INOM EXAMENSARBETE BIOTEKNIK,AVANCERAD NIVÅ, 30 HP

, STOCKHOLM SVERIGE 2016

PRODUCTION OF PLANT DEFENSE COMPOUNDS IN CELL CULTURES AND THEIR EFFECTS ON BACTERIAL GROWTH

JUNE WINBLAD

KTHSKOLAN FÖR BIOTEKNOLOGI

www.kth.se

PRODUCTION OF PLANT DEFENSE

COMPOUNDS IN CELL CULTURES AND

THEIR EFFECTS ON BACTERIAL GROWTH

Master degree thesis report

School of Biotechnology, KTH, Stockholm

Supervisors:

Docent Anna Ohlsson

Docent Gunaratna Kuttuva Rajarao

Examiner:

Professor Pål Nyrén

June Winblad

January 2016

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Abstract

The textile industry is using antimicrobial substances to prevent the growth of

microorganisms during storage and transport. Most antimicrobials in use are synthetic

chemical compounds, many of which have proved to be toxic to human health and to the

environment. Some of these substances are not allowed to be used as biocides in the EU. A

prerequisite for an effective ban is the availability of feasible alternatives. One possible

solution would be to identify safe antimicrobial agents from nature.

The purpose of this study was to treat plant cell cultures with elicitors (defense inducing

substances or preparations) and natural stress signal compounds in order to increase their

production of antimicrobial substances. A shoot differentiated Pisum sativum (garden pea)

culture and an undifferentiated fine suspension Populus trichocarpa (black cottonwood)

culture were selected for the experiments. Solutions of the stress signalling compounds

nicotinamide, nicotinic acid, salicylic acid and methyl jasmonate and the elicitor chitosan

were used to induce defense metabolism. Phenolic compounds constitute a group of

secondary metabolites in plants, which are utilized as markers of an activated defense. Cell

extracts from control (untreated) and treated cultures are tested for their effects on bacterial

growth.

The amount of total phenolic substances was analyzed in cell material as well as in culture

medium after harvest of the cultures. The largest effect was a 7.5 times increase in phenolic

content in cell material after methyl jasmonate treatment. Salicylic acid caused increased

phenolic content in Populus culture medium, but overall the excretion of phenolic substances

to the culture medium was not influenced to any great extent. The major part of the phenolics

was retained in the cell material.

Extracts from untreated Populus cells proved to have antibacterial properties against Bacillus

subtilis in an agar plate assay. The effect was even stronger with extracts from salicylic acid

treated culture.

Chitosan did not affect production of phenolic compounds in Populus cells but extracts from

the same cells had antibacterial properties when B. subtilis was grown both on solid (agar)

and in liquid medium, when compared to extract from untreated culture. Also extract from

cells treated with a combination of nicotinamide and methyl jasmonate had an inhibiting

effect in liquid medium, while nicotinamide alone had the opposite effect, leading to

stimulated growth of B. subtilis.

From this study there is no direct evidence to show that phenolic compounds are causing the

antibacterial properties of the plant Populus. The results, however, point at a possibility to

utilize plants or pant cell cultures for the production of substances with antibacterial activity

for use in various applications.

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Our suggestion for further study is to establish the minimum inhibitory concentration (MIC)

and minimum bactericidal concentration (MBC) of Populus cell extracts on Gram-positive

bacteria in agar medium. The natural plant Populus should be tested against both Gram-

positive and Gram-negative bacteria. The use of liquid or agar mediums for the bacterial tests

should be considered.

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Sammanfattning

Textilindustrin använder antimikrobiella ämnen för att förhindra tillväxt av mikroorganismer

under lagring och transport. De flesta antimikrobiella medel som används är syntetiska

kemiska föreningar varav många har visat sig vara giftiga för människors hälsa och för miljön.

Några av dessa ämnen är inte tillåtna som biocider inom EU. En förutsättning för effektiva

förbud är tillgången på genomförbara alternativ. En möjlig lösning skulle vara att identifiera

icke toxiska antimikrobiella ämnen från naturen.

Syftet med den här studien är att behandla växtcellsodlingar med elicitorer (försvars-

inducerande substanser och preparationer) och naturliga stressignalsubstanser för att öka

cellernas produktion av antimikrobiella ämnen. En skott-differentierad kultur av Pisum

sativum (trädgårdsärt) och en odifferentierad finsuspensionskultur av Populus trichocarpa

(jättepoppel) valdes ut för experimenten. Lösningar av stressignalsubstanserna nikotinamid,

nikotinsyra, salicylsyra och metyljasmonat samt elicitorn kitosan användes för att påverka

försvarsmetabolismen. Fenolföreningar är en grupp sekundärmetaboliter i växter som

utnyttjas som markörer för ett aktiverat försvar. Cellextrakt från obehandlade respektive

behandlade kulturer testades avseende effekt på bakteriertillväxt.

Mängden totala fenoliska substanser analyserades i cellmaterialet och i odlingsmediet efter

skörd av kulturerna. Den största effekten var en 7,5 gånger ökad halt av fenoliska substanser i

cellmaterialet efter behandling med metyljasmonat. Salicylsyra orsakade ökad halt av totala

fenoler i Populus odlingsmedium, men totalt sett påverkades inte utsöndringen av fenoliska

substanser till odlingsmediet i någon större utsträckning. Den största andelen fanns kvar i

cellmaterialet.

Extrakt från obehandlade Populus-celler visade sig ha antibakteriell effekt mot Bacillus

subtilis odlad på agarplatta. Effekten var ännu starkare med extrakt från kulturer behandlade

med salicylsyra.

Kitosan påverkade inte produktionen av fenoliska föreningar i Populus-celler, men extrakt

från samma celler hade antibakteriella egenskaper vid odling av B. subtilis både på fast

medium (agar) och i flytande medium, vid jämförelse med odlingsextrakt från obehandlad

kultur. Även extrakt från celler behandlade med en kombination av nikotinamid och

metyljasmonat hade en hämmande effekt i flytande medium, medan nikotinamid ensamt hade

motsatt effekt, nämligen stimulerad tillväxt av B.subtilis.

Från den här studien finns inga direkta bevis för att fenoliska föreningar är ansvariga för de

antibakteriella egenskaperna hos Populus-kulturen. Men resultaten pekar på en möjlighet att

utnyttja växter eller växtcellskulturer för framställning av ämnen med antibakteriell aktivitet

för användning i olika tillämpningar.

Vårt förslag till vidare studier är att fastställa den minsta hämmande koncentrationen (MIC)

och den minsta baktericida koncentrationen (MBC) av Populus cellextrakt på grampositiva

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bakterier odlade på agarmedium. Naturligt växande poppel bör testas mot både grampositiva

och gramnegativa bakterier. Man bör också jämföra resultaten från bakterietester på flytande

respektive fast odlingsmedium.

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List of abbreviations

dH2O: deionized water

IZ: inhibitory zone. That is the area on an agar plate where there is no bacterial growth

because a plant extract is placed.

MBC: minimum bactericidal concentration. That is the lowest concentration of plant extract

resulting in killing ≥ 99.9% of an original inoculum of microorganisms.

MeJA: methyl jasmonate

MeOH: methanol

MIC: minimum inhibitory concentration. That is the lowest concentration of plant extract

resulting in visible inhibition of microorganism growth after overnight incubation.

NIA: nicotinic acid

NIC: nicotinamide

OD: optical density

PS-S: Pisum sativum shoot

SA: salicylic acid

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Table of contents

Abstract ...................................................................................................................................... 1

Sammanfattning ......................................................................................................................... 3

List of abbreviations ................................................................................................................... 5

1. Introduction ............................................................................................................................ 7

1.1 Problem ............................................................................................................................ 7

1.2 Literature review .............................................................................................................. 7

1.3 Plant cell culture ............................................................................................................. 11

1.4 Aims and Goals .............................................................................................................. 13

2. Materials and methods ......................................................................................................... 14

2.1 Treatment of cell cultures ............................................................................................... 14

2.2 Extraction of secondary metabolites and analysis of phenolic production .................... 17

2.3 Antibacterial assay ......................................................................................................... 18

3. Results .................................................................................................................................. 22

3.1 Cell mass and pH ............................................................................................................ 22

3.2 Production of phenolic compounds ................................................................................ 24

3.3 Effects on Bacterial growth ............................................................................................ 27

4. Discussion ............................................................................................................................ 35

4.1 Cell growth, pH and stress response .............................................................................. 35

4.2 Phenolic production and defense response ..................................................................... 35

4.3 Added compounds and defense response ....................................................................... 35

4.4 The NIC effect ................................................................................................................ 36

4.5 Cell extracts and bacterial growth .................................................................................. 36

4.6 Defense compounds and antibacterial effects ................................................................ 37

5. Conclusions .......................................................................................................................... 38

6. Future studies ....................................................................................................................... 39

Appendixes ............................................................................................................................... 40

References ................................................................................................................................ 42

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1. Introduction

1.1 Problem The textile industry is often using antimicrobial substances to prevent the growth of

microorganisms during storage and transportation. Today a significant part of textiles and

ready-made clothes sold in Europe and worldwide are manufactured in the warm and humid

areas of East and Southeast Asia. Most antimicrobials in use today are synthetic chemical

compounds, such as silver compounds, triclosan and triclocarban, many of which have proved

to be toxic to human health as well as to the environment. These compounds are spread via

the food chain, and often not degraded. They may cause cell death and endocrine disruption,

affect reproduction, pollute water and soil, and damage aquatic organisms. (KEMI 2012, 2013;

Bergman et al. 2012)

To counter the application of toxic antimicrobials, EU has issued strict regulations. Some

chemical antibacterial substances are no longer allowed to be used as biocides in the EU.

