Trang 1 BUI THI THU PHUONG Topic title: DEVELOPMENT OF DNA EXTRACTION METHOD OF CHEMICAL, INTENSIVE CHEMICAL AND TRANSITION SOIL AND THE EFFECT Trang 2 BUI THI THU PHUONG Topic title
INTRODUCTION
Background
Jasmine rice, officially known as Khao Dawk Mali 105 (KDML105), is a renowned aromatic rice originating from Thailand This popular rice variety was first discovered by a local farmer in 1945 in Lam Pradoo district, Chonburi province, and was later recognized in 1951.
199 panicles of the local variety were selected from a nearby district of Chachearngsoa province for pure line selection [2]
Figure 1.1 Long grain white Jasmine rice
(https://www.dreamstime.com/stock- photo-long-grain-uncooked-white- jasmine-rice-close-up-food-background- image65134089)
Jasmine rice originated in Bang Khla District, Chachoengsao Province, a region characterized by saline, sandy soil with no flooding This area has undergone significant transformation, evolving into a thriving industrial and residential hub, while also becoming a prominent rice-producing region in Northeast Thailand.
Jasmine rice is distinguished by its unique characteristics and size, featuring long, white grains that are at least 7.0 mm in length and 3.0 mm in width In terms of chemical composition, Jasmine rice contains between 12% and 19% amylose at a 14% humidity level Its desirable properties have made it a popular genetic resource for rice breeding, contributing to its widespread use in cultivating new rice varieties.
Jasmine rice is a nutrient-rich food, boasting an impressive array of vitamins and minerals, including vitamin B1, B2, niacin, carbohydrates, protein, iron, calcium, and phosphorus This versatile grain can be consumed in various ways, such as whole grain consumption, offering a range of health benefits The preparation methods of jasmine rice also vary across cultures, with some Asian countries cooking it in ample water to achieve a desirable texture, while Western cultures often boil it in excess water until the grain is fully cooked As a vital crop in Thailand, jasmine rice plays a significant role in both domestic consumption and economic growth, serving as a primary export commodity.
[8] It was produced annually at approximately 9.4 M ton of paddy rice from 4.3 M ha
[9] The annual export quantities of aromatic rice were as high as 1.06–1.45 million tons, which represented 20–27% of Thailand’s total rice export [10]
1.1.2 Jasmine rice growing 1.1.2.1 Life cycle of the rice plant
Figure 1.2 Growth phases and stages of the rice plant (https://nature-and- farming.blogspot.com/2014/10/rice-production-chapter-2-growth-stages.html)
The growth duration of a rice plant typically spans 3-6 months, influenced by factors such as variety and environment Throughout this period, the plant undergoes three distinct growth phases: vegetative, reproductive, and ripening The vegetative phase is marked by active tillering, gradual plant height increase, and regular leaf emergence In contrast, the reproductive phase is characterized by culm elongation, decline in tiller number, and the emergence of the flag leaf, booting, heading, and flowering of spikelets The ripening phase, which follows fertilization, is subdivided into milky, dough, yellow ripe, and maturity stages, based on grain texture and color, with varying lengths among different varieties.
Jasmine rice, also known as Hom Mali rice, is characterized by its tall and lanky architecture, reaching an average height of approximately 140 cm at harvest As a photo-sensitive variety, it can only be grown once a year, specifically during the period of August to December due to its photoperiod sensitivity However, planting in December is recommended for optimal growth and yield, resulting in high-quality paddy grains that meet the standard Notably, the Northeast region of Thailand is currently the best area for growing premium Hom Mali rice, with the country's total plantation area covering around 7.6 million acres and yielding 1125-1500 kg per acre.
Microbiota refers to the collective community of microorganisms inhabiting a specific environment, ubiquitous in nature, from soil and oceans to the atmosphere and the human body Present wherever the body interfaces with the external world, microbiota plays a vital role in sustaining life on Earth, with a profound impact on human, animal, and plant health.
The human microbiota has a profound impact on host physiology, comprising a diverse array of microbes including bacteria, archaea, viruses, and eukaryotic microbes Research suggests that the microbiota plays a crucial role in promoting immune cell maturation and normal immune function development However, an imbalance of the microbiota has been linked to various diseases, including infections, liver and respiratory diseases, and autoimmune disorders In contrast, a balanced microbiota is essential for maintaining homeostasis, with microorganisms in the gut, oral cavity, and skin communicating with the body's organ systems Similarly, plant-associated microorganisms are key drivers of plant health, productivity, and ecosystem functioning, and have been shown to promote plant growth and provide protection against pathogens.
