Nghiên cứu ứng ảnh hưởng của chất điều hòa sinh trưởng thực vật brassinolide đến khả năng chịu mặn của lúa cao sản vùng đồng bằng sông cửu long tt tiếng anbh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Crop Science Code: 62 62 01 10 LE KIEU HIEU EFECTS OF BRASSINOLIDE PLANT GROWTH REGULATOR ON SALINE TOLERANCE OF HIGH YIELD RICE IN THE MEKONG DELTA Can Tho, 2020 THE THESIS HAS BEEN COMPLETED AT CAN THO UNIVERSITY Instructor 1: Prof Dr Nguyen Bao Ve Instructor 2: Assoc Prof Dr Pham Phuoc Nhan The thesis is defended in front of the University Examination Council in Can Tho University Place: Time:………… ……………Date:…………….…….… Reviewer 1: Reviewer 2: Further information of the thesis could be found at: Learning Resource Center of Can Tho University National library of Vietnam LIST OF PUBLISHED PAPERS Le Kieu Hieu, Nguyen Bao Ve and Pham Phuoc Nhan 2019 Effects of brassinolide on the restriction of salinity damage on rice plants under nethouse conditions Vietnam Agricultural Science and Technology Journal, No 1: 32-39 Le Kieu Hieu Pham Phuoc Nhan and Nguyen Bao Ve 2019 Effects of brassinolide on physiological and biochemical characteristics of salinity tolerant rice (6‰) in the seedling stage Vietnam Agricultural Science and Technology Journal, No 2: 44-49 Le Kieu Hieu, Nguyen Bao Ve and Pham Phuoc Nhan 2019 Effects of brassinolide on restriction of salt harmfulness on field rice in Bac Lieu province Vietnam Agricultural Science and Technology Journal, No 8: 62-66 CHAPTER 1: INTRODUCTION 1.1 The urgency of the thesis In recent years, the saline intrusion has become more and more complicated In some coastal provinces of the Mekong Delta, saline water deeply penetrated into the paddy rice fields, disturbed seriously people livelihood and crop production activities Especially, in the first quarter of 2016, saltwater intrusion in the Mekong Delta was evaluated to be the most serious in the past 100 years and was forecasted to be worse in the following years (Luong Xuan Dinh et al., 2016) According to Tanwar (2003), the salinity tolerance threshold of rice was 3.0 mS/cm of soil and 2.0 mS/cm for irrigation water, the rice yield will plummet when the salinity exceeds this value Rice is very susceptible to salinity at the seedling, tillering and panicle initiating stage At repening, most of rice cultivars are less sensitive to salinity (Lauchli and Grattan, 2007) Currently, there are some strategies that could enhance rice tolerance to salinity such as using resistant varieties, cultivation techniques or using brassinosteroids – a plant growth regulator, which have currently been studied and applied Many results showed that brassinolide (BL) (C28H48O6 - a natural lactone steroid discovered in 1979, belonging to brassinosteroids) could increase salinity tolerance of plant by stimulating growth (El-Feky and Abo-Hamad, 2014), accumulating proline (Vardhini, 2012; Nguyen Van Bo et al., 2014), stabilizing chlorophyll (Nithila et al., 2013), increasing activity of antioxidant enzymes (El-Mashad and Mohamed, 2012), on some upland crops However, there are limited studies on effects of this substance on the biochemical and physiological characteristics, growth and productivity of rice in saline areas of the Mekong Delta Based on practical needs of rice production in this region and BL application could improve potentially rice tolerance and reduce yield loss under saline condition, the thesis titled "Effects of brassinolide on saline tolerance of high yield rice in the Mekong delta" was conducted 1.2 Aims of the thesis Determine the effects of brassinolide applications on physiological and biochemical characteristics of high yield rice under salt-stressed condition; Find out the effectiveness of brassinolide treatments at seedling, tilling, panicle initiating, and flowering stage when rice grown under salt stress in the Mekong Delta 1.3 Content of the thesis The content of the thesis includes investigation of some biochemical and physiological characteristics of rice treated with brassinolide and effects of brassinolide treatments on rice at different stages of rice grown in net house and application of the most effective brassinolide-treated level on field trials 1.4 The new findings of the dissertation - When growing rice under saline condition treated with brassinolide contributed to increase proline, photosynthetic pigments, protease, catalase activity, content of total nitrogen, phosphorous and sodium in shoots which enhanced rice tolerance to salinity - The thesis has determined the concentrations of brassinolide treatment when rice grown under saline condition as follows: (1) At the ‰-salted condition: Growing rice treated with brassinolide of 0,05 mg/L at the seedling or tillering stage improved rice yield, while at stages of panicle initiating and flowering, brassinolide of 0,10 mg/L should be used (2) At salinity of ‰, application of brassinolide of 0,05 mg/L improved rice yield, in contrast brassinolide of 0,10 mg/L should be applied at stages of tilling or panicle initiating and flowering - In the natural-salted field (3.2‰ and 4.82‰) in Bac Lieu province, times brassinolide application of 0,05 mg/L at the seedling stage and of 0,10 mg/L at the tilling stage and panicle initiating improved growth and rice yield from 21% to 29% 1.5 Meaning of the thesis - The scientific significance of thesis: Determine the effects of brassinolide applications on physiological - biochemical characteristics, rice growth and yield under salt-stressed condition - The practice of thesis: Help farmers reduce rice yield loss in salted condition CHAPTER 3: RESEARCH METHOD 3.2 Research materials and facilities - The thesis was conducted from 2015 to 2018 The experiments were carried out in the laboratory of biochemistry, in net house at College of Agriculture, Can Tho University and trials on the paddy rice field in Phuoc Long district and Gia Rai town, Bac Lieu province - Main experimental materials: Rice varieties: OM2517 and OM5451; Brassinolide (BL) plant growth regulator produced by the Merck company; Sodium chloride was the substance used to create a saline environment in the laboratory and net house 3.3 Research methods 3.3.1 The effects of brassinolide applications on some physiological and biochemical characteristics of rice seedlings 3.3.1.1 Experiment 1: Effects of BL applications on physiological and biochemical characteristics of rice seedlings under 3‰ salt-stressed condition a) Design of experiment: Experiment was completely randomized design, one factor, treatments The brassinolide concentrations of 0; 0.05; 0.10; 0.20; 0.40 mg/L were used for the experiments at laboratory Each treatment included replications, growing trays for each replication b) Experimental procedure - Preparation of growing tools: Holed foam sheet (10 holes in a row and 10 rows per sheet) was floating inside the rectangular plastic tray containing nutrient solution Three germinated rice seeds were planted in each hole The underside of the foam sheet was covered with a net to keep the rice from falling into the growing