Agrobacterium-mediated transformation is effected by many physical and chemical factors. In the present study both physical and chemical parameters were evaluated by using
gus as a reporter gene. The transient expression of
gus gene can be easily measured from the transformed plant cells
(Batra and Kumar (2003). Genetic transformation by
Agrobacterium tumefaciens EHA-105 can be improved by evaluating physical and chemical factors which was resulted in developing an efficient method of gene transfer to several plants like tobacco
(Kutty 2010), Sorghum
(Indra Arulselvi et al., 2010), Rice
(Tripathi et al., 2010).
Bacterial growth phase and infection period
The results of transformation using
Agrobacterium tumefaciens strain EHA 105 at various bacterial concentrations ranged from A
600nm 0.17-1.2 and infection periods analysed at 15, 30 and 60 min were shown in Table 1. Of four different bacterial densities, the highest numbers of GUS-positive spots were found at A
600nm 0.17, 30 min (9.33±0.13b). The transient activity and transformation frequency were decreased with increased bacterial concentration and infection period.
The percentage of GUS positive explants from bacterial density at A
600nm 0.17 to 0.56 cultures were increased 28.33% and decreased at A
600nm 1.2 cultures. Transformation frequency decreases as necrotic damage increases from lower bacterial density (A
600nm 0.17) to higher density (A
600nm 1.2). This indicates that explants viability has direct effect on transformation efficiency.
Higher concentrations of
Agrobacterium were used to transform recalcitrant plants such as rice
(Chan et al., 1992), Sweet potato
(Gonzalez et al., 2008) and pepper
(Ismail et al., 2006). Low bacterial density was also used to transform plants such as in Brocolli
(Metz et al 1995), Wheat
(Cheng et al 1997) and tobacco
(Kutty PC 2010). But in our study early log phase was found to be the best for transforming
Euphorbia tirucalli L. Bacterial density at A600nm 0.17 growth phase was found to be most effective in producing high transformation efficiency. In order to minimise the necrotic damage of explants, culture was diluted to 1:10 before infection of explants. Similar approach was reported
(Chakrabarty et al., 2002), in which bacterial inoculums was diluted before infection. The transformation efficiency was found at A6
00nm 0.56, 0.8 and their mean differences were statistically insignificant. Necrotic damage was absent in bacterial density at A600nm 0.17 and infection period 30 minutes and it was chosen in this study.
Co-cultivation temperature
The effects of different co-cultivation temperatures on transient GUS expression were studied and the result were shown in Table 2. The explants which were precultured for two days followed by co-cultivated at three different temperatures like 22, 25 and 28°C and the highest GUS positive spots were found at 25 and 28°C with the mean value 42 (±05.17a) and 39 (±04.13a) respectively. There were no visible spots were observed at 22°C. This indicates that co-cultivation temperature has direct effect on
Agro bacterium-mediated transformation. The mean differences of GUS positive spots at 25 and 28°C were statistically insignificant.
The percentage of GUS positive explants were found to be approximately 40% at both temperature 25 and 28°C (Table 2). After analysing with trinocular microscope, 42 spots were found per explant incubated at 25 and 28°C. There were several reports on higher transformation efficiency where explants Co-cultivated at 22°C and obtained more number of GUS – positive spots
(Dillen et al., 1997) (Kutty 2010). Efficient DNA delivery into plant cells at 22 °C was also reported in Sweet potato
(Gonzalez et al., 2008), Cotton
(Sunil kumar and Rathore 2001) and cauliflower
(Chakrabarty et al., 2002). It was reported in the earlier studies that size of crown gall tumour decreased when Co-cultivation temperature was increased
(Braun (1947). Another study found that Ti- Plasmids were lost in
Agrobacterium tumefaciens when the culture was grown over 36 hours at elevated temperatures (Watson B1975). Low temperatures from 20 to 22°C were found to be promoting pilus assembly and it was influenced by
VirB gene at low temperature which is required for conjugal transfer of TDNA into plant cells
(Fullner et al., 1996).
Based on the results obtained, Co-cultivation temperature either at 25 or 28°C was found to be equally effective to get more number of GUS-positive spots as well as more number of GUS-positive explants. Hence we have chosen 25°C as co-cultivation temperature and it was introduced in further experiments. We have chosen 25 instead of 28°C since low temperatures are favourable for efficient T-DNA transfer
(Salas et al., 2001) compare to high temperatures.
Acetosyringone in cocultivation medium
The effect of Acetosyringone (AS) on
Agrobacterium-mediated transformation was studied using six different con centrations from 50-800 μM. These different concentrations were added seperately to the Cocultivation medium and results were shown in Table 3. Of six different concentrations of AS 100 μM concentration gave highest number of GUS positive spots with the mean value (163.33±44.36c). The lowest numbers of GUS positive spots were found at AS concentration from 200-800 μM. The mean differences between 100 μM and rest of the AS concentrations were statistically significant. The positive explants at 25°C were 40% without AS (Fig 3C) and increased to 72% after the addition of AS 100 μM (Fig 3D). The number of GUS-positive spots were counted using Trinocular microscope and one of such explant with microscopic view was shown in (Fig 3E).
There was no increase in the number of GUS positive explants after adding AS into the bacterial inoculums (data was not shown). Other concentrations of AS were not shown any increase in the transient GUS expression when compared with AS concentrations at 50 and 200-800 μM. It reveals that
Agrobacterium cells may have been induced to maximum towards virulent stages at 100μM concentration of AS. The same concentration was also reported in other plants of
Euphorbiaceae family
(Li et al., 2007; Kumar et al., 2010). The optimum concentration of AS for higher transient expression varies based on genotype and cultivar of plant
(Gonzalez et al., 2008).
GUS histo chemical analysis
The method of
Jefferson (1987) was used for a GUS histochemical assay. The positive results were observed with naked eye and each blue spot was counted using stereo microscope, irrespective of its size (Fig 3). The explants co-cultivated at 25°C and the number of GUS-positive explants obtained was 15 per 25 explants (Fig 3C). Whereas the explants co-cultivated at 25°C and 100 µM AS resulted GUS-positive explants were 18 per 25 explants (Fig 3D). An individual explant of Control (Fig 3A) and GUS positive explant (Fig 3B) were shown with magnification. A close-up image of microscopic view of GUS-positive explants with blue spots were shown (Fig 3E).
pre-cultured explants at room temperature for 2 days showed low transient expression when compared with explants without pre-culture. Increasing the number of days from 2 to 8 days did not show any increased transient GUS expression (data not shown). Similar finding was reported
(Cervera M, et al., 1998), where pre-cultured explants of citrus showed decreased transient GUS-expression when compared with explants without pre-culture.