The rhizosphere, a critical zone surrounding plant roots, is influenced by the roots, affecting the soil's biological and chemical parameters Root exudates play a key role in this process, enabling plants to sustain their microbiota, and are categorized into low molecular weight compounds, such as amino acids and sugars, and high molecular weight exudates, including mucilage and proteins The rhizosphere also harbors nitrogen-fixing bacteria that release nod factors, facilitating their attachment to plant roots, which in turn, enhances nutrient uptake and triggers the release of antimicrobial compounds to combat pathogens.
Figure 1.3 Root exudation in the rhizosphere
1.1.4 Microbiota application for plant growth promotion
The intricate relationship between plants and microbes plays a vital role in determining the health, productivity, and overall condition of the plant This complex interaction can range from beneficial to pathogenic, with varying outcomes depending on factors such as plant species and nutrient conditions Harnessing the power of microbiota, microbiome engineering has emerged as a promising approach to enhance plant growth by artificially selecting and engineering microbial communities with specific effects on host fitness Through multigenerational selection, microbiome engineering can restore balance to perturbed ecosystems, promoting high functional redundancy among soil microorganisms, enhanced phenotypes, and increased resistance to disease, ultimately leading to improved host fitness, growth, and productivity.
Figure 1.4 Microbiome engineering improves plant growth
Microbiome engineering is a rapidly expanding field of study and application globally, with a primary focus on plant-microbiome engineering aiming to optimize the interaction between plants and microorganisms for enhanced beneficial outcomes By leveraging this synergy, plant-microbiome engineering offers numerous advantages, including improved plant health and increased ecosystem diversity However, one of the notable drawbacks of this approach is the time-consuming nature of selection experiments, which can hinder its widespread adoption and implementation.
1.1.5 The characteristics of silty, sandy and clay soil
Soil particles vary significantly in size, with sand particles being the largest at 0.05 to 2 mm in diameter, silt particles falling in the middle range of 0.002 mm to 0.05 mm, and clay particles being the smallest at less than 0.002 mm in diameter.
Figure 1.5 Soil particle size (a) Sandy soil (b) Silty soil (c) Clay soil
(https://support.rainmachine.com/hc/en-us/articles/228001248-Soil- Types?mobile_site=true)
Sandy soil's large particle spaces make it dry and unable to retain water, while silty soil's moisture-retentive quality leads to poor drainage and cold temperatures In contrast, clay soil's unique texture allows it to hold onto water and nutrients, making it rich in plant food for better growth Although silty soil retains water longer than sandy soil, it struggles to hold onto nutrients, whereas clay soil's slower drainage enables it to maintain a tighter grip on essential plant nutrients.
Objectives
- To observe the effect of transition, chemical and intensive chemical soil to Jasmine rice growth
- To develop DNA extraction method suitable for silty, sandy and clay soil.
Scope of study
- Collect Jasmine rice seeds and the field soil samples, planting in pots
- Count the number of rice tillers once a week
- Compare the best rice plant and the worst rice plant (depend on the number of rice tillers) in each soil group and between 3 soil groups
- Collect the pictures of the rice plant
- Develop DNA extraction method from the field soil samples.
MATERIALS AND METHODS
Equipment and materials
2.1.1 Equipment Table 2.1 Equipment of studies
No Name of device Type Country
3 Deep freezer VXE 380 Czech Republic
16 PCR machine T100 TM Thermal Singapore
A total of 27 soil samples were collected from the field, comprising three distinct types: silty, sandy, and clay soil These samples were categorized into three groups, including chemical soil, intensive chemical soil, and transition soil, with nine samples in each group, as presented in Tables 2.2, 2.3, and 2.4, respectively All samples were stored at a temperature of -80°C to preserve their integrity.
Table 2.2 9 samples of chemical soil
Malai Tongjarern, a seasoned rice farmer from Khon Kean, Thailand, has been cultivating the land for over 20 years Located in Kokpochai, Bankok, his farm employs a combination of sustainable and conventional practices, including the application of green manure once and chemical fertilizer 16-8-8 at a rate of 25 kg per 1.6 square meters The soil type on this farm is predominantly silt, which supports the growth of rice crops.
