Khóa luận tốt nghiệp Công nghệ sinh học: Evaluation of the effect of some growth regulators on callogenesis and somatic embryogenesis of Paramignya trimera (Oliv.) Guillaum

Over the course of the study fromMarch to December 2023, the aim was to identify the optimal type and concentration ofPGRs for effective callus formation and embryo development in this p

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY- HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATION OF THE EFFECT OF SOME GROWTH REGULATORS ON CALLOGENESIS AND SOMATIC EMBRYOGENESIS OF Paramignya trimera (Oliv.) Guillaum

Major : BIOTECHNOLOGYName : TRAN NGUYEN HAI THOStudent’s ID : 19126171

School year : 2019-2023

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY- HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

UNDERGRADUATE THESIS

EVALUATION OF THE EFFECT OF SOME GROWTH REGULATORS ON CALLOGENESIS AND SOMATIC EMBRYOGENESIS OF Paramignya trimera (Oliv.) Guillaum

Advisors Student

Nguyen Vu Phong, A/Prof Ph.D Tran Nguyen Hai Tho

Ha Thi Truc Mai, M.Sc

Thu Duc City, 03/2024

I would like to express my special thanks and gratitude to my supervisor, Nguyen

Vu Phong, A/Prof Ph.D., and Ha Thi Truc Mai, M.Sc., who allowed me to do this thesis,which also helped me in doing a lot of experiments and thesis They provided me with

the tools that I needed to choose the right direction and complete my thesis I am really

thankful to them

Secondly, I would like to thank the teachers of the Faculty of Biological Sciences

at Nong Lam University, my friends from the BIP teams, and especially Ms DangHuynh Thuy Vy and Ms Cao Thi Cam Huong for their help, suggestions, andencouragement during the time I was working on the thesis

Besides, I would like to thank my parents for their wise counsel and sympatheticears You are always there for me

Finally, I would like to sincerely thank everyone for the best things they havegiven me You are always there for me

DECLARATION OF AUTHORSHIP

My name is Tran Nguyen Hai Tho, ID: 19126171, Class: DH19SHB of

Biotechnology major at Nong Lam University in Ho Chi Minh City I declare: This is agraduation thesis written directly by myself; the data and information in the study are

completely honest and objective I am fully responsible to the board for these

The study conducted at Nong Lam University’s Laboratory of Plant Integrated

Biology focused on the influence of plant growth regulators (PGRs) on callogenesis and

somatic embryogenesis in Paramignya trimera Over the course of the study fromMarch to December 2023, the aim was to identify the optimal type and concentration ofPGRs for effective callus formation and embryo development in this plant species.Initial experiments involved sterilizing seed explants with Javel solution and using

different types of explants such as zygotic embryos, cotyledons, hypocotyls, andradicles These explants were cultured on Woody Plant Medium (WPM) supplementedwith various combinations of NAA and BA for callus induction, and later, for somatic

embryo induction, explants were transferred to WPM with TDZ The results highlightedthat sterilization with 20% Javel solution for 20 minutes provided a 70% survival rateand a 44.79% germination rate for seeds Callus induction was most successful with

zygotic embryo and cotyledon explants, showing an 86.61% and 93.75% formation rate,respectively, when cultured on WPM with 2.0 mg/l NAA and 0.2 mg/l BA Thesecalluses were nodular, firm, and capable of embryo generation Additionally, the studynoted the formation of adventitious roots in some explants.For somatic embryo

development, the highest success rate (88.89%) was achieved with young leaf calluscultured on WPM containing 0.07 mg/L TDZ However, higher concentrations of TDZ(0.3 mg/L) resulted in a higher rate of abnormal embryo development (83.33%) This

research provides valuable insights into the conditions necessary for effective somaticembryogenesis and callogenesis in Paramignya trimera, potentially contributing to theconservation and propagation efforts for this species

Keywords: Callogenesis, Paramignya trimera, plant growth regulators, somaticembryogenesis, tissue culture

TÓM TẮT

Nghiên cứu được thực hiện tại Phòng thí nghiệm Sinh học tổng hợp thực vật của Đại học

Nông Lâm tập trung vào ảnh hưởng của các chất điều hòa sinh trưởng thực vật (PGR)

đến quá trình hình thành mô sẹo và tạo phôi soma ở cây Paramignya trimera Trong suốt quá trình nghiên cứu từ tháng 3 đến tháng 12 năm 2023, mục đích là xác định loại và nồng độ PGR tối ưu để hình thành mô sẹo và phát triển phôi hiệu quả ở cây Paramignya trimera Các thí nghiệm ban đầu liên quan đến việc khử trùng mẫu hạt giống bằng dung dịch Javel và sử dụng các loại mẫu cấy khác nhau như phôi hợp tử, lá mam, trụ dưới lá

mam và rễ mầm Những mẫu này được nuôi cấy trên Woody Plant Medium (WPM) được

bổ sung nhiều sự kết hợp khác nhau của NAA va BA dé tạo mô sẹo, và sau đó dé tao phôi soma, các mẫu được chuyền sang WPM bổ sung TDZ Kết quả nhân mạnh rằng việc khử trùng bằng dung địch Javel 20% trong 20 phút mang lại tỷ lệ sống sạch là 70%

và tỷ lệ nảy mầm của hạt là 44,79% Việc tạo mô sẹo thành công nhất với các mẫu phôi hợp tử và lá mam cho thấy tỷ lệ hình thành lần lượt là 86,61% và 93,75% khi nuôi cấy trên WPM với 2,0 mg/l NAA va 0,2 mg/l BA Những mô seo này có dang nốt san, chắc

và có kha năng tạo phôi Ngoài ra, nghiên cứu còn ghi nhận sự hình thành rễ bat định ở

một số mẫu mô sẹo Ở nghiên cứu phôi soma, tỷ lệ phát triển phôi hình cầu cao nhất (88,89%) đạt được với mô sẹo lá non được nuôi cay trên WPM chứa 0,07 mg/L TDZ Tuy nhiên, nồng độ TDZ cao hơn (0,3 mg/L) dan đến tỷ lệ phôi phát triển bat thường cao hơn (83,33%) Nghiên cứu này cung cấp những hiểu biết có giá trị về các điều kiện cần thiết cho quá trình tạo phôi soma và hình thành mô sẹo hiệu quả ở Paramignya Trimera,

có khả năng góp phần vào nỗ lực bảo tồn và nhân giống loài này.

Từ khóa: Hình thành mô sẹo, Paramignya Trimera, chất điều hòa sinh trưởng thực vật,

tạo phôi soma, nuôi cây mô

1V

Trang

ACKNOWLEDGEMENT - - SH HH HH ng 1

DECLARATION OF AUTHORSHIP - - 5 52.22 re iiASTER SPR OP ors ese EEE sree ees se en see ee 11

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CHAPTER 1 INTRODUCTION sáng 4216 16660153638481801610350395015500440001484158 1

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2.4.4 Application of somatic embryogenesis ccceeceeecceeeceeteceeseceeceeeeceseeeeeeaees 13

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2.6.1 Studies on the callus formation of P frie@frd 2222 << << <+++++zzzzzzxese+ 142.6.2 Studies on the somatic embryogenesis formation from the callus of P trimera.16

CHAPTER 3 MATERIALS AND METHODS - -+5-<S2<<2<+zcserrrreerrrrree 173.1 Places and duration for conducting eXperiImenifS -++++£++s<c++ecezs+ 173.2 Equipment and tool ce lý

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2;3» L; BlØlop16al:Trial6TiaÌSeesesessasaeseseeibsbeisusssllikl3eixessssereitEoksSilSÐsuEtieEgotssedficgptsrfehgiEEk 17B28 MS GIT -zszz>szxszszss>ssatbsisfc4atEio0i482kiogadgb3ig:BmSGiGE2GGSSE0/5.4000:3đ2đãiu-3G/E.Gg880đ01cb0đi019/98/09930E0/SS0U2IERHX8 17

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3.4.1 Sterilize the Ð trimera seed explant ce ceccecee cece eee eeceeeescesceeeeseesenseneenes 18

3.4.2 Investigate the effects of explant type and concentration of NAA combined with

BA Oi CALS INCU CHOP seeeaseseesikieinbdeiiiaiadabaduaslickikeoagsulokeagsualclsginbudsiuodideskung ii grcgudzcianrbk 19

