Extended Abstract : Biology and Biodiversity

Faculty of Science, Thaksin University
Phatthalung (August 26-29, 2022)
Biology and Biodiversity

12th SCiUS Forum

12th SCiUS Forum
August 26 – 29, 2022
at Thaksin University

organized by
Ministry of Higher Education, Science, Research and Innovation

and Thaksin University



12th SCiUS Forum

คำนำ

คณะกรรมการบริหารโครงการห้องเรียนวิทยาศาสตร์ในโรงเรียน โดยการกำกับดูแลของมหาวิทยาลัย
(โครงการ วมว.) เห็นชอบให้มีการจัดกิจกรรม 12th SCiUS Forum ในระหว่างวันที่ 26 – 29 สิงหาคม 2565
เพ่ือให้นักเรียนโครงการ วมว. ระยะที่ 2 ชั้นมัธยมศึกษาปีท่ี 5 ประจำปีการศึกษา 2564 ได้นำเสนอผลงานโครงงาน
วทิ ยาศาสตรแ์ ละแลกเปลี่ยนองค์ความรกู้ ับเพื่อนๆ นักเรียนต่างโรงเรียนในโครงการ วมว. จำนวน 19 แห่ง ไดแ้ ก่

1. โรงเรยี นสาธิตมหาวทิ ยาลัยเชยี งใหม่ – มหาวิทยาลัยเชียงใหม่
2. โรงเรยี นมธั ยมสาธติ มหาวิทยาลยั นเรศวร – มหาวิทยาลยั นเรศวร
3. โรงเรียนราชสีมาวิทยาลยั – มหาวิทยาลยั เทคโนโลยสี ุรนารี
4. โรงเรียนสาธิตมหาวทิ ยาลยั ขอนแก่น ฝ่ายมธั ยมศกึ ษา (ศกึ ษาศาสตร)์ – มหาวทิ ยาลยั ขอนแกน่
5. โรงเรียนสาธติ มหาวทิ ยาลัยมหาสารคาม (ฝา่ ยมัธยม) – มหาวทิ ยาลยั มหาสารคาม
6. โรงเรียนดรณุ สิกขาลยั – มหาวิทยาลยั เทคโนโลยีพระจอมเกลา้ ธนบรุ ี
7. โรงเรียนสาธิตแห่งมหาวิทยาลัยเกษตรศาสตร์ วิทยาเขตกำแพงแสน ศูนย์วิจัยและพัฒนาการศึกษา –

มหาวิทยาลยั เกษตรศาสตร์ วิทยาเขตกำแพงแสน
8. โรงเรียนสาธิต "พิบูลบำเพ็ญ" มหาวทิ ยาลยั บูรพา – มหาวิทยาลยั บรู พา
9. โรงเรียน มอ.วทิ ยานุสรณ์ – มหาวิทยาลัยสงขลานครินทร์ วิทยาเขตหาดใหญ่
10. โรงเรยี นสาธิตมหาวทิ ยาลยั สงขลานครนิ ทร์ – มหาวทิ ยาลัยสงขลานครนิ ทร์ วทิ ยาเขตปตั ตานี
11. โรงเรยี นปา่ พะยอมพิทยาคม – มหาวิทยาลยั ทักษิณ
12. โรงเรียนสาธติ มหาวทิ ยาลยั พะเยา – มหาวทิ ยาลยั พะเยา
13. โรงเรยี นลือคำหาญวารนิ ชำราบ – มหาวทิ ยาลัยอุบลราชธานี
14. โรงเรยี นสริ นิ ธรราชวทิ ยาลัย – มหาวทิ ยาลยั ศิลปากร
15. โรงเรยี นสวนกุหลาบวิทยาลัย รงั สติ – มหาวิทยาลยั ธรรมศาสตร์
16. โรงเรยี นสาธติ มหาวิทยาลัยขอนแกน่ ฝา่ ยมธั ยมศกึ ษา (มอดินแดง) – มหาวทิ ยาลัยขอนแก่น
17. โรงเรยี น มอ.วิทยานุสรณ์ สรุ าษฎรธ์ านี – มหาวทิ ยาลยั สงขลานครินทร์ วทิ ยาเขตสรุ าษฎร์ธานี
18. โรงเรยี นสุรวิวัฒน์ – มหาวทิ ยาลัยเทคโนโลยีสรุ นารี
19. โรงเรยี นสาธติ อิสลามศกึ ษาฯ – มหาวทิ ยาลัยสงขลานครินทร์ วทิ ยาเขตปตั ตานี

กิจกรรม 12th SCiUS Forum ดำเนินการภายใต้มาตรการป้องกันการระบาดของโรคติดเชื้อไวรัส
โคโรนา 2019 (COVID-19) และแนวปฏิบัติในการเข้าร่วมกิจกรรม 12th SCiUS Forum ซ่ึงในการนำเสนอ
ผลงานประกอบด้วยการนำเสนอโครงงานประเภท Oral presentation และ Poster presentation จำแนก
สาขาวิชาโครงงานวิทยาศาสตร์ออกเป็น 7 สาขา ได้แก่ สาขาวชิ าเคมี สาขาวชิ าชีววิทยาและความหลากหลาย
ทางชีวภาพ สาขาวิชาฟิสิกส์และดาราศาสตร์ สาขาวิชาวิทยาศาสตร์ส่ิงแวดล้อมและนิเวศวิทยา สาขาวิชา
คณิตศาสตร์และสถิติ สาขาวิชาเทคโนโลยีและคอมพิวเตอร์ และสาขาวิชาสะเต็มและนวัตกรรม สำหรับ
เอกสารเล่มนี้เป็นการรวบรวม Extended Abstract ของโครงงานวิทยาศาสตร์ประเภท Oral
presentation สาขาวชิ าชีววิทยาและความหลากหลายทางชีวภาพ

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12th SCiUS Forum

คณะผู้จัดทำหวังเป็นอย่างย่ิงว่า เอกสารฉบับน้ีจะเป็นประโยชน์ต่อนักเรียน ครู คณะกรรมการตัดสิน
ผู้เข้าร่วมกิจกรรม รวมถึงคณะทำงานจากทุกหน่วยงานที่เกี่ยวข้อง และขอขอบพระคุณผู้เก่ียวข้องทุกท่านท่ีได้
ให้ความรว่ มมือสนบั สนุนการจดั กิจกรรม 12th SCiUS Forum ในครัง้ น้ี

คณะผ้จู ดั ทำ
กรกฎาคม 2565

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สารบญั 12th SCiUS Forum

คำนำ หน้า
สารบัญ ก
รายชือ่ โครงงานวทิ ยาศาสตร์ ค
ประเภท Oral presentation สาขาชีววิทยาและความหลากหลายทางชีวภาพ
1
กลุ่มท่ี 1 วันท่ี 27 สิงหาคม 2565 58
กลมุ่ ท่ี 1 วนั ที่ 28 สงิ หาคม 2565 83
กลมุ่ ที่ 2 วนั ท่ี 27 สิงหาคม 2565 133
กลุม่ ที่ 2 วนั ท่ี 28 สิงหาคม 2565 154
กลุ่มท่ี 3 วันท่ี 27 สงิ หาคม 2565 212
กลุ่มที่ 3 วันท่ี 28 สิงหาคม 2565 237
กลมุ่ ท่ี 4 วันที่ 27 สงิ หาคม 2565 294
กลุ่มที่ 4 วนั ที่ 28 สงิ หาคม 2565 318
กลุ่มท่ี 5 วันท่ี 27 สงิ หาคม 2565 376
กลมุ่ ที่ 5 วันท่ี 28 สิงหาคม 2565

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12th SCiUS Forum

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12th SCiUS Forum

List of Science Projects 12th SCiUS Forum
Oral presentation

Biology and Biodiversity Group 1
Saturday August 27, 2022

No. Code Title Author School
Naresuan University
1 OB1_03_11 Survey of the potent Miss Thanatchaporn Sumitsawan Secondary
Demonstration School
prebiotics extracted from Miss Laksika Duangpila
Demonstration School,
some kitchen herbs and food University of Phayao

plants compared with Chiang Mai University
Demonstration School
Jerusalem artichoke
Piboonbumpen
2 OB1_02_06 Application of bioinformatics Miss Kasiwara Suksup Demonstration School,
Burapha University
and system biology in Miss Wouthjira Kiddee Princess Sirindhorn's
College
Gardenia sootepensis Hutch
Demonstration School,
for antivirus and inhibitors of University of Phayao

SARS-CoV-2 main protease PSU.Wittayanusorn
School
studies
Demonstration School,
3 OB1_01_03 The study of increasing Mr. Kritsana Thinjan University of Phayao

phenolic compound in Miss Chiraphat Yanarueng

Lymnophila aromatica using Mr. Pranote Juntranimit

stress conditions

4 OB1_13_03 The Study of Protein Content Miss Pimpisa Sewaka

and Microbiological Quality Miss Zazie Bertho

of Rice Milk-Based Kefir

5 OB1_12_01 Classification of Miss Puntira Boonpen

economically important Miss Titivorada Rodsai

Jasmine cultivars by Mr. Patiphan Krumprasert

morphological characters and

DNA barcode

6 OB1_02_03 Polyphenols content and Miss Boontharika Pala-Aud

antioxidant capacity of Miss Jetpreya Chisala

Tilicora triandra (Colebr.) Miss Panvira Khunprom

leaf extract

7 OB1_15_04 Effects of pollen Miss Yanisa Traiprakong

morphology, fabrics, and Miss Saruta Chumpong

time on the retention of

pollen on clothes

8 OB1_02_02 Induction Mutation of Plant Miss Saisahwan Kamoi

Growth Promoting Bacteria Miss Wasita Injai

by Cold Plasma Technology Miss Boonraksa Mangmee

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No. Code Title Author School
Miss Thanatchaya Toom-Ariya Kasetsart University
9 OB1_10_02 Antimicrobial and Mr. Natchanon Kongsut Laboratory School
Miss Ployrawee Phet-Ngam Kamphaeng Saen
Antibiofilm activity of Campus Educational
Research and
actinobacteria isolated from Development Center
Demonstration School
rhizosphere soil and of Khon Kaen
University
mangrove sediments Demonstration School,
University of Phayao
10 OB1_04_02 Using thermal camera to Miss Chonnipa Sasiwilasakon
Chiang Mai University
assess phenotype of corn Mr. Jetbodin Piata Demonstration School

seedling under water deficit Islamic Science
Demonstration School
11 OB1_02_05 Repellent activity of Mr. Jukkraphob Khatsri
Piboonbumpen
Cannabis on Tenebrio Miss Pusanisa Sawangpakdee Demonstration School,
Burapha University
Molitor larvae and

bioinformatics of defense

mechanism related abiotic

stress tolerance gene

12 OB1_01_08 Organic formula created for Miss Tamonwan Fannuwong

dendrobium orchids to help Miss Thammajaree Inyavileart

with mericlonal propagation

Dendrobium orchids

(Dendrobium Sonia)

13 OB1_19_02 Increasing qualities of Miss Warada Yusoh

amaranth microgreens under Miss Wachiya Malaeh

plant factory system by LED

light

14 OB1_13_01 In vitro shoots and roots Miss Anyamanee Tanpradubsing
induction of Philodendron Miss Chotiros Taorat

erubescens cv. pink princess

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12th SCiUS Forum

Title : Survey of the potent prebiotics extracted from some OB1_03_11

Field : kitchen herbs and food plants compared with Jerusalem artichoke
Biology

:Author Miss Thanatchaporn Sumitsawan

Miss Laksika Duangpila

:School Naresuan University Secondary Demonstration School

:Advisor Dr. Songkran Chuakrut (Naresuan University)

Abstract
Nowadays inulin, which is a prebiotic, is added to various food products to help promote the

growth of probiotics in the colon for their consumers’ health. However, some probiotics, such as Bifidobacteria,
have been reported to be unable to use or can be used inefficiently for growth. Thus, this research aims to
survey Thai herbs and some food plants which can promote the growth of two types of probiotics with anti-
colon cancer strains, Streptococcus thermophilus TISTR 894 (ATCC 19258) and Bifidobacterium bifidum
TISTR 2129 (ATCC 29521), using Jerusalem artichoke (Helianthus tuberosus) which has high inulin content
as a control. In the first experiment, finding the best conditions to extract prebiotics by using Jerusalem
artichoke as the test plant. It was found that the result was extracted with water at 60°C for 4 hours could extract
the prebiotics the most, which was checked with the growth of S. thermophilus TISTR 894. In an experiment
part 2, 30 plants samples were extracted for prebiotics under optimum condition. The prebiotic extracts were
evaporated and dried under vacuum condition at 50°C for approximately 2 days. Then, 2% (w/v) of each extract
from all plant samples was mixed with MRS broth without glucose. The 2% (v/v) growing culture of each
probiotic was inoculated in the medium in triplicates. The cultures were incubated at 37°C for 48 hours and
sampled at 0, 24 and 48 hours for growth measurement in term of optical density at 600 nm. The results revealed
that Jerusalem artichoke extract had the highest activity to promote the growth of S. thermophilus whereas Thai
yacon and coconut shoot extracts could promote as well. In addition, the galangal extract had the highest
activity among the herbal recipes but its activity was half of the coconut shoot. Interestingly, the maximum
growth of B. bifidum was observed in the medium supplemented with the white radish and Chinese yacon
extracts respectively. The extract from Jerusalem artichoke had lower effect on B. bifidum about half growth
when compared to S. thermophilus. It indicated that B. bifidum could not utilize inulin, a major prebiotic, as a
carbon source for growth. Moreover, the lemon grass extract could promote the highest growth of B. bifidum
when compared to those herbal recipes but its activity was half of the white radish extract.