Chemical soil group 2: Name of owner: Chuen Laokonka Address: Bankok, Kokpochai, Khon Kean, Thailand Grow rice more than 20 years Use: Green manure; Chemical fertilizer 15-15-15: 35 kg/1.6 m 2 (1 time) Soil type: silt
Chuen Laokonka, a seasoned rice farmer from Bankok, Kokpochai, Khon Kean, Thailand, has been cultivating rice for over 20 years His agricultural practices involve incorporating green manure once and applying chemical fertilizer 16-8-8 at a rate of 25 kg per 1.6 square meters, also once The soil type in his farm is primarily silt, which supports his rice cultivation endeavors.
Table 2.3 9 samples of intensive chemical soil
Intensive chemical soil group 1: Name of owner: Soodjai Prajummueang Address:
In Khon Kaen, Thailand, specifically in Kokpochai, Bangkok, an 8-year rice farm has been utilizing a combination of chemical and organic fertilizers to optimize crop yields The farm's rice cultivation process involves the application of 50 kg of 15-15-15 chemical fertilizer per 1.6 square meters, supplemented by two rounds of insecticide and three rounds of liquid bio-fertilizer Following the rice harvest, the farm transitions to corn planting, which also relies heavily on chemical fertilizers and insecticides Notably, the farm's soil type is predominantly silt, a crucial factor influencing its agricultural practices.
Intensive chemical soil group 2: Name of owner: Sombul Wongchaileng Address:
Moolnak, Pochai Kokpochai, Khon Kean, Thailand Use: Weed killer (1 time); Herbicide (1 time); Insecticide (1 time); Urea: 12.5 kg/1.6 m 2 (1 time); Fertilizer formulate 15-15-15: 12.5 kg/1.6 m 2 Soil type: silt
Intensive chemical soil group 3: Name of owner: Jirapon Jaikachem Address:
Moolnak, Pochai Kokpochai, Khon Kean, Thailand Use: Herbicide (1 time);
Insecticide (1 time) Urea: 20 kg/1.6 m 2 (1 time); Fertilizer formulate 15-15-15: 20 kg/1.6 m 2 (1 time) Soil type: silt
Table 2.4 9 samples of transition soil
Transition soil group 1: Name of owner: Jongrak Jarupunngam Address: Jorakhe,
Nongruea, Khon Kean, Thailand Start organic rice farm for 2 years Use insect dung: 30 kg/1.6 m 2 Soil type: clay
Transition soil group 2: Name of owner: Pattareeya Srisopon Address: Lerngphak,
Koodrung, Mahasarakam, Thailand Start organic rice farm for 1 year Use photosynthetic bacteria (1 time) Soil type: sand
Transition soil group 3, owned by Kidsanatchai Sawadsitung, is a notable example of an organic rice farm in Tapra, Mueang, Khon Kean, Thailand After three years of transitioning to organic farming, the farm has seen significant improvements To achieve this, the farm utilizes a combination of natural fertilizers, including 150 kg of chicken dung per 1.6 square meters, as well as regular applications of fermented banana shoot and photosynthetic bacteria every month The clay soil type has responded well to these organic methods, showcasing the potential for sustainable farming practices in the region.
Jasmine rice seeds were used in this study Seeds were collected from 25 Moo 11 Ban Pongchueag, Namakhuea, Sahadsakhan, Kalasin 46140, Thailand (Fig 2.1)
Figure 2.1 The seed of Jasmine rice
For DNA isolation, a DNA extraction buffer consisting of 0.1 M Tris-HCl, 0.1 M EDTA at pH 8, and 1.5 M NaCl was utilized The buffer was sterilized through autoclaving at 121°C for 20 minutes to ensure aseptic conditions This study employed three variations of the buffer, including one with the addition of 10% SDS and 1% activated charcoal, another with only 1% activated charcoal, and a control with no added SDS or activated charcoal.
Figure 2.2 Three types of DNA extraction buffer a b c
Methods
2.2.1 Preparing soil mixture for planting
This study utilized a customized soil mixture, comprising 4.5 kg of sterilized standard soil, 48 g of compost derived from rice bran and cow dung, and 300 g of field soil samples To account for varying water concentrations, the field soil samples underwent drying experiments, with the resulting weights recorded in Table 2.5 The soil mixture was then thoroughly combined and allocated to 30 pots, with 5 kg of mixture and added water in each, and divided into 27 planting pots and 3 control pots that did not contain field soil samples.