solution - Treatment of rice seeds and sowing: seeds of OM2517 variety was soaked in water for 24 hours and then incubated When the seeds just cracked, depending on the treatments, spray BL solution at the concentrations described above Continue incubating the seeds until germination and then sowed (3 seeds per hole) For the first days, distilled water was used as growing solution - Salinization of nutrient solutions: The Yoshida nutrient solution (Yoshida et al., 1976) was salted by dissolving g of NaCl in liter of nutrient solution The salinity of solution was measured by machine Each tray contained liters of salted nutrient solution - Saline treatment: After days when seedlings grew well, replaced distilled water by 3‰-salted nutrient solution The growing soltuions were renewed with the interval of days c) Data recording: Experimental data were collected at days after salt treatment as following: chlorophyll content in leaves (Wellburn, 1994), proline content (Bates et al., 1973), catalase (Barber, 1980), protease (Kunit, 1974), total N, P, K, Ca, Mg, Na content in plants (Ngo Ngoc Hung et al., 2004) Some growth parameters were also recorded including plant height, root length, fresh weight and dry weight according to the method of the Ministry of Agriculture and Rural Development (2011) 3.3.1.2 Experiment 2: Effects of BL applications on physiological and biochemical characteristics of rice seedlings under 6‰ salt-stressed condition The same experiment procedure was applied but replacing 3‰ saline nutrient solution by 6‰ saline nutrient solution 3.3.2 Effects of brassinolide applications on growth and yield of rice in net house condition There were several independent experiments were carried out 3.3.2.1 Effects of applied BL applications on rice growth and yield a) Experiment 3: Effects of BL applications on rice growth and yield under 3‰ saltstressed condition in net house * Design of experiment: Experiment was completely randomized design, one factor, included treatment and the brassinolide concentrations of 0; 0.05; 0.10; 0.20; 0.40 mg/L were used for the experiments in net house Each treatment included replications, each replication included pot * Experimental procedure - Soil preparation: Coastal rice soil was taken at a depth of 0-20 cm, dried naturally in the air Each pot contained kg of crushed soil After being submerged for 14 days, the pots were drained and 15 germinated rice seeds were sown into each pot Rice cultivar OM2517 was used in this experiment - Brassinolide application: germinated rice seeds were incubated with BL solutions day before sowing - Saline treatment: Fresh water in pots were drained before sowing After sowing, 3‰-salted water was filled into pots Salt water was applied once for crop season - Fertilization: The formula of 100N - 60P2O5 - 30K2O kg/ha was applied in the experiment The amount of fertilizers per pot was calculated equivalently to million kg of dry soil/ha At 15 days after sowing, plants per pot were left to grow until harvest The water level in the pot was maintained stably about -7 cm from the ground * Data recording: Growth parameters and yield components were followed the evaluation method of the Ministry of Agriculture and Rural Development (2011), proline content in rice after days of salt treatment (Bates et al., 1973) b) Experiment 4: Effects of BL applications on rice growth and yield under 6‰ saltstressed condition in net house The experiment was conducted in the same procedures of experiment but the salinity was 6‰-salted water 3.3.2.2 Brassinolide application at tillering stage a) Experiment 5: Effects of brassinolide treatments on rice growth and yield under 3‰-salt-stressed condition at tillering stage in net house * Design of experiment: Similar to experiment (Section 3.3.2.1) * Experimental procedure: The same procedures as above description The differences were as followed: - Brassinolide application: BL was sprayed day before salinity treated to rice plants (at 18 days after sowing) - Saline treatment: Saltwater (1 liter) was filled into pots once at tillering stage (at 19 days after sowing) Before being flooded with with 3‰ salt water, pots were drained to remove remaining fresh water in each pot * Data recording: Similar to experiment (Section 3.3.2.1) b) Experiment 6: Effects of brassinolide treatments on rice growth and yield under 6‰-salt-stressed condition at tillering stage in net house Experiment was carried out in the same way as experiment but the salt level of 6‰ was applied 3.3.2.3 Brassinolide application at panicle initiating stage a) Experiment 7: Effects of brassinolide treatments on rice growth and yield under 3‰-salt-stressed condition at panicle initiating stage in net house * Design experiment: Similar to experiment (Section 3.3.2.1) * Experimental procedure: The same procedures as above description The differences were as followed: - Brassinolide application: BL was sprayed day before salinity treated to rice plants (at 48 days after sowing) - Saline treatment: Saltwater (1 liter) was filled into pots once at tillering stage (at 49 days after sowing) Before being flooded with with 3‰ salt water, pots were drained to remove remaining fresh water in each pot * Data recording: Similar to experiment (Section 3.3.2.1) b) Experiment 8: Effects of brassinolide treatments on rice growth and yield under 6‰-salt-stressed condition at panicle initiating stage in net house Experiment was carried out in the same way as experiment 7, excepting for the salt level of 6‰ 3.3.2.4 Brassinolide application at flowering stage a) Experiment 9: Effects of brassinolide treatments on rice growth and yield 3‰-salt-stressed condition at flowering stage in net house * Design of experiment: Similar to experiment (Section 3.3.2.1) * Experimental procedure: The same procedures as above description The differences were as followed: - Brassinolide application: BL was sprayed day before salinity treated to rice plants (at 60 days after sowing) - Saline treatment: Saltwater (1 liter) was filled into pots once at tillering stage (at 61 days after sowing) Before being flooded with with 3‰ salt water, pots were drained to remove remaining fresh water in each pot * Data recording: Similar to experiment (Section 3.3.2.1) b) Experiment 10: Effects of brassinolide treatments on rice growth and yield under 6‰-salt-stressed condition at flowering stage in net house Experiment 10 was carried out in the same way as experiment 9, excepting for the salt level of 6‰ 3.3.3 Effects of brassinolide treatments on rice growth and yield under saltstressed condition on the paddy rice field Using the BL levels had most effective to eliminate salt stress for rice at each growth stage (from experiments in net houses - Section 3.3.2) 3.3.3.1 The experiment in Phuoc Long district, Bac Lieu province Experiment 11: Effects of BL applications by spraying on rice growth and yield under 4.82‰-salt-stressed in Phuoc Long district, Bac Lieu province Experiment was laid out in randomized complete block design including treatments and replications (Table 