3.4.3 Examination of somatic embryo 1nducf1oni - 52552 +52 =+sc+sc+sczsccxe 203.5 Observe anatomical morphOÌOV - + + + +*++*£+*£+E+£zk£zkrrkrrrrrerrrrerkerkee 213/6 ata) PROCESSING cs ong ti660185G251G0050G30IA SLãNt8QGfStR0bsifSgGRdlcSgtrkatiBsgstditS5iGaiv3800%/G0038G76/62 5458 21

CHAPTER 4 RESULTS AND DISCUSSION sissssssssesssssassssssonsssrsssseacxvoeanssnassnsvezneesenes 224.1 Efficacy of sterilizing P trimera seed explants with Javel Solution DB.4.2 Influence of kinds of explants and NAA, BA concentrations on callus formation 234.3 Examination of somatic embryo induction - 55-5555 *S+<£++c£+ezzcrseeeres 304.3.1 Examination of somatic embryo induction from zygotic embryo 30

4.3.2 Investigating the effects of TDZ on somatic embryo Induction 31

AAs DISSCUSSION se eeeevaxediinllnesgiiiBianisrauvEnasuioh E0dkishEnllloitsoslkueosiuooosedlseeokdleorossssllrodninllreebie 374.4.1 Efficacy of sterilizing Ð trimera seed explants with Javel Solution 374.4.2 Influence of explant types and NAA, BA concentrations on callus formation 374.4.3 Examination of somatic embryo induction -+5-+<+<++s<<+scescc+ 38CHAPTER 5 CONCLUSIONS AND RECOMMENDATION 393,1 CONCLUSIONS 0ic0cs.s-scoresnascasosssconsnsiionssenetcnsastubnnanasebasnanostessudastentenishentnantinnsnntsasanennen 39

5.2, RECOMMMENAIAUONS sercesercnsacomseransennnn T000 16050015130 54358489188010000104060001801005903038350881 39

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LIST OF ABBREVIATIONS

BA Benzyl andenine

PIB Integrative Plant Biology

ME Malt extract

MS Murashige and Skoog, 1962

NAA a-Naphathalene acetic acid

PGRs Plant growth regulators

TDZ Thidiazuron

WPM Woody plant medium, 1980

LIST OE TABLES

TrangTable 2 1 Some disinfectants are used in plant tissue cuÏture -«++ hTable 3 1 Time and rate of Javel solution used in treatmens - - 18

Table 3 2 The rate of callus induction due to the effect of NAA combined with BA 19

Table 4 1 Efficacy of sterilizing xao tam phan seed explants with Javel Solution 22Table 4 2 Influence of types of explants and NAA, BA concentrations on callusDTV H[GULOTÏL <i-c<62621862101E066n20bit0izgi2gi2iSsiilielidccikcgiokgRuiocsjgkrbidgaosptkcloiatiisaoosizlistioekilietktegtgilssBisskgaexidbrigssiobsbi 24Table 4 3 Callus morphology after 4 weeks and 8 weeks of culture - 26Table 4 3 (Next) Callus morphology after 4 weeks and 8 weeks of culture 2]Table 4 4 Effect of explant type and concentration of NAA and BA on adventitious

root formation from callus after 4, 6, and 8 weeks of cuÌfure - -s=+-<s 28Table 4 5 Percentage of explants forming globular-shaped embryos after 4 and 8 weeks

OR GIEUEGGBHBSEHIGGIEGHRISILS.SSHIGGGBDNHSGHIENESRSSRIEBGGS3880030G03803GGGBR/013H888 52Table 4 6 Percentage of explants with abnormal embryo formation after 4 and 8 weeks0i eee ecccceeeeecccccccceceeeceececsessseececececcceceseesssssssceceeccececersrsssssssececeeeeeeeeesetenees 34

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Figure 4 4 Adventitious roots formed from callus in all types of explants on WPM + 2mg/L NAA + 0.2 mg/L BA after 8 weeks of culture - - 29Figure 4 5 Organogenesis from zygotic eMbryo6 : :ecceeeeeeee eee eeeeeeeeeeeeeeeenees 31

Figure 4 6 Embryos form from the callus after 8 weeks of culture - 32Figure 4 7 Somatic embryos were formed after 18 weeks of culture in treatment S1(WPM + 0.07 mg/L TDZ), .ceceecescesceseeseeseeseeseecesceecesceseenecaecaeeaceaeeseeseeaeeceececeeeeseeaeens 33

Figure 4 8 Somatic embryos were formed after 10 weeks of culture on treatment S4WPM + 0.7 mg/L TDZ The circular cavity positions are the positions where the explant

is under different objective stereoscopic TmICTOSCOD€S - - 5 55552552522 c+<c+<s+ 35Figure 4 9 Embryo explants developed buds after 22 weeks of culture in treatment S1WPM + 0.07 7000215 36

Figure 4 10 Embryo explants were formed after 22 weeks of culture in treatment S1

MW EM + 0:07 ie Me, TID bác cocz sis econ te vai tastrvr i nice ch a wo do bbb aa tet 36

CHAPTER 1 INTRODUCTION

1.1 Background information

P trimera is a plant belonging to the genus Paramignya, family Rutaceae, with

the scientific name Paramignya trimera (Oliv.) Guillaum In oriental medicine, it is often

called "Don Diep Dang Thich” In the human world, it is called the "re moi" plant or the

"than xa" plant Ð trimera has high medicinal value and is an important medicine intraditional medicine because it contains groups of rare substances such as coumarin

triterpenoids, flavonoids, saponins, alkaloids, and glycoside derivatives These groups ofsubstances have been shown to have anti-inflammatory effects, treat liver disease anddiabetes, and help repel many cancer cell lines It is because of the excellent effect of P.trimera in the treatment and reverse of cancer that, in recent years, the price of Ð trimera

has been inflated Therefore, this medicinal plant is being hunted by people and exploited

to the point of exhaustion to make medicine, so its distribution range is increasingly

narrowed and in danger of extinction in the wild if no measures are taken for effectiveconservation Therefore, in the current market of P trimera medicinal herbs, it is always

in a state of real and fake; there are many business addresses of fake Ð trimera, and P

trimera of unknown origin mixed with fake medicinal herbs has very low or notherapeutic value, seriously affecting users' confidence in the effects of P trimera

In order to maintain, preserve, and develop the seed source, as well as create a

large number of plants to serve the pharmaceutical industry, people have propagated by

traditional methods such as cuttings, grafting, and branches However, these methodsstill face many difficulties, such as complex growth characteristics, a slow growth rate,difficult rooting from cuttings, mortality due to stem rot from 2 — 11% (Tran Nam Thangand Nguyen Thi Thu Ha, 2016), being difficult to tame when brought from the mountain,

and requiring a large number of seedlings to propagate To solve this problem, the

method of in vitro propagation of callus, somatic embryogenesis, and regeneration of

complete plants from somatic embryos is an outstanding method that has many

meanings in terms of both conservation and science and economics The advantage ofthis method is the rapid multiplication of plant varieties and the generation of geneticallyhomogenous crops, which ensure the production of a variety of plants of uniform origin

and good quality This is also a method commonly used in conservation, either in the

1

production of medicinal biomass or secondary compounds of rare medicinal speciessuch as Panax vietnamensis, Panax vietnamensis var fuscidiscus, Schefflera octophylla

(Lour.) Harms, Dioscorea collettii Hook f., Taxus wallichiana, Eurycoma longifolia Sofar, a few studies aimed at rapid in vitro multiplication of P trimera have been published,

but the results are limited (Tran Trung Hieu et al., 2017; Ngo Thi Xuyen et al., 2018)

The project "Evaluation of the effect of some growth regulators on callogenesisand somatic embryogenesis of Paramignya trimera (Oliv.) Guillaum” was carried out

to establish the process of somatic embryogenesis in P trimera for further investigation

Evaluation of PRGs on somatic embryogenesis 1n P trimera

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction of Paramignya trimera (Oliv.) Guillaum

2.1.1 Classification

P trimera is also known as xao

tam phan, don diep dang thich, re moi

plant or than xa plant

Scientific name: Paramignya

trimera (Oliv.) Guillaum

Synonym names: Severinia

trimera (Oliv) Swingle, Atalantian

Genus :Paramignya Figure 2 1 Paramignya trimera (Oliv.)

