Field of the invention
The present invention relates to a method and a dispersible composition for enhancing leaf growth of tobacco plants. More particularly, the present invention relates to a method of application of thiourea at a specific amount to enhance the overall growth and leaf yield in Nicotiana tabacum. The present invention further relates to dispersible compositions comprising thiourea and methods of treating Nicotiana tabacum therewith.
Background and prior art
In the agricultural and horticultural fields, attention is directed to plant growth regulators, which accelerate or suppress the growth of plants when used in trace amounts and which can regulate plant growth in desired conditions. Primarily, the terms "plant growth regulation" and "plant growth regulator" refer to acceleration, they sometimes refer to suppression of plant growth (in overdose use) in a broad sense. Plant growth regulation refers to either 'promotion' or 'inhibition' of growth in true sense. It implies that hormones depending on developmental phenology either promotes or inhibits/retards growth. However, out of 5 plant growth hormones, 3 (Auxins, Cytokines & Gibberellins) have been classified as 'promoters' and remaining 2 (ABA & Ethylene) as 'inhibitors'. A few (auxin and/or cytokine based) plant growth regulators (PGRs) have 'dual-effect' which are concentration dependent (for ex. 2, 4-D at lower dosage act as hormone and at higher dosage act as herbicide).
Globally, emphasis in crop science•research has been to enhance the economic yield either through improved varieties, higher inputs or more efficient use of photosynthates. There are good evidences in many crops that the demand for photosynthates can directly influence net photosynthetic rate and translocation patterns. Several growth regulators (PGR) and/or growth chemicals (GC) have been under continuous testing and field use to improve desirable growth/yield traits from time immemorial.

Thiourea (TU) is one such compound classified as growth chemicals, though less popular, has potential to enhance growth of plants. TU, a sulfhydryl compound having formula I, is an organic compound containing carbon, nitrogen, sulfur and hydrogen. Even though TU is structurally similar to urea yet it significanlly differs from the functional properties of urea.

TU is a non-biological thiol and plays several bio-regulatory roles and hence implicated in diverse biological activities such as phloem transport, substrate binding site of amino acid carrier, phloem loading of sucrose, and hence translocaiion of photosynthates. Several literatures have reported the possible role of the SH-group in the bio-regulator. Further, TU has also been reported to increase the photosynthetically active leaf surface during grain filling in cereals. It is known to increase the photosynthesis in plant by improving leaf chlorophyll content and enzymes (RuBisCO, Sucrose synthase, etc) activities. In addition, it enhances nitrogen metabolism through increasing Nitrate Reductase (NR) activity and increases total soluble protein and starch content. It is known to mimic plant hormone,, namely, gibberillic acid and cytokinin action. It has also been used to break winter bud dormancy in grapes and other horticutture fruit crops and germination in potato.
Even though TU application is well documented in several crop plants and other solanaceous species like potato, it has been rarely used in tobacco, except for a few reports on seed germination at nursery stage.
Vrea based nitrogenous fertilizers is also known to be used to increase the leaf yield of Nicotiana tabacum. However such leaves have higher nicotine content since nitrogen directly influences the leaf nicotine. Such higher nicotine content leads to enhanced

nicotine to nor-nicoiine ratio subsequenlly deteriorating the leaf quality parameters as per the CTRI reports 2002-2007. Use of excess fertilizers than recommended by CTRI is non productive from leaf chemistry point of view.
US4279639 teaches plant growth and suppression regulators of the N-(2-substituted-4-pyridyl) ureas and thioureas as well as plant growth regulators, which provide effects equivalent to or superior to known plant growth regulators, agricultural compositions comprising the same and methods of controlling plant growth using the same. According to the prior art, the amount of the compound used when applied by directly spraying plants is generally 10 to 100 liters per 10 area as a solution of a concentration of 0.0001 to 10,000 ppm as effective ingredient, preferably 0.01 to 10,000 ppm. When applied to the soil, an amount 5 to 10 times that given above is required. Further according to the prior art, it goes without saying that the amount applied will differ according to the object of the control and the plant to which applied.
Therefore there exists a need for a plant growth regulator, which could be used within the recommended dose and also exhibit the desired increase in growth.
Accordingly, the present invention provides a composition comprising TU within recommended amounts thereby improving the overall growth rate and the leaf yield.
Objects of the invention
It is therefore an object of the present invention to provide a method of enhancing the overall growth rate of Nicotiana tabacum plant by the application of thiourea, at a specific concentration and at a specific time during plantation.
It is another object of the present invention to provide a growth rate component for enhancing the leaf yield of Nicotiana tabacum.

