A method of developing stress tolerant plants exhibiting self- glucogenic properties for use in bioethanol production from

lignocellulosic biomass

FIELD OF THE INVENTION

The present invention relates to the method of development of stress tolerant plants, which also exhibit self-glucogenic properties and thus can be utilized in the bio ethanol production from the lignocellulosic biomass.

BACKGROUND OF THE INVENTION

Ethanol is gradually becoming one of the chosen fuels for the automotive industry both as a fuel and as a fuel additive in blends. Its easy availability combined with its suitability in spark-ignited internal combustion systems as a fuel and as a fuel additive in petrol blends makes it one of the renewable carbon source of choice. This proposal is geared towards providing farmers with a non food-competitive crop adapted to the biofuel industry for marginal and rainfed areas. This proposal draws on the expensive nature of agricultural crop waste digestion by externally applied cellulose degrading enzymes rendering the process non-feasible at remote locations and thus the expensive transportation cost of an otherwise low priced post-harvest plant waste. Pearl millet has been selected as the target of this technology as it is a marginal crop with about 9.3 million Ha cultivation and a good adaptation to arid drought-prone cultivation.

This present application will entail the development of Agrobacterium mediated plant transformation vectors encoding a peroxisomal or vacuolar-targeted cellulase, cellobiohydrolase and xylanase. Specifically pearl millet (Pennisetum glaucum (L.)) is transformed with a peroxisomally or vacuolar- targeted cellulase, cellobiohydrolase β-glucosidase and xylanase towards storing the hydrolytic enzymes for post-harvest glucogenesis and with a rice glutamate decarboxylase for enhancing biomass under stress conditions.

Currently pearl millet cultivation yields around 8.3 million tons of grain annually and the stovers are used as fodder for farm animals. It is a preferred crop in areas where irrigation is not available and scanty rainfall around planting time is sufficient to assure the farmer of some yield. While a number of technologies are centered around the use of grain for glucogenesis, the competition of this process with food production makes it unattractive. This technology will thus facilitate the decentralized conversion of bulk farm agricultural residue into high glucose broths that can further be fermented to ethanol at local centers. The fuel thus produced may be directly used locally or used in the national transportation grid. Pearl millet is a commonly cultivated marginal land crop providing both feed and fodder to farmers. It has been largely ignored by the biofuel community, which concentrates on maize, an input intensive crop, for bioethanol production. This invention entails the enhancement of leaf biomass under marginal conditions to enhance the recovery of sugars after degradation of the cell wall components by glycolases expressed and sequestered in the peroxisome/vacuole.

Roughly 10 % of India's area is considered marginal, arid or unsuitable for cultivation. Developing crops for these areas automatically increases the food production of the country, arrests the further deterioration of the existing soil surface and enhances the living standard of peoples dependent on these lands. The value of this invention lies in producing a food crop with biofuel opportunities on marginal lands.

Increases in biomass are reported when plants are treated with gamma amino butyric acid, a common non-protein building amino acid. Glutamate decarboxylase is also a known alleviator of salinity stress. Taken together, glutamate decarboxylase is a valid target to enhance biomass under stress conditions. Glycolysis by enzymes in transgenic plants has been reported in maize. The combination of these enzymes is targeted for crops under environmental stress.

Expression of peroxisomally or vacuolar -targeted cellulase, cellobiohydrolase β-glucosidase and xylanase along with glutamate decarboxylase for enhancing biomass under marginal cultivation conditions will result in improved biomass Pearl millet with self degrading glucolases for post harvest biofuel feedstock production from the non-edible plant matter.

BACKGROUIVD OF THE INVENTION

This is an area of active research internationally and some preliminary work is in progress in India. Maize is the target crop in most cases as the American acreage of Maize covers 33 million hectares. The expression of Cellulase El from Acidothermus cellulyticus has been carried out in a number of plants including tobacco, Arabidopsis, rice, maize and duckweed. The localization of this enzyme has been evaluated in various organelles including the chloroplast, the apoplast, the vacuole and the cytosol. Xylanase has also been evaluated in various crops including rice, barley, Arabidopsis, potato, and tobacco. Its localization has been evaluated in the cytosol, chloroplast, peroxisome, and apoplast. A method to express ligninases and cellulase in maize has been described.

