Extended Abstract : Biology and Biodiversity

12th SCiUS Forum

2.4. Put a start point as we used circle paper then
put 10 of T. molitor with 2.0-2.2 cm. and average
mass is 0.8 g. then count for 1 minute and take
off the paper.
2.5. Observe T. molitor movement for 5, 10, 20,
30, 40, 50, 60, 120, 160, 180, 240, 300 minutes,
respectively. Then, record and analyze.

Results and Discussion

Figure 1: Cannabis Callus in tissue culture

Culturing Cannabis calluses have survived at a rate

of 20-30% and grew strongly in ½ MS medium

combined with sugar and agar. Calluses in liquid

medium of ½ MS, 2,4-D, sugar, and agar. Calluses

are induced within 1-2 weeks.

The Cannabis in tissue culture shows the rate of

survival as in cultural jars the C. Sativa survives 15 jars
within ½ MS medium. Cannabis species “ESK132-2” survived 30 jars in ½ MS. Cannabis species “ESK 1914”

could not survive in ½ MS medium. The results reveal a survival chance within 2 weeks in ½ MS which shows a

suitable medium for Cannabis cultivation.

Table 1: Number of T. molitor on each side with Repellent Index

Time (min) 5 10 20 30 40 50 60 120 160 180 240 300
Without
Cannabis 3 43 5 4 4 5 4 4 4 4 6
Cannabis 5 55 4 4 5 4 5 4 5 4 3
No
movement 2 12 1 2 1 1 1 2 1 2 1
Repellent
Index 0.2 0.1 0.2 -0.1 0 0.1 -0.1 0.1 0 0.1 0 -0.3

Figure 2: Repellent activity in the graphic statistic
This table displays the number of T. molitor inside a box with Repellent Index
Repellent index, RI, shows repellent from Cannabis to T. molitor or mealworm. It’s calculated from:

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− ℎ


The result illustrates a few differences in the T. molitor movement despite Cannabis. The repellent index
demonstrates a little change between Cannabis and without Cannabis, this could occur in the case of T. molitor does
not have a receptor for cannabinoids. But at some point, T. molitor rashly moves away from Cannabis. In addition,
the consumption of chicken feed obviously observed that the without Cannabis side is greater than the Cannabis
side in comparisons of ingested quantity. The repellent index depicts ambiguous results. Therefore, Cannabis might
have the ability to repel T. molitor away from the chicken feed.

Figure 3: 5 Figure 4:
minutes of 300 minutes
observation of
observation

Conclusion
The result represents unreliable movement observation as the repellent index reveals a slight change of

movement between -0.3 to 0.2. However, the consumption depicts Cannabis likely affects T. molitor, by less
consuming chicken feed that surrounds the Cannabis. The experiment needs metabolites analyze with HPLC and
add abiotic stress tolerance experiment in the future.

Acknowledgments
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by the Ministry of Higher Education, Science, Research, and Innovation. This
extended abstract is not for citation.

References
1. Jin D., Dai K., Xie Z., and Chen J. 2020. Secondary Metabolites Profiled in Cannabis Inflorescences,

Leaves, Stem Barks, and Roots for Medicinal Purposes. Retrieved from https://doi.org/10.1038/s41598-
020-60172-6
2. John M. McPartland, J. Agraval, D. Gleeson, K. Heasman, and M. Glass. 2006. Cannabinoid receptors in
invertebrates. Retrieved from https://pubmed.ncbi.nlm.nih.gov/16599912/
3. John M. McPartland. 2005. Cannabis as repellent and pesticide. Retrieved from
https://doi.org/10.1111/j.1420-9101.2005.01028.x
4. Tanney CAS, Backer R, Geitmann A and Smith DL. 2021. Cannabis Glandular Trichomes: A Cellular
Metabolite Factory. Retrieved from https://www.frontiersin.org/articles/10.3389/fpls.2021.721986/full

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:Title Organic formula created for dendrobium orchids OB1_01_08

to help with mericlonal propagation Dendrobium orchids

(Dendrobium Sonia)

Field : Biology and Biodiversity

Author : Tamonwan Fannuwong , Thammajaree Inyavileart

:School Chiang Mai University Demonstration School

:Advisor Mr. Kusol Raungprataungsuk

Mr. Tanuchai Smitasiri

Abstract
Orchids are a celebrated symbol of tropical paradise, and are a strategic product in Thailand’s

efforts to grow its exports. Top selling species include Dendrobium Sonia. In order to cultivate orchids, it is
necessary to rely on plant culture tissue , so as to propagate orchids rapidly. Plant tissue culture theory has
been shown to maintain the same genetic characteristics of the cultivated orchid species. In addition to the
large Dendrobium orchid family, D. Sonia genus and subspecies have become a key commercial orchid
subspecies for export as potted plants and as cut flowers. This type of orchid is popularly cultured with
protocorm-like bodies (PLBs) as documented by Vacin & Went, 1949 (VW 1949) and Murashige & Skoog
medium, 1962 (MS 1962). The cultivation method was developed by comparing two organic formulae
created for D. Sonia. Organic formula 1 was composed of spent water used to boil soybeans, and organic
formula 2 was composed of ground soybean pulp that coalesced for 42 days. The results showed that PLBs
cultured in organic formula 1 and organic formula 2 had average growth rates of -0.35 and 0.13 square
millimeters per day respectively, while the VW 1949 and MS 1962 formulae were controls formulae, which
showed average growth rates of 0.34 and 0.19 square millimeters per day respectively. It was found that the
organic formula 2 and MS 1962 formula had similar growth rates. Therefore, organic formula 2 can be used
to propagate D. Sonia orchids as an effective substitute of the MS 1962 formula. It is also a simple
preparation process; furthermore, the ingredients used for formula 2 are cheap, can be applied to other
orchids’ subspecies and also apply to other kinds of plant.

:Keywords Dendribium Sonia , Tissue culture , Organic formula

Introduction
Dendrobium orchids are one of the most important export orchids of Thailand. Therefore, it is

necessary to propagate by a tissue culture method, which is a method that produces large quantities and
doesn’t mutate. In general, the formulas used in orchid tissue culture are Murashige & Skoog (MS) and
Vacin & Went (VW) which are chemically formulated. For that reason, the researcher was interested in

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developing a formula with all organic ingredients. which has soybean as the main ingredient raw materials
can be found in the market. There is an easy way to prepare and to compare its performance with MS and
VW formulas.

Methodology
The experiment was divided into 3 parts as follows,
Part 1 : Cooking food for tissue culture.

1.1 Prepare equipment and raw materials for tissue culture cooking.
Prepare ingredients such as cavendish bananas, tomatoes, potatoes, pandan leaves, water

obtained from boiling soybeans and ground soybean pulp.
1.2 Plant tissue culture cooking process
1.2.1 The study of the growth rate of PLBs of D. Sonia using food cultivated plant tissues

whose main ingredient is water obtained from boiling soybeans.
1. Mix the ingredients according to prepared proportions.
2. Add water obtained from boiling soybeans, sugar and egg yolks.
3. Microwave for 5 minutes or until potatoes are cooked.
4. Filter using a thin white cloth.
5. To be filtered in a filter set with a vacuum.
6. Add coconut water and gel.
7. Add distilled water and adjust the volume.
8. Put it in the microwave for about 5 minutes.
9. add eggshell extract.
10. Adjust the pH to about 5.6-5.8.
11. Place into test tubes of 5 milliliter each and cover with aluminum foil or plastic cap.
12. Put in a hot plastic bag, squeeze out the air, and secure it with a rubber band.
13. Placed in a vapor pressure cooker at 18 pounds per square inch for 20 minutes or a

pressure drop to 0 pounds per square inch.
1.2.2 The second experiment was to study of the growth rate of PLBs of D. Sonia using

food cultivated plant tissues whose main ingredient is ground soybean pulp.
1. Do the same as 1.2.1, but change from water obtained from boiling soybeans to ground

soybean pulp. obtained from grinding, spinning and filtering by using thin white cloth.
2. Do the concentration -10 times, 1 times and 10 times.
1.2.3. Preparation of MS and VW

Part 2 : PLBs of D. Sonia in a tissue culture medium.
2.1 Prepare the equipment.
2.2 Put the PLBs of D. Sonia in a test tube.
2.3 Putting the ruler in the test tube.

Part 3 : Measuring the surface area of PLBs of D. Sonia
3.1 Prepare equipment and take pictures.

3.2 Measure the surface area of the PLBs of D. Sonia in Image J program.

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Results
1. D. Sonia tissue culture in an organic formula 1

1.1 Survival rate of PLBs of D. Sonia in organic formula 1
From the experimental results, the results showed that PLBs of D. Sonia cultured in organic
formula 1 had 4 of the 31 plants that survived, representing a survival rate of 12.00 percent.

1.2 Growth rate of PLBs of D. Sonia formula 1
Figure 1. Shows the relationship between time and surface area of PLBs of D. Sonia in organic
formula 1.

From the figure 1, When PLBs of D. Sonia were put in
organic formula 1 on the first day, it was found that the mean
surface area of PLBs of D. Sonia was 30.28±5.13 mm2, Day 14
21 35 and 42 had mean surface area of 30.23±11.34
17.05±13.33 19.88±4.42 and 15.95±6.16 mm2, respectively.
The average growth rate of PLBs of D. Sonia was -0.35 mm2
per day.

2. D. Sonia tissue culture in an organic formula 2
2.1 Survival rate of PLBs of D. Sonia in organic formula 2
From the experimental results, the results showed that PLBs of D. Sonia cultured in organic

formula 2 had 12 of the 18 plants that survived, representing a survival rate of 66.67 percent.

2.2 Growth rate of PLBs of D. Sonia in organic formula 2
Figure 2. Shows the relationship between time and surface area of PLBs of D. Sonia in organic
formula 2.

From the figure 2, When PLBs of D. Sonia were put in
organic formula 2 on the first day, it was found that the mean
surface area of PLBs of D. Sonia was 9.93±4.67 mm2, Day 14
21 35 and 42 had mean surface area of 7.24±2.76 11.52±4.30
10.59±5.79 and 15.96±12.23 mm2, respectively. The average
growth rate of PLBs of D. Sonia was 0.13 mm2 per day.

3. PLBs of D. Sonia tissue culture in MS formula
3.1 Survival rate of PLBs of D. Sonia in MS formula
From the experimental results, the results showed that PLBs of D. Sonia cultured in MS formula

had 18 of the 18 plants that survived, representing a survival rate of 100.00 percent.

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3.2 Growth rate of D. Sonia in MS formula
Figure 3. Shows the relationship between time and surface area of PLBs of D. Sonia in MS formula.

From the figure 3, When PLBs of D. Sonia were put in
MS formula on the first day, it was found that the mean surface
area of PLBs of D. Sonia was 10.98±3.19 mm2, Day 14 21 35
and 42 had mean surface area of 11.71±5.30 16.75±6.65
13.90±8.06 and 20.68±14.48 mm2, respectively. The average
growth rate of PLBs of D. Sonia was 0.19 mm2 per day.

4. PLBs of D. Sonia tissue culture in VW formula
4.1 Survival rate of PLBs of D. Sonia in VW formula
From the experimental results, the results showed that PLBs of D. Sonia cultured in VW

formula had 18 of the 18 plants that survived, representing a survival rate of 100.00 percent.

formula. 4.2 Growth rate of PLBs of D. Sonia in VW formula
Figure 4. Shows the relationship between time and surface area of PLBs of D. Sonia in VW

From the figure 4, When PLBs of D. Sonia were put in
VW formula on the first day, it was found that the mean surface
area of PLBs of D. Sonia was 13.45±7.51 mm2, Day 14 21 35
and 42 had mean surface area of 12.06±5.38 18.74±7.99
18.71±9.80 and 29.25±11.09 mm2, respectively. The average
growth rate of PLBs of D. Sonia was 0.34 mm2 per day.

Conclusion
When PLBs of D. Sonia were sterile and cultured on four different mediums for 42 days, the VW

medium was found to grow PLBs of D. Sonia with a mean maximum growth rate of 0.34 mm2 per day
followed by MS food formula organic 2 organic 1, respectively, with an average growth rate of 0.19 0.13
and -0.35 mm2 per day, respectively.

The survival rate of MS and VW tissue culture medium was 100.00 percent, and the survival rate of
organic formula 2 and organic formulation 1 was 66.67 and 12.00 percent.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation.

References
Anupan Kongbangkerd, Sangduean Wannachart. Effect of medium components on growth and development
of in vitro shoot culture of Dendrobium ochreatum Lindl. NU Science Journal 2011; 8(2): 67-76.

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Title : Increasing Qualities of Amaranth Microgreens OB1_19_02

Under Plant Factory System by LED light

Field : Biology and Biodiversity

Author : Ms. Warada Yusoh

Ms. Wachiya Malaeh

School : Islamic Sciences Demonstration School, Prince of Songkla University, Pattani Campus

Advisor : Dr. Eaknarin Ruangrak

Department of Agriculture, Faculty of Science and Technology, Prince of Songkla

University, Pattani Campus

Abstract
Microgreens are seedlings grown from the seeds of vegetables, herbs, and grains that are regarded as

functional food with high mineral contents and helpful bioactive compounds. Nowadays, the research on the
effects of light factors affecting the growth and quality of microgreens is usually confined. Therefore, this study
was focused on determining the optimal light spectrum that enhanced the growth and nutritional values of Thai
amaranth microgreens with growth LED artificial light. There were cultured under different light spectra for 8
days provided by illuminated continuously 16 hours for a day. The experiment was replicated three times using
statistics of a randomized complete block design (RCBD). Thai amaranth microgreens gave different light
spectrums: red, blue, red + blue (70:30), and white. The results revealed that the light spectrum played an
important role in the production of phytochemicals in Thai amaranth microgreens. Although the
monochromatic blue spectrum had the worst overall performance, it provided the highest antioxidant capacities
of Thai amaranth microgreens. For the overall performance, a red and blue ratio of (70:30) LED spectrum can
increase the nutritional value of Thai amaranth microgreens, according to the findings.

Keywords : Microgreens, Light spectrum, Plant factory

Introduction
The covid-19 pandemic has made people around the world that has turned their attention to health

and food consumption. Microgreens have become one of the options for nutritional food. Most of the scientific
research has found that plants grown in the microgreen stage have the highest nutritional value, such as
carotenoids, vitamins, antioxidants, and phenols. Most people are interested in growing microgreens because
their nutritional value is higher than that of mature vegetables up to 40 times. This result in high market value
due to higher demand. The amaranth is one of the most commonly used vegetables for microgreens. They are
known for their different flavors and textures for each variety of amaranth. Amaranth can produce a good
nutritional food, flavors and textures under suitable light spectrums. LED light source is one of light source
that can produce specific spectrum, electrical saving and low heat. Therefore, the LED lighting system is used
as the light source for microgreens production system, recently. The lighting spectrum can be set to suit the

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needs of the plants. It allows plants to grow better than they get from sunlight and the yield is higher quality.
Research into the effects of light factors on microgreen growth and quality is usually confined. Thus, this study
was focused on identifying suitable light spectrum for enhancing the growth and nutrition of Thai amaranth
microgreens under LED light source.