The Swedish government in 2013 introduced a government bill “Towards a non-toxic

everyday life”, to strengthen the implementation of the regulations. (ECHA 2012;

Regeringskansliet 2013; Mossialos et al. 2010).

Implementation of political decisions requires technically feasibly solutions. The textile

industry therefore needs access to safe antimicrobial agents to replace the toxic chemical ones.

Otherwise these regulations would not be implemented or accepted by the industry. One

possible solution would be to develop safe antimicrobial agents from nature.

1.2 Literature review Naturally safe antibacterial substances from plants have been used since ancient times,

especially as medicine. Some natural substances from plants are toxic to insects and

microorganisms but not necessarily to humans. Some of this ancient knowledge has been

ignored in industrialized societies. The reasons are that synthetic antimicrobials often are very

effective and can be produced at low cost. The danger of these chemicals to human health and

the environment indicates the necessity to identify nontoxic natural substances to replace the

toxic chemicals in use today.

During recent years researchers in different regions have begun investigating plants, from

herbs to trees, testing for antimicrobial effects. Most of these studies are for medical purposes,

basically because resistant bacterial strains have become a major problem due to overuse of

antibiotics. These studies indicate that the extracts of some plants have inhibitory effects

against a range of bacteria and fungi. Some of them have even stronger effects than some

commercial antibiotics.

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1.2.1 Effects against pathogenic microorganisms

Extracts of some Mediterranean plants, Olea europaea, mastic gum, and Inula viscosa,

showed excellent antibacterial properties against bacteria causing oral diseases (Karygianni et

al. 2014).

Jadhav et al. (2014) found that extract of some Indian herbs, Terminalia paniculata,

Terminalia crenulata Roth, Cuscuta reflexa Roxb, Bridelia retusa Spreng and Syzygium

cumini Linn, had inhibitory effects against sexually transmitted pathogens, Neisseria

gonorrhoeae, Haemophilus ducreyi and Candida albicans.

Some plant essential oils and compounds had antibacterial effects against Escherichia coli

O157:H7 and Salmonella enterica in apple juice. (Friedman et al. 2004). Some of the most

effective essential oils and compounds which were active against both bacteria species were

oregano oil, cinnamon leaf oil, lemongrass oil, lemon oil, carvacrol, geraniol, eugenol and

citral.

Essential oils from the Turkish plants Satureja spicigera and Thymus fallax have shown

excellent antibacterial effects against 25 plant pathogenic bacteria strains and seed infections

(Kotan R. et al. 2010). The pathogens’ host plants include cotton, apple, pear, apricot, tomato,

lettuce, cabbage, radish, pepper, zinnia and geranium.

Both antioxidant and antimicrobial effects against aerobic bacteria and mould for lamb patties

preservation in Egypt were reported for extracts from four natural plants, ginseng, jatropha,

jojoba and ginger (Ibrahim et al. 2011).

It is important to realize that even though some plant extracts show antibacterial effects, the

same extracts may also have either antagonistic or synergistic effects together with

conventional antibiotics tested against E. coli causing human infections. (Ushimaru et al.

2012).

While this report was being written, the Nobel Committee awarded half of the 2015 Nobel

Prize in Physiology or Medicine to the Chinese researcher Tu Youyou. She rediscovered and

purified the effective compound, Artemisinin, from the herb Artemisia annua (sweet

wormwood) to cure malaria. Her discovery shows that there is an effective plant-based

solution when synthesized drugs failed due to development of drug resistance in the malaria

parasite.

1.2.2 Effects against Gram-positive bacteria

Many plant extracts have showed inhibitory effects against Gram-positive bacteria. Nostro et

al. (2012) found that extracts of the natural plant or the cell culture of Limonium avei, an

endangered herbal species from the central Mediterranean coastal region, have showed good

inhibitory effect against Gram-positive bacteria, Staphylococcus aureus, Staphylococcus

epidermidis, Listeria monocytogenes, Bacillus subtilis, Enterococcus hirae, as well as the

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methicillin resistant and sensitive strain S. aureus. The natural plant extract has better effects

than those of the cultured plant cells.

Gonçalves et al. (2009) found that the extract of the Mediterranean plant Drosophyllum

lusitanicum showed strong antimicrobial effects against S. epidermidis, S. aureus,

Streptococcus pyogenes and Enterococcus faecalis.

Extracts from two Australian plants, Eremophila neglecta (Ndi et al. 2007) and Eremophila

microthec (Barnes et al. 2013), showed antibacterial effects against S. aureus, S. pyogenes

and Streptococcus pneumoniae.

Tussilago farfara and Urtica dioica are both found in Europe and other parts of the world and

have since ancient times been used in traditional medicine. T. farfara has for example been

used to treat cough and U. dioica for treatment of a range of diseases. Turker et al. (2008)

found that leaf extracts from these plants, as well as extracts from Helichyrsum plicatum

flowers and from Solanum dulcamara aerial parts showed strong inhibitory effects on S.

pyogenes, S. aureus and S. epidermidis.

Joray et al. (2011) reported that the plant-derived compounds, 23-methyl-6-O-

desmethylauricepyrone and (Z,Z)-5-(trideca-4,7-dienyl) resorcinol, had effects against

resistant S. aureus. The synergistic effect of desmethylauricepyrone with erythromycin or

gentamicin had decreased the usage of the two antibiotics by 300 and 260 times respectively.

1.2.3 Effects against both Gram-positive and Gram-negative bacteria

Most reports regarding effects of plant extracts against Gram-negative bacteria also include

Gram-positive bacteria. However, Chang et al. (2008) reported that the essential oil of

Cinnamomum osmophloeum leaf has shown good inhibitory effects against Gram-negative

bacteria Legionella pneumophila, without including any Gram-positive bacteria in their

investigation. The researchers also found that cinnamaldehyde was the dominate component

(>90%) in the essential oil and was the important compound responsible for the antibacterial

properties.

Bartfay et al. (2012) found that extract of the whole herb Epilobium angustifolium showed

excellent antibacterial properties, with great statistical significance, against a variety of both

Gram-positive and Gram-negative bacteria, for instance Micrococcus luteus, S. aureus, E. coli

and Pseudomonas aeruginosa. Moreover, the inhibitory effects of the extract in culture have

shown better results than those of vancomycin and tetracycline.

Another comparison with commonly used antibiotics was done by Soberón et al. (2007) who

found that the alcoholic extracts of the Argentinian plant Tripodanthus acutifolius had better

effects against Gram-positive bacteria S. aureus and Gram-negative bacteria P. aeruginosa

than those given by the commercial antibiotics cefotaxin and oxacillin.

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The plant Eremophila neglecta had good effects against a long row of Gram-positive bacteria,

such as S. pneumonia, S. pyogenes, B. subtilis, Bacillus cereus, Enterococcus faecium, E.

faecalis, Mycobacterium fortuitum, Mycobacterium chelonae, and Erysipelothrix

rhusiopathiae, and also the Gram-negative bacterium Moraxella catarrhalis (Anakok et al.

2011).

It has been reported that both the shape and the membrane surface of bacteria S. aureus and E.

coli were damaged after the bacteria were treated with extracts of the medicinal plant

Melastoma candidum (Yong et al. 2014).

Interesting for the present investigation is the production of terpenoid and phenolic

compounds studied by Lv et al. (2012). It was reported that the essential oil of the Chinese

plant Chimonanthus praecox showed antioxidant effects and excellent inhibitory effects

against Gram-positive bacteria B. subtilis and Gram-negative bacterial Salmonella typhi. The

researchers identified that the main compounds in the essential oil were sesquiterpenoids and

their derivatives (35.97%), monoterpenes and their derivatives (15.87%) and phenolic

compounds (0.35%). The authors suggest that the synergistic action of the three types of

compounds contributes to the antibacterial effect of the essential oil.

A study with relevance for the long term goal of the present investigation is the report by

Baliarsingh et al. (2013) in which they show that the extract of Mangifera indica leaves from

plants grown in India gave good antibacterial effects against both Gram-positive and Gram-

negative bacterial strains, S. aureus, S. pyogenes, E. coli and Klebsiella pneumoniae and when

the extract was used to dye silk and cotton yarns with cationic surfactant, the colors were

strengthened and bacterial growth in the yarns was inhibited.