Nationally this concept has been explored as a process study with agricultural waste being processed into bioethanol by various processes including the ITT Delhi process which is a two-step process involving saccharification followed by fermentation to ethanol. The United Nation has adopted this method evaluated at a 50 Liter daily output scale. The method is geared towards the removal of ethanol as it is produced during fermentation. Very little work has been carried out on the expression of these enzymes in plants using transgenesis.

The use of the peroxisome as a localization organelle for a cellulase has not been reported so far nor has the dual expression and targeting of cellulases and xylanase to the peroxisome or vacuole been reported so far. The use of biomass enhancement under stress conditions in Pearl millet is again a novel concept. The application of glutamate decarboxylase in this process is entirely new. Taken together this is a novel way to generate auto-glucogenic lines with a biomass enhancement trait in any crop and more specifically in pearl millet

The use of glutamate decarboxylase to enhance biomass has never been reported before and is novel. This enzyme has been addressed as a modulator of stress, as a modulator of nitrogen metabolism. Its application towards enhancing biomass under stress conditions has not been reported and is novel. To further tie this application to biofuel production using endogenously expressed cellulases and xylanase for autogluconeπesis has not been reported and is novel.

India is moving towards blending petrol fuel with ethanol at levels of 5% or 10%. Such a step calls for a projected demand of 2.3 billion liters of ethanol by 2009-2010 at 5% blending levels. The corresponding level for 10% would then be approximately double. It has been estimated that if the entire molasses production of the country is diverted to ethanol production then the requirement in 2009-2010 will touch 10.4 million tons at 5% blending and 13.8 million tons at 10% blending. Considering the peak molasses production of 2002-2003 at 8.9 million tons, a significant shortfall in production will be encountered. This shortfall may be partially met by pearl millet production in marginal areas, which cannot be used for crop production, let alone sugarcane production. The combined increased biomass under marginal cultivation and the auto-glucolytic nature of the crop waste will enable both increased feed stock production and reduced processing costs towards ethanol production.

Pearl Millet is globally grown in 32 M Ha, of which about 1OM Ha is covered in India alone, as the lead country under area for Pearl Millet. At a meager 40% technology penetration rate in Pearl Millet, about 13 MHa will be under GM Pearl Millet with various uses. The additional value created is about 5000 rupees per hectare, means 200 crores @ 40% penetration. Thus the market value for such GM pearlmillet is stated at 200 crores in India alone.

A major part of the country is classified as marginal (around 10% of the total area) and is not cultivated by farmers. Populations dependent on such areas have little or no options towards a meaningful agrarian lifestyle. This leads to a rapid degradation of these areas and hopelessness in the lives of the farmers. As fuel ethanol will be a high demand product any reasonable source of feedstock will be used in its production. The contribution of these areas towards the national need will thus be significant and provide a new window of opportunity in an otherwise bleak future.

A significant level of novelty is included in this invention where for the first time pearl millet has been selected as a target for industrial applications for BioEthanol production. The use of the peroxisome/vacuole as a target organelle for targeting of cellulase is novel. The combined use of xylanase and cellulases is also novel. Finally introducing all these traits in a background of biomass enhancement through the use of glutamate decarboxylase is entirely novel

The expression of cellulase in maize has resulted in a 2.1% yield of cellulase. Similar levels have been achieved in numerous other plants indicating that the expression of cellulase in plants results in active protein, which is capable of degrading cellulose to smaller saccharides. The targeting of this hydrolase to organelles has resulted in significant protection of the plant cell from inhibitory responses during the growth of the plant and no untoward results have been reported indicating that plants expressing in an organelle, limited cellulases are not affected in their growth patterns.