; Guillaum plant in Mr Van Khon

Species :Paramignya trimera Nguyen’s garden Trang Bom — Dong Nai.

There is currently disagreement among plant taxonomists regarding the

taxonomic status of only Paramigaya According to data from many research

organizations and botanists at Kew Royal Botanical Garden (UK) and Missouri

Botanical Garden (USA), as of 2013, there were 30 species believed to belong to this

genus (Nguyen Thi Dieu Thuan, 2015); according to records in the Plant List Database

(www.theplantlist.org, 2020), there are 30 plant records matching the query

"Paramignya" in different geographical locations and environment live differently (Phi

et al., 2020)

2.1.2 Morphological characteristic

P trimera is described as a small plant, climbing bush, soft and tough bark, 3—5

m tall, about 10 cm in diameter There are hard thorns on the trunk and branches, andthe bark is yellow-brown Leaves are simple, thick, and grow in clusters or in 3 clusters;

eaf edges are curved down, narrow, 8 — 2 cm long, and 1 - 3 cm wide The top of the leaf

is darker green than the bottom of the leaf The leaf blade is about the same size The

size gradually gets smaller from the stem to the tip The side veins of the leaves have

about 8 — 10 pairs, and the petiole is about 2 — 3 mm long The flowers grow in clusters

in the leaf axils, the flower stalk is about 2mm long; the white flower includes 3 sepals,

3 petals about 4 mm long, and 4 — 3 separate stamens The fruit is round, usually yellow,

and about 15 - 20 mm in diameter The hard roots are a dark yellow on the outside and

a light yellow on the inside Every part of the plant has essential oils, mainly in the roots.The essential oil has a light scent like ginseng When chewed, the roots have a bitter andthen sweet taste on the tip of the tongue, a bit like ginseng The plant has a fruitingseason from May to October, often growing on rocky slopes in arid places

2.1.3 Distribution

The genus Paramignya belongs to the Rutaceae family It is widely distributed in

southern Vietnam, the southern Philippines, Thailand, Malaysia, Java, Indonesia,Australia, and the dry and humid regions of Sri Lanka (Ninh The Son, 2018) InVietnam, there are 7 species of the Paramignya genus, including P armata Oliv., etc.andamanica King, P griffithii Hook F., P hispida Pierre ex Guillaum, P monophyllaWight, P pefelofii Guillaum, P scandens (Griff.) Craib, and P trimera (Oliv.) Guillaum(Pham Hoang Ho, 1999) Among them is the species Paramignya trimera (Oliv.)Guillaum, locally known as “xao tam phan” (Tang ef a/., 2015)

In Khanh Hoa, P trimera has many places, such as Hon Heo Mountain (NinhVan commune), Da Ban, Le Cam, Ninh Giang (Ninh Hoa town), Phuoc Dong (NhaTrang city), Khanh Vinh, Cam Binh and Cam Phuoc Dong (Cam Ranh City) This is aplant that grows wild in nature and is very small in number Currently, the tree is not onthe list of rare plants that need to be protected It only grows on bare land, not inprotective forests That makes it difficult to prevent people from exploiting it (Tran Nam

2.1.4 The medicinal value of P trimera

In traditional medicine, people use species in the genus Paramignya to treatcancer, hepatitis, diabetes, and nasal infections Many of their nutritional activities canextract a variety of chemical components, such as coumarin, triterpenes, alkaloids, andglycoside derivatives These components have anti-tumor, anti-oxidant, and anti-inflammatory effects (Ninh The Son, 2018) In addition, they are resistant to rot diseases

and plant-pathogenic fungi (Krueger and Navarro, 2007) They are typical species for

this genus, and they also show many interesting biological activities such as antioxidant,anti-cancer, anti-inflammatory, and alpha-glucosidase inhibitory activities (Nguyen ThiKieu Oanh ef al., 2022)

According to the Vietnam National Institute of Medicinal Materials (NIMM), P.trimera is active against many types of cancer cells, such as liver, breast, colon, ovarian,and cervical cancer (Nguyen Thi Dieu Thuan, 2015) Researchers have also proven that

a methanol extract from the leaves and roots of P trimera 1s toxic to 12 cancer cell lines.These cell lines include MiaPaCa2 (pancreas), HT29 (colon), A2780 (ovary), H460(lung), A431 (skin), Dul45 (prostate), BE2-C (neuroblastoma), Michigan Cancer

Foundation (MCF)-7 (breast), MCF-10A (normal breast), U87, SJ-G2, and SMA

(glioblastoma) (Nguyen ef a/., 2017) Besides, P trimera bark essential oil has strongcytotoxic activity against HepG2 liver cancer cells It works in a dose-dependentmanner, with a maximum inhibitory concentration of 21 g/mL (Nguyen Thi Kieu Oanh

et al., 2022) Besides, P trimera roots also contain antioxidants such as phenolics,flavonoids, etc (Nguyen ef al., 2015) In rat experiments, an aqueous extract of P.trimera at a dose of 10 g/kg body weight (kgP)/day reduced serum AST and ALTenzyme concentrations, total cholesterol, and body mass The liver damage in mice waspartially limited when using Paracetamol 400 mg/kgP within 7 days (Do Thi Thao etal., 2015) Also in mice, P trimera fractions include P trimera methanol, P trimerahexane, P trimera ethyl acetate, P trimera butanol, and an active P trimera 6 (3,7-dimethyl-2,6-ooctadienyl) We screened 7-hydroxy-2H-1 benzopyran-2-one for toxicity

on 5 cancer cell lines: liver cancer (Hep G2), colon cancer (HTCI 16), breast cancer(MDA MB231), ovarian cancer (OVCAR 8), and uterine cancer (Hela) The resultsobtained showed that only 3 fragments (P trimera n-hhexane, P trimera methanol, andthe active ingredient P trimera) showed toxicity on all 5 cancer cell lines: the P trimera

n-hhexane fragment and the active ingredient P trimera exhibits moderate toxicity onliver cancer cell lines (IC 50 = 39.61 g/ml) and breast cancer cell lines (IC 50 = 18.4g/ml) The P trimera ethyl] acetate fraction only showed toxicity on breast cancer lines.Other fractions showed relatively weak cytotoxic effects on cancer cells (Nguyen MinhKhoi ef ạ., 2013).

According to Nguyen Manh Cuong ef a/ (2016), they isolated and determined thechemical structures of 10 compounds from P trimera, including six new compounds.From there, create an extract and evaluate the hepatoprotective effects of water extractand methanol on roots and stems using a model of hepatotoxicity with paracetamol

Research results show that P trimera root extract at a dose of 10 g/kg body weight/day

has a liver - protective effect This effect reduces AST and ALT levels and partially limitsliver damage caused by paracetamol in the BALB/c white mouse model Also, P trimeramethanol extract at a dose of 10 g/kg B in mice has a liver protection effect almost equal

to that of the reference control substance (silymarin at a dose of 50 mg/kg B) In anotherstudy, Nguyen Manh Cuong ef ai (2018) demonstrated that P trimera extract at aconcentration of 250 ug/ml has the ability to strongly inhibit the proliferation of human

breast cancer cells (MCF-7), effectively limiting the invasion of MCF-7 cells and causing

these cells to undergo programmed death

In addition, the methanol extract of P trimera roots also has stronger antioxidanteffects than extracts using other organic solvents, such as water, acetonitrile, ethylacetate, and hexane (Nguyen ef a/., 2015) The methanol extract of P trimera root also

helps effectively support the treatment of type 2 diabetes through its high a-glucosidase

enzyme inhibitory activity with an ICso = 36.6 ug/mL (Dang ef al., 2017) Methanolextract from the leaf powder can capture free radicals with an ICso = 6,434 mg/mL andresist the Gram-positive bacterial strain Staphylococcus aureus, with an antibacterialring about 12 mm in diameter (Ngo Thi Xuyen ef a/., 2018) Or for the essential oil fromthe bark of the P trimera plant, it also shows the strongest inhibitory activity againsttwo strains of Pseudomonas aeruginosa with a minimum inhibitory concentration(MIC) of 256 g/mL, two strains of Staphylococcus aureus, and two strains ofSalmonella sp with MIC values of 384, 480, and 512 ug/mL, respectively (Nguyen Thi

