Khóa luận tốt nghiệp Công nghệ sinh học: Investigation of some cultural conditions and media on the growth of in-vitro propagation of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY-HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESINVESTIGATION OF SOME CULTURAL CONDITIONS AND MEDIA ON THE GROWTH OF IN-VITRO PROPAGAT

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

INVESTIGATION OF SOME CULTURAL CONDITIONS AND MEDIA ON THE GROWTH OF IN-VITRO PROPAGATION OF NGOC LINH GINSENG (Panax vietnamensis Ha et Grushv.)

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

UNDERGRADUATE THESIS

INVESTIGATION OF SOME CULTURAL CONDITIONS AND MEDIA ON THE GROWTH OF IN-VITRO PROPAGATION OF NGOC LINH GINSENG (Panax vietnamensis Ha et Grushv.)

Advisor Student

MSc Pham Van Hieu Nguyen Thi Truc My

Thu Duc City, 08/2023

First, I would like to express my deep gratitude to the Biotechnology Center of HoChi Minh City for providing all budget, machinery, equipment, and chemicals for theexperiment thesis

I would like to express my gratitude to MSc Pham Van Hieu, who dedicatedlyguided me with a lot of knowledge, corrections, and I also would like to thank Dr.Nguyen Xuan Dung, who created favorable conditions for me during the process ofmaking my thesis

I wish to thank the Board of Directors of Nong Lam University - Ho Chi MinhCity, the administrators of the Faculty of Biological Sciences, and all the lecturers forproviding me with the necessary resources to pursue my studies there Thanks to theguidance and teaching of yours, I can apply it to solve problems related to the majorduring the thesis

Finally, I would like to the staff and friends in Department of Plant Biotechnology,who have supported and helped me throughout the learning and research process

Sincere thanks to all!

AFFIRMATION AND COMMITMENT

My name is Nguyen Thi Truc My, Student ID: 18126228, Class: DH18SHC ofBiotechnology major, Nong Lam University, Ho Chi Minh City I declared that allresults presented in this graduate thesis were conducted by myself All the data andinformation are entirely accurate and honest I fully accept responsibility for thesecommitments in front of the committee

Thu Duc City, August , 2023

Student’s signature

NGUYEN THI TRUC MY

ll

This study was conducted to determine some growth conditions such as lightintensity, day-night temperature and medium of culture that affect the growth anddevelopment of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) by plant tissueculture method To investigate the appropriate light mtensity for the growth of Ngoc

Linh ginseng, the experiment was set with different light intensities (5-20 pmol.m?.s"),

the in-vitro plantlets were cultured in MS and SH medium supplemented with 0.2 mg/Lkinetin and 13% coconut water After finding out the appropriate light intensity, thestudy continuously conducted to investigate day-night temperatures by screening theday-night temperature (23-13/15/18 °C) under the existing light intensity from theprevious experiment /n-vitro plantlets were also cultured in the same media as above.All were cultured at day and night humidity of 55 and 65%, respectively, 12 hours oflighting per day After 12 weeks of culture, the results of the comparison of parameterssuch as number of leaves, number of roots, shoot height and chlorophyll content showed

that conditions of light intensity at 20 umol.m~”.s!, temperature day and night at 23 and

15 °C, respectively, were suitable for the growth of Ngoc Linh ginseng Besides, theparameters and morphological characteristics showed that ginseng in SH culturemedium was more suitable growth than that in MS medium

Keywords: Panax vietnamensis, light intensities, day - night temperature, MS and SHmedium

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TABLE OF CONTENTS

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LIST OF TABLES

PageTable 4.1 Effect of light intensities on growth of in-vitro VG in MS medium 16Table 4.2 Effect of light intensities on growth of in-vitro VG in SH medium 18Table 4.3 Effect of day-night temperature on growth of in-vitro VG in MS medium

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Table 4.4 Effect of day-night temperature on growth of in-vitro VG in SH medium

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LIST OF FIGURES

PageFigure 4.1 Growth of in-vitro VG under different light intensities after 12 weeks in MS

Figure 4.3 Growth of in-vitro VG under various day-night temperature after 12 weeks

I MS medium PA 21Figure 4.4 Growth of in-vitro VG under various day-night temperature after 12 weeks