The expression of xylanase likewise has resulted in 2-2.5% expression levels in a number of crop plants, though the list is not as extensive as that of cellulase, it is impressive in its own right. The role of this enzyme is to degrade hemicellulose, a major component of the cell wall with a significant pentose composition. Again the success of this strategy lies in few indications of intolerance by the plant in spite of the relative high level of expression.

The combined role of these enzymes in a biomass-enhanced background should result in significant steps towards biomass based biofuel prospects. In house indications with tobacco systems expressing glutamate decarboxylase have shown that the biomass enhancement is stable from generation to generation and is clearly induced under stress conditions. Taken together this project should result in significantly viable results.

OBJECT OF THE PRESENT INVENTION

The primary object of the invention is to develop stress tolerant plants exhibiting self-glucogenic properties for use in bioethanol production from lignocellulosic biomass.

Another object of the invention is to make the lignocellulosic biomass self-glucogenic by employing a combination of genes namely cellulase (1-3 endoglucanase), cellobiohydrolase, β-glucosidase and xylanase.

Further, the object of the invention is to develop a method wherein the transformed plant expresses glutamate decarboxylase (GAD), cellulase, xylanase, cellobio hydrolase(CBH), beta glucosidase enzymes, at significantly higher level than the level of the enzymes expressed by a non-transformed plant of the same species under the same conditions. Yet another object is to select the target plants from the group consisting of monocots, dicots, cereals, forage crops, legumes, pulses, vegetables, fruits, oil seeds, fiber crops, flowers, horticultural, medicinal and aromatic plants and transform it with the DNA construct mediated by Agrobacterium-meάiated transformation.

Yet another object of the instant invention is to obtain a transformed plant with a DNA construct comprising a promoter operably linked to a nucleotide sequence that encodes a functional glutamate decarboxylase (GAD), cellulase, xylanase, cellobio hydrolase(CBH), beta glucosidase enzymes, wherein the plant exhibits significantly improved growth characteristics, yield, reproductive function and other morphological or agronomic characteristic compared to a non-transformed plant in addition to self glucogenic properties.

SUMMARY OF THE INVENTION

The present invention relates to a method of increasing stress tolerance in plants (monocotyledons and dicotyledons) with a glutamate decarboxylase gene and making the lignocellulosic biomass self- glucogenic. A method employing the glutamate decarboxylase gene from rice, cellulase (1-3 endoglucanase) gene from A. cellulyticus, cellobiohydrolase gene from T. reesei, β-glucosidase gene from Λ. nαeslundii and xylanase gene from T. reesei and store these enzymes in the peroxisome or vacuole, for enhancing the stress tolerance of the plants that generate a self glucogenic biomass has been demonstrated.

This strategy in the instant invention will sequester the hydrolytic enzymes from the cellulose fibril polymerizing machinery and protect the plant from auto-degradation during the growth and maturation of the crop. After harvest and threshing of the grain, the crop residue will be homogenized to release the enzymes from the peroxisome or vacuole. It is expected of this technology to digest the crop residue without further addition of cellulose digesting enzymes using minimal resources that may be implemented at the level of the farm.

BRIEF DESCRIPTION OF SEQUENCE LISTING:

SEQ ID No. 1 GAD Nucleotide sequence from Oryzα sαtivα cv. Indica variety Rasi (1479 bps)

SEQ ID No. 2 Cellulase (Endoglucanase) nucleotide sequence from Acidothermus (1410 bps)

SEQ ID No. 3 Codon optimized cellulase (Endoglucanase) nucleotide sequence from Acidothermus (1410 bps)

SEQ ID No. 4 Xylanase nucleotide sequence from T. reesei (1398 bps)

SEQ ID No. 5 Codon optimized Xylanase nucleotide sequence from T. reesei (1398 bps)

SEQ ID No. 6 Cellobiohydrolase-I nucleotide sequence from T. reesei ( 1542 bps)