:Keywords prebiotic, extraction, growth promotion, Streptococcus thermophilus, Bifidobacterium bifidum

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12th SCiUS Forum

Introduction
Colorectal cancer is a disease caused by abnormal cell divisions. That are uncontrollable and can even

result in life-threatening conditions. According to research, colorectal cancer is most common between the ages
of 50 and 70 and it is found that males are more likely than females. Colorectal cancer is ranked 4th among
cancers in the world. Although, there are methods or treatments such as radiation, chemotherapy, and surgery.
However, therapies may affect the state of mind and body. The authors have recognized the importance and
seen that colorectal cancer can be prevented. According to the studies, probiotics can reduce the risk of
colorectal cancer. It is a bacterium that is beneficial to the body. For example, Lactobacillus can produce lactic
acid that can inhibit the growth of pathogenic bacteria. Bifidobacterium helps break down cholesterol, inhibits
absorption through the intestinal wall, reduces constipation and stimulates intestinal peristalsis. Moreover, it
causes balance in the colon, resulting in a reduced risk of colon cancer. For probiotics to be in our intestines,
they need nutrients to grow and multiply, which nutrients are prebiotics. Prebiotics are food that cannot be
digested and absorbed but can travel up to the large intestine unchanged. It can be fermented in the colon by
beneficial bacteria such as Bifidobacterium and Lactobacillus. It also has to promote the growth of beneficial
bacteria in the digestive tract but not promote the expansion of pathogenic bacteria. Prebiotics can be found
naturally in some vegetables, fruits, grains, and herbs. Likewise, Thailand is in the tropics and can cultivate
herbs that have many properties, such as medicine. This research will focus on Thai herbs and some food plants
that are used for cooking because they are readily available and grown throughout the year. Some Thai herbs
contain prebiotics that can promote the growth of Bifidobacterium bacteria, which are beneficial bacteria that
are in balance in the large intestine and will help prevent colorectal cancer.

Methodology

The experiments were divided into 2 parts as follows,

Part 1: Finding the best conditions to extract prebiotics by using Jerusalem artichoke as the test plant. By
extracted with water or ethanol at 60°C for 30 minutes1, 2, 3 and 4 hours. It was
found that the result was extracted with water at 60°C for 4 hours could extract
prebiotics the most.

Part 2: Extracted prebiotics from 22 herbal recipes and 8 food plant samples. First,
cut samples into small pieces then dried in the hot air oven for 70°C and ground
them. After that, extracted herbs and some food plants in the water bath at 60°C
for 4 hours and evaporated and dried under vacuum condition at 50°C for
approximately 2 days. Then, mixed 2% (w/v) of each extract from all plant
samples with MRS broth without glucose and the 2% (v/v) growing culture of

each probiotic was inoculated in the medium in triplicates. The cultures were incubated at 37°C for

48 hours. Finally, sampled at 0, 24 and 48 hours for growth measurement in term of optical density at 600 nm
and compared the results.

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Results
It was found from part 1 experiment that extracted with water at 60°C for 4 hours could extract

prebiotics the most. Furthermore, in experiment part 2 revealed that Jerusalem artichoke extract had the highest
activity to promote the growth of S. thermophilus whereas Thai yacon and coconut shoot extracts could
promote as well among food plants. Besides, the galangal extract had the highest activity among the herbal
recipes but its activity was half of the coconut shoot. While, the maximum growth of B. bifidum was observed
in the white radish and Chinese yacon extracts respectively. The lemon grass extract could promote the highest
growth of B. bifidum when compared to those herbal recipes but its activity was half of the white radish extract.
In contrast, the extract from Jerusalem artichoke had lower effect on B. bifidum about half growth when
compared to S. thermophilus.

Conclusion
The highest activity to promote the growth of S. thermophilus was found in Jerusalem artichoke

extract, which is considered an herb. Likewise, Thai yacon and coconut shoot extracts, which are considered
food plants, could promote as well. The white radish and Chinese yacon respectively, promoted the growth of
B. bifidum the most. The lemon grass extract, which is an herb, can help promote B. bifidum too, but its activity
was half of the white radish extract. Furthermore, the extract from Jerusalem artichoke had lower effect on B.
bifidum about half growth when compared to S. thermophilus. Therefore, it implied that B. bifidum could not
make use of inulin as a carbon source for its growth but S. thermophilus could.

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. Paiboon Thammarutwasik, Kongkarn Kijroongrojana, Tipparat Hongpattarakere, Suphitchaya Chantachum,

Arunporn Itharat, Wantana Reanmongkol, et al. Prebiotics – A Review. Songklanakarin Journal of Science and
Technology 31(4) : 401-408; 2009.

2. Natcha Niljantuk, Samruey chuekram, Suthida kangkuntod, Jirawan Oonmetta-aree and Jittra Singthong.

Prebiotic Activities of Jerusalem artichoke. Science and Technology Research Journal Nakhon Ratchasima
Rajabhat University 4(2) : 18-24; 2019.

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Title : Application of bioinformatics and system biology in OB1_02_06
Gardenia sootepensis Hutch. For antivirus and inhibitors

of SARS-CoV-2 main protease studies

Field : Biology and Biodiversity

Author : Ms.Kasiwara Suksup

Ms.Wouthjira Kiddee

School : Demonstration school University of Phayao, University of Phayao

Advisor : Dr.rer.nat. Chatchawal Wongchai ( Division Biology, School of Science, University of Phayao,
Phayao 56000, Thailand )

Abstract
Gardenia sootepensis Hutch. (Rubiaceae) is a potent medicinal plant and found in Northern to

Southern Thailand. Ascertain is a chemotype in G. sootepensis which inhibits the reverse transcriptase (RT)
enzyme of various viruses. The epidemic of the novel coronavirus disease (COVID-19) has evoked an urgent
demand for finding new potential therapeutic agents. Therefore, this study aims to evaluate regions of
similarity between biological sequences of G. sootepensis and other antivirus herbs and protease inhibitors of
SARS-CoV-2 from plants. Using matK, nucleotide sequences in the chloroplast genome were assessed as
potential and suitable for use as standard DNA. The study found that Nigella sativa L. has the most similar
nucleotide sequence demonstrating a close evolutionary line and researching the nature of anti-CoV
substances in G. sootipensis. Flavonoid, Myricetin, Lycorine, and Quercetin-3-B-galactoside have been
reported against SARS-CoV. The comparisons between proteins (LPO, LTF, RIP) that inhibit SARS-CoV in
G. sootepensis found low similarity at 11%, 12%, and 17% respectively. Moreover, the Phyto-stem cell of
G. sootepensis is developing for various bioactive compound extracts which in this study callus
induction. G. sootepensis callus was fast developed in the solid medium than growing in the liquid medium
(MS+BA+2,4-D). Further experiments, the HPLC, and molecular genetics analysis are essential to support
this work and conduct the pharmaceutical development.

Keywords : Gardenia sootepensis, Phyto-stem cell, Protease inhibitors, SARS-CoV-2

Introduction
The pandemic of the novel coronavirus disease (COVID-19) has evoked an urgent demand for

finding new potential therapeutic agents. From recent and past studies when performed a molecular docking
of anti-HIV drugs to refine HIV protease inhibitors and nucleotide analogues to target COVID-19 indicated
that antiviral drugs for HIV have the best binding affinity for the 3-chymotrypsin-like protease of COVID-19
and one of target for the antiviral treatment of AIDS is the reverse transcriptase (RT) of HIV.[1]
Furthermore, Gardenia sootepensis Hutch. is an important medicinal plant which present the chemotype
(Coronalolide) of inhibitory effect on RT enzyme of the HIV. It was reported that, Ribosome-inactivating
protein (RIP) is plant protein that have potential to inhibits HIV-1.[2] while Lactotransferrin (LTF) and
Lactoperoxidase (LPO) against SARS-CoV.[4] However, the quality of standard medicinal compounds
isolated from plants often depends on seasonal variations. Thus, plant biotechnology and plant cell culture

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12th SCiUS Forum

provides a better way than in the generation of phycochemicals. Bioinformatics reported that matK is a
nucleotide sequence in the chloroplast genome that has been assessed to be sufficiently potent and suitable
for use as a standard DNA.[3] Basic Local Alignment Search Tool (BLAST) is the tool for calculating
sequence similarity. BLAST can be used for a lot of purposes, such as can infer the functions and
evolutionary relationships of whole genomes. Therefore, this work aims to provide the researchers with a
comprehensive profile of the method of making Phyto-stem cells and the medicinal potential of
G. sootepensis for the development of anti-CoVs and study close evolutionary relationship between
G. sootepensis and another plants or protein inhibitors to highlight some present issues and future.

Methodology
The experiments were divided into 2 parts as follows,

Part 1 : Tissue Culture Part 2 : Blast (Basic Local Alignment Search Tool) Bioinformatics

Results and Discussion

Tissue culture can be divided into 3 main
steps: preparation of culture medium, sterilization
and cultivating. The culture medium was prepared
by weighing MS powder, sugar and agar, then
mixed MS powder and sugar into distilled water.
Divide the solution into 2 portion with first
portion was adjusted to volume and then adjusted
to pH at 5.7-5.75 and second part added BA
solution and 2,4-D solution then adjust the
volume and adjust the pH at 5.7-5.75 then add

agar powder and heat it. Pour it into a bottle and take it to the clave. In the sterilization process plants are
washed with running water for 1 hour, then disinfected with warm water at 40 degrees Celsius, 70% alcohol,
15% and 5% Clorox concentrations, respectively, when the plant parts are soaked in 5% Clorox, they will be
brought into the incubator for further cultivation.

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Figure 2. showed regions of similarity between
biological sequences of G.sootepensis and others
antivirus herbs and protease inhibitor of SARS-
CoV-2 from plants 10 samples. From figure 2
found Nigella sativa L. has the most similar
to the nucleotide sequence demonstrates a close
evolutionary line. This can indicate a trend of
close evolutionary relationship.

Since 2004, it was reported that 22 compounds active from plants against SARS-CoV along with their
reported anti-SARS-CoV mechanism of action [4] while G. sootepensis shows 4 similarity compounds that
can inhibit SARS-CoV2; Flavonoid, Lycorine (Lycoris radiata), Myricetin (Myrica rubra) and Quercetin-3-
B- galactoside (Ginkgo biloba), Table 1.

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Table 2. showed G.sootepensis protein
comparisons with protein LPO, LTF and RIP
were found to be 11%, 12% and 17% respectively.

Conclusion
G. sootepensis callus was fast developed in solid medium than growing in liquid medium

(MS+BA+2,4-D). BLAST analysis presented low-probability of RIP, LTF and LPO in G. sootepensis.
Biochemical and molecular approach may need to improve the quality and quantity of phytochemicals and
test of anti-viruses is necessary for futher experiment.

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. Minh Hien Nguyen et al.. Evaluation of COVID-19 protease and HIV inhibitors interactions. Acta

Pharmaceutica [Internet].2022[cited 2022 Mar 4];72:1-8. Available from: https://doi.org/10.2478/acph-
2022-0010
2. S Lee-Huang et al.. Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. GENE
[Internet]. 1995 [cited 2022 May 25];161:151-6. Available from: https://doi.org/10.1016/0378-1119(95)
00186-A
3. Narumol Thanananta et al.. Assessment of Genetic Relationship and Identification of Dendrobium
Section Callista Using Nucleotide Sequences of rpoC1 and matK Genes. Thai Journal of Science and
Technology [Internet]. 2018 [cited 2022 Jan 14];7:81-8. Available from: https://doi.org/10.14456/
tjst.2018.12
4. Tariq Khan et al.. Plant in vitro Culture Technologies. A Promise Into Factories of Secondary
Metabolites Against COVID-19. Plant Science’s Contribution to Fighting Viral Pandemics: COVID-19
as a Case Study [Internet]. 2021 [cited 2022 Jan 14];12: unpaged. Available from: https://doi.org/
10.3389/fpls.2021.610194

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Title: The study of increasing phenolic compound in OB1_01_03

Field: Limnophila aromatica sp. using stress condition
Author:
Biology and Biodiversity
School: Mr. Kritsana Thinjan
Advisor: Ms. Chiraphat Yanarueng
Mr. Pranote Juntranimit
Chiang Mai University Demonstration School
Asstant.Prof. Dr. Usawadee Chanasut
Mr. Kusol Raunprataungsuk

Abstract
Antioxidants help inhibit or slow the destruction of body cells. The simplest antioxidant intake is from

eating food. Limnophila aromatica sp. is a vegetable with high antioxidants that is commonly found in Thailand.
The aim of this study was to increase the amount of antioxidant compounds such as phenolic substance by
growing under abiotic stress condition including carbon dioxide fumigation for 1, 3 and 5 hours and soaked
in saline solution with 0.1 molar NaCl concentration for 12 and 24 hours and measured the changes in
phenolic content, antioxidant activity, vitamin C content, dry weight, height and the number of leaves
for two weeks after the stress conditions were applied. The result was found that the plant that soaked
in the saline solution for 12 hours had the highest increase in vitamin C content. and had the highest antioxidant
activity in the first week. Plants that fumed with carbon dioxide for 1 hour subsequently increased its vitamin C
content and antioxidant activity. However, this plant had the highest phenolic content when compared to the other
plants growing under different conditions There was an increase in antioxidant activity on day 12, comparing
the relationship between the phenolic content and their antioxidant activities, they were not positively related.
This may be due to the time difference between the quantification of phenolic compounds and antioxidant
activities. As a result, the antioxidant activities were lower than expected and affected the relationship between
both values.

Keywords: Limnophila aromatica sp., Stress, Antioxidant, Phenolic

Introduction
Nowadays, most of Thai people died and suffers from cancers. This is partly due to the lack of

antioxidants that slow down the destruction of body cells. By the way, the easiest way to get antioxidants is through
consumption. Authors see that Thai food is often eaten with vegetables as a side dish which are rich in antioxidants
and easy to access. Therefore, Authors wanted to increase the units of antioxidants using stress conditions by
choosing an easy, effective quickly, and actually increased the units of antioxidants. In this experiment, we mainly
focus on the increase of phenolic compounds and vitamin C, allowing consumers to receive more antioxidants per

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serving of Limnophila aromatica sp. and being an alternative choice for farmers to increase the value of
agricultural products.

