; respectively) the highest compared with other treatments after weeks of ex vitro culture (Fig 3.21 F) Image 3.21 The morphology of the Sacol brown strain plants obtained from different in vitro microprogagules types after weeks of ex vitro culture Scale bar: cm A: Type 1; B: Type 2; C: Type 3; D: Type 4; E: Type 5; F: Type After growing in ex vitro conditions, the plants with a weight of 50 – 70 g were transferred to the field to evaluate the quality of the in vitro microprogagules The results showed that the fresh and dry weight of seaweed (420.57 g, 51.53 g; respectively) was 5.25 times higher than the fresh and dried seaweeds of natural origin (80.17 g; 8.50 g; respectively) after 12 weeks of culture in Van Phong Bay The cumulative GR of plants in vitro origin reached 2.57%/day, which was 4.42 times higher than that of plants of natural origin (0.57%/day) (Fig 3.14) 22 In addition, the fresh dried rate of seaweed of in vitro origin (12.50%) was higher than that of seaweed of natural origin (10.81%) after 10 weeks of culture (Fig 3.15) Thus, plants were harvested after Growth rate (%/ngày) 2 -2 10 12 Fresh dried rate (%) 10 weeks of culture to evaluate carrageenan content and quality 15 10 weeks Plants in vitro Giốngfrom in vitro weeks Plants in vitro Giốngfrom in vitro Plants natural Giốngfrom tự nhiên Fig 3.14 GR of in vitro origin and natural origin after 10 weeks of culture in the field 10 12 Plantstự from natural Giống nhiên Fig 3.15 Fresh dried rate of in vitro origin and natural origin after 10 weeks of culture in the field The results of the analysis of carrageenan content also showed that the in vitro origin had a higher carrageenan content (28.83%/w) than the natural seaweed (24,33%) after 10 weeks of field culture (Fig 3.16) The strength and viscosity of carrageenan extracted from seaweeds of in vitro origin (928.67 g.cm-2; 34.17 cps; respectively) were higher than the strength and viscosity of carrageenan of natural origin (909.00 g.cm-2; 28.50 cps; respectively) (Fig 3.17) 23 950 Strength (g.cm-2) Carrageenan content (%/w) 40 30 20 10 920 890 860 830 800 Plants from Giống in vitroGiống nhiên Plants tự from in vitro natural Giống in vitro Plants from in vitro Giống tự nhiên Giốngfrom tự Plants nhiên natural Fig 3.16 Carrageenan content of in Fig 3.17 Strength of carrageenan vitro origin and natural extracted from seaweeds of origin after 10 weeks of in vitro origin and natural culture in the field origin after 10 weeks of culture in the field From the results, the schematic summary of the in vitro propagation process of the Sacol brown strain is carried out as follows: Image 3.22 Schematic summary of the in vitro propagation process of the Sacol brown strain (1) Culture development (2) Axenic material (3) Callus induction (4) Callus proliferation (5) Somatic embryogenesis induction (6) Single somatic (7) cm micropropagules regeneration (8) Ex vitro cultivation (9) Field cultivation 24 CHAPTER CONCLUSIONS AND RECOMMENDATIONS 4.1 CONCLUSIONS Currently, there are two strains (Payaka brown strain and Sacol brown strain) being cultivated in Van Phong bay and Cam Ranh bay The Sacol brown strain cultivated in Van Phong bay had better biological characteristics Therefore, it was selected for in vitro propagation research This study proposes a potential procedure for effective regeneration of the Sacol brown strain of K striatus This study was also the first to investigate the effect of AgNPs on the antibacterial ability and induction of callus in the Sacol brown strain of K striatus The adaptability of in vitro seedlings was evaluated to the natural environment as well as the content and quality of carrageenan Seedlings derived from in vitro have good growth ability The biomass obtained is 5.25 times higher and the carrageenan content is higher than that of natural seedlings The research results have given the in vitro breeding process of the Sacol brown strain by the in vitro culture method, opening up new research directions in the future 4.2 RECOMMENDATIONS Continue to evaluate the growth and genetic stability of seaweed derived in vitro after – generations Research on seed production on a larger scale to provide seeds for commercial production will contribute to improving seedling quality LIST OF WORKS OF THE AUTHOR Vu Thi Mo, Tran Van Huynh, Le Trong Nghia, Hoang Thanh Tung, Nguyen Ngoc Lam, Duong Tan Nhut, Callus induction from Kappaphycus striatus under different culture conditions, Journal of Biotechnology, 2018, 16 (2), 301–309 Vu Thi Mo, Le Kim Cuong, Hoang Thanh Tung, Tran Van Huynh, Le Trong Nghia, Chau Minh Khanh, Nguyen Ngoc Lam, Duong Tan Nhut, Somatic embryogenesis and plantlets regeneration from seaweed Kappaphycus striatus, Acta Physiologiae Plantarum, 2020, 42, 104 Vu Thi Mo, Vo Thanh Trung, Le Trong Nghia, Hoang Thanh Tung, Vu Quoc Luan, Đo Manh Cuong, Hoang Đac Khai, Nguyen Thi Nhu Mai, Phan Minh Thu, Nguyen Ngoc Lam, Duong Tan Nhut, Growth rate, concentation and quality of carrageenan in two strains of seaweed Kappaphycus striatus (F Schmitz) Doty ex P C Silva, 1996 grown in Khanh Hoa waters, Vietnam Science and Technology, 2021, Accepted ... rate (%) Size (mm) 41, 1c ± 2,8* Necrotic Necrotic MS Necrotic Necrotic Necrotic ½ MS Necrotic Necrotic Necrotic MS ½ PES ½ PES PES ½ MPI ½ MPI MPI ½ Necrotic 82,2a ± 0,6 70,0b ± 1,9 72,2b ± 3,4... brown in color, and unscratched (400 g, 30 – 40 cm) They were collected at Van Phong bay and Cam Ranh bay, Khanh Hoa province 2.2 Contents research Biological and ecological characteristics of strains... nâu Payaka (VânPhong) Phong) Sacol brown Dòng nâu strain Sacol(Van (VânPhong) Phong) Dòng brown nâu Payaka (CamRanh) Ranh) Payaka strain (Cam Dòng nâu strain Sacol(Cam (CamRanh) Ranh) Sacol brown