Table 2.5 The real weight of the field soil samples for planting (g)
Real weight soil for planting (g)
Real weight soil for planting (g)
Real weight soil for planting (g)
The selection of high-quality jasmine rice seeds is a crucial step before planting, as illustrated in Fig 2.3 This process involves submerging the seeds in a water flask, allowing for a simple yet effective separation of viable and non-viable seeds within just 2-3 minutes.
Figure 2.3 The process of germinating rice seeds
The germination process of Jasmine rice seeds commenced within 3-4 days and continued for a period of three weeks Following this initial phase, the best three plants were selected based on leaf length, while the two weakest plants were removed The chosen plants were then nurtured for 10 weeks, with the sole addition of water throughout the growth period Notably, water levels were meticulously controlled, maintained at a consistent 5 cm per pot to optimize the growth conditions for the Jasmine rice plants.
2.2.3 Development of DNA extraction method
Metagenomic DNA was extracted from field soil samples utilizing three distinct methods Notably, method 1, which involved sonication, was a novel approach developed specifically for this study to optimize DNA extraction from soil, as outlined in Table 2.6.
Take rice seeds into a water flask
Remove the bad seeds (above surface of water)
Select the good seeds (below surface of water)
Put 5 seeds for each pot (total: 27 pots)
Table 2.6 Metagenomic DNA extraction from soils using sonication
Weighed 5 g of soil sample and 5 g of small glass beads, transferred into 50 mL tube Added 5 mL of DNA extraction buffer, use two types of buffer: (1) added 1% of activated charcoal; (2) no added 10% of SDS and 1% of activated charcoal
Vortexed for 10 min and kept cool the soil samples for 10 min Sonicated at 30% amplitude, 40% amplitude, 50% amplitude and 60% amplitude, and duration of
5 times (the 30s for each time)
3 Centrifuged at 8000 rpm for 10 min at 4 o C Transferred 2.5 mL of the supernatant to 15 mL tube Repeated for 2 times
To precipitate the desired components, 500 μL of 3 M sodium acetate (CH3COONa) and 2 mL of polyethylene glycol (PEG) were added to the supernatant, followed by incubation at -20°C for 20 minutes The mixture was then centrifuged at 8000 rpm for 15 minutes at 4°C, resulting in the separation of the pellet from the supernatant, which was subsequently discarded The pellet was resuspended in 500 μL of distilled water and transferred to a fresh 1.5 mL Eppendorf tube for further processing.
Added 500 àL of phenol:chloroform: isoamyl alcohol (25:24:1) Centrifuged at
13500 rpm for 2 min at 4°C Collected the aqueous phase and transferred to a fresh 1.5 mL eppendorf Repeated for 2 times
Added 400 àL of chloroform: isoamyl alcohol (24:1) Centrifuged at 13500 rpm for 2 min at 4°C Collected the aqueous phase and transferred to a fresh 1.5 mL eppendorf Repeated for 3 times
Added 500 àL of ice-cold isopropanol Precipitated at 4°C for 10 minutes in a deep freezer Centrifuged at 13500 rpm for 15 min at 4°C Discarded the supernatant
8 The pellet was dried and dissolved in 30 àL of distilled water
Building on the foundation of Method 1, two additional approaches were developed: Method 2, which utilizes a combination of large and small glass beads, and Method 3, which incorporates small glass beads and three types of buffer Notably, the weight of the field soil samples in each of these methods remains consistent, ensuring comparable results across the board.
In method 2, the process began with the addition of large glass beads and an 0.85% Sodium chloride (NaCl) solution, which was then vortexed for 30 minutes to ensure thorough mixing Following this, small glass beads were added to the mixture, and sonication was omitted from this step The subsequent steps in method 2 were carried out in accordance with the protocol outlined in method 1.
In method 3, cell disruption was achieved using small glass beads in combination with three distinct buffer solutions: one containing 10% SDS and 1% activated charcoal, another with 1% activated charcoal only, and a third with no added SDS or activated charcoal, followed by vortexing to ensure thorough mixing.