SEQ ID No. 7 Codon optimized cellobiohydrolase-I nucleotide sequence from T. reesei (1542 bps) SEQ ID No. 8 Betaglucosidase from Actinomyces naeslundii (1467 bps)

SEQ ID No. 9 Codon optimized Betaglucosidase from Actinomyces naeslundii (1467 bps)

SEQ ID No. 10 GAD amino acid sequence from Oryza sativa cv. Indica variety Rasi

SEQ ID No. 11 Cellulase (Endoglucanase) amino acid sequence from Acidothermus, 469 aa

SEQ ID No. 12 Xylanase amino acid sequence from T. reesei, 465 aa

SEQ ID No. 13 Cellobiohydrolase-I amino acid sequence from T. reesei, 513 aa

SEQ ED No. 14 Betaglucosidase amino acid sequence from A. naeslundii,

SEQ ID No. 15 Nucleotide sequence of Peroxisomal target peptide

SEQ ED No. 16 Amino acid sequence of Peroxisomal target peptide

SEQ ED No. 17 Nucleotide sequence of 3' (C-terminal) vacuolar target peptide

SEQ ID No. 18 Amino acid sequence of C-terminal vacuolar target peptide

SEQ ED No. 19 Nucleotide sequence of maize vacuolar target peptide, 129bp

SEQ ED No. 20 Amino acid sequence of maize vacuolar target peptide

SEQ ID No. 21 Ubiquitin Promoter nucleotide sequence (1989 bp)

SEQ ED No. 22 CaMv Promoter nucleotide sequence (808 bp)

SEQ ID No. 23 2X CaMv Promoter nucleotide sequence (770 bp)

SEQ ID No. 24 CvMv Promoter nucleotide sequence (522 bp)

SEQ ED No. 25 Eucalyptus grandis COMT Promoter nucleotide sequence (534 bp)

SEQ ID No. 26 Rice RUBISCO Promoter nucleotide sequence (260 bp)

SEQ ID No. 27 ZM-VB-OMT Promoter nucleotide sequence ( 1675 bp)

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

FIGURE 1: Shows Assembly of each gene cassette is as follows: Schematic representation of each cassette (i-v). i) a rice glutamate decarboxylase (GAD) gene driven by 35S CaMv promoter; ii) cellulase (Endoglucanase) gene driven by a RUBISCO promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; iii) Cellobiohydrolase (CBH-I) gene driven by UBIQUITIN promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; iv) β-glucosidase (BGL) gene driven by the ZMVBO promoter and a C-terminal vacuolar targeting sequence terminated by a proteinase inhibitor terminator; v) Xylanase (XynZ) gene driven by COMT promoter .

FIGURE 2: Shows vector map (pAGTSG) of binary plant transformation vector harboring the vacuolar targeted - cellulase gene driven by a RUBISCO promoter, xylanase gene driven by COMT promoter, cellobiohydrolase driven by UBIQUITIN promoter, β-glucosidase gene driven by the ZMVBOMT promoter and a GAD gene driven by 35S CaMv promoter within a single T-DNA region. FIGURE 3:Shows growth performance of three cultivars of pearl millet ABI13B, ABI56B and 843B (L- R); seed germination in three cultivars (A); Plant growth (B); Leaf size (C)

FIGURE 4: Shows callogenic potential of different explants in pearl millet; callus growth from immature embryo, arrows denote embryogenic calli (A); callus growth from seeds (B); inset shows the explant. Multiple shoot regeneration from embryogenic calli in pearl millet; Shoot regeneration on SRM with activated charcoal (C); root proliferation on charcoal free medium (D); different stages of shoot regeneration (E & F)

FIGURE 5: Shows xylose sensitivity Assay in pearl millet; 2% xylose concentration was effective selection for seed germination (A); 0.5% xylose concentration was effective selection for callus growth (B)

FIGURE 6: Shows successful transformation of pearl millet was achieved and confirmed with transient GUS assay in transformed immature embryo (A-D) using Agrobacterium strain GV3101.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aid those skilled in the art in practicing the present invention. Even so, the following detailed description of the invention should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein may be made by those of ordinary skill in the art without departing from the spirit or scope of the present invention.

