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HPLC method was developed to separate the polyphenolics found in the hairy root extracts of O. basilicum and to study their variation. The representative HPLC chromatograms (Figure 3.14) obtained from the three hairy root lines derived from the three different cultivars of O. basilicum showed variable number of peaks in all the samples. The HPLC chromatogram of HR 2 (derived from B 3) was observed to have a lesser number of polyphenolic peaks at 280 nm in comparison to HR 4 (derived from B 12) and HR 5 (derived from B 13) at all ages. This observation clearly indicated that variation in number of polyphenolics existed between the hairy root lines derived from the different cultivars of O. basilicum. HR 4 was identified to have highest number of polyphenolics than HR 2 and HR 5. The number of peaks in extracts of same hairy root line at different ages showed similar number of peaks with difference in their peak area. RA presence in all the samples was confirmed with the standard RA chromatogram by HPLC analysis.
RA was detected in all hairy root lines as a major peak in all samples at retention time; 15.28 min. A shift in retention time of RA in this chapter from 14.5 min in the Figure 2.4 of chapter 2 of this thesis was found. Shift in retention time was attributed to the use of a different column make and its dimensions (Apollo TM C 18 150 mm
× 4.6 mm × 5μm to C18 Phenomenax, Gemini- NX250 mm × 4.6 mm × 5 μm) in this study. Caffeic acid was also identified with the commercial standard in the hairy root extracts at retention time 12.08 min.
134 Figure 3.14. Representative HPLC chromatograms of three hairy root lines. a) HR 2 (derived from B 3 cultivar of O. basilicum). b) HR 4 (derived from B 12 cultivar of O. basilicum) and c) HR 5 (derived from B 13 cultivar of O. basilicum).
Individual run for extracts at five different ages after harvest are compiled in each subfigure. Different colors show different ages after harvest. The prominent peak in all samples was identified as RA (15.28 min).CA (12.08 min) was also found in all samples.
135 3.9.2. Rosmarinic acid
In the previous chapter of this thesis, Rosmarinic acid (RA) was identified as the major metabolite in O. basilicum cultivars and was selected as the targeted metabolite in this chapter for the screening of developed hairy root lines. Developed hairy root lines were quantified for the RA content using HPLC analysis. RA standard curve with equation y = 19014x - 9029.3 and R² = 0.9994 was used for its quantification in the unknown hairy root extracts.
Variation in the levels of RA content was found in extracts of all hairy root lines. RA content was found to increase till 40 d in HR 1, 2 and 3 (all derived from B13 cultivar) while in HR 4 (derived from B12) its value increased till 50 d (Table 3.4 a).
HR 5, 6 and 7 (all derived from B13 cultivar) showed highest RA content at 60 d.
The highest RA content in HR 1 (40 d; 71.02 ± 12.67 mg/ g DW), HR 4 (50 d; 69.49
± 1.79 mg/ g DW) and HR 5 (60 d; 76.40 ± 3.70 mg/ g DW) were found to be significantly similar which clearly indicated that RA synthesis is age dependent in hairy root lines derived from the three different cultivars of O. basilicum.
On comparison between hairy root lines derived from the same cultivar, no significant difference (p ≤ 0.05, p ≤ 0.01) was found between HR 1, 2 and 3 (derived from B3) at 40 d and 60 d and similar observation was made for HR 5, 6 and 7 (derived from B3) at 40 d, 50 d and 60 d. 40 d can be considered as optimum age for RA production in HR 1, 2, 3 and 4 because of high RA content. HR 2 showed significantly highest levels of RA at 20 d (28.83 ± 1.65 mg/ g DW) and 30 d (42.31 ± 2.21 mg/ g DW) in comparison to all other hairy root lines. HR 2 from B3, HR 4 from B 12 and HR 5 from B 13 can be selected due to higher RA levels.