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CHAPTER 1 INTRODUCTION

1.1 Problem statement

Panax vietnamensis (Panax vietnamensis Ha et Grushv.) is an endemic species

of ginseng in Viet Nam belonging to the family Araliaceae, has long been known andused as kinds of a rare and precious herb in traditional medicine with its therapeuticeffects such as anti-stress, antioxidant, immune system booster, cancer prevention, liverprotection, and so on Through many previous studies, Ngoc Linh ginseng has manypharmaceutical compounds in which the saponin compounds are the most important,particularly, the dammarane saponin in Ngoc Linh ginseng has a high content, inaddition, in the ocotillol saponin group, and there is the M-R2 fraction accounting for50% of the total saponin content, the most comparison to the species of the genus Panax

in the world (Hoang et al, 2012) Based on the above characteristics, Viet Nam ginsenghas become a precious medicinal species not only in Viet Nam but also around the world

However, Ngoc Linh ginseng today is over-exploited, with a high risk ofextinction recorded in 250 species located in Vietnam's Red Data book that needs to beprotected (Duong et al, 2013) The propagation of Ngoc Linh ginseng is still difficultbecause it only grows well on Ngoc Linh mountainous area and the harvest time is verylong - from 5 to 8 years, because that the ginseng roots have enough active ingredients

as desiring, sexual propagation conventional seeding methods will not give high resultsfor many reasons such as seeds germinating for a long time, so they will be destroyed

by many insects or animals, and the germination rate is low (Vu, 2018) In-vitropropagation with similar culture conditions to the natural conditions of Ngoc Linhmountainous (including soil, shade, climate, humidity, and so on) so that ginseng cangrow well, especially roots in order to improve the ability to survive when grown in thewild is also a difficult issue to consider From the above practical issues, the study wascarried out to determine some culture conditions and culture medium affecting thegrowth of Ngoc Linh ginseng with the aim of helping the plant adapt to naturalconditions and has high vitality, contributing to the preservation of precious medicinalherbs for Viet Nam

1.2 Research objectives

This study aimed to investigate some suitable culture conditions susch as lightintensity, temperature, and medium on the growth of in-vitro Ngoc Linh ginseng.

in-CHAPTER 2 LITERATURE REVIEW

2.1 Panax vietnamensis

2.1.1 Origin and distribution

Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) is one of 11 types ofginseng belonging to the Araliacease family, also known as Viet Nam ginseng, KhuNam ginseng (K5), and Truc ginseng This is a precious ginseng known for its 52 types

of saponins and other compounds It has long been used as a herb that contributes to thetreatment of diseases including hepatitis, diabetes, and cancer (Nguyen and Phuong,2019) in the top 5 best types of ginseng discovered in the world

Ngoc Linh ginseng 1s distributed in the central highlands of Viet Nam, they growconcentratedly on the Ngoc Linh mountainous area of Tu Mo Rong district, Kon Tumprovince, and Nam Tra My district, Quang Nam province at altitudes from 1.200 to2.100 m below the forest canopy and cloud covered up to 80% or more, along moiststreams on humus soil

Ngoc Linh ginseng was firstly discovered in Viet Nam in 1973 by pharmacistsKim Long Dao, Chau Giang Nguyen, and Thi Le Nguyen In 1985, it was officiallyknown and scientifically named Panax vietnamensis Ha et Grushv., by Dr Ha Thi Dungand Professor Grushvisy (Nguyen, 2015)

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5 leaflets/stem The leaflets are inverted ovate, 3 - 5 cm wide, serrated, leaf surface hairy

on both front and back Inflorescences single, crowned, with long stalks The fruit isovoid shape, the young is green, and when ripe it is dark red with black spots appearing

at the top of the fruit, kidney-shaped white seeds (Do et al, 2006; Dinh et al, 2021) Theunderground part of cultivated Viet Nam ginseng is divided into rhizome, radix, and fine

root.