Methodology
Part 1: Stress experiments.

The stress condition kits were divided into
1. Control variable kits: water. Arrange the plant pots evenly apart in the basin, add 15 liters of water

and change the water every three Days.
2. saline-soaked kits: 12-hour and 24-hour soaks in 0.1 molar saline solution, the amount of saline

solution used is 15 liters.
3. 1 hour, 3 hour and 5 hour-carbon dioxide fumigation experiment kits, there will be at least 0.006 molar

CO2 in the bag (not involve the amount of carbon dioxide from plant respiration, etc.), which is 2.5 times more
than in the air.
Samples collected in this experiment for

1. Measuring the amount of vitamin C: Collected 2 sample shoots, 10 cm long for once a week.
2. Measuring phenolic and antioxidant activity: Collected 2 samples, 10 cm long from every pot in each
condition, collect every other day in the first week and in the second week collected every three days.
3. Samples for observing growth: Collected 2 sample shoots, 10 cm long for once a week.
Part 2: Measurements and calculations.
1. The quantification experiment of phenol compounds
Make standard graphs of gallic acid at different concentrations using a spectrophotometer. The samples
were extracted with 70% ethanol, diluted with distilled water to a ratio of 1:10. Take 1 ml of the solution, add 2
ml of sodium carbonate and 2.5 ml of 10% Folin solution, and shakes for 1 hours then put it in the
spectrophotometer with a wavelength of 725 nanometer.
2. Antioxidant activity by DPPH method
Make standard graphs of gallic acid at different concentrations using a spectrophotometer. The samples
were extracted with 70% ethanol, diluted with distilled water to a ratio of 1:10. Take 1 ml of the solution, Add the
DPPH diluted to a ratio of 1:4 then shake for 1 hours then put it in the spectrophotometer with a wavelength of
517 nanometer.
3. Vitamin C determination
Prepared a standard vitamin solution. Take 10 ml of the extracted solution, add 2 drops of starch water,
titrated with iodine solution. Until it turns purplish green.

Result
Quantity of phenolic was shown in figure 1. In the control kit has the highest quantity on day 7 of the

experiment. In a kit that has been soaked in saline solution for 12 hours has the highest quantity on day 5 of the
experiment. In a kit that has been soaked in saline solution for 24 hours has the highest quantity on day 9 of the

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experiment. In the 1 hour-carbon dioxide fumigation kit has the highest quantity on day 12 of the experiment. InUnits of phenolic compound
the 3 hours-carbon dioxide fumigation kit has the highest quantity on day 5 of the experiment. In the(mgGAE/gFW)
5 hours-carbon dioxide fumigation kit has the highest quantity on day 12 of the experiment.

60
50
40
30
20
10

0
1 3 5 7 9 12
Day

control saline 12 hrs. saline 24 hrs. CO2 1 hr. CO2 3 hrs. CO2 5 hrs.

Figure 1: Phenolic contents of each stress condition’s samples.
Quantity of antioxidant was shown in figure 2. In the control kit has the highest quantity on day 12 of the
experiment. In a kit that has been soaked in saline solution for 12 hours has the highest quantity on day 1 of the
experiment. In a kit that has been soaked in saline solution for 24 hours has the highest quantity on day 9 of the
experiment. In the 1 hour-carbon dioxide fumigation kit has the highest quantity on day 12 of the experiment. In
3 hours-carbon dioxide fumigation kit has the highest quantity on day 3 of the experiment. In the 5 hours-carbon
dioxide fumigation kit has the highest quantity on day 9 of the experiment. The quantity of vitamin C is as follows.
In the first and second weeks the control kit has the highest quantity. Therefore, other experimental kits have a
negative effect on the quantity of vitamin C.

Units of antioxidant compound 35
(mgGAE/gFW) 30
25
20 .
15
10

5
0

1 3 5 7 9 12
Day

control saline 12 hrs. saline 24 hrs. CO2 1 hr. CO2 3 hrs. CO2 5 hrs.

Figure 2: Units of antioxidant activity of each stress condition’s samples.

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Conclusion
In part of phenolic, the 5 hours carbon dioxide fumigation kit has the highest quantity by using 12 days

but on the first day a kit that has been soaked in saline solution for 12 hours has the highest quantity. And part of
antioxidant, the control kit has the highest quantity by using 12 days but the first day a kit that has been soaked in
saline solution for 12 hours has the highest quantity. Comparing the relationship between the phenolic content and
their antioxidant activities, they were not positively related. This may be due to the time difference between the
quantification of phenolic compounds and antioxidant activities. As a result, the antioxidant activities were lower
than expected and affected the relationship between both values.
Acknowledgements

This project was supported by Science Classroom in University Affiliated School (SCiUS) under
Chiang Mai University and Chiang Mai University 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
Gorai D, Jash SK, Singh RK, Gangopadhyay A. Chemical and Pharmacological Aspects of Limnophila aromatica
(Scrophulariaceae). AJPCT 2014;2:348-356.
Petrussa E, Braidot E, Zancani M, Peresson C, Bailon MT, et al. Plant Flavonoids—Biosynthesis, Transport and
Involvement in Stress Responses. MDPI 2013;14:14950-14973.
Calles MA, Estev ́z I, Gomez A, Felix JD, Patui S, et al. Rhizobium laguerreae Improves Productivity and
Phenolic Compound Content of Lettuce (Lactucasativa L.) under Saline Stress Conditions. MDPI 2020;9:2-15.

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Title: The Study of Protein Content and OB1_13_03
Microbiological Quality of Rice Milk-Based Kefir

Field: Biology and Biodiversity
Author: Ms. Pimpisa Sewaka

Ms. Zazie Bertho
School: Piboonbumpen Demonstration School, Burapha University
Advisor: Mr. Warapong Senapak (Faculty of Science, Burapha University)

Abstract
This study aimed to determine the acidity, protein content and microbiological quality of rice milk-

based kefir samples which were produced from jasmine rice milk and rice berry milk. The acidity was
measured by using titration method every 3 hours for 24 hours. The results showed that the acidity of jasmine
rice milk-based kefir and rice berry milk-based kefir increased. The protein content of jasmine rice milk-based
kefir and rice berry milk-based kefir was 0.39±0.02% and 0.28±0.01% (w/v), respectively. The data of the
microbiological quality was compiled with the standard quality of drinking yogurt product of Ministry of
Public Health’ s Announcement Vol. 353(2013). The result of jasmine rice milk-based kefir and rice berry
milk-based kefir showed that total bacteria, yeast enumeration and mold enumeration followed the standard,
but coliform bacteria were above 3 per gram of sample which was in excess with the standard.

Keywords: Kefir, Jasmine rice, Rice berry, Microbiological quality

Introduction
Rice is an important industrial drop in Thailand. From the fact that Thai people focus on consuming

rice as a main dish because it has enough nutrients and easy to find. Kefir is a combination of bacteria and
yeast. Appearance is white granules and when it fermented with milk. Then it will be a food product with high
nutritional value include main nutrients from milk such as protein and essential amino acids (lysine, isoleucine,
phenylalanine, valine, threonine, methionine and tryptophan). And in research the fermentation of kefir in
black carrot juice, mulberry juice, pomegranate juice, and strawberry juice to determine the increase of
anthocyanin and antioxidant. Therefore, we will consider it in the next step by adding acidity titration, protein
valuation and microbiological quality testing. And switch from fruit juice to rice milk-based kefir instead.

We hope that fermenting kefir with rice milk-based kefir will create a new product that is more
valuable in terms of nutrition. And it is expected that this product will make consumers pay more attention to
their health.

Methodology
These experiments were splitted up into six parts respectively.
Part 1: Active kefir
There is no standard way to active kefir. Although the suggest activation step is activating it in

pasteurized milk maturing at room temperature approximately 25 °C for 30 days. After that filter out and
collect only kefir grains.

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Part 2: Make rice milk
Bring 75 g of rice then boiled in 1 liter of boiling water (100 degrees) for 30 minutes. Whereupon
filter out and collect only fluid. By this method, approximately 600 ml of rice water is obtained. Do this method
with both types of rice (rice berry and jasmine rice).

Part 3: Make sample of rice milk-based kefir
Separate rice milk into 40 mL per one container. It’s total of 9 samples per type of rice according to
the maturing time. Then add 2 g of kefir in all samples. After that mature in room temperature for 0, 3, 6, 9,
12, 15, 18, 21 and 24 hours then check the other values when it on time.

Part 4: Total acidity titration
Titration acidity test was tested every hour of maturing (9 sample per a type of rice) by drain 5 mL
of rice milk-based kefir and diluting it with 10 mL of water, then add 2-3 drops of the indicator, in this case
will be used as phenolphthalein. Then, it was titrated with 0.063 N sodium hydroxide solution. Repeat it 3
times to find one acidity value and repeat again to look for averaged of total acidity. Do this step with both
types of rice.

Lactic acid = N x V x Mw g/L
Vsample

N= Normality of standard NaOH (0.063 N)
V= Volume of standard NaOH (mL)
Mw = Molecular weight of lactic acid (90.08 g/mol)
Vsample = Volume of the sample (15 mL)

Part 5: Protein valuation by submitting the 24 hours samples through Faculty of Science
lab Burapha University.

Use Kjeldahl method.

Part 6: Microbiological quality testing by submitting the 24 hours samples through Faculty of
Science lab Burapha University.

6.1 Check the total of yeast by count on Sabouraud dextrose agar (SDA) and DRBC agar.
6.2 Check the total of mold by use Bacteriological Analytical Manual Chapter 18.
6.3 Check the total of bacteria by use Bacteriological Analytical Manual Chapter 3.
6.4 Check the total of coliform by use Bacteriological Analytical Manual Chapter 4.

Results, Discussion and Conclusion
The time interval for jasmine rice milk-based kefir and rice berry milk-based kefir were every 3 hours

total time 24 hours Jasmine rice milk-based kefir and rice berry milk-based kefir affected on total acidity
(Figures 1).

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Total acidity 0.8
0.7
0.6 5 10 15 20 25
0.5
0.4
0.3
0.2
0.1

0
0

Time (h)
Figure 1 Total acidity of jasmine rice milk-based kefir and rice berry milk-based kefir.

The comparison of total acidity of jasmine rice milk-based kefir and rice berry milk-based kefir was showed
in Figure 1. It was found that the total acidity increased with time of fermentation and rice berry milk-based
kefir have higher amounts of acidity.

Protein content in samples were analyzed by Kjeldahl method. Results indicated in Table 1 revealed
that the content of protein in cow milk-based kefir has the highest number of proteins as expect.
Table 1 Protein Content of cow milk-based kefir jasmine rice milk-based kefir and rice berry

milk-based kefir.

Sample Result
Protein content (%)
cow milk-based kefir
jasmine rice milk-based kefir 3.25 ± 0.01
rice berry milk-based kefir 0.39 ± 0.02
0.28 ± 0.01

The data of the microbiological quality indicated in Table 2. The result was compiled with the
standard quality of drinking yogurt product of Ministry of Public Health’ s Announcement Vol. 353(2013).
The result of cow milk-based kefir, jasmine rice milk-based kefir and rice berry milk-based kefir showed that
total bacteria, yeast enumeration and mold enumeration followed the standard, but coliform bacteria were
above 3 per gram of sample which was excessed with the standard, maybe because of using wrong container
that couldn’t be disinfected.

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Table 2 Microbiological quality of cow milk-based kefir jasmine rice milk-based kefir and rice berry milk-
based kefir compared with the standard

Microbiological cow milk-based jasmine rice rice berry milk- standard
Quality kefir milk-based kefir based kefir
2.5 × 105 > 1 × 107
Total bacteria (cfu/g) 8.5 × 108 7.2 × 106 < 10 cfu/g > 1 × 104
Yeast (cfu/g) < 10 cfu/g < 10 cfu/g 4.0 × 107
Mold (cfu/g) 1.4 × 109 6.0 × 107 < 100

Coliform (MPN/G) >1600 79 31 < 3 per gram of sample

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

Burapa University of Piboonbumpen Demonstration 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. ศานต์ เศรษฐชยั มงคล และอญั ชิสา กลุ ทวสี ุข. (2557). นมเปร้ียวคีเฟอร์: เทคโนโลยชี ีวภาพจากมุมมองวทิ ยาการดา้ นโอมิกส์. ฉบบั ท่ี 1.มหาวทิ ยาลยั สยาม
2. มนตรา ศรีษะแยม้ , et al. (2561). ฤทธ์ิทางชีวภาพของคีเฟอร์เวยจ์ ากนมถวั่ เหลืองและนมงาดาํ . มหาวิทยาลยั ราชภฏั พิบูลสงคราม
3. Sümeyye Alagöz Kabakcıa, et al. (2020). Changes in the quality of kefir fortified with anthocyanin-rich
juices during storage. Ankara University

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Title: Classification of economically important jasmine cultivars OB1_12_01
by morphological characters and DNA barcoding

Field: Biology and Biodiversity

Author: Titivorada Rodsai, Puntira Boonpen and Patiphan Kramprasert
School: Princess Sirindhorn’s College and Silpakorn University

Advisor: Assist. Prof. Dr. Thanyanan Wannathong Brocklehurst and Dr. Pakkapol Thaowetsuwan
(Department of Biology, Faculty of Science, Silpakorn University)

Abstract:

Jasmine (Jasminum sambac (L.) Aiton) is an important flowering plant for Thais used in local tradition,
as an ornamental plant, and also for the perfume industry. However, plant sellers and consumers often confuse the
jasmine cultivars due to the similarity of their morphological characters. In addition, dependable information on
Thai-jasmine cultivar identification is not available online. In the present study, eighteen jasmine samples were
selected from eleven cultivars obtained from various locations to study their morphologies for cultivar
identification. DNA barcoding was then applied for molecular classification and grouping. Our results indicate
that the morphological characters of jasmine leaves greatly overlap between cultivars, whereas the flower
morphological traits are unique in each cultivar. However, since some of the jasmine cultivars do not bloom all
year round, it is difficult to identify the cultivars by their flower morphologies alone. For the DNA barcoding
results, the DNA samples were amplified using Polymerase Chain Reaction (PCR) for the psbA-trnH site and 500
bp DNA fragments were obtained. When the nucleotide sequence data were analyzed, using MEGA11® to create
the phylogenetic tree with the maximum parsimony method, it was found that the psbA-trnH nucleotide sequences
accurately identified all samples to species level. Eighteen jasmine samples were classified into two groups: the
first was Maluli Si Chomphu (J. multiflorum (Burm.f.) Andr.), which was the comparative species, and the second
was the remaining samples of Mali jasmine (J. sambac). Interestingly, in the psbA-trnH nucleotides of two samples
from Mali Son cultivar (Grand Duke of Tuscany), single nucleotide polymorphism (SNP) was recorded. Therefore,
it can be concluded that flower morphology can be used to identify jasmine cultivars, but psbA-trnH nucleotide
sequences cannot distinguish the differences between most cultivars, except Mali Son.

