Evaluation of tissue culture and particle bombardment procedure for Sauropus androgynus and plasmid...

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Molecular Techniques in Biosciences

(D224P6 MY)

______________________________________________________________________________

26 MARCH 2013

Evaluation of tissue culture and particle bombardment procedure for

Sauropus androgynus and plasmid DNA transformation from Escherichia

coli through alkaline lysis.

By student ID – 009921

School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor.

Abstract – Different plant species might require different protocol to be used when culturing and

performing particle bombardment. The aim of this study is to test the protocols for tissue

culture, plastid DNA extraction and isolation from Escherichiacoliand biolistic parameter for

Sauropus androgynous. Surface sterilisation used in this study were inadequate to prevent

fungual and bacterial contamination but the contamination still low at 16.67%. Alkaline lysis

method used to obtained plasmid DNA from Escherichiacoli are heavily contaminated with 2.19

ratio of A260/A280 from nanodrop and not able to be used for biolistic. Particle bombardment

procedure was done through Biolistic ® PSD-1000/He and successfully produced organogenesis

(roots) and healthy growing callus.



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1.0 INTRODUCTION

The recombinant technology has allowed the researchers to break the gene barrier to created

transformed or genetically modified (GM) organism to fulfil some beneficial purpose like

enhanced nutritional value, pest resistance, increase yield and many other examples. Many

protocols have been established in order to find a better way to deliver the recombinant DNA

like particle bombardment. Particle bombardment or biolistic, utilize high velocity

microprojectiles to deliver foreign DNA into the target cells or tissues. The original concept come

from Sanford etal . (1987) whom invent the term itself – ‘biolistic’, which stand for ‘biological

ballistics’. It is a versatile methodology to deliver foreign DNA particularly plasmid because of its

wide range of target cells from diverse organisms. However different organism might have

different parameter to be used for particle bombardment and this paper is taken an approach to

test the parameter for the targeted plant.

Plasmid transformation has been first established on as early as 1980s by Boynton etal .

(1988) in Chlamydomonasreindhartii . Since then, many researcher has applied the technology

and became popular approach due to their own transcription-translation machinery. Plasmid is

usually obtained from bacteria and plasmid DNA extraction and isolation is not an easy task to be

carry out. Hence, many protocols have been developed to enhance the quality of extracted DNA

plasmid. In this writing, an approach is taken using alkaline lysis to observe the successfulness of

the method to extract and isolate plasmid DNA from Escherichiacoli.

The chosen target plant for the transformation is Sauropusandrogynus because of its

nutritional values. S.androgynous is an underutilized green herb and known among the local

Malaysians as ‘cekur manis’ or ‘asin-asin’. Many researches of this plant is related to Bronchiolitis

Obliterans (BO) syndrome that cause an outbreak at Southern Taiwan on 1995 (Ger etal . 1997).

Unfortunately, not many researches have been done related to nutrition aspect of the species.

One study (Subhasree etal. 2009) proved that the plant is a rich source of antioxidants like

vitamin A, B and C. This paper is aimed to prepare the culture of S.androgynousthrough indirect

somatic embryogenesis. Here surface sterilization is also tested to see either the plant survive or

not than can be observed by the growth of the culture. The developed culture then further

undergoes plastid transformation using plastid obtained from E. coli . Plasmid extraction and

isolation procedure is done through alkaline lysis. For this purpose, the recombinant protein



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candidate is green fluorescent protein (GFP) and done through biolistic transformation where

the parameter will be tested parameter to suit the culture.

2.0 METHODOLOGY

2.1 Establishment of aseptic culture of S. androgynus

Healthy juvenile leaves of S.androgynus is selected from the shade house and washed using tap

water to remove dirt or any surface contaminants. The washed leaves are cut into 12 pieces with

size approximately 1.5 cm x 1.5 cm using scissors. The excised leaves then undergoes surface

sterilization, the sterilants used are; 70% (v/v) ethanol solution and 20% (v/v) sodium

hypochloride – all performed in sterile Schott bottle for 20 minutes to reduce risk of

contamination. The damaged area or edges that cause by the sterilants is trimmed into not

larger than 1 cm x 1 cm. The six trimmed explants then transferred into each petri dish with MS +

2 mg/l NAA and 1.0 mg/l Kinetin with the adaxial surface of each explants is gently pressed

against the agar to ensure a good contact. Each petri dishes are sealed with parafilm to maintain

moisture and incubated at 25oC in 16 hour light / 8 hour dark for 14 days. After 7 days, the

explants are examined for contamination. If there is a contamination, subculturing is needed for

non-contaminated explants by transferring into other sterile petri dish with the same medium.