The stem of ginseng has scars left by the annual birth and death, based on whichone can calculate the age of the ginseng One of the best example, Kim Long Dao andChau Giang Nguyen have obtained samples of ginseng with rhizomes 1 meter long and

Ngoc Linh ginseng grows on fertile, low-erosion soils with high humus content,containing a lot of water, the average annual humidity is from 85% to 87% and theaverage annual rainfall is from 2.600 to 3.200 mm Ginseng populations were foundwith many plants of different ages, which indicates that Ngoc Linh ginseng has goodregenerative capacity from seeds (Hanh and Pham, 2020)

According to Do et al (2006), P vietnamensis grows strongly in spring - summer.The fruit will develop from May to October, after falling fruit will wait 4 months ofwinter and will sprout new spring next year Ginseng roots should be harvested from 3years of age and older, and preferably in winter

2.1.5 The value of Panax vietnamensis

2.1.5.1 The medicinal value

e Saponin compounds

According to previous research, 52 types of saponins are concentrated mainly inNgoc Linh ginseng roots, underground There are 26 saponin compounds known in thegenus Panax ginseng species and 26 newly isolated saponin compounds found only in

Vietnamese ginseng New compounds included majonoside R2, ginsenoside Rh5, ginsenoside R1-R25 (Dinh et al, 2023) VG (Vietnamese ginseng) has three groups ofsaponin compounds, which is protopanaxadiol containing 22 representative compounds,ginsenoside-Rb1, -Rb3, -Rd In the protopanatriol group, there are 17 representativecompounds, ginsenoside-Re, Rgl, and finally, the ocotillol group contains 11representative compounds, maJoside-RI and -R2 (Nguyen et al, 2007) A notablesaponin among these is majonosode R2, which accounts for 5.29% of Ngoc Linhginseng, which is a derivative of OCT and it does not exist in Panax ginseng species.M-R2 has the highest concentration in the rhizome (93.5 mg/g) and radix (70.6 mg/g).Second, is the compound V-R2 saponin In contrast, the content of M-R2 and V-R2 infine root is much less than that in rhizome and radix (Le et al, 2015) There have beenmany studies showed that majonoside-R2 saponin is the main compound of Ngoc Linhginseng with many effects similar to ginseng Such as anti-stress, anti-cancer, anti-depressant, antioxidant, liver-protective, and resistance-enhancing (Do, 2003).

vina-The stem and leaves of Vietnamese ginseng have a total of 19 isolated saponinsincluding 11 known saponins and 8 novel saponins named vina-ginsenoside (L1 - L8).The saponin content in the PPD group was higher than that in the PPT and OCT group(Vu, 2018)

e Other compounds

According to research by Vu (2018) showed:

Fatty acids: 17 fatty acids have 8-20 carbondioxide, the highest proportion islinoleic acid with 40.04% and the lowest 1s linolenic acid with 2.61%

Polyacetylene: There are 7 compounds isolated from the roots of VG

Micro and macro elements: 20 elements have been identified in the undergroundpart of VG

Amino Acids: 18 amino acids have been identified with anti-aging properties.2.1.5.2 The economic values

According to preliminary calculations, if planting | ha of ginseng, the profit will

be over 2 billion VND/ha after 8 years Because of its high cost, the Prime Minister ofViet Nam approved Ngoc Linh ginseng to become a national product in 2017 (Le et al,2018)

In addition, according to Hanh and Pham (2020), due to the scarcity and danger ofextinction of Ngoc Linh ginseng, the government decided to establish a nationalforbidden zone, focusing on planting in Kon Tum and Quang Nam provinces Based onthat, it is possible to improve the quality of life of households in Nam Tra My district,Quang Nam province, and Tu Mo Rong district, Kon Tum province, created jobs forpeople, eliminate hunger and reduce poverty for households in highlands, socio-economic development.

2.1.6 Factors affecting the growth of Ngoc Linh ginseng

2.1.6.1 The light

Light affects plant morphogenesis through plant receptors and is involved in thebiosynthesis of primary and secondary metabolites (Jie, 2003) Besides, some plantspecies under high light conditions will reduce their photosynthetic ability and viceversa, if the light is too low, it will also affect photosynthesis physiology, so each plantspecies will have optimal light intensity (Junqian and Baolin, 2016)

Ngoc Linh ginseng is a shade-loving plant Therefore, they grow naturally underthe forest canopy covered by more than 80%, direct sunlight is about 25,000 - 30,000lux

2.1.6.2 Temperature

Temperature directly affects the distribution and growth of plants Too high atemperature will affect the chlorophyll content in the leaves, affecting the photosyntheticfunction, and vice versa, if the temperature is too low, it will lead to a halt in metabolicactivity Therefore, some species will be limited to a certain temperature range to besuitable for the growth of each different plant species