2.2 Safety of investment samples

Sterilize the tissue explant obtained from the field nursery, the tissue explant itselfcarries many microorganisms The result of this sterilization process is to provide a sterile

sample source or reduce the infection rate, this stage depends on the sterilization time,

kind of disinfectant and disinfectant concentration (Nguyen Quang Thach et al., 2004)

Materials used for plant tissue culture can be any part of the plant, including root

and stem segments, leaf parts (petiole and leaf - blade), embryonic structures such ascotyledons, epicotyl, hypocotyl, pollen grain, ovule, and underground storage organs

Depending on the purpose of culturing and the characteristics of the plant species, the

appropriate kind of explant will be determined (Vu Van Vu et al., 2009) However, the

basic principle is that the explant must contain living cells from young tissues with alarge proportion of rapidly dividing cells, easily forming a callus The original tree musthave good quality, a high yield, no signs of disease, and not be in dormancy Sterile

explants are obtained from seeds after surface sterilization, sowing seeds in sterile

conditions to grow into plants, and taking explants, which are taken from fresh plants

and treated by soaking in disinfectant solution (Pham Thanh Ho, 2005)

The current common disinfection method is to use chemicals that kill fungi and

bacteria, such as sodium hypochloride solution, detergent or ethanol 70° The ability ofdisinfectants to kill microorganisms depends on the concentration, time, and degree ofpenetration on the tissue explant surface Ethanol 70° can be used (time depends on the

kind of explant used) to increase disinfection efficiency and then treated in a disinfectantsolution For surface tissue explants covered with a layer of wax, a few drops of surfacetension-reducing agents such as tween 20, tween 80, and tepol should be added to thedisinfectant solution For disinfectant processing, they must be completely submerged in

the disinfectant solution Parts of the tissue explants that are blanched by the sterile agentmust be removed before placing the explant current in the medium To avoid the sterile

agent affecting the explant current directly, leave a covering layer when the tissue enters

the disinfectant solution Before placing the explant in the medium, we will cut off or peeloff this last layer (Vu Van Vu et al., 2009)

Table 2 1 Some disinfectants are used in plant tissue culture (Vu Van Vu et al., 2009)

Disinfectant solution Chemical formula Concentration (%) Time (mins)

Calcium hypochlorite Ca(Cl1O)2 5-15 10 - 30

Sodium hypochlorite NaClo 0.5 —2 10 - 30

Hydrogen peroxide H202 10-12 5-15

Mercuric (II) chlorite HgCh 0.1-1 2-10

Bromine solution HBr + HbrO 1-2 10 - 30

Disinfectants such as Ca (C1O)2 and NaC]O contain the element Cl When dissolved

in water, they produce strong oxidizing agents such as hypochlorite ions (OCI) and

hypochlorous acid (HCIO) These agents inactivate microorganisms The followingequations describe the reaction:

NaClO > Na*+ OCI

Ca (CIO) — 2Ca?* + 2OCI

HCIO © H* + OCT

In water, OCI and HCIO will convert to each other depending on the pH value

In water environments with low pH, the proportion of HCIO is high, and in high pHenvironments, the proportion of OCI ions is high Because HCIO is uncharged, it canpenetrate the cell walls and membranes of microorganisms, resulting in a betterdisinfecting effect compared to OCI Therefore, it is advised not to use disinfectantcompounds containing Cl when pH > 7.5 HCIO inactivates microorganisms through

oxidation, hydrolysis, and deamination reactions The reaction mechanism of HCIO

includes binding to proteins to form N-chloro compounds, binding to SH (sulfhydryl)radicals of proteins, and oxidizing a-amino acids to nitrile (R-C°N) and aldehyde (R-CHO) When penetrating cells, HCIO first causes physical damage to the cell walls andmembranes of microorganisms Inside the cytoplasm, HCIO acts on the mitochondria,destroying cytochrome enzymes that catalyze redox reactions, which play an important

role in cellular respiration The result of cytochrome destruction is a decrease in theamount of glucose and ATP inside the cytoplasm Besides, HCIO also disrupts

metabolism and protein synthesis The reaction of HCIO inside cells produces freeradicals OH, which have strong oxidizing properties and change purines

2.3 Callus culture

The purpose of in vitro propagation is to regenerate complete plants directly from

the original tissue explant Not only can plants be obtained quickly, but the plants arealso quite genetically identical However, often cultured tissue does not regenerate

plants Instead, it develops into a callus mass Primary callus cells help regenerate plants

to create uniformly regenerated plants Through the callus stage, virus-free individualscan also be obtained (Le Tran Binh, 1997)

2.3.1 Callus cells

Callus is an unorganized mass of cells, formed from highly differentiated tissuesand organs under special conditions (wounds, treatment plant growth regulators, etc.)(Nguyen Duc Luong and Le Thi Thuy Tien, 2006) Callus tissue is the starting material

for other important research such as cell and tissue differentiation, cell line selection,stem cell culture, single cell culture, somatic embryo culture, and the production ofbiologically active secondary compounds, etc However, there has been researchshowing that callus is not a mass of unorganized cells; instead, callus has a structuresimilar to the sprouts of the lateral roots and has gene expression very similar to the root

meristem (Atta et al., 2008)

Callus, when formed, can be spongy callus or hard callus In particular, thespongy callus contains many spongy cells with small nucleus, thin cytoplasm, and largevacuoles Hard callus tissue contains firm cells, large nuclei, dense cytoplasm, and small

vacuoles (Vu Van Vu et al., 2009) Depending on the purpose of culture, callus in generaland embryogenic callus in particular have specific shapes, textures, and characteristics

of constituent cells, especially pigmented callus (green, purple), which is always ofinterest to research by scientists because this type of tissue often contains higher levels

of secondary compounds than non-pigmented callus due This is due to profound andpositive changes resulting from photosynthesis and the extreme physiological andbiochemical states of materials (Mai Truong et al., 2013)

In callus induction, if the callus is continued to be maintained in a mediumcontaining auxin, it will proliferate rapidly, but if transferred to a medium with complete

nutrients without the presence of auxin, the proliferation will increase and the callus willslow down (Gautheret et al., 1967)

2.3.2 Stages of callus development

Callus in vitro belongs to one of the following three processes, thanks to auxin

The first is the dedifferentiation of parenchyma cells, more or less deep inside the organ:wood and pipeline, cortical parenchyma, or core The second is the division of stratumcorneum cells (phloem layer - wood) Most dicotyledons' stratified cells divide easilyunder auxin's influence Grasses and vines, in contrast, need exogenous auxin fordivision Finally, there is a disturbance of the initial meristems (buds or roots); thisprocess is preferentially applied in monocotyledons because these plants do not havetypical stratification like dicotyledons, and parenchymal cells find it difficult todifferentiate Callus formation is related to the physiological condition of the explant

culture, the use of auxin alone or in combination with cytokinin, and the nature andconcentration of auxin (Bui Trang Viet, 2000)

2.3.3 Factors affecting callus

2.3.3.1 Culture organ

The age of the explant is an important factor that affects the ability to

photosynthesize and callus induction Callus is a cluster of undifferentiated cells withstrong division properties, often created due to disturbances in the organogenesis

process Therefore, young plants or young stem pieces of mature plants easily producecallus under tissue culture conditions (Bui Trang Viet, 2000) Hypocotyl, radicle, and

cotyledons are commonly used in callus research due to their rapid growth ability and

in vitro explant sources

to the physiological condition of the explant tissue and to the use of auxin alone or in

combination with cytokinin In most cases, callus induction often requires the

combination of auxin and cytokinin, of which auxin can have opposing effects,stimulating or hindering depending on the concentration Optimal auxin concentrations

will activate some enzymes, leading to increased DNA and RNA content to help thedivision of callus cells (Bui Trang Viet, 2000) Too high auxin concentrations will induce

ethylene biosynthesis; the accumulation of even a small amount of ethylene in theculture vessel can inhibit the growth and development of many plant cultures

(Machakova et al., 2008) However, the presence of cytokinin in the culture medium isessential Cytokinin stimulates cell division in the presence of auxin Cytokinin acts on

both steps of cell division: nuclear division and cell division (Bui Trang Viet, 2000)

Cytokinin combines with auxin to stimulate cell division, promote mRNA transcription,and stimulate the synthesis of specific proteins and enzymes in specific tissues to create

calluss (Scot, 1972) Endogenous plant growth regulators are derived from intermediateproducts of the glycolytic cycle and affect callusing ability when combined with

exogenous growth regulators (Hopkins, 1995)