1.2.4 Effects against fungi

Some plant extracts have inhibitory effects against fungi. Gonçalves et al. (2009) found that

the extract of Drosophyllum lusitanicum had not only effects against Gram-positive bacteria

as mentioned earlier but also against the fungi, C. albicans, Candida famata, Candida

catenulata, Candida guilliermondi, Yarrowia lipolytica, Trichosporon mucoides,

Trichosporon beigelii, and Cryptococcus neoformans.

Extracts of the plants Tagetes minuta, Lippia javanica, Vigna unguiculata and Amaranthus

spinosus grown in South Africa were reported to have inhibitory effects against the

mycotoxigenic fungi Fusarium verticillioides and Fusarium proliferatum (Thembo et al.

2010).

In Nepal, Paudel et al. (2014) found that the essential oil of a parasitic vine, Cuscuta reflexa

Roxb., had moderate inhibitory effect against Aspergillus niger.

The vegetable-pathogenic fungus Colletotrichum lagenarium can cause anthracnose of

cucumbers, with several damaging symptoms on leaves and fruits. Chen et al. (2012) reported

11

that extracts of Cinnamomum camphora (L.) has excellent inhibitory effect against this

fungus and that the fungus hyphae became shortened after the treatment with the extracts.

1.3 Plant cell culture

1.3.1 Induction of plant defense response

In some studies mentioned in Section 1.2 the researchers have tried to identify the effective

compounds in plant extracts responsible for the antimicrobial activities. These compounds are

different kinds of secondary metabolites in plant cells. Such compounds can be produced and

released when plants defend themselves against biotic or abiotic stresses in nature. Such

secondary metabolites can be toxic to insects and microorganism but not necessarily to

humans. (Berglund et al. 2015)

Phenolic compounds are one class of secondary metabolites in plants (Cheynier 2012) and are

well-studied as markers of plant defense system (Ruiz-García and Gómez-Plaza 2013). They

have also been found to have antimicrobial properties (Puupponen-Pimiä et al. 2001; Alves et

al. 2013). Moreover, phenolic compounds can be easily analyzed and compared with results

of other researchers as standard methods are available (Swain and Hillis 1959). In this project

the production of total phenolic compounds was thus studied as an indicator of plant defense

response.

The production of phenolic compounds and other secondary metabolites can be induced by

exposure of plants or plant cells to chemical elicitors or stress signaling compounds (Berglund

et al. 2015). In the present study, the stress signalling compounds nicotinamide (NIC),

nicotinic acid (NIA), salicylic acid (SA) and methyl jasmonate (MeJA) and the elicitor

chitosan have been added to plant cell cultures with the purpose to increase the production of

antimicrobial substances.

NIC and NIA, as the common forms of Vitamin B3, have been found to be able to induce

defense-related metabolism as stress signaling compounds for defense response in plant cells

(Berglund et al. 1993a, 1993b, 2015; Berglund and Ohlsson 1995; Louw et al. 2000; Ohlsson

et al. 2008). NIC has also been suggested to have priming effect, that is, NIC may strengthen

a second stress-signal and thus result in a higher level of defense response (Berglund et al.

2015).

SA itself, originally purified from the bark extract of willow tree, has for years been known

for its anti-inflammatory effect in humans. SA is also an important plant hormone in

regulating the defense response in plants against microbial pathogens (Kumar 2014). When

plant cells were induced with SA it resulted in the accumulation of reactive oxygen species

and expression of defense genes (Vasyukova and Ozeretskovskaya 2007). SA has been shown

to stimulate phenolic synthesis in Salvia miltiorrhiza cell culture (Dong et al. 2010) and in

peas combined with a low pH treatment (McCue et al. 2000).

As an elicitor, chitosan stimulated plumbagin formation in the cell cultures of Plumbago

rosea L. and enhanced permeability of plumbagin from cell to culture medium (Komaraiah et

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al. 2002). Chitosan has been found to stimulate the defense response of plants by inducing

accumulation of phytoalexins (Hadrami et al. 2010). Chitosan has also showed the effect of

increasing total polyphenols in grapes and strawberries (Ruiz-García and Gómez-Plaza 2013).

MeJA has been demonstrated to effectively induce lipid peroxidation and lipoxygenase and to

play an important role in signaling and activating defense responses in tobacco cells (Dubery

et al. 2000). MeJA has also been found to enhance plant defense response after subsequent

stress (Delaunois et al. 2014). MeJA is a derivative from jasmonic acid (JA) and has similar

activity as JA in increasing the biosynthesis of polyphenols in plants (Ruiz-García and

Gómez-Plaza 2013). JA has showed the eliciting effect of increasing production of

phenylpropanoids and naphtodianthrones in Hypericum perforatum L. cell culture

(Gadzovska et al. 2007).

NIC, NIA, SA, chitosan and MeJA, were therefore used in this project with the purpose to

induce plant defense systems and to stimulate production of antimicrobial substances in plant

cell cultures.

1.3.2 Treatment of plant cell cultures

In the studies mentioned in Section 1.2, it can be seen that the researchers often choose to

study the common plant species in their regions. In this degree project the species Pisum

sativum (garden pea) and Populus trichocarpa (black cottonwood) were used for studies as

representatives as they are common plant species in many parts of the world. Populus belong

to Salicaceae Family, also called as Willow Family.

Extract of pea peel has been found to have antioxidant and antimicrobial effects (Hadrich et al.

2014). Extract of pea seed has been used to control biofilm formation by inhibiting P.

aeruginosa and the tannins and phenolic compounds were found to be responsible for its

antibacterial property (Dazal et al. 2015). This leads to a suggestion of using pea peel waste

for production of antibacterial compounds. Antimicrobial flavonoids were found from the

twigs of Populus and effective against plant pathogens, Pseudomonas lachrymans, Ralstonia

solanacearum, Xanthomonas vesicatoria and Magnaporthe oryzae. (Zhong et al. 2012).

From the buds of Populus anitibacterial dihydrochalcone derivatives were found to be

effective against S. aureus (Lavoie et al. 2013).

Cell cultures of these two plants (P. sativum and Populus) have been well-studied for their

defense mechanisms by a research group at the School of Biotechnology, KTH. This degree

project was the first attempt to investigate the production levels of phenolic compounds by the

two plant cell cultures after treatment with NIC, NIA, SA, chitosan and MeJA, as well as

resulting effects on bacterial growth. The experiments were carried out with simple

combinations to obtain experience and initial screening.

The P. sativum culture was treated with NIC, NIA and SA. The Populus culture was treated

with SA, chitosan and MeJA. When the outcomes showed that Populus cell had antibacterial

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activities, more additions (NIC, SA, chitosan and MeJA) were made to the Populus culture.

The suggested priming effects of NIC were also investigated.

According to findings from previous research in plant defense mechanisms at KTH the plant

cells need some time, in this case three days, to perform the defense response after being

stressed. Some defense reactions may be missed if it takes a longer time before the cells are

harvested. NIC as a priming factor also needs time, in this case one day, to “prime” the cells

so that they could become more sensitive to stress factors. In this degree project the rest of the

additions to the cell cultures were therefore done three days before harvesting and NIC as a

priming factor was added one day earlier.

The technology of plant cell cultivation was used in this project. The advantage is that the

researcher can obtain a simplified and controlled experimental system and get quick results

within a short period (Berglund et al. 1993; Ohlsson et al. 2006). Once the potential

compounds are identified from the cells it is possible to produce them in large scale under

controlled conditions. In this way the compounds can be produced without modifying their

structure and functionality. The disadvantage is that the result obtained from plant cell

cultures may not in all cases reflect the effects of natural plants (Nostro et al. 2012).

1.3.3 To investigate effects on bacterial growth

Gram-positive bacteria B. subtilis and Gram-negative E. coli were used in the project. The

two bacteria are well-studied and often used as representatives of the two Gram strains. They

are also relatively safe when being used in a non-clinic laboratory.

To investigate bacterial growth, agar plate assay and microtiter (96-well) plate assay were

used in this project. The advantage of agar plate assay is that it is simple and quick way to

obtain qualitative results. A disadvantage is that one doesn’t know what might have happened

during incubation if, for instance, the bacteria were inhibited first but then came back to take

over. The advantage of microtiter plate assay is, however, that the dynamic process can be

observed by recording the growth curve of the microorganisms, and that it can provide

quantitative results. The disadvantage is that it is more complicated and very much dependent

on accurate and sensitive equipment.

1.4 Aims and Goals

This Master degree project is the first step towards the long term goals. The specific aim of

the degree work is restricted to stimulating production of defense compounds marked by

phenolic compounds from plants, and investigating their effects on bacterial growth.

