PLANT GENETIC ENGINEERING [SHGB 6108] PRACTICAL REPORT

BY

AHMAD MOHAMMED GUMEL

April 9th,2010

LECTURER:

PROFESSOR NORZULAANI KHALID

The effect of explant wounding in Agrobacterium tumefaciens (LB4404) Transformation of invitro cultivated Tobacco explant (Nicotiana benthimiana): hands on practical study based on GUS reporter gene histochemical assay, DNA extraction and PCR analysis.

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ABSTRACT:

Agrobacterium mediated transformation of invitro cultivated Nicotiana benthimiana was carried out in this practical. A. tumefaciens (LB4404) hovering pCAMBIA1304 plasmid was used to demonstrate the effect of explant wounding in relation to Agrobacterium transformation induction; β-glucuronidase gene was used as a reporter gene. Transformed explant were molecularly analyzed by PCR to detect the presence of GUS gene and qualitatively analyzed by histochemical assay. Putative transformed explants were judged by their ability to grow on Hygromycin-MS selective media. 5- adaxial surface cuts parameter was found to be probably the best parameter in Agrobacterium transformation induction of explants with size 5mm X 5mm, having putative transformed percentage of 75%.

INTRODUCTION:

Plant plasticity and tetipotency are among the main concepts for driving plant geneticist and molecular biologist towards cell culture and regeneration. Plants are sessile and have long life span; this gives them an advantage to endure extreme conditions and predation than animals. With plasticity characteristics in hand, plants have the ability to initiate cell division from almost any tissue of the plant and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli. This regeneration power goes hand-in-hand with maintenance of genetic potential as another feature described as tetipotency.

Plant transformation has become a widely accepted method for observing the main physiological functions in plants as well as improving the plant crop quality and characteristics. This procedure solely depends on the introduction of foreign gene or transgene that is commonly referred to as Gene of Interest (GOI) into the genome of the plant. Various methods have being developed to achieved plant transformation with many success been reported in different plant species (Hui-Yin, 2008; Kapila, De Rycke et al., 1997; Kumar and Rajam, 2005; Kushikawa, Hoshino et al., 2001; Li, Volrath et al., 2003; Norzulaani Khalid, 2003; Norzulaani Khalid, 1989; Pena, Cervera et al., 1995; Sanyal, Singh et al., 2005; Sharma and Anjaiah, 2000; Slater A, 2008). These methods include Direct gene transfer, which is the transfer of the naked DNA into the plant by different processes like particle bombardment, electroporation, silicon carbide fibers, microinjection etc; or it can be by indirect gene transfer that involve the cloning of gene of interest into T-DNA of a dicotyledonous plant pathogenic, gram negative soil bacteria called Agrobacterium spp. (A. tumefeciens and A. rhizogenes), that are responsible for causing Crown-gall disease in plant (Banerjee, Prat et al., 2006; Chaudhury, Madanpotra et al., 2007; Dang and Wei, 2007; Diemer, Jullien et al., 1998; Ditt, Nester et al., 2005; Frame, Shou et al., 2002; Gelvin, 2009). Though, before there were a lot of problems when using Agrobacterium transformation in monocot, but now a days, advent in recombination techniques and plasmid technology made it possible to be used in transformation of many monocotyledons plants such as

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Zea mays, Barley and Oryza sativa as reported in different (Gould, Devey et al., 1991; Ozawa, 2009; Shrawat, Becker et al., 2007).

Extensive genetic analyses conducted in the 1980s identified key Agrobacterium genes involved in virulence. During the past decade, however, genomic technologies have revealed numerous additional bacterial genes that more subtly influence transformation. The results of these genomic analyses allowed scientists to develop a more integrated view of how Agrobacterium interacts with host plants. In a similar manner, genomic technologies have identified numerous plant genes important for Agrobacterium-mediated genetic transformation. Knowledge of these genes and their roles in transformation has revealed a lot of insight on how Agrobacterium manipulates its hosts and helps in increasing the probability of a successful transformation outcome(Gelvin, 2009).Different optimization parameters such as cell density, cocultivation time, wounding effect, cell culture growth phase and media composition were studied by many researchers and were reported in many literature(Yong, Abdullah et al., 2006).

LITERATURE REVIEW:

The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome in 1983 (Wikipedia, 2009, November 30th). Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants (Joos H, 1983; Schell J, 1977).

In the 1980s, scientists learned to disarm the oncogenes, usually the opine synthase genes of virulent Agrobacterium strains, such that tissues infected by the bacteria could regenerate into normal plants (Bevan M W, 1983; Fraley RT, 1983; Gelvin, 2009; Herrera-Estrella L, 1983).Substituting genes of interest for oncogenes and opine synthase genes by deletion of the tumor inducing oncogenes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats i.e Left and Right borders respectively, at least one of which is needed for plant transformation, resulted in plants expressing these novel transgenes and, thus, novel phenotypes, this greatly increased the utility of Agrobacterium as a vehicle for gene transfer in plant biology laboratories(Gelvin, 2009).  The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance genes that confers resistance to antibiotic like Hygromycin or Kanamycin) to enable selection, and a reporter gene ( like GUS uidA gene or gfp gene) to confirm the transformation by expression of the reporter enzymes in plants that have been successfully transformed..

Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-explants can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture.

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Agrobacterium genetically transforms its host by transferring a well-defined DNA segment from its tumor-inducing (Ti) plasmid to the host-cell genome(Tzfira and Citovsky, 2006). The vir region, located on the Agrobacterium Ti plasmid, encodes most of the bacterial virulence (Vir) proteins used by the bacterium to produce its T-DNA and to deliver it into the plant cell. In wild-type Agrobacterium strains, the T-DNA region (defined by two 25 base pair direct repeats termed left and right T-DNA borders) is located in cis to the vir region on a single Ti plasmid. In disarmed Agrobacterium strains, where the native T-DNA region has been removed from the Ti plasmid, a recombinant T-DNA region usually resides on a small, autonomous binary plasmid and functions in trans to the vir region (Tzfira and Citovsky, 2006). The transformation process begins with the bacterium–plant attachment (Figure 1; step 1), followed by induction of the expression of the vir region by specific host signals (Figure 1; steps 2 and 3). A single-stranded (ss) T-DNA molecule (T-strand) (Figure 1; step 4) is then produced by the combined action of the bacterial VirD1 and VirD2 proteins [7]. In bacterial cells, the T-DNA exists as a ssDNA–protein complex (immature T-complex) with one VirD2 molecule covalently attached to the 50 end of the T-strand [8]. This complex, along with several other Vir proteins [9], are exported into the host cell (Figure 1; step 5) by a VirB/D4 type IV secretion system [10], a step that requires interaction of the bacterial Tpilus with at least one host-specific protein [11]. Once inside the host-cell cytoplasm, the T-DNA is thought to exist as a mature T-complex (T-complex), in which the entire length of the T-strand molecule is coated with numerous VirE2 molecules. These molecules confer to the T-DNA the structure [12] and protection [13] needed for its travel (Figure 1; step 6) to the host-cell nucleus. It is mainly during the last steps of the transformation process — namely, transport through the cytoplasm (Figure 1; step 6), nuclear import (Figure 1; step 7), intranuclear transport (Figure 1; step 8), T-DNA uncoating (Figure 1; step 9) and integration (Figure 1; step 10) — that the Agrobacterium utilizes various cellular mechanisms to accomplish the genetic transformation of its host(Tzfira and Citovsky, 2006).

FigureI: Mechanisms of Agrobacterium gene transfer in plans ( adopted from Tzfira & Citovsky, 2006)

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Sometimes in Agrobacterium-plant cell interaction, the transfer of T-DNA-complex is not integrated into the plant genome. The non-integrated T-DNA copies remain transiently present in the nucleus, which can be transcribed, leading to transient expression of the T-DNA genes(Kapila et al., 1997).

Reporter genes are widely used in plant transformation vectors, both as a means of accessing gene expression by promoter analysis (Chalermsri and Seiichi, 2005; Dang and Wei, 2007; Ding, Aldao-Humble et al., 2003; Honda and Moriguchi, 2006; Pena et al., 1995); or as easily scored indicators of transformation. A widely used reporter gene in plants is the uidA, or gusA, gene that encodes the enzyme β-glucuronidase (GUS). Since the ß-glucuronidase gene (gusA) was first isolated from Escherichia coli (Jefferson 1987), the GUS assay has become a widely used reporter system in the study of bacteria, animals, plants and, more recently, in yeasts and filamentous fungi. This is mainly because of enzyme stability and the high sensitivity and amenability of the assay to detection by fluorometric, spectrophotometric, or histochemical techniques.

This β-glucoronidase gene, is perhaps the most widely used reporter gene in plant transformation vectors. GUS activity in tobacco leaf extracts was analyzed by fluorometric and histochemical assays(Jefferson R A, 1987). Qualitative GUS assay can be obtained following histochemical assays using chromogenic 5-bromo-4-chloro-3-indolyl-β-D-glucoronide popularly called “X-gluc”. This enzyme can cleave the chromogenic (color-generating) substrate X-gluc (5-bromo-4-chloro-3-indolyl-β-D- glucuronide; refer to chemical equation below), resulting in the production of an insoluble bluish-indigo color of 5’5’-dibromo-4’4’-dichloro-indigo in those plant cells displaying GUS activity. Plant cells themselves do not contain any GUS activity, so the production of a blue color when stained with X-gluc in particular cells indicates the activity of the promoter that drives the transcription of the gusA-chimeric gene in that particular cell; there by identifying the precise location of the expression effectively(Karcher, 2002 ).