Keywords: Jasminum, Jasminum Sambac, Plant morphology, Genetic diversity, DNA barcode

Introduction

Jasmine (Jasminum sambac (L.) Aiton) is an important economic flower in Thailand that has long
generated income for farmers. It is used in Thailand on any occasions such as Mother's Day and Songkran Day,
and is also used in various industries. The plant is a small shrub or creeper that can reach a height of 3 meters and
stretch more than 10 meters (Chalermglin, 2013). Leaves are green, simple and oppositely arranged on the stem
(Sampath et al., 2008; Chalermglin, 2013). The flowers are borne single or in a group (inflorescence) at the end of
the shoots. Flowers of jasmine are complete flowers with sepals, petals, stamen, pistil, with the presence of several
green sepals containing 4-10 petals that fuse forming a tubular shape (Chalermglin, 2013). The propagation of
jasmine in Thailand is mainly done by grafting, layering and cuttings (Chaitanya, 2018). In Thailand, there are
6 reported cultivars: Mali Chanthabun, Mali Chat Dok Bua, Mali Chat Phikun, Mali La, Mali Son and Mali Thot
(Chalermglin, 2013). They were selected and named after their beautiful and unique flower characteristics.
Generally, morphological characters are primarily used to identify cultivars; however, there are limitations in doing
so. Cultivars, for example, cannot be identified by leaf characters, due to their high variability, and jasmine is not
always in its flowering season. Recently, new jasmine cultivars were brought into the Thai ornamental plant
market, such as Mali Sedthi and Mali Yipun. Without scientific records or proper explanations of these newly
named plants, confusion is unavoidable among Thai sellers and buyers.

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DNA barcode identification is a technique which involves the use of one or more nucleotide regions to
identify an organism (Ahmed et al., 2022). The optimal region must have high genetic variability and a low
volatility adjoining region to serve as a primer binding site for DNA fragment proliferation by polymerase chain
reaction (PCR) (Hasancebi, 2022). DNA barcoding is a simple, quick, and inexpensive technique. According to
Sang et al. (1997), the psbA-trnH region has been successfully used as a DNA barcode in Paeonia (Paeoniaceae),
resulting in DNA fragments of approximately 281–324 bp. Moreover, Chang (2015) investigated the use of the
psbA-trnH site as a DNA barcode in three jasmine species and found that the DNA sequences could be used to
classify the jasmines to the species level. Therefore, the present research aimed to investigate and compare the
differences between some Thai jasmine cultivars by using their morphological characters along with their DNA
information in order to identify them correctly and hopefully reduce the confusion of farmers, sellers and
consumers.

Methodology

Sample collection and morphological examination: Living samples of 10 jasmine cultivars, i.e., Mali
La, Mali Chanthabun, Mali Chat Dok Bua, Mali Chat Phikun, Mali Thot, Mali La Gleep Riaw, Mali La Bai Glom,
Mali Sedthi, Mali Yipun and Mali Son, and a sample of Maluli Si Chomphu were collected from 8 different sources
and planted in the greenhouse at the Department of Biology, Faculty of Science, Silpakorn University. Code names
and numbers were assigned to the plant samples. Dried specimens were prepared following the directions in the
book "Guide to collecting herbarium specimens in the field” (Royal Botanic Garden Edinburgh, 2017). For
preservation of jasmine flowers, flower samples were immerged in Formalin-Aceto-Alcohol solution (FAA) for
3-4 days until they were well penetrated. They were then transferred into 70% Ethanol solution for permanent
preservation. Leaf and flower morphological characters were examined and compared with the information from
the book "Jasmine in Thailand" (Chalermglin, 2013).

DNA extraction and PCR: The DNA from all jasmine samples was extracted and amplified at the
psbA-trnH sites using PCR technique with a total volume of 20 µl, at a concentration of 25 ng/µl and a temperature
programmed to initial denaturation at 94°c for 3 minutes. Denaturation was carried out at 94°c for 30 seconds,
annealing at 55°c for 30 seconds, elongation at 72°c for 1 minute, and the final extension was completed at 72°c
for 10 minutes using the T100™ thermal cycler. The PCR-derived DNA was then diluted to 50 ng/µl in a volume
of 50 µl. The resulting products were sent for nucleotide sequencing and analysis with BTseq™ technology at
Celemics in South Korea.

Bioinformatics and phylogenetic analyses: The nucleotide sequences were compared with a data set
derived from GenBank using the program BLASTn (www.blast.ncbi.nlm.nih.gov/Blast.cgi). DNA multiple
sequence alignment was performed using ClustalOmega (http://www.ebi.ac.uk/Tools/msa/clustalo). The aligned
DNA sequences were then used to reconstruct phylogenetic trees with maximum parsimony algorithm in the Mega
11® program (Tamura et al., 2021) following the default settings. Bootstrap analysis was also carried out with 100
cycles.

Results

Morphology: Jasmine is a small scandent or shrub. All samples have simple leaves arranged oppositely
on the branch. Several leaf shapes were observed, e.g., elliptic, obtuse and ovate. The apex of the leaves in most
samples are acute. Flowers were found to be either single or borne in cymose inflorescence. Petal numbers are
highly variable ranging from 4-40. Samples from Mali La and Maluli Si Chomphu have one layer of petal while
samples from other cultivars have several layers of petals (Fig. 1).

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DNA data: The psbA-trnH region was amplified in all samples, and 500 bp DNA fragments were
obtained. After comparing the nucleotide sequences in the NCBI database using the BLAST technique, it was
found that the nucleotide sequences of all jasmines in the present study matched the psbA-trnH region of the
Jasminum genus. However, Maluli Si Chomphu, a comparative species, has the nucleotide sequence close to
J. multiflorum (acc. no. MG754892) instead, while the rest of nucleotide samples matched with previously reported
sequences of J. sambac (acc. no. MH069934; MG754896; MG754898).

Phylogenetics: The 10 shortest phylogenetic trees were obtained from the phylogenetic reconstruction
with Maximum Parsimony algorithm. All of them had the same topology; therefore, only one tree was chosen for
further examination (Fig. 2). It was found that all samples were separated into two groups J. multiflorum, and
J. sambac. As expected, the first group consisted of J. multiflorum and a sample of Maluli Si Chomphu, which is
a comparative species. The remaining samples were placed into the group of J. sambac that was distinctively
monophyly with maximum support of the bootstrap value (Fig. 2). Unfortunately, it was not possible to
differentiate the cultivars in this group from each other. The two samples of Mali Son were interestingly grouped
together, as shown in Figure 2.

Discussion

Morphology: The leaf morphological characters of jasmine were highly variable even on the same plant.

Most leaves had elliptic shapes, similar to those reported by Sampath et al. (2008). However, it was also found
that some of the planted varieties had a different leaf shape than what was stated in the book “Jasmine in Thailand”.
In this report leaf shapes of jasmine cultivars are elliptic or ovate. In addition, four jasmine types that were not
mentioned in “Jasmine of Thailand” (Chalermglin, 2013) were included in this study. The leaf width and length
of Mali Sedthi and Mali Yipun were found to be similar to those of cultivar Mali Chanthabun. While leaves from
Mali La Bai Glom had a similar width and length to cultivar Mali Thot, they were not like the variety Mali La,
with a similar name. However, Mali La Bai Glom leaves also have some features similar to cultivar Mali
Chanthabun, such as a subcordate shape in some leaves and tips. Moreover, it was found that Mali La Gleep Riaw
has the same leaf morphology as Mali La cultivars.

According to Chalermglin (2013), there are multiple variations of jasmine cultivars. The petals of Mali
Chanthabun are large and flat, stacked in 2-3 layers, similar to jasmine blooms; however, Mali La has only one
layer of petals. Mali Chat Dok Bua has five layers of thickly heaped petals and a tightly wrapped petal in the center
when it is in bloom. Mali Chat Phikun contains more layers of petals than Mali Chat Phikun, but the petals are
smaller. Mali Son, has more than five layers of firmly piled petals, but no petals wrapped around the middle. Mali
Chanthabun and Mali Chat Dok Bua mutated from Mali Son, while Mali Chat Phikun mutated from Mali Son.
Because it is not yet the blossoming season, the flower section of Mali Thot could not be compared.

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DNA analysis: From the analysis of the psbA-trnH sequences of 18 jasmine samples, 17 samples of
J. sambac were grouped together separated from one sample of Maluli Si Chomphu. Therefore, it can be concluded
that the psbA-trnH sites can identify jasmine in detail at the species level, but cannot distinguish between cultivars
of jasmine (Chang, 2015; Jeyarani et al., 2018). However, within the J. sambac group, cultivars could not be
differentiated or grouped together following their name (Fig. 2), except for the grouping of two Mali Son samples.
This could be explained as a result of propagation by grafting or cuttings, which is a popular propagation method
since jasmine in Thailand is seedless (Chaitanya, 2018). Therefore, the sequence of nucleotides at the same location
may be the same. In the Mali Son group, it was found that they were grouped together, shown by the presence of
the single nucleotide polymorphism (SNP) on the 214th base along the sequence. Further investigation revealed
that this SNP was present in some samples of J. sambac in Genbank (acc. no. GU135379; LC461808: not included
in this study) at the same nucleotide position.

Chalermglin (2013) stated that Mali La cultivar is the parental cultivar of all jasmines. He also mentioned
two possible hypotheses about the origin of other jasmine cultivars. The first hypothesis is that Mali Chanthabun
cultivar mutated from Mali La cultivar, while the second hypothesis indicated that Mali Chat Dok Bua, Mali Thot,
and Mali Chat Phikun, mutated from Mali Son cultivar. In our study, the comparison of nucleotide sequence data
and phylogenetic analysis found that Mali La and Mali Chanthabun had 100% similarity of nucleotide sequences
in the psbA-trnH site which supports the first hypothesis. On the contrary, psbA-trnH sequences of Mali Chat Dok
Bua, Mali Thot, and Mali Chat Phikun do not have the SNP as recorded in Mali Son cultivar. Our results, therefore
do not support the second hypothesis of Chalermglin (2013). However, the results still support the statement that
Mali La is highly likely to be the be the parental strain of other cultivated jasmine cultivars.

Conclusion
Identification of jasmine cultivars in general cannot be done using only leaf morphology due to their

variability. On the other hand, the flower's characters are more informative for cultivar identification. In addition,
the psbA-trnH region as a DNA barcode can be used to identify jasmine to the species level, but cannot successfully
classify J. sambac to the cultivar level, except in Mali Son. From all of the above, it was possible to identify the
cultivars of jasmine, without the need for an expert, by using their flower morphologies. However, using the
psbA-trnH region for DNA barcoding, can only classify species of jasmine, but not identify cultivars except in
Mali Son.

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

Silpakorn University of Princess Sirindhorn’s College. The funding of SCiUS is provided by the Ministry of
Higher Education, Science, Research and Innovation. This extended abstract is not for citation.

References
Chalermglin, P. (2013). Jasmine in Thailand. Bangkok: Amarin Printing and Publishing Company Limited.
Ahmed, S., Ibrahim, M., Nantasenamat, C., Nisar, M.F., Malik, A.A., Waheed, R., Ahmed, M.Z., Ojha, S.C. and

Alam, M.K. (2022). Pragmatic applications and universality of DNA barcoding for substantial
organisms at species Level: A review to explore a way forward. 2022: 1-19.
Chaitanya, H.S., Nataraja, S. and Krishnappa, M. (2018). Review on propagation techniques of Jasmine
(Jasminum sambac (L.)). Journal of Pharmacognosy and Phytochemistry 7(6): 593-596.
Chang Kek Ning. (2015). DNA barcoding Jasminum sambac, Jasminum nitidum and Nyctanthes arbor-tristis by
using ITS2 and trnH-psbA genes. Faculty of Science, Technology, Engineering and Mathematics,
Inti International University, 1-15.
Jeyarani, J.N., Yohannan, R., Vijayavalli, D., Dwivedi, M.D., and Pandey, A.K. (2018). Phylogenetic analysis
and evolution of morphological characters in the genus Jasminum L. (Oleaceae) in India. Journal of
Genetics, 97:1225.
Hasancebi, S. (2022). Identification of Lens Cultivars in Market by Molecular Tool: DNA Barcoding and SSRs.
Research Square, 1 – 14, https://doi.org/10.21203/rs.3.rs-1202399/v1.
Sampath, M., Narayanappa, S.B., Sondur, S.N. and Simon, L. (2008). Analysis of genetic diversity among
Jasminum sambac (Linn.) Ait. and J. Grandiflorum Linn. varieties using Morphological and molecular
markers. Floriculture and Ornamental Biotechnology. 2(2): 60-64.
Sang, T., Crawford, D.J. and Stuessy T.F. (1997). Chloroplast DNA phylogeny, reticulate evolution, and
biogeography of Paeonia (Paeoniaceae). American Journal of botany, 84(8): 1120 – 1136.