3.1) The area of each replicate was 20 m2 Table 3.1: Different treatment on the field condition Treatment Time points of BL spraying Control untreated (spraying with water) Treatment of rice varieties (seedling) Seedling + tillering Seedling + tillering + panicle initiating Seedling + tillering + panicle initiating + flowering Concentration of solution Seedling: 0.05 mg/L Tillering: 0.10 mg/L Panicle initiating: 0.10 mg/L Flowering: 0.10 mg/L - The farming techniques of rice cultivation according to local farmers' practices were as follows: Using rice variety OM2517, sowing density of 120 kg/ha The amounts (in kg) of fertilizers for 10.000 m2 were 91.5 N – 73.5P2O5 – 65.5 K2O - Time points of brassinolide application: Germinated rice seeds were incubated with BL and spraying BL at tillering (18 days after sowing), at panicle initiating (41 days after sowing), and at flowering stage (60 days after sowing) * Data recording: Soil pH and EC were taken by portable pH and EC meter Growth parameters and rice yield components were followed the evaluation method of the Ministry of Agriculture and Rural Development (2011) including plant height, the number of tillers at 10, 30, 50 and 70 days after sowing, number of spikelets per m 2, number of spikelets per panicle, the ratio of filled spikelets per panicle, weight of 1000-grain, theoretical yield and actual yield (tons/ha) 3.3.3.2 The experiment in Gia Rai town, Bac Lieu province Experiment 12: Effects of BL applications by spraying on rice growth and yield under 3.2‰-salt-stressed in Gia Rai town, Bac Lieu province * Design of experiment: Similar to experiment 11 (Section 3.3.3.1) Treatments in experiment were shown in Table 3.2 Table 3.2: Different treatment on the field condition Treatment Time points of BL spraying Control untreated (spraying with water) Treatment of rice varieties (seedling) Seedling + tillering Seedling + tillering + panicle initiating Seedling + tillering + panicle initiating + flowering Concentration of solution Seedling: 0.05 mg/L Tillering: 0.05 mg/L Panicle initiating: 0.10 mg/L Flowering: 0.10 mg/L - Using rice variety OM5451, sowing density with 120 kg/ha The amounts (in kg) of fertilizers for 10.000 m2 were 84 N – 75P2O5 – 51 K2O - BL treatment: germinated rice seeds were completely wetted by BL solutions and incubated at room temperature for 24 hours and BL solutions were sprayed on rice field at tillering (20 days after sowing), at panicle initiating (45 days after sowing), and at flowering stage (65 days after sowing) * Data recording: Similar to experiment 11 (Section 3.3.3.1) 3.4 Data analysis method: Data were analyzed for variance to find differences between treatments in each experiment Duncan’s multiple range test at significant level of 1% or 5% were used to compare the treatment means CHAPTER 4: RESULTS AND DISSCUSSION 4.1 The effects of brassinolide treatments on some physiological and biochemical characteristics of rice seedlings 4.1.1 The proline content At the or 6‰-salted condition, the proline content of rice stems was improved under seed incubation with BL 0.2 mg/L (Table 4.1) The same result was also found in study of Samia et al (2009) on maize, incubating seeds with BL 0.25 mg/L increased proline production capacity under 50 mM or 100 mM saline (NaCl) conditions The high accumulation of proline in saline crisis condition adjusted osmosis, increasing water absorption, limited the uptake and transport of Na+ from roots to stems, thereby increasing tolerance in salty conditions Table 4.1: Effects of BL treatments on proline content in rice seedlings at days after salt stressed at 3‰ and 6‰ Proline (µmol/g FW) 3‰-salted 6‰-salted 34.05 c 38.08 c 35.62 bc 45.96 b 36.48 b 44.69 b 40.65 a 52.02 a 35.79 b 47.84 b ** ** 2.43 3.66 Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV (%) Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) by Duncan's multiple range test 4.1.2 The photosynthetic pigments in leaf - Experiment at salinity of 3‰: In saline conditions at the seedling stage, incubating seeds with BL at different concentrations increased the content of photosynthetic pigments, was significantly higher compared to the control (Table 4.2 and Table 4.3) Particularly, brassinolide application of 0.20 mg/L showed the highest carotenoids (59.42 µg/g FW) The similar results were also found in studies of Bera et al (2006) and Prakash et al (2007), the total amount of chlorophylls, soluble proteins were found in high amounts when treated with BL Table 4.2: Effects of BL treatments on chlorophyll a and b (µg/g FW) content in plants days after saline treatment at 3‰ and 6‰ Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV (%) Experiment 3‰-salted Chlorophyll a Chlorophyll b 127.29 c 43.58 b 170.77 b 56.87 a 189.30 ab 63.28 a 193.64 a 54.32 a 207.76 a 63.67 a ** ** 6.24 9.35 6‰-salted Chlorophyll a Chlorophyll b 48.93 b 18.47 45.60 b 18.57 58.36 ab 22.16 72.61 a 25.34 72.77 a 25.28 * ns 15.78 17.47 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% (**) and 5% (*) level, respectively, by Duncan's multiple range test; ns: not significant difference - Experiment at salinity 6‰: The results of Table 4.2 and Table 4.3 showed that in the high salinity (6‰) experiment, the highest content of photosynthetic pigments was found under 0.10-0.40 mg/L of BL treatments and it was significant difference from control and 0.05 mg/L of BL treatments (higher than 9.43-23.84 µg/g FW and 9.43-23.84 µg/g FW compared to those of the control, respectively for chlorophyll a and carotenoids) Table 4.6: Effects of BL treatments on Nts (%N) and Pts (%P2O5) in plants days after saline treatment at 3‰ and 6‰ Experiment Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV (%) 3‰-salted Nts 3.78 3.93 4.07 4.01 3.92 ns 2.78 6‰-salted Pts 2.19 2.30 2.35 2.36 2.32 ns 4.75 Nts 3.19 b 3.36 ab 3.54 a 3.38 ab 3.35 ab * 3.26 Pts 1.48 b 2.06 a 2.16 a 1.96 a 1.41 b ** 10.46 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% (**) and 5% (*) level, respectively, by Duncan's multiple range test; ns: not significant difference Table 4.7: Effects of BL treatments on K (%K2O) and Ca (%Ca) in plants days after saline treatment at 3‰ and 6‰ Experiment Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV (%) 3‰-salted Kts 2.84 3.23 3.44 3.26 2.75 ns 8.76 6‰-salted Cats 0.20 0.24 0.23 0.25 0.22 ns 0.00 Kts Cats 2.24 2.72 2.93 2.95 2.64 ns 11.06 0.25 0.27 0.27 0.26 0.26 ns 0.78 Note: ns: not significant difference Table 4.8: Effects of BL treatments on Na (%Na) and Mg (%Mg) in plants days after saline treatment at 3‰ and 6‰ Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV (%) Experiment 3‰-salted Nats 1.55 1.58 1.53 1.42 1.55 ns 8.03 Mgts 0.22 0.23 0.23 0.23 0.22 ns 0.00 6‰-salted Nats 1.88 a 1.66 b 1.59 b 1.63 b 1.70 b ** 3.74 Mgts 0.27 0.27 0.28 0.27 0.26 ns 1.26 Note: In a column, the numbers followed by the same letter were not significantly different at 1% level (**) by Duncan's multiple range test; ns: not significant difference 10 4.2 Effects of brassinolide treatments on rice growth and yield under 3‰ salt-stressed