The long term goals of this research is to find and test natural antimicrobial substances from

plant cell cultures, develop feasible methods for production of such substances, and to

develop alternatives for industrial application to enable for instance the textile industry to

abandon the use of toxic antimicrobials.

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2. Materials and methods

The experiments performed in the degree project consist of three major parts: 1) to treat plant

cell cultures with elicitors and stress signalling compounds, 2) to extract secondary

metabolites from the cell material and to analyze the production of phenolic compounds from

the extracts, and 3) to investigate the growth of bacteria when they are exposed to the extracts.

2.1 Treatment of cell cultures 2.1.1 Plant material

Pisum sativum shoot (PS-S) tissue differentiated culture and Populus trichocarpa (Populus)

fine suspension culture were available in the laboratory at the School of Biotechnology, KTH.

The PS-S culture was cultivated in modified Murashige-Skoog (MS) medium MS-III GA3

(for formula of the medium see Appendix 1), and sub-cultivated every second week for

maintenance (Berglund et al. 1993b).

The Populus cell culture was cultivated in modified MS-I medium (for formula of the

medium see Appendix 1), and sub-cultivated weekly for maintenance in the same way as

described for hybrid aspen suspension culture (Ohlsson et al. 2006).

Both cultures were grown at room temperature at a photoperiod of 12 h fluorescent light and

12 h dark. All work with plant cultures until the point of harvest were carried out under sterile

conditions.

2.1.2 Chemicals

The compounds used were NIC (Merck), NIA (Merck), SA (Analar), chitosan (Aldrich) or

MeJA (Aldrich). The preparation of their stock solutions is specified in Table 1. Chitosan was

solubilized in 0.1 mM HCl at 65°C for a short time, whereafter pH was adjusted to

approximate 5 with 1 M NaOH. The chitosan stock solution was sterilized by autoclaving.

The other stock solutions were filter-sterilized with 0.20 µm non-pyrogenic sterile filters.

Table 1 Preparation of stock solutions.

Compound Weight (g)

Solvent Stock concentration (mM)

NIC (122.13 g/mol) 0.2443 100 ml dH2O 20

2.4426 100 ml dH2O 200

NIA (123.11 g/mol) 0.2462 100 ml dH2O 20

SA (138.13 g/mol) 0.1381 100 ml 10% MeOH 10

0.3453 100 ml 10% MeOH 25

Chitosan(low molecular) 0.6700 100 ml 0.1 mM HCL (65 oC) 6.7 mg/ml (pH5)

Chitosan (6.7 mg/ml) 1.5 ml 8.5 ml dH2O 1 mg/ml

MeJA (224.30 g/mol or 1.03 g/ml)

22 µl 1 ml pure MeOH + 1 ml

dH2O

50

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2.1.3 Treatments of plant cell cultures

Treatment 1

1. Preparing 12 sterilized erlenmeyer flasks of modified MS-III GA3 medium, each

containing 50 ml.

2. Selecting a flask of well-grown PS-S cell culture from previous sub-cultivations.

Filtering the culture to get the shoot tissues, carefully dividing the tissues into tiny

pieces, weighing and inoculating 2.5 g (wet weight) of fresh parts into each of the 12

flasks.

3. Incubating the 12 flasks of cultures for 14 days. On the 11th

day, adding NIC, NIA or

SA into the cultures in triplicates. The additions are specified and the cultures are

coded in Table 2.

4. Harvesting the tissue cultures on the 14th

day.

Table 2 Additions to cell cultures and codes given to the cultures in Treatment 1.

Culture no. Addition Final concentration (mM)

Code*

1 5 ml dH2O - 1-PS-untr

2 5 ml dH2O -

3 5 ml dH2O -

4 5 ml 20 mM NIC 2 1-PS-NIC

5 5 ml 20 mM NIC 2

6 5 ml 20 mM NIC 2

7 5 ml 20 mM NIA 2 1-PS-NIA

8 5 ml 20 mM NIA 2

9 5 ml 20 mM NIA 2

10 5 ml 10 mM SA 1 1-PS-SA

11 5 ml 10 mM SA 1

12 5 ml 10 mM SA 1

(* the code consists of three parts: the first part, ‘1’, means it is from Treatment 1. The second part,

‘PS’, means PS-S culture. The third part indicates which substance was used. In the third part ‘untr’

means untreated, that is with no addition.)

Treatment 2

1. Preparing 12 sterilized erlenmeyer flasks of modified MS-I medium, each containing

50 ml.

2. Selecting a flask of well-grown Populus cell culture from previous sub-cultivations.

Filtering the culture to get the cells, inoculating 2.5 g (wet weight) of cells into each of

the 12 flasks.

3. Incubating the 12 flasks of cultures for 7 days. On the 4th

day, adding SA, chitosan or

MeJA into the cultures in triplicates. The additions are specified and the cultures are

coded in Table 3.

4. Harvesting the cell cultures on the 7th

day.

16

Table 3 Additions to cell cultures and codes given to the cultures in Treatment 2.

Culture no. Addition Final concentration (mM)

Code*

1 5 ml dH2O - 2-PO-untr

2 5 ml dH2O -

3 5 ml dH2O -

4 5 ml 10 mM SA 1 2-PO-SA

5 5 ml 10 mM SA 1

6 5 ml 10 mM SA 1

7 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml 2-PO-chi

8 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml

9 0.75 ml 6.7 mg/ml Chitosan 0.1 mg/ml

10 0.2 ml 50 mM MeJA 0.2 2-PO-MeJA

11 0.2 ml 50 mM MeJA 0.2

12 0.2 ml 50 mM MeJA 0.2

(* the code consists of three parts: the first part, ‘2’, means it is from Treatment 2. The second part,

‘PO’, means Populus culture. The third part indicates which substance was used. In the third part ‘untr’

means untreated, that is with no addition, and ‘chi’ means chitosan.)

Treatment 3

1. Preparing 15 sterilized erlenmeyer flasks of modified MS-I medium, each containing

50 ml.

2. Selecting a flask of well-grown Populus cell culture from previous sub-cultivations.

Filtering the culture to get the cells, inoculating 2.5 g (wet weight) of cells into each of

the 15 flasks.

3. Incubating the 15 flasks of culture for 7 days. On the 3rd

day, adding NIC as a priming

factor into 8 of the 15 flasks of culture. On the 4th

day, adding SA, chitosan or MeJA

into the cultures in duplicates. The additions are specified and the cultures are coded

in Table 4.

4. Harvesting the cell cultures on the 7th

day.

17

Table 4 Additions to cell cultures and code of cultures in Treatment 3.

Culture no.

Addition on the 3rd day

Final concentration

(mM)

Addition on the 4th day

Final concentration

(mM)

Code*

1 0.5 ml dH2O - - 3-PO-untr

2 0.5 ml dH2O - -

3 0.5 ml dH2O - -

4 0.5 ml 200 mM NIC 2 - 3-PO-NIC

5 0.5 ml 200 mM NIC 2 -

6 0.5 ml dH2O - 2 ml 25 mM SA 1 3-PO-SA

7 0.5 ml dH2O - 2 ml 25 mM SA 1

8 0.5 ml dH2O - 0.75 ml 1 mg/ml

chitosan

0.015 mg/ml 3-PO-chi

9 0.5 ml dH2O - 0.75 ml 1 mg/ml

chitosan

0.015 mg/ml

10 0.5 ml 200 mM NIC 2 2 ml 25 mM SA 1 3-PO-

NIC+SA 11 0.5 ml 200 mM NIC 2 2 ml 25 mM SA 1

12 0.5 ml 200 mM NIC 2 0.75 ml 1 mg/ml

chitosan

0.015 mg/ml 3-PO-NIC+chi

13 0.5 ml 200 mM NIC 2 0.75 ml 1 mg/ml

chitosan

0.015 mg/ml

14 0.5 ml 200 mM NIC 2 0.2 ml 50 mM

MeJA

0.2 3-PO-

NIC+MeJA

15 0.5 ml 200 mM NIC 2 0.2 ml 50 mM

MeJA

0.2

(* the code consists of three parts: the first part, ‘3’, means it is from Treatment 3. The second part,

‘PO’, means Populus culture. The third part indicates which substance(s) were used. In the third part

‘untr’ means untreated, that is with no addition, and ‘chi’ means chitosan.)

2.1.4 Harvesting of cultures

Plant cell cultures were harvested by vacuum filtration. The mass weight was measured. The

plant cell material was collected into Falcon tubes and quickly frozen in liquid nitrogen. The

tubes were then stored in a refrigerator at - 18 oC for later analysis.

The pH of the filtered medium was also measured.

2.2 Extraction of secondary metabolites and analysis of phenolic

production 2.2.1 Extraction of secondary metabolites from plant cell material

The harvested cell material was first homogenized in liquid nitrogen and then extracted with

80% MeOH as follows (Swain and Hillis 1959):

1. Place 100 mg of the homogenized cell material in a 1.5 ml Eppendorf tube.

2. Add 1 ml 80% MeOH into the tube, and vortex the mixture thoroughly for ca 2

minutes.