136 3.9.3 Caffeic acid
Other than RA, caffeic acid was also detected in all samples at retention time 12.08 min. The presence of caffeic acid is reported for the first time in hairy root samples of O. basilicum (Figure 3.15 b). CA standard curve with equation; y = 2E + 06x + 222605 and R² = 0.9965 was used for its quantification in the unknown hairy root extracts. The trend of CA content found in all hairy root lines was different. An increase in the level of CA was observed in HR 2, 5, 6 and 7 while decrease in its content after 40 d in HR 1 and 3 and after 50 d in HR 4 was found. As observed the amount of CA found was much less in comparison to RA and thus it can be concluded that CA is the minor metabolite in the developed hairy root lines. HR 4 was observed to have significantly (p ≤ 0.05, p ≤ 0.01 and p ≤ 0.001) higher concentration of CA at all ages (0.93 mg/g to 1.74 mg/g DW) in comparison to the other hairy root lines.
137 Table 3.4. Levels of Rosmarinic acid and Caffeic acid (mg/ g DW) detected in hairy root lines at five different ages after subculturing on M medium.^
(a) Rosmarinic acid content (mg/ g DW) Hairy root
lines (HR)
Days after subculturing (d)
20 30 40 50 60
HR 1 10.53 ± 1.76b 22.62 ± 2.39b 71.03 ± 12.67a, ** 47.06 ± 3.63b 38.22 ± 4.62cd HR 2 28.83 ± 1.65a 42.31 ± 2.22a 69.02 ± 2.78a, *, ** 67.21 ± 5.95a 41.44 ± 5.82bcd HR 3 4.63 ± 0.65c 23.76 ± 2.20b 66.24 ± 5.48ab, *, ** 42.92 ± 1.27b 24.23 ± 1.32c HR 4 11.10 ± 1.75b 23.31 ± 1.89b 68.93 ± 3.97a, *, ** 69.49 ± 1.79a 57.98 ± 4.24abc HR 5 11.34 ± 1.03b 15.54 ± 2.09bc 45.03 ± 7.32abc, *, ** 55.15 ± 1.67ab 76.41 ± 3.71a HR 6 4.11 ± 0.14c 5.48 ± 0.21d 29.76 ± 4.61c, ** 48.82 ± 1.24b 67.17 ± 9.38ab HR 7 3.13 ± 0.21c 9.70 ± 2.24cd 36.75 ± 3.07bc, *, ** 49.59 ± 2.44b 68.47 ± 7.21ab
(b) Caffeic acid content (mg/ g DW) Hairy root
lines (HR)
Days after subculturing (d)
20 30 40 50 60
HR 1 0.15 ± 0.02b 1.05 ± 0.05b 1.02 ± 0.01b 0.72 ± 0.04b 0.15 ± 0.01d HR 2 0.27 ± 0.03b 0.62 ± 0.04c 0.67 ± 0.01cd 0.73 ± 0.04b 0.74 ± 0.04c HR 3 0.12 ± 0.02b 0.44 ± 0.02cd 0.93 ± 0.07bc 0.15 ± 0.00c 0.11 ± 0.01d HR 4 0.93 ± 0.16a, #, ## 1.30 ± 0.07a, #, ## 1.61 ± 0.14a, #, ## 1.74 ± 0.17a, #, ## 1.53 ± 0.01a, #, ##
HR 5 0.14 ± 0.02b 0.41 ± 0.01d 0.50 ± 0.01d 0.70 ± 0.02b 0.78 ± 0.01c HR 6 0.12 ± 0.01b 0.26± 0.04de 0.78 ± 0.08bcd 0.89 ± 0.04b 1.03 ± 0.03b HR 7 0.11 ± 0.00b 0.18 ± 0.00e 0.51 ± 0.05d 0.78 ± 0.01b 0.98 ± 0.01b
138 Data represented as mean ± SEM of each of three replicates (n = 3). Section a and b of the table
represents RA and CA content. Different letters indicate significant differences (p ≤ 0.05, p ≤ 0.01) according to Tukey’s HSD between different hairy root lines at specific age for RA and CA content. No significant difference (p ≤ 0.01 and p ≤ 0.001) in the levels of RA at 40 d of all root lines except HR1 and HR6 was found and represented as “*” and “**”in the section a. HR 4 showed significantly higher CA content in comparison to all other hairy root lines at all ages (p ≤ 0.01 and p ≤ 0.001) and is represented as “#” and “##”in the section b of the table. Highlighted figures show highest value for each root line.
