FIELD OF THE INVENTION
The present invention relates to the application of Gibberelic Acid (GA) signal molecule for enhancing concentration of flavor compounds of tobacco leaves. More particularly, the present invention relates to a method and composition for significant increase in the concentration of flavor compounds, number of trichomes, and terpene specific glandular trichomes for enhancing flavor of tobacco leaves along with change in said leaf morphology.
BACKGROUND OF THE INVENTION
Brazilian tobacco is known to be having more flavor than that of the Indian tobacco. The same cultivar/variety grown in Brazilian environment is more flavorful than when grown in Indian conditions. It was clearly hypothesized that the expression of genes and proteins controlling flavor component in tobacco is largely influenced by environmental and cultural practices than the genetic makeup.
GA treatments are known to stimulate trichome initiation and branching on leaves in relation to defense of the plant with reference to pathogen infection and insect or herbivore repulsion. Conventionally volatile compounds production is induced by wounding by mechanical damage or by herbivore- or pathogen infection (Dudareva et al., 2004 and Pichersky et al., 2006). Review of literature clearly shows application of signaling molecules such as salicylic acid, methyl jasmonate etc., results in the expression of the cascade of the genes involved in production of the secondary metabolites majority of which have been reported for defense of the plant against pests and pathogens.
Gibberellic acid is a naturally occurring hormone found in all plants and used to stimulate or control the growth rate of plants. As of today, over 100 gibberellins have been isolated.
Gibberellic acid (GA3) is the GA most often used commercially, since it can be readily obtained in large quantities from fermentations of the fungus Gibberella fujikuroi. Other GAs, for example GA4 and/or GA7, are used for specific crops or specific purposes for

which they are more effective than GA3, though GA4/7 are produced in lower yields by commercial fermentations and are therefore more expensive than GA3.
US 7453024 relates to the field of genetic engineering of flavor, fragrance and bio-control agent development. More specifically it relates to a process for production of natural flavors, fragrances or bio-control agents by the control of one or more genes implicated in that process. The invention provides an isolated or recombinant nucleic acid or functional fragment thereof encoding a proteinaceous molecule essentially capable of flavor, fragrance and/or bio-control agent synthesis when provided with a suitable substrate under appropriate reaction conditions. The invention further provides a nucleic acid or functional fragment thereof encoding a proteinaceous molecule essentially capable of synthesizing at least a monoterpene alcohol linalool when contacted with geranyl diphosphate (GPP) and/or at least a sesquiterpene alcohol nerolidol when contacted with farnesyl diphosphate (FPP) under appropriate reaction conditions.
A journal publication Plant Physiol, November 2001, Vol. 127, pp. 1256-1265 Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits show that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri 5-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as γ- and α-tocopherols, lycopene, β-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.
Both the prior art documents specifically relates to the process for the production of specific isoprenoid compounds viz., Linalool & Nerolidol in US 7453024 and Linalool and 8-hydroxy linalool in journal publication by metabolic engineering through genetic modification by transforming with specific genes (US 7453024) / gene journal publication enabling to produce the enhanced levels of specific compounds.

The disadvantage of the above-mentioned prior art is that earlier studies were all focused on epidermal cell fate differentiation forming the trichomes especially in Arabidopsis and effect of gibberellic acid regulatory elements in controlling the same and the trichomes as such for defense of the plant with reference to pathogen infection and insect or herbivore repulsion. There was no report of GA involved or used for increase in concentrations of flavor / volatile compounds in the literature. There was no report of GA involved in change of leaf morphology especially narrowing of the leaves upon application in the literature, above all there were no reports of use of GA in tobacco for increase of flavour/volatile compounds. Further in the prior art genetic modification/metabolic pathway modification has been conducted which are complicated, time consuming procedure and not cost effective.
Thus there is a need to provide a method of GA induced significant increase in the concentrations of many of the flavor compounds along with a definite and significant increase in the number of trichomes and terpene specific glandular trichomes (as GA is involved in terpenoid pathway) along with the change in the leaf morphology.
OBJECTS OF INVENTION
It is an object of the present invention to overcome the disadvantages of the prior art.
Another object of the present invention is to produce tobacco leaves adapted to increase production of several metabolites for enhancement in concentration of the flavor compounds of said tobacco leaves.
Another object of the present invention is to produce tobacco leaves adapted to change in the leaf morphology.
It is another object of the present invention to produce tobacco leaves having 35 to 40% increase in number of trichomes.
Another object of the present invention is to produce tobacco leaves having 51 to 87% increase in number of terpene specific glandular trichomes.