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Title: Polyphenols content and antioxidant capacity of Tilicora triandra (Colebr.) leaf extract

:Field Biology and Biodiversity OB1_02_03

Author: Ms. Boontharika Pala-Aud
Ms. Jetpreeya Chisala
School: Ms. Panvira Khunprom
Demonstration school, University of Phayao

:Advisor Asst. Prof. Dr. Komsak Pintha (School of Medical Science, University of Phayao)

Asst. Prof. Dr. Payunsak Tantipaiboonwong (School of Medical Science, University of Phayao)
Abstract

Leaves of Yanang (Tiliacora triandra (Colebr.) Diels) were traditionally used as antipyretic and
detoxification. This study purposed to study the effect of boiling time extraction of Yanang leaf. Total phenolic,
total flavonoid, antioxidant activities by DPPH, and ABTS of those were compared with ethanol extraction.
The experiment was performed by boiling fresh Yanang leaves at 90°C for 5, 10, 15, and 20 minutes. Then,
removed the undissolved solid by centrifugation and lyophilized solution until dry. For ethanol extraction, the
leaves were soaked in 95 percent ethanol for 4 hr. Ethanol was removed by rotary evaporator and dried by
lyophilized. The results showed that all boiling water extracts of Yanang leaves had total phenolic, flavonoid
content, and antioxidant activities by DPPH and ABTS higher than ethanolic extract. The extract boiled for 5
min had the highest total phenolic content with 96.01 ± 3.17 mg gallic acid equivalent (GAE)/g extract, total
flavonoid content with 55.48 ± 1.95 mg catechin equivalent (CTE)/g extract, and the scavenging of DPPH
radical with inhibition concentration (IC50) of 75.14 ± 1.55 µg/ml and the scavenging of ABTS radical with
IC50 of 12.31 ± 0.93 µg/ml. This preliminary study determined the suitable boiling time of Yanang leaves for
daily consumption.

:Keywords Polyphenols, Free radicals, Antioxidants, Yanang leaves

Introduction
Yanang (Tilicora triandra (Colebr.) Diels) is a Thai traditional herb in Menispermaceae and is used

as an ingredient to make drinks and local food. Traditional medicine can use as an anti-pyrectic, antibacterial,
detoxification, and immune modulator agent. It has a high nutrition value and vital nutrients such as protein,
carbohydrates, lipid, vitamin A, vitamin C, vitamin E, etc. Ready-to-drink beverages made from the Yanang
leaf have become popular as functional drinks. Yanang leaf also has biological activities such as anti-
inflammation, in vitro anticancer activity, anti-diabetic, antioxidant activity, etc. A free radical can be defined
as an atom or molecule containing a lone pair of an electron in the valence shell, so it is an unstable molecule.
Free radicals have two sources, the first from outside the body e.g., infection, stress, and air pollution,

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Including food. And the second is from inside the body e.g., cell growth, immune system and signaling system
between cells, etc. When the body gets a lot of free radicals, the body can not destroy them in time. They will
attack Our cells, resulting in inflammation of tissue and the cause of various chronic diseases. Therefore, the
prevention of free radicals must use antioxidants because they will provide electrons to free radicals, and they
won’t become free radicals. The objectives of this study purpose to compare the polyphenols content and
antioxidant activity by DPPH and ABTS assay of Yanang leaves extracted with boiled water at 90oC for 5, 10,
15, and 20 minutes compared with the extracted of Yanang leaves by 95% ethanol.

Methodology

Part1: Prepare a Tiliacora triandra (Colebr.) (TT) extract

1.1. Extraction by 95% ethanol (TTE). Take 20 grams of Yanang leaf and blend them by blender, soak in 95%

ethanol for 4 hours, when the time up to take it to filtrate by cotton and bring it to evaporate ethanol by rotary

evaporator, finally make it dry by freeze dryer.

1.2. Extraction by water (H2O) at 90°C for 5, 10, 15 and 20 minutes (TTB5, TTB10, TTB15, TTB20).

Take 20 g. of Yanang leaf and chop it, measuring a water 200 ml. Put a leaf of Yanang and water into a pot

boiled them at 90°C for 5 minutes. And make it same but the time of boiling is a change to 10, 15 and 20

minutes. When due, put it on Erlenmeyer flasks for filterate, it gonna be the TTB5, TTB10, TTB15 and TTB20.

Take it on a tube and bring them in a centrifuge for the extract to silt in, finally making it dry in a freeze dryer.

Part 2: Determination

2.1. Determination of total phenolic contents (TPC) by folin-ciocalteu assay

Take the sample extracts curing with foiln compound for 15 minutes, add sodium carbonate (Na2CO3), and put

in the dark for 15 minutes. And measuring the absorbance at the wavelength of 750 nm., In the final take the

result to compare with the galic acid standard graph.

2.2. Determination of total flavonoid contents (TFC) by aluminum chloride assay

Take the sample extracts curing with 5% sodium nitrite (NaNO3), put in the dark for 5 minutes, and added a

10% aluminum chloride (AlCl3) with 1 M sodium hydroxide (NaOH) and taken to the dark for 10 minutes.

And measuring the absordance at the wavelength of 510 nm., In the final take the result to compare with the

cataechin standard graph.

2.3. Determination of antioxidant activity by DPPH radical scavenging assay

The antioxidant activity of DPPH radical scavenging was obtained by mixing the sample extracts with DPPH

solution in the dark for 15 minutes. When the time is up, take it to measure the absorbance at the wavelength

of 517 nm.

% DPPH radical inhibition = [" $%&'(%)*" +,-.)/]×233
" $%&'(%)

2.4. Determination of the antioxidant activity of ABTS radical scavenging assay
The antioxidant activity of ABTS radical scavenging was obtained by mixing the sample extracts with ABTS
solution in the dark for 6 minutes. When the time is up, take it to measure the absorbance at the wavelength
of 735 nm.

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% ABTS radical inhibition = [" $%&'(%)*" +,-.)/]×233
" $%&'(%)

Results
1. Determination of total phenolic contents (TPC)

The result showed that TTB5, TTB10, TTB15, and TTB20 have a total phenolic more than TTE.
TTB5 has the most total phenolic of all, it has total phenolic contents of 96.01±3.17 mg gallic acid equivalent
(GAE)/g extract. TTB10, TTB15, and TTB20 have the total phenolic contents in the range of 77 – 83 mg gallic
acid equivalent (GAE)/g extract, as shown in table 1 and figure 1.

Table 1: Showed the computation of total phenolic contents (TPC) (mg GA/g extract) Figure 1: Showed the computation of total phenolic contents (TPC) (mg GA/g extract)

2. Determination of total flavonoid contents (TFC)

The result showed that TTB5, TTB10, TTB15, and TTB20 have a total flavonoid more than TTE.

TTB5 has the most total flavonoid of all, it has a total flavonoid of 55.48±1.95 mg catechin equivalent (CTE)/g

extract. TTB10, TTB15, and TTB20 have the total flavonoid contents in the range of 40 – 43 mg catechin

equivalent (CTE)/g extract, as shown in table 2 and figure 2.

75

Total Flavonoid content 55.48
(mg CT/g extract)
50 40.85 43.35 43.10

Table 2: Showed the computation of total flavonoid contents (TFC) (mg CT/g extract) 25
10.85

0
TTE TTB5 TTB10 TTB15 TTB20

Figure 2: Showed the computation of total flavonoid contents (TFC) (mg CT/g extract)

3. DPPH radical scavenging assay

The result showed that TTB5, TTB10, TTB15, and TTB20 have an antioxidant capacity more than

TTE. The highest is the TTB5, it has the scavenging of DPPH radical with an inhibition concentration (IC50)
of 75.14 ± 1.55 µg/ml. TTB10, has 76.21 ± 0.77 µg/ml. TTB15, has 75.56 ± 0.88 µg/ml. and the less is TTB20,

it has 81.53 ± 0.55 µg/ml.

100 IC50 = 208.59±1.02 µg/ml 88 AŒ100 IC50 = 75.14±1.55 µg/ml 93 AŒIC50 = 76.21± 0.77µg/ml

% DPPH radical inhibition % DPPH radical inhibition 75 73 % DPPH radical inhibition 100 93
75 74
75
58

50 50 50
32 35 37

25 17 25 15 25 18
8 6 7
0 0 0
0 25 50 100 200 400 0 10 20 40 80 160 0 10 20 40 80 160

0 0 0

TTE concentration (µg /ml) TTB5 concentration (µg /ml) TTB10 concentration (µg/ml)
Figure 3: Showed the DPPH radical scavenging of TTE Figure 4: Showed the DPPH radical scavenging of TTB5 Figure 5: Showed the DPPH radical scavenging of TTB10

IC50 = 75A.5Œ6±0.88 µg/ml IC50 = 81.53±0.55 µg/ml 93 IC50 = 3.13±1.14 µg/ml

100 100 100 94

% DPPH radical inhibition 94 % DPPH radical inhibition % DPPH radical inhibition

75 70 75 75 70
57
50 50
35 50 35

25 16 25 27 25 16
12
6 0 5 0 6
0 0
0 10 20 40 80 160
0 0 0 0.625 1.25 2.5 5 10

0 10 20 40 80 160

TTB15 concentration (µg/ml) TTB20 concentration (µg/ml) Vitamin C concentration (µg/ml)
Figure 7: Showed the DPPH radical scavenging of TTB20 Figure 8: Showed the DPPH radical scavenging of vitamin c
Figure 6: Showed the DPPH radical scavenging of TTB15

DPPH radical scavenging activity of Tiliacora triandra extract

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4. ABTS radical scavenging assay
The result showed that TTB5, TTB10, TTB15, and TTB20 have an antioxidant capacity more than

TTE. The highest is the TTB5, it has the scavenging of ABTS radical with an inhibition concentration (IC50)
of 12.31 ± 0.93 µg/ml. TTB10 has 13.44 ± 0.74 µg/ml. TTB15 has 13.78 ± 0.86 µg/ml. and the less is TTB20,
which has 23.57 ± 1.28 µg/ml.

%ABTS inhibition 100 IC50 = 28.57±1.14 µg/ml 83 %ABTS inhibition 100 IC50 = 12.31±0.93 µg/ml 81 %ABTS inhibition 100 IC50 = 13.44±0.74 µg/ml

75 75 75 74

50 48 50 46 50 37
29
25 26 25 21
25 18 15 12
00 4
00 0 2.5 5 10 20 00

0 6.25 12.5 25 50 40 0 2.5 5 10 20

TTE concentration (µg/ml) TTB5 concentration (µg/ml) TTB10 concentration (µg/ml)
Figure 10: Showed the ABTS radical scavenging of TTB5 Figure 11: Showed the ABTS radical scavenging of TTB10
Figure 9: Showed the ABTS radical scavenging of TTE

%ABTS inhibition IC50 = 13.78±0.86 µg/ml 71 %ABTS inhibition IC50 = 23.57±1.28 µg/ml 79 %ABTS inhibition IC50 = 2.67±1.04 µg/ml

100 100 100 100
75 75 66
75

50 38 50 43 50
32
25 21 25 15 22
11 25 18

00 00 00
0
0 2.5 5 10 20 2.5 5 10 20 0 0.3125 0.625 1.25 2.5
TTB20 concentration (µg/ml)
TTB15 concentration (µg/ml) Vitamin C concentration (µg/ml)

Figure12: Showed the ABTS radical scavenging of TTB15 Figure 13: Showed the ABTS radical scavenging of TTB20 Figure 14: Showed the ABTS radical scavenging of vitamin c

ABTS radical scavenging activity of Tiliacora triandra extract

Conclusion
From this study, we found, that the Tilicora triandra (Colebr.) extract by 95% ethanol has total

phenolic, total flavonoid, and antioxidant capacity less than extract by water at 90˚C for 5, 10, 15, and 20 min.
TTB5 has the most total phenolic, total flavonoid, and antioxidant capacity, the secondary is TTB10, TTB15,
and TTB20 respectively.

Extracts TPC TFC DPPH ABTS

TTE 37.34±1.42 10.85±0.67 208.59±1.02 28.57±1.14
TTB5 96.01±3.17 55.48±1.95 75.14±1.55 12.31±0.93
TTB10 83.54±5.38 40.85±0.89 76.21±0.77 13.44±0.74
TTB15 77.93±2.56 43.35±1.08 75.56±0.88 13.78±0.86
TTB20 79.32±2.17 43.10±2.27 81.53±0.55 23.57±1.28

Table 3: showed the computation of total phenolic (TPC) (mg GA/g extract), total flavonoid (TFC) (mg CT/g extract), DPPH and ABTS radical scavenging

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. Chemical Constituents and in vitro anticancer activity of Tiliacora triandra leaves Pharmacognosy
Journal,2016,8,1,1-3. DOI:10.5530/pj.2016.1.1.
2. Tiliacora triandra extract possesses antidiabetic effects in high-fat diet/streptozotocin-induced diabetes in

rats. Available from: https://doi.org/10.1111/jfbc.13239.

3. Effects of Tiliacora triandraLeaf Water Extract in High-Fat Diet Fed Mice. J Med Assoc Thai 2017; 100
(Suppl. 5): S78-S84.
4. Some Phytochemicals and Anti-inflammation Effect of Juice from Tiliacora triandra Leaves. journal of Food
and Nutrition Research, 2018. 6(1);32-38.