Summary of the invention
The present invention relates to a dispersible composition for enhancing overall growth rate of Nicotiana tabacum comprising thiourea at a concentraiion range of 500-1000ppm wherein the composition is sprayed at least 35 days after field planting and not later than 45 days.
Another aspect of the present invention is to provide a method for enhancing overall growth rate of Nicotiana tabacum by the application of only thiourea at a concentration range of 500-1000ppm as spray and at a specific time of plantation which is at least 35 days after field planting and not later than 45 days.
Brief description of the accompanying Drawings
Figure 1 provides the bar diagram of the effect of different concentraiions of thiourea on leaf area (LA) and plant height (PI Ht) (In tobacco, emphasis is more always on the LA and yield).
Figure 2 provides a bar diagram of the improvement in physiological traits in thiourea treated plants.
Figure 3 provides the C02 response (A/Ci) curves in (a) thiourea treated and (b) untreated tobacco plants.
Figure 4 provides the bar diagram of the genotypic response to yield improvement (% increase over their respective control plants) differs to applied chemical (A1 and A3: breeding lines and K326: variety) [field study, July-Sep 2009, KLS region, Mysore].
Figure 5 provides a bar diagram representation of the effect of thiourea on improvement in yield attributes and biomass/yield (TDM) (percent over untreated control).

Figure 6 provides the bar diagram represeniing difference (kg/Ac) in leaf yield (percent values in inset) in control and TU treated plants of both the genotypes under field conditions (NLS-2008-09).
Figure 7 provides a differential genets expression (NR and RA) profile in TU treated and control plants (Log 10 value>0.5 indicates statistical significance).
Figure 8 provides efficient biochemical efficiency (A/R-RuBssCO efficiency, ratio of C-fixation rate to RuBisCO content) in TU treated plants compared to control.
Figure 9 provides flavour profile of TU treated T-position leaves.
Figure 10 provides control (untreated) GC profile and TU treated GC profile.
Detailed Description of the Invention
The present invention provides method for the application of only TU for regulating the physiological activities of Nicotiana tabacum when applied in optimum amounts as recommended by CTRL
More specifically, it has been found that TU treated plants have higher intrinsic physiological efficiencies, coupled with higher carbon & nitrogen contents thereby markedly improving the growth and yield attributes of the plant. The leaf yield of FCV tobacco is also significanlly improved on application of TU. The yield advantage due to TU is significant and to a tune of 23% increase (2008). This result was consistent for subsequent two seasons (2008-09 and 2009), depicting around 21% yield increase. TU by virtue of its involvement in C and N metabolism coupled with higher enzyme activities would have resulted in higher growth and yield gain response.
The cured leaves of treated plants when subjected to leaf quality and flavor analysis, the quality (nicotine, total sugars and chlorides) and flavor profile were essentially the same as that of untreated tobacco, suggesting TU application doesntt change curing and post curing chemistry.

For any hormones/PGRs to elicit bio-physiological response, certain concentration level is must. To understand at what concentration any PGR is active (in each crop species), one needs to develop 'dosage response' curve. As concentration of TU used earlier (by other researchers) in a few solanaceous and other species was known (around 750ppm as foliar spray), the present invention provides the same with two additional levels (±250ppm) in tobacco to be more precise on working concentration.
The optimal concentration of TU for application ranges between 500 to 750 ppm and do not affect the leaf quality and flavor profile, unlike nitrogenous growth promoters used in prior art.
Thus, the application of TU substantially enhances the leaf yield of tobacco without any alteration in chemical parameters.
The following examples illustrate the composition comprising TU to increase the growth and yield attributes according to the present invention.
Example 1
A composition comprising TU composition at a concentration of 500 ppm is prepared by reacting TU with a solvent/carrier. Preferred solvent for dissolving TU is water.
Plants of two FCV genotypes (K-326 and CH- 1) were sprayed with said TU composition, thrice at an interval of 7 days. TU treated plants (2 genotypes) showed considerable increase in growth and physiological traits (Table.1). Increase in Specific Leaf Weight (SLW) and chlorophyll content (TCC) indicates higher leaf thickness with more pigments that associates with better leaf N, might result in enhanced growth and yield components in TU treated plants. Increase in Relative Water Content (RWC) signifies maintaining relatively better tissue water status may result in turgor linked changes in leaf expansion and growth. Interestingly, the percent improvement in leaf

biomass/dry matter was apparent in TU treated plants in both genotypes. Further, the C and N content analyzed in dried leaf samples in also increased in both genotypes.