in net house condition 4.2.1 Plant height at harvest - Experiments at seedling stage: measured height of the plants under salt stress showed no difference between BL applications at seedling stage (Table 4.14) Other results that supported what has been shown here, were those by Anuradha (2002), application of BL in salinity condition could minimize negative effects on rice germination and growth at seedling stage Table 4.14: Effects of BL treatments at different rice growth stages on plant height (cm) at harvest Concentration of BL (mg/L) Experiment at seedling stage Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage Control 0.05 0.10 0.20 0.40 F CV(%) 81.95 84.23 85.72 84.61 81.66 ns 4.02 76.53 b 81.84 a 81.40 a 80.80 a 79.49 ab * 3.46 78.52 c 84.81 a 83.25 ab 80.10 bc 79.80 bc * 3.7 78.77 b 82.51 a 81.62 a 81.82 a 80.68 ab * 2.12 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 5% level (*) by Duncan's multiple range test; ns: not significant difference - Experiments at tillering and flowering stage: Results presented in Table 4.14 shown a positive effect of BL in tillering stage The application of BL concentrations of 0.05; 0.10; 0.20 mg/L contributed to enhance the plant height (Table 4.14) - Experiment at panicle initiating stage: It was obvious that there was a positive effect on the height of rice by BL application BL Spraying on rice plants at the concentration of 0.05 mg/L under salted-growing condition showed the highest result (increased plant height by 8,01% when compared to that of the control) 4.2.4 The numbers of panicle per pot - Experiments at seedling stage: The results of panicle/pots was significantly different at 1% level Usage of different BL concentrations increased the number of panicles/pot from 4.17 to 8.33% (Table 4.20) - Experiment at tillering stage: Spraying BL on rice plants under salt stress at tillering stage showed that the number of panicles/pot between treatments were statistically significant differences at 1% level (Table 4.20) One of the studies that supported these results also, was a study by Nguyen Minh Chon et al (2010), applying BL could increase cell division through inducing chlorophyll accumulation and the transport of photosynthetic products would be stimulating for the treated plants in tillering stage 11 Table 4.20: Effects of BL treatments at different rice growth stages on number of panicle/pot at harvest Concentration of BL (mg/L) Experiment at seedling stage Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage Control 0.05 0.10 0.20 0.40 F CV(%) 19.20 c 20.80 a 20.2 ab 20.2 ab 20.0 b ** 2.66 17.00 c 20.00 a 19.80 a 19.00 ab 17.60 bc ** 7.05 20.00 c 21.60 ab 22.40 a 21.20 b 19.20 c ** 3.66 25.60 26.00 27.60 27.80 25.60 ns 6.83 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) by Duncan's multiple range test; ns: not significant difference - Experiment at panicle initiating stage: The number of panicles/pot varied and the difference was statistically significant at 1% level between treatments In particular, using BL 0.10 mg/L was mostly effective in increasing the number of panicles/pots (22.40 panicles/pot), 12% higher when compared to the control When rice plants at panicle initiating being stressed by salt, their production would be affected, especially the degradation of flower buds and reduction number of branches on panicles (Counce et al., 2000) - Experiments at flowering stage: At the flowering stage, the number of effective shoots were not much variation, so spraying BL at this stage did not affect to the number of panicles/pot (Table 4.20) 4.2.6 The number of filled spikelets per panicle - Experiment at seedling stage: Under salinity condition, incubating rice with BL showed no change in the number of filled spikelets In this period, rice plants had the ability to recover after salinity was lower, so the BL treatments did not affect to the number of filled grains/panicle at harvest (Table 4.22) Table 4.22: Effects of BL treatments at different rice growth stages on the number of filled spikelet/panicle at harvest Concentration of BL (mg/L) Experiment at seedling stage Experiment at tillering stage Control 0.05 0.10 0.20 0.40 F CV(%) 45.24 44.45 46.30 46.18 45.51 ns 2.15 39.92 b 44.48 a 44.14 a 44.20 a 42.40 ab * 4.90 Experiment at panicle initiating stage 25.68 c 32.07 ab 34.16 a 33.73 ab 31.81 b ** 5.04 Experiment at flowering stage 28.22 c 34.38 b 37.76 ab 38.18 a 37.40 ab ** 7.27 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) and 5% (*) level, respectively, by Duncan's multiple range test; ns: not significant difference 12 - Experiment at tillering stage: The number of filled spikelets/panicle in the BL treatments by spraying at different concentrations increased the number of filled spikelets/panicle (ranging from 6.21 to 11.42%) and significant difference at 5% level by statistical analysis (Table 4.22) More precisely, spraying BL at the concentration of 0.05 mg/L showed the highest number of filled spikelets/panicle (44,48 filled spikelets/panicle) - Experiment at panicle initiating stage: The number of filled spikelets/panicle in the BL-applying treatments at different concentrations increased the number of filled spikelets/panicle, it was significant increase from 23.87 to 33.02% compared to those of the control plants (Table 4.22) - Experiment at flowering stage: The result presented in Table 4.22 showed that BL spraying enhanced the number of filled spikelets/panicle from 23.60 to 35.29%, it was significant difference when BL used at the concentration of 0.20 mg/L, which improved the number of seeds on the panicles most effectively Regardless to salt stress at different growing stages such as tillering, panicle initiating or flowering, rice plants treated with BL at appropriate concentrations led to improve the number of filled spikelets/panicle These results were in agreement with the report of Fujii and Saka (2002), brassinosteroids play a vital role in increasing of starch accumulation in seeds, contributing to increase the percentage of filled grains on the crop, through increasing the size of the mature leaves and inducing the transport of carbohydrates to the seed 4.2.7 The weight of 1000 grains The weight of 1000 grains in the experiments did not show any significant differences (Table 4.23) According to Yoshida (1981), the weight of 1000 grains was mainly determined by the genetic characteristics of varieties and the grain size was strictly controlled by the husk Table 4.23: Effects of BL treatments at different rice growth stages on the weight of 1000 grains at harvest Concentration Experiment at of BL (mg/L) seedling stage Control 0.05 0.10 0.20 0.40 F CV(%) Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 26.38 26.13 26.59 26.56 26.23 ns 1.98 26.09 26.02 26.02 26.10 26.14 ns 2.12 26.53 25.86 26.62 26.63 26.56 ns 3.03 26.01 26.66 26.61 26.75 26.10 ns 2.10 Note: ns: not significant difference 4.2.8 The rice yield per pot The effects of BL treatments on rice plants at different development stages peresented in Table 4.24 showed that: 13 - At seedling stage: The rice yield/pot in BL treatments were higher than those of the control (increasing from 5.23 to 10.50%) and were statistically significant differences at 1% level - At tillering stage: The weight of grain/pot had statistical significance (1%) between treatments Treating BL (concentration of 0.05; 0.10; 0.20 mg/L) increased the weight of grain/pot from 29.58 to 30.02% compared with control plants Table 4.24: Effects of BL treatments at different rice growth stages on rice yield (g/pot) Concentration Experiment at of BL (mg/L) seedling stage Control 0.05 0.10 0.20 0.40 F CV(%) 22.58 b 24.65 a 24.89 a 24.95 a 23.76 ab ** 3.76 Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 17.92 b 23.22 a 23.24 a 23.30 a 19.61 b ** 9.09 13.40 d 18.00 b 19.90 a 18.64 b 15.94 c ** 4.30 19.16 d 23.10 c 27.71 a 28.26 a 25.24 b ** 6.36 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) by Duncan's multiple range test - At panicle initiating and flowering stage: The rice yield/pot when spraying BL in all treatments were higher than those in the control At the panicle initiating stage, the yield were increased from 18.96 to 48.51%; at the flowering stage, the yield increase ranged from 20.56 to 47.49% Application of BL concentration at 0.10 mg/L had the most effective improvement the rice yield/pot at both developmental stages of panicle initiating and flowering Generally, BL application contributed to improve rice yield under salt stress Treating BL at appropriate concentrations could help to improve weight of grain/pot at different growth stages of rice under stress conditions (Das and Shukla, 2011), but not all the dosages of BL could be effective for rice plant to withstand salinity Whenever the high concentration of BL was used, they could be more sensitive for rice plants to grow and yield under salt stress conditions 4.2.9 Proline accumulation in rice plant After days of BL treatmenta, the proline contents were significant differences (Table 4.25): - Experiment at seeding, tillering and panicle initiating stage: Applying BL at a concentration of 0.10 mg/L showed the highest proline content (seedling stage increased to 58.92%; tillering period increased to 26.43% and the panicle initiating period increased by 50.81% compared to the control) - Experiment at flowering stage: Proline content in plants increased from 48.97 to 64.98% in BL treatments (concentration of 0.10, 0.20, and 0.40 mg/L) compared to those of the control In which, spraying BL concentration of 0,20 mg/L led to induce the highest proline content in the shoots (12.06 µmol/g) 14 Other studies supporting these results including those of Phap (2006), brassinosteroids in salty environment enhanced proline accumulation in leaf cells as an adaptive trait of rice related to stress tolerance Table 4.25: Effects of BL treatments at different rice growth stages on proline accumulation (µmol/g) in plant Concentration of BL (mg/L) Control 0.05 0.10 0.20 0.40 F CV(%) Experiment at seedling stage Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 6.11 c 8.73 b 9.71 a 9.46 a 6.03 c ** 5.03 8.59 c 10.01 b 10.86 a 9.76 b 8.81 c ** 4.29 6.20 c 8.03 b 9.35 a 9.54 a 7.47 b ** 7.15 7.31 c 7.05 c 10.96 b 12.06 a 10.89 b ** 7.83 Note: In a column, the numbers followed by the same letter were not significantly different at 1% level (**) by Duncan's multiple range test 4.3 Effects of brassinolide treatments on rice growth and yield under 6‰ salt-stressed condition in net house 4.3.1 Plant height at harvest - Experiment at seedling stage: Similarly to 3‰-salinity, when BL applications for rice grown under 6‰ salt-stressed condition at the seedling stage did not affect on the height at the harvest (Table 26) Table 4.26 Effects of BL treatments at different rice growth stages on plant height (cm) at harvest Concentration Experiment at of BL (mg/L) seedling stage 0.05 0.10 0.20 0.40 F CV (%) 77.88 79.57 79.08 82.66 78.51 ns 4.36 Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 76.53 b 81.84 a 81.40 a 80.80 a 79.49 ab * 3.46 71.28 75.33 75.39 78.25 74.14 ns 4.47 78.87 79.90 81.46 80.62 78.96 ns 3.82 Note: In a column, the numbers followed by the same letter were not significantly different at 5% level (*) by Duncan's multiple range test; ns: not significant difference - Experiment at tillering stage: Treatment of BL at this growing stage showed that plant height between different treatments had statistically significant differences at 5% level at harvest Spraying rice with BL concentration of 0.05; 0.10; 0.20 mg/L at tillering stage improved the height of the plants under saline condition If BL was used at higher concentration (0.4 mg/L), the height of rice plant at harvest was inhibited (Table 4.26) 15 - Experiments at panicle initiating and flowering stage: The plant height in BL sprayed treatments tended to be higher than the control, but statistically significant difference was not found (Table 4.26) According to Nguyen Van Bo et al (2016), when rice affected by salinity at 45-60 days after transplanting, the difference in height at harvest would be insignificant 4.3.4 The number of panicles per pot - Experiments at seeding and flowering stage: under saline condition rice treated with BL at these two stages showed an increase in the number of panicles/pot from 1.0 to 1.2 panicles/pot and from 1.0 to 1.6 panicles/pot, respectively in comparison to the control, but the differences were not statistically significant (Table 4.32) When saline water was applied to rice at later stages (from panicle initiating stage and onward) there would be little or no effect on number of panicles However, salt-stressed occurrence at the panicle initiating stage, rice would delay flowering time (Choi et al., 2003) and prolong the growth time of about - 10 days (Phap, 2006) - Experiment at tillering stage: Saline treatment and BL spraying when rice entered the tillering stage showed that the number of panicles/pot had a statistically significant change at 1% level between treatments Spraying BL for rice at the concentration of 0.05; 0.10; 0.20 mg/L gave the best number of panicles/pot (Table 32) - Experiment at panicle initiating stage: The number of panicle/pot had a statistically significant difference at 1% level between treatments (Table 4.32) The number of panicles was lower at high salinity caused by accumulating less anabolism in the reproductive system (Hasamuzzaman et al., 2009) Although, BL had a role in increasing the number of effective tillers (Abe, 1989), there was no increase or difference between treatments when BL applications at 50 or 55 days after sowing Table 4.32 Effects of BL treatments at different rice growth stages on number of panicle/pot at harvest Concentration Experiment at of BL (mg/L) seedling stage 0.05 0.10 0.20 0.40 F CV (%) 17.80 18.80 19.20 19.00 18.80 ns 7.85 Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 15.60 b 17.40 a 17.20 a 17.20 a 16.00 b ** 4.96 18.20 c 21.20 b 21.80 ab 22.80 a 21.20 b ** 4.29 26.40 27.40 27.20 28.00 27.00 ns 3.34 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) by Duncan's multiple