3. Centrifuge the mixture at 13 000 rpm for 10 minutes at room temperature.

4. The supernatant is the extract and is transferred into a new Eppendorf tube.

5. The concentration of the extract is 0.1 mg/µl. (The concentration is defined as the

amount of extracted plant cell material per unit volume of the used solvent.)

18

When a large amount of cell material, for instance 5 g, was extracted the procedure was as

follows:

1. Place 5 g of cell material into a 50 ml Falcon tube.

2. Add 20 ml 80% MeOH into the tube, and vortex the mixture thoroughly for ca 2

minutes.

3. Centrifuge the mixture at 4750 rpm for 20 minutes at 4 oC.

4. Transfer the supernatant into a new 50 ml Falcon tube.

5. Add 20 ml 80% MeOH into the sediment, vortex thoroughly for ca 2 minutes.

6. Centrifuge the secondary mixture at 4750 rpm for 20 minutes at 4 oC.

7. Transfer the supernatant again into the same Falcon tube as in step 4.

8. Add another 10 ml 80% MeOH into the sediment again, vortex thoroughly for ca 2

minutes.

9. Centrifuge the third-time mixture at 4750 rpm for 20 minutes at 4 oC.

10. Transfer the supernatant again into the same Falcon tube as in step 4 and 7.

11. The supernatant is the extract. The concentration of the extract is 0.1 mg/µl.

When the primary extract had to be concentrated, a rotative evaporation method was used to

concentrate the extract at 150 mbar for 25-30 minutes at 40 oC. The volume could be reduced

from 50 ml to about 10 ml.

2.2.2 Analysis of production of phenolic compounds from cell material

Phenolic compounds were analyzed according to the Swain and Hillis method (1959). A

standard curve was created each time when samples were analyzed as follows:

1. For a standard curve, take a series of volumes such as 0, 10, 25, 50, 100 and 200 µl,

respectively, of 0.1 mg/ml chlorogenic acid into a 1.5 ml Eppendorf tube. For cell

extract samples, take a suitable volume.

2. Add dH2O to a total volume of 700 µl in each tube.

3. Add 50 µl Folin-Denis reagent into each tube, mix and then let it stand still for 3

minutes for reactions.

4. Add 100 µl saturated Na2CO3 and mix.

5. Add 150 µl dH2O and mix, then let it stand still for 1 hour for reactions.

6. Centrifuge the mixtures at 13 000 rpm for 10 minutes.

7. Transfer 700 - 800 µl the supernatant into a cuvette.

8. Measure absorbance at 765 nm.

2.3 Antibacterial assay Antibacterial assay in this project included agar plate assay and microtiter (96-well) plate

assay.

2.3.1 Bacterial strains

The extracts of plant cell material were tested against two bacterial strains, Bacillus subtilis

ATCC 21332 (Gram-positive) and Escherichia coli ATCC 25404 (Gram-negative). The two

bacterial strains were available in the laboratory.

19

Both bacteria were grown with Nutrient Agar medium. B. subtilis was incubated overnight at

30 oC while E.coli was incubated overnight at 37

oC. Two agar plates of each bacterial strain

were kept at 4 oC and subcultured every 2 weeks for maintenance.

2.3.2 Agar plate assay

Both bacterial strains were tested with the agar plate method. The procedure was as follows:

1. Autoclaved Nutrient agar was poured to each petri plate to a depth of 4 mm,

equivalent of 25 ml liquid agar to a 100-mm plate. The agar plates were left to solidify

until there was no visible liquid on the surface.

2. Take the bacterial overnight culture, dilute the culture with sterile saline solution to an

OD of 0.1 at 600 nm.

3. Spread 150 µl the diluted culture evenly on each Nutrient Agar plate. Leave the

culture to soak into the agar.

4. Use a sterile metal cylinder to punch 4 wells with a diameter of 6.5 mm in each agar

plate. The wells should be placed evenly on the agar surface as shown in Figure 1.

5. Add 30 µl solvent into a well as negative control, load cell extract samples with a

series of 3 doses into the other 3 wells. The load with lower dose was diluted with

dH2O. One agar plate for each sample of the cell extracts. The load of samples is

illustrated in Figure 1. The doses added for each agar plate assay are specified in Table

5.

6. Incubate the agar plates overnight.

7. Observe the agar plate next morning if there is any inhibitory zone(s). Inhibitory zone

(IZ) is the zone where there is no bacterial growth around the well. The larger the

zone is, the better the inhibitory effect against bacteria is. IZ examples are shown in

Figure 1.

8. Use a ruler to measure the diameter of IZ(s) if it appears. When measuring, round up

to the next millimeter including the diameter of the well.

Figure 1 Example of agar plate test design. Inhibitory Zones are shown.

20

Table 5 Doses of cell extracts used in Agar plate assay and bacteria tested.

Samples Concentration (mg/µl)

Dose in different load (mg) 30 µl 20 µl 10 µl

Tested bacteria

1-PS-untr 1-PS-SA

0.1

-"-

3

-"-

2

-"-

1

-"-

B.subtilis

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

0.25

-"-

-"-

-"-

7.5

-"-

-"-

-"-

5

-"-

-"-

-"-

2.5

-"-

-"-

-"-

B.subtilis

2-PO-untr 2-PO-MeJA

0.1

-"-

3

-"-

2

-"-

1

-"-

B.subtilis

2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA

0.25

-"-

-"-

-"-

7.5

-"-

-"-

-"-

5

-"-

-"-

-"-

2.5

-"-

-"-

-"-

B.subtilis

E.coli

3-PO-untr 3-PO-NIC 3-PO-SA 3-PO-chi 3-PO-NIC+SA 3-PO-NIC+chi 3-PO-NIC-MeJA

0.4

-"-

-"-

-"-

-"-

-"-

-"-

12

-"-

-"-

-"-

-"-

-"-

-"-

8

-"-

-"-

-"-

-"-

-"-

-"-

4

-"-

-"-

-"-

-"-

-"-

-"-

B.subtilis

2.3.3 Microtiter 96-well plate assay

Populus cell extracts from Treatment 3 were tested against B. subtilis with the 96-well plate

method. The procedure was as follows:

1. Dilute the Nutrient Broth medium with sterile dH2O to 1:10.

2. Take the bacterial overnight culture, dilute it with 1:10 medium to an optical density

(OD) of 0.1 absorbance at 600 nm.

3. Add the cell extract to a sterile microtiter 96-well plate (Sarstedt) (see the example

design in Figure 2), the total volume of each well was 100 µl, as detailed below:

1:10 medium blank: 100 µl.

Culture control (OD=0.1): 100 µl.

Sample, dose 1: 5 µl cell extract + 5 µl dH2O + 90 µl culture.

Sample, dose 2: 10 µl cell extract + 90 µl culture.

Solvent control: 10 µl solvent + 90 µl culture.

The cell extracts from Treatment 3 had a concentration of 5 mg/µl.

The doses applied were 25 or 50 mg.

4. Add 100 µl dH2O into each of the remaining wells to keep moisture in the plate,

5. Incubate the plate with lid overnight in a FLUOstar Optima device (BMG Labteck) at

30 oC with orbital shaking.

6. Absorbance (OD value) of each well was read and recorded every half an hour with an

A-620 filter.

21

Sample1 Sample2 Sample3 Sample4 Sample5 Sample6 Sample7

1 2 3 4 5 6 7 8 9 10 11 12 A dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O

B dH2O Medium Blank

Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control

dH2O

C dH2O Medium Blank

Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control

dH2O

D dH2O Medium Blank

Culture control

Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Dose 1 Solvent control

dH2O

E dH2O Medium Blank

Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control

dH2O

F dH2O Medium Blank

Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control

dH2O

G dH2O Medium Blank

Culture control

Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Dose 2 Solvent control

dH2O

H dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O dH2O

Figure 2 Example of microtiter 96-well plate test design. Each dose is at least in triplicate.

Controls and blanks are at least in duplicate.

22

3. Results

This chapter presents the results from 1) measurement of cell mass and pH in plant cell

culture medium, 2) analysis of content of phenolic compounds in the cell extracts and in plant

cell culture medium and 3) investigation of the effects of the cell extracts on bacterial growth.

3.1 Cell mass and pH In treatment 1, all additions to the PS-S culture caused a small decrease in growth and a clear

increase in pH in used culture medium compared to the control culture (Figure 3). The

strongest effect was seen after SA addition, from 9.7 g to 8.1 g cell mass and from pH 5 to pH

6.2.

In Treatment 2, the cell mass of Populus culture (Figure 4) was affected only by MeJA, which

caused a decrease from 12.8 g to 10.6 g. In this culture, medium pH in the SA treated culture

(pH 6.2) was lower than in the control (pH 6.5), but it was higher in the chitosan and MeJA

treated cultures, pH 6.9 and pH 6.7, respectively.