Methodology
The methodology is divided into 4 parts as follows,
Part 1: Growing microgreens
Put the coconut coir in the tray water the coconut dust with moisture,
then sow Thai amaranth seeds into a tray after that controlled at 25 ±
2 °C and leave it for 1-2 days.
Part 2: Experiment
The experiments: The study used a randomized complete block
design (RCBD) with 3 replications consisting of 4 light spectrums
include: red (600−700 nm), blue (400−500 nm), red + blue ratio of

(70:30), and white (430−780 nm) with the lights on for 16 hours/day to Thai amaranth microgreens all over the
experiment period for 8 days.
Part 3: Preparation of yield
At harvest time, cut the stem and leaves, wash the coconut dust from the Thai amaranth microgreens and keep
it at -20 ° C for biochemical determination.
Part 4: Biochemical determination
Biochemical determination is divided into 4 determinations as follows:

4.1 Determination of chlorophyll a, b, total chlorophyll, and total carotenoids content
1 g of fresh Thai amaranth microgreens were ground with 5 mL of 95% ethanol. Then sample 15 min, then
filtrated using a filter paper (no.1), after that centrifuge, 3,000 rpm for15 min at 4 °C, transferred to 96 - cell
and measured with a microplate reader at wavelengths of 440, 480, 495, 649 and 665 nm.

4.2 Determination of total vitamin C contents
7 g of fresh Thai amaranth microgreens were immediately macerated in 14 mL of oxalic acid (0.5% w/v). The
mixture was then filtrated using filter paper (no. 1). The filtrates (1 mL) were mixed with 4 mL of 10%
trichloroacetic acid, then immediately placed on ice for 5 min. Centrifuge at 8,000 rpm for 5 min, 3 mL of the
supernatant was mixed with 0.2 mL of 0.2 M folin-ciocalteu reagent and left at room temperature for 1 hour
and measured using a spectrophotometer at 760 nm.

4.3 Sample preparation for determination of phenolic contents and antioxidant capacity
0.5 g of dry Thai amaranth microgreens were ground as powder, then soaked in 25 mL 40 % ethanol at room
temperature for 2-3 hours and filtrated using a filter paper (no.1). Take the filtered part for the determination
of total phenolic content and total antioxidant capacity.

4.3.1 Determination of total phenolic contents
0.2 mL of extracted sample, distilled water 2.5 mL, then add 0.2 mL of 10 % folin-ciocalteu reagent and 2 mL
of 7.5 % Na2CO3 that keep in dark for 90 min and measure using a spectrophotometer at 765 nm.

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4.3.2 Determination of total antioxidant capacity
Diluted filtered water in ratio 1:10, take sample 0.3 mL, then add 1.5 mL of 0.1 mM DPPH after that keep it in
dark for 40 min, and measure using a spectrophotometer at 517 nm.

Results
The effects of light spectra on chlorophyll b, total chlorophyll, and total carotenoids content of Thai

amaranth microgreens were affected by light spectra treatments, chlorophyll a content was not affected by light
spectra, and it was the lowest under the blue spectrum, chlorophyll b, and total chlorophyll contents were found
under red + blue (70:30) spectrum, even if not different from the white spectrum. However, the total carotenoid
content of Thai amaranth microgreen was found under the red + blue (70:30) spectrum did not different from
white light, and it was the lowest under the blue spectrum (Table 1.).

Table 1. Effect of light spectrum on chlorophyll a, b and carotenoids content (mg/g) of Thai amaranth

microgreens

Light spectrum Chlorophyll a Chlorophyll b Total Chlorophyll Total carotenoids
(a+b)
Red (660 nm) 0.100±0.00 0.073±0.01b 0.054±0.00b
Blue (447 nm) 0.097±0.01 0.070±0.02b 0.173±0.01b 0.053±0.04b
Red: Blue (70:30) nm 0.110±0.00 0.110±0.02a 0.167±0.04b 0.056±0.00a
White (430-780 nm) 0.110±0.01 0.105±0.02a 0.220±0.02a 0.058±0.03a
0.215±0.02a

RCBD ns * * *

Note: * significant difference at p <0.05 level, respectively.

The effect of light spectrum on vitamin C, total phenolic content, and antioxidants capacities by DPPH
of Thai amaranth microgreens, the vitamin C of Thai amaranth microgreens was not affected by light spectrum,
and it was the lowest under the blue spectrum. The total phenolic content of Thai amaranth microgreens was
affected by light spectra treatments, the highest total phenolic contents were found under the white spectrum,
and it was the lowest under the blue spectrum and the highest antioxidant capacities were detected under the
blue spectrum, although the lowest was found under white spectrum (Table 2.).

Table 2. Effect of light spectrum on vitamin c contents, total phenolic content (TPC)

and antioxidants capacities by DPPH of Thai amaranth microgreens

Light spectrum Vitamin C TPC Antioxidant Capacities
(mg/100g) (mg GAE/g DW) (%Inhibition)

Red (660 nm) 1.075±0.01 17.65±0.09b 57.55 ± 0.17b
Blue (447 nm) 1.020±0.02 7.98±0.09b 80.14 ± 0.19a

Red: Blue (70:30) nm 1.170±0.01 16.84±0.09b 61.20 ± 0.21b
White (430-780 nm) 1.050±0.01 21.05±0.09a 52.91 ± 0.22b

RCBD ns * *

Note: * significant difference at p <0.05 level, respectively.

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Conclusion
The results of the study findings revealed that the light spectrum played an important role in the

production of phytochemicals in Thai amaranth microgreens. Although the monochromatic blue spectrum had
the worst overall performance, it provided the highest antioxidant capacities of Thai amaranth microgreens.
For the overall performance, a red and blue ratio of (70:30) LED spectrum, it can increase the nutritional value
of Thai amaranth microgreens. Thai amaranth microgreen had better antioxidant capacities in the blue spectrum
than in the white spectrum, while the white spectrum had higher total phenolic content than the blue spectrum
for Thai amaranth microgreen.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Prince of Songkla University and Islamic Sciences Demonstration School. The funding of SCiUS is provided
by Ministry of Higher Education, Science, Research and Innovation, which is highly appreciated. This
extended abstract is not for citation.

References
1. Meas, S., Luengwilai, K., & Thongket, T. Enhancing growth and phytochemicals of two amaranth

microgreens by LEDs light irradiation. Scientia Horticulturae 2020; 265: 109204.
2. Jagota, S. K. and Dani, H. M. A new colorimetric technique for the estimation of vitamin C using Folin

phenol reagent. Analytical Biochemistry; 1982; 127: 178-182.
3. O. V. Bulda; V. V. Rassadina; H. N. Alekseichuk; N. A. Laman. Spectrophotometric measurement of

carotenes, xanthophylls, and chlorophylls in extracts from plant seeds. 2008; 55(4): 544-551.

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Title : In vitro shoots and roots induction of Philodendron OB1_13_01
erubescens cv. pink princess
Field :
Author : Biology and Biodiversity

School : Ms. Chotiros Taorat
Advisor :
Ms. Anyamanee Tanpradubsing

Piboonbumpen Demonstration School, Burapha University, Chon Buri

Ph.D. Sirasatiyakorn Banharn, Department of Biology, Faculty of Science, Burapha University,

Chon Buri

Abstract
For shoot and root induction of Philodendron erubescens cv. pink princess in vitro was launched

by culturing 1 cm. of nodal explants on MS media with plant growth regulator in the cytokinin group i.e. BA
at concentration of 0, 0.5, 1.0 mg/L and auxin group i.e. IAA at concentration of 0, 0.1, 0.5 mg/L for 4 weeks.
It was found that MS media containing BA 0.5 mg/L and IAA 0.1 mg/L was a suitable medium for shoot
induction with the highest shoot number (8.00±2.65 shoot/explant) and highest leaf length. (1.62±0.25 cm).
For root induction launched by culturing nodal explants on MS media with plant growth regulator in the auxin
group i.e. IAA, IBA and NAA at concentrations of 0, 0.1, 0.5, 1.0 mg/L with 0.4 g of activated charcoal for 4
weeks. The results were found that MS media with IAA 0.5 mg/L, IBA 0.1 mg/L and NAA 0.5 mg/L were
optimal media for root induction which having highest number of roots, respectively (3.67±0.58., 3.67±0.58
and 3.67±1.15 root/explant) and it was found that MS media with IBA 0.1 mg/L + 0.4 g activated charcoal had
the highest root length (0.49±0.04 cm). Moreover, the study of comparative anatomy of seedlings cultured in
vitro and in vivo showed that stoma was found in both upper and lower epidermis of the in vitro seedlings, the
chloroplast density in the ground tissue was less, and the vascular bundles were not fully developed. For in
vivo seedlings, it was found that stoma could be found only in the lower epidermis, the density of chloroplast
was intense, the vascular bundles were more developed than the in vitro seedlings. Besides these, raphide
crystals were found in epidermis. And also in vitro seedlings were found druse crystals in epidermis. After
obtaining seedlings with well-developed shoots and roots, they were transplanted into 4 different planting
materials i.e. peat moss, coconut dust, peat moss with coconut dust ratio 1:1 and peat moss with perlite ratio
3:1 for 30 days. For this study, it was found that all planting material had 100% survival percentage and peat
moss with coconut dust ratio 1:1 was the optimal planting material that gave the highest shoot length, total leaf
area and shoot dry weight.

Keywords : shoot and root induction, plant growth regulator, Philodendron erubescens cv. pink princess,

Introduction
Philodendron erubescens cv. pink princess is the plant in the family of Araceae which originated

in South America, near Columbia. Philodendron pink princess has a red stem and heart-shaped leaves. Its
young leaves are dark green , when fully developed it will appear as green leaf with pink spotted on each leaf.
From its outstanding characteristics of leaves, P. pink princess is currently popular as an ornamental plant for
decorating homes and buildings, resulting in the popularity of commercial propagation, and the increasing in

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trading price. In addition to its beauty appearance, P. ‘pink princess’ also has the ability of purifying air by
absorbing toxic substances in the air via the transpiration process (Junhui, et al., 2011). The most conventional
propagation for P. ‘pink princess’ is nodal cuttings which provides the product quickly, but has limitations in
terms of quality control, and the amount of product is not enough to meet demands. Another way of culturing
is sowing, but it takes long time, requires expertiseand sometime unable to maintain its original appearance.
According to these informations, plant tissue culture technique is used for propagation in this study which can
propagate a large quantity of plants in short period of timest, sterile plant. The resulting offspring are identical
to the mother plant in all respects espically no mutation. So, this in vitro culturing technique is considered to
be more effective compared to conventionalpropagation methods. From the research of Chen et al. (2012)
studied the effect of plant growth regulator in cytokinin group. It was found that Kn and BA at concentration
of 0.5, 1 mg/l were more optimal concentration for root induction than TDZ at concentrations of 0.5 and 1
mg/l. Additionally, BA was more effective than Kn for root induction in three species of Philodendrons.
Alawaadh et al. (2020) studied the effect of cytokinin and auxin increased shoot multiplication compared
between cytokinins only and the combination of cytokinins and auxins.It was found that the combination of
cytokinins and auxins yielded more shoots than cytokinins alone, besides the greatest number of roots induction
and roots fresh weight is in MS medium supplemented with NAA at concentration of 1-2 mg/l. When the
plantlets had were successfully acclimatized, it was found that the survival percentage was 100% and the
seedlings that were morphologically similar to the mother plant. From this research this made the researcher
interested in studying the protocol of shoot and root induction and propagation of P. erubescens cv. pink
princess. including focus on the development of an efficient in vitro propagation method of P.‘pink princess’,
which this method is expected to produce the large amounthealthy plants including applied to a wide variety
of plants. Therefore, the objective of this research was study a different types and concentrations of plant
growth regulators (PGR) on shoots and roots induction including study a different appropriate planting
materials for P. cv.pink princess. Data from this study, it can be used to apply micropropagation of
Philodendron species, as well as to establish a future source of income.

Methodology
In vitro shoots and roots induction of P. erubescens cv. pink princess was separated into 4

experiments as follows.
1. The effect of the plant growth regulator such as : cytokinin (BA) and auxin (IAA) on shoot induction of P.
erubescens cv. pink princess

1 cm Nodal explants from sterile seedling were cultured on Murashige and Skoog media (MS,
1962) with plant growth regulators (PGR) in the cytokinin group i.e. BA at concentrations of 0, 0.5, and 1.0
mg/L and auxin group i.e. IAA at concentrations of 0, 0.1, and 0.5 mg/L by using only one type of PGR, and
combination of them, total of 9 experiments with 5 replications and each replicate includes one specimens.
After 4 weeks of cultivation, experimental data such as shoot number, shoot length, root number, root length,
leaf number and leaf length were collected.
2. The effect of the auxin (IAA, IBA and NAA) on root induction of P. erubescens cv. pink princess

1 cm Nodal explants were cultured on MS media with PGR in the auxin group i.e. IAA, IBA and
NAA at concentrations of 0, 0.1, 0.5, and 1.0 mg/L with the addition of 0.4 g activated charcoal, total of 10

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experiments with 5 replications and each replicate includes one specimens . Experimental data such as shoot
number, shoot length, root number, root length, leaf number and leaf length were collected after 4 weeks of
cultivation.
3. The study of comparative anatomy of seedlings cultured in vitro and in vivo.

Select fully developed leaves from seedlings cultured in vitro and in vivo. Studied leaves anatomy
by a free-hand section using a razor blade to cross the leaves through the midline. Select the thinnest tissue of
leaves floated in petri dish, then wet mount on the slides. Observe cells under a light microscope using a low-
power objective lens (10X) and high-power objective lens (40X). Collect experimental data and compare the
anatomy of two condition.
4. A study of different appropriate planting materials for survival percentage and growth of P. ‘Pink Princess’
seedling

Acclimatiztion process of seedling in vitro by unscrewing the glass bottle of well developed plants
and placed at room temperature for 15 days. Then the rooted (in vitro) plantlets were rinsed with water
thoroughly and transplanted into 4 different planting materials i.e. peat moss, coconut dust, peat moss with
coconut dust ratio 1:1 and peat moss with perlite ratio 3:1.There were 4 experiments with 5 replications and
each replicate includes one seedling. After planting for 1 month, the experimental data were collected such as
survival percentage, shoot length, total leaf area per plant and shoot dry weight.

Statistical analysis
The experimental design was CRD with five replicates and each replicate includes one specimens.

The results were subjected to analysis of variance and the significance of differences among mean values was
carried out using Tukey's range test at P\0.05 using Minitab software, version 17.0.

Results and conclusion

1. From the study the effect of the cytokinin (BA) and

auxin (IAA) on shoot induction of P. erubescens cv. pink

princess, it was shown that MS medium containing BA 0.5 mg/L

MS BA 0.50 BA 1.0 BA 0.5 BA 0.5

+ IAA 0.1 + IAA 0.5 and I AA 0.1 mg/L was optimal media for shoot induction with

the highest shoots number

BA 1.0 BA 1.0 IAA 0.1 IAA 0.5 per explant of 8.00±2.65

+ IAA 0.1 + IAA 0.5 shoots/explant, and the
highest leaf length of
Fig. 1 : Shoots induction from nodal explant of Philodendron ‘pink MS IBA 0.1 IBA 0.5 IBA 1.0
princess’ on MS media enriched with BA and IAA at different
concentrations for 4 weeks 1.62±0.25 cm.