Exampee 2
Using a single FCV genotype:
Pot grown plants were treated four times with different compositions having distinct concentrations of 500 ppm, 750 ppm and 1000 ppm as spray of TU, in a period of 30 days. Growth and physiological traits were recorded at the end of 45 days with destructive plant sampling. Growth and physiological parametess were relatively higher in TU treated plants compared to control as evident from Table 2 below.


Index: Pt Ht - Plant Height (em), Root L- Root Length (em), TLA -Total Leaf Area (cm2pl-1), Leaf DW - Leaf Dry Weight (g pl-1), Stem DW - Stem Dry Weight (g pl-1), Root DW - Root Dry Weight (g pl-1), RWC - Relative Water Content (%), SLW -Specific Leaf Weight (g cm-2), TCC - Total Chlorophyll Content (mg g-1Fresh Weight). DM/LA - Dry Matter/Leaf Area (ratio)
As is clearly seen from the results above, treated plants showed apparent growth traits improvement. The leaf and total biomass was relatively on higher side compared to control. 500 ppm provides apparent improvement in yield and growth attributes. (Fig. 1 and 2).
Intrinsic physiological traits such as carbon fixation and transpirational rates measured in treated plants showed considerable increase over untreated plants. Further, to examine intrinsic carbon fix efficiency (CE, RuBisCO driven), CO2 response (dA/dCi) curves

were generated (Fig. 3). TU treated plants had higher CE compared to control as well as mesophyll efficiency as substantiated by lower Ci/gs ratios.
The C02 compensation point (CCP, at which photosynthesis or carbon fixation process begins) for TU was ≈ 45ppm and for untreated plants was ≈ 150ppm, suggesting TU triggers early initiation of carbon fixation process in tobacco. The intrinsic carboxylation efficiency (CE), driven by RuBisCO was higher in TU treated plants (0.65µmolm-2s-1) compared to untreated plants (0.58µmolm-2s-1), which signifies involvement of TU in activating photosynthetic process. Lower Ci/gs ratio (an indicator of intrinsic mesophyll efficiency) in TU treated plants (0,41) compared to untreated control (0.75) further substantiated TU's involvement in efficient carbon fixation mechanism.
Crop seasons (2008, 08-09 and 2009)
A pilot study was carried out in the field comprising 200 plants of single (I), two (II) and three (III) FCV genotypes. TU sprayed at a concentration of 500 ppm, 4 times at grand growth stage at an interval of 7 days. Treated plants had higher intrinsic physiological efficiencies, coupled with higher C and N contents. The yield advantage due to TU was significant and to a tune of 23% (2008). The results on yield improvement were consistent for further two consecutive seasons (08-09 and 2009), depicting around 21% increase.
C-N analysis (combined results of all seasons) in cured leaves revealed that N content was markedly high in TU treated leaves which substantially reduced the C/N ratio, indicating efficient utilization of carbon source at a given N input.
TU also appeared to decrease M/L (midrib to lamina) ratio of potassium (considered as a quality element in tobacco industry) through more uptake and mobilization in leaves.


The cured leaves of TU treated plants were subjected for leaf chemistry as well as flavour profile. The range in leaf qualities such as nicotine, total sugars and chlorides was normal that generally found in tobacco of that traditional region, suggesting TU application doesn't change leaf quality parameters.
From the flavour front, the GC profile of untreated and TU treated didntt differ much; indicating TU's role in non-altering favorable flavour profile.
Example 3
TV and gene/s expression
Primarily based on sequence homology for key enzymes of C and N metabolism, reported genes in the public data base of tobacco/related solanaceous family (RuBisCO (R), R-activase, NR, Chlorophyll aIb ect), oligos (primers) were synthesized. Prior to this, cDNAs from the control and TU treated fresh green leaf samples were prepared and kept in refrigerated condition until further use. Rt-PCR was performed to examine the expression profile of genets in leaf sample for these key genes keeping 18s rRNA as check. The genes such as NR (nitrate Reductase) and RA (rubisco activase) were found to be up-regulated in TU treated leaves (Fig. 7).
Further as NR is the key enzyme for nitrogen assimilation in plants and is sensitive to water deficits, hence maintenance of higher NR activity through TU application assumes greater significance under water-limited environments.