To optimize the precipitation process, modifications were made to the original method In the first step, polyethylene glycol (PEG) was substituted with absolute ethanol, while in the second step, isopropanol was replaced with a combination of 3 M sodium acetate (CH3COONa) and absolute ethanol Furthermore, distilled water was swapped with nuclease-free water (NEB, UK) to enhance the purity of the process.
RESULTS
Compare the tiller number of the rice plant
Figure 3.1 Averages of tiller number per pot in chemical soil
The chart in Fig 3.2 reveals a notable trend in tiller number per pot, with averages increasing from week 4 to week 8 before declining after week 9 across all samples Notably, group 1 outperformed groups 2 and 3 in terms of tiller number after week 8, indicating a significant difference in growth patterns The standard errors of the means are represented by error bars, which were generated using Microsoft Excel 2010 to provide a clear visual representation of the data variability.
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 week 4 week 5 week 6 week 7 week 8 week 9 week 10
Figure 3.2 Averages of tiller number per pot in intensive chemical soil
The growth patterns of tiller numbers in transition soil were observed across three groups Notably, group 1 and group 3 exhibited an increase in tiller numbers from week 4 to week 9, followed by a decline at week 10, while group 2 showed an increase from week 4 to week 8, with a decrease after week 9 Initially, groups 1 and 3 outperformed group 2 in terms of tiller numbers, but by week 8 and week 10, group 3 surpassed both groups 1 and 2 The standard errors of the means are represented by error bars, which were generated using Microsoft Excel 2010.
Figure 3.3 Averages of tiller number per pot in transition soil
0 10 20 30 40 50 60 70 80 week 4 week 5 week 6 week 7 week 8 week 9 week 10
A v e ra g e o f ti ll er n u m b er
0 10 20 30 40 50 60 70 week 4 week 5 week 6 week 7 week 8 week 9 week 10
The growth patterns of tiller number per pot varied across three soil groups, with transition soil exhibiting an increase from week 4 to week 9, followed by a decline at week 10 In contrast, chemical soil showed an increase from week 4 to week 8, before decreasing after week 9 Notably, intensive chemical soil and transition soil initially outperformed chemical soil in terms of tiller number averages, although chemical soil ultimately achieved the highest average tiller number by week 8.
Figure 3.4 Averages of tiller number in three soil groups
Rice plants growth observation
The selection of the best and worst rice plants in each group was based on the tiller number results Notably, observations of rice plant growth revealed a rapid growth spurt from week 4 to week 8, but a decline was observed at week 10, as indicated by the yellowing of rice leaves A comparative analysis of rice plant growth across different soil groups and within each group is presented in tables 3.1, 3.2, 3.3, and 3.4, providing valuable insights into the impact of soil type on rice plant development.
0 10 20 30 40 50 60 70 week 4 week 5 week 6 week 7 week 8 week 9 week 10
Table 3.1 Rice plants growth in chemical soil
Table 3.2 Rice plants growth in intensive chemical soil
Table 3.3 Rice plants growth in transition soil
Table 3.4 Rice plants growth in three soil groups
Extraction of metagenomic DNA from different methods
Metagenomic DNA was extracted from transition soil using three distinct methods: sonication (method 1), a combination of big and small glass beads (method 2), and a combination of small glass beads and three types of buffer (method 3) Notably, transition soil group 1 and group 2 were utilized in method 1, while all groups of transition soil were employed in method 2, allowing for a comprehensive comparison of DNA extraction efficiency across different methods and soil groups.
Method 1, 2, and 3 were employed to assess DNA quantity and quality, with the latter two methods utilizing groups 1 and 3 DNA analysis was conducted using a nanodrop spectrophotometer, where a 260/280 ratio indicated protein contamination and a 260/230 ratio signified the presence of contaminants Notably, method 1 yielded low DNA quantity for transition soil group 1, but higher than group 2, whereas method 2 showed low DNA quantity across all samples In contrast, method 3 demonstrated good DNA quantity, as presented in Tables 3.5, 3.6, and 3.7 However, DNA quality was consistently low for all samples, with ratios below 1.8.