2. From the study of the effect of the auxin (IAA, IBA, NAA) NAA 0.1 NAA 0.5 NAA 1.0
on root induction of P. erubescens cv. pink princess, it was found that MS IAA 0.1 IAA 0.5 IAA 1.0
media with IAA 0.5 mg/L, IBA 1.0 mg/L, and NAA 0.5 mg/L were
suitable for root induction with the highest root number respectively NAA 0.1 NAA 0.5 NAA 1.0
(3.67±0.58 roots/explant, 3.67±0.5 roots/explant, and 3.67±1.15
roots/explant) but it can be seen that MS media with IBA 0.1 mg/L Fig.2 : Root induction from nodal explant of Philodendron ‘pink
provided the highest root length (0.49±0.04 cm.) princess’ was cultured on MS media with different concentrations of

IAA, IBA and NAA for 4 weeks.

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3 . According to the study of comparative anatomy of seedlings cultured in vitro and in vivo, the

results revealed that in vitro seedlings, cutin was not found in both upper and lower epidermis, cells in the

epidermis layer are small and orderly arranged, stomata were found in both upper and lower epidermis, and

druse crystals were discovered in dermal tissue andground tissue. As of in vivo seedlings, revealed that upper

and lower epidermis had cutin cover both of adaxial surface and abaxial surface and also found the bulliform

cell. Stoma could be found only in lower epidermis and its density was intensive. Furthermore, raphide crystals

were found only on the abaxial surface . Chloroplast density in the mesophyll layer of in vitro seedlings was

lower than in vivo seedlings. The cell shape in the vascular tissue was uncertain, and the boundary between

xylem and phloem tissue could not be distinguished. Meanwhile, in vivo seedlings could distinguish the

boundary between xylem and phloem tissue, with xylem on upper side and phloem on lower side of vascular

bundle. Overall, it can be seen that tissue development of in vivo seedlings was better than in vitro seedlings.

4. A study of different appropriate planting materials
for survival percentage and growth of P. ‘pink princess’. It was

found that all planting material had 100% survival percentage,

however peat moss with coconut dust ratio 1:1 was the best

planting material that gave the highest shoot length (2.33 ± 0.208 peat moss coconut peat moss with peat moss
cm.), total leaf area (1.53±0.1176 cm2) and shoot dry weight dust coconut dust with perlite

(0.01386±0.0023 g.) Fig. 3 : Growth of Philodendron ‘pink princess’ seedling after
planting in different e planting materials for 1 month

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Burapha University and Piboonbumpen Demonstration School. The funding of SCiUS is provided by
Ministry of Higher Education, Science, Research and Innovation, which is highly appreciated. This extended
abstract is not for citation.

References
Junhui J. Z., Feifei Q., Jie S., Jian-wu L. & Hui-lian X.. (2011). Purification of formaldehyde-polluted air by

indoor plants of Araceae, Agavaceae and Liliaceae. Journal of Food Agriculture and Environment, 9,
(3&4): 1012-1018.
Chen F.C., Wang C.Y.,  J.Y. Fang. (2012) . Micropropagation of self-heading Philodendron via direct
shoot regeneration. Scientia Horticulturae, 141, 23-29.
Asma A. A. (2020). Micropropagation of Lacy Tree Philodendron (Philodendron bipinnatifidum Schott ex
Endl.). Hort Science, 55(3): 294-299.

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Oral presentation
Biology and Biodiversity Group 1

Sunday August 28, 2022

No. Code Title Author School
Chiang Mai University
1 OB1_01_01 Comparison of dust capturing Miss Kunyakorn Sathongsirikul Demonstration School

efficiency of ornamental Miss Photsinee Juengsamretkarn Kasetsart University
Laboratory School
plants with different leaf Kamphaeng Saen
Campus Educational
physical characteristics Research and
Development Center
2 OB1_10_03 Nutraceutical properties of Miss Wissuta Thawichsung Princess Sirindhorn's
College
red holy basil Mr. Pachara Chongbunyatcharoen
Demonstration School
Miss Pimnipa Rattanasing of Khon Kaen
University
3 OB1_12_03 The effects of chemical Mr. Kobboon Yousawad Demonstration School,
University of Phayao
fertilizer formulas on the Mr. Harit Yanatchara
PSU Wittayanusorn
growth and flowering of the Surat Thani School

duckweed (Wolffia globosa)

4 OB1_04_04 Effects of nitrogen on Miss Suphakarn Kietchalermkun

Burkholderia pseudomallei Miss Nichaphat Srirompotong

and rice growth

5 OB1_02_09 Conservation and Miss Boonyaporn Autama

Micropropagation of Miss Kununya

Thrixspermum sp. In vitro Chalermponevonggul

Mr. Buranas Faikrue

6 OB1_17_07 Applying silage as mulch: A Miss Jutamart Chaeaul

case study of Lycopersicon Miss Pawitra Payakhapiban

esculentum Mill.

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Title : Comparison of Dust Capturing Efficiency of OB1_01_01
Ornamental Plants with Different Leaf
Field : Physical Characteristics
Author : Biology
Ms.Photsinee Juengsamretkarn
School : Ms.Kunyakorn Sathongsirikul
Advisor : Chiang Mai University Demonstration School
Asst. Prof. Dr.Sutthathorn Chairuangsri
Mr.Kusol Raungprataungsuk

Abstract
This study was to assess dust capturing capacity in ornamental plants with three different physical

characteristics of leaves, which were waxy leaves, rough-surfaced leaves, and hairy leaves. In total, nine
species were investigated. The powder with a particle size of <2.5 microns, 2.5-10 microns and >10 microns
were used in the experiment to represent dust particles PM2.5, PM10 and larger than PM10, respectively. An
electric fan was placed in a 1-cubic-meter chamber to allow particle diffusion during the experiment. In each
experiment, plants were placed in the chamber for 5 minutes after those twelve leaves of each plant species
were randomly picked and soaked in distilled water for 90 minutes to wash out the dust. After taking the leaves
out, the samples were dried in the oven and the beakers were then weighed to determine the amount of dust
that leaves had captured. Stereo Microscope was used to examine the physical characteristics of each leaf as
well as particle sizes. The results revealed that Pinto peanut had the highest ability to capture >10 microns
dust, which was 0.3609 mg/cm2, Cape honeysuckle had the highest ability to capture 2.5-10 microns dust
which was 1.1222 mg/cm2 and Blue Salvia had the highest ability to capture <2.5 microns dust, which was
0.9076 mg/cm2. All three of them have rough-surfaced leaves, while waxy leaves and hairy leaves have lower
ability to capture dust.

Keywords : Dust capturing, Dust, Leaf surface, Ornamental plant

Introduction
Thailand is heavily facing the problem of haze. The particulate matter in haze can be divided into

different groups depending on particle size. PM2.5 is a very small particle, caused by incomplete combustion
of fossil fuels, biomass burning, construction, and human activities in daily life. Once PM2.5 pass into the
body, the homeostasis could be disturbed, and it stimulates free radicals. Moreover, nowadays people are
interested in planting ornamental plants for decoration. If these plants could help reduce the dust, it would
have a positive effect on the environment and humans. Therefore, this study was to assess dust capturing
capacity in ornamental plants with three different physical characteristics of leaves, which were waxy leaves,
rough-surfaced leaves, and hairy leaves. The physical characteristics of each leaf were examined by a stereo
microscope and dust capturing capacity was tested by a particle size of <2.5 microns, 2.5-10 microns and >10
microns in a 1-cubic-meter chamber.

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Methodology
1. Powder particle size determination

The particle size of 3 different kinds of power (cooling powder, baby powder, press powder) was

determined under light microscope. One hundred particles from each kind of powder were measured using a

micrometer. The average particle sizes and standard deviation were calculated.
2. Leaf Physical Characteristics

Nine ornamental plants were studied; Blue salvia, Cape honey suckle, Pinto peanut, Banyan, Croton,
Brush cherry, little yellow star, Chinese fringe, and Begonia. The picture of upper and lower leaf surfaces was
taken under a stereomicroscope.
3. Leaf dust capturing capacity

Leaf dust capturing capacity was measured by the gravimetric method (Huixia Wang, 2011). The
experiments were set up separately for each type of powder. All 9 ornamental plants were used for dust
capturing capacity for baby powder and cooling powder (particle size 2.5-10 microns and >10 microns) and 3
species with highest dust capturing capacity were used for experiment with press powder (particle size <2.5
microns). To determine the dust capturing capacity for each plant species, 3 plant samples were placed in the
chamber, the fan was turn on and 10g of powder was poured into the chamber. After 5 minutes, the fan was
turn off and the plants were gently taken out. Four leaves were randomly picked from each plant, the leaves
were immersed in 50ml of distilled water in a beaker (dry and weighted before (W1)) for 2 hours. Then the
leaves were taken out carefully and beakers with the solution were oven dried for 24 hours and weighted (W2).

The dust capturing capacity was calculated using the equation

W=(W2-W1)/A , where W is dust capturing capacity (mg/cm2)
W1 is the weight of beaker
W2 is the weight of beaker plus dust sample
A is surface area of the selected leaves (cm2).

Results
1. Powder particle size

The average particle size of cooling powder, baby powder and press powder were 35.9±2.77 microns,
8.75±0.19 microns and <2.5 microns. Therefor the powder of cooling powder, baby powder and press
powder were use as representative of particulate matter >10 microns, 2.5-10 microns and <2.5 microns,
respectively
2. Leaf characteristic
The leaf of the nine ornamental plants could be separated into 3 groups which are waxy leaves, rough-
surfaced leaves, and hairy leaves as shown in Table 1.
3. Dust capturing capacity
In the experiment with powder with particle size >10 microns and 2.5-10 microns, the 9 ornament plants
have different capacities for dust capturing. In overall, the plant could capture 2.5-10 microns particle
higher than the >10 microns particles. The rough-surfaced leaves seem to have a higher capacity to capture

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particles larger than 10 microns, followed by hairy leaves and waxy leaves. For particle size 2.5-10
microns, the capturing capacity were varied among plant species with the same leaf characters.
Three plants species, including Pinto peanut, Blue salvia and Chinese fringe were selected for the
experiment with particle <2.5 microns from their high capturing capacity and low variation (SD value).
The Blue salvia had the highest ability to capture <2.5 microns dust, which was 0.9076 mg/cm2. Followed
by pinto peanut for 0.7199 mg/cm2 and Chinese fringe for 0.2823 mg/cm2. (Table 2)

Table 1 : Leaf surface characteristic of 9 ornamental plants

Characteristic Plants

Salvia spp. Tecoma capensis Arachis pintoi

rough-surfaced leaves

Ficus annulate Syzygium austral Codiaeum variegatium.
Blume.

waxy leaves

Melampodium Loropetalum chinense Begonia spp.
divaricatum

hairy leaves

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Table 2 : Dust capturing capacity of each plant

Plants Dust capturing capacity (mg/cm^2)
>10 microns 2.5-10 microns <2.5 microns

Blue salvia Salvia spp. 0.29±0.09 0.76±0.79 0.91±0.65

rough- Cape Tecoma capensis 0.32±0.14 1.12±1.36
surfaced honeysuckle (Thunb.) Lindl.

leaves

Pinto peanut Arachis pintoi 0.36±0.14 1.01±0.71 0.91±0.41

Banyan Ficus annulata 0.24±0.08 0.45±0.39
0.13±0.19 0.83±2.24
waxy Croton Codiaeum 0.18±0.07 0.85±0.86 0.28±0.17
leaves variegatium. Blume. 0.29±0.12 0.51±.022
Brush cherry Syzygium australe 0.33±0.29 0.80±0.58
hairy 0.27±0.14 0.23±0.04
leaves Little yellow (J.C.Wendl. Ex
star Link) B.Hyland.
Melampodium
Chinese
fringe divaricatum
Loropetalum
chinense (R. Br.)

Oliv.

Begonia Begonia spp.

Conclusion
The study showed that different physical characteristics of leaf effect on different ability to capture

dust. Pinto peanut had the highest ability to capture >10 microns dust, which was 0.3609 mg/cm2, Cape
honeysuckle had the highest ability to capture 2.5-10 microns dust which was 1.1222 mg/cm2 and Blue Salvia
had the highest ability to capture <2.5 microns dust, which was 0.9076 mg/cm2. All three of them have rough-
surfaced leaves, while waxy leaves and hairy leaves have lower ability to capture dust.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation.

References
Huixia W, Hui S, Yangyang L. Leaf Dust Capturing Capacity of Urban Greening Plant Species in Relation
to Leaf Micromorphology. China: National Natural Science Foundation of China & State Forestry
Administration; 2011.

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Title : Nutraceutical properties of Thai red holy basil OB1_10_03
Field : Biology and Biodiversity 00
Author : Mr.Pachara Chongbunyatcharoen

School : Miss Pimnipa Rattanasing

Advisor : Miss Wissuta Thawichsung

Kasetsart University laboratory School Kamphaeng Saen Campus

Educational Research and Development Center

Assistant Professor Dr.Siriporn Sripinyowanich

Abstract
Non-communicable diseases (NCDs) have become important public health problems in many parts

of the world including Thailand. NCDs can contribute a cumulative output loss and the use of commercial
drugs used to treat NCDs can cause unpleasant side effects. To overcome these problems, the use of plant
extracts is probably a better way to prevent these diseases. Leaves of red holy basil (Ocimum sanctum L.)
contains various bioactive substances that have antioxidant activity, anticonvulsive effect on the intestinal
tract, and reduce the level of fat and cholesterol in the body. Therefore, this study aimed to study the effect of
water extracts of Thai red holy basil on inhibitory activities of pancreatic lipase (PL), -glucosidase, and
acetylcholinesterase (AChE), in vitro to examine the biological properties of the extracts as therapeutic drugs
for anti-NCDs. Antioxidant activities were also determined by Folin-Ciocalteu method. Five accessions of
Thai red holy basil (OS02, OS03, OS04, OS7, and OS08) with various amounts of eugenol and anthocyanin
contents were candidate of red holy basil for this study comparing to a white holy basil (OS10). Water extracts
of Thai red holy basil leaves contained low amount of total phenolic content (6.17-16.78 mg gallic acid/g dry
weight). Interestingly, the extracts of red holy basil could inhibit the activities of pancreatic lipase, -
glucosidase, and acetylcholinesterase almost higher than that of white holy basil extract, especially accession
OS07. The IC50 of OS07 leave extract on pancreatic lipase, -glucosidase, and acetylcholinesterase inhibitory
activities were 3.74 g/ml, 2.57 mg/ml, and 0.56 g/l, respectively. Overall, over findings suggest that the
leave extracts of Thai red holy basil have potential as a nutraceutical agent prove beneficial to prevent of
chronic consumption such as Alzheimer disease, obesity, and diabetes mellitus.

Keywords : Anthocyanin; Non-communicable diseases; Nutraceutical properties; Red holy basil

Introduction
Non-Communicable diseases (NCDs) are largely caused by unhealthy lifestyles or risky behaviors

such as drinking alcohol, tobacco use, insufficient physical inactivity, and having stress for a long time can
raise blood glucose, lipids, and pressure leadings to NCDs such as dementia, diabetes, atherosclerosis, cancer,
and obesity. These NCDs replaced acute infections as the major causes of death for adults and elderly. [1]

In fact, many herbal plants possess that ability to prevent some NCDs, e.g., diabetes and Alzheimer’s
disease. [1] Red holy basil (Ocimum sanctum Linn.) is widely used in traditional medicine in different cultures
and is also known for its culinary uses. It belongs to the Lamiaceae which is recognized as the unique aromatic
plant that are grown widely and distributed throughout tropical and temperate regions, including Thailand.