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Chalermsri and Seiichi (2005), observed Agrobacterium mediated genetic transformation of Carnation (Dianthus caryphyllu L.) using leaf explants derived from invitro plants; they reported that Gus-positive area was located only on the cut end of the explants. Although they used leaf explants with large wound area, the transformed cells were located only on the cut ends of the explants. They however, observed that when the cut ends of the leaf explants were at a distance from the leaf base, the explants failed to produce GUS-positive shoots, while the explants whose cut ends were close to the leaf base had GUS-positive cells at the leaf base and produce GUS-positive shoots(Chalermsri and Seiichi, 2005).

Tania Jachinto et al (1998), reported a comparison between wounding effect and Methyl Jasmonate inducible GUS activity in Wound-regulation and tissue specificity of the tomato prosystemin promoter in transgenic tobacco plants, they observed a low constitutive GUS activity in the vascular bundle tissues that, after wounding, had increased several fold (Fig. 2A and B). When plants were exposed to MJ, a similar tissue-specific expression pattern was observed (Fig. 2C and D) they observed that In Fig. 2A and 2B are shown the expression patterns of GUS in leaf tissue before and after wounding. Induction of GUS activity was also found in the vascular bundles of petioles of wounded and MJ treated leaves(Taˆnia Jacinto, 1999). Similar observation was also reported by (Chalermsri and Seiichi, 2005; Chaudhury et al., 2007; Chen, Hsiang et al., 2003; Diemer et al., 1998)

Beside GUS uidA gene, Many plant reporter genes were reported to be used in assessing plant transformation process this include gfp gene, unlike GUS histochemical assay which is destructive here the test is nondestructive as it involves the use of UV light to view the

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expression of the protein in the transformed explant(Blumenthal, Kuznetzova et al., 1999; Chen et al., 2003; Chiu, Niwa et al., 1996; Liu, Bugos et al., 2001; Peckham, Bugos et al., 2006).

Plant DNA extraction and subsequent polymerase chain reaction (PCR) is a very powerful technique, now used in many areas of molecular biology, which allows in vitro amplification of specific DNA sequences from undetectable quantities of target DNA. It is something that is commonly employed by many researchers in assessing the transformation of the plant as reported by many papers (Doyle JJ, 1987; Doyle JJ, 1990; Edwards K, 1991; Gould et al., 1991; He, Zhang et al., 2007; Hirsikorpi, Kämäräinen et al., 2002; Kushikawa et al., 2001; Murray HG, 1980; Ozawa, 2009; Perrin and Hull, 1999; Sharma and Anjaiah, 2000; Stewart CN Jr, 1993; Wang H, 1993). John et al (1991) studied parameters affecting PCR in the transformation of root lines of Nicotiana species, containing nptII and Gus genes, as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. They observed that in tobacco species the PCR reaction worked efficiently with up to 2 µg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10ng of genomic DNA was present in the reaction. The advantage of PCR, of course, is that target sequences can be amplified to levels which are clearly visible on agarose/EtBr gels, the visible bands of the DNA can be compared with the corresponding base pairs of the expected gene of interest, there by assessing the status of transformation either positive or negative(John D. Hamill, 1991).Sharma and Anjaiah (2000) in their research on An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation used PCR amplification of 700-bp fragment of nptII in accessing the Integration of the transgenes and stable genetic transformants in the progeny (Sharma and Anjaiah, 2000). Pena and Cervera also used PCR in assessing citrus genetic transformation in High efficiency Aguobacterium-mediated transformation and regeneration of citrus (Pena et al., 1995).

In general, several effects were found to have an influence in Agrobacterium transformation efficiency in plants; influence of cocultivation time, Agrobacterium cell density in the culture media, the explants size and level of wounding for the plant phenolic release and Agrobacterium passage were among the different parameters cited by researchers (Hirsikorpi et al., 2002; Honda and Moriguchi, 2006; Joubert, Beaupère et al., 2002; Kapila et al., 1997; Kumar and Rajam, 2005; Kushikawa et al., 2001; Liu et al., 2001; Ozawa, 2009; Pena et al., 1995; Rafat, Aziz et al., 2009; Sanyal et al., 2005; Sharma and Anjaiah, 2000; Shrawat et al., 2007; Sunilkumar, Vijayachandra et al., 1999; Tzfira and Citovsky, 2006; Tzfira, Frankman et al., 2003; Wang and To, 2004).

In this practical, we studied the effect of explants wounding in the transformation of Nicotiana benthimiana using Agrobacterium LB4404 carrying pCAMBIA1304. The transformation was assessed using GUS histochemical assay and DNA extraction as well as PCR analysis.

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Objective of the practical study:

In order to study Agrobacterium- mediated transformation in plants and to investigate the wounding effect on transformation efficiency

To learn the techniques of plant DNA extraction and molecular analysis of the transgenic plant genome by PCR analysis

To be able to observe the procedure of assessing the transformation by reporter gene analysis i.e. GUS uidA histochemical analysis.