Table 3.5 DNA quantity and quality by method 1 for transition soil Sample DNA concentration (ng/àL) A 260/280 A 260/230
Table 3.6 DNA quantity and quality by method 2 for transition soil Sample DNA concentration (ng/àL) A 260/280 A 260/230
Table 3.7 DNA quantity and quality by method 3 for transition soil Sample DNA concentration (ng/àL) A 260/280 A 260/230
Analysis of soil samples on 0.8% agarose gels revealed varying results across three methods Method 1 yielded bright bands for DNA extracted from transition soil group 2, although the DNA was significantly degraded In contrast, Method 2 failed to produce bright bands for any of the samples Method 3 showed promise, with bright bands and minor impurities observed for sample 1 of transition soil group 3, as well as sample 2 of transition soil group 1, but was unsuccessful for the remaining samples.
Figure 3.5 Metagenomic DNA was extracted from transition soil (a) Method 1;
Land 1: T1 was sonicated at 30% amplitude; Land 2: T1 was sonicated at 50% amplitude; Land 3: T2 was sonicated at 60% amplitude; Land 4: T2 was sonicated at 40% amplitude (b) Method 2 (c) Method 3 Land M: 1 Kb DNA ladder Samples were electrophoresed on 0.8% agarose in 1X TAE buffer
Metagenomic DNA was successfully extracted from intensive chemical soil groups 2 and 3 using two distinct methods, with method 2 employing big and small glass beads for group 2, and method 3 utilizing small glass beads and three types of buffer for group 3 The extracted DNA was then assessed for quantity and quality using a nanodrop spectrophotometer, which revealed good DNA yields of over 100 ng/μL across all samples However, the DNA quality was found to be lower than the desired threshold of 1.7, as indicated by the A260/280 and A260/230 ratios, which are indicative of protein contamination and the presence of other contaminants, respectively.
Table 3.8 DNA quantity and quality by method 2 for intensive chemical soil Sample DNA concentration (ng/àL) A 260/280 A 260/230
Table 3.9 DNA quantity and quality by method 3 for intensive chemical soil Sample DNA concentration (ng/àL) A 260/280 A 260/230
Analysis of the extracted soil samples on 0.8% agarose gels revealed bright bands for all samples using methods 2 and 3 Notably, method 2 produced tight bands with minimal smearing and high DNA concentration across all samples In contrast, method 3 yielded tight bands with light smearing, but with varying molecular weights - high for samples 1 and 3, and low for sample 2 However, RNA contamination was still evident on the gel.
Figure 3.6 Metagenomic DNA was extracted from intensive chemical soil group 2 and group 3 (a) Method 2 (b) Method 3 Land M: 1 Kb DNA ladder Samples were electrophoresed on 0.8% agarose in 1X TAE buffer
Metagenomic DNA was successfully extracted from soil samples belonging to chemical groups 1 and 3, as well as transition group 2, utilizing a method that involved small glass beads and three types of buffer The extracted DNA was then evaluated for quantity and quality using a nanodrop spectrophotometer The results showed that the DNA samples were assessed for purity, with the A260/280 ratio indicating protein contamination and the A260/230 ratio signifying the presence of other contaminants.
All of the samples have good DNA quantity (more than 200 ng/àL) However, DNA quality is lower than 1.5 (Table 3.10)
Table 3.10 DNA quantity and quality by method 3 for chemical soil and transition soil
Sample DNA concentration (ng/àL) A 260/280 A 260/230
Analysis of the extracted soil samples using 0.8% agarose gels revealed that method 3 yielded high-quality results, characterized by bright bands with minimal smearing and high molecular weight across all samples, as shown in Fig 3.7 However, RNA contamination was still present in the gel.
Figure 3.7 Metagenomic DNA was extracted by method 3 (a) Chemical soil group 1 and group 3 (b) Transition soil group 2 Land M: 1 Kb DNA ladder Samples were electrophoresed on 0.8% agarose in 1X TAE buffer.
CONCLUSIONS AND DISCUSSION
a) Method 2 (b) Method 3
DNA Deoxyribonucleic acid mm Millimeter m 2 Square meter
% Percentage àL Microliter mL Milliliter g Gram kg Kilogram min Minutes s Second ºC Degree centigrade or Celcius rpm Revolutions per minute ng Nanogram
Jasmine rice, officially known as Khao Dawk Mali 105 (KDML105), is a renowned aromatic rice originating from Thailand Its rich history dates back to 1945 when a local farmer in Lam Pradoo district, Chonburi province, discovered the exceptional local variety, which was later recognized in 1951.