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The diversity of red holy basil accessions in Thailand could also affect levels of bioactive profiles, none the
less, there is limited data on their nutraceutical profiles particularly of enzymatic inhibitory analysis related to
NCDs diseases among red holy basil accessions available in Thailand. The aim of this study is therefore to
investigate the nutraceutical properties of Thai red holy basil extracts against NCDs, such as the anti-diabetes,
anti-Alzheimer’s disease, and potential against arteriosclerosis disease activity under in vitro. A novelty of
this study is the determination of properties of various Thai red holy basil accessions that have not yet been
reported in linked with the purple pigments (anthocyanins).

Methodology

1. Tissue and extract preparation

1.1 Plant tissue preparation: In this

study, five cultivars of red holy basil leaves

from Thai genetic sources namely OS02,

OS03, OS04, OS07 and OS08 (Figure 1), and

study in comparing to a white holy basil Figure 1: Investigated Thai holy basil used in this study.
(OS10) as the control.

1.2 Leave extract preparation: Take 10 g of dried leaves tissue, add 30 ml of distilled water into

titration flask, shake at room temperature for 48 h, then filter with filter paper size 11 µm. The extracts were

distilled by a rotary evaporator at 60oC, and the crude extracts were collected and weighed to calculate the

percentage of results.
1.3 Extraction and quantitative analysis of anthocyanin [2]: Prepare a solution of pH 1 (KCl-HCl

buffer) and pH 4.5 (CH3COONa-HCl), then pipette 0.3 ml of 0.1 mg/ml of extract into a 2.7 ml of pH buffer
solution, and leave for 10 min. The absorbance was measured at 510 and 700 nm wavelengths.

2. In vitro enzymatic inhibitory studies of the red holy basil leaves extracts
2.1 α-glucosidase inhibitory activity test [3]: Add the sample (20 µl) with 50 mM of sodium

phosphate buffer (pH 6.8) solution, 100 µl with enzymatic solution α-glucosidase (1.0 U/ml) in 20 µl of buffer

was placed in a well plate, incubated at room temperature for 10 min, and the solution was added 20 µl PNP-
G (2 mM), incubate at room temperature for 5 minutes. Finally, 40 µl of 1.0 mM Na2CO3 was added and the
absorbance was measured at a wavelength of 405 nm. The percentage of α-glucosidase enzyme inhibition was

calculated.

2.2 Analysis of the efficacy of pancreatic lipase inhibition [3]: 30 µl enzyme solution was added to

850 µl of Tris buffer. After that, 100 µl of the extract was added. The mixture was heated to 37oC for 15 min

and added p-NPB 10 mM in dimethyl formamide. The absorbance was measured at a wavelength of 400 nm.

2.3 Analysis of Acetylcholinesterase (AChE) inhibitory activity [3]: Prepare a 200 µL mixture
containing the enzyme AChE and place it in a test tube containing the substance. 5,5-́ dithiobis and sample
extracts After that, 500 μl of phosphate buffer was added, the mixture was incubated at 25oC for 20 min, 100
μl of acetylthiocholine iodide was added, the mixture was placed at 25oC for 3 min, and the absorbance was

measured at a wavelength of 412 nm.

2.4 Total phenolic content analysis [4]: Mix the gallic acid standard solution or the sample to be

tested in volume 0.3 ml with Folin-Ciocalteu reagent solution (0.7 ml) and incubated at room temperature for

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5 min. Then add 2.5% solution of Na₂CO₃ 1.0 ml, incubate at room temperature for 20 min. Absorbance was
measured at a wavelength of 760 nm. TPC was determined from the gallic acid standard curve.

3. Statistical Analysis
The variance was analyzed according to the complete randomized design trial and the mean was

compared by Duncan's multiple range test at 95% confidence level. The results were submitted to multivariate
principal component analysis (PCA).

Results
The water extracts from leaves of five Thai red holy basil accessions (OS02, OS03, OS04, OS07 and

OS08) and a white holy basil (OS10) were studied for their effect against the NCDs-related enzyme activities
under in vitro as shown in Table 1. The extracts of red holy basil leaves were grouped by total anthocyanin
content; high (22.76 and 22.25 mg l-1 of OS03 and OS07), medium (12.95 and 11.96 mg l-1 of OS02 and OS04)
and low (2.1238 mg l-1 OS08) content, whereas 3.86 mg l-1 in OS10 leave extract.

The potential inhibitory activity of the extracts against enzymes involved in the pathogenesis of
diabetes was evaluated based on α-glucosidase inhibitory activity and expressed as IC50 value (Table 1). The
inhibitory of α-glucosidase were determined from 5.64 mg ml-1 for OS02 and 2.27 mg ml-1 for OS04. It should
be noted that the content of anthocyanins was not caused an important increase in this activity exhibited by
the extracts of red holy basil.

High PPL inhibitory activity was detected in the extract at the concentration of 200 µg ml-1 of OS07
(28.51+0.8%, IC50=3.74) (Table 1). Most of these plant extracts were potent of PPL as the results represented
their low concentration able to produce 50% inhibition of PPL.

In addition, the water extract of OS08 leaves was the most potent plant inhibitor of AChE with IC50
value of 0.1 µg µl-1 (Table 1). The anti-AChE activity of the extracts of OS07 and OS02 had not significantly
different when compared to the control.

Table 1: IC50 values of the water extracts of red holy basil leaves potently inhibited -glucosidase, pancreatic

lipase (PPL), and acetylcholinesterase (AChE) compared with that of the white holy basil extract (n = 3).

Holy basil Source Yield of IC50 on the inhibitory activity of the extracts
accession crude
-glucosidase PPL (µg/ml) AChE (g/l)
OS02 extract (%) (mg/ml) 6.30 0.92
5.64
Nakhon Pathom 26.2

OS03 Nakorn Nayok 15.0 2.41 5.47 1.36

OS04 Ratchaburi 15.2 2.27 4.48 2.88

OS07 Songkhla 31.8 2.57 3.74 0.56

OS08 Songkhla 12.2 2.43 4.40 0.10

OS10 Nakhon Pathom 17.6 NA 5.13 0.56

The antioxidant capacity of the extracts was determined by Folin-Ciocalteu method. Total phenol
content (TPC) of the water extracts of holy red basil were found in ranging from 6.17 to 16.78 g of GAE
gDW-1 as Gallic Acid equivalents. The maximum and minimum amounts of TPC were recorded in OS02 and
OS08, respectively.

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According to the obtained results, it can be confirmed

that anthocyanin contents are not responsible for most of the

observed in vitro enzymatic inhibitory studies of the

investigated Thai red holy basil extracts. The score plot (Figure

2) showed the projection of six plant extracts in the

multivariate space of the first two PCs. The clear separation of

the extracts pointed out the main similarities between them.

The one extract grouped on the left (OS08) were characterized

by low anthocyanins with high anti-AChE. OS03, OS04 and

OS07 extracts, positioned on the lowed right, were

characterized by high anthocyanins with high anti-α- Figure 2: Principal component analysis (PCA)

glucosidase, high anti-PPL activities, and high anti-AChE describing data sets obtained from the
(only OS03 and OS07), but OS04 had low anti-AChE. In turn, spectrophotometric determinations of total
OS02 and OS10, which are preferable to cook, had low anthocyanin content, results of the inhibitory activity
activities against α-glucosidase and PPL but high activities on (IC50) of PPL, α-glucosidase, AChE and antioxidant
antioxidant and against AChE. activity of the water extracts of five red holy basil
and a white holy basil.

Conclusion
The water extracts of Thai red holy basil leaves have potential biological activity against non-

communicable diseases, e.g., the potential anti-diabetes mellitus, anti-Alzheimer’s disease, and anti-obesity
activities. The red holy basil OS07 was found to be most effective as it contains high anthocyanins with high
inhibitory activity of α-glucosidase, acetylcholinesterase, and pancreatic lipase.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Kasetsart University Kamphaeng Saen Campus and Kasetsart University Laboratory School Kamphaeng Saen
Campus Educational Research and Development Center. The funding of SCiUS is provided by Ministry of
Higher Education, Science, Research and Innovation, which is highly appreciated. This extended abstract is
not for citation.

References
1. Panchal P, Parvez N. Phytochemical analysis of medicinal herb (Ocimum sanctum). Int J Nanomater
Nanotechnol Nanomed. 2019;5(2): 008–11.
2. Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R. Analysis and biological activities of anthocyanins.
Phytochemistry. 2003;64(5): 923-933.
3. Mousavi L, Salleh RM, Murugaiyah V. Phytochemical and bioactive compounds identification of
Ocimum tenuiflorum leaves of methanol extract and its fraction with an antidiabetic potential. Int J
Food Properties. 2018;21(1): 2390–2399.
4. Yingngam B, Monschein M, Brantner A. Ultrasound-assisted extraction of phenolic compounds from
Cratoxylum formosum ssp. Formosum leaves using central composite design and evaluation of its
protective ability against H2O2-induced cell death. Asian Pac J Trop Med. 2014;7: 497–505.

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Title: The effects of chemical fertilizer formulas on the growth OB1_12_03

Field: and flowering of the duckweed (Wolffia globosa)
Author:
Biology and Biodiversity
School:
Advisor: Mr. Harit Yanatchara

Mr. Kobbon yousawad

Princess Sirindhorn's College, Silpakorn University

Dr. Kampanat Tharapoom and Dr. Panupong Thongprem

(Department of Biology, Faculty of Science, Silpakorn University)

Abstract
Wolffia globosa is one of the smallest flowering plants which is very popular on the food market in

these years, as it has recently been considered as a ‘Superfood’. However, the knowledge of wolffia biology
is still poorly understood. According to previous studies and our observation on wolffia farmers, flowering of
the wolffia has yet to be discovered in Thailand. The wolffia productions are still insufficient when compared
to the high demand on wolffia consumption throughout the year. This experiment, thus, investigated the impact
of chemical fertilizer formula on wolffia growth and flowering using three types of common fertilizers; 1 )
Equal NPK fertilizer (N:P:K = 16:16:16), 2) Nitrate-base fertilizer (N:P:K = 46:0:0), and 3) Phosphate-base
fertilizer (N:P:K = 15:30:15+TE), at three concentrations, i.e., i) the concentration at 50 percent lower than a
recommended ratio, ii) the concentration at the recommended ratio, and iii) the concentration at 5 0 percent
higher than the recommended ratio on the label. Wolffia were cultivated in Nestlé Pure Life drinking water
mixed with the fertilizer at three different concentrations, as described above, for 15 days with 12 hr of light
exposure. The results indicated that the number and the size of wolffia thalli were gradually decreasing when
they were exposed to the fertilizers at higher concentration. The number of wolffia thalli were not significantly
different among the three fertilizer types. Nevertheless, number of the wolffia in the Nitrate-base group was
fewer than the number in the control group (T-test: t(10) = 3.54, p = 0.0053 ). Flowering events of the wolffia
were not observed in this study. Moreover, it is interesting that the control group with only Nestlé Pure Life
drinking water seemed to induce the highest number of wolffia thalli without adding any fertilizers.

:Keywords Wolffia globosa, Plant propagation, Chemical fertilizer, Superfood

Introduction
Wolffia is the world's smallest flowering plant which is living in freshwater. The shape is oval, made

it looks like a tiny green egg floating on the water. The size is between 0.1 – 1.0 mm in diameter. It can absorb
nutrients in the water through the thallus surface directly [1]. It's a perfect flower in monocotyledon type.
There are no petals, sepals, and vascular systems [2]. Wolffia are one of the most popular plants on the food
market. However, the biology of them is not well studied. With the previous studies [1-2] and our observation
on wolffia farmers indicated that flowering of the wolffia has yet to be discovered in Thailand. In addition, it
is difficult to grow wolffia in all seasons and to compete the high demand on the food market. Wolffia's growth

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also varies with the environment and ecological factors (e.g., water minerals, light, and temperature, etc.)
which can affect nutrient storages in wolffia [3].

In this research, we studied the growth and flowering event of Wolffia globosa using common
chemical fertilizers that are easily found on the market by following these three hypotheses; i) the high level
of phosphorus concentration would stimulate the flowering of wollfia due to the nutrient could induce
maturation of plants, ii) the high level of nitrogen concentration would enlarge the thallus size due to the
nutrient could enhance the growth of plants, and iii) higher nutrient concentration would increase the growth
rate of wolffia depend on the plant physiological efficiency. Some ecological factors were also observed and
conducted alongside the experiments. The results might be useful for the wolffia farmers and other plant
breeders whereas this can be a biological information to further wolffia studies.

Methodology
Preparing wolffia: The stock of Wolffia globosa were bought from The Saengsakulrung Farm in Dan Makham
Tia District, Kanchanaburi Province, Thailand. Then, DNA barcoding were done to confirm the wolffia
species.
Wolffia treatments: Fifty-four 100 ml of clear plastic cups were filled with 80 ml of Nestle Pure Life Drinking
Water mixed with chemical fertilizers 3 formulas; A.) Equal NPK formula:16-16-16, Charlie's© B.)
Nitrogenous formula: 15-30-15+TE, Vesco©, C.) Phosphorous formula: 46-0-0 Vesco©, varied at 3
concentrations (Table 1). The control group was only 80 ml Nestle Pure Life Drinking Water without
fertilizers. Five perfect thalli (the same size of complete oval shape with no attached budding thallus) were
carefully selected and put in each cup. The experiment was conducted for 15 days with 12 hr light exposure.
At the 15th day, total thalli were counted in each cup. Then we carefully placed a 5 x 5 mm transparency
plastic square on the water's surface near the wolffia and photographed entire of the cub with the CU Smartlens
20X attached to the smartphone camera (Xiaomi 10T pro and Samsung A71). The size (width and length) of
the 10 largest thalli were measured in ImageJ softwear version 1.53, using the plastic square as the reference
scale. Flowering events were observed every 3 days alongside the observation of some ecological factor, i.e.,
humidity, water temperature and water pH. The experiments were conducted between 9th - 24th January 2022.
Statistical analysis: Number and size of wolffia thalli were compared among the treatments and control using
One-way ANOVA at p-value 0.05. If the ANOVA indicated a significant difference between the treatments,
we further tested for the multiple comparisons with a conventional T-test at the adequate p-value 0.0083. All
the data with more than 6 replicates were tested for a normal distribution prior to conduct under the parametric
tools. All the statistic were undergone with Microsoft Excel version 2203.

Results
Growth of wolffia culture in each treatment within 15 days showed that wolffia tended to grow better

in the groups of fertilizer at the lowest concentration (50 percent lower concentration than recommended ratio)
as shown in Table 1. Therefore, the statistical comparison of number and size of the wolffia cultured in each
treatment would be further conducted at this concentration.

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Table 1 The growth of wolffia after 15 days. Each treatment was varied at 3 different concentrations; 50 percent lower
than the recommended concentration (A1, B1 & C1), at the recommended concentration (A2, B2 & C2) and 50 percent
higher than the recommended concentration (A3, B3 & C3). Number and size of the thalli were showed in mean±SE.