MATERIALS AND METHODS

Plant material and Plant culture media:

Invitro plants of Nicotiana benthamiana was provided to us during the practical.

MSO medium was prepared by laboratory scientist based on Murashige and Skoog (1962) composition and was provided during the laboratory study, this media we were told that is has no antibiotic but do contained macronutrients in different proportions such as calcium chloride di hydrate, ammonium nitrate, potassium nitrate, magnesium sulphate hepta hydrate. Micronutrients include potassium iodide, cobalt chloride hexa hydrate, hydrogen borate, sodium molybdate, manganese sulphate tetra hydrate, copper sulphate penta hydrate and zinc sulphate hepta hydrate also in different concentration too. Ferum compound incorporated are iron sulphate hepta hydrate, and sodium EDTA, contained vitamins like Glisin, nicotinic acid, piridoksin, thiamine HCL, and inositol while plant growth regulator BAP 1mg/L, 3% sucrose and 0.8% agar was included after medium pH was adjusted to 5.8.

MS- antibiotic medium was also prepared by laboratory scientist based on Murashige and Skoog (1962) composition and was provided during the laboratory study, this media we were told to contained in addition to MSO medium components 3mg/L plant growth regulator BAP, and an antibiotic Hygromycin 10mg/ml (for putative transform selection) and Cefotaxime 25mg/ml (for killing Agrobacterium on the explants) that were added after the autoclave media was cooled down to 50-60oC.

It is important to mention that all the chemicals and compounds used in these media preparations are of laboratory grade and high purity.

Bacterial Strain and Vector:

We are provided with an overnight culture of disarmed hyper virulent A. tumefaciens strain LB4404 hovering vector plasmid pCAMBIA1304. These Bacteria were cultured overnight at 28°C and 200 rev./min in liquid LB medium (1% (w/v) tryptone, 0.5% (w/v) yeast extract and 1% (w/v) sodium chloride, pH 7.0) containing 25 mg/L kanamycin and 25 mg/L nalidixic acid.

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Based on spectrophotometric optical density at λ= 550nm; the Agrobacterium cell culture density was found to contained 14 x 108 cfu/ml.

Vector plasmid pCAMBIA1304 has GUS gene driven by the cauliflower mosaic virus (CaMV) 35s promoter and poly A signal, which was used as reporter gene, Hygromycin resistance gene, expected to conferred antibiotic resistance to putative transformed explants used as a selectable marker, pBR322 Origin of replication and mgfp5 green fluorescence protein gene as another reporter gene, this plasmid is 12361bp picture shown below.

Transformation and regeneration:

5mm x 5mm leaf explants were cut on the Adaxial sides of the leaves of invitro tobacco plants. Three wounding parameters were set, that is explants with 3 cuts, 5 cuts and 10 cuts respectively, and two controls were set as control-negative having explants without the cut and no Agrobacterium incubation, and a control-positive having explants without cut but were subjected to Agrobacterium incubation as the other experimental samples. All experimental samples were set in duplicates, and both explants in each sample including that of positive control were incubated in Agrobacterium culture for 30seconds to allow the bacteria to infect explants, after that they are blotted with tissue paper to remove excess Agrobacterium on the explants surface. This work was done under sterile condition in laminar flow with sterile forceps and blades. After the incubation and blotting, each respective sample was placed on MSO medium and

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cocultivated at 28oC for 3 days at 16 hours/8 hour photo/dark period, this allowed time for Agrobacterium to transfect the explants and introduce the GOI into the plant cells.

After three days of cocultivation, we observed that the explants still appeared green in colour no any Agrobacterium growth on the explants. The explants were transferred after 3 days cocultivation to another media i.e. MS-antibiotic media, which contained cefotaxime antibiotic and hygromycin for 9 weeks at 28oC, 16 hours/8 hour photo/dark period, while the samples were transferred to fresh media after every two weeks. At this stage the media was expected to kill any Agrobacterium on the explant by having the cefotaxime antibiotic and allow the growth of only putative transformed explants that acquire the selectable marker gene of hygromycin resistance from the transformation process, while those that lack the gene will die on the medium due to presence of hygromycin within the medium composition.

Here at week five we observed milky colonies of Agrobacterium growth around most of the explants, most especially those with higher cut numbers (10 cuts/explant), there by contaminating the samples, this could possibly be due to either high Agrobacterium cell concentration in the culture broth or low concentration of cefotaxime in the media that was ineffective in killing the Agrobacterium in the medium. This made us to repeat over the experiment again.