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Title : Effects of pollen morphology, fabrics, and time OB1_15_04

on the retention of pollen on clothes
Field : Biology and biodiversity
Author : Miss Saruta Chumpong

Miss Yanisa Traiprakong
School : PSU.Wittayanusorn School , Prince of Songkhla University
Advisor : Asst. Prof. Dr.Wongkot Phuphumirat ( Prince of Songkhla University )

Mr.Krittanai Panyang ( PSU.Wittayanusorn School )

Abstract

Nowadays people are interested in criminal cases. When there is a murder case, there will be a variety
of physical evidence to prove. In particular, forensic palynology uses pollen and spores as physical evidence.
So, the organizers are interested in studying and comparing the persistence of pollen on fabrics. The
preparation of this project is intended. 1. To study the effect of pollen morphology on the retention of pollen
on clothes. 2.To study the effect of type of fabrics on the retention of pollen on clothes. 3.To study the effect
of time on the retention of pollen on clothes. Conduct by pollen selection and acetolysis mixture to put in
microtubes. Put in the fume hood and boil. Then look for the pollen morphology, pour the pollen mixture on
clothes and test with the volunteers at different intervals. The clothes are washed and the remaining water is
centrifuged until sediment is obtained. Pollen is counted under a microscope.

Study results and project preparation. Study pollen morphology of plants affecting retention on fabric.
It was found that pollen of lily showed the highest retention to fabric. Lily pollen is reticulate, which is
inconsistent with this hypothesis, echinate pattern such as daisy has the best retention. To study the effect of
time on the retention of pollen on clothes, it was found that the longer pollen attaches on fabric, the more the
number of pollen decreases because wind blows pollen. To study the effect of type fabrics on the retention of
pollen on clothes, it was found that from the experiment, denim had a retention of pollen than other fabrics
due to the complex weave of fibers.

Keywords : Pollen morphology , forensic palynology , fabrics , acetolysis mixture , microscope

OB1_15_04/1
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12th SCiUS Forum

Introduction
Crime is a major problem facing the world causing damage to life and property and it is likely to

increase in the future. Therefore, it is necessary to investigate the process of suspects entering the judicial
process. At present, scientists use evidence to verify theoretical knowledge. Solving legal problems and
evidence of cases that occur in a variety of problems is called forensic science. Evidence found at the crime
scene, including blood, hair, sperm, fingerprints, etc. also have forensic applied biological knowledge, such
as Forensic palynology is an applied science of spores. Evidence in a legally effective case Forensic
evidence can be connected to the crime scene of people or objects. Spore and pollen formation and
completion of plants are very small and have different shapes in the durability of each genus or species.

From the study of Mildenhall holmium (2006), it can be concluded that the use of forensic evidence is
very effective. The study took place in Australia. Two men entered a woman's house without locking the
door. When the woman woke up, she heard the sound inside and saw all the intruders running away. One of
the bastards left jacket at home. They hurried back, so they accidentally walked through the Canary bushes
at the door. After the suspect was arrested, the scientists collected evidence, clothing, and anther analysis.
The results showed the high doses of hypericum indicate that it is in direct contact with hypericum. Samples
taken from the crime scene. From these analysis results to be more accurate.

In addition, forensic pollen studies on the retention efficacy of pollen on three fabrics: cotton, denim
and fleece were studied by Julia et al. (2017). The plant can cling to both fresh and dry pollen on clothing by
pollinating plants that carry insects for pollination. Insect-pollinated plants were more effective at adhering
to clothing than wind-borne plant pollen. In addition, a Mildenhall study (1990) showed that pollen tends to
be more adhesion and deposition in fabrics with relatively complex weaves such as denim.

Methodology
The experiments were divided into 2 parts as follows
Part 1 : Study the pollen morphology
1.1 Explore and select 8 types of animal-pollinated flowers. For example , Lily , Tulip , Peach lily ,

Daisy , Gerbera , Protea, Stock and Carnation. Then separate anthers for pollen extraction by acetolysis.
Ripe and unbroken anthers are selected ,placed in a microtube and add acetolysis mixture prepared by
mixing acetic anhydride and conc. sulfuric acid (9:1) to flood anthers. Put in a fume hood and then tong the
centrifuge tubes to boil them in a beaker using the hotplate stirrer for about 5 minutes.

1.2 Study the pollen morphology under a light microscope. use a teasing needle with a nose hair rake
pollen for looking for pollen morphology. Count only 1 anther.

Part 2 : Study the retention efficiency of pollen on clothing
2.1 Prepare pollen mixture. Then take 3 types of fabrics ( denim , spandex , cotton ) , 12 of each type.
Mark a square of 10 x 10 cm². Use a micropipette to suck the pollen mixture ( there are about 1000 pollen
grains) and place it on every fabric 10 microliters in the middle of the fabric. Divided 3 types of fabrics ,
4 pieces of each type , but 3 times for attaching , total 36 pieces. Put them on the volunteers every 2 days,
10 days and 20 days.

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2.2 Bring the fabrics that have been attached for 2 days, 10 days and 20 days to wash without mixing
by putting them in water mixed with detergent. Then use a toothbrush to scrub 20 times, put the fabrics to
rest, put the remaining water into the centrifuge tube and centrifuge for about 6000 times per 10 minutes
until the pollen settles at the bottom of the tube and all the remaining water is exhausted. Drop 1 drop of
glycerin in each tube and take the sediment to a light microscope. Count the remaining pollen after
experimenting with the volunteers.

Results
Table 1 : The table shows the pollen morphology of different plant

Name Lily Daisy Gerbera Stock Tulip Peace Lily Protea Carnation

Pollen monad monad monad monad monad monad monad monad
unit

Pollen size medium small medium small large small medium medium

Pollen prolate oblate prolate prolate oblate oblate oblate spheroidal
shape

Aperture mono- tricol- tricol- tricol- mono- - triporate porate
colpate porate porate porate sulcate

Ornamen- reticu- echi- psilate psilate psilate plicate psilate microechi-
tation late nate nate

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Conclusion
From the study and comparison of the pollen persistence efficiency of each flower on different fabrics.

In conclusion, pollen can last on different fabrics differently. Each type of fabric has different durability. The
2 days period showed the most persistent pollen, the 10 days period had pollen persistence close to 20 days.
The pollen of the plant that was best tolerated on the fabric was the patterned lily. And the fabric with the
most persistence of pollen is denim because denim has a more complex weave of fibers than other fabrics.
Therefore, it can be assumed that the faster the witness object is found, the more pollen will be found.

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

Prince of Songkla University and PSU.wittayanusorn 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
Chanwatcharachot, T. (2020). Jeans VS Denim How are jeans and denim different?. Retrieved September

28, 2021, from https://www.mcshop.com/blog/?
Chotmanee, T. (2018). 360 Concepts in Biology. Bangkok: Chulalongkorn University Press.
Eadprapan, N. (2018). Spreading and potential of forensic evidence in the community and testing how

to collect forensic evidence from the locale. Master thesis, M.S., Prince of songkhla University,
Songkhla.
Graduates of Suan Sunandha Rajabhat University. (2018). Management and forensic evidence collection in
the ministry of justice of the central institute of Forensic Science. The Second National Conference
and Research Presentation. Bangkok.
House and garden publishing. n.d. flower. Retrieved April 3, 2022, from https:// www.baanlaesuan.com
I-Tim. [Pseudonym]. (2019). 3 types of fabrics that women should know and advantages of different
fabrics. Retrieved September 28, 2021, from https://www.sistacafe.com/summaries/64695
Julia C.W., Harriat A.B., Hannah T., Anne E.G. (2018). Differential retention of pollen on clothing and
the effectiveness of laboratory retrieval methods in forensic settings. Forensic science international,
288, 36-45.
Mildenhall D.C., (1990). Forensic palynology in New Zealand. Review of Palaeobotany and Palynology.
64(1-4), 227-234.
Na Songkhla, N. (2020). What is cotton? What are the features?. Retrieved September 28, 2021, from
https://www.mcshop.com/blog/?
Noonpradech, P. (2013). The role of judges in crime prevention. n.p.
Srithammarong, T. (2016). Assessing crime-risk areas by analyzing spatial statistics. Master thesis, M.S.,
Silpakorn University, Nakornprathom.

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Extended Abstract 12th SCiUS Forum
Title : Induction Mutation of Plant Growth-Promoting
OB1_02_02
Bacteria by Cold Plasma Technology

Field : Biology
Author : Miss Boonraksa Mangmee

Miss Wasita Injai
Miss Saisawhan kamoi

School : Demonstration school University of Phayao, University of Phayao
Advisor : Dr.Kanta Sangwijit, Plasma Bioengineering Unit

Abstract
Microorganisms in soil plays a crucial role in the agriculture section. Plant growth promoting bacteria

(PGPB) provide several benefits in agriculture such as secretion and modulation of phytohormones, nutrient
acquisition and improvement of soil texture. In additional, PGPB can also enhance plant growth by transform
the nutrients into absorbable form for plants such as fixing the nitrogen and changing phosphorus to dissolvable
forms. Furthermore, PGPB are used as biostimulants or fertilizers to control the abundance of nutrients in soil.
Improving properties of PGPB is a promising strategy for enhancing plant growth and productivity. Cold
plasma technology is one of popular tools for microorganisms’ property improvement. In this study 20 isolates
of bacteria were screened from soil in Phayao. The 16S rRNA gene sequences of selected bacteria showed high
similarity to Bacillus subtilis, Burkholderia ambifaria, Burkholderia contaminans, and Staphylococcus
hominis. B. subtillis is a nonpathogenic bacterium which can help promoting plant growth. Therefore, B.
subtiilis was selected for increasing its efficiency by induction mutation using cold plasma. The bacterial cells
were treated under plasma of helium or argon with energy of 120 W for 1 min to 10 min. The bacterial isolates
which extibited lower pH in culture media compered to the control were obtained under argon plasma treatment
at 5 and 10 min. Subsequently, evaluating growth promotion effect on plants by B. subtillis and its mutant will
be further studied.

Keywords : PGPB, Cold Plasma Technology, mutation

Introduction
The soil consists of small living organisms including bacteria, fungi, protozoa, and algae (Ubonrat J. et

al.,2018). Plant growth promoting bateria (PGPB) are the microorganism or bacteria inhabit in the host plant
around plant roots and promote productivity including resistance of plant disease, adjustment of hormones,
phosphate solubilization, nitrogen fixation and capability use by plant (Glick, 2012).

Long-term and continuosly using of chemical fertilizers affects the soil microorganisms, nutrients and
other living things, such as losing the balance of microorganism in soil, the erosion of nutrients in soil and the
outbreak of plant disease. So, in order to reduce the using of chemical fertilizers, PGPB is another way to solve
these problems (Suthisa W. and Wapi B.,2019). Plant growth promoting bateria (PGPB) which can promote
plant growth and productivity has been used worldwide since 1950s as an alternative biofertilizer. However,
the inconsistent efficiency and vitality of PGPB are the obstacles in the agricultural application leading to the

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limitation of PGPB utilization. Genetics engineering has been used to overcome this problem. Cold plasma
technology is a novel and effective mutagenesis tool which is an environmental-friendly technique capable of
induction mutation in many microorganisms (Naito K.,2005). Therefore, aim of the study is to screen PGPB
and enhancing its efficiency by induction mutation using cold plasma technology.
Methodology
1. Screening of phosphate solubilizing bacteria

A Soil sample was collected from 2 experiment fields including Avocado farm at Chiang Rai and
University of Phayao in depth of 30 cm. The suspensions of soil sample were spread on Pikovskaya medium
(PVK) and incubated at 37 °C for 5 days to observe the halo zone. Any colonies with clear zone formation
around the colonies were selected and streaked to obtain pure isolates.
2. Identification of bacteria

Genomic DNA of the selected bacteria were isolated and used as a DNA template for 16S rDNA
amplification. Two primers, forward primer 5’-AGT TTG ATC CTG GCT C-3’ and and reverse
primer 5’-GGC TAC CTT GTT ACG A-3’ were used for gene amplified by PCR. Subsequenly, the 1500 bp
PCR products were obtained and purified using GeneJET Gel Extraction Kit (Thermo Scientific™), and 16S
rDNA sequences were analyzed online with NCBI blast (https ://www.ebi.ac.uk).
3. Induction mutation of bacteria

Plasma treatment of the selected bacteria was operated using atmospheric pressure plasma jet (APPJ)
at Plasma Bioengineering Unit, School of Science, University of Phayao. The selected bacterial was grown
overnight at 37 °C, then cells were harvested by centrifugation at 4,200 rpm for 10 min. Bacterial cells were
resuspended with 100 µl of sterile water and dropped in a holder for plasma treatment. Helium and argon gas
were chosen to generate plasma, the electrode was connected to the power supply with power of 120 W and
gas flow rate of 2 SCCM. The effect of treatment time the was studied ranging from 1 to 10 min. After plasma
treatment, each treated sample was grown on PVK agar and incubated at 37 °C for 5 days.
4. Screening of bacterial mutant

After the bacterial cells were treated by plasmas, the treated bacterial cells were incubated at 37 °C
for 5 days. The indirect phosphate solubilizing ability was invaluated by pH measurement at 3, 5 and 7 days
after incubation. The mutant with the lowest pH was selected and named UP13-M while the control was named
UP13

Results
1. Characterization and Identification of bacteria isolated form soil

Soil samples were collected from several areas to find microorganisms that can use inorganic
phosphates. These bacteria were grown on medium containing inorganic phosphates. After growing bacteria on agar
plates with inorganic phosphates for 5 days at 37°C, bacterial colony of different morphology were chosen. The
phosphate solubilization was exhibited with a halo zone formed around the bacterial colony. As a consequence, 7
isolates, named AF03, AF15, AF28, UP01, UP07, UP13 and UP46 which produced visible halo zone on PVK agar
plates were seleceted for molecular identification. Analysis of a 16S rRNA sequences of the selected isolates showed
the highest homology to Bacillus spp., Burkholderia contaminans and Staphylococcus hominis as shown in
Table1.

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Table 1. Identification of bacteria isolates for phosphate solubilization

isolates phosphate solubilization pathogenesis

AF03 + - Bacillus subtilis
Bacillus ambifaria
AF15 + + Bacillus ambifaria
Burkholderia contaminans
AF28 + + Bacillus subtilis
Bacillus subtilis
UP01 + + Staphylococcus hominis

UP07 + -

UP13 + -

UP46 + +

Molecular analysis based on 16S rRNA homology in Table 1 indicated that isolates AF03, UP07 and

UP13 are non-pathogenic bacteria. Among 3 isolates, isolated UP13 was chosen for further study owing to its

widest halo zone on PVK agar plate compared to other B. subtilis.