Yet another object of the present invention is to enhance levels of several of the flavor compounds by the application of composition comprising gibberrelic acid at a specific time and concentration.
SUMMARY OF THE INVENTION
The present invention relates to a composition for enhancing concentration of flavor compounds of tobacco plant leaf wherein said composition comprises giberrellic acid at a concentration of 50 to 250µM and adjuvant.
A further aspect of the present invention is to provide a method for enhancing concentration of flavor compounds of tobacco plant leaf wherein said method comprises application of composition comprising giberrellic acid to the plant at 30-40 days after planting and at a concentration of 50 to 250µM.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
Figure 1 provides a graphical representation of the effect of GA spraying on trichome
number.
Figure 1A provides a graphical representation of effect of GA spraying on nicotine
content.
Figure 1B provides a graphical representation of the effect of GA spraying on total sugar content.
Figure 2 provides the microscopic view of the density of trichomes in tobacco leaves treated with (a) gibberellic acid (b) Control.
Figure 3 provides a comparative study data by way of graphical representation of various flavor compounds upon application to GA vs. Control along with the bench mark Brazil sample.

Figure 4 provides a graphical representation of the GC-MS overlay of GA treatment with control in cured leaves across different regions.
Figure 5 illustrates the biosynthetic pathway of GA.
Figure 6 provides a bar diagram representation of the effect of different concentrations of
GA( viz., 50, 100 and 250 µM) at 30 DAT on enhancement of flavor compounds in FCV
tobacco K326 at (A). KLS (Mysore), (B). NLS (Rajamundry) and in Burley tobacco
Blanket A1 at (C). Guntur
Figure 7 provides GC-MS profile of different concentrations of GA (viz., 50, 100 and
250 µM) at 30 DAT in FCV tobacco K326 at (A). KLS (Mysore), (B).NLS(Rajamundry)
and in Burley tobacco Blanket A1 at (C). Guntur
Figure 8 provides effect of GA on enhancement of flavor compounds in green leaves of
FCV tobacco K326 (A). GA 50, 100 and 250 µM at 30 DAT (B). GA 100 µM at 2 days
before transplanting (DBT) Vs 30 days after transplanting (DAT) and (C). GA 100 µM at
30 DAT Vs 60 DAT
Figure 9 provides the GC-MS profile of GA in green leaves of FCV tobacco K326 (A). GA 50, 100 and 250 µM at 30 DAT (B) GA 100 µM at 2 days before transplanting (DBT) Vs 30 days after transplanting (DAT) and (C). GA 100 µM at 30 DAT Vs 60 DAT.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a method to enhance concentration of flavor compounds in tobacco plant leaves by the application of Gibberellic Acid (GA) along with the adjuvant at a specific concentration and at a specific period of the plant growth. Further the method has resulted in significant increase in the concentrations of many of the flavour compounds along with a definite and significant increase in the number of trichomes and terpene specific glandular trichomes, which has resulted in increase in the flavor of the tobacco.

GA application has resulted in increased production of many of the flavour compounds in both green and cured leaves. Application of GA enhanced the concentration of flavor compounds in green and cured leaves when compared to control. In green leaves, concentration of solanone, megastigmatrienones, neophytadiene, phytol, scopoletin and myristaldehyde are increased while several new flavour compounds like vanillin, oxo-ionol, solavetivone, rishitin, longifolenaldehyde, thunbergol, farnesol and cembrene are observed, which are not detected in the control. The sharp peak of the compound 4,8,13-duvan 1,3-diol in GA in green leaves which is completely absent in the control. This duvane diterpene 4,8, 13-duvan 1,3-diol constitutes almost 30% of the total constituents in the GC.
Further the glandular heads are the primary and the only site of the diterpene 4,8,13-duvan 1,3-diol biosynthesis in tobacco. The degradation or pyrrolytic products of duvane have distinctive flavor and aromatic properties that affect tobacco quality. In a biogenetic scheme for duvane terpenes, solanone is the degrading product during curing. Phytol is associated with chlorophyll in green leaves. During the process of curing and aging, it forms phytadiene (neophytadiene). Interestingly, neophytadiene is also observed in the chromatogram, suggesting breakdown of phytol already in the green leaf. The olefin neophytadiene (3-methylene 7,11, 15-trimethyl 1-hexadecene) does not impart flavor to a cigarette but is preferred by a majority of the smokers, because of a 'smoothing' effect. Since a majority of the smokers prefer cigarette with high neophytadiene content, it is conceivable that addition of this material to tobacco is considered. Megasigmatrienone I and Megastigmatrienone II, flavor compounds, so far were seen only upon curing. Similarly in the cured leaves, concentration of solanonone, megastigmatrienones,oxo-ionol, ionone, hydroxy damascene, p-vinyl guaiacol, geranyl acetone neophytadiene solavetivone, rishitin , scopoletin, fatty acids (palmitic acid, stearic acid) and phytosterols (squalene, tocopherol, Vitamin E, campesterol, lanosterol, fucosterol, beta-amyrin) were increased upon GA application as compared to control.

