Molecular Techniques in Biosciences (D224P6 MY)

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15 APRIL 2013. Approximately 1489 words

Methodology review for transformation of Citrullus vulgaris Schrad (watermelon) through biolistic
By student ID 009921
School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor.

Research summary
This writing is an evaluation of the methodology of a study conducted by Suratman et al (2010) to find the optimum particle bombardment parameter for Citrullus vulgaris Schrad (watermelon). The study used DNA-coated 1.0 m gold particle for all transformations. Parameters analyzed were the explant ages (4 and 5 days old), different concentration of osmotic treatment using sorbitol or mannitol (0.2, 0.4 and 0.6 M) for 24 hours, different DNA concentration (0.6, 1.2 and 1.8 g), rupture disk pressure (650, 900 and 1100 psi) and target distance from stopping plate (6, 9 and 12 cm). 4-day-old explants were used in all transformation parameters testing other than explant ages. Plasmid used is pRQ6 and pCAMBIA both carrying both gusA and hpt genes and grown in liquid medium (LB liquid media for pRQ6 and YEP broth for pCAMBIA) containing ampicillin and kanamycin respectively. The study found that the optimized parameters are 4 days old explant, 1100 psi helium pressure, 6 cm target distance, DNA concentration at 1.2 g and osmotic treatment with 0.6 M prior to bombardment. The parameters is tested using different plasmid vesctors; pAHG11 (carrying gusA and hpt genes), pCAMBIA 1301 (carrying gusA and hpt genes), pCambar (carrying bar gene) and cotransformation of pCAMBIA 1301 and pAHG11 (carrying gusA, bar and hpt genes) and grown in selection medium for more than 2 months. Transformants then undergo -glucuronidase (GUS) histochemical assay and selected using hygromycin at 2 or 5 mg/L (for pRQ6 and pCAMBIA 1301) or 2 mg/L PPT (pHAG11). The survived explants were cultured in antibiotic- and PPT-free medium for 4 weeks and recultured in selection medium. Polymerase chain reaction (PCR) analysis is done to confirm the integration of transgene. Only about 14 - 23 % of the explants survived the selection process and only 1 out of 60 (1.67%) regenerated explants show GUS positive. However, PCR results shows 1 - 17 % of the genes presence in the transformed explants. This proved that the exogenous DNA has been integrated into the genome and possibly the gusA gene has fused with selectable marker resulting the marker gene not to be expressed. Statistical analysis has been done using SSPS software for Windows version 12. The data was analyses using one way ANOVA and HSD value and compare at p<0.05.

Methods review
In the study, a lot of protocols has been used; plasmid DNA isolation, particle bombardment, histochemical GUS assay and molecular analysis including PCR and gel electrophoresis. The choice of using plant seedlings of yellow watermelon are advantageous compare to the mature plant. Study in Arabidopsis discovered that transcription level of plastid genes significantly reduced in older leaves compare to young leave (Zoschke et al 2007) which might lead to the lower expression level of desired gene in transformed plant. Photosynthesis rate is also reported to decline with increase age of plant (Miller 1995). These shows that older plants are less responsive, not readily to adapt the changes, has more phenolic compounds and less cells growth rate that may lead to the less gene transformation and making the parameter testing more challenging as more strict protocols may needed. Watermelon is well known for a rich sources of beneficial plant compounds like lycopene that found to inhibit cancer cells growth (Hall 2004) and citrulline used for cardiovascular and immune fuction (Collins et al 2007). In a research perspective, choosing watermelon for transformation increases the value of the species for further study as there are less research done for transformation of watermelon. Plasmid DNA isolation and extraction conducted is to obtain plasmids from Escheria coli (pRQ6, pAHG11 and pCamBar) and Agrobacterium tumefaciens (pCAMBIA). Ampicillin with 100 g/mL concentration has been chosen for E. coli plasmid DNA and 50 g/mL kanamycin for A. tumefaciens. Both concentrations are acceptable as minimum inhibitory concentration (MIC) of ampicillin for the microbe are 1.1 to 5.7 g/mL (Thonus et al 1982) and 50 g/mL for kanamycin is commonly used (Wang 2007). Using proper kit (QIAprep Spin Miniprep Kit in the experiment which can prepare up to 20 l of DNA) for small quantities of plasmid DNA extraction is a good choice to increase quality, cut cost and save time (QIAprep Spin Miniprep Kit 2013). The option of using GUS as reporter gene rather than GFP give some limitations because the assay is destructive and not suitable for in vivo detection (Jefferson 1987) while GFP is nondestructive (Molinier et al 2000) so more culture can be preserved. As the results, some of the culture (120 in total) has been sacrifice which is necessary for histochemical assay. Depends on ones choice, GUS is more preferable by the authors probably due to its robustness, versatility,