Vietnamese ginseng is a plant that lives in cool and low temperatures Grows anddevelops strongly at temperatures from 15 °C to 25 °C (Tordoff et al, 2000)

2.1.6.4 Plant Growth Regulator

Plant growth regulators have impacts on physiological and biochemical processes

of growth and development at different concentrations of plants in general (Sabagh et

al, 2021) and Ngoc Linh ginseng in particular

e Auxin

Auxins are groups of plant growth regulators widely used in plant tissue culture,which can be used alone or in combination with cytokinins to stimulate callus growth,and cell suspension, and regulate morphogenesis (Le and Trinh, 2008) There are 2 types

of auxins: auxins of endogenous origin synthesized in stem tips, meristems, and youngleaves with similar structures to IAA and synthetic auxins created by man such as IBA,NAA, 2,4-D, etc (Duong, 2007) This group often has the common characteristics ofcell division, which helps to increase stem length, internodes, directionality (light,earth), apical dominance, rooting, and vascular differentiation

In 2011, Nguyen et al had a study on the ability of callus formation and unstablerooting of Ngoc Linh ginseng in-vitro cultured when supplemented with 2,4-D, IBA,and NAA

e Cytokinin

Cytokinin is a derivative of adenine, which is a hormone involved mainly in celldivision, change in apical dominance, and shoot differentiation in tissue culture.Cytokinins are necessary for cell division and differentiation of shoots from callus ororgans, induce embryogenesis, enhance extra shoots, induce chloroplast development,and enlarge leaves Cytokinins include substances such as kinetin, benzoic acid, TDZ,

BA (BAP), ete (Duong, 2006)

The ratio of auxin and cytokinin utilization is also extremely important whenadding to the culture media A higher concentration of auxin than cytokinin leads toembryogenesis that forms callus and roots In the opposite case, the concentration ofauxin lower than cytokinin will increase the production of shoots and axillary shoots (Leand Trinh, 2008)

In 2007, according to a study by Mukherjee and Kumar, the effect of using kinetinsignificantly increased the change in leaf number, number of branches, leaf area, height,and reduced chlorophyll loss in Cajanus cajan L Also in another study, it was shownthat the addition of Kn (kinetin) at appropriate doses resulted in the greatest increase in

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biomass in the growth regulator trials of Ngoc Linh ginseng cell suspension culture(Tran et al, 2017).

e Gibberellin

Gibberellin was found in the 1930s, nowadays more than 136 different gibberellinmolecules have been detected, of which the most commonly used is GA3 GA3stimulates shoot elongation and germination of asexual embryos In fact, in plant lifegibberellin plays an important role in many physiological processes such as thephysiological dormancy of seeds and shoots, the physiological development of flowers,and increasing the length of plants But in plant cell and tissue culture conditions, theeffect of gibberellic acid is not very clear Therefore, compared with auxins andcytokinins, gibberellin is rarely used (Le and Trinh, 2008)

e Axit abscisic (ABA)

ABA is a group of naturally occurring growth inhibitors, that cause shootdormancy, opening and closing stomata, and delaying seed germination and flowering.When facing adverse conditions, ABA enhances the resistance of plant cells, so ABA isused in culture medium and has certain effects such as: helping to create asexualembryos, stimulating embryo maturation, and stimulating shoot formation in manyplants (Le and Trinh, 2008)

2.1.7 Studies of Viet Nam ginseng in domestic and the world

2.1.7.1 Domestic studies

In 2012, Le et al Reported on the influence of monochromatic light on the growthand ability to accumulate saponin content through the callus culture of in-vitro NgocLinh ginseng The results showed that the maximum mass of fresh callus was 0.748 gand dry callus was 0.064 g at a 50:50 ratio of blue and red light combination This provedthat blue and red light were suitable for the growth in-vitro of Ngoc Linh ginseng

In 2013, Truong et al Studied callus culture capable of somatic embryogenesisfrom leaf callus of Ngoc Linh ginseng in a liquid medium The results showed that calluscapable of embryogenesis rapidly increased biomass in liquid culture medium MS + 0.5mg/L 2,4-D + 0.5 mg/L NAA and embryo tissue rapidly proliferated in liquid medium

⁄2 MS + 0.2 mg/L NAA + 0.2 mg/L BA from which shoots or roots would be formeddepending on the next culture medium

In 2015, Duong et al Conducted a study on light-emitting diodes on the growth ofcallus, seedlings, and saponin accumulation during somatic embryogenesis of NgocLinh ginseng The results showed that callus increased from 70 mg cultured to 1197 mgunder yellow LED after 3 months Plant formation was most effective when culturedwith the combination of 60% red and 40% green LEDs For the highest MR2 saponincontent when combining 20% red LED and 80% green LED Thereby, it is possible toshowed the potential influence of different LEDs on different purposes.