Wolffia A B C
growth Equal NPK formula: Nitrogenous formula: Phosphorous formula:

16-16-16 46-0-0 12-24-12 Control

A1 A2 A3 B1 B2 B3 C1 C2 C3

Number 7.17±1.76 5.33±1.04 5.00±1.32 7.5±1.80 6.0±1.32 5±0.87 12.33±2.57 8.33±3.18 5.67±1.53 14.17±1.76
of thalli (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6) (n=6)

width 0.30±0.03 0.24±0.11 0.23±0.14 0.33±0.03 0.28±0.13 0.27±0.14 0.38±0.06 0.33±0.06 0.21±0.11 0.36±0.37
(mm) (n=29) (n=18) (n=22) (n=34) (n=27) (n=30) (n=35) (n=33) (n=20) (n=60)

length 0.51±0.77 0.42±0.18 0.38±0.21 0.49±0.05 0.44±0.19 0.43±0.22 0.57±0.06 0.49±0.10 0.35±0.19 0.57±0.61
(m m) (n=29) (n=18) (n=22) (n=34) (n=27) (n=30) (n=35) (n=33) (n=20) (n=60)

The average number of wolffia thalli in each treatment were significantly different (ANOVA; F
=4.27, df = 3, p = 0.05). Only B1 group had a significant lower number of thalli (7.5±1.80 thalli) than control

group (14.17±1.76 thalli) (Fig 1A). For the size of the thallus, there was a statistically difference in the thalli
width between the treatments (ANOVA; F =3 . 6 1 , df = 3 , p = 0 . 0 3 2 , Fig 1B) . In A1 group fertilizer had an
average thalli width (0.30±0.03 mm) significantly smaller than the C1 group (0.38±0.06, T-test: t(55) = 3.79, p
= 0.00038) and the control group (0.36±0.37, T-test : t(43) = 4.22 , p = 0.00012, Fig. 1B). Additionally, A1
group also had an average length of thalli (0 . 5 1 ± 0 . 7 7 mm) significantly smaller than the control group
(0.57±0.61, T-test: t(50) = 3.15, p = 0.0027). However, flowering was not observed in this study.

(A) (B)

Figure 1 The growth of wolffia in each fertilizer treatment at the 15th day. A) number of wolffian thalli, B) size (length
and width) of wolffian thalli. A1 = the lowest concentration of equal NPK formula fertilizer, B1 = the lowest concentration
of nitrogenous-based formula fertilizer, C1 = the lowest concentration of equal phosphorus-based formula fertilizer. The
lowercase alphabets ‘a and b’ indicate the group that has a statistical difference at p = 0.0083.

Environmental factors throughout the experimental period were relatively stable. The average water
temperature was 26.63±0.68 oC, average air temperature was 27.54±1.27oC, average relative humidity
70.5±10.9 % and average pH of water was 6.03±0.05, 6±0, 5.53±0.05, 7.64±0.17, 7.81±0.05, 8.06±0.10,
6.06±0.10, 6±0, 5.86±0.13, 7.61±0.05 in A1, A2, A3, B1, B2, B3, C1, C2, C3 and control group respectively.

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Discussion
The result did not follow the hypothesis as we expected that the higher concentration of fertilizer

would increase the number of wolffia thalli. In contrast, the lowest concentration was likely to increase the
growth of wolffia better. This would be because the higher concentration would induce a hypertonic effect on
wolffia cells. Moreover, in the control group that we used only Nestlé Pure Life drinking water was likely to
increase the highest growth of wolffia in both thalli number and size, which could be inplied that some hidden
factors in this drinking water might be alternate nutrients (i.e., Calcium and Magnesium) which are suitable
for the growth [4]. The results also indicated that no flowering was observed during the experiment even we
used the phosphorus-based formula. Interestingly, the group under this treatment was likely to increase the
size of the thalli. We hypothesized that the low phosphorus concentration could enhace cell poriferation and
plant budding [5]. In addition to this, the phophorus-based fertilizer that we used in this experiment also
cantained a supplementation of some trace elements (TE).

Conclusion
Wolffia globosa that grew in the low concentration of all fertilizer formulas were likely to grow better

than the higher concentrations. Eventhough, the flowering was not observed in this experiment but the plants
that were treated with the phosphorus-based fertilizers seems to enhance the size of the thalli. However, using
only Nestlé Pure Life drinking water seems to increase the highest growth in both number and size of the plant
thalli. These may be underlined as one of factors in further wolffia biology studies regarding to the effect of
these treatments on the growth of a wolffia.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The

funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This
extended abstract is not for citation.

References
1 Romano E. L. and Aronne G. (2021). The World Smallest Plants (Wolffia sp.) as Potential Species

for Bioregenerative Life Support Systems in Space. Plant volume 10.
2 Deepanya, W. (2012). Full research report: The development of products water meal (Wolffia arrhiza

(L.) Wimm.) leather. Faculty of Science and Technology, Rajabhat Phetchabun.
3 Meetam M. (2021). ‘Associate Professor Doctor in Biology’Faculty of Science, Mahidol University.

(11 June 2021). An interview via electronic mail.
5 Chia TaI Group. (2020). Soil and nutrition management for paddyfield [Online], Access from:

https://www.chiataigroup.com/article-detail/fertilizerforrice.
4 Nestle Pure Life Thailand. (Published date is not available). 12 steps to control the water quality,

Access from: https://www.nestlepurelife.com/th/th-th/water-purification-process.

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Title :
Effects of nitrogen on Burkholderia pseudomallei OB1_04_04
Field : and rice growth
Authors :
Biology and Biodiversity
School :
Advisors : Miss Nichaphat Srirompotong

Miss Suphakarn Kietchalermkun

Demonstration School of Khon Kaen University, Khon Kaen University

Prof. Dr. Surasak Wongratanacheewin

Department of Microbiology, Faculty of Medicine, Khon Kaen University

Mrs. Kamonwannaporn singhamatr

Demonstration School of Khon Kaen University

Abstract
The number of Melioidosis patients among rice agriculturists has rapidly increased, especially in the

Northeast of Thailand. The disease is caused by a Gram-negative bacterium, Burkholderia pseudomallei (Bp.).
Recently, Burkholderia has been reported as a plant enhancing genus. Meanwhile, Bp. also has been found in
wetlands and nitrogen deficiency environments. This evidence contributes to a postulation that Bp. might be
considered as plant nitrogen-fixing bacteria, though it was suppressed by 7 mg/kg of nitrogen in soil. The main
purpose of this study was, therefore, to find the most optimal concentration of nitrogen that would significantly
decrease the average number of Bp. in the agricultural soil but still productively support RD16 rice growth. The
study was designed to explore 3 factors including nitrogen concentration, the Bp. growth, and the rice growth. The
objectives of the investigation were as follows: 1) the impact of nitrogen concentration on the bacterial growth and
its ability to stimulate the rice growth, 2) the impact of bacteria to the rice growth, and 3) the effects of nitrogen
concentration to the growth of both Bp. and the rice. The study was performed in both microbiology laboratories
and a greenhouse of agriculture faculty. There was comparison of different concentrations of nitrogen — N-15
(47.425 mg/kg), N-21 (64.675 mg/kg), and N-46 (136.55 mg/kg) — to the bacterial growth and the rice growth
separately. The finding revealed that the concentration at N-21 provided the best result with the least number of
bacteria and the highest growth of the rice. In addition, the bacterial growth did not show a significant effect on
the rice growth. The combination of 3 factors was then investigated in the greenhouse. The results demonstrated
that although there were no significant correlations among nitrogen concentration, the Bp. growth, and the growth
of the rice, the data showed that nitrogen concentration at N-21 tended to promote the highest height of the rice
with the least number of Bp. as found in the laboratories while N-15 gave the similar height of the rice as in N-21,
but the higher number of bacteria were observed. In contrast, N-46 granted the highest number of bacteria with
the lowest growth of the rice. In conclusion, as Bp. was found normally in soil of rice fields in the Northeast of
Thailand, the use of N-21 should benefit the rice growth but allow less chances to promote the bacteria, leading to
less chances of Burkholderia pseudomallei infections.

Keywords : Burkholderia pseudomallei, Melioidosis, RD16 Rice, Nitrogen, Fertilizer

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Introduction
Melioidosis is an infectious disease caused by a Gram-negative bacterium, B. pseudomallei (Bp.). Patients

mostly got the infection by interacting or consuming the bacterial contamination soil and water (Chuah JC,
Tan KE, Sermswan WR, Ziegler DA, 2017). With these means, cultivators, especially in the Northeast of Thailand,
are the most risk targets. According to a report from Khon Kaen hospital and so on between 2012 and 2015, the
patients at least 1700 people got Melioidosis. Moreover, at least 700 people died each year due to these bacteria

(ดเิ รก ลมิ้ มธรุ สกลุ และคณะ, 2021). Symptoms of disseminated Melioidosis infection may be presented with chronic

wounds at skin, parotid abscess, and labored respiration, which can range from mild bronchitis to severe
pneumonia. Additionally, the genus Burkholderia is liable to be nitrogen fixing bacteria (P Estrada-De Los Santos,
R Bustillos-Cristales, J Caballero-Mellado, 2001) which can transform dinitrogen along atmosphere to usable
compounds for plants such as ammonium and nitrate forms. In addition, there is an assumption that soil with high
nitrogen concentration (7 mg/kg) inhibited the Bp. growth, while less nitrogen (1.8 mg/kg) supported the Bp.’s
(Sermswan WR, Wonggratanacheewin S, 2017).

Vital nutrients for plants are nitrogen (N), phosphorus (P), and potassium (K). To fulfill the objectives,
we controlled the concentration of nitrogen in fertilizer at different ratios which are 15:0:0 to 21:0:0 and 46:0:0
for N, P, and K to set them as independent variables. Furthermore, a rice cycle is basically divided into 3 phases,
including vegetation, reproduction, and ripeness. In this study, we attended the vegetative phase (Day 42-49) by
the reason that we measured the growth of the rice by the height, not by the rice products.

Methodology
Part 1: Studying for the effect of nitrogen concentration on the Bp. growth

- Part 1.1 Culturing Bp. with different concentration of nitrogen in 10 g soil (small scale) in laboratory
The bacteria were cultured in 10 g soil with 15:0:0 (N-15), 21:0:0 (N-21), and 46:0:0 (N-46) NPK

fertilizer in the test tubes as experiment groups. The 0:0:0 (N-0) NPK fertilizer was used as a control group. The
number of the bacteria was detected by spreading plate every two weeks for two months.

- Part 1.2 Culturing Bp. with different concentration of nitrogen in 2 kg soil (plot scale) in a greenhouse
Likelihood, the methodologies of 1.2 were the same as 1.1 methods, but it was done in the pots located

in the greenhouse of Faculty of Agriculture, Khon Kaen University instead of test tubes.
Part 2: Studying for the effect of the number of Bp. on the growth of the RD16 rice

The 102, 104, and 106 colony-forming unit (CFU) per milliliter of the bacteria were initially added to the
pots of the seven days RD16 rice sprouts with N-21 in soil in the greenhouse. The control group had 0 CFU of the
bacteria. The height of the above-ground rice was then measured every two weeks for two months.
Part 3: Studying for the effect of nitrogen concentration on the RD16 rice growth

Nitrogen concentrations were varied from N-15, N-21, to N-46 which were considered as 47.425, 64.675,
and 136.55 mg/kg soil nitrogen for planting the seven days RD16 rice sprouts in the pots in the greenhouse,
compared to 0 mg/kg soil nitrogen (N-0). The height of the above-ground rice was then collected every two weeks
for two months.

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Part 4: Studying for the most optimal nitrogen concentration that promote the appropriate height of the
RD16 rice but the least number of Bp.

The equivalent number of Bp., 102 CFU per milliliter, was initially added into the pots of the seven days
RD16 rice sprouts with different amount of nitrogen: N-15, N-21, and N-46. The N-0 treatment was used as a
control group. The height of the above-ground rice and the number of Bp. were collected every two weeks for two
months.

Results and Discussion
Part 1: Effect of nitrogen concentration on the Bp. growth

The effect of nitrogen concentration on the Bp. growth is shown in Figures 1 and 2. On Day 42, we found
that N-21 granted the least number of Bp. in the laboratory compared to N-15 (Fig. 2) and the plot scale.

a = N-0
b = N-15
c = N-21
d = N-46
Day 42 (b,c) ; p < 0.01

Figure 1 Effect of nitrogen concentration Figure 2 Effect of nitrogen concentration
on the number of Bp. in laboratory. on the number of Bp. in plot scale.



Part 2: Effect of the number of Bp. on the growth of the RD16 rice
The effect of the bacterial number on the rice growth is shown in

the bar chart (Fig. 3) that the heights of the rice were equal for every
treatment from Day 7-49. In other words, the number of Bp. did not relate
to the rice growth.

Figure 3 Effect of the number of Bp.
on the growth of the RD16 rice.

Part 3: Effect of nitrogen concentration on the RD16 rice growth
Figure 4 on Day 49 illustrates that N-21 provided RD16 rice with

the highest heights, compared to the others. Additionally, the heights of
the rice on N-15 is significantly lower than N-0 (p < 0.01).

Figure 4 Effect of nitrogen concentration
on the height of rice.

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Part 4: Effects of Nitrogen on the Bp. growth and the rice growth
Different amounts of nitrogen in fertilizer did not grant the distinct number of Bp. (Fig. 5). Meanwhile,

the bar chart in Fig. 6 demonstrates that the height of the rice peaked both in N-15 and N-21 (p > 0.05) on Day 49.
From the result in Part 3 that the treatment with only N-21 in the condition gave the highest height of the rice, it
shows that Bp. still interacted with nitrogen and the rice to a certain extent.

Figure 5 Effect of nitrogen on the FiFgiugruere6 6EfEfeffcetcotfonfintriotrgoegnenononthteheRDRD1616
number of Bp. with RD16 rice. ricriecegrgorwowththwwithithBpB.p.

Conclusion

We found for the first time that N-21fertilizer with nitrogen concentration at 64.675 mg/kg could be the

most effective nitrogen concentration which gave the least number of Bp. in soil but still promoted the highest

height of RD16 rice, compared with the others. In addition, there was no significant correlation between the

number of Bp. and the rice growth. Therefore, a ratio of fertilizer at N-21 could be eligible to apply in RD16 rice

agriculture, increasing the rice growth but decreasing the infectious bacteria.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The funding

of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation and Melioidosis research
center, Khon Kaen University. This extended abstract is not for citation.

References

1. ร.ศ. ดร. นพ. ดิเรก ลมิ้ มธรุ สกุล และคณะ. คูม่ อื โรคเมลิออยด.์ พมิ พค์ รั้งที่ 1. กรุงเทพฯ: อกั ษรกราฟฟคิ แอนด์ดไี ซน;์ 2564.

2. Chuah JC, Tan KE, Sermswan WR, Ziegler DA. Hydrological connectivity and Burkholderia pseudomallei
prevalence in wetland environments: investigating rice-farming community’s risk of exposure to melioidosis
in North- Thailand. Springer 2017;189:287-300.l

3. P Estrada-De Los Santos, R Bustillos-Cristales, J Caballero-Mellado. Burkholderia, a genus rich in plant-
associated nitrogen fixers with wide environmental and geographic distribution. ASM 2001;67:2790-2798.