2.3.3.3 Plant tissue culture medium

The nutritional medium will help provide all the mineral elements necessary for

cell or tissue growth In experiments to create callus in citrus trees, researchers often use

MS medium (Murashige and Skoog, 1962); and WPM medium (Woody Plant Medium)(Gmitter et al., 1991; Phi et al., 2017; Tran Trung Hieu et a/., 2017)

2.3.3.4 Other factor

Light slows down callus induction because light easily decomposes the auxin

group Therefore, in most cases, people often incubate callus tissue in a dark room Lightcan also stimulate the production of phenolic compounds These phenolic compoundscan bind to enzymes involved in cell growth and prevent their activity (Nguyen DucLuong and Le Thi Thuy Tien, 2006)

The seed’s physiological state affects germination To germinate, a seed mustaccumulate the necessary substances and be fully ripe, not exceeding its lifespan Seed

maturity is not simultaneous with fruit maturity, but there are also some types of seeds

that ripen very early and have the ability to germinate right in the fruit (legumes,

jackfruit, etc.) There are cases where the seeds are ripe but encounter unfavorableconditions (the shell is too hard or the embryo has many germination inhibitors, etc.),

and then the seeds will enter a state of dormancy On the contrary, if the seed is too old,

it will lose its ability to germinate The longevity of the boundary particles varies but isoften related to the chemical nature of the storage material Dormancy in seeds may be

10

caused by endogenous inhibitors, light requirements, low temperatures, dry storagerequirements, and embryo immaturity (Yeung et al., 1981) Seed dormancy may be

localized in the seedcoat, the endosperm, or both

Technical manipulation is important For example, placing leaves face down on

the medium surface helps the leaves absorb and metabolize nutrients At the same time,

the lower surface of the leaf is in direct contact with the air in the culture vessel,

facilitating gas exchange and promoting nutrient transport When placed face up, theabsorption and transport of nutrients occur by directing the upper surface of the leafdown towards the environment, which causes bending of the leaf sample (Duong TanNhut et al., 2012)

2.4 Somatic embryos culture

2.4.1 Somatic embryos

Somatic embryo is an indeterminate bipolar structure consisting of a shoot poleand a root pole, which, under appropriate conditions, can develop into a fully functionalorganism (Duong Tan Nhut, 2010)

2.4.2 Somatic embryogenesis in plant

Embryogenesis from somatic cells is a process by which one or more somatic

cells can divide in a certain order to create an embryo This process is similar or nearlyidentical to embryogenesis from a zygote under experimental conditions (including theuse of PGRs) (Bui Trang Viet, 1994)

There are two pathways of somatic embryogenesis: direct somatic embryogenesisand indirect somatic embryogenesis In direct somatic embryogenesis, somatic embryos

are initiated directly from the explant without an intermediate callus stage Indirectsomatic embryogenesis involves intermediate callus stages and clumps, pre-embryonic

(PEM), before somatic embryo formation (Guan ef al., 2016) In indirectembryogenesis, cells need reprogramming Mainly, they need to go back to an

undifferentiated state to become capable of embryogenesis (Fracisco et al., 2006)

2.4.3 Factors affecting somatic embryos

2.4.3.1 Explant source

Regarding explant source, ovules explants usually give rise to direct somaticembryogenesis (Gmitter and Moore, 1986; Carimi et a/., 1998), whereas non - ovules

explants undergo indirect somatic embryogenesis with an intermediate callus stage.(Carimi et al., 1998).

Somatic embryos can develop on all organs of carrots or Alfalfa that carry certaingenotypes that regulate embryogenesis, indicating a wide range of expression of

embryogenic potential However, in most plant species, embryogenesis is restricted to

certain tissues of the above genotype Tissue culture experiments show that an

embryogenic response gradient exists between different plant organs Embryogenic

tissues have the highest embryogenic potential, and this ability decreases in hypocotyls,

petioles, leaves, and roots (Neumann, 2000) But even if embryogenic potential has been

lost in plant vegetative cells, it can still be restored These indirect embryogenesis

pathways need to go through an intermediate callus stage to express their embryogenicpotential

2.4.3.2 Growth regulators

Auxin plays a role in the formation of embryogenic cells because it affects cell

polarity and stimulates subsequent asymmetric division (Nguyen Duc Luong and Le Thi

Thuy Tien, 2006) However, in some cases, the reduction or removal of auxin in themedium helps maintain the embryogenic cells necessary for embryogenesis (Komamine

et al., 1992)

Besides, the type and concentration of cytokinin required for somatic embryo

induction vary depending on the species Cytokinin acts on both steps of cell division:

nuclear and mitotic division So in cytokinin - poor cultures, auxin stimulates

chromosome division, even in binucleate cells, but not wall division Wall division onlyoccurs when exogenous cytokinin is present (Bui Trang Viet, 2000)

Besides, according to Gosal and colleagues (1995), the somatic embryogenesis

process of citrus plants will be rapidly promoted by factors such as ơ- Naphthaleneacetic

acid (NAA), 6-Benzylaminopurine (BAP), Kinetin (KIN), Gibberellic acid (GA3), andother complex compounds such as malt extract (ME), yeast extract, coconut water, andsome carbohydrates such as sucrose, galactose, casein, and glycerol

2.4.3.3 Plant tissue culture medium

WPM mineral medium (Lloyd and McCown 1980) will provide all the mineral

elements necessary for cell or tissue growth This is a commonly used medium to create

somatic embryos in citrus plants (Balwinder et al., 2011)

12

2.4.4 Application of somatic embryogenesis

Currently, cloned embryos are considered a more effective technique for plant

propagation Classical asexual propagation techniques have limitations, especially forwoody plants such as fruits, and forest plants

The most important benefit of the somatic embryogenesis technique is the ability

to keep somatic pre-embryo cells of superior lines in freeze-dried conditions for decades

or even hundreds of years without affecting the genetics of the material when reborn.Creating somatic embryos is also used to create green plants of desired lines in largequantities under in vitro conditions to provide for production (Phan Thi Thu Hien et al.,2015) Somatic embryos are also raw materials for gene transfer in plants and protoplast

culture technology

2.5 Studies on citrus

In India, 1n 2011, research by Paul et.al on curry plants (Murraya koenigii)investigated the effects of GPRs, 2,4-D, NAA, and BA individually or in combination.combine to form a callus capable of generating embryos from cotyledons (COT) andimmature zygotic embryonic columella (ZE) Research has demonstrated that callus can

give rise to embryos more easily on medium that combines both auxin and cytokinin,

specifically after 4 weeks of culture on medium supplemented with BA (2.22 nM-8,88uM) and NAA (2,675 HM) with discrete callus structure and callus color from green tolight yellow At concentrations of BA 4.44 uM and NAA 2.675 uM, the highest rate ofcallus formation was 90% in ZE and 70% in COT Meanwhile, the combination of 2,4-

D and BA also forms callus, but at a lower rate, and if only auxin 1s added separately orGPRs are not added, there is no callus induction In the embryogenesis experiment, threeGPRs, BA, KN, and TDZ, were examined individually to find the growth regulator withthe highest embryogenesis rate The results showed that TDZ at a concentration of 4.54

uM gave the highest rate of embryonic induction for ZE As for cotyledons, the TDZ

concentration at 9.08 uM is suitable for a high rate of embryogenesis BA and KN also

showed the ability to generate embryos, but at a lower rate than TDZ

Ha Thi Thuy e¢ a/ (2013) conducted research on creating embryogenic callus and

cloned embryos from ovule culture in some citrus fruit plant varieties And they

concluded that the rate of embryogenic callus creation from ovule and young seedcultures depends on fruit age and that for unfertilized ovules, embryogenic callus form

has not yet been obtained The suitable medium for creating embryogenic calluses is

MT medium supplemented with malt extract 500 mg/L, sugar 5%, agar 5 g/L, and BAPwith different concentrations in different varieties Van Du orange is most optimal at a

concentration of 2.0 mg/L; orange Sanh at 2.0 mg/L; Chum tangerine at 1.0 mg/L; and