stability and simplicity. Limitation using GUS like increasing staining intensities over time even the gene expression is low (Mantis and Tague 2000) has been countered by quickly staining the cell 24 hours after bombardment. Suggestion on improvement using GUS assay is to use Bacillus sp. GUS gene rather than E. coli which reported to have more superior properties (Jefferson et al 1987); more thermostable with half-life of 10 minutes at 650C and enzymatically stable (Jefferson and Mayer 2003). On top of that, there is one component missing on how blue spot from the GUS histochemical assay is counted. Klein et al (1988) has described that whether a single cell or a group cells that exhibit blue spot it is considered as one expression unit and this is not define by the author which may leads to the data inaccuracy and invalidity if the counting did not follow the description. For particle bombardment, the choice of gold itself is beneficial compare to other microcarrier like tungsten. Gold is non-toxic and inert towards catalytic degradation while tungsten might destroy the culture through acidification of the media (Sanford et al, 1993). Tungsten is also possibly cause agglomeration during DNA coating procedure because of its irregular structure (Hunold et al 1994). However using 1.0 m gold size nano particles in the experiment can be further optimize by using 0.6 m that have smaller size. One study (RandolphAnderson et al 1995) reported that by using smaller microprojectile molecule, less damage is being inflicted to the cells giving them a better chance of survivality. The choice to use 4-days-old explants for the biolistic transformation (for other parameters than plant age) seems to be avail when later experiment proven the expression of GUS is highest at this age. The stages of the cells growth is crucial that determine the competency for both transformation and regeneration (Kikkert et al 2005). Other studies also support this findings as the efficiency of the gene expression decreases along with plant age in tobacco (Predaja et al 2010), japonica rice (Ramesh and Gupta 2005), tomato (Ruma et al 2009) and cotton (zyiit and Gzkirmizihigh 2008). Osmotic treatment is also a contributing factor to the transformation rates by plasmolyzing the cells prior to the bombardment (Vain et al 1993). The study chose to use mannitol and sorbitol which found to be the best osmoticum treatment for biolistic transformation study (Shark et al 1991; Armaleo et al 1990). Different concentration has been used: 0.2, 0.4 and 0.6 M and found that treatment with 0.6 M shows the highest average number of spots per plant. Another type of

plants also shows similar results like 0.4-0.6 M for maize (Vain et al 1993) but through other researches, it is found that different plants might have different concentration depending on the treatment duration and possibly depends on the plant species (refer Table 1.0).

Table 1.0: Different concentration and duration of mannitol and sorbitol used in different type of plant for osmotic treatment that give high expression in biolistic transformation Concentration 0.4 - 0.6 M 0.25 M 0.5 M 0.5 M 0.4 M Duration 41, 162 h 24 h 24 h 4h 41, 452 h Plant type Maize Basmati rice Malaysian indica rice Coffea Wheat Sources Vain et al 1993 Jain et al 1996 Zuraida et al 2010 Rosillo et al 2003 Ingram et al 1999

1 pre-transformation duration; 2 post-transformation duration

Study on indica rice (Zuraida et al 2010) founds that the shorter period treatment (12-24 hours) with osmotic agent resulting the higher expression. The study conducted by Suratman et al (2010) did not investigate the different duration of the osmotic treatment and used 24 h treatment for the whole experiment and this can further investigated. Pre- and post-transformation of osmotic treatment can also to be taken into account although study in maize shows no different of post treatment (Vain et al 1993).

Conclusion
To conclude, the study has taken most of crucial details into account when planning the experiment. Some minor improvement might needed but might be not necessary and some things need to be clarified. The protocols in the study might become a pioneer for further research in C. vulgaris or any other watermelon species especially in transformation to produce better yield and quality of the fruits.

References
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Websites and other resources Hall, C.V. (2004) Watermelons as Food in the 22 Century. In: Food Security and Vegetables: A Global Perspective. http://www.public.iastate.edu/~taber/Extension/Watermelons%20as%20food-Hall.pdf. (Access date 27/04/2013) Jefferson, R. A. and Mayer, J. E. (2003) Microbial -glucuronidase genes, gene products and uses thereof. U.S. Patent no. 6,641,996, 103.

QIAprep

http://www.qiagen.com/Products/Catalog/SampleTechnologies/DNA-Sample-Technologies/Plasmid-DNA/QIAprep-Spin-Miniprep-Kit. (Access date 27/04/2013)

Spin

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Kit.

(2013).