4. Sermswan WR, Wonggratanacheewin S. Physicochemical and Biological Factors of Soil and the Potential Use
of Antagonistic Microbes for Biocontrol of Burkholderia pseudomallei . Springer 2017.

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Title : Conservation and Micropropagation of Thrixspermum sp. OB1_02_09
In vitro

Field : Biology and Biodiversity

Author : Ms. Kununya Chalermponevonggul

Ms. Boonyaporn Autama

Mr. Buranas Faikrue

School : Demonstration School, University of Phayao
Advisor : Mrs. Thanyaporn Tangjaroenchai (Divison of Biology School of Science, University of Phayao)

Assoc. Prof. Dr. Kongsak Promthep (Demonstration School University of Phayao School of

Science, University of Phayao)

Ms. Hathairat Laksuk (Demonstration School University of Phayao School of Science, University
of Phayao)

Abstract
At present, the number of orchids in nature has decreased due to the forest destruction, and they

are smuggled out of the wild for commercial purposes. Thrixspermum is an epiphytic orchid, 160 species have
been reported. In Thailand, it is evenly distributed in all regions but in small numbers. It has short flowering
period that is risk of extinction. The objectives of this study were to conservation and micropropagation
Thrixspermum sp. to return to natural habitats by evaluating seed germination and the influence of growth
regulators on protocorm. Six-month-old pod seeds were sterilized for 15 minutes with a 30 percent v/v Clorox
solution containing 2 drops Tween-20 for 15 minutes. The sterilized seeds were cultured on Vacin and Went
(1949) (VW) medium. The results showed that seeds could germinate and develop into protocorms for
5 months. These protocorm segments were transferred to induced shoot and root on Vacin and Went (1949)
(VW) medium supplemented with various concentrations of NAA (1-Naphthaleneacetic acid) 0, 0.5, 1.0 and
1.5 mg/L and BA (6-Benzylaminopurine) 0, 1, 3 and 5 mg/L for 16 weeks. The cultures were kept under light
at 3,000 lux, 16 hours photoperiod. The results showed that Vacin and Went (1949) (VW) medium
supplemented with 0 mg/L NAA with 0 mg/L BA showed the most average on number of leaves and height
were 2.33 ± 1.07 per explant and 1.18 ± 0.23 cm, respectively.

Keywords : Thrixspermum sp., Micropropagation, Conservation

Introduction
Orchids are among the most diverse of the flowering plant families, with over 800 described genera

and 25,000 – 30,000 species. They are member of the family of monocots and distributed in the warm and
tropical region. Many of different orchid can variety that thrive in so many different area growing conditions
for instance, wood, soil, rock and damp area. In Thailand, Wild orchids have been surveyed 168 described
genera and 1170 species. They are very popular because the species that blooms with beautiful bright color
and grow in the forest. At present, they have been popular and have been used to expand the species in various
forms and currently they have become ornamental plants and great demand. Some of them are endemic and
nowadays some are rare or endanger species because of they are take out from natural habitets, climate change.

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Tissue culture techniques are propagation methods that can increase the number in a short period
of time and reduce the limitations that exist naturally Therefore, the objective is to propagate Thrixspermum
sp. by tissue culture technique by studying from the seed germination stage. Development as a protocorm in
different formulations for the benefit of conservation and then continue propagating orchids on other genera.

Methodology
The experiments were divided into 2 parts as follows,

Part 1 : Study on germination development stages of Thrixspermum sp. seeds.
Thrixspermum sp. capsules in yellow green color were collected and rinsed in tap water to remove

debris. Afterward, Immersing in 30% (v/v) Clorox solution and tween-20 2 drops for 15 min. The sterilized
capsules were transferred to a Biological Safety Cabinet (BSC). Dip in 95% ethanol and heat on Alcohol burner
for a few seconds. Let the heated capsule’s part cool for 10 sec before use. The capsules were cut in half
longitudinally by sterile scalpel. Seeds on a sterile Petri dish were transferred on Vacin and Went (1949) (VW)
medium. Take seeds into sterile room and kept under the light condition at 3,000 lux, 16 hours photoperiod
and temperature at 25±2 °C. for 5 months. Record results of seed germination under a stereo microscope
Part 2 : Study the growth of Thrixspermum sp. supplemented with plant growth regulators.

Five months old seedlings (size 4 mm) were cultured on VW (Vacin and Went, 1949)
supplemented with 0, 0.5, 1 and 1.5 mg/L NAA combined with 0, 1, 3 and 5 mg/L BA. Take the seedlings into
sterile room and kept under the light condition at 3,000 lux, 16 hours photoperiod and temperature at 25±2 °C.
for 16 weeks. Record growth, Number of leaves and height. Sort data in Microsoft excel and analyze by IBM
SPSS Statistics version 26

Results
1. Study on germination development stages of Thrixspermum sp. seeds.

From the study on germination development stages of Thrixspermum sp. seeds found that seeds
germination could be devided into 4 stages as shown in Figure (a-d) (adapted from Stewart and Kane, 2006)

(a) (b) (c) (d)

Figure 1 (a-d) Germination development stages of Thrixspermum sp. seeds
(a) Stage 0 : Mature seed.
(b) Stage 1 : No germination, swollen embryo.
(c) Stage 2 : Continued embryo enlargement.
(d) Stage 3 : Appearance of promeristem showing a pointed vegetative.

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2. Study the growth of Thrixspermum sp. supplemented with plant growth regulators
Effects of VW medium supplemented with organic additives namely, coconut water, mashed

potato and mashed bananas and supplemented with different concentrations of plant growth regulators.
The result found that VW medium supplemented with 0 NAA mg/L combined with 0 BA mg/L showed the
most average on number of leaves was 2.33±1.07 per explant and VW medium supplemented with 0 NAA
mg/L combined with 0 BA mg/L showed the highest of seedling was 1.18±0.23 cm as shown in Table 1 and
Figure 2
Table 1 Effect on the growth and development of Thrixspermum sp. (16 weeks after culturing).

Plant Growth Regulators (mg/L)

Treatment NAA BA Number of leaves Height (cm)
per explant
VW 1 0 0 2.33 ± 1.07a 1.18 ± 0.23a
VW 2 0 1 1.54 ± 0.50b 1.14 ± 0.46ab
VW 3 0 3 1.25 ± 0.45bc 1.00 ± 0.29abcd
VW 4 0 5 1.58 ± 0.51b 0.96 ± 0.24abcd
VW 5 0.5 0 1.33 ± 0.49bc 1.03 ± 0.34abcd
VW 6 0.5 1 1.25 ± 0.45bc 1.08 ± 0.19abc
VW 7 0.5 3 1.17 ± 0.39bc 0.94 ± 0.16abcd
VW 8 0.5 5 1.25 ± 0.45bc 0.92 ± 0.19abcd
VW 9 1 0 1.25 ± 0.45bc 0.84 ± 0.21cd
VW 10 1 1 1.00 ± 0.00c 0.91 ± 0.35abcd
VW 11 1 3 1.25 ± 0.45bc 0.83 ± 0.23cd
VW 12 1 5 1.25 ± 0.45bc 0.79 ± 0.20d
VW 13 1.5 0 1.00 ± 0.00c 0.88 ± 0.15bcd
VW 14 1.5 1 1.25 ± 0.45bc 1.03 ± 0.39abcd
VW 15 1.5 3 1.00 ± 0.00c 1.02 ± 0.41abcd
VW 16 1.5 5 1.08 ± 0.29c 1.14 ± 0.46ab

The means values by different letters within the column are significantly different based on Duncan’s multiple
range test (P< 0.05)

(a) (b) (c) (d)

Figure 2 (a-d) Example of the effect on Vacin and Went medium supplemented with Plant growth regulators

(NAA with BA) on number of leaves and seedling height (a) N0B0 (NAA 0 mg/L+ BA 0 mg/L), (b) N1B1

(NAA 1 mg/L+ BA 1 mg/L), (c) N1B5 (NAA 1 mg/L+ BA 5 mg/L), (d) N1.5B0 (NAA 1.5 mg/L+ BA 0 mg/L)

Scale bar = 2 mm

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Discussion
In this study, Thrixspermum sp. seeds were able to germinate and develop into seedling on VW

medium supplemented with organic additives including coconut water, mashed potatoes and mashed bananas
and mix of these substances (free - plant growth regulator). Seedling can grow better than other treatments.
Tawatchai et al. (2013) reported the protocorms of Dendrobium aphyllum (Roxb.) Fischer cultured on
a medium without the addition of growth regulators were able to increase the number of protocorms better than
those cultured on medium supplemented with BA at a concentration of 0.5-2.0 mg/L. While leaf parts of
Brasiliorchis picta cultured on a medium without the addition of growth regulators can induce protocorm like
bodies (PLBs) due to the influence of cytokinins within the orchid parts themselves (Santos et.al, 2016)

Conclusion
From the study of Conservation and Micropopagation of Thrixspermum sp. In vito from pods in

Vacin and Went (VW) for 16 weeks, showed that the germination development stages of Thrixspermum sp.
seeds in 4 stages from embryo to seedling stages. Development in Plant Growth Regulators found that Vacin
and Went (1949) (VW) medium supplemented with 0 mg/L NAA with 0 mg/L BA showed the most average
on number of leaves and height is 2.33 ± 1.07 per explant and 1.18 ± 0.23 cm, respectively.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS).

The funding of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation.
This extended abstract is not for citation.

References
Chen Xinqi and Jeffrey J. Wood. THRIXSPERMUM. Flora of China. 2009, 25: 466–470.
Santos, S.A., Smidt, E.C., Padial, A.A. & Ribas, L.L.F. Asymbiotic seed germination and in vitro

propagation of Brasiliorchis picta. African Journal of Biotechnology. (2016), 15(6), 134-144.
Stewart, S.L., and M.E. Kane. Symbiotic seed germination of Habenaria macroceratitis (Orchidaceae),

a rare Florida terrestrial orchid. Plant Cell Tiss. Organ Cult. 2006, 86: 159-167.
Tawatchai Subthira, Suphap Suntaranond and Sumontip Bunnag. Effects of Plant Growth Regulator on in vitro

culture of Dendrobium aphyllum (Roxb.) Fischer. KKU Res J (GS) 13 (1) : January - March 2013.
Wen-Hong Chen and Yu-Min Shui. A New Species of Thrixspermum (Orchidaceae) from China. Brittonia,

Vol. 57, No. 1 (Jan. - Mar., 2005), pp. 55-58

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Ti tl e: Applying silage as mulch: A case study of OB1_17_07
Lycopersicon esculentum Mill.
Fi eld:
Author: Biology and Biodiversity
School : Miss Jutamart Chaeaul
Advi sor: Miss Pawitra Payakhapiban

PSU. Wittayanusorn Suratthani School
Prince of Songkla University, Surat Thani Campus

Associate Professor Dr. Theera Srisawat (Prince of Songkla University, Surat Thani
Campus)

Miss Phimphanit Khongrueang (PSU. Wittayanusorn Suratthani School)

Abstract
Evaluation of suitable mulch material for organic tomato production is an important issue in organic

farming. The present study aimed to compare the effect of silage mulching on the yield, quality, and growth of
tomato. A field experiment was conducted at a private farm in Khiansa district, Surat Thani, from October 2021
to February 2022 to evaluate the mulching performance on tomato production. To do this, plastic PE, straw and

silage, and control group without mulch, were tested to find the best materials that can help plant grow and
improve production of tomato. We found that all the mulches significantly influenced fruit quality, but there
was no difference on the plant growth and yield. The results indicated that the highest yield of fruit number
(918) and fruit weight (9,265 g) was found in the mulch with plastic PE. The total fruit number and fruit weight
were no significant differences in all mulches. The highest number of leaves (138 leaves) were observed in the
plastic PE mulch treatment, but the highest plant height (71 cm) was observed in the control group. Based on

our univariate approach, the silage can be an effective, more environmentally friendly alternative to
polyethylene and straw mulches. Among three mulch materials, silage- based mulch has effective on plant
development and yield production. Its advantages due to the environmental and cost issues make silage an
attractive option for mulch material in organic farming.

Keywords: Tomato; Mulch; Plastic PE; Straw; Silage

Introducti on
Tomato (Lycopersicum esculentum Mill.) is commonly grown vegetable in the world because it is

cultivated for consumption on all continents. The essential component in tomato is considered for high level of

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dietary source of the antioxidant lycopene, resultingin benefits to human health. In modern agriculture, overuse
of plastic mulch is concerned, resulting in ecosystem problems. Although straw has been widely used in
agriculture, it is adding cost of material. Silage is organic material that is bothbiodegradable and compostable,
which farmers can prepare by themselves. By the way, the effect of silage on growth and crop yield has never
been described. In this study, we used silage as mulch material compared to plastic PE and straw to find out
efficiencies on plant growth and yield of tomato.

Methodology

1. Experimental description

The experiment was conducted under field conditions at a private farm of Khiansa district, Surat
Thani. The experiment was carried out during the rainy season (from October 2021 to February 2022). The
experiment was designed with four treatments. The treatments consisted of three different materials for soil
mulches: 1) plastic PE, 2) straw and 3) silage, whereas without mulching used as control. The plants were
carefully grown for 122 days.

2. Plant growth and tomato yield

Evaluations of plant growth were carriedout at 3-day intervals, based on plant height and number
of leaves with three replicates. The total number of fruit and fruit weight/ plant( g) were recorded. Fruit
Circumference(cm) and fruit length(cm) were randomly evaluated as fruit quality.

3. Soil temperature and moisture

Soil temperature and moisture were recorded, at 7- day intervals, from the beginning day of
transplanting until 35 days. The collection of data was monitored with a Flower care smart monitor (Beijing
Huahua Caocao Technology, Beijing, China).

4. Statistical analysis

We used statistical analysis (analysis of variance, ANOVA) to test the effect of mulching on
development and productivity variables. If statiscally significant effects of each factor were reported (P < 0.05),
the means were thus compared by the Scheffer test at 5%probability.

Resul ts
1. Plant growth and Tomato yield

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Among mulching materials, there was no difference in plant height and number of leaves (Table
1). For the number and weight of fruit, the treatments with the plastic PE had the highest average of the total
number of fruits and fruit weight per plant (Table 2). Fruit qualities significantly increased in all mulching
treatments. The measured large tomato fruit by circumference was 8 cm with the widest fruit on the treatment
of straw (8.83 cm), silage (8.66 cm) and plastic PE (8.36 cm), respectively.

Table 1. Plant height and number of leaves under different mulching, at 44 days after transplanting

Treatment Plant Height (cm) Number of leaves
Plastic PE 61.00±4.58 138.33±12.58
Straw 57.00±12.53 71.00±13.53
Silage 67.33±2.31 98.00±2.65
Control 71.67±26.50 121.67±59.23

Table 2. The number and weigth of fruit under different mulching

Treatment Fruit Fruit Fruit Fruit weight Total Number
Circumference(cm) Length(cm) weight/Fruit (g) /Plant(g) of Fruit

Plastic PE 8.36±0.38ab 3.77±0.42 10.09 386.04 918

Straw 8.83±0.50a 3.78±0.38 10.66 161.28 363

Silage 8.66±0.56ab 3.77±0.30 10.57 155.07 352

Control 8.07±0.36b 3.38±0.35 7.94 202.02 611

Values within the same columns followed by different letters were significantly difference at P<0 .05 (Scheffe)

2. Soil temperature and moisture
The highest soil moisture was found in the treatment of straw mulching (Table 3). The lowest soil

temperature (32.00°C) was observed by using silage material.

