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

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

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:

1 Learning Resource Center of Can Tho University

2 National library of Vietnam

LIST OF PUBLISHED PAPERS

1 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

2 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

3 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

2

- The thesis has determined the concentrations of brassinolide treatment when rice grown under saline condition as follows: (1) At the 3 ‰-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 6 ‰, 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, 3 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

a) Design of experiment: Experiment was completely randomized design, one

factor, 5 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 3 replications, 4 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 3 days, distilled water was used as growing solution

- Salinization of nutrient solutions: The Yoshida nutrient solution (Yoshida et al.,

1976) was salted by dissolving 3 g of NaCl in 1 liter of nutrient solution The salinity of solution was measured by machine Each tray contained 3 liters of salted nutrient solution

- Saline treatment: After 3 days when seedlings grew well, replaced distilled

water by 3‰-salted nutrient solution The growing soltuions were renewed with the interval of 4 days

c) Data recording: Experimental data were collected at 8 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‰ stressed condition in net house

salt-* Design of experiment: Experiment was completely randomized design, one

factor, included 5 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 5 replications, each replication included 1 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 5 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 1 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

* 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 5 days of salt treatment (Bates et al., 1973)

b) Experiment 4: Effects of BL applications on rice growth and yield under 6‰ stressed condition in net house

salt-The experiment was conducted in the same procedures of experiment 3 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 3 (Section 3.3.2.1)

* Experimental procedure: The same procedures as above description The

differences were as followed:

- Brassinolide application: BL was sprayed 1 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 3 (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 6 was carried out in the same way as experiment 5 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 3 (Section 3.3.2.1)

* Experimental procedure: The same procedures as above description The

differences were as followed:

- Brassinolide application: BL was sprayed 1 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 3 (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 8 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 3 (Section 3.3.2.1)

* Experimental procedure: The same procedures as above description The

differences were as followed:

- Brassinolide application: BL was sprayed 1 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 3 (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

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 5 treatments and 3 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 Concentration of solution

1 Control untreated (spraying with water)

2 Treatment of rice varieties (seedling) Seedling: 0.05 mg/L

4 Seedling + tillering + panicle initiating Panicle initiating: 0.10 mg/L

5 Seedling + tillering + panicle initiating + flowering 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

6

- 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 m2, 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 Concentration of solution

1 Control untreated (spraying with water)

2 Treatment of rice varieties (seedling) Seedling: 0.05 mg/L

4 Seedling + tillering + panicle initiating Panicle initiating: 0.10 mg/L

5 Seedling + tillering + panicle initiating + flowering 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 3 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 8 days after salt stressed at 3‰ and 6‰

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 8 days after saline treatment at 3‰ and 6‰

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)

4.1.3 Catalase enzyme activity

- Experiment at salinity of 3‰: Incubating seeds with BL at different concentrations

showed that the catalase enzyme content between treatments was not significantly different

by statistical analysis (Table 4.4)

- Experiment at salinity of 6‰: Saline treatment and incubating seeds with BL at

different concentrations at the seedling stage showed that the catalase activity content between treatments was significantly different by statistical analysis (Table 4.4) Among them, spraying BL with a concentration of 0.10 – 0.40 mg/L gave the best catalase activity (increased from 74.06 to 81.33%), and the lowest was found in the control treatment

According to Gao et al (2008), under the salted conditions, catalase enzyme was

reported as an adaptive trait related to its ability to enhance plant tolerance to salinity The BL promoted the hydrogen peroxide cleavage faster by the catalase-boosting effect of plant cell peroxisomes (Halliwell, 1977) so that plants could absorb more water under stress conditions

Table 4.4: Effects of BL treatments on catalase activity (µmol H 2 O 2 /minute.mg protein ) in plants

8 days after saline treatment at 3‰ and 6‰

Concentration

of BL (mg/L)

Enzyme activity

Increase compared to control (%)

Enzyme activity

Increase compared to control (%)

4.1.4 Protease enzyme activity

- Experiment at salinity of 3‰: Experimental results showed that the

protease activity had a statistically significant difference (1%) between the

treatments (Table 4.5) At the 3‰-salted condition, spraying BL increased the protease enzyme activity from 0.081-0.227 Tu/mgprotein, respectively, comparing to that of the control Among them, BL application of 0.05 mg/L showed no increase

of protease activity

- Experiment at salinity of 6‰: The results of the 6‰ salinity experiment

tended to be similar to the 3‰ salinity experiment When incubating BL treatments showed that the protease activity was higher (0.031 – 0.062 Tu/mgprotein) compared

to the control (Table 4.5) Among them, BL application of 0.05 – 0.20 mg/L resulted in higher protease activity

Table 4.5: Effects of BL treatments on protease activity (Tu/mg protein ) in plants 8 days after saline treatment at 3‰ and 6‰

4.1.5 Mineral composition in plants

- Experiment at salinity of 3‰: The results of Table 4.6, Table 4.7 and

Table 4.8 showed that at the 3 ‰-salted condition, all analysed elements in plants were not different among BL treatments

- Experiment at salinity 6 ‰: When growing rice in 6 ‰-salted condition,

the accumulating mineral content (N, P) in the plants increased significant difference between the treatments (Table 4.6) Among them, N increased from 5.02

to 10.97% and the best Nts content (3.54% N) was found under 0.10 mg/L of BL concentration The incubation of seeds with BL of 0.05 – 0.20 mg/L increased P content from 32.43 to 45.95% compared to the control.The Na mineral content in plant (Table 4.8) had a significant decrease and there w ere statistically significant differences (1%) between treatments found When seeds were incubated with BL,

Na content decreased from 9.57 to 15.43% compared to the control At the same time, after 8 days of salt treatment, the content of K, Ca and Mg in plant were not significant difference between treatments (Table 4.7)

Note: ns: not significant difference

Table 4.8: Effects of BL treatments on Na (%Na) and Mg (%Mg) in plants 8 days after saline treatment at 3‰ and 6‰

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