In the ninth week, here most of the samples are green in colour, no much Agrobacterium contamination, but no callus growth in the sample, this could possibly be due to the reason that we started it at week 5 after contamination of the first samples, so the time is not enough for the explant to start developing callus as it was only 4-weeks post infection at week nine but still 2 out of 20 explants shows some sign of calli. However, we observed in some samples with callus grow from the explant and appeared creamy-white to light green in colour with rough bead like

structures on the explant surface texture.

We also observed in some explant like those with 3 cuts/explant, though there is no Agrobacterium contamination, but they turn brownish in colour, showing that they are dead, this could be explain may be they were untransformed explant, therefore they lack the hygromycin resistance gene and as such they all die on the media.

Since we don’t have callus growth on our explants, explant-samples with best parameters and growth appearance on the media were selected and assayed for Histochemical analysis of GUS and PCR/ electrophoresis analysis of gfp gene presence.

HISTOCHEMICAL GUS ASSAY:

As mentioned earlier, due to high level of Agrobacterium contamination of our samples, and the limited time constraint, we were unable to be opportune to undertake the GUS histochemical assay, but based on cited literature as mentioned in introduction and literature review parts; ß-glucuronidase gene (gusA) was first isolated from Escherichia coli (Jefferson 1987 Plant Mol. Biol. Rep. 5:387-405), the GUS assay has become a widely used reporter system in the study of

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bacteria, animals, plants and, more recently, in yeasts and filamentous fungi. This is mainly because of fast analysis, non-radioactive, enzyme stability and the high sensitivity and amenability of the assay to detection by different methods such as fluorometric, spectrophotometric, or histochemical techniques. In general, histochemical alnalysis is done insitu. Several papers cited used of GUS histochemical assay to assess the transformation in Agrobacterium mediated transformation of different plant species(Jefferson R A, 1987; Jefferson, 1987; Shrawat et al., 2007; Tzfira and Citovsky, 2006; Welsh and Kay, 1997; Yancheva, Golubowicz et al., 2005) andamong those that reported the GUS histochemical assay specifically in tobacco(Jefferson R A, 1987; Jefferson, 1987; Suehara, Takao et al., 1996; Taˆnia Jacinto, 1999; Thomasset, Ménard et al., 1996)The procedure employed in most papers relied on the Jefferson et al (1987) modified protocols, which involves the use of a colourless substrate of 5-bromo-4-chloro-indolil-β-glucuronide (x-gluc), that can easily be distinguished on lower concentration by forming a precipitate product known as dichloro-dibromo-indigo (ClBr-indigo) as an unstable intermediate product that undergo further dimerisation oxidative process to form bluish indigo colour of 5,5’-dibromo-4,4’-dichloro-indigo, which is seen at the location of enzyme activity within the tissue of transformed explants.

Composition of GUS staining solution 0.1M sodium phosphate buffer [pH 7.0] 0.5 mM potassium ferricyanide 0.5 mM potassium ferrocyanide 10mM EDTA 0.1% Triton X-100 1.0mM 5-bromo-4-chloro-3-indolyl-β-D-glucuronide

FAA fixative solutions composition:Ethanol 45mlGlacial acetic acid 5mlFormaldehyde 5mlDistilled water 45ml

PROCEDURE FOR TOBACCO EXPLANT GUS HISTOCHEMICAL ASSAY (based on Jefferson et al; 1987) Histochemical assay First the sample is deep in GUS staining solution, and incubated overnight at 37oC. the GUS staining solution is then removed, and the sample undergo 70% ethanol washin at room temperature for three to four times to remove chlorophyll, it is then followed by FAA fixative solution treatment; this fixed the colour and allow clear visualization of the bluish indigo colour of 5,5’-dibromo-4,4’-dichloro-indigo at the site of gene activity within the explant tissue.

Below is the overall reaction of x-gluc enzyme in GUS histochemical assay:

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MOLECULAR ANALYSIS OF TRANSGENIC TOBACCO EXPLANTS

DNA Extraction:

Transgenic explant DNA extraction was done based on Doyle and Doyle 1987 modified protocols based on CTAB DNA extraction techniques. Similar methodology was reported by a lot of literature on transgenic DNA extraction process (Doyle JJ, 1987; Doyle JJ, 1990; Edwards K, 1991; Murray HG, 1980; Slater A, 2008; Stewart CN Jr, 1993; Wang H, 1993)

Buffers and Chemicals:

2x CTAB buffer:

100ml 1M Tris (pH 8.0) 0.1M

280ml 5M NaCl 1.4M

40ml 0.5M EDTA (pH 8.0) 20mM

20g CTAB 2% (w/v)

1g PVP 1% (w/v)

Distilled water 1L

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Β-mecaptoethanol 0.2-1% (v/v)

CI reagents: Chloroform/ isoamyl alcohol 24:1

RNase: 10mg/ml

76%9v/v) Ethanol/0.2M NaAc: 790ml 96% (v/v) Ethanol, 80ml 2.5M NaAc (pH 5.0), 130ml sdH2O