199 panicles of the local variety were selected from a nearby district of Chachearngsoa province for pure line selection [2]
Figure 1.1 Long grain white Jasmine rice
(https://www.dreamstime.com/stock- photo-long-grain-uncooked-white- jasmine-rice-close-up-food-background- image65134089)
Jasmine rice originated in Bang Khla District, Chachoengsao Province, an area initially characterized by saline, sandy soil and no flooding Over time, this region has undergone significant transformation, evolving into a thriving industrial and residential hub Today, it stands as the most renowned and expansive rice production area in Northeast Thailand, solidifying its reputation as a premier hub for Jasmine rice cultivation.
Jasmine rice is distinguished by its unique characteristics and size, featuring long, white grains with an average length of at least 7.0 mm and an average width of at least 3.0 mm In terms of chemical attributes, Jasmine rice contains between 12 and 19 percent amylose at a 14 percent humidity level, contributing to its desirable properties These characteristics have made Jasmine rice a valuable genetic resource for rice breeding, with its cultivar frequently utilized in the development of new rice varieties.
Jasmine rice is a nutrient-rich food, boasting an impressive array of vitamins and minerals, including vitamin B1, B2, niacin, carbohydrates, protein, iron, calcium, and phosphorus This versatile grain can be consumed in various forms, such as whole grain, offering a range of health benefits Across cultures, the cooking methods for jasmine rice vary, with some Asian communities preferring to cook it in ample water to achieve the desired texture, while Western cultures often boil it in excess water until the grain is fully cooked As a staple crop in Thailand, jasmine rice plays a vital role in both domestic consumption and economic growth, serving as a primary export commodity.
[8] It was produced annually at approximately 9.4 M ton of paddy rice from 4.3 M ha
[9] The annual export quantities of aromatic rice were as high as 1.06–1.45 million tons, which represented 20–27% of Thailand’s total rice export [10]
1.1.2 Jasmine rice growing 1.1.2.1 Life cycle of the rice plant
Figure 1.2 Growth phases and stages of the rice plant (https://nature-and- farming.blogspot.com/2014/10/rice-production-chapter-2-growth-stages.html)
The growth duration of a rice plant typically spans 3-6 months, influenced by factors such as variety and environmental conditions During this period, the plant undergoes three distinct growth phases: vegetative, reproductive, and ripening The vegetative phase is marked by active tillering, gradual increase in plant height, and regular leaf emergence, while the reproductive phase is characterized by culm elongation, decline in tiller number, and the emergence of the flag leaf The ripening phase, which follows fertilization, can be subdivided into milky, dough, yellow ripe, and maturity stages, with the length of ripening varying among varieties.
Jasmine rice is characterized by its tall and lanky architecture, typically reaching a height of approximately 140 cm at harvest As a photo-sensitive variety, it can only be grown once a year, with optimal growth during the period of August to December In Thailand, December is considered the ideal time for planting Jasmine rice, resulting in high-quality paddy grain that meets the standard Notably, the Northeast region of Thailand is renowned for producing the best Hom Mali rice, with the country's total plantation area spanning around 7.6 million acres and yielding 1125-1500 kg per acre.
Microbiota refers to the diverse community of microorganisms present in various environments worldwide, including soil, oceans, atmosphere, and the human body, particularly in areas where it interfaces with the external world The omnipresence of microbiota underscores its significance in sustaining life on Earth, as it has a profound impact on the health and well-being of humans, animals, and plants.
The human microbiota has a profound impact on host physiology, comprising a vast array of microorganisms including bacteria, archaea, viruses, and eukaryotic microbes Research has shown that a balanced microbiota is crucial for the maturation of immune cells and the development of normal immune functions, while an imbalance can contribute to various diseases, including infections, liver and respiratory diseases, and autoimmune disorders Similarly, the microbiota of animals and plants plays a vital role in maintaining homeostasis and overall health, with plant-associated microorganisms driving plant growth, productivity, and ecosystem functioning, while also providing protection against pathogens.