After 14 days, the culture will undergoes biolistic.

2.2 Isolation and purification of plasmid DNA

For plasmid extraction, alkaline method is used based on protocol developed by Birnboim

et al . 1979. A single, well-isolated colony of Escherichiacoli from a recent sub-cultured Luria

Bertani (LB) agar plate is inoculated and incubate overnight (not longer than 16h to prevent

plasmid loss) at 37oC. 10ml of the bacterial cultured is centrifuged and the pellet is resuspended

in pre-chilled Solution I and incubate at room temperature. A fresh prepared Solution II is added

and gently inverted before incubated on ice. Then, a pre-chilled Solution III is added and gently

inverted several times before incubated on ice. The solution is centrifuged. The top phase is

transferred to the new tube and phenol-chloroform-isoamyl alcohol (25:24:1) is added before

centrifuge again. The top phase is transferred into a new tube and incubate at room



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temperature after 100% absolute ethanol is added. The solution is centrifuged and the pellet is

washed with 70% pre-chilled ethanol incubate at least 15 minutes at -20OC. After the incubation,

the solution is centrifuge, supernatant is removed and let the pellet dry. The pellet then

resuspend in TE buffer. Spectrophotometer is used to quantify the amount of DNA obtained and

gel electrophoresis is done to identify any contamination.

2.3 Microprojectile-mediated DNA delivery into culture of S. androgynus

Healthy explants that have most callus growing after 2 weeks of culturing is chosen for

biolistic. The device used is Biolistic ® PSD-1000/He and sterilized by wiping the apparatus and

biolistic chamber with 70% (v/v) alcohol. Gold microcarrier 0.6 µ in size is also being sterilized by

washing in 100% ethanol suspension and vortex. The supernatant is removed and washing step

is repeated for two before resuspend on sterile water. 5 µg/µl DNA is added into the gold

suspension and vortex at maximum speed. Calcium chloride and spermidine is added. The

mixture is centrifuge and the supernatant is discarded. The pellet is resuspended in 100% (v/v)

ethanol and vortex at maximum speed for 1 min and centrifuge again. The supernatant is

discarded before resuspended in 100% (v/v) ethanol. Particle bombardment done at 9 cm target

distance at 1100 psi with vacuum pressure used 26 in.Hg. The culture is left for a week and

observed for any changes in growth and contamination.

3.0 RESULTS AND FINDINGS


After a week, not all of the explant shows distinctive callus growth. Callus appear to be clear in

colour with irregular shape appeared at the edge of the explants. There is 16.67% of

contamination of fungus on the explant and one contamination on medium for both petri dishes

(refer Figure 1.0). Browning colouration occurred as high as 75.0% (nine explants) for both petri

dishes. On average, 56.3% of the surface area of the explants shows browning. Subculture is

done to save the five selected explant that sterile or free from contamination to the new plate

and no contamination of any kind observed after a week. The culture then is chosen as candidate

for biolistic.



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Figure 1.0 : (A) Explants of S.androgynusin both petri dishes in day 1 (B) The contamination occurred in both petri

dishes after a week. The arrow C1 indicateds the area of contamination of fungus on the growth media and arrow C2

indicates fungus contamination on the explant. There were some callus growth observed in some explants (G 1 and

G2). (C) Week 2 of the subculture chosen from week 1 (that contain contamination of fungus). Explants in week 2

exhibit zero contamination both on media and explants (D) The close up of callus growth (G1 and G2) exhibit in some

of the explant in week 1.


Plasmid extraction and isolation is done using alkaline lysis method. The results of gel

electrophoresis shows neither smearing nor present of any DNA (refer Figure 1.2, well M4).

However the ratio of A260 to A280 (260/280) from nanodrop result shows 2.19 (refer Table 1.0)

indicates there is contamination of proteins probably from RNA (ratio ≈1.8 indicates pure DNA).

In this experiment, the methodology give high amount of contamination and unable to give the

desired DNA amount. The DNA obtained did not used for the biolistic due to these limitation

instead using the prepared DNA sample from the lab.

A C

C

G2

C2

G1

C

G1

G2

B D



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Table 1.0 : Nandrop reading obtained from spectrophotometer. Absorbance at 260nm (A260) shows the DNA

concentration and absorbance at 280nm (A280) indicates the protein contamination. A good quality DNA should

have a ratio 260/280 about 1.7-2.0, larger value indicates the contamination of proteins and other molecules.

Sample ID ng/µl A260 A280 260/280

4 203.51 4.070 1.858 2.19

Figure 1.3 : Gel electrophoresis of the plasmid DNA (well M4) shows unclear smearing and no clear DNA fragments is

being detected. Lambda Hind III was used DNA ladder.