Table 3. Average soil temperature and moisture under different mulching.

Treatment Soil moisture (%) Temperature (°C)
Plastic PE
Straw 33.25±10.40 32.18±1.94
Silage 39.25±14.38 32.08±2.22
Control 30.25±14.31 32.00±2.36
37.00±4.08 32.03±1.80

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Di scussion
The lowest temperature effect of silage produced on soil caused by the material reduced

evaporation rate from soil surface and increased soil protection from solar radiation. We found that the
application of straw mulch resulted in highly increased soil moisture, enhancing water infiltration. The largest
size by width of tomato fruit produced by straw and silage muching, respectively. Previous authors reported
that the effect of grass residue mulching on plant height was more significant effective because of its ability to
keep soil temperature and moisture at optimum level ( Mendonça et al. , 2021) . This is the first report that
comfirmed the increased effect of silage mulching on tomato yield and fruit quality.

Concl usion
All mulchmaterials helpedto increase the soil moisture retentionandoptimize the soil temperature

for tomato palnt growth resulting in increased yield. It is possible to conclude that silage material has an effect
on tomato plant development and yield, resembled those obtained by the others. The study thus reveals that
silage can be used effectively as representative mulching material in agriculture.

Acknow l edgements
This project was supported by Science Classroom in University Affiliated School (SCiUS) under

Prince of Songkla University, Surat Thani Campus and PSU. Wittayanusorn Surat thani School. The funding of
SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This extended abstract
is not for citation.

References
1. Goel, L., Shankar, V. and Sharma, R. (2020). Effect of organic mulches on agronomic parameters – a case

study of tomato crop (Lycopersicon esculentum Mill.). International Journal of Recycling of Organic Waste
in Agriculture. 9(3): 297-307.
2. Hudu, A. I., Futuless, K. N., amp, Gworgwor and N. A. (2002). Effect of mulching intensity on the growth and
yield of irrigated tomato (Lycopersicon esculentum Mill.) and weed infestation in semi-arid zone of Nigeria.
Journal of Sustainable Agriculture. 21: 37-45.
3. Mendonça, S. R., Ávila, M. C., Vital, R. G., Evangelista, Z. R., Pontes, N. de, &amp; Nascimento, A. dos.
(2 0 2 1 ). The effect of different mulching on tomato development and yield. Scientia Horticulturae. 275:
109657.

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List of Science Projects 12th SCiUS Forum
Oral presentation

Biology and Biodiversity Group 2
Saturday August 27, 2022

No. Code Title Author School
Rajsima Witthayalai
1 OB2_06_03 Histochemistry and Miss Natnaree Srisuk School

Immunocytochemical studies Miss Chalisa Sangaprakhon PSU.Wittayanusorn
School
of leptin-like peptide in the Miss Suchanat Chamnandee
Lukhamhanwarinchamra
secretory cells from the b School
Naresuan University
salivary gland of Pomacea Secondary
Demonstration School
canaliculata PSU Wittayanusorn
Surat Thani School
2 OB2_15_03 Ground foraging ant Miss Sidarat Nakpalad
Paphayomphittayakom
communities at the Plant Miss Naphason Srakhao School

Genetic Conservation Area of Demonstration School
of Khon Kaen
Prince of Songkla University, University

Songkhla Province.

3 OB2_08_01 Feeding Behaviors in Miss Yanisa Jaroenporn

Cellular Slime Molds from Miss Juthakan Phiwton

Ubon Ratchathani province Miss Kwanjiraporn Sukkasem

4 OB2_03_05 Vasorelaxant effect on rat Miss Trongkwun Wichai

isolated aorta of Shiitake Miss Narissara Singanusong

mushroom (Lentinula

edodes) protein hydrolysate

5 OB2_17_03 Isolation and characterization Mr. Phongsatorn Ousakulwattana

of probiotics from mangrove Miss Rinrada Suwandee

sediments for application in

inhibition biofilm formation

of bacterial pathogens in

aquaculture

6 OB2_14_02 Antimicrobial Resistance and Miss Tidarat Khunchit

Virulence Factors of Miss Pinmanee Srethongkaew

Pseudomonas aeruginosa

Isolated from

Slaughterhouses in

Phatthalung Province

7 OB2_04_03 Curcumin-loaded Miss Chawanluk

nanocomplexes alleviate Ruangchayajatuporn

adenine-induced acute kidney Miss Yada Pratipanawatr

injury in hamsters

83

No. Code Title Author 12th SCiUS Forum

8 OB2_17_04 Prevalence of pedunculate Mr. Napat Saejiw School
PSU Wittayanusorn
barnacle (Octolasmis spp.) Miss Natthanicha Ragpinij Surat Thani School

infection in mud crab (Scylla Mahasarakham
University
paramamosain) and effect of Demonstration School
(Secondary)
salinity in vitro Engineering Science
Classrooms
9 OB2_07_03 The attractiveness of different Miss Sasinan Muenhawong (Darunsikkhalai School)

common household sugar Miss Pattarathida Khunthula Rajsima Witthayalai
School
types to the ghost ant Mr. Tanawut Hancherngchai
Demonstration School
(Tapinoma melanocephalum) of Khon Kaen
University
10 OB2_09_04 The study of Morphology and Miss Kanganid Aswanuwath
Demonstration School,
Probiotic Characterization of Miss Chotchanit Boonchuay University of Phayao

Lactic Acid Bacteria Isolated Mr. Arthapol Tharananithikul

from fermented food

11 OB2_06_02 Separation and Miss Koranan Panpim

characterization of Miss Phattaratida Navachat

antibacterial compounds from Miss Sasikan Wangklang

soil bacteria in animal farm

12 OB2_04_08 Sequence comparison of Miss Yathida Lopphanthong

human collagen type I in Miss Kunanchaya Boonrom

mollusk databases and

characterization of acid

soluble protein in freshwater

snail genus Pomacea

13 OB2_02_07 Effects of 20- Miss Anyada Wankaew

hydroxyecdysone on protease

activity in ovary of the red

flour beetle, Tribolium

castaneum

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Title : Histochemistry and Immunocytochemical studies of leptin- OB2_06_03
like peptide in the secretory cells from the salivary gland of
Field : Pomacea canaliculata
Author :
Biology and Biodiversity
School :
Advisor : Chalisa Sangaprakhon

Natnaree Srisuk

Suchanat Chamnandee

Rajsima Wittayalai School, Suranaree University of Technology

Asst. Prof. Dr. Naruwan Saowakon, School of Preclinical Sciences, Institute of Science,
Suranaree University of Technology

Abstract

The golden apple snail (Pomacea canaliculata) has been classified as an invasive species to destroys
crops in agriculture, especially rice and orchid fields. It eats the young plants in the seedling stage until tillering
by biting the stems of those plants. It makes an economic loss. Leptin is the hormone that controls or regulates
digestive enzymes in feeding and fasting behaviors. The enzymes in the feeding behaviors are produced and
secreted from several organs such as the salivary gland, stomach, and intestine. The secretory cells in the digestive
organs secrete enzymes and hormones. Therefore, the objective of this study was to investigate the secretory cells
of the salivary glands of the adult P. canaliculata during feeding and fasting by cytochemistry and
immunolocalization of leptin. In the H&E staining in salivary glands of P. canaliculata, three different secretory
cell types were detected in the glandular epithelium, interlobular duct, intralobular duct, and salivary duct. They
contained mucocytes, granular cells, and vacuolated cells. The mucocytes were the most cell types located at the
end of the secretory part and granular cells were between vacuolated cells. The acid and neutral
mucopolysaccharides are detected by Alcian blue pH 2.5 and PAS-Alcian blue pH 1.0. The mucocytes produced

both acid and neutral mucopolysaccharides while vacuolated cells produced only mucin to lubricant the digestive
tract. The mucocytes of fed snails presented less acid and neutral mucopolysaccharides than fasted snails. In the
salivary gland, strong leptin-immunoreactive(ir) was detected in granular cells and moderated leptin-ir was found
in mucocytes of the fasted group more than in the fed group. The findings on the presence of leptin hormone
indicated that it may inhibit the mucin and enzymatic secretion of a salivary gland. The basic knowledge obtained
from the finding in this work is beneficial in controlling for the overpopulation of the golden apple snail.

Keywords: Leptin hormone, Mucin, P. canaliculata, Salivary gland, Secretory cell

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Introduction
Pomacea canaliculata or golden apple snail is classified as one of the serious pests spread-out in the

economic field due to its high consumption rate, short life cycle, and smaller number of enemies. Generally,
feeding and fasting behaviors in the animal are regulated by the balancing of leptin- ghrelin hormones. Leptin in
the mammal is an adipocyte-derived hormone, that is produced from various organs such as the salivary gland
nervous tissues, stomach, and reproductive organs. The salivary gland is one of the accessory organs in feeding
behavior that secretes mucin and digestive enzymes. The extensive studies of leptin in the mammal reported that
leptin involves food intake and the animal reproductive behavoir whereas the studies about leptin in the
invertebrates has been still uncleared. We focus on the leptin in the salivary gland of the golden apple snail during
feeding and fasting changes. Therefore, the objective of this study was to investigate the morphology of secretory
cells and the distribution of leptin-like peptide hormone from the salivary glands of the P. canaliculata by
histochemistry and immunocytochemical techniques.

Methodology
The experimental framework of this study is shown in Fig. 1. Briefly, golden apple snails were collected

from a freshwater pond. After acclimatization, snails were divided into 2 groups by feed and fast for 24 hours,
then the salivary glands in each group were collected and processed for morphological study, and the existence of
leptin-like peptides study by H&E, Alcian blue pH 2.5, PAS-Alcian blue pH 1.0, and immunoperoxidase methods.
The secretory cells were identified, counted, compared, and analyzed by Turkey’s post hoc test.

Fig. 2 The gross anatomy of
salivary gland (SG) and
esophagus (E) of P. canalicalata
after dissection.

Fig. 3 (a) the embedded
cells section by microtome.
(b) Floating in water-bath.

Fig. 1 Experimental framework of this experiment

Result and discussion
When stained with hematoxylin and eosin (H&E), two salivary glands opened into the buccal cavity through

a main salivary collecting duct that runs by the side of the esophagus as shown in Fig. 4. They were classified base
on the morphological appearance of the secretory part and ductal part, respectively. A salivary gland of P.
canaliculata was classified in the compound tubuloacinar type, which contained many lobe.

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Each lobe consisted of numerous lobules connected between them by a ductal part. The extractor duct of the
salivary gland was classified into 3 types; (i) interlobular duct, (ii) intralobular duct, and (iii) salivary duct which
glandular epithelium lined with simple columnar epithelium with cilia, simple high cuboid, and simple low
cuboidal epithelium, respectively. The secretory part of the salivary gland of P. canaliculata was composed of
three cell types: (i) mucocytes, (ii) granular cells, and (iii) vacuolated cells. The mucocytes presented an eccentric
nucleus with numerous small vacuoles. They were acini- liked in shape and located at the end of a secretory part
(Fig. 5a and b) The granular cells were found between mucocytes and vacuolated cells. They revealed an eccentric
nucleus with pink granules in the cytoplasm. The vacuolated cells contained the large vacuoles in the cytoplasm.
The characteristic of vacuolated cells seemed like goblet cells in the intestine.

Fig. 4 (a) The longitudinal section of the Fig. 5 (a and b) Sections of the salivary gland of P. canaliculata stained with H&E,
salivary gland of P. canaliculata stained with showing mucocytes (MC), granular cell (GC), and vacuolar cell (VC). (c and d) secretory
H & E, showing secretory cell type (I, II, III), cells of salivary gland showing the different cell types by Alcian blue pH 2.5 and Periodic
scale bar = 100 m. (b and c) High acid Schiff (PAS)-Alcian blue pH 1.0 before changing feeding. (e and f) the secretory cells
of the fed group showed a weak intensity of Alcian blue and PAS-Alcian blue (pH 1.0) than
magnification of salivary gland from fasted group (g and h) Scale bar= 25 m.

rectangular showing the different cell types I Alcian blue and Periodic acid Schiff (PAS) stainings were used to
(mucocyte, arrowhead), II (granular cell,
arrow), III (vacuolated cells, VC), classify the type of mucin. The acid mucin was detected by Alcian blue
(interlobular duct, D), (esophagus, E), (H&E),
while PAS detected the neutral mucin, showed in blue and purple,
25 m.

respectively. The mucocytes presented strong staining of Alcian blue (AB) pH 2.5, while the vacuolated cells were

unstained. The granular cell contained the pink granules in the cytoplasm (Fig. 5c). Sections stained with a

combined technique of PAS-Alcian blue (pH 1.0) reaction showed a strong blue-purple color in secretory cells.

Some of the mucocytes were stained pink color (Fig. 5d). As the result, it implied that the secretory cells in the

salivary gland can be produced both neutral and acid mucin. In the relative comparison of mucin-type production

between fed and fasted groups, the positive staining of Alcian blue pH 2.5 and PAS-Alcian blue pH 1.0 showed a

strong result in the fasted group than in the fed group (Fig. 5e-h). It showed a greater significance of neutral mucin

(purple and pink) in the fasted group than in the fed group (p<0.05); (44.56 ± 5.03% vs 24.29 ± 7.33% and 53.32

± 6.12 vs 18.59 ± 8.77%), while the acid mucin (blue) was hardly different (23.48 ± 6.83% vs 13.07 ± 7.38%) as

shown in Fig. 6.

Fig. 6 The percentage of mucous

cells in the secretory cell types in
the salivary glands. A different

character on the graph showed
significant differences (p <0.05).

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Those mucins of P. canaliculata might act similar to characterize like other gastropods, they were used
to lubricate food and kill pathogens (1,2). The immunohistochemistry method revealed that the existence and
localization of leptin-liked peptides were found in granular cells and mucocytes while vacuolated cells did not
express. The leptin immunoreactivity (ir) of granular cells in the fasted group showed intensive stronger stained
than in fed group, and moderately stained in mucocytes (Fig. 7). The presence of leptin in secretory cells of the
salivary gland may relate to food intake function and may be inhibited the secretory cell from releasing acid and
mucin during fasting to prevent their life from danger.

Fig. 7 Leptin-liked immunoreactive (ir) in the secretory cells
of the salivary gland of P. canaliculata between fasted group
(a) and fed group (b). The strong leptin-ir was observed in
granular cells (GC), moderate staining in mucocyte (MC), and
unstained in vacuolated cells. Scale bar =25 m.

Conclusion

The secretory cells in the salivary glands of P. canaliculata are classified into 3 types; mucocytes,
granular cells, and vacuolated cells joined with the intralobular duct, interlobar, and main salivary duct through
the buccal cavity. Mucocytes and granular cells produced neutral and acid mucin while vacuolated cells produced
only mucin to lubricate food in the digestive tract. Mucocytes and granular cells of salivary glands in the fasted
snails were found the intensive immunoreactivity of leptin- like peptides more than in the fed snails. Mucocytes
and granular cells would act as endocrine. The findings on the presence of leptin hormone indicated that it may
inhibit the mucin and secretion of a salivary gland. The knowledge from the finding is beneficial in controlling for
overpopulation of the golden apple snail.