76%(v/v) ethanol/ 10mM NH4Ac: 790ml 96% (v/v) Ethanol, 10ml 1M NH4Ac, 200ml sdH2O

Procedure:

The parameter selected for DNA extraction were the best samples parameters from:-

Negative control

Positive control

Explants with 5 cuts

2g of fresh leaves of best parameters explant were placed in a motar, liquid nitrogen was added to freeze the tissue, once the liquid nitrogen was added we observed that there is a kind of boiling reaction that takes place, while the liquid nitrogen evaporating, so the grinding was done quickly and carefully while adding the liquid nitrogen at intervals. It was then gently grinded using pistil, the finely grinded sample that look whitish to greenish-yellow in colour was added to eppendorf tube that contained 2µl mercaptoethanol and the tube was shaken gently in order not to destruct the DNA. The tube was incubated for 15minutes at 55oC after which were cooled at room temperature and 2.5ml CI was added and the tube was shaken by invert shaking for 10minutes. The tubes were then centrifuged at 10,000rpm for 10 minutes. The upper aqueous phase was taken and added to a new eppendorf tube. 10µl of RNAse was added an incubated for 30 minutes at room temperature, this degrade any contaminating RNA leaving only the DNA. Isopropanol was added to the supernatant to precipitate the DNA and it was then cold-incubated for 10 minutes to enhance the precipitation, and was later centrifuged at 13000rpm for 10minutes. The clear aqueous supernatant was drained leaving a milky white pallet at the bottom of the tube. 50µl of TE was added to dissolve the pallets and was stored at -20 before commencing to PCR analysis.

PCR ANALYSIS:

Within these recent years PCR has been a major tool at hand to molecular biotechnologist, its simplicity, and high copy number of the known segment of genomic DNA within a short time gives it an impressive advantage in molecular analysis. PCR involves the use of segment of the

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genomic DNA and two oligonucleotide primers (here GUS gene primer was used) that match a specific sequence within the genomic DNA ( in this study GUS-gene), deoxynucleotide triphosphate as the building blocks of nucleotide residues and a thermostable Taq DNA polymerase as the builder. The reaction is carried out in a suitable buffer (PCR- buffer) within a specialized machine called thermal-cycler, it involves about 30 repetitive cycles of temperature differentiation, first the reaction temperature is raised to 94oC for 5minutes, this denatured the DNA, it is then lowered to 50oC for 2 minutes, to allowed the annealing of the primers by base pairing of the sequences on the flanking side of genomic DNA, further raising of the temperature to 72oC for 3minutes caused the primer to initiate DNA synthesis and Taq DNA polymerase to build on the DNA copy using the deoxynucleotide triphosphates. This reaction of three cycles is repeated about 30times, producing millions of copies of DNA within a short period of time, in short PCR can be seen as an efficient molecular photocopy machine that molecular scientist has ever had in history of science.

So we were given the PCR products of our respective samples and genomic DNA sample for running on Agarose Gel/ETBr electrophoresis.

Agarose Gel/ETBr Electrophoresis:

8µl of the PCR product and was mixed with 2µl loading dye (ethidium bromide) on a paraffin paper, this dye influence the DNA density and enhance the DNA band colour for visibility on the gel plate. Pipette was used to transfer the 10µl of the PCR product- loading dye mixture into one of the 1% (w/v) agarose gel ( 1% is used because the PCR DNA are smaller as such they can move easily) well, Vivantis VC100bp DNA-plus ladder was loaded into wells at both edge of the gel, besides the samples well which serve as a standard reference of the base pair number as it known from the manufacturer’s label, the same procedure was followed as above for the genomic DNA sample only that 0.8% agarose gel was used for genomic DNA, due to its size bigger than the PCR DNA.

The gels were run for 35minutes at 120volts, after which the separated band were visualized using computerized photometric UV light camera, and the gel picture was presented below in the result section.

In this practical our group “C” gel wells are the last three and last two from the right side of the gels before the right side-ladder in both genomic and PCR gels in the following order:-

For genomic gel electrophoresis: The specifications of the 3 wells are presented below:

lane 1: Positive control

lane 2: explants with 3 cuts

lane 3:explant with 5 cuts

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For PCR product gel electrophoreis :  the specifications of the two wells are:

lane 1: positive control

lane 2:Explant with 5 cuts.

Results and Discussions:

Agrobacterium mediated transformation of Nicotiana benthimiana using bacterial strain LB4404 hovering plasmid pCAMBIA1304 was studied in this practical, though all protocols were followed according to the practical manual given, however there was a lot of bacterial contamination of the experimental samples, which led us to repeat over the experiments, and as such we were un able to get callus during result analysis, hence we only conduct all the result analysis on putative transformed explants, i.e. those explants that were able to grow on the MS-selective media. Therefore all results presented herein are based on putative transformed explants analysis not on callus analysis.