In treatment 3, growth was inhibited by all additions, except for chitosan, which stimulated

growth to some degree (Figure 5). Chitosan also caused an increased medium pH, while the

rest of the additions resulted in lower pH, especially SA. In SA-treated culture, pH was 5.2,

much lower than in the control culture (pH 6.4).

23

Figure 3 Cell mass and pH value of PS-S cultures after addition of different substances in

Treatment 1. Averages from triplicate samples with standard deviations are shown.

Figure 4 Cell mass and pH values of Populus cell cultures after addition of different substances

in Treatment 2. Averages from triplicate samples with standard deviations are shown.

Figure 5 Cell mass and pH value of Populus cell cultures after additions of different substances

in Treatment 3. Values are from only one sample for each treatment.

4,5

5,0

5,5

6,0

6,5

7,0

0

2

4

6

8

10

12

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

Ave

rage

pH

of

cult

ure

m

ed

ium

Ave

rage

ce

ll m

ass

(g)

Cell cultures

Mass pH

6,0

6,2

6,4

6,6

6,8

7,0

7,2

0

2

4

6

8

10

12

14

2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA

Ave

rage

pH

of

cult

ure

m

ediu

m

Ave

rage

ce

ll m

ass

(g)

Cell cultures

Mass pH

5,0

5,2

5,4

5,6

5,8

6,0

6,2

6,4

6,6

6,8

7,0

0

2

4

6

8

10

12

14

16p

H v

alu

e o

f cu

ltu

re m

ediu

ms

Cel

l mas

s (g

)

Cell cultures

Mass pH

24

3.2 Production of phenolic compounds In PS-S culture medium from Treatment 1 the content of phenolic substances seemed to

increase by all treatments, although not to a significant degree (Figure 6).

In cell extracts from the same cultures (Figure 6), no differences were seen, except for a small

decrease caused by SA.

When looking at total content of phenolic substances in the cultures (Figure 7), a clear

lowering effect can be seen after SA addition, dependent partly on the lower cell mass of this

culture. Figure 7 also illustrates that the major part (64 - 82 %) of the phenolic substances

were found in the cell material.

Figure 6 Contents of phenolic compounds in PS-S cultures after addition of different

substances in Treatment 1. Averages from triplicate samples with standard deviations are shown.

Figure 7 Production and distribution of phenolic compounds in PS-S cultures in Treatment 1.

05

1015202530354045505560

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

Ph

eno

lic c

on

ten

ts (

µg/

ml)

PS-S culture medium

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

Ph

eno

lic c

on

ten

ts (

µg/

mg)

PS-S cells

0

2

4

6

8

10

12

1-PS-untr 1-PS-NIC 1-PS-NIA 1-PS-SA

Ph

eno

lic p

rod

uct

ion

(m

g)

PS-S cultures

In culture medium In tissues

25

In Treatment 2, no effects on phenolic content in the culture medium from the Populus culture

were observed (Figure 8). However, in the cell material a large increase in phenolics was

measured in extracts from MeJA treated cultures, from 0.7 µg/mg to 6 µg/mg, which

represents a 8.5 times increase.

The distribution of phenolic compounds between culture medium and cell material is

illustrated in Figure 9. Approximately 90 % of the phenolics were found in the cell extracts

from the control, SA-treated and chitosan-treated cultures, while in the MeJA-treated culture

the large increased amounts of phenolic substances produced were retained in the cells, i.e.

98 % of total phenolics found in the cell material.

Figure 8 The total phenolic contents in Populus cell cultures after addition of different

substances in Treatment 2. Averages from triplicate samples with standard deviations are shown.

Figure 9 Production and distribution of phenolic compounds in Populus cultures in Treatment 2.

Averages from triplicate samples with standard deviations are shown.

In Treatment 3, the content of phenolic substances in the medium from the Populus culture

(Figure 10) was increased by some of the treatments. SA caused the largest increase from 17

µg/ml to 52 µg/ml. This increase seemed to be partially inhibited by a previous addition of

02468

10121416182022242628

Ph

en

olic

co

nte

nts

(µ

g/m

l)

Populus culture mediums

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

Ph

en

olic

co

nte

nts

(µ

g/m

g)

Populus cells

0

10

20

30

40

50

60

70

80

2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA

Ph

en

olic

pro

du

ctio

n (

mg)

Populus cultures

In culture medium

In cells

26

NIC, resulting in a content of 35 µg/ml. On the other hand, a combination of NIC and

chitosan resulted in higher phenolic content than either NIC or chitosan alone. By themselves

they did not differ much from the control. Also a combination of NIC and MeJA gave a

higher content than the control.

Similarly there was an increased phenolic content also in cell extracts (Figure 10) after SA

addition. The most prominent effects on phenolics in cell extracts were caused by NIC and

MeJA in combination, 2.8 µg/mg compared to the control value 0.9 µg/mg.

The distribution of phenolic substances between culture medium and cells (Figure 11)

followed approximately the same pattern as for cell extracts as such. In this experiment, like

in the other two, the highest quantity of phenolic substances was found in the cell material.

Figure 10 Phenolic contents in Populus culture after addition of different substances in

Treatment 3. Values from only one sample for each elicitation are shown.

Figure 11 Production and distribution of phenolic compounds in Populus cultures, Treatment 3.

Values from only one sample for each elicitation are shown.

0

10

20

30

40

50

60

Ph

en

olic

co

nte

nt

(µ

g/m

l)

Populus culture mediums

0,0

0,5

1,0

1,5

2,0

2,5

3,0

Ph

en

olic

co

nte

nt

(µ

g/m

g)

Populus cells

0

5

10

15

20

25

Ph

en

olic

pro

du

ctio

n (

mg)

Populus cultures

In culture medium

In cells

27

3.3 Effects on Bacterial growth Plant cell extracts were tested for effects on bacterial growth. One reason to choose extracts

rather than culture medium was that most of the phenolic production in cell cultures was

found in the cells. Another reason was that plant extract is the major study objective in most

of other relevant researches so that the outcome of this project could be compared with results

of other studies.

3.3.1 Agar plate assay

Cell extracts from Treatment 1

Among the four PS-S cell extracts from Treatment 1, the 1-PS-untr and the 1-PS-SA were

first selected for tests on bacteria since the addition of SA resulted in the largest differences in

cell mass, pH, and phenolic production compared to the untreated.

The two extracts had a concentration of 0.1 mg/µl and were tested on B. subtilis cultured on

agar plates. After overnight incubation no inhibitory zone was observed.

The four PS-S cell extracts were concentrated to 0.25 mg/µl. All four extracts were tested on

B. subtilis cultured on agar plates. After overnight incubation no inhibitory zone was observed.

Cell extracts from Treatment 2

Among the four Populus cell extracts from Treatment 2, the 2-PS-untr and the 2-PS-MeJA

were tested first on B. subtilis agar plates because the addition of MeJA resulted in an extra

high production of phenolic compounds compared to that of the untreated one.

The two extracts had a concentration of 0.1 mg/µl. After overnight incubation no inhibitory

zone was observed.

The four Populus cell extracts were concentrated to 0.25 mg/µl and tested on B. subtilis agar

plates. After overnight incubation inhibitory zones were observed. The size of the inhibitory

zones are shown in Figure 12.

The samples with the highest dose, 7.5 mg, all showed inhibitory effects on bacteria B.

subtilis. The 2-PO-MeJA and the 2-PO-SA cell extracts both resulted in a 10.5 mm of

inhibitory zone, and 2-PO-untr gave 7.5 mm. With medium dose, 5 mg, the 2-PO-chi and the

2-PO-SA cell extracts had inhibitory zones of 7.5 mm and 8.5 mm respectively. The lowest

dose, 2.5 mg, and the negative control gave no inhibitory effects.

Figure 13 is a photo of an incubated B. subtilis agar plate. The inhibitory zones were caused

by the 2-PO-SA cell extract.

28

Figure 12 Size of inhibitory zones by Populus cell extracts on B. subtilis cultured agar plates.

The cell extracts were from Treatment 2. The dose of 2.5 mg and the negative control gave no

inhibitory effects.

Figure 13 Photo of a B. subtilis cultured agar plate. Two inhibitory zones were observed around

two wells marked as 5(mg) and 7.5(mg). The sample was a cell extract of the 2-PO-SA.

6,5

7

7,5

8

8,5

9

9,5

10

10,5

11

2-PO-untr 2-PO-SA 2-PO-chi 2-PO-MeJA

Dia

me

ters

of

inh

ibit

ory

zo

ne

s (m

m)

(Dia

me

ter

of

we

lls w

as 6

.5 m

m)

Populus cell extracts

Negative control 2.5mg 5mg 7.5mg

29

Cell extracts from Treatment 3

The seven Populus cell extracts from Treatment 3 had a concentration of 0.4 mg/µl and were

tested on B. subtilis cultured agar plates. After overnight incubation inhibitory zones were

found on five of the seven agar plates. The sizes of the inhibitory zones are shown in Diagram

A of Figure 14. Negative controls gave no inhibitory effect.