Acknowledgments

This project was supported by Science Classroom in University Affiliated School ( SCiUS) under
Suranaree University of Technology and Rajsima Wittayalai School. The funding of SCiUS is provided by the
Ministry of Higher Education, Science, Research, and Innovation, which is highly appreciated. This extended
abstract is not for citation.

References

(1) Lo-da-Cunha A, Alves Â, Oliveira E, Calado G. Comparative study of salivary glands in carnivorous and
herbivorous cephalaspideans (Gastropoda: Euopisthobranchia). Journal of Molluscan Studies 2016; 82: 43-54.

(2) Poljaroen J, Tinikul Y, Tinikul R, Anurucpreeda P, Sobhon P. Leptin-like immunoreactivity in the central
nervous system, digestive organs, and gonads of the giant freshwater prawn, Macrobrachium rosenbergii. Acta
Histochemica 2017; 119: 569-581.

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Title : Ground foraging ant communities OB2_15_03

Field : at the Plant Genetic Conservation Area of Prince of Songkla University, Songkhla Province.
Author : Biology and Biodiversity
Miss Naphasorn Srakhao
School : Miss Sidarat Nakpalad
Advisor : PSU.Wittayanusorn School, Prince of Songkla University
Mr. Surawut Saengmanee
Miss Apinya Boonkhum
Dr. Nawee Noon-anant (Division of Biological Science, Faculty of Science,
Prince of Songkla University)

:Abstract

Ants are insect belonging to the order Hymenoptera, family Formicidae and play various roles in the
terrestrial ecosystem, including one of the bioindicators of the environmental change. This study aimed to
investigate species composition and species diversity of the ground foraging ants, to determine some physical
and biological factors, and to determine the relationships between some physical and biological factors and
ant species richness at the Plant Genetic Conservation Area of Prince of Songkla University, Songkhla
Province during September to October 2021. Two study sites (the plant communities along the ridge with
shallow soils and rock outcrops and the plant communities along the valley slopes and near water) were chosen
and each plant community was set up with three parallel line transects. Ants were collected by three methods
(honey bait traps, pitfall traps, and leaf litter sifting) along each line transect. Some physical and biological
factors (soil temperature, soil pH, soil humidity, litter humidity, canopy cover, and land cover) were recorded.
Sorensen similarity index, t-test, and Spearman’s rank correlation coefficient were analyzed. A total of 50 ant
species belonging to 29 genera and 6 subfamilies were identified based on the morphological characteristics
of the workers. Myrmicinae was found the most species (20 species) followed by Ponerinae (15 species). The
top two dominant ant genera were Pheidole (8 species) followed by Tetramorium (5 species). The number of
ant subfamilies, genera, and species in the plant communities along the valley slopes and near water was higher
than in the communities along the ridge with shallow soils and rock outcrops (6 subfamilies, 25 genera and 39
species and 5 subfamilies, 18 genera and 28 species, respectively). The similarity of ants in two plant
communities was 51 percent and 17 ant species were found in both plant communities. Regarding some
physical and biological factors, the results showed that soil pH was the only one factor significantly different
in September and October. In October, the species richness of Tetramorium was significantly positively
correlated to soil temperature in plant communities along the ridge with shallow soils and rock outcrops, on
the other hand, Tetramorium was significantly negatively correlated to soil temperature in plant communities
along the valley slopes and near water

Keywords : ground foraging ants, ant species composition, ant species diversity,
Plant Genetic Conservation Area, Prince of Songkla University

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Introduction
The ants are belonging to the order Hymenoptera, family Formicidae and play an important role at many

levels in the ecosystem, such as herbivores, carnivores and predators (Alonso, 2000). Moreover, ants can be
used to be the bioindicator, due to the sensitivity to the habitat fragmentation and disturbance (Alonso, 2000).
Diversity of ants at the Plant Genetic Conservation Area of Prince of Songkla University are a few reports, at
the same time, the determination of the relationships between some physical and biological factors and ground
foraging ant species richness are no report (Watanasit et al., 2005; Ruadreo, 2014). Thus, this study aimed to
investigate species composition and species diversity of the ground foraging ants, to determine some physical
and biological factors, and to determine the relationships between some physical and biological factors and
ant species richness.

Methodology

Three parallel line transects, 40 meters long and 20 meters apart (Figure 1), were set up in each plant

community in the Plant Genetic Conservation Area of Prince of Songkla University, Songkhla Province: the

plant communities along the ridge with shallow soils and rock outcrops and the plant communities along the

valley slopes and near water.

Ants were collected by three

sampling methods along each line

transect: honey bat traps (30

minutes), pitfall traps (24 hour) and

leaf litter sifting (1 hour). Some

physical and biological factors (soil

temperature, soil pH, soil humidity,

litter humidity, canopy cover and

land cover) were recorded at the Figure 1: The three line transects with the position of the ant sampling methods,

physical and biological factors
Points of each line transect (Figure 1).

Ants were identified to the subfamilies, genera, and species based on the morphological characteristics

of the workers. The ant specimens were taken with the Leica M205 FCA fluorescence stereo microscope and

multi-focused montage images were produce using Helicon Focus (Pro) 8.1.0 from a series of photographs.

Sorensen similarity index (ant diversity between the two plant communities based on the qualitative
data), t-test (the physical and biological factors between the two plant communities) and Sperman’s rank

correlation coefficient (two dominant ant genera, and the physical and biological factors) were analyzed. In

all cases, a P-value of 0.05 or less was considered statistically significant.

Results and Discussion
Composition and diversity of ground foraging ant

A total of 50 ant species belonging to 29 genera and 6 subfamilies were found. Myrmicinae had the
highest number of species (20 species) (Table 1), consistent with the previous studies in this area (Watanasit
et al., 2005; Ruadreo, 2014). Most species of Myrmicinae and Ponerinae were ground foraging ants which
found in the leaf litter and soil layer, as expected from the previous studies in this area and the lowland area

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of the Southern Thailand (Watanasit et al., 2005; Watanasit et al., 2008; Ruadreo, 2014). This study found
Lioponera which is the new ant genus record in the Southern Thailand (Khachonpisitsak et al., 2020). The
genus Pheidole found the highest number of species (8 species), as expected from the previous studies in this
area and the lowland area of the Southern Thailand (Watanasit et al., 2005; Watanasit et al., 2008; Ruadreo,
2014). Moreover, the Phidole is the second largest genus in the world (Antwiki, 2022).

Table 1: Composition and diversity of ground foraging ants in the plant communities along the ridge with

shallow soils and rock outcrops (A) and the plant communities along the valley slopes and near water (B)

A B Total of Total of

Subfamilies Genera Species Genera Species genera Species

1. Dolichoderinae 3 4 3 4 3 5

2. Dorylinae 1111 2 2

3. Ectatomminae - - 12 1 2

4. Formicinae 3 5 2 2 3 6

5. Myrmicinae 5 10 8 17 9 20

6. Ponerinae 6 8 10 13 11 15

Total 18 28 25 39 29 50

The number of ant subfamilies, genera and species in the plant communities along the valley slopes and
near water higher than in the communities along the ridge with shallow soils and rock outcrops (6 subfamilies,
25 genera and 39 species and 5 subfamilies, 18 genera and 28 species, respectively) (Table 1), consistent with
the previous studies in this area (Ruadreo, 2014). The similarity of ants in two plant communities was 51
percent and 17 ant species were found in both plant communities. The habitat complexity in the plant
communities along the valley slopes and near water, such as the plant diversity, might be higher than in the
communities along the ridge with shallow soils and rock outcrops, then this might be the main factor effect to
the composition and diversity of the ground foraging ants.

Some physical and biological factors
In the September, soil temperature and canopy cover in the communities along the ridge with shallow

soils and rock outcrops, were higher than in the plant communities along the valley slopes and near water. At
the same time, land cover and soil pH in the plant communities along the valley slopes and near water, were
higher than in the communities along the ridge with shallow soils and rock outcrops. In the October, soil
humidity and litter humidity in the plant communities along the valley slopes and near water, were higher than
in the communities along the ridge with shallow soils and rock outcrops. At the same time, soil pH in the
communities along the ridge with shallow soils and rock outcrops was higher than in the plant communities
along the valley slopes and near water.

This resuts indicated that the most physical factors between the two plant communities were different
between the September and October. These factors might not be the main factors effect to the composition and
diversity of the ground foraging ants in this area.

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Relationships between some physical and biological factors and ant species richness
In October, the species richness of Pheidole in the plant communities along the ridge with shallow soils

and rock outcrops, was significantly positively correlated to land cover (r = 0.99, p = 0.04). At the same time,
the species richness of Tetramorium in the plant communities along the ridge with shallow soils and rock
outcrops, was significantly positively correlated to soil pH (r = 1.00, p = 0.00) and soil temperature (r = 1.00,
p = 0.00), but Tetramorium in plant communities along the valley slopes and near water, was significantly
negatively correlated to soil temperature (r = -1.00, p = 0.00). This study not consistent with the previous
studies in the other area of the Southern Thailand, which Pheidole was significant correlated to the soil
temperature and Tetramorium not significant correlated to the soil temperature (Watanasit et al., 2008).

Conclusion
The plant communities along the valley slopes and near water found the number of ant subfamilies,

genera and species, higher than the communities along the ridge with shallow soils and rock outcrops. In
September and October, soil pH was the only one factor significantly different between the two plant
communities. Pheidole was significantly positively correlated to land cover in the plant communities along
the ridge with shallow soils and rock outcrops. While, Tetramorium was significantly positively correlated to
soil pH and soil temperature in the plant communities along the ridge with shallow soils and rock outcrops,
but significantly negatively correlated to soil temperature in the plant communities along the valley slopes and
near water.

Acknowledgements
This project was supported by Science Classroom in University Affiliated School (SCiUS). The funding

of SCiUS is provided by Ministry of Higher Education, Science, Research and Innovation. This extended
abstract is not for citation.

References
Antwiki. Diversity by taxonomic group [Internet]. 2022 [cited 2022 May 29]. Available from: https://www.
antwiki.org/wiki/Diversity_by_Taxonomic_Group.
Alonso LE. Ants as Indicators of Diversity. In: Agosti D, Majer JD, Alonso LE, and Schultz TR, editors. Ants:
Standard Methods for Measuring and Monitoring Biodiversity. Washington and London: Smithsonian
Institution Press; 2000. p. 103-11.
Khachonpisitsak S, Yamane S, Sriwichai P, Jaitrong W. An updated checklist of the ants of Thailand
(Hymenoptera, Formicidae) ZooKeys 2020;998: 1-182.
Ruadreo N. Ant communities in lowland semi-evergreen foret at a reserve area of Prince of Songkla
University, Songkhla Province [Undergraduate Degrees]. Songkhla: Prince of Songkla University, Songkhla
Province; 2014.
Watanasit S, Noon-anant N, Binnima N. Preliminary survey of ants at a reserve area of Prince of Songkla
University, Songkla Province, Southern Thailand 2005;27(1): 39-46.
Watanasit S, Noon-anant N, Phlappueng A. Diversity and ecology of ground dwelling ants at Khao Nan
National Park, southern Thailand. Songklanakarin J. Sci. Technol 2008;30(6): 707-12.

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Title : Feeding Behaviors in Cellular Slime Molds from OB2_08_01

Field : Ubon Ratchathani province
Author : Biology and Biodiversity
Miss Kwanjiraporn Sukkasem
School : Miss Juthakan Phiwton
Advisor :
Miss Yanisa Jaroenporn
Lukhamhanwarinchamrab School, Ubon Ratchathani University
Asst. Prof. Dr. Kanchiyaphat Ariyachaokun (Ubon Ratchathani University)

Abstract
Cellular slime molds (dictyostelids) are single-celled eukaryotic, phagotrophic bacterivores in

soil. Often, these organisms are grown in the laboratory with Escherichia coli as the food bacterium. In this
study, we evaluated the feeding preferences of two species of dictyostelids isolated from Ubon Ratchathani
university campus, Thailand. Our results showed that the isolated dictyostelids preferred gram-negative
bacteria and gram-positive bacteria. E. coli remained the food of choice by the most of the isolated cellular
slime molds. Our study is the first attempt to evaluate the feeding preferences of locally isolated
dictyostelids.

Keywords : Cellular slime molds, dictyostelids, bacteriovores and phagocytes

Introduction

The amoebozoa are diverse and abundant group of organisms that remain largely unexplored. Cellular
slime molds or dictyostelids (social amoebae) are single-celled, phagotrophic micropredators that feed on
bacteria. They are common soil dwelling amoeba most often isolated from the leaf litter decomposition zone
of forest soils [1]. The first known dictyostelid was isolated by Brefeld [2]. Recently reported the number of
dictyostelid species to be more than 150 species which belongs to eight major groups based on phylogenetic
analyses of both morphological and molecular data [3]. Dictyostelids are also characterized mainly by their
unusual life cycle. During the asexual component of the life cycle, spores germinate releasing amoebae. These
feed on bacteria and produce more amoebae by binary fission. If the food supply becomes depleted, the
amoebae enter their social phase. They aggregate, or come together, thus producing a multicellular organism.
These multicellular aggregates differentiate into mature fruiting bodies [4]. The fruiting body is a resting
structure for the organism. Unless food becomes available, the amoebae will remain dormant within the spores
[5]. As part of their fascinating life cycle, cellular slime molds exhibit a variety of cellular behaviors that has
made them a very popular experimental system for studying basic questions about cell biology, gene
expression, development, ecology, and evolution [6]

Cellular slime molds are commonly cultivated in the lab as two-membered cultures. They are typically
grown on Hay Infusion (HI) agar in the presence of a selected bacterium, typically E. coli, which acts as their
food source [7]. Cavender and Raper’s isolation method for the isolation of Acrasieae is considered by most
researchers to be the standard method for cellular slime mold isolation and cultivation [7]. In this study, the
feeding preferences of the dictyostelid species isolated from Ubon Ratchathani university campus, Thailand
was assessed to determine how well these species would grow when provided with different species of bacteria.
The present study would provide information that could help optimize techniques used for the isolation and
cultivation of cellular slime mold species for future research. Understanding their feeding preference also gives
insights into their physiology as well as their potential applications in medical biotechnology.

Methodology

1. Sample isolation and culture and morphological assessment.
A total of 9 soil samples were collected from study sites in Ubon Ratchathani university campus, Thailand.
The samples were collected from soil/humus layer at each site. They were placed in sterile plastic bags and
sent to the laboratories at faculty of science, Ubon Ratchathani university for processing. Each sample was
weighed and sufficient sterile distilled water added to obtain an initial soil/water dilution of 1:10. This mixture
was shaken to disperse the material and to suspend the cells of dictyostelids. A 5.0 mL volume of this initial
dilution was added to 7.5 mL of sterile, distilled water to create a 1:25 dilution of sample material. Aliquots
(each 0.5 mL) of this suspension were added to each of three 90 × 15 mm culture plates prepared with HI agar
[1]. This produced a final dilution of 0.02 g of soil per plate. Approximately 0.4 mL heavy suspension of 12–
24 hours pregrown E. coli was added to each culture plate, and plates were incubated under diffuse light at
20–25 oC. Each plate was examined at least once a day for up to 2 weeks. After appearance of initial

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