We counted the number of putative transformed explants per each replicate plate and calculated the percentage of putative transformed explants, the result is presented in the below table:

Table I: number of putative transformed explants per replicates and percentage of putative transformed

Parameter Replicate A % of putative transformed explants in A

REPLICATE B % of putative transformed explants in B

Average % of putative transformed explants

Negative control

0/6 0 0/6 0 0

Positive control

4/6 66.7 3/6 50 58.4

3 cuts 4/6 66.7 3/6 50 58.45 cuts 4/6 66.7 5/6 83.3 7510 cuts 2/6 33.3 2/6 33.3 33.3

% of putative transformed (λ) = putative transformed explants

totalnumber of expalnts x 100

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Analyzing Table I aboveNegative control

0/6 0 0/6 0 0

Negative control has zero number of survived explants and as such zero putative transformation; WHY?.....................

This is because the Negative control samples only contained the explants that were not incubated in the Agrobacterium culture, so they are not infected neither are they transfected, but they are grown on the antibiotic MS media, hence when we observed plates before the result analysis, the samples were brownish in color, and the color transcend to the medium vicinity, no any signed of grows, we knew that probably the samples are dead. Positive control

4/6 66.7 3/6 50 58.4

Positive controls have the explants that are incubated in the agrobacterium culture but they lack surface wounding parameter. Though they are infected but the degree of infection is not as much as in those samples that are wounded based on the parameters, this result in some (7/12) surviving explants on the media with 58.4% of putative transformed explants. Similar observation was reported by CARL D. LARUE (1941).

3 cuts 4/6 66.7 3/6 50 58.4

3 cuts parameter here also behave just like in positive control, having 7 out of 12 explants as putative transformed, with 58.4% of putative transformed; shows that probably the number of cuts here are not much enough to cause any observed difference in transformation induction as compared to the positive control.

5 cuts 4/6 66.7 5/6 83.3 75

But moderate increase in the number of wounds to 5 cuts show a surprising increase in the number of putative transformed with 9 out of 12 explants i.e. 75% putative transformed as the highest % transformed, increase in number of wounding above this level result in decrease in number of putative transformed this can be seen in 10 cuts parameters

10 cuts 2/6 33.3 2/6 33.3 33.3

This could probably be the number of wounding per the size of the explant 5mmX 5mm is high which may result in high expression of Vir gene protein as well as too much phenolic sugars hindering the bacteria to be properly attracted and attached to the wounding area, or too much wound could possibly encourage cell regeneration rather than allowing the bacterial passage to effect genetic transformation. similar observation was reported by Tania Jacinto et al 1998.

Hence, for a clearer understanding of the above table I analysis a graphical Comparison based on the percentage of putative explants by nuber of cuts in presented below:

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0

10

20

30

40

50

60

70

80

90 Comparison of Explant wounding effect in two Replicates A & B

Number of wounding per expalnt

% o

f put

ative

tra

nsfo

rmed

exp

lant

Figure I: comparison of explants wounding effects by percentage of putative transformed in both replicates A and B using Agrobacterium LB4404 mediated transformation of N. benthimiana

Negative control Positive control 3 cuts 5 cuts 10 cuts0

10

20

30

40

50

60

70

80

Explant wounding effect

Number of wound per explant

Aver

age

% o

f put

ative

tran

sfor

med

exp

lant

s

FigureII: Average percentage of both experimental replicates putative transformed explants by wounding cuts in Agrobacterium LB4404 mediated transformation of N. benthimiana

AB

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By observing figure one we can see that there are some variations in number of putative transformed explants between the replicates samples, with samples in replicates B showing more responsive to experimental treatments, while it is expected to be similar as it all have the same treatments and the same parameters, this could possibly be due to some experimental errors in either human error in experimental settings and runinig or medium concentrations, incubation procedures infection time difference with some seconds or difference in even

However, when the overall average percentage putative transformed (figure II) is plotted against the wounding cuts we will see that in tobacco explants of 5mmX5mm size the optimum cut for Agrobacterium induction could probably be 5 cuts parameter , as it has the highest putative transformed explants percentage of 75%.

Molecular Analysis:

Molecular analysis was carried out to check DNA integration by PCR analysis and GOI expression by GUS histochemical assay in the following steps:

DNA EXTRACTION:

Based on the Doyle and Doyle (1987) protocols mentioned in methodology above; the parameter selected for DNA extraction were the best samples parameters:

Negative control

Positive control

Explants with 5 cuts

For genomic gel electrophoresis :  Our group wells on gel is the last 3 well before the DNA ladder from right edge of the gel figure I in next slide.

The specifications of the 3 wells are presented below:

lane 1: Positive control

lane 2: explants with 3 cuts

lane 3:explant with 5 cuts 

For PCR product electrophoreis :  our group wells are the last two wells from the right edge of the gel before the ladder figure II in next slide:

lane 1: positive control

lane 2:Explant with 5 cuts.