2. Induction mutation of bacteria and mutant screening

The mutants of the selected bacteria, UP13, were screened by spotting on PVK agar plates and

incubated at 37 °C for 5 days. The control, UP13, was grown on the same PVK agar media as the control. The

qualitative phosphate solubilization ability of each plasma treated bacterial was evaluated by the halo diameter

of each colony. The mutant with the widest halo diameter was selected for further analysis. Further, indirect

quantitative estimation of phosphate solubilization ability was done in PVK broth culture by pH measurement.

Analysis of organic acids producing by bacteria, bacterial culture was filtrated through Whatman® filter paper,

then pH value of each filtrate was carried out with pH meter. Table 2 showed pH value of culture filtrate from

the screened mutant at 3, 5 and 7 days. The mutant with the lowest pH value (UP13-M 110) was selected and

named UP13-M. The mutant (UP13-M) and the control (UP13) will be cultivated in PVK borth for evaluating

growth promotion effect on plants.

Table 2. pH measurement at different days of cultivation.

Sample 3 days 5 days 7 days

PVK broth 5.86 5.96 5.91

UP13 5.24 5.38 5.08

UP13-M 15 5.28 5.02 4.97

UP13-M 16 5.11 4.93 4.84

UP13-M 18 5.26 5.17 5.05

UP13-M 110 5.07 4.85 4.66

UP13-M 224 5.09 5.15 4.91

UP13-M 312 5.22 4.90 5.00

UP13-M 455 5.27 4.99 4.94

UP13-M 496 5.21 5.13 5.02

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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. K Naito, M Kusaba, N Shikazono, T Takano, A Tanaka, T Tanisaka and M Nishimura.

Transmissible and nontransmissible mutations induced by irradiating Arabidopsis thaliana
pollen with gamma-rays and carbon ions. Genetics 2005; 169, 881-9. [22] KP Gopinath, S
Murugesan, J Abraham and K Muthukumar. Bacillus sp. mutant for improved biodegradation of
congo red: Random mutagenesis approach. Bioresour. Technol. 2009; 100, 6295-300.

2. Ubonrat J., Moonmangmee N., Kunnajak N. and Moonmangmee D. Isolation and screening of

plant growth promoting bacteria (PGPB) from rice (Oryza sativa) and rhizosphere soil.

3. Glick, B. R. Plant growth-promoting bacteria: mechanisms and applications. Scientifica 2012,

963401, https://doi. org/10.6064/2012/963401 (2012).

4. K. Naito, M Kusaba, N Shikazono, T Takano, A Tanaka, T Tanisaka and M Nishimura.

Transmissible and nontransmissible mutations induced by irradiating Arabidopsis thaliana
pollen with gamma-rays and carbon ions. Genetics 2005; 169, 881-9.

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Title : Antimicrobial and antibiofilm activity of actinobacteria OB1_10_02
isolated from rhizosphere soil and mangrove sediments

Field : Biology and Biodiversity

Author : Miss Thanatchaya Toom-ariya

Mr. Natchanon Kongsut

Miss Ployrawee Phet-ngam

School : Kasetsart University Laboratory School Kamphaeng Saen Campus Educational Research and

Development Center

Advisor : Dr. Ratchanee Mingma, Faculty of Liberal Arts and Science, Kasetsart University, Kamphaeng

Saen Campus, Nakhon Pathom

Abstract : Actinobacteria are gram-positive bacteria that have an important role in producing of bioactive
compounds against various microorganisms and have the ability to inhibit the formation of biofilms. Bacterial
biofilms increase the resistance of microorganism for antimicrobial agents and developed the human infection.
The proposed of this research was to isolation of actinobacteria from mangrove sediments and rhizosphere
soil of cherry and cannon ball (sala) tree. Antibacterial activity and antibiofilm formation were evaluated. The
result showed that a total of actinobacteria 25 isolates were obtained: 17 isolates from mangrove sediment and
7 isolates from rhizosphere soils. Out of these, 9 isolates showed inhibition activities against at least one tested
pathogen. Isolates KK22 and KK27 showed the highest activity against Bacillus cereus and Staphylococcus
aureus, respectively. Isolate KK27 was further selected for antibiofilm activity. The results showed that isolate
KK27 reduced 31.1% and 68.5% biofilm formation of S. aureus and B. cereus, respectively. Based on 16S
rRNA sequence analysis, isolate KK27 was the most closely related to Streptomyces daghestanicus NRRL B-
5418T with 99.14% similarity.

Keywords : Anti-biofilm; mangrove sediments; rhizosphere soil; actinobacteria

Introduction
Bacterial biofilm formation layer causes a serious problem in public health. These bacteria produce

extracellular polymeric substance (ESP) covering their cells and becoming the layer of the biofilm. Biofilms
production causes microorganisms more resistant to drugs and have an important role in the chronic infections.
The removal of biofilm can be done by using chemical substances such as chlorine, ozone, formaldehyde, etc.,
which are costly and couldn’t effectiveness 100 percent. The natural substances produced by microorganisms
is getting more attention for protection and inhibition of biofilm formation. Mangrove forest ecosystem is one
of the most natural resources that contains various plants, animals and contain a great variety of
microorganisms. Actinobacteria are Gram-positive bacteria that widely distributed in marine and terrestrial
habitat. Actinobacteria are abundant surrounds the high level in rhizosphere soil approaching 40% in the total
of soil microflora. Actinobacteria can produce a variety of biologically active secondary metabolites and have
an important role in pharmaceutical, agricultural and industrial.

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The objective of this research was to isolate actinobacteria from rhizosphere soils and mangrove
sediments. The antimicrobial activities against pathogenic microorganisms and anti-biofilm formation
activities were evaluated.
Methodology
The experiments were divided into 2 parts as follows,
Part 1: Isolation of actinobacteria from rhizosphere soils and mangrove sediments

1.1 Soil sample collection
Rhizosphere soils were collected from cherry trees (Prunus avium) and sala trees (Shorea robusta),

while mangrove soil sediments were collected from Klong Khone Mangrove Forest at Samut Songkhram
province. The samples were collected in clean plastic bag and transported to Laboratory. Soil samples were
air dried (not exposed to sunlight) for one week.

1.2 Isolation of actinobacteria
Ten gram of soil sample was suspended in 90 ml sterile distilled water and serially diluted up to

10-2 to 10-5. Aliquots (0.1 ml) of each dilution was spread on starch casein (SC) agar (1) supplemented with
antibiotics ketoconazole (100 µg/ml) and nalidixic acid (25 µg/ml) to inhibit fungi and bacteria, respectively.
The plates were incubated for 14-21 days at 28 ˚C. The appearance and growth of actinobacteria were observed
and picked up using sterile toothpick and streaked on International Streptomyces project medium 2 (ISP-2)
(2). The purified colonies were sub-cultured on ISP-2 slant.
Part 2: Determination of antimicrobial activity against pathogenic microorganisms and anti-biofilm formation
activity

2.1 Antimicrobial activity
Escherichia coli ATCC 25922, Pseudomonas aeruginosa, Staphylococcus aureus ATCC 27853 and

Bacillus cereus TISTR 687 were used as tested strains and antimicrobial activity were evaluated by agar
overlay method (3). The width of inhibition zone and colony diameter were measured. The ratio of inhibition
zone per colony size were recorded.

2.2 Anti-biofilm formation activity
Actinobacteria were cultured in starch casein broth (SCB) at 30 °C, 150 rev/min for 7 days. Cultures

were centrifuged to precipitate the cells and the supernatant were collected and filtered using membrane pore
size 0.45 µm. Antibiofilm activity was determined using method described by Leetanasaksakul and
Thamchaipenet (4) with some modification.

2.3 Identification of actinobacteria by 16s rRNA gene sequencing
Genomic DNA extraction and amplification of the 16s rRNA gene were performed according to the

methods of Take et al. (5). The sequencing of purified PCR products was performed by a commercial
sequencing company at Bionics (Korea). The sequences were compared and blasted with other microbial
sequence in EzTAXON-e database (6).

Results
A total of actinobacteria 25 isolates were obtained, 17 isolates from mangrove sediments, 2 and 6

isolates from rhizosphere soil of cherry and sala tree, respectively (Table 1). The appearance of actinobacterial
colonies are shown in Figure 1.

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Table 1 Number of actinobacteria isolated from soil samples

Soil sample Number of isolates Isolate name

Mangrove sediments 17 KK01, KK04, KK06, KK11, KK17, KK20,
KK22, KK24, KK25, KK27, KK28, KK33,
Rhizosphere soil of cherry tree 2 KK36, KK37, KK38, KK40, KK42
Rhizosphere soil of sala tree 6 CH03, CH04
25
Total SR02, SR10, SR11, SR13, SR17, SR18

The antimicrobial activity against pathogenic bacteria using agar overlay method showed that out of
25 actinobacterial isolates, 9 isolates (36%) were found to inhibit at least one tested Gram-positive bacteria
consisting of B. cereus (inhibition ratio from 1.53 to 4.13) and S. aureus (inhibition ratio from 1.7 to 3.30).
All isolates could not inhibit Gram-negative bacteria. The antibacterial activity of actinobacteria isolated from
mangrove sediments was higher than from rhizosphere soils. The isolates KK11 and KK27 had highest
inhibition activity against B. cereus and S. aureus, respectively. Isolate KK27 was selected for biofilm
inhibition assay due to it had highest activity against S. aureus (Figure 2). Staphylococcus aureus has become
resistant to all antibiotics. Biofilm production of this bacteria is an important factor that causing human
infection and leading to treatment failures.

Figure 1 Actinobacterial colonies growing on starch Figure 2 Antibacterial activity against Staphylococcus
casein agar after incubated at 30 oC for 2 weeks. aureus ATCC 27853 of isolate KK27 using agar
overlay method.

The supernatant of isolate KK27 inhibited the biofilm formation of S. aureus and B. cereus with 31.1
and 68.5 percent, respectively (Figure 3), but could not inhibit the biofilm formation of E. coli and P.
aeruginosa. Analysis of 16S rRNA gene sequencing showed that isolate KK27 was most closely related to
Streptomyces daghestanicus NRRL B-5418 with 99.14% similarity.

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Figure 3 Bacterial biofilms inhibition assay
with crystal violet staining by the 96 well
microtiter plate.

Conclusion
In this study, actinobacteria 25 isolates were isolated from rhizosphere soil and mangrove sediments.

There were 9 and 5 isolates could inhibit Bacillus cereus and Staphylococcus aureus, respectively. Isolate
KK27 which was isolated from mangrove sediment had the highest inhibition activity against S. aureus. The
biofilm inhibition assay showed that KK27 inhibited the biofilm formation of S. aureus and B. cereus at 31.1
and 68.5 percent, respectively. The 16S rRNA gene sequence analysis revealed that isolate KK27 belong to
the genus Streptomyces, with highest similarly to S. daghestanicus (99.14% similarity).

Acknowledgment
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.

Reference
1. Küster E, Williams ST. Selection of media for isolation of streptomycetes. Nature. 1964; 202: 928-929.
2. Shirling EB, Gottlieb D. Method for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 1966;

16: 313-340.
3. Anand TP, Bhat AW, Shouche YS, Roy U, Siddharth J, Sarma SP. Antimicrobial activity of marine

bacteria associated with sponges from the waters off the coast of South East India. Microbiol. Res. 2006;
161(3): 252-262.
4. Leetanasaksakul K, Thamchaipenet A. Potential anti-biofilm producing marine actinomycetes isolated
from sea sediments in Thailand. Agric. Nat. Resour. 2018; 52: 228-233.
5. Také A, Inahashi Y, Ōmura S, Takahashi Y, Matsumoto A. Streptomyces boninensis sp. nov., isolated
from soil from a limestone cave in the Ogasawara Islands. Int. J. Syst. Evol. Microbiol. 2018; 68: 1795–
1799.
6. Yoon SH, Ha SM, Kwon S, Lim J, Kim Y, Seo H and Chun J. Introducing EzBioCloud: A taxonomically
united database of 16S rRNA and whole genome assemblies. Int. J. Syst. Evol. Microbiol. 2017; 67: 1613-
1617.

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Title : OB1_04_02Using thermal camera to assess phenotype of corn seedling

Field : under water deficit
Authors : Biology and Biodiversity
Mr. Jetbodin Piata
School : Miss Chonnipa Sasiwilasakon
Advisor : Demonstration School of Khon Kaen University, Khon Kaen University
Asst. Prof. Dr. Watanachai Lontom
Department of Biology, Faculty of Science, Khon Kaen University

Abstract
Drought is one of the abiotic stresses that cause deleterious effects on growth and yield in many crop

plants. Several physiological and biochemical parameters, e.g., water status, greenness and membrane stability
were affected by drought; moreover, drought directly affects transpiration of plants. In drought conditions, plants
are induced to close their stomata to preserve water that increases leaf temperature. Change in leaf temperature
detected by thermography can be used to evaluate crop water stress index (CWSI) and index of stomatal
conductance (IG) in plants. Therefore, the effects of drought on the changing temperature together with some
physiological and biochemical responses in Nakhon Sawan 5 and Samlee Isan maize seedlings were studied. Maize
seedlings at V3 stage of both varieties were subjected to drought by withholding irrigation until their leaves rolled.
Then, thermal images of seedlings were taken. Relative water content (RWC), SPAD chlorophyll meter reading
(SCMR), electrolyte leakage (EL), malondialdehyde (MDA) content and seedling growth were also determined.
The results demonstrated that most of parameters were significantly different between control and drought
conditions. Based on the growth and physiological data, Samlee Isan was slightly drought tolerant than Nakhon
Sawan 5. Different temperature changes between well-watered and drought stressed seedlings of both maize
varieties could be detected by thermography.