139 3.10 Comparison between non-transformed and transformed hairy root lines The advantage of hairy root lines over non-transformed roots for RA production from the selected cultivars of O. basilicum was also studied. The hairy root lines were compared with the normal (non-transformed) roots for the RA content and age selected for this comparison was based on the highest amount of RA obtained for the hairy roots of the three cultivars. Forty days was selected as the age for B 3, fifty days for B 12 while sixty days was selected for B 13. Significant difference in RA content between non-transformed and transformed root lines was found for all cultivars of O. basilicum (Figure 3.15 a). It was found that compared with roots of normal plants grown in M medium, HR 1, 2 and 3 showed five times more production of RA. For B 12 and B 13 derived hairy root lines, the production rate was double that of normal roots of the same age.
Another advantage of hairy root lines over non-transformed roots lines is their capacity of higher biomass production. This parameter was also studied for the developed hairy root lines and non-transformed roots obtained from plants grown in an in vitro whole plant (as described in chapter 2 of this thesis) on M medium.
Significant difference was found between the non transformed roots and transformed hairy root lines of the same cultivar for all the three cultivars of O. basilicum (Figure 3.15 b). HR 1, 2 and 3 showed twice the biomass of normal roots while in rest of the root lines more than 1.5 times biomass was found in comparison to non-transformed roots. These results clearly showed that hairy root cultures have a higher capacity for production of secondary metabolites and a higher biomass than normal roots and their suitability for large scale production.
140 Figure 3.15. Comparison between non-tranformed and transformed hairy root lines of three cultivars of O. basilicum. Solid black line show boundary between three different cultivars. a) Comparison of RA (mg/ g DW) between non-transformed and transformed roots of B 3, B 12 and B 13. Significant difference at p ≤ 0.05 was found between non-transformed and transformed roots of all cultivars. b) Comparison of Dry weight (mg) between non-transformed and transformed roots of B 3, B 12 and B 13. Different letters indicate significant difference at p ≤ 0.05 was found between non-transformed and transformed roots of all cultivars.
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3.4 Discussion
The study was set for the development of hairy root lines from the selected cultivars of O. basilicum. The cultivars used were B 3 (low RA producing), B 12 and B 13 (high RA producing). Developed hairy root lines were used for screening and selection of the hairy root lines against their morphology and RA content. This chapter identified the explant type, their age and A. rhizogenes strains for transformation of selected cultivars of O. basilicum. Correlation between developed root phenotype, endogenous auxin content and molecular make up was also established. The distinct relationship between root growth and secondary metabolite production was also found in this study. Selection of the developed hairy root lines from the three selected cultivars for mycorrhization studies on the basis of their morphology and RA content was thus reported in this study.
The establishment of hairy roots in the three cultivars of O basilicum using different A rhizogenes strains showed that root induction is dependent on the type of explants and bacterial strain used as previously observed (Chriqui et al. 1996; Bansal et al.
2014; Nourozi et al. 2014; Thiruvengadam et al. 2014). Cotyledons have been reported to be the best explants for A rhizogenes transformation studies in comparison to young leaves (Fattahi et al. 2013; Thimmaraju et al. 2008; Triplett et al. 2008) but this is contrary to the results obtained in the present study. Results presented in this study showed similarity to a number of other studies (Nourozi et al.
2014; Bansal et al. 2014; Vyas and Mukhopadhyay 2014) who also found young leaf to be the most suitable explant for transformation and they have suggested this suitability as the result of the physiological state of the plant cell or its higher
142 regeneration capacity and larger wounding surface availability in the case of leaves.