range test; ns: not significant difference 4.3.5 The number of filled spikelets per panicle - Experiment at seeding stage: germinated rice seeds incubated with BL and sown under saline condition resulted in no change the number of filled spikelets/panicle at harvest (Table 4.33) Due to the short-term saline effect at this growing time, the rice 16 plants had ability to recover, therefore BL spraying with did not affect the number of filled spikelets/panicle Table 4.33 Effects of BL treatments at different rice growth stages on the number of filled spikelet/panicle at harvest Concentration of BL (mg/L) Experiment at seedling stage Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 0,05 0,10 0,20 0,40 F CV (%) 37.80 39.09 39.36 40.55 39.01 ns 3.25 36.36 c 38.53 abc 40.34 ab 41.52 a 37.76 bc * 6.64 21.74 c 23.35 b 28.39 a 25.48 b 23.72 bc ** 7.46 20.01 d 24.83 c 28.54 a 26.80 b 26.81 b ** 4.11 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) and 5% (*) level, respectively, by Duncan's multiple range test; ns: not significant difference - Experiment at tillering stage: The number of filled spikelets per panicle in the sprayed BL treatments increased number of filled spikelets per panicle (3.85–14.19%) when comparing to the control and the difference was statistically significant at 5% level (Table 4.33) Among them, spraying rice with BL concentration of 0.20 mg/L gave the highest number of filled spikelets per panicle (41.52 number of filled spikelets/panicle) - Experiments at panicle initiating and flowering stage: rice plants under high salinity conditions (6‰) in combination with BL applications, both experiments resulted in significant increases of the number of filled spikelets per panicle in contrast to those of the controls (Table 4.33) When rice salt-stressed at panicle initiating and at flowering stage treated with brassinolide of 0.10 mg/L showed the most effective on number of filled spikelets per panicle According to Arteca (1995), rice treated with BL contributed to increase number of filled spikelets per panicle This improvement could cause by the better leaf size which promoted the transport of carbohydrates to the seeds 4.3.6 The weight of 1000 grains - Experiments at seedling, tillering and panicle initiating stage: The weight of 1000 grains was not statistically different between the treatments in all three experiments (Table 4.34) - Experiment at flowering stage: Saline treatment and spraying brassinolide at flowering stage showed statistically significant difference in the weight of 1000-grain at 1% level between treatments (Table 4.34) Among them, BL applications at 0.10 and 20 mg/L gave the best weights of 1000-grain, the lowest value was found in the control treatment (25.51g) According to Anuradha and Rao (2003), BL had the effect on increasing the cell division rate in the chloroplasts of leaves, increasing the ability to accumulate chlorophyll, maintaining the leaf green index, which was one of the significances for a better photosynthetic ability of rice after flowering, thereby improving weight of grain under saline condition 17 Table 4.34 Effects of BL treatments at different rice growth stages on the weight of 1000 grains at harvest Concentration Experiment at of BL (mg/L) seedling stage 0.05 0.10 0.20 0.40 F CV (%) 26.15 26.55 26.40 26.22 26.05 ns 1.82 Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 25.27 25.83 26.00 26.64 25.99 ns 2.85 25.72 25.71 26.19 26.38 25.96 ns 1.69 25.51 b 25.52 b 26.88 a 26.85 a 26.62 ab * 3.28 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 5% (*) level, by Duncan's multiple range test; ns: not significant difference 4.3.7 The rice yield - Experiment at seedling stage: There was no statistically significant difference found in rice yield between treatments when rice grown under 6‰-salted condition and BL application at this stage (Table 4.35) The early stressed occurrence and good farming practices would help rice to recover completely at later stages of growth and development - Experiment at tillering stage: The rice yield per pot was statistically significant difference at 1% level between treatments (Table 4.35) Among them, BL application at 0.10 and 0.20 mg/L showed the most improvement on rice yield - Experiment at panicle initiating stage: When growing rice in salted condition and treated plants with BL of 0.10 or 0.20 mg/L at the panicle initiating stage improved effectively on rice yield per pot (Table 4.35) - Experiment at flowering stage: Treatments in which BL applied, rice yield per pot was higher than the untreated control (increased from 28.88 to 54.79%) (Table 4.35) Among them, BL application of 0.10 mg/L resulted in the best rice yield (20.85 g/pot) Table 4.35 Effects of BL treatments at different rice growth stages on rice yield (g/pot) Concentration Experiment at of BL (mg/L) seedling stage 0.05 0.10 0.20 0.40 F CV (%) 17.58 19.50 19.94 20.19 19.10 ns 7.75 Experiment at tillering stage 14.34 c 17.38 ab 18.03 a 19.04 a 15.72 bc ** 9.66 Experiment at panicle initiating stage 10.19 c 13.80 b 16.20 a 15.29 a 13.04 b ** 7.30 Experiment at flowering stage 13.47 d 17.36 c 20.85 a 20.14 ab 19.25 b ** 4.76 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) by Duncan's multiple range test; ns: not significant difference 18 4.3.8 Proline accumulation Experimental results (Table 4.36) showed that proline contents between treatments had significant differences - Experiment at seedling stage: BL application at 0.05 – 0.20 mg/L increased effectively on proline content (improvement from 44.41 to 59.21% when comparing to those of the controls) - Experiment at tillering stage: Similar to other experiments, BL application at 0.05 mg/L to rice at seedling stage showed the highest proline content (15.58 µmol/g fresh weight) - Experiment at panicle initiating stage: Proline accumulation in rice plants increased significantly at 1% level between treatments Among them, the treatments sprayed with BL at 0.10 – 0.20 mg/L showed the highest proline content (increased 77.67 – 92.40%, respectively in contrast to those of the controls) Table 4.36 Effects of BL treatments at different rice growth stages on proline accumulation (µmol/g) in plant Concentration Experiment at of BL (mg/L) seedling stage 0.05 0.10 0.20 0.40 F CV (%) 16.01 b 23.59 a 23.12 a 25.49 a 14.58 b ** 9.68 Experiment at tillering stage Experiment at panicle initiating stage Experiment at flowering stage 11.40 c 15.58 a 15.20 ab 15.31 ab 13.50 b ** 9.76 8.42 c 10.57 bc 14.96 a 16.20 a 11.48 b ** 14.25 9.29 c 10.00 bc 12.96 a 12.21 ab 11.95 ab * 14.08 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% (**) and 5% (*) level, respectively, by Duncan's multiple range test - Experiment at flowering stage: BL application at 0.10 mg/L showed the highest proline content (12.96 µmol/g fresh weight) Experimental results showed that appropriate BL applications might play a role in increasing proline accumulation to help rice tolerate better to salinity According to Phap (2006), brassinosteroids enhanced the proline accumulation in leaf cells as an