Example 4
TU and intrinsic biochemical (enzyme and hormon) assays:
The actual (in vivo) enzyme (RuBisCO and NR) contents were determined along with total cytokinin in treated and control leaves of tobacco (Table. 3). It was apparent that TU treated leaves showed higher enzyme and CK contents compared to control. An increase of 23,17.7 and 23.4 % was observed in NRA, RuBisCO and total CKs respectively over untreated control leaves, suggesting TU plays a key role in increasing growth-linked traits in tobacco, hence can be promoted for field application for leaf yield improvement.
Increased RuBisCO efficiency (deduced as a ratio of C-fixation rate to RuBisCO content per se) to a tune of 7.2 percent in TU treated plants (Fig. 8) compared to control, further substantiated the efficacy of this PGC in enhancing growth and yield traits in FCV-tobacco.

Index: NRA (Nitrate Reductase Activity), RuBisCO (Rubilose 1,5 Bis phosphate Carboxylase/Oxygenase, mglgProtein), Total Cytokinins (picomol/g Fresh weight)

Example 5
TU and Leaf chemistry/Quality:
As a routine measurement, leaf chemical parameters such as nicotine, total sugars (TS) and chlorides were analyzed in TU treated at a concentration of 500 ppm, and control cured leaf samples. It was observed that there were no detectable changes in these leaf chemistry parametess upon TU application (Table. 4), indicating that the applied chemical doesntt alter the leaf chemistry as such. The range was normal that generally expected in tobacco of that traditional region.

In another set of experimenss (different from reported earlier), the trend on C, N contents, C/N ratio apart from K+ content in cured leaves were similar. TU treated leaves showed no difference in contents of both the macromolecules. TU reduces the C/N ratio, indicating efficient utilization of carbon resource at a given N input. However, potassium (K4) content in lamina was relatively higher compared to midrib in TU treated leaves as indicated by lower M/L ratio (Table. 5), suggesting, TU would efficiently promote transport of K+ from midrib to lamina (M/L) (activation of efflux K+ channel).


Example 6
TU and flavour profile:
The cured leaves of TU treated along with control were subjected for flavour profiling (3 FCV genotypes, A1, A3 and K326). The GC profile of untreated and TU treated had no difference (when superimposed,, indicating TU has no role in altering favorable flavour profile (Fig. 9).
Example 7
Residual effect of appiied TU in tobacco leaves:
Ten day old TU sprayed (500 and 1000ppm) leaf samples were oven dried and homogenized and 0.5 g was fed to GC to examine chemical profile. Standard curve suggested Rt of TU was 0.788 min. However, in the GC profile no such peak was detected at this time scale (Fig. 10), suggesting absence of TU traces in treated leaves (10 days post foliar spray).
Inference: Free TU was undetected in tobacco leaves treatedwith TU (500 to 1000ppm)

We claim:
1. A dispersible composition for enhancing overall growth rate of Nicotiana tabacum comprising thiourea at a concentration range of 500-1000 ppm wherein the composition is sprayed at least 35 days after field planting and not later than 45 days.
2. The dispersible composition as claimed in claim 1 for enhancing overall growth rate of Nicotiana tabacum wherein the preferred concentration range is 500 to 750 ppm.
3. A dispersible composition as claimed in claim 1 for enhancing overall growth rate of Nicotiana tabacum wherein the composition particularly improves leaf yield of the plant.
4. A method for enhancing overall growth rate of Nicotiana tabacum by the application of composition comprising thiourea at a concentration range of 500-1000ppm as spray and at a specific time of plantation which is at least 35 days after field planting and not later than 45 days.
5. A method as claimed in claim 4 for enhancing overall growth rate of Nicotiana tabacum wherein the composition is sprayed at a composition range of 500 to 1000 ppm at least 35 days after field planting and not later than 45 days.