Duong tangerine at 0.5 mg/L Besides, culturing on shaking liquid medium with MT

medium supplemented with 500 mg/L malt extract, 3% sugar, and BAP is suitable forrapid multiplication of embryogenic callus biomass For somatic embryos, they are

formed in an environment with low BAP content If the BAP concentration is higher,

the embryos tend to develop abnormally Finally, cloned embryos with developedcotyledons germinated into healthy seedlings when cultured on MS medium without

of abnormal structures during the evolution of somatic embryos, which he named

abnormal embryos Abnormal structures include multiple embryos with attached budand root poles clearly observed at the heart-shaped stage, or just a large mass of swollen

tissue with only differentiation of the root pole and no shoot pole The embryo has manycotyledons or forms a rosette with many buds and roots In this form, many normal budscan also arise Topoonyanont (1999) and Marcio et al (2001) have found many types ofabnormalities during the formation of somatic embryos They classified them into threemain groups: hypophyseal abnormalities (changes in size and shape), including thenumber of cotyledons; apical meristem; and shoot development Perhaps this is a unique

characteristic of species in the genus Citrus during somatic embryogenesis Normal

somatic embryos reached the highest rate in the 50 g/l sucrose treatment (2.3%) (Pham

Thi Bich Thuy and Nguyen Bao Toan, 2008)

2.6 In vitro studies on P trimera

2.6.1 Studies on the callus formation of P trimera

Phi et al (2017) researched the micropropagation of Paramignya trimera andinvestigated that WPM medium was the most suitable for rapid in vitro propagation

14

compared to MS and Knudson medium In investigating callus formation from 0.5 cm

in vitro shoot segments with growth regulators IBA, TDZ, and 2,4-D, after 12 weeks of

culture in dark conditions on WPM medium containing supplementing TDZ 1.0 mg/Land 2,4-D 3.0 mg/L, explants formed callus at the highest rate of 86.67% The callus

grew quickly and was green Meanwhile, callus formation tests with individual growth

regulators showed lower callus rates, specifically TDZ 1.0 mg/L, IBA 3.0 mg/L, and

2.4-D 3.0 mg/L, which gave callus formation rates of 70%, 68.33%, and 63.33%,

respectively

In the same year, Tran Trung Hieu et al researched and established an in vitropropagation process for the Ð trimera During the shoot multiplication process, the

explants experience leaf loss after 8-12 weeks of culture This phenomenon is common

in many woody species and has been reported to be due to the ethylene production ofthe implant and the presence of ethylene negatively affecting callus proliferation,embryogenesis, and shoot regeneration To overcome the above phenomenon, the

authors added STS (silver thiosulfate) to the medium, which can inhibit ethylene activityand help increase shoot height, promoting tissue morphogenesis The formation of callusfrom leaf explants was investigated on WPM medium supplemented with growth

regulators BA, IBA, and 2,4-D, resulting in WPM medium supplemented with STS 3,

BA 5 mg/l, and IBA 5 mg/l giving the highest callus formation (47%) from mature leaf

samples after 12 weeks of culture Most of the callus is spongy white at the cut surface

at both ends of the leaf's main veins, then spreads to the entire main vein surface Of the

callus formed, 21% of the leaf samples produced firm, glossy, opaque white callusblocks at the cross-section of the leaf blade These callus blocks continue to grow andform small clusters shaped like an embryo (or bud) on the surface of the callus

In 2018, Ngo Thi Xuyen e¢ al investigated the effect of sample disinfection timewith 0.1% HgCl2 on the survival rate of P trimera samples The experiment showed that

the best disinfection time for leaf samples is 8 minutes with a survival rate of 76.67%,

and for stem samples, it is 12 minutes with a survival rate of 33.33% At the same time,the authors investigated the effects of individual growth regulators NAA and TDZ on the

ability of leaf samples to form calluses In MS medium supplemented with only NAA,

the sample did not respond to the culture medium in all treatments (the sample remainedgreen and did not respond) However, in MS medium supplemented with 0.6 mg/l TDZ,

the highest rate of callus formation reached 76.19% after 60 days of culture.Morphologically, most calluses were spongy and colored dark yellow.

In recent years, research team PIB (Plant Integrative Biology) has conducted

surveys on a number of factors affecting callus formation Nguyen Thi My Duyen

(2019) investigated callus formation on medium supplemented with 2,4-D (0.2, 0.4, 0.6,

0.8, and 1.0 mg/L) and NAA (1.0 and 2.0 mg/L) combined with BA (0.0, 0.2, and 0.5mg/L) The results showed that the sample rate of callus formation was very low onmedium supplemented with only 2,4-D and highest at 68.5% when adding 2 mg/L NAAcombined with 0.2 or 0.5 mg/L BA The rate of callusing samples will increase 2.47times when the auxin concentration is from 1.0 mg/L to 2.0 mg/L NAA Tran Thi

Nguyet Nga (2021) continued to investigate the rate of callusing samples on WPMmedium supplemented with NAA (1.0, 2.0, 3.0, and 4.0 mg/L) combined with BA (0, 2mg/L) and obtained results of 1.0 mg/L NAA or 2.0 mg/L NAA combined with 0.2 mg/L

BA, both giving a high callus formation rate of 75% Cao Thi Cam Huong (2022)

continued to investigate the rate of callusing samples on MT and WPM mediasupplemented with NAA concentrations (1.0 mg/L; 2.0 mg/L; 3.0 mg/L; 4.0 mg/L) and

BA (0.2 mg/L) As a result, leaf samples from one-year-old plants cultured on WPM

medium supplemented with 0.2 mg/L BA and 2 mg/L NAA had a callusing rate of 100%after 6 weeks The callus tissue was nodular, hard, and capable of seedling generation.2.6.2 Studies on the somatic embryogenesis formation from the callus of P

trimera

In 2021, Tran Thi Nguyet Nga investigated somatic embryogenesis from the leaf

callus of the P trimera plant on WPM medium supplemented with NAA (0.5 and 1.0mg/L) combined with BA (0.2 and 0.5 mg/L) The results showed that at 1.0 mg/L BA

and 1.0 mg/L NAA, 75% of explants created somatic embryos after 4 weeks of culture

In 2022, Cao Thi Cam Huong investigated the effects of mineral medium and

GPRs on the induction of callus and cloned embryos of the P trimera in three mineralmediums (MS, MT, and WPM) supplemented with malt extract (500 mg/L) and BA(3.0 mg/L; 4.0 mg/L) after 6 weeks of culture The results showed that MT mediumsupplemented with 500 mg/L ME combined with 4 mg/L BA gave the highest rate ofsomatic embryo induction at 83.8% The embryo develops a globular-shape and heart-

shaped stage

16

CHAPTER 3 MATERIALS AND METHODS

3.1 Places and duration for conducting experiments

The project was carried out from March 2023 to the end of December 2023, at

the Plant Integrated Biology Laboratory, Faculty of Biological Sciences at Nong LamUniversity in Ho Chi Minh City

3.2 Equipment and tool

Equipment: analytical balance, pH meter, electric stove, sterile culture cabinet,

drying cabinet, chemical storage refrigerator, autoclave, water distiller, shaker

Tools: 100 ml glass bottle; Erlenmeyer flask (250 ml, 500 ml); glass cup (100 ml, 250

ml, 500 ml, 1000 ml); volumetric flask (20 ml, 100 ml, 250 ml, 500 ml); petri dish;measuring cylinder; pipetman; glass chopstick; transplanting knife; clip 25-30 cm long;

alcohol lamp; alcohol spray; waterproof cotton balls and other tools

3.3 Materials

3.3.1 Biological materials

The fruit explant Paramignya trimera, was collected from Dong Nai

3.3.2 Medium

The media used in the study were Woody Plant Medium (WPM) and Murashige

and Skoog (MS) Depending on the purpose of each experiment, the medium will besupplemented with growth regulators and other compounds at different concentrations

The medium is adjusted to pH = 5.7 + 0.1, put into a 250 ml glass bottle, and

autoclaved in an autoclave at a temperature of 121°C and a pressure of 1 atm for 20

minutes Explants were grown in a growth room at a temperature of 25 + 2°C and ahumidity of 60 — 65%.