adaptive trait for rice in relation to stress tolerance under saline condition 4.4 Effects of brassinolide treatments on rice growth and yield under saltstressed condition on the paddy rice field in Phuoc Long district, Bac Lieu province 4.4.1 pH and EC changes in soil solution The soil pH values in the experiment at Figure 4.1 showed that pH values were relatively stable and ranged from 4.6 to 5.6 According to Ngo Ngoc Hung et al (2004), pH value ranging from 6.0 – 7.5 considered as the best pH range for the growth and development of rice plants 19 Soil EC values at the experimental time reached the highest point (7.5 mS/cm) from the beginning of cultivating season and decreased gradually to harvest time (Figure 4.1) Figure 4.1: pH and EC changing in the soil during rice cropping season 4.4.3.1 The number of panicle/m2 The results in Table 4.39 showed that the number of panicle/m2 fluctuated from 412 – 473 panicles/m2 in treatments and had a statistically significant difference at 5% level Table 4.39 Effects of BL application on panicles/m2 and filled spikelets/panicle in the field experiment in Phuoc Long district, Bac Lieu province Concentration The increase The increase Weight of Number of Number of of BL compared to compared to 1000 grains panicles/m spikelet/panicle (mg/L) control (%) control (%) (g) 412 b 58.43 c 26.27 416 b 0.97 61.27 bc 4.60 25.07 432 ab 4.85 63.77 abc 9.14 25.67 463 a 12.38 69.93 a 19.68 26.25 473 a 14.81 67.67 ab 15.81 26.09 F * * ns CV (%) 5.01 6.25 3.15 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 5% (*) level, by Duncan's multiple range test; ns: not significant difference Treatments in which BL sprayed at seedling and at flowering stage had the highest number of panicles (473 panicles/m 2, increased 14.81% in comparison to that of the control treatment), no significant difference with treatments in which BL sprayed two times at seedling and at panicle initiation stage (463 panicles/m2, increasing 12.38% compared to the control treatment) The number of panciles in the control treatment without spraying BL was lowest in this research (412 panicles/m2) According to Grattan et al (2002) had revealed that salinity had significant effects on panicles generated The stress of rice plant in saline condition lead to slower 20 development in the height of rice plant and dramatically decrease the number of effective tillers 4.4.3.2 The number of filled spikelets per panicle The results (Table 4.39) showed that the number of filled spikelets/panicle fluctuated from 58.43 to 69.93 filled spikelets/panicle and had a significant difference at 5% by statistical analysis among treatments The number of filled spikelets/panicle was lowest in control treatment (58.43 filled spikelets/panicle) According to Abdulah et al (2001), the accumulation of high concentration Na + on rice flowers led to the decrease of pollen vitality and incompleted of seed formation; Treatments in which BL spraying at seedling and at tillering stages produced the highest number of filled spikelets/panicle (69.93 filled spikelets/panicle, increased 19.68% and 14.13% compared to control and seedling stage treatment, respectively) According to Hasamuzzaaman et al (2009), the number of filled spikelets/panicle decreased significantly when salinity increased The number of filled spikelets/panicle decreased at a salinity of 3.4 mS/cm (equivalent to 2.18‰) or higher (Zeng and Shannon, 2000) 4.4.3.3 The ratio of filled spikelet per panicle Data in Table 4.41 showed that the ratio of filled spikelet/panicle varied from 60.97 to 70.50% and was statistically significant difference at 1% level between treatments While the lowest ratio of filled spikelet was observed in the control treatment (non-treated BL), the highest percentage was obtained in treatment with BL applied at germination and at flowering stage Yoshida (1981) stated that the ratio of filled spikelet was determined by the capacity of carbohydrate reception and the transportation of other compounds from leaves to seeds Mohammadi et al (2010) reported that unfilled spikelet ratio was greatly affected by the environment and saline condition Effect of BL as growth regulator supported plants to increase leaf size and maintain leaf colour to maximize light efficiency, leading to an increase of filled spikelet ratio 4.4.3.4 The weight of 1000-grain The weight of 1000-grain among treatments varied from 25.07 – 26.27 gram and were insignificant difference by statistical analysis (Table 4.40) The decrease in weight of 1000 grains because of the limitation of photosynthetic capacity led to less accumulation of carbohydrate and other components (Hasamuzzaman et al., 2009) Khatun and Flowers (1995) reported that the decrease of 1000-grain weight coincides with the increase of salinity level 4.3.3.6 Rice yield Actual rice yield fluctuated from 3.59 to 5.5 tons/ha (Table 4.41) and statistically significant differences at 5% level were found between treatments Maintaining the growth ability of rice plants by spraying BL at seedling and at flowering stage had the highest actual yield (5.5 tons/ha, increasing 53.20% compared to the control treatment Similarly, twice of BL spraying at seedling and at panicle initiation period led to relatively good result (5.34 ton/ha, increasing 48.75% compared to that of the control) The control treatment was lowest in actual yield, only 3.59 tons/ha, and was insignificant difference from the treatment in which BL applied once at the seedling stage According to Singh (2006), 21 when rice plants cultivated continuously in saline conditions, salinity affected the formation of panicle, pollen germination, and flower fertilization, consequently, leading to increase the number of unfertilized panicles The effects of BL showed that this hormone, which stimulates the increase of leaf area and leaf age, increased the ability of photosynthesis and assimilation of nutrients to transport starch into seed Especially, when BL sprayed at the flowering stage enhanced rice growth and vitality of pollens, increasing the number of branch on rice panicle (Nguyen Minh Chon, 2005), and the increase of formation of filled spikelets Table 4.41 Effects of BL application on filled spikelet ratio (%) and rice yield (ton/ha) in the field experiment in Phuoc Long district, Bac Lieu province Concentration Filled spikelet of BL ratio (%) (mg/L) 60.97 c 63.95 bc 66.55 ab 67.52 ab 70.50 a F ** CV (%) 3.18 The increase compared with control (%) 4.89 9.15 10.74 15.63 Actual yield (ton/ha) 3.59 c 3.77 bc 4.35 b 5.34 a 5.50 a ** 8.41 The increase compared with control (%) 5.01 21.17 48.75 53.20 Note: Means in the same column followed by the same letter are not significantly different by Duncan test, ns: non-significant difference, (**): significant difference at the 1% level 4.5 Effects of brassinolide treatments on rice growth and yield under saltstressed condition on the paddy rice field in Gia Rai town, Bac Lieu province 4.5.1.1 pH and EC changes in soil solution Table 4.42 showed that EC value in experimental soil fluctuated from 2.65 mS/cm