Differences in the level of virulence between different strains of A. rhizogenes can also be put forward to explain the difference in transformational efficiency (Nourozi et al. 2014). The agropine-producing A. rhizogenes A4 strain was identified as the most potent bacterial strain in the current study. It is likely that wild origin of this strain accounts for such an observation. Some A. rhizogenes inoculated explants of B 12 and B 13 showed callus formation near to the hairy root induction site, a response probably due to wound induced phytohormone production as suggested by Triplett et al. (2008). Low infection potential of A. rhizogenes strain 11325 may be related to its different chromosomal virulence genes as discussed by Tiwari et al. (2008). It can be thus concluded from the current study that hairy root generation is explant specific and virulence dependent.
A. rhizogenes is reported to induce the differentiation of genetically transformed roots in dicotyledonous plants (Amselem and Tepfer. 1992). Two primary groups of pRi plasmid play roles in the root induction process. The TL region harbors the rol genes and the TR region carries the aux genes that are responsible for hairy root formation. Transformation of the selected O. basilicum cultivars produced profusely growing, highly branched, negatively geotropic roots growing on the hormone independent medium. At initial stage, two distinct morphotypes (long and ramified and short and ramified) showing similarity to the two hairy root phenotypes of cucumber obtained by Chriqui et al. (1996) were also observed in my study. The second type was not found to grow on subsequent subcultures and a likely explanation is the presence of only the TR sub-fragment and functional aux genes
143 but not TL- DNA transfer region which is clearly important for differentiation and morphogenesis of hairy roots. Roots of O. basilicum cultivars showing long and ramified morphology were thus selected for the current study.
Roots that were produced following inoculation with A. rhizogenes strain ARqua1- pTSC5 were confirmed to be transformed by use of the GUS assay as proposed by Jefferson et al. (1987). As observed by Cseke et al. (2007), root hairs in the current study were also found to show higher GUS activity and enabled the rapid screening of transformed roots. Highest transformation efficiency and similarity in the T-DNA region of A4 and ARqua1- pTSC5 were the reasons for selection of A4 derived hairy root lines for further confirmation and characterization studies. Seven different axenic A4 derived transformed root lines from the three different cultivars of O.
basilicum were selected. The integration of rol b, rol a, aux 1 and aux 2 confirmed integration of complete TL-DNA region and the transgenic nature of all hairy root lines similar to the results obtained by Mallol et al. (2001); Batra et al. (2004);
Bandyopadhyay et al. (2007); Thimmaraju et al. (2008); Fattahi et al. (2013).
Phenotypic variation and variation in the levels of growth were noted in the hairy root lines derived from different cultivars as also observed in many studies (Amselem and Tepfer 1992; Chiriqui et al. 1996; Mallol et al. 2001; Thimmaraju et al. 2008; Triplett et al. 2008). These variations are due to factors such as difference in the copy number of inserted gene, its type and position in the host, methylation of genome and chromosomal changes (Triplett et al. 2008). In many studies phenotypic variation is correlated to the expression of aux genes (mainly to aux 2 as proposed by Croes et al. 1989; Amselem and Tepfer 1992; Chiriqui et al. 1996) and thus their
144 presence was also confirmed in this study. Contrary to the results obtained by Alpizar et al. (2008), the presence of both aux 1 and aux 2 was found in the current study similar to the results presented by Amselem and Tepfer (1992) and can further support the fact that a correlation can exist between aux genes and morphology of hairy root lines.
The production of callus by all the roots on NAM supplemented medium further confirmed the presence of functional aux 2 genes and the highest response achieved for HR 4 can be correlated with its thick morphology (Amselem and Tepfer 1992;
Chiriqui et al. 1996). Auxin also have a significant influence on root length, branching correlated pattern and number of laterals as reported for hairy root cultures of Beta vulgaris by Thimmaraju et al. (2008). Here also a positive correlation between endogenous auxin content and root phenotype was found for sixty days old root lines (length; r 2 = 0.7271 and number of tips; r 2 = 0.2821). The thin morphotype observed for HR 1 and 5 may also be correlated with low endogenous auxin concentration. The rooty callus morphology of HR 5, 6 and 7 can be explained by difference in the copy number and insertion site of TL and TR-DNA as discussed by Bandyopadhyay et al. (2007). Further distinct variation in root morphology in this study was found to be cultivar specific as generally similar morphotypes were obtained from the same cultivar.