Post biolistic explant shows more browing in colour compare to prebiolictic (refer Figure 1.1).

Observation showed most of them exhibit somatic embryogenesis callus formation and one

explant exhibit organogenis of roots. Calluses at this stage are more distinctive with whitish

colour in irreglobular structure aroung the edges of the explants. No contamination was

observed.



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Figure 1.1 : (A) Explants of S. androgynus arrangement before biolistic (B) Explants a week after particle

bombardment shows browning in colour. Organogenesis of roots (arrow R) and more callus growth (arrow G) can be

observed in every explant compare to pre-biolistic. (C) Close up picture showing the organogenesis of roots.

4.0 DISCUSSION


Contamination that occurred after a week in tissue culture is also probably due to inadequate

aseptic technique and lack of surface sterilisation. Inadequate aseptic technique usually happen

when the tissue culturist is lack of skills and experiences to sterile the equipment properly and

lack of proper equipment (in example; face mask). Contamination is transmitted mainly through

airborne and contact. It is encourage for the handler to regularly clean and sterile the equipment

and work place especially before and during the process of culturing with 70% ethanol and

reduce verbal communication during culturing or wear face mask. With the adequate aseptic

technique, the improvement can be shown with subculture result where no contamination of

fungus or bacteria was observed.

Surface sterilisation used in this experiments was; 70% (v/v) ethanol solution and 20%

(v/v) sodium hypochloride for 20 minutes. The culture observed during the period of the

experiment grow very well but contamination occurred only in first week of the culture growth.

It is assume that the surface sterilants used is compatible for the culture to grow but inadequate

A B CG

G



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for removing the contaminants. It is suggested to sterilize the plant for longer period of time or

used mercuric chloride (HgCl2). One study (Ramakrishna etal ., 1991) proved the usage of HgCl2 -

can significantly reduce the risk of many bacterial and yeast contamination including Alternaria

alternata, Fusarium spp. Aureobasidium pullulans, Bacillus spp., and Epicoccumpurpurascens,

when used for 3-10 minutes. 80% (v/v) of ethanol concentration can be used instead 70% (v) in

this experiment as one study able to get as high as 90% aseptic culture (Giladi et al ., 1979).

However, both suggested method might reduce the culture growth as shown in both studies.

In this experiment however, the contaminants can only be suspected because no

approach was taken to identify the contamination and contamination within the tissues remains

unknown. It is suggested to take a safety precaution to culture the explants with antibiotics and

antifungal to prevent bacterial and fungal growth. The antibiotics rifampicin, tetracycline,

cefotaxime and polymyxin B are the four proven antibiotics known to be effective against

bacteria in tissue culture (Young etal ., 1983).


Alkaline lysis for plasmid extraction required certain crucial solution which represent by Solution

I, II and III. Solution I contains glucose, Tris-HCL (pH 8.0), EDTA (pH 8.0) and autoclave at 121°C

for 15 min stored at 4°C. EDTA or ethylenediaminetetraacetic acid plays an important role in

Solution I to stabilize DNA phosphate backbone and chelate divalent metal cation (Mg2+ and

Ca2+) for DNAses thus prevent it from function properly. In other words, EDTA protects the DNA

by deactivating metallopeptidase or metal-dependant enzyme including DNAses from digesting

the DNA by chelating the metal ion that required by the enzymes (Auld, 1995). Another effectivemethod is pretreatment of the explant into antibiotic achromycin that could control up to 100%

contamination at 100 mg/l (Geier, 1997).

Solution II is also alkaline as it contains Sodium hydroxide (NaOH) and 1% (w/v) Sodium

dodecyl sulphate (SDS). SDS have hydrophobic properties that able to breakdown the plasma

and nuclear membrane and destroy the histones conformation by removing the negative ions

from the proteins. Meanwhile NaOH will denature plasmid and chromosome into single strands.

Solution III is an acidic solution to neutralize the previous solutions used. It contains potassium



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acetate, glacial acetic acid and water. The main purpose of the Solution III is to aggregate the

unwanted molecules like bacterial DNA and RNA, proteins, contaminants, and others so that it

can easily discarded. The action of potassium acetate and acetic acid help to precipitate the

dodecyl sulphate (from SDS) bound proteins.