to 5.02 mS/cm The highest value of EC was recorded from the beginning day and 20 days after sowing, indicating its effect on rice plant growth and development, while at harvesting time EC value on the field was the lowest EC values tended to reduce gradually because of increasing precipitation to dilute the salinity of experimental soil Table 4.42 Soil pH and EC value at the different stages of rice growth Time point Data recording pH EC (mS/cm) Before sowing 5.24 5.02 20 DAS 40 DAS 60 DAS The harvest 5.40 5.48 5.70 5.84 4.58 3.15 2.97 2.65 pH values in soil solution was between 5.24 and 5.84 with the highest value at harvest time (Table 4.42) This range of pH was normal for rice growth and development (Ngo Ngoc Hung et al., 2004) 22 4.5.4.1 The number of panicles per m2 Data of Table 4.46 showed that the number of panicles per m2 was affected by times of BL spraying In BL sprayed treatments, the number of panicles per m2 were higher than that of the control, increasing from to 57 panicles/m2 compared to control treatment Statistic analysis showed significant difference at 1% level between treatments The treaments with more than two times of BL spraying at seedling and at later stages produced the highest number of panicle per m2 Table 4.46 Yield components and rice yield of experiment in Gia Rai, Bac Lieu province Concentration Number of of BL panicles/m2 (mg/L) 413 b 421 b 455 a 468 a 470 a F ** 3.59 CV (%) Number of grains/panicle 63.70 c 65.80 bc 67.90 ab 70.07 a 70.73 a * 2.92 Filled spikelet ratio (%) 40.83 c 41.70 c 44.60 b 46.79 a 47.00 a ** 2.15 Weight of 1000 grains (g) 25.66 25.92 26.16 25.88 25.88 ns 2.07 Yield (tons/ha) 4.05 b 4.26 b 4.91 b 5.24 a 5.27 a ** 5.07 Note: In a column, the numbers followed by the same letter(s) were not significantly different at 1% level (**) and 5% (*) level, respectively, by Duncan's multiple range test; ns: not significant difference 4.5.4.2 The number of filled spikelets/panicle The number of filled spikelets/panicle ranged from 63.70 to 70.73 filled spikelets/panicle and statistically significant differences at 5% level were found between treatments (Table 4.46) Spraying BL in treatments 3, and exhibited a significant different increase number of grains per panicle, from 4.2 to 7.03, compared with control treatment BL application might imrpove photosynthetic pigments such as chlorophyll, carotenoids, and therefore it could promote photosynthetic activity leading to the production of a mass amount of carbohydrate in rice plants Carbohydrate biosynthesis might depend upon the intensity of photosynthetic activity, leaves area and accumulation process (Nguyen Dang Hung and Vu Thi Thu, 2006) 4.5.4.3 The number of filled spikelet/panicle The number of filled spikelets/panicle in the treatments with BL spraying at seedling, at tillering and at later growing stages obtained an increase from 3.77 to 6.17 filled spikelets in contrast to that of the control (Table 4.46) BL application by spraying times at seedling, tillering, panicle initiation and flowering stage resulted in the highest number of filled spikelets/panicle (47 filled grains/panicle) but the difference from the treatment in which BL applied three times was not recognized Fujii and Saka (2002) reported that photosynthetic production reserved in the leaves was later transported into rice seeds at milking stage More than 80% of dry mass storage in rice grain was produced at the late flowering stage Applying brassinosteroids by spraying could be useful for pollination, resulting in more starch accumulation and contributing to the increase of filled spikelet ratio 23 4.5.4.4 The weight of 1000 grains The weight of 1000 grains varied from 25.66 g to 26.16 g and showed no significant differences between treatments (Table 4.46) Yoshida (1981) stated that grain weight was mostly decided by inheritance and the size of grain was controlled tightly by rice husk 4.5.4.5 Rice yield The loss of rice yield caused by salinity was extremely depended upon the growing stages and salt tolerance of rice variety Data from table 4.46 showed that BL treatments enhanced significantly actual rice yield between treatments The more times of BL application resulted in higher yield Das et al (2011) reported that BL addition in salt solution increased amount of dissolved carbohydrates, free amino acids, proline, and Na+/K+ ion ratio in GR-7, GR-11, GR-12, Dandi and Gurjari rice varieties after 5, 10 and 15 days after sowing, strongly supporting the idea of increasing rice yield in saline stress condition CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions When rice plants living under saline condition treated with brassinolide contributed to increase proline, photosynthetic pigments, and protease activity which enhanced rice tolerance to salinity At the 6‰-salted condition, brassinolide of 0.10 mg/L had also positive effects on catalase, total nitrogen content in shoots (10.97% higher than the control) Rice treated by brassinolide level of 0.05 mg/L increased 39.19% of total phosphorous content but decreased 11.7% of total sodium content in shoots in comparison to those of the control When growing rice in salted condition treated with brassinolide contributed to improve rice growth and yield The brassinolide application varies depending on saline levels and growing stage of rice as follows: (1) Rice at the seedling or tilling stage in 3‰-salted condition and treated plants with brassinolide of 0.05 mg/L improved rice yield, while growing rice at stages of panicle initiating and flowering, rice should be treated with brassinolide of 0.10 mg/L; (2) At salinity of 6‰, rice at the seedling stage and treated plants with brassinolide of 0.05 mg/L improved rice yield, while at stages of tilling or panicle initiating, and flowering, brassinolide should be sprayed at the concentration of 0.10 mg/L In the natural-salted fields (from 3‰ to 5‰) in Bac Lieu province, brassinolide application times per crop season for rice (0.05 mg/L BL at the seedling stage, 0.10 mg/L BL at the tilling stage and panicle initiating) improved rice growth and yield from 21% to 29% 5.2 Recommendations Continue to study on other rice cultivars and on different saline soils to draw general conclusions Continue to study: salt treatment at the beginning of the crop (for Summer -Autumn crop) and salt treatment at the end of the crop (panicle initiating + flowering stage) for the Winter-Spring crop in the Mekong Delta 24 ... characteristics of rice treated with brassinolide and effects of brassinolide treatments on rice at different stages of rice grown in net house and application of the most effective brassinolide- treated level... panicle initiating and flowering, brassinolide of 0,10 mg/L should be used (2) At salinity of ‰, application of brassinolide of 0,05 mg/L improved rice yield, in contrast brassinolide of 0,10 mg/L should... currently been studied and applied Many results showed that brassinolide (BL) (C28H48O6 - a natural lactone steroid discovered in 1979, belonging to brassinosteroids) could increase salinity tolerance