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Based on the above mentioned; below present the electrophoresis gel UV- photograph for both PCR and genomic sample

Figure III: genomic DNA gel

1 2 3

Figure IV: PCR product gel

1 2

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When we compare the DNA bands on gel and the corresponding reference on Vivantis VC100bp DNA-plus ladder bands, we will be able to see that though positive control was forgotten to be run together in the PCR product gel figure IV above, but still we manage to get a gene band around 1.5kbp in both lane I and II of positive control and 5cuts parameters respectively, but the bands are too bright and sharp to be the GUS gene band. Possibly it may be bacterial or plant plasmid since we grinded the whole explants in the DNA extraction, so probably the brightness in the band is due to plasmid concentration not the GUS gene as GUS gene supposed to appear light (faintly) not as in the figure IV presented above. However, further molecular analysis like Southern blotting and sequencing the sample by for example Sanger’s sequencing can be done on the PCR sample and compare it to GUS sequence databases to confirm the presence of the GUS- gene.

Though positive control was run on genomic DNA gel sample (figure III above), the bands are mostly smeared and not directly readable, this smearing could be due to the genomic DNA base pairs density (molecular wieght i.e the genomic DNA fragments are un digested so are heavy compared to the PCR DNA fragments) or it could be due to RNA contamination, that is to say probably the RNAse concentration used wasn’t enough to degrade all the extracted RNA in the samples as such causing contamination in the gel especially in lane 2 and 3 in figure III above. Despite this fact the band appeared to be faint in color (lane 1 in figure III above) and we observed a band corresponding to 1.2 kbp; however, could this be a GUS band or not? This question can only be answered by further molecular analysis as mentioned above supposed to be undertaken on both the genomic DNA samples and the PCR DNA samples, in order to confirmed GUS gene.

GOI Expression analysis by GUS histochemical assay:

Though we were un opportune to do the GUS histochemical assay, but we were presented with the already assayed sample based on Jefferson R. A. 1987 modified protocols, and we have observed physically the nature of the bluish-indigo coloration as a result of x-gluc reaction which leads to the formation of insoluble 5,5’-dibromo-4,4’-dichloro-indigo. As represented in the below reaction:

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SUGGESTION AND CONCLUTION:

Suggestion:

Though, we were able to present our results above based on the provided practical proforma and followed procedures as described in different literature, still it is not enough based on what we have presented to confirmed the presence of the GOI in the transformation process, it could be probable that we are only amplifying plant or bacterial plasmid, thinking that it is our gene of interest, hence for this result to be confirm, we suggest that a further analysis should be carried on the extracted genomic DNA and PCR DNA samples, and this may include running the PCR again using the positive control for validation, GUS flourometric, spectrometric and histochemical assays as well as using Southern transfer to blot nitrocellulose for the presence of the gene if possible sangers sequencing can be done and compare the

result against the database GUS gene sequence.

I suggest that it is also important before commencing further analysis after the isolation of nucleic acids, DNA purity should be check, as the solution is still contaminated with proteins. These are removed by extraction with phenol. To check the success of the removal, a purity determination is performed, which is based on the different absorption characteristics of the proteins and the nucleic acids. Nucleic acids have an absorption maximum at 260 nm, whereas proteins absorb at 280 nm. The purity of the nucleic acids is assessed by determining the quotient

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of the absorption data measured at 260 nm and 280 nm. If the quotient is between 1.8 and 2, the purity is 70 to 95 % and is considered to be o.k. The DNA purity can also be checked by electrophoresis, as if there is any contamination will definitely show in the bands.I also suggest that for all these presented data to be fully comprehended and to establish a significant difference that exist among the parameters a statistical analysis such as ANOVA needs to make.

We suggest that though all the instructors did their best to see the realization of this practical, which we are grateful, we advise that either Agrobacterium density in the culture or Cefotaxime concentration should be carefully revised in order to reduce the effect of Agrobacterium contamination, which happen in almost all the practical samples.

Conclusion:

In conclusion, we generally know that wounding of explant prior transformation is necessary in order to provide accessibility for Agrobacterium to transform the competent cells that may locate more deeply in the tissue. The secretion of phenolic compounds by wounding effect of metabolically active cell also improves the transformation efficiency as it acts as a chemotactic agent upon wounding which attracts the Agrobacterium towards the wound in order to effect the transformation. In this practical we learnt the methods of transformation and optimization of the transformation induction by wounding the explants. Upon what we have presented we have seen that in explants of N. benthimiana of size 5mm X 5mm under our practical condition the possible optimize wounding parameter is 5 cuts per explants; which led to the putative transformed explants percentage of 75% as the highest percentage among the samples either above 5 cuts or below 5 cuts the percentage is not attractive. But further molecular and experimental analysis as well as statistics needs to be done for confirmation of this assumption.

Thank you.

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