Also in 2015, Nguyen's Master's thesis showed the development of Ngoc Linhginseng when it was acclimatized to locations with similar ecosystems on Ngoc Linhmountains These are Tam Dao, Lac Duong and Sapa The results showed that the ability

to grow and accumulate saponins when acclimatized Ngoc Linh ginseng out of theoriginal region, the two sites of Lac Duong and Tam Dao had higher adaptability than

Sa Pa

In 2019, Nguyen et al Reported on the improvement of accumulated saponincontent in the indeterminate root culture over the stages of culture and propagation ofNgoc Linh ginseng The results demonstrated that the combination of 7 mg/L IBA and0.5 mg/L BA were the best for roots growth The biomass yield of the roots started todecrease on the 70th day of culture, but the saponin MR2 and Rb1 contents continued

to Increase However, root biomass growth was still important for saponin productionand root biomass was highest in a medium containing 150 mg/L YE cultured for 56days

In addition in 2019, Pham et al Studied the ecology and saponins of Panaxvietnamensis var fuscidiscusus 1n Lai Chau Viet Nam was found in 2003 and namedLai Chau ginseng They also have saponin compounds similar to those of ginseng foundpreviously and provide many economic improvement benefits to the mountainouspeople in Lai Chau

About acclimatization, in 2021, there was also a study by Truong et al Thatreported evaluating the saponin content in Ngoc Linh ginseng when it acclimatized toLam Dong province by HPLC-UV/CAD method The results showed that whenanalyzing the main saponin content of ocotillol types such as MR1-2, etc in plants from

2 to 5 years old, the saponin content increased gradually with the age of ginseng.Research has shown promise in the acclimatization of Ngoc Linh ginseng

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In 2022, in the report of Duong et al It was successful in the regeneration oftransgenic seedlings from hairy roots of somatic embryogenesis and seedling culture byadding nano iron of Ngoc Linh ginseng The results obtained in this study showed thathairy roots formed 100% callus on SH + 0.5 mg/L NAA combined with 2.0 mg/L BAmedium after 6 weeks was cultured Then callus was cultured in SH + 1 mg/L 2,4-D +

2 mg/L BA medium and the rate of somatic embryogenesis also reached 100% Embryosprogressed through each stage and became seedlings in SH medium without PGRs Theaddition of nano-iron supported the seedlings to develop in-vitro roots derived fromhairy roots to help the seedlings survive growing in the soil

2.1.7.2 World studies

In 2014, Chen et al Studied the photosynthetic efficiency and nitrogen partitioning

in the shade-demanding plant Panax notoginseng under different growth irradiationlevels The results showed that the stomata's slow and limited photosynthetic responsewas observed at low irradiance (low light conditions) On the contrary, chlorophyllcontent in leaves increased significantly when radiation was reduced, but carboxylationand bioenergetics content decreased Therefore, it is concluded that increasing leaf area

is more important than changing the biochemical physiology of shade-demandingspecies such as P.notoginseng

Another study of Vasyutkina in 2018 on an analyzed comparing the differentgenetic diversity between Panax vietnamensis Ha et Grushv and P ginseng C.A Meyervia ISSR Marker The results showed that all the parameters in this reported of VG aresignificantly higher than that of P ginseng, P vietnamensis has limited distribution innature along with high genetic diversity, so the conservation strategy of this ginsengspecies 1s still very urgent

In 2020, Jang et al There was a study on the physiological response andginsenoside production of Panax ginseng cultures grown under red-to-blue light-emitting diodes at different rates The results showed that when compared with themonochromatic treatments R (red) or B (blue), the overall growth rate of ginsengincreased in treatment R50B50 The higher the R ratio, the higher the ginoside content

in the leaves, while the roots were less affected However, induction withmonochromatic R causes leaf malformation and senescence The combination of R and