3.4 Methods

3.4.1 Sterilize the P trimera seed explant

Experiment objectives: Determine the Javel ratio and appropriate processing time

to sterilize the P trimera seed explants

Table 3 1 Time and rate of Javel solution used in treatments

Treatment Time (mins) Javel (%)

Al 15 20A2 20 25A3 15 20A4 20 25

Choose ripe fruits that are intact, undamaged, and have no signs of pests ordiseases, and remove the skin The seeds are washed under running water to remove thefruit juice Seed explants were pre-sterilized by soaking in a 5% diluted soap solution

(15 minutes) Then soak the seeds with a fungicide solution (Mancozeb, India) for 10minutes, then wash them with clean water The seed explants are transferred to the Clean

Bench to be sterilized with 70% alcohol for 30 seconds and washed three times with

sterile distilled water for 3 minutes each time Next, sterilize the explant with (20; 25%)Javel solution (Sodium hypochloride, NaClO 5%) supplemented with tween 20 for (15;

20 minutes) Then rinsed the sample with three times sterile distilled water for 3 minuteseach time Finally, sterilize the explant with tetracycline antibiotic combined withampicillin in a 1:1 ratio for 30 minutes, then rinse three times with sterile distilled water.Seed explants, after sterilization, were placed on MS medium

Setting up experiment: The experiment was constructed at random (Table 3.1).Each treatments includes 10 bottles of medium, each bottle contains 2 explants, theexperiment is repeated 4 times

The monitoring sterilization ability indicators were recorded on the 7th, 14th,21st, and 28th days after transplanting, and germination rate indicators were recorded

on the 35th, 42nd, 49th, and 56th days

3; number of bacterial infection explants

Percentage of infection explants (%) = x 100

3; number of cultured explants

18

number of live explants

ẽ 3 100

Percentage of live and clean explants (%) =8 P ( 0) 3; number of cultured explants

> number of germinated explants

x 100Percenta f germinated explants (%) =e Be ĐÁ x P (%) > number of live and clean explants

3.4.2 Investigate the effects of explant type and concentration of NAA combinedwith BA on callus induction

Experiment objectives: To determine the appropriate concentration of NAAcombined with BA for callus formation from cotyledons, hypocotyl, radicles and zygoticembryo P trimera explants

Table 3 2 The rate of callus induction due to the effect of NAA combined with BA

Treatment Explant NAA (mg/L) BA (mg/L)

Cl 0 0C2 Zygotic embryo 1 0.2C3 2 0.2C4 0 0C5 Cotyledons 1 0.2

Có 2 0.2C7 0 0

C8 Hypocotyl 1 0.2

C9 2 0.2C10 0 0

Cll Radicle 1 0.2

C12 2 0.2

Methodogy: Jn vitro cotyledons explants were cut crosswise into pieces about 2

mm x 10 mm in size; hypocotyl and radicle explants from in vitro seeds were cut into

5mm sections; and sexual embryo explants were separated from seeds Explants were

placed on WPM medium supplemented with NAA and BA according to each treatment

(Table 3.2)

Experimental design: The experiment of 12 treatments consists of arranged in acompletely randomized fashion with 2 repetitions, each with 2 bottles, each with 4 explants

Monitoring criteria were recorded at the 4, 6 and 8-week time points, includingtime of callus appearance, rate of callus formation, and callus morphological

characteristics including:

Including time of callus appearance

3; number of callusing explants

Rate of callus formation (%) = x100

ÿ number of explants

Callus morphological characteristics (shape, color)

3.4.3 Examination of somatic embryo induction

3.4.3.1 Examination of somatic embryo induction from zygotic embryo

Experiment objectives: Investigating the effects of WPM medium supplemented

with 50 mg/L sucrose, 500 mg/L ME and 2 mg/L BA on the induction of somatic

embryos from zygotic embryos

Methodogy: Select clean living seeds from experiment 1 Separate the seeds to

remove the zygotic embryos of the P trimera seeds and transplant them into the medium

Experimental design: The experiment was arranged completely randomly with 5

cell-culture dishes containing 4 explants each

Criteria monitored and recorded after 4 weeks, 8 weeks, and 12 weeks include

induction and embryo formation time, rate of embryo formation, and embryo morphology.3.4.3.2 Investigating the effects of TDZ on somatic embryo formation from youngleaf callus explants

Objective of the Experiment: To identify the optimal TDZ concentration forinducing somatic embryogenesis in callus tissue derived from young leaves

Methodology: Leaf explants from one-year-old plants were sterilized and slicedinto 2 mm by 10 mm segments These explants were then cultured on WPM enrichedwith 2 mg/L NAA and 0.2 mg/L BAP to promote callus formation Following an 8-weekperiod of culture, the embryogenic callus underwent a series of somatic embryoinduction treatments included TDZ concentrations of 0.07 mg/L (S1), 0.1 mg/L (S2),

0.3 mg/L (S3), and 0.7 mg/L (S4)

Experimental design: The study was organized in a completely randomized

design comprising four treatments, each with three replicates Every replicate included

one dish containing three explants, totalling 36 explants for the experiment

20

Measurements: Key indicators such as the time to embryo induction, embryoinduction rate, and embryo morphology were systematically recorded at 4 and 8 weeksinto the experiment.

3.5 Observe anatomical morphology

Embryo explants at different stages of development from different explants werecollected and microscope slides created The explant was cut into thin slices and then

soaked in a 5% Javel solution for 17 minutes Continue soaking the slices in a 1% aceticacid solution for 2 minutes to remove the remaining javelin Soak the slice in a methylene

blue solution for 5 - 10 seconds Soak in an acetocarmine solution for 15 - 30 minutes

and gently heat over an alcohol lamp After each step, rinse with distilled water 4 - 6

times Place the explant on the microscope slide, add a drop of water, and cover the slide.Observe under an optical microscope with 4X, 10X, and 40X objectives

3.6 Data processing

The data were calculated using Excel software and statistically processed using

Minitab 16 software Treatments were classified according to Tukey's HSD with a

P-value < 0.05 For percentage data, the data are converted according to the formula y=

ASIN(Vx/10) x 180/3.14 before statistical processing, where x is the percentage before

statistical processing Besides, if x has value 0%, replace it with 1/4n; otherwise, if xhas value 100%, replace it with 100 - 1/4n (with n being the denominator when

calculating the ratio (%)) (Gomez and Gomez, 1984) Data numbers are presented as themean + Standard Error

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Efficacy of sterilizing P trimera seed explants with Javel Solution

The study highlighted the effectiveness of Sodium hypochlorite (NaClO),commonly known as Javel solution, for sterilizing P trimera seed explants The primaryaim of this sterilization process is to remove bacterial and fungal infections withoutcompromising the explant's growth potential and viability (Nguyen Duc Luong and LeThi Thuy Tien, 2006) The experimental findings revealed that adjusting the Javel

solution's concentration and exposure time significantly influenced the cleanliness and

germination rate of the explants, while effectively reducing infection rates

Table 4 1 Efficacy of sterilizing P trimera seed explants with Javel Solution

Rate of Rate of cleanJavel ; ¬ Rate of

Time bacterial living a

value > 0.05); Data are processed according to the formula asin(x/10)x180/3.14; Time (A);

exposure time to achieve effective sterilization without harming explant viability After

28 days of culture, most seed samples remained green with minimal damage, suggesting

that Javel solution is an appropriate sterilant for P trimera seeds, considering bothenvironmental and human health impacts The shift from Mercury (II) chloride toSodium hypochlorite not only reduces health and environmental risks but also maintainshigh sterilization efficiency, offering new avenues for plant explant management

Table 4.1 demonstrates that using a 20% Javel solution for 15 minutes resulted in

a 46.25% rate of bacterial infection and a relatively high germination rate of 37.24%.However, extending the exposure time to 20 minutes significantly reduced bacterialcontamination rates to 22.50% and increased both the rate of clean living explants to70% and the germination rate to 44.79% Increasing the Javel concentration to 25%

further reduced contamination rates but significantly decreased the germination rate,

illustrating the adverse effects of high NaClO concentrations on seed viability due to itsstrong oxidizing properties, which can damage plant cell structures, includingchlorophyll This damage complicates the explant's recovery, leading to higher mortalityand reduced germination capability

In summary, while Javel solution is effective for sterilizing P trimera seedexplants, the sterilization efficiency hinges on carefully balancing the disinfectant'sconcentration and exposure time to minimize damage to the explants

4.2 Influence of kinds of explants and NAA, BA concentrations on callus formation

Results from Tables 4.2 and 4.3 clearly reflect the positive impact of adding NAA

and BA to the cultivation medium on callus formation from different kinds of explantssuch as cotyledons, hypocotyls, radicle roots, and zygotic embryos The increasing rate

of callus formation over time shows that the presence of growth regulators like NAA

and BA is necessary to stimulate the development of callus, an important step inregenerating plants from explants Previous studies have proven the role of NAA and