Cell extracts from the following cultures showed antibacterial effects: the 3-PO-untr, the 3-

PO-NIC, the 3-PO-SA, the 3-PO-chi, and the 3-PO-NIC+chi, with inhibitory zones between

7.5 and 10 mm. The 3-PO-SA cell extract resulted in inhibitory effects with all doses. The

lowest dose of the other four samples did not show any inhibitory effect.

This test was repeated nine days later. Inhibitory zones were observed again, and still from

the five samples. The sizes are shown in Diagram B of Figure 14.

In the repeated test the 3-PO-SA and the 3-PO-NIC+chi samples showed inhibitory effects at

medium and higher doses, with inhibitory zones between 7 and 10 mm. None of the lower

doses showed any inhibitory effect this time.

Figure 15 shows photo of two B. subtilis cultured agar plates. The inhibitory zones were

caused by the 3-PO-SA cell extract. The left-hand plate was from the first test and the right-

hand one was from the repeated test. The inhibitory zones on the right-hand plate are not as

clear as the ones in the left-hand due to lack of back light.

The seven Populus cell extracts from Treatment 3 were tested on E.coli cultured agar plates.

After overnight incubation no inhibitory zones were found. This test was repeated with the

same result.

30

Figure 14 Size of inhibitory zones by Populus cell extracts on B. subtilis cultured agar plates.

The cell extracts were from Treatment 3. Diagram A: the first test. Diagram B: the repeated test.

Figure 15 Photos of two B.subtilis cultured agar plates. Inhibitory zones were observed around

three wells marked as 10(µl), 20(µl) and 30(µl) on the left-hand plate, and around two wells

marked as 20(µl) and 30(µL) on the right-hand. The left-hand plate was from the first test and

the right-hand from the repeated test. The samples were the 3-PO-SA cell extracts.

6,5

7

7,5

8

8,5

9

9,5

10

10,5

11

11,5D

iam

ete

rs o

f in

hib

ito

ry z

on

es

(mm

) (D

iam

ete

r o

f w

ells

was

6.5

mm

)

Populus cell extracts

Negative control 4 mg 8 mg 12 mg A

6,57

7,58

8,59

9,510

10,511

11,5

Dia

me

ters

of

inh

ibit

ory

zo

ne

s (m

m)

(Dia

me

ter

of

we

lls w

as 6

.5 m

m)

Populus cell extracts

(Repeated 9 days later)

Negative control 4mg 8mg 12mgB

31

3.3.2 Microtiter 96-well plate assay

Based on the outcomes of agar plate assay the seven Populus cell extracts from Treatment 3

were concentrated to 5 mg/µl and tested on B. subtilis with the microtiter 96-well plate assay.

Figure 16 shows a photo of one microtiter plate test.

The growth curves of bacteria B. subtilis presented in Figures 17- 22 are based on average

values. Each figure includes growth curves of the bacteria exposed to an cell extract from a

treated culture v.s the 3-PO-untr cell extract, both in two different doses, and the growth curve

of the bacteria in the control culture.

In Figure 17, the curve with 50 mg dose of 3-PO-chi cell extract run below the curve of the

culture control from the 5th

hour to the end of the incubation period. The other three curves

stayed above that of the culture control all the time. Moreover the two curves from exposure

to 3-PO-untr were above the other three from the 4th

hour.

Figure 18 shows a similar situation for bacteria growth exposed to the 3-PO-NIC+chi. The

curve with 50 mg dose of 3-PO-NIC+chi cell extract went below the one of the culture control

from the 5th

hour to the end. The two curves from exposure to the 3-PO-untr were above the

other three from the 4th

hour.

This indicates that the increased dose of 3-PO-chi or 3-PO-NIC+chi cell extract inhibited the

bacterial growth while the two samples in lower dose and 3-PO-untr samples in both doses

stimulated the bacteria growth by different degrees.

The findings shown in Figures 17 and 18 are consistent with the observed results from the

microtiter plate, Columns 7 and 9, in Figure 16.

In Figure 19 the two growth curves of bacteria exposed to the 3-PO-NIC+MeJA cell extracts

went below the curves of bacteria exposed to the 3-PO-untr cell extracts from the 5th

hour to

the end but slightly over the curve of the culture control. The two curves of the 3-PO-

NIC+MeJA started from a higher OD than the other three curves which shows that the 3-PO-

NIC+MeJA cell extract may contain some kinds of compounds causing a higher absorption at

the wave length. The 3-PO-NIC+MeJA cell extract did not inhibit the bacterial growth, nor

did it stimulate it.

The other Populus cell extracts from the 3-PO-NIC, the 3-PO-SA or the 3-PO-NIC+SA all

had an effect of stimulating the bacterial growth shown in Figures 20-22 respectively. The 3-

PO-NIC cell extracts had the strongest stimulation. The 3-PO-SA cell extracts had similar

stimulation effect as that of the 3-PO-untr samples. The increased dose of the three samples

resulted in stronger simulation than the lower dose. The lower dose caused growth curves to

go down below the curves of the 3-PO-untr in all three cases.

32

Figure 16 Photo of one microtiter 96-well plate test after overnight incubation.

33

Figure 17 Growth curves of B. subtilis exposed to the 3-PO-chi and the 3-PO-untr cell extracts.

The culture control is included.

Figure 18 Growth curves of B. subtilis exposed to the 3-PO-NIC+chi and the 3-PO-untr cell

extracts. The culture control is included.

Figure 19 Growth curves of B. subtilis exposed to the 3-PO-NIC+MeJA and the 3-PO-untr cell

extracts. The culture control is included.

0,2

0,4

0,6

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-chi, 25mg 3-PO-chi, 50mg

0,2

0,4

0,6

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-NIC+chi, 25mg 3-PO-NIC+chi, 50mg

0,2

0,4

0,6

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-NIC+MeJA, 25mg 3-PO-NIC+MeJA, 50mg

34

Figure 20 Growth curves of B. subtilis exposed to the 3-PO-NIC and the 3-PO-untr cell extracts.

The culture control is included.

Figure 21 Growth curves of B. subtilis exposed to 3-PO-SA and 3-PO-untr cell extracts. The

culture control is included.

Figure 22 Growth curves of B. subtilis exposed to the 3-PO-NIC+SA and the 3-PO-untr cell

extracts. The culture control is included.

0,2

0,4

0,6

0,8

1,0

1,2

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-NIC, 25mg 3-PO-NIC, 50mg

0,2

0,4

0,6

0,8

1,0

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Cultrue control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-SA, 25mg 3-PO-SA, 50mg

0,2

0,4

0,6

0,8

1,0

0,0 2,0 4,0 6,0 8,0 10,0 12,0

OD

Time (h)

Culture control 3-PO-untr, 25mg 3-PO-untr, 50mg

3-PO-NIC+SA, 25mg 3-PO-NIC+SA, 50mg

35

4. Discussion

The outcome of this study is discussed from different angles in this chapter.

4.1 Cell growth, pH and stress response Changes in cell mass and pH in the culture medium can give information regarding stress

response in plant cell cultures. In comparison with the 1-PS-untr culture the cell mass of PS-

S decreased slightly in all three cultures after the stress signalling compounds were added

(Figure 3). In most cases the cell mass from treated Populus cultures decreased to a different

extent (Figure 4-5). This indicates that the plant cell culture responded to the stress caused by

the added substances. The response made the culture reallocate its basic capacity for growth

or other cell functions as an effect of growth-defense tradeoffs (Huot et al. 2014). A part of

such capacity could then be used to activate its defense mechanism. For example, growth was

inhibited while the production of phenolic substances was increased in the MeJA treated cell

cultures 2-PO-MeJA and 3-PO-NIC+MeJA.

Change of pH in the cultures varied to a large extent. Culture medium pH was clearly

influenced by SA, increasing in PS-S culture and decreasing in Populus cultures. The

opposite reactions may be due to the large difference in tissue differentiation between the

cultures. However, the effects indicate distinct responses to SA addition. McCue et al. (2000)

found that SA in combination with a low environmental pH stimulated phenolic production in

peas. In the present study, the 1-PS-SA culture had a decreased production of phenolic

compounds with an increased pH in comparison to the 1-PS-untr culture. The 3-PO-SA

culture had an increased phenolic production with a decreased pH in comparison to the 3-PO-

untr culture. If McCue et al. are correct, it would be possible that the higher pH caused the SA

effect of lower phenolic production in PS-S. Or, the lowered pH might have contributed to the

SA effect of stimulating phenolic biosynthesis in Populus.