B is important in morphological characteristics and ginoside content of ginseng

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In study of Zheng et al 2020, has reported comparing the accumulation ofdynamics in Panax notoginseng of Chinese ginseng, with P vietnamensis, P.stipuleanatus and P bipinnatifidus In this report, 17 elements are studied such as B,

Na, Mg, Al, P, K, Ca, Cr, Mn, Fe, Co, Zn, Mo, Cd, and Pb The results showed that P.vietnamensis and P stipuleanatus had more similarities than P notoginseng and P.bipinnatifidus

2.2 Introduction to plant tissue culture

2.2.1 Difinition

Plant tissue culture is considered the most effective method to study theorganogenesis and morphology of plants According to a 2014 report by Bui et al, thestudied about morphology and anatomy of embryos during embryogenesis of Ngoc Linhginseng (Panax vietnamensis Ha et Grushv.) Tissue culture techniques have helpedplants to grow rapidly and create a phenotypically and genetically homogeneouspopulation, greatly contributing to the rapid multiplication of new and endangered plantvarieties (Vu et al, 2009)

2.2.2 Scientific establishment

2.2.2.1 The totipotency of plant cells

In 1902, a scientist, named Haberlandt proposed that every single cell of amulticellular organism has the potential to develop into a complete individual, and sincethen this view has been taken as the theoretical basis for plant tissue culture From a cellthat develops into a complete tree, it is the expression of the cell's omnipotence, andeach cell has the ability to metabolize specific tissues and organs to perform differentfunctions (Ngo, 2009)

2.2.2.2 Differentiation and dedifferentiation of plant cells

Cell differentiation is the transformation of primary embryonic cells intospecialized tissue cells that perform various functions There are about 15 different types

of tissues in the plant with different functions, but they all originate from embryos thathave undergone differentiation (Ngo, 2009) For example, epidermis tissue isresponsible for protection, etc

Cell dedifferentiation is cells that have undergone differentiation but have notcompletely lost their ability to transform In necessary cases and under certain

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conditions, they can return to the form of embryonic cells and have the ability to dividestrongly (Binh, 2009) For example, during tobacco tissue culture, the differentiatedcells of the leaves under suitable conditions of the medium, will dedifferentiate andcontinuously divide, continuously forming callus The cells of the callus are no longerfunctional cells like leaf cells If the callus is transferred to another medium, depending

on the composition of the medium, callus cells can differentiate towards the formation

of shoots and roots and can form a complete plant (Ngo, 2009)

2.2.3 Methods of plant tissue culture

There are several methods of plant tissue culture, such as growth apical culture,callus culture, single cell, protoplast culture, haploid pollen culture, tissue culture, andseparate organs (Vu, 2009)

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CHAPTER 3 MATERIALS AND METHODS

3.1 Time and location

This study was conducted at Department of Plant Biotechnology — BiotechnologyCenter of Ho Chi Minh City from June, 2022 to June, 2023

3.2 Materials

3.2.1 Research materials

In-vitro Ngoc Linh ginseng with size 1.5 — 2 cm at 6-month-old (Panaxvietnamensis Ha et Grushv.) provided by Deparment of Plant Biotechnology —Biotechnology Center of Ho Chi Minh City

3.2.2 Medium and chemicals used

Cultured medium for the growth of P vietnamensis was MS medium (Murashigeand Skoog, 1962) (Cat#M524, United States) including vitamins supplemented with 30g/L sucrose, 2 g/L Polyvinyl Pyrrolidone (Cat#PB0436, Canada), 0.1 g/L myo-inositol(Cat#IB0536, Canada), 130 mL/L coconut water, 2.20 g/L phyto agar (Cat#P1003,GoldBio), and 1 mg/L vitamins combined with 0.2 mg/L kinetin (Kn) (Cat#KB0745,Biobasic) from stock (Img/mL)

Other cultured medium for the growth of P vietnamensis was SH medium (Schenkand Hildebrandt, 1972) including vitamins supplemented with 30 g/L sucrose, 2 g/LPolyvinyl Pyrrolidone, 0.1 g/L myo-inositol, 130 mL/L coconut water, 2.20 g/L phytoagar and 1 mg/L vitamins combined with 0.2 mg/L kinetin (Kn) from stock (1mg/mL)

All cultured meida were adjusted to pH 5.8 and autoclaved at 121 °C, 1 atm for 20min

3.2.3 Equipments and tools