BA in stimulating cell division and development in plant cells, and this result continues

to affirm their role The distinct difference in the rate of callus formation among the

kinds of explants and concentrations of NAA, BA indicates a complex relationshipbetween explant type, growth regulators, and cultivation medium, requiring a deepunderstanding to optimize the regeneration process

Table 4 2 Influence of types of explants and NAA, BA concentrations on callus induction

PGPs (mg/L) Rate of callus induction (%)Treatment Explant

NAA BA 4 weeks 6 weeks 8 weeks

C5 Cotyledons 1 02 31.25543.88 6250°+000 62.50°+9.85

C6 2 02 50.00°40.00 56.25°+362 93.75°+0.00C7 0 0 0.00°+0.00 0.00°+0.00 0.00°+0.00C8 Hypocotyl l 02 43.75°43.62 62.50°+7.50 68.75°43.88

value > 0.05); Data are processed according to the formula asin(/x/10)x180/3.14; Explant

(A); NAA mg/L (B)

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The results showed that the rate of callus explants gradually increased over 4, 6,

and 8 weeks of culture No callus formation occurred in the treatments without GPRs(C1, C4, C7, and C10) The best rate of callus explants reached 93.75% when culturingcotyledons and hypocotyl, 75% when culturing zygotic embryo explants, and 56.25%

when culturing radicle explants on WPM medium supplemented with 2 mg/L NAA and

0.2 mg/L BA (Treatments C6, C9, C3, and C12) However, the callus morphology ofcotyledons (treatment 6) is a firm, glossy white callus at the cut that mainly forms nearthe zygotic embryo site and develops tissue clusters with a structure like embryo blocks

At the surface of the callus, the callus morphology of the propagules (treatment C9) ispale yellow, firm, and has the ability to give rise to embryos, while the callusmorphology of the zygotic embryos (treatment C3) has the shape of callus tissue Smallyellow-white blocks and growing in tissue clusters with a structure like somatic embryoblocks, the callus morphology of the radicle (treatment C12) callus is pale yellow, shiny,firm, and spongy (Figure 4.2)

Figure 4 2 Callus induction from 4 types of explants after 8 weeks on WPM+ 2

mg/L NAA+0.2 mg/L BA medium (a) Zygotic embryo; (b) Cotyledons; (c)

Hypocotyl; (d) Radicle

The rate at which explants form calluses is influenced by both explant type andNAA concentration Both of these factors interact with each other with a p-value=0.006,

demonstrating a statistically significant difference Considering each factor separately,

the explant rate of callus induction of zygotic embryos, cotyledons, hypocotyl, andradicle roots on WPM medium supplemented with 2 mg/L NAA and 0.2 BA (Treatments

C3, C6, C9, and C12) was high 1.33 times, 1.5 times, 1.36 times, and 1.5 times higher

than the same explant on WPM medium supplemented with 1 mg/L NAA and 0.2 mg/L

BA (Treatments C2, C5, C8, and C11) time Regarding the concentration of NAA and

BA, when not added to the medium, the explants did not form callus and gradually died;

this proves that BA and NAA are necessary for callus induction When adding NAA and

0.2 mg/L BA, the rate of callus explants increased the most at the concentration of 2mg/L NAA in all 4 types of explants This result is similar to the results of Nguyen Thi

My Duyen (2019) and Cao Thi Cam Huong (2022) when surveying young leaf explants

of the Ð trimera

Although all treatments had no change in callus formation time, there was a clearchange in the morphology and development of the formed callus The impact of factors

also creates changes in callus morphology after 4 and 8 weeks of culture (Table 4.2)

After 2 weeks of culture, the explants were induced to form calluses After 4 weeks, the

explants were still green; calluses mainly formed at the cut site, and a few callus samplesformed both on the stem and around the cut After 8 weeks, the callus morphology in

the treatments had obvious changes

Table 4 3 Callus morphology after 4 weeks and 8 weeks of culture

Morphological characteristics of the explant after cultureTreatment

4 weeks 8 weeks

Cl Green gold sample, non-touch Green gold sample, non-touch

Greenish-yellow explant, small Greenish-yellow explant, small

C2 yellowish-white callus, firm, shiny, yellowish-white callus, firm, shiny,

formed in blocks formed in blocksC3 Greenish-yellow explant, small Pale greenish yellow explant, callus-

yellowish-white callus, firm, shaped like small yellow-whiteshiny, formed in blocks blocks, firm, shiny, and numerousC4 Green gold sample, non-touch Green gold sample, non-touch

C5 A green explant with a small, A green explant with a small,

opaque white callus forms mainly opaque white callus forms mainlynear the embryonic site near the embryonic site

Có A green explant with a small, A green explant with a small,

opaque white callus forms mainly — opaque white callus forms mainlynear the embryonic site near the embryonic site

C7 Green gold sample, non-touch Green gold sample, non-touch

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Table 4 3 (Next) Callus morphology after 4 weeks and 8 weeks of culture

Morphological characteristics of the explant after cultureTreatment

4 weeks 8 weeksC8 The green explant induced a callus The green explant induced a callus

at both ends of the cut; the callus is at both ends of the cut; the callus islight yellow and small light yellow, small, and few

C9 Green explant The callus, formed Green explant The callus, formed

mainly around the cut at both ends, mainly around the cut at both ends,

is yellow and small is yellow, small, and solid

C10 Green gold sample, non-touch Green gold sample, non-touch

The green explant feels slightly

en The green explant feels slightly swollen at both ends of the cut

swollen at both ends of the cut The callus is light yellow, small,

and fewThe explant is green, and the callus The explant is green, and the callusC15 formed mainly around the cut at formed mainly around the cut at

both ends is light yellow, small, both ends is light yellow, small,

spongy, and very little spongy, and very little

Figure 4 3 Callus formed from the explants after 8 weeks C7 Zygotic embryo (WPM),C2 Zygotic embryo (WPM + I mg/L NAA + 0.2 mg/L BA), C3 Zygotic embryo (WPM + 2 mg/LNAA + 0.2 mg/L BA), C4 Cotyledons (WPM), C5 Cotyledons (WPM + I mg/L NAA + 0.2 mg/LBA), C6 Cotyledons (WPM + 2 mg/L NAA + 0.2 mg/L BA), C7 Hypocotyl (WPM), C8Hypocotyl (WPM + I mg/L NAA + 0.2 mg/L B4), C9 Hypocotyl (WPM + 2 mg/L NAA + 0.2mg/L BA),C10 Radicle (WPM), CIl Radicle (WPM + I mg/L NAA + 0.2 mg/L B4), C12Radicle (WPM + 2 mg/L NAA + 0.2 mg/L BA)

Figure 4.3 shows the morphology of 12 treatments of a large, nodular, firm, paleyellow callus composed of large interconnected blocks (C2, C3, C8, C9, C11, and C12)

and explant type Small, nodular, and firm, interspersed with white spongy tissue (C5,C6) Le Van Hoa et al (2012), when studying bamboo plants, found that high auxin

concentrations stimulate the formation of discrete calluses, but when auxinconcentrations are reduced, the calluses become nodular and firm

Table 4 4 Effect of explant type and concentration of NAA and BA on adventitious

root formation from callus after 4, 6, and 8 weeks of culture

embryo

ZygoticC3 2 0.2 0.00°+0.13 0.00°+0.06 0.00°+0.00

embryoC5 Cotyledons 1 0.2 58.34%+44.87 50.002+5.77 50.002°+5.77

Có Cotyledons 2 0.2 62.50°+7.50 90.00221260 86.61°+0.76 C8 Hypocotyl l} 02 41.67%+44.87 50.002+0.00 45.00°+2.88 c9 Hypocotyl 2 0.2 50.00%+4.11 74.11%+7.78 86.61°+0.75 Cll Radicle 1 0.2 66.67°+25.90 3333°+0.00 50.00°+9.74

value > 0.05); Data are processed according to the formula asin(/x/10)x180/3.14; Explant

(A); NAA mg/L (B)

During the process of investigating the effects of explant type and concentration

of NAA and BA on callus induction, a number of callus cells appeared to formadventitious roots Results recorded root emergence time and root formation rate incallus treatments (Table 4.4)

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