The external addition of GA increased the level of phytol, carotenoids, phytoene monoterpenes which is in line with the finding that the such external addition regulates the endogenous GA.
A composition comprising GA at a concentration of 50 -250 µM is prepared by mixing along with the adjuvant. The adjuvant mainly consists of Tween 20 at 0.1 %.
Leaf Morphology in GA treated plants
It is observed that the morphology of the GA treated plant leaves changes and the leaves become narrower and more bodied. This was the first report of GA affecting the morphology of leaf.
Smoke test of GA treated Tobacco:
Having seen the positive volatile profiles using the GC-MS analysis in GA treated plants compared to control the best treatment of GA was used for smoke test. It was the first report on GA increasing the flavour of the tobacco.
Application of GA signal molecule along with the adjuvant at a specific concentration and at a specific period of the plant growth period only once has resulted in significant increase in the concentrations of many of the flavour compounds (Fig.4) along with a definite and significant increase in the number of trichomes (to an extent of 35-40 % over the control) (Fig. 1 and 2) and terpene specific glandular trichomes (to an extent of 51-87 % over the control) as is evident from Table.1, which resulted in increase in the flavor of the tobacco.



From table 1 it is observed that the number of secretory trichomes shows a 83% increase over the control at a concentration of 100 µM.
Comparison of flavor profile of GA treatments and control across regions with reference to our bench mark sample i.e. Brazil samples clearly indicates the increase in flavour compounds near to that of Brazil profiles as shown in figure 3.
The GC-MS profile clearly shows the significant increase in the concentrations of several flavour compounds in GA treated plants along with a definite and significant increase in the number of trichomes.



It is found that the number of leaves per kg of cured leaves is less in GA treated plants as compared to the control. The average number of leaves per kg of cured leaves were found to be 133.4 leaves in case of GA treated plants whereas it was 137 leaves in case of control plants. The difference in the number of cured leaves are found to be 3.6 leaves lesser in GA treated plants when compared to control as provided in Table 3. This clearly indicates that GA treated plants resulted in production of more bodied leaves when compared to control.

A marginal increase in yield of cured leaves in GA treated plants to an extent of 3.6 % is demonstrated when compared to control as shown in Table.4.

Grade out turns were also found to be more in case of GA treated plants when compared to control as shown in Table.5. GA treatment has resulted in production of brighter grades when compared to control. GA treated plants produced 21% more brighter grades than control.


GA treatment has resulted in higher Grade Index of about 75 when compared to 71 in
control as shown in Table.6. This in turn resulted in higher value for GA treated tobacco
when compared to control as shown in Table.6
The leaf surface of Nicotiana tabacum L. produces a number of natural products,
primarily trichome exudate secondary metabolites that have known or potential economic
value.
Application of GA on tobacco has resulted in several folds increase in production of
various beneficial metabolites, which can be used for different purposes as mentioned in
Table.7 which can be commercially exploited.

The different concentrations of GA( viz., 50, 100 and 250 µM) at 30 DAT on enhancement of flavor compounds in FCV tobacco K326 at (A). KLS (Mysore), (B). NLS (Rajamundry) and in Burley tobacco Blanket A1 at (C). Guntur was tested and the results are in Figure 6 which provides a bar diagram representation of the effect of different concentrations of GA( viz., 50, 100 and 250 µM) at 30 DAT on enhancement of flavor compounds
GC-MS (Gas Chromatography - Mass chromatography) profile of different concentrations of GA (viz., 50, 100 and 250 µM) at 30 DAT in FCV tobacco K326 at (A). KLS (Mysore), (B).NLS(Rajamundry) and in Burley tobacco Blanket A1 at (C). Guntur was studied and the results are provided in Figure 7
The effect of GA on enhancement of flavor compounds in green leaves of FCV tobacco K326 (A) at different concentratiorls of GA 50, 100 and 250 µM at 30 DAT (B). GA 100 µM at 2 days before transplanting (DBT) Vs 30 days after transplanting (DAT) and (C). GA 100 µM at 30 DAT Vs 60 DAT were studied. The results are depicted as bar diagram in Figure 8.
The GC-MS profile of GA in green leaves of FCV tobacco K326 (A). GA at 50, 100 and 250 µM at 30 DAT (B) GA 100 µM at 2 days before transplanting (DBT) Vs 30 days after transplanting (DAT) and (C). GA 100 µM at 30 DAT Vs 60 DAT was studied. The results are shown in Figure 9.

We claim:
1. A composition for enhancing concentration of flavor compounds of tobacco plant leaf wherein said composition comprises giberrellic acid at a concentration of 50 to 250µM and adjuvant.
2. The composition as claimed in claim 1, wherein said adjuvant comprises Tween 20 at 0.1%.
3. The composition as claimed in claim 1, wherein the said composition is adapted to provide 35 to 40% increase in number of trichomes of the tobacco leaf.
4. The composition as claimed in claims 1 and 2, wherein the said composition is adapted to provide 51 to 87% increase in number of terpene specific glandular trichomes of the tobacco leaf
5. A method for enhancing concentration of flavor compounds of tobacco plant leaf wherein said method comprises application of composition comprising giberrellic acid to the plant at 30-40 days after planting and at a concentration of 50 to 250µM.
6. The method as claimed in claim 4, wherein the said method results in 35 to 40% increase in number of trichomes of the tobacco leaf.
7. The method as claimed in claims 4 and 5, wherein said method results in having 51 to 87% increase in number of terpene specific glandular trichomes of the tobacco leaf.