Manual extraction protocol have been used required skilful and experienced handler to

be performed. Findings from nanodrop reading shows that the DNA obtained may heavily

contaminated with proteins and RNA. One limitation using the nanodrop is the absorbance at

260nm indicates both concentration of both plasmid and genomic DNA and also RNA so the

reading will not able to give the true concentration of DNA. However, gel electrophoresis have

been done to justify the contamination of RNA, proteins and genomic DNA. One quirk is, the

result of gel electrophoresis does not shows any smearing that supposed to indicate the present

of the contaminants. It is suspected poor pipetting technique is man cause of this situation

especially when one is doing manual extraction (will discuss below). It is suggest for DNA

extraction and isolation to use kit that may give better result compare to manual. Another

improvement that can be done is to replace NaOH with arginine buffer (in range pH 11.4 to 12.0)

that proved to aid to obtain plasmid DNA suitably pure for both sequencing and also stabilize pH

during the lysis (Cloninger etal ., 2008).

Handling technique is probably the most crucial part when conducting experiment.

Pipetting technique applied in this experiment is in doubt since the results of gel electrophoresis

and 260:280 from nanodrop indicates contamination of other proteins or RNA. Pipette is a

precision instrument that a small mistake (especially in liquid handling) can lead to the large

error in final results. The pipette tip should be prewet first with the aliquot of the sample

solution at least three time. One study (Zeman etal., 1974) proved that without prewetting, an

error up to 15% of rated volume dispensed from pipette. When aspirating the solution, the

plunger should depressed until the first stop and when dispensing the solution, the plunger need

to slowly depressed past the first stop to ensure the whole delivery of the solution.



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In biolistic, gold is preferable compare to tungsten because gold is homogenous, non-toxic and

inert towards catalytic degradation (Sanford et al., 1993). Meanwhile tungsten might cause

acidification of the agar medium that may lead to cell death thus destroying the culture (Sanford

etal., 1993). 0.6 µ gold is chosen compare to 1.0 µ gold because small size microprojectile is

more efficient for particle bombardment to give less tissue damage thus give a better chance of

survival for tissue to grow (Randolph-Anderson et al ., 1995). Theoretically, spherical shape

particles should cause less damage to the cells. In this case, spherical structure of gold

microprojectile may lessen the cell injury. Calcium chloride and spermidine used to help the

adhesion of the DNA towards gold particles to be bombarded. Compare to tungsten, that have

irregular structure can cause agglomeration during the DNA coating procedure while gold remain

separated (Hunold etal ., 1994).

Based on the post-biolistic culture result, there were high amount of growth of calluses

exhibit on the explants. This is a crucial findings because in study conducted using Nicotiana

tabacum, gas blast and acoustic shock from the device introduced mechanical cell injuries that

that take longer time to recover and even worst cannot be recover at all (Russell etal ., 1992).

The parameter used in this methodology proved to suit the S.androgynousculture. To further

enhance the parameter, vacuum level can be increased to reduce more air friction that interfere

with the bombardment. One study (Genga etal ., 1991) shows the three fold increase using 28 in

Hg of the beta-glucuronidase (GUS) reporter gene in tobacco.

Browning that occurred during the growth of the culture and after particle bombardment

is a neutral process that occurred due to oxidation of the phenolic compound that leached out

from explants. Quinone that produced from oxidized phenolic compound are responsible for the

discolouration (Murata et al., 2002). However, the products from phenols oxidation can be

phytotoxic that lead to necrosis of the cells (Bhojwani and Razdan, 1996). Browning that

occurred in the culture in this experiment is considered tolerable that may not cause severe

necrosis of the tissue. In case the browning colouration needs to avoid or longer period time is

needed for culture to growth, adding activated charcoal is proven to reduce this effect (Zhu et

al., 1997).



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5.0 CONCLUSION

This writing have successfully optimized some protocols while some needs further adjustment.

Tissue culture of S.androgynus have been able to develop successfully and improvements on

culturing mainly focus on how to reduce and identify the contamination. Future study on the

effect of surface sterilisation towards the culture need to be done with proper aseptic technique

applied so then it will not interfere with the results.

Plasmid extraction and isolation through alkaline lysis was challenging especially to

newcomer. Rigorous training and exposure on the essential laboratory technique seems

necessary especially pipetting skill for one that will use this protocol. Contamination and

unsuccessful attempt will cause time delay, increase cost and cause poor reputation of

performer.

Biolistic parameter of S.androgynus culture is probably the most crucial in this finding

that can be further optimize to enhance the delivery of DNA. Further study could be done to

further show the GFP gene expression in the culture. Another possible study that can be

conducted is to optimize another parameters of the unsuccessful protocols so that any

differences or similarities can be observed and finds which give a better results.



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http://www.ncbi.nlm.nih.gov/pubmed/12596843







Evaluation of tissue culture and particle bombardment procedure for Sauropus androgynus and plasmid...

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