4.2 Phenolic production and defense response A broad spectrum of defense related substances in plants can be found within the phenolic

metabolism. The analysis of total phenolics is therefore often used as a measure of plant stress

response. The most prominent effect on phenolic content in the cultures was seen after MeJA

treatment (Figures 8 and 9). Also SA addition to the Populus culture resulted in increased

phenolic content (Figure 10). The increased phenolic content caused by MeJA and SA is in

line with the inhibitory effects on bacterial growth in the agar plate assay (Figures 12, 14) by

extracts from the treated cultures.

4.3 Added compounds and defense response The stress signaling compounds SA and MeJA are well-known for their ability to induce

defense responses in plants (Huot et al. 2014). As discussed in Section 4.2, SA or MeJA could

trigger a stronger Populus defense response by activating much more biosynthesis of phenolic

compounds in its cells. This finding is supported by an earlier study review by Ruiz-García

and Gómez-Plaza (2013) for their statements on MeJA, and a finding by Delaunois et al.

(2014) that MeJA can enhance defense response after stress.

36

The elicitor chitosan did not show any clear effect on phenolic production or defense response

for Populus. In accordance with this, the culture grew well. However, extracts from chitosan

treated cultures showed a clear inhibiting effect on bacterial growth (Figures 12, 17) which

illustrates that the analysis of total phenolics is not always a reliable method to indicate

defense induction in plant cells. Other pathways within secondary metabolism might have

been induced by chitosan.

4.4 The NIC effect

The effect of NIC in this study was interesting, because it appeared to give rise to substances

that stimulated bacterial growth in the microtiter plate assay (Figure 20). In combination with

SA, NIC also caused increased growth, while SA itself did not influence growth considerably

in this assay (3-PO-NIC+SA; Figure 22). In the agar plate assay, NIC erased the inhibitory

effect of SA. The 3-PO-NIC+SA culture also had lower phenolic production than the 3-PO-

SA culture. These results show that NIC weakened the response caused by SA.

The situation was similar for the 3-PO-NIC+chi culture. NIC counteracted the chitosan effect

on culture pH, growth and phenolic production. However, in the microtiter plate assay,

extracts from the NIC treated culture could not override the growth inhibiting effect of extract

from chitosan treated culture.

The purpose of using NIC was to prepare, or "prime" the cells so they would respond more

strongly to the following treatments. Obviously the cultures responded, even though the

results from the parameters analyzed here instead pointed towards an opposite response,

which is a new finding regarding effects of NIC.

4.5 Cell extracts and bacterial growth

We should keep in mind that the two growth assays used had different outcomes. In the agar

plate assay a solid growth medium is used and an endpoint result is recorded. In the microtiter

plate assay a liquid medium is used and the growth can be followed continuously. Cell

extracts from untreated as well as treated Populus cultures all showed inhibitory effects on

Gram-positive bacteria in the agar plate assay. In the microtiter plate assay, the clearest results

were growth inhibition by extracts from chitosan cultures and growth stimulation by extracts

from NIC cultures, when compared to extracts from untreated cultures. MeJA was not tested

alone, but in combination with NIC it resulted in growth inhibition.

According to a study by Nostro et al. (2012), plants from nature may have even stronger

inhibitory effects than the cultured plant cells. If this is true also for Populus, the natural plant

of Populus should in the future studies be tested on Gram-positive bacteria and also on Gram-

negative bacteria.

The antibacterial substances in cell extracts may lose effect over time since the inhibitory

effects in the repeated test (Figure 14) were weaker.

37

4.6 Defense compounds and antibacterial effects

The outcome of this study shows no direct correlation between phenolic production and the

plant’s antibacterial properties. This, however, does not mean that the phenolic compounds

did not contribute to the antibacterial effects. Puupponen-Pimiä et al. (2001), Nostro et al.

(2012), Lv et al. (2012) and Alves et al. (2013) have detected that phenolics have effective

antimicrobial properties in different plant species. Lv et al. (2012) also stated that the

antimicrobial properties were most likely based on synergistic actions of many kinds of

compounds in plant cells. The results of this study indicate that the real situations and actions

in plant cells are much more complicated, and that there might be other key compounds

working together with the phenolics in Populus cells responsible for the antimicrobial

activities.

38

5. Conclusions

This is an initial study regarding production of antimicrobial substances by plant cell cultures.

Treatment of Populus trichocarpa fine suspension cell cultures with elicitors and stress signal

substances was performed to increase the production.

The following main conclusions can be drawn:

Cell culture growth and culture media pH as well as the production of phenolic substances

were influenced by the treatments, which indicate stress responses of the P. trichocarpa

culture. This kind of responses can often be connected to the production of defense substances,

for example antimicrobial compounds.

Extracts from untreated P. trichocarpa cultures could inhibit growth of B. subtilis on agar

plates.

The production of bacterial growth inhibiting compounds was influenced by treatment of

P.trichocarpa cell cultures with elicitors and stress signal substances:

- inhibition of B. subtilis growth on agar plates by salicylic acid, methyl jasmonate and

chitosan treatments.

- inhibition of B. subtilis growth in liquid media by chitosan treatment.

- stimulation of B. subtilis growth in liquid media by nicotinamide treatment.

These results point at a possible strategy for production in plants or plant cell cultures of

natural and non-toxic substances with antimicrobial properties.

39

6. Future studies

Bacterial growth curve

In the microtiter plate assay the growth curve did not show that the bacteria in the control

culture had grown much. However, a certain bacterial growth was clearly visible. There was

also an aggregation of bacteria at the bottom of the culture control wells. It is possible that the

measured values were too low due to some technical problems of the plate reader. This

experiment should be repeated with an accurate plate reader.

Qualitative results

The outcome of the microtiter plate assay did not provide the expected quantitative result. All

results in this study are qualitative. A continuation of this study should establish the minimum

inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Populus

cell extracts on Gram-positive bacteria in agar medium.

MeJA v.s NIC+MeJA

MeJA was not used as a single addition in Treatment 3. Therefore cell extract from MeJA

treated Populus culture could not compare with NIC+MeJA effects in agar plate assay and

microtiter plate assay. This should be done in future studies.

Bacteria exposed to extracellular substances

The plant cell culture medium was not tested on bacteria in this project. The culture medium

is, however, an interesting object for future studies as it could be a convenient source of

excreted products in case of scale-up of the plant cell culture system.

Morphological investigation

In further studies, the bacteria exposed to cell extracts should be observed with light

microscopy to give a morphological investigation of how the bacteria are affected.

40

Appendixes

Appendix 1 The modified MS medium

Medium stocks:

Part 1: 1 L

Chemicals Amount Unit

CaCl2·2H2O 4.44 g

KH2PO4 3.4 g

Na2EDTA·2H2O 0.37 g

MnSO4·H2O 0.17 g

ZnSO4·7H2O 0.09 g

H3BO3 0.06 g

KI 8 mg

Na2MoO4·2H2O 2.5 mg

Deionized water Up to 1 L

Part 2: 1 L

Chemicals Amount Unit

KNO3 45.9 g

NH4NO3 9.09 g

MgSO4·7H2O 3.7 g

FeSO4·7H2O 0.28 g

Deionized water Up to 1 L

Part 3: 1 L

Compounds Amount Unit

m-inositol 10 g

Thiamine 0.05 g

Pyridoxine 0.05 g

Nicotinamide (niacin-amide) 0.1 g

Deionized water Up to 1 L

Part 4: 2 L

Amino acides (L-forms) Amount Unit

Alanine 5.94 g

Arginine 0.62 g

Asparagine 0.76 g

Aspartic acid 0.34 g

Glutamine 0.06 g

Glutamic acid 3.15 g

Glycine 0.94 g

Histidine 0.01 g

Oxyproline (hydroxyproline) 0.25 g

Leucine 0.99 g

41

Lysine 0.39 g

Methionine 0.01 g

Phenylalanine 0.01 g

Proline 0.39 g

Serine 2.55 g

Threonine 0.82 g

Tyrosine 0.01 g

Valine 0.46 g

Deionized water Up to 2 L

Hormones stocks

Hormones Stock concentration Solvent

2,4-D 1 mg/ml Ethanol

Kinetin 1 mg/ml Deionized water

BA (benzyladenine) 0.4 mg/ml Deionized water

GA3 (gibberellic acid) 1 mg/ml Deionized water

To make 10 L of the modified MS medium:

1. Mix the stocks: part 1 1 L

part 2 1L

part 3 100 ml

part 4 100 ml

2. Add sucrose 300 g.

3. Fill up with deionized water to 10 L.

4. Adjust pH to 6.0 with 1M NaOH.

5. Add 2,4-D stock 10 ml and kinetin stock 200 µl for medium I, or BA stock 5 ml and

GA3 stock 3.5 ml for medium III GA3.

6. Check and adjust pH.

7. (Add 80 g of agar if agar medium is needed.)

8. Distribute the medium to smaller Erlenmeyer flasks with 50 ml or 100 ml per flask.

9. Autoclave the medium for 20 minutes at 121 oC.

10. Keep the autoclaved medium in cool for experimental use.

42

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