The invention is directed to generating stress tolerant plants, which express one or more exogenous cellulose-degrading (cellulase) enzymes. The invention is further drawn to a method of producing cellulases in plants. The invention is further drawn to a method of sorting the produced cellulases in plants to organelle compartments like vacuole or peroxisomes. The invention is further directed to generating stress tolerant plants whose biomass is amenable for cellulose digestion without addition of external cellulose-degrading (cellulase) enzymes. The invention allows the production of cellulases using the means and methods of large-scale agriculture rather than the conventional route of large-scale fermentation of the bacteria or fungi, which are native producers of the cellulases.

The recombinant plants are produced by incorporating into a plant host genome one or more expression constructs comprising a DNA sequence which encodes a protein having cellulose-degrading activity. Introduction of the exogenous gene or genes into the plant is accomplished by any means known to the art. The expression constructs described herein below enable the stable transformation of plants with one or more genes, which encode cellulose-degrading enzymes. The constructs include a DNA coding sequence which encodes a cellulase (as that term is described herein) which is operatively linked to regulatory sequences which direct constitutive, stage-specific, or tissue-specific expression of the cellulase DNA.

Cellulose-Degrading Enzymes (Cellulases) and Genes: As noted above, the term "cellulase" shall be used herein to refer to any and all enzymes, which catalyze the cleavage of cellulosic or lignocellulosic materials. As used herein, "cellulase"is synonymous with "cellulose-degrading enzymes." Explicitly, but not exclusively, included within the term cellulases are those enzymes, which fall under the Enzyme

Classification, heading EC 3.2.1.

The functionality of these particular enzymes is summarized as follows: EC 3.2.1.4 enzymes (β-1, 4- endoglucanases) hydrolyze internal 1, 4 glycosidic bonds of the polysaccharide chain, thereby yielding new chain ends at the surface of cellulose crystals.

EC 3.2.1.6 enzymes (β-1, 3-endoglucanases) hydrolyze internal 1,3 glycosidic bonds of the polysaccharide chain, which also results in the formation of new chain ends at the surface of cellulose crystals.

EC 3.2.1.21 enzymes (β-glucosidases) hydrolyze cellobiose into glucose, a readily fermentable substrate.

EC 3.2.1.91 enzymes (1, 4-exocellulases) cleave cellobiosyl residues (cellobiose is a glucose dimer) from the chain ends of cellulose.

Particularly preferred enzymes (and hence particularly preferred genes) for use in the present invention are glutamate decarboxylase gene from rice, cellulase (1-3 endoglucanase) gene from Λ. cellulyticus, cellobiohydrolase gene from T. reesei, β-glucosidase gene from A. naeslundii and xylanase gene from T. reesei.

"Stress" as used herein refers to a factor, which externally causes a change in the growth of plants.

"Environmental stress" refers to a stress provided by a change in an external environment, including salts, high osmotic pressure, drying, high temperature, low temperature, intense light, air pollution, and the like.

Expression Constructs: Once the protein coding sequence of the desired proteins (i. e., the glutamate decarboxylase gene and cellulase genes) has been identified and isolated, it must be inserted into an appropriate expression construct containing regulatory elements to direct the expression of the gene and to direct secretion of the gene product or targeting of the gene product to a particular sub-cellular location or organelle.

Manipulation of oligonucleotide sequences using restriction endonucleases to cleave DNA molecules into fragments and DNA ligase enzymes to unite compatible fragments into a single DNA molecule with subsequent incorporation into a suitable plasmid, cosmid, or other transformation vector are well-known to the art.