The rhizosphere, the zone surrounding a plant root, is a dynamic interface where the root's biological and chemical activities significantly impact the surrounding soil environment Root exudates play a crucial role in this process, enabling plants to sustain their microbiota and facilitating a symbiotic relationship between the plant and microorganisms These exudates are primarily composed of two types of compounds: low molecular weight compounds, such as amino acids, sugars, and phenolics, which contribute to the diversity of root exudates, and high molecular weight exudates, including mucilage and proteins, which make up a substantial proportion of root exudates This complex interaction also involves nitrogen-fixing bacteria, which release nod factors to aid in the attachment to plant roots, ultimately allowing plants to enhance nutrient uptake and defend against pathogens by releasing antimicrobial compounds.
Figure 1.3 Root exudation in the rhizosphere
1.1.4 Microbiota application for plant growth promotion
The plant-microbe relationship is crucial for plant health, productivity, and overall condition, with interactions ranging from beneficial to pathogenic and varying outcomes depending on plant species and nutrient conditions Microbiome engineering, a form of microbiota application, aims to improve plant growth by engineering a community of culturable and unculturable microbes with specific effects on host fitness This experimental method involves multigenerational, artificial selection to rebalance ecosystems disrupted by perturbations, which can lead to decreased microbial diversity, increased disease rates, and reduced host fitness By restoring balance to ecosystems, microbiome engineering can enhance phenotypes, increase resistance to pathogens, and promote high functional redundancy in soil microorganisms, ultimately improving soil fertility and plant productivity.
Figure 1.4 Microbiome engineering improves plant growth
Microbiome engineering has become a widely studied and applied field globally, with a significant focus on plant-microbiome engineering The primary objective of this field is to manipulate the plant-microbiome interaction to achieve enhanced beneficial outcomes for the plant, resulting in improved plant health and increased ecosystem diversity However, one of the major drawbacks of plant-microbiome engineering is the time-consuming nature of selection experiments, which can hinder the progress of research and applications in this field.
1.1.5 The characteristics of silty, sandy and clay soil
The size of soil particles varies significantly, with sand particles being the largest, ranging from 0.05 to 2 mm in diameter Silt particles fall in the middle, measuring between 0.002 mm and 0.05 mm in diameter In contrast, clay particles are the smallest, with diameters of less than 0.002 mm, making them a crucial component in determining soil texture and composition.
Figure 1.5 Soil particle size (a) Sandy soil (b) Silty soil (c) Clay soil
(https://support.rainmachine.com/hc/en-us/articles/228001248-Soil- Types?mobile_site=true)
Sandy soil's dry nature is attributed to the large spaces between its particles, resulting in poor water retention In contrast, silty soil is smooth to the touch and retains water longer, although it has limited capacity for holding nutrients Conversely, clay soil boasts a unique characteristic, being sticky when wet and smooth when dry, which enables it to drain slowly and tightly hold onto essential plant nutrients, ultimately making it rich in plant food for optimal growth.
- To observe the effect of transition, chemical and intensive chemical soil to Jasmine rice growth
- To develop DNA extraction method suitable for silty, sandy and clay soil
- Collect Jasmine rice seeds and the field soil samples, planting in pots
- Count the number of rice tillers once a week
- Compare the best rice plant and the worst rice plant (depend on the number of rice tillers) in each soil group and between 3 soil groups
- Collect the pictures of the rice plant
- Develop DNA extraction method from the field soil samples
PART II MATERIALS AND METHODS 2.1 Equipment and materials
2.1.1 Equipment Table 2.1 Equipment of studies
No Name of device Type Country
3 Deep freezer VXE 380 Czech Republic
16 PCR machine T100 TM Thermal Singapore
A total of 27 soil samples, comprising three distinct types - silty, sandy, and clay soil, were collected and categorized into three groups: chemical soil, intensive chemical soil, and transition soil, with nine samples in each category These samples were then stored at a temperature of -80°C for further analysis.
Table 2.2 9 samples of chemical soil
Malai Tongjarern, a seasoned rice farmer from Khon Kean, Thailand, has been cultivating rice for over 20 years Based in Bankok, Kokpochai, Malai's farming practices involve using green manure once and chemical fertilizer 16-8-8 at a rate of 25 kg per 1.6 square meters, also applied once The soil type in this chemical soil group 1 is predominantly silt, which supports Malai's rice cultivation efforts.
Chemical soil group 2: Name of owner: Chuen Laokonka Address: Bankok, Kokpochai, Khon Kean, Thailand Grow rice more than 20 years Use: Green manure; Chemical fertilizer 15-15-15: 35 kg/1.6 m 2 (1 time) Soil type: silt