Keywords : Crop water stress index, Drought, Maize seedling, Physiological parameters, Thermography

Introduction
Corn is one of Thailand's important economic crops used in the livestock industry and for consumption.

In Thailand, corns are grown mostly in northern, northeastern, and central parts respectively. One of limiting
factors in growing corn, especially in the Northeast of Thailand, is the lack of water due to inconsistent rainfall
and long drought periods. Drought has a huge impact on growth and productivity of corn. The reduction of corn
yield depends on the duration and severity of drought stress and the developmental stage at which the crop is
affected. Understanding the effect of drought stress and methods to identify its occurrence are essential for further
crop management. However, monitoring crop water stress by measuring soil moisture content and determining
plant growth and physiological changes is destructive, time consuming and labor intensive. Recently, infrared (IR)
thermography has been used to monitor plant-environment interactions by visualizing surface temperature

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distribution of plants. Plant temperature varies with transpiration, stomatal conductance, and water status;
moreover, it shows some relationships with parameters indicating crop stress. Therefore, this study aims to
investigate the effects of drought on the changing temperature together with some physiological and biochemical
responses in Nakhon Sawan 5 and Samlee Isan maize seedlings.

Methodology
Plant material and experimental set up

Seeds of Nakhon Sawan 5 and Samlee Isan corn varieties were soaked in water and germinated separately
in plastic pot containing 400g of dry soil. Each pot was irrigated with equal amount of water and maintained in a
greenhouse until corn seedlings were at V3 stage. After that, seedlings were divided into 2 groups; control group
was kept under well-watered condition throughout the experimental period and stress group was subjected to
drought stress by withholding irrigation for 7 days or until their leaves rolled. The experiment was arranged in
completely randomized design with 5 replicates.
Measurements of soil moisture, growth, and physiological parameters

Soil moisture content was measured using the gravimetric method. Soil was sampled at 5 cm beneath soil
surface, weighed and oven-dried at 105°C for 72 h. Soil moisture content was calculated as follows:

Soil moisture content (%) = [(Wet weight - Dry weight)/Dry weight] x 100
Growth of corn seedling was analyzed by measuring fresh weight and dry weight of seedling shoot and
root. Leaf greenness that was indicated by SPAD chlorophyll meter reading (SCMR) was determined in the most
fully expanded top leaf using a SPAD-502 chlorophyll meter (Spectrum Technologies, USA). Two pieces of
2 x 2 cm2 leaf blade were cut at mid-leaf section. Leaf samples were immediately weighted for fresh weight (FW)
measurement. Then, they were submersed in deionized water for 8 h and weighed. Leaf turgid weight (TW) was
recorded. Subsequently, leaf samples were oven-dried at 70°C for 48 h and weighed for dry weight (DW)
measurement. Relative water content (RWC) was calculated using the following formula:

RWC (%) = (FW-DW) / (TW-DW) x 100
Electrolyte leakage, a parameter indicating membrane injury, was investigated in leaf tissue using the
method of Filek et al. (2012). Malondialdehyde (MDA), one of the products from lipid peroxidation, was
determined using thiobarbituric acid reactive substance (TBARS) assay according to the modified method of Heath
and Packer (1968).
Thermal image acquisition and image analysis
Thermal images of control and stressed plants were taken by using FLIR C2 thermal camera (FLIR
Systems, Inc., U.S.A.). Wet and dry cotton sheets were used as wet and dry artificial references, respectively.
Canopy temperature (Tcanopy), wet reference temperature (Twet) and dry reference temperature (Tdry) were analyzed
using FLIR Tools software. Crop water stress index (CWSI) and index of stomatal conductance (IG) were
calculated by the following equations (Costa et al., 2013):

CWSI = (Tcanopy – Twet) / (Tdry – Twet)
IG = (Tdry – Tcanopy) / (Canopy – Twet)

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Statistical analysis
The data were subjected to analysis of variance (ANOVA) and treatment means were compared by

Duncan's New Multiple Range Test (DMRT) at p<0.05 using SPSS software.

Results

Soil Moisture Content 70 During 7 days of water deficit treatment, the soil moisture contents in
(%) Control Stress stress group were significantly dropped to 16.96  1.98% and 18.46 
4.94% in Nakhon Sawan 5 and Samlee Isan corn varieties, respectively
60 (Figure 1). This result indicated that seedlings in stress group were
exposed to water deficit condition.
50

40

30 *

20 *

10

0 Samlee Isan
Nakhon Sawan 5

Figure 1 : Soil moisture content in control and drought groups of Nakhon Sawan 5 and Samlee Isan corn varieties. Vertical bar indicates

 SD. *Significant difference from control group in the same variety at p<0.05.

6 0.6 Water deficit caused significant reduction in
A B shoot fresh weight of corn seedlings in both
varieties (Figure 2A); however, significant
5 0.5 change of shoot dry weight due to drought was
Shoot fresh weight (g) 0.4Shoot dry weight (g) not observed (Figure 2A). In Nakhon Sawan 5
4 * 0.3 variety, root fresh weight and dry weight were
3* 0.2 significantly decreased under drought condition,
0.1 but these changes were not recorded in Samlee
2 Isan (Figure 2C and 2D).
0
1 Nakhon Sawan 5 Samlee Isan

0 D 0.4
Nakhon Sawan 5 Samlee Isan
0.3
C 1.6 Root fresh weight (g) *Root dry weight (g)
1.4 *
1.2 0.2
1
0.8 0.1
0.6
0.4 0
0.2 Nakhon Sawan 5 Samlee Isan
0
Nakhon Sawan 5 Samlee Isan

Figure 2 : Shoot fresh weight (A), shoot dry weight (B), root fresh weight (C), and root dry weight (D) of Nakhon Sawan 5 and Samlee Isan

corn seedlings grown under control and drought stress conditions. Vertical bar indicates  SD. *Significant difference from

control group in the same variety at p<0.05

RWC of seedlings in both varieties was significantly affected by drought. Under drought stress condition,

RWC of Nakhon Sawan 5 and Samlee Isan seedlings was significantly lower than those in well-watered condition
*

(Figure 3A). The effects of drought stress on leaf greenness and MDA content were differed between Nakhon

RWC (%) A120 Control Stress B 25 ** Sawan 5 and Samlee Isan seedlings. Under drought
100 ** 20 stress condition, significant changes in SCMR and
MDA content were observed only in Nakhon Sawan
80 15 5 seedling (Figure 3B and 3D). In this study, both
SCMR corn varieties did not show the significant increment
60 10 of electrolyte leakage in the drought group (Figure
3C).
40 5

20

0 0
Nakhon Sawan 5 Samlee Isan
Nakhon Sawan 5 Samlee Isan

14 60 **
C 12 D 50
Electrolyte MDA
Leakage (%) 10 (nmol/gFW) 40
8
6 30
4
2 20
0
10

0

Nakhon Sawan 5 Samlee Isan Nakhon Sawan 5 Samlee Isan

Figure 3 : Relative water content (A), SPAD chlorophyll meter reading (B), electrolyte leakage (C), and malondialdehyde content (D) of
Nakhon Sawan 5 and Samlee Isan corn seedlings grown under control and drought stress conditions. Vertical bar indicates  SD.
*Significant difference from control group in the same variety at p<0.05

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Thermal camera clearly detected the difference of canopy temperature between well-watered and drought
stressed corn seedlings (Figure 4A and 4B). The canopy temperature and CWSI of the drought group were
significantly higher than the control group in both varieties. In contrast, control group showed significantly higher
IG than the stress group (Figure 4B-4D). Seedlings of Nakhon Sawan 5 and Samlee Isan showed similar change in
the canopy temperature when they were subjected to drought. Therefore, this study could not observe their
temperature differences using thermography.

T canopy (°C)B 30.5 Control Stress
30.0 **
29.5 Nakhon Sawan 5 Samlee Isan
29.0
28.5
28.0
27.5
27.0
26.5

C 1.00 * * D 3.50
0.90
0.80 3.00
0.70
0.60 2.50
CWSI
0.50 IG2.00

0.40 1.50
0.30
0.20 1.00
0.10 0.50 ** **

0.00 0.00

Nakhon Sawan 5 Samlee Isan Nakhon Sawan 5 Samlee Isan

Figure 4 : Thermal image (A), canopy temperature (B), crop water stress index (C) and index of stomatal conductance (D) in control and

drought groups of Nakhon Sawan 5 and Samlee Isan corn varieties. Vertical bar indicates ± SD.

*Significant difference from control group in the same variety at p<0.05.

Conclusion
Drought condition due to soil water deficit had adverse effects on shoot fresh weight and water status in

V3 seedings of Nakhon Sawan 5 and Samlee Isan corn varieties. Under drought condition, only Nakhon Sawan 5
seedling showed significant changes in root weight, leaf greenness and lipid peroxidation. These results imply that
Samlee Isan slightly tolerates to drought than Nakhon Sawan 5 at V3 seedling stage.

This study shows the potential of thermography for studying crop plants under drought condition.
Thermal imaging clearly provides data to discriminate well-watered and drought stressed seedlings. This
technique can be used as a non-destructive approach to explore plant-environment interactions. However, the
difference of temperature change between corn genotypes due to drought could not observed in this study.
Therefore, the possible approaches to overcome this limitation will be study in the future.

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
Pradawet C, Khongdee N, Pansak W, Spreer W, Hilger T, Cadisch G. Thermal imaging for assessment of maize
water stress and yield prediction under drought conditions. Journal of Agronomy and Crop Science 2022;00:
1-15.

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Title: Repellent activity of Cannabis on Tenebrio molitor OB1_02_05
larvae and bioinformatics of defense mechanism
Field: related abiotic stress tolerance gene
Author: Biology and biodiversity

Pusanisa Sawangpakdee

School: Jukkraphob Khatsri
Advisor: Demonstration School University of Phayao

Dr. rer.nat Chatchawal Wongchai (Division Biology, School of Science, University of Phayao,

Phayao 56000, Thailand)

Abstract:
T. molitor or Mealworm is an invertebrate with a special antenna function as a chemoreceptor In vitro

culture of Cannabis was used to study the feeding of T. molitor. Apical buds and nodes of Cannabis were
selected and sterilized for micropropagation and callus induction. During plant development, after two weeks
move to ½ MS + 2,4-D medium. The repellent experiment starts with a specific box designed into divided 2
sections for feeding food with Cannabis and without Cannabis mixed. We observed the movement of T. molitor
at 5, 10, 20, 30, 40, 50, 60, 120, 160, 180, 240, and 300 minutes, respectively. 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 in feeding food. In conclusion, the
experiment with Cannabis repellent activity during feeding time may need analyzing metabolites with HPLC
and an abiotic stress tolerance approach is necessary for further testing.

:Keywords Abiotic stress; Cannabis; Cannabis defensive genes; Repellent activity; Tenebrio molitor

Introduction

Cannabis genus plants are widely known for the medical properties of hemp. The plants are widely
distributed in central, eastern, and southern Asia. There are 3 recognized species: Cannabis indica, Cannabis
sativa, and Cannabis ruderails. The beginnings of this plant in medicine are around 2800 BC when it was used
to cure a variety of health problems and was listed in the pharmacopeia of Emperor Shen Nung. Nowadays,
Cannabis is widely used in medicine. The propagation of Cannabis using the tissue culture method is better in
the study because the product of Cannabis callus is purified and grows in a short time compared to the normal
cultivation. The main reason for using Cannabis is that it contains many secondary metabolites, terpenes,
steroids, and flavonoids. (1) The most important secondary metabolites are Cannabidiol (CBD) and

For internal use in the 12th SCiUS Forum only. Not for citation.

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12th SCiUS Forum

Tetrahydrocannabinol (THC). These metabolites are used in many medicinal products, mostly found in the
female plant's sugar leaves, fan leaves, and flower buds. Not only do these metabolites act as medicinal agents,
but some metabolites in Cannabis also serve as defense mechanisms to protect Cannabis plants from herbivores
and pests. Cannabis defense mechanisms include the combination of Cannabinoids and terpenes,
Cannabigerolic acid (CBGA), and Tetrahydrocannabinolic acid (THCA), which act as toxic to insects for the
defense mechanism in the volatile oil. (4) However, as this mechanism of Cannabis could deter insects, there
is a concern in most insects as some Cannabinoid receptors are absent in insects, e.g., T. molitor has
chemoreceptors to recognize different chemical substances, but there is concern about cannabinoid recognition
in the defense mechanism. (2), (3) To discover the insect repellent effect and insect-plant interaction in
Cannabis, our project presents the study of insect repellent effect and insect-plant interaction in Cannabis
callus using T. molitor and the study of Cannabis propagation in tissue culture.

Methodology
Cannabis culturing
1.1. Collect Cannabis segments from indoor garden

1.2. Clean, cut for apical buds and nodes, then bleach the segments as steps
follow
1.2.1. Sterilize the segments with dish soap, rub it 2-3 times to remove the
dirt
1.2.2. Wash the segments in running water for 45 minutes
1.2.3. Puts segment in container pour 40 ℃ water, stir for 1 minute
1.2.4. Add Clorox in 10% v/v in the container and stir for 15 minutes, pour
out the liquid and wash in clean water
1.2.5. Add Clorox in 5% v/v in the container and stir for 10 minutes, pour
out the liquid, and wash in clean water.
1.3. Take the segments into the tissue culture chamber and put the
segments in ½ MS tissue medium
1.4. Wait until the tissues grow for 2 months, then take out the tissues and
put them into ½ MS + 2,4-D medium + sugar + agar and water this became the liquid medium
1.5. put it in the shaker of 230 r/m until calluses were induced

Repellent activity experiment
2.1. Craft a test box, this box divided into 2 sides of with Cannabis side and
without Cannabis side
2.2. Put 1.1 g. of Cannabis calluses in filter papers to each hole in the box for
the Cannabis side.
2.3. Mixed 1.4 g. of chicken feed food on each side.

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