The purpose of developing hairy root lines in this study was to select them for co-culture development, thus M media was used as the selection substrate. Nutrient composition and concentration impacts secondary metabolite production (Tada et al.
1996; Thimmaraju et al. 2008) and this was well observed in this study also. Hairy
145 root lines growing on M medium produced higher levels of rosmarinic acid than normal roots of the same cultivar that were grown in MS medium but were one month younger (when compared with data of Chapter 2). This observation of high production of metabolites in hairy roots further supports the notion that secondary metabolite production is effected by substrate used. A high metabolic content and excessive growth rates have been defined as the advantages of transformed root cultures over normal plant roots (Mallol et al. 2001; Bais et al. 2002; Grzegorczyk et al. 2006) and similar observation of increased RA content and biomass have also been made in this study when grown on same medium. Low content of caffeic acid in the developed hairy root lines and non-transformed also supports the previous observation (Chapter 2) that caffeic acid is a minor polyphenolic in hairy root lines also as found in the non-transformed roots of O. basilicum cultivars. A notable decrease in the content of RA with increased age in HR 1, 2, 3 and 4 can be correlated to the cultivar type, excessive growth or nutrient limitation and also shows that RA production is nutrient and growth dependent. The RA content of hairy roots of the same cultivar were approximately the same at their optimal RA production age but differed between cultivars and their age. A positive though low correlation (r 2 = 0.5423) was also found between total phenolic and RA content.
To, my knowledge there are only two reports (Tada et al. 1996; Bais et al. 2002) which have reported A. rhizogenes mediated transformation of O. basilicum and RA production. Hairy root growth kinetics for RA production in transformed roots of O.
basilicum grown in different mediums has been studied by Tada et al. (1996). High RA production of 14.1 % D.W. in MS medium by J1 clone was reported after eight
146 weeks of growth which is twice the highest amount in the current study. This difference in RA production may be due to use of different medium (MS) by Tada et al. (1996) or different strain of bacterium. In a study conducted by Grzegorczyk et al.
(2006) in Salvia officinalis, RA levels were found similar to the amount reported in the current study. In a different study conducted by Lee et al. (2010) in Nepeta cataria optimal levels of RA and biomass were found at a specific age showing that there is considerable variation in RA content and biomass from genus to genus of the same family. Recent report by Fattahi et al. (2013) on transformation of Dracocephalum kotschyi Boiss for RA production have identified 1500 μg/g DW as highest yield when compared with similar studies. However if one calculates the concentration of RA found in hairy roots of this study or in several of the other studies cited by Fattahi et al. (2013) it was observed that they are incorrect in stating that their yield was much high. In fact the yield of RA found in the current study is more than fifty times greater than that claimed by Fattahi et al. (2013). My study also found similar levels of RA to that reported by Weremczuk-Jezyna et al. (2013) in transformed roots of D. moldavica L.
In conclusion this study for the first time demonstrated the utility of hairy root lines derived from the three cultivars of O. basilicum as efficient resource of RA on M medium. RA was found as the major metabolite in all hairy root lines. Inter-cultivar variability in terms of morphological diversity and RA content was also remarkably observed in this study. B 3 (for two out of three root lines) and B 12 produced thick roots while B13 yielded thin and callus hairy root morphotypes. Although, no significant difference was found in RA content for hairy root lines derived from the
147 same cultivar at its optimal production age, but there were differences in RA content of roots derived from the different cultivars at all ages. A distinct relationship between age and RA content was also found here which advocates the fact that RA synthesis in these hairy root lines is age dependent and can be further correlated with growth potential and nutrient availability also. The amount of RA produced is higher in comparison to that produced by untransformed roots in same (this study) or different medium as reported in chapter 2, thus proving advantage of transformed hairy root cultures for enhanced secondary metabolite production. HR 2, HR 4 and HR 5 were selected as they represented variability and produced high RA content.
They were taken forward in the next chapter for mycorrhization studies to understand potential of mycorrhiza as an elicitor.
148