A transcription regulatory sequence must be included in the expression construct in order to direct the transformed plant cells to transcribe the inserted coding sequences. Transcriptional regulators may be inducible or constitutive. Inducible transcription regulators direct transcription of the downstream coding sequences in a tissue-specific or growth-stage specific manner. Constitutive regulators provide for sustained transcription in all cell tissues. For purposes of the present invention, constructs, which provide constituitive expression of the coding sequence, are preferred.

It is also preferred that the expression construct contain a transcription initiation sequence from the tumor-inducing plasmid (Ti) of Agrobacterium. Several T-DNA transcription initiation sequences are well known and include, without limitation, the RUBISCO, actin, 35S Cauliflower mosaic virus (CaMv), Cassava vein mosaic virus (CvMv), octopine synthase, nopaline synthase, and mannopine synthase initiators.

Either upstream or downstream of the coding sequence and fused to the coding sequence, the expression construct may be manipulated to contain a signal sequence which directs the resulting polypeptide to a particular organelle or targets the expressed product for secretion (or to signal post-transcriptional or post- translational modification of the gene product). Likewise, the expression construct should also include a termination sequence to signal transcription termination.

To facilitate selection of successfully transformed plants, the expression construct should also include one or more selectable markers. The neomycin phosphotransferase gene (NPT EI) is a well-characterized and widely employed antibiotic resistance selection marker. This marker provides resistance to kanamycin. A large number of other markers are known and can be used with equal success (e. g., other antibiotic resistance markers, dihydrofolate reductase, hygromycin phosphotransferase and the like). Many non-anti biotic selection markers like luciferase, β-glucuronidase and the like are also widely employed. A previously described positive selection marker - xylose isomerase, which employs the xylose sugar as a selection means is preferred in this invention.

Fig. 2 depicts schematic representations of suitable expression construct for transformation of plants. These construct is intended for use with Agrobacterium- mediated transformation using the binary vector approach. However, similar constructs can be coated onto micro-projectiles for transformation by particle bombardment. Fig. 2 is a schematic diagram of binary vector T-DNA for an expression construct to transform plants to contain glutamate decarboxylase gene from rice, cellulase (1-3 endoglucanase) gene from A. cellulyticus, cellobiohydrolase gene from T. reesei, β-glucosidase gene from A. naeslundii and xylanase gene from T. reesei.

In Fig. 2., promoters and structural genes are depicted as arrows, which indicate the direction of transcription and terminators, are depicted as boxes. See the "Brief Description of the Figures" for a legend to the abbreviations. The construct also contains a constitutive Xi expression cassette to allow for positive selection using xylose isomerase. Transformation of Plants: Transformation of the plants can be accomplished by any means known to the art, including Agrobacterium-mediated transformation, particle bombardment, electroporation, and virus- mediated transformation. The method of transformation is not critical to the functionality of the present invention insofar as the method chosen successfully incorporates the oligonucleotide construct containing the desired protein-encoding region and any accompanying regulatory sequences into the plant host. The nature of the plant host to be transformed has some bearing on the preferred transformation protocol. For dicots, Agrobacterium-mediated transformation utilizing protoplasts or leaf disks is most preferred. Although the Examples disclose the use of pearl millet and tobacco as systems for generating stress tolerant self-glucogenic plants, any crop plant, including monocots, can be utilized. Transformation of monocots is typically achieved by particle bombardment of embryogenic cell lines or cultured embryos. See, for instance, Vasil et al. (1993) and Castillo et al. (1994). Recent developments in"super- binary"vectors, however, also allow for the use of Agrobacterium-mediated gene transfer in most of the major cereal crops. See, for instance, Ishida et al. (1996). In this case, the explant source is typically immature embryos.

Agrobacterium-mediated transformation of the plant host using explants is preferred for its relative ease, efficiency, and speed as compared to other methods of plant transformation. For example, disks are punched from the leaves of the plant host and cultured in a suitable medium where they are then exposed to Agrobacterium containing the expression construct and (preferably) a disarmed tumor-inducing (Ti) plasmid. Agrobacterium tumefaciens LBA 4404 is the preferred strain for transformation. The preferred binary vector is the pCGN1578 binary vector (McBride and Summerfelt (1990)).

The binary vector transformation method is well known and needs only be briefly described herein. The T-DNA portion of the Ti plasmid is flanked by two border regions (the right and left borders), which act as recognition sites for the excision of the T-DNA from the plasmid prior to its transfer to the plant host. Excision of the T-DNA is mediated by the vir genes of the Ti plasmid and involves nicking of the right and left borders of the T-DNA, which frees a single-stranded oligonucleotide fragment. This fragment is then mobilized out of the Agrobacterium and into the plant host target.

In the binary vector method, the T-DNA with its right and left border regions is cloned into E. coli in known fashion, and the wild-type genes normally found between the two border regions is excised. The expression construct encoding the genes of interest are inserted between the right and left border regions. This construct is designated the "binary plasmid." Construction of the binary plasmid is accomplished utilizing the well-characterized recombinant genetic methods applicable to E. coli. Successful transformants are selected utilizing a co-transformed marker appropriate for E. coli.

The binary plasmid is then mobilized back into Agrobacterium. This is accomplished by direct transformation procedures well known to those skilled in the art. The Agrobacterium itself, such as the preferred GV3101 strain, is genetically manipulated to contain a Ti plasmid (called the helper plasmid) which lacks the T-DNA and the tumor-inducing regions (i. e., the Ti plasmid is"disarmed") but which still encodes the virulence proteins necessary for DNA transfer. By cooperation between the helper plasmid and the binary plasmid, the length of DNA between the two border regions of the binary plasmid is excised and mobilized into the plant host, where it is incorporated into the plant host genome. The binary method derives its name from the fact that the plasmid containing the expression construct to be transferred is maintained within Agrobacterium as a distinct and independently replicating vector from the Ti plasmid itself.

Selection of successful transformants is accomplished using the co-transformed selection marker discussed above. If the marker is Xi, selection is accomplished by growing the transformants on a media supplemented with xylose sugar.

For the present invention, the most preferred plants for transformation are pearl millet and tobacco.

However, any plant species will function with comparable success.

Included among the plant species which can be utilized in the present invention are cauliflowers, artichokes, apples, bananas, cherries, cucumbers, grapes, lemons, melons, nuts, oranges, peaches, pears, plums, strawberries, tomatoes, cabbages, endive, leeks, lettuce, spinach, arrowroot, beets, carrots, cassava, turnips, radishes, yams, sweet potatoes, beans, peas, soya, wheat, barley, corn, rice, rapeseed, millet, sunflower, oats, tubers, kohlrabi, potatoes, and the like.

The plants to be transformed are preferably common green field plants, such as the preferred alfalfa and tobacco, as well as soya, corn, and the like. Equally preferred are plant hosts, which are grown specifically for "biomass energy," such as switchgrass, poplar, and the like. In this instance, the enzymes would not be recovered from the plants. The plants are then transformed and regenerated into whole plants, which express fully functional, cellulose-degrading enzymes in economically significant quantities.

Pearl millet is one of the most preferred plant species for use in the present invention because pearl millet is a hardy plant, which grows well with minimal fertilization and irrigation. In the tropics pearl millet is a commonly cultivated marginal land crop providing both feed and fodder to farmers. It has been largely ignored by the biofuel community, which concentrates on maize, which is an input intensive crop, for bioethanol production. This invention entails the enhancement of leaf biomass under marginal conditions to enhance the recovery of sugars after degradation of the cell wall components by glycolases expressed and sequestered in the vacuole or peroxisome.

The cellulase enzymes described in the instant invention are most preferred because they are native to thermo-tolerant bacteria and are relatively heat stable. This allows to maintain the enzyme activity intact of the cellulases expressed in the plant material during the bio-ethanol production, where the process includes relatively rigorous heat treatments which other wise would adversely effect the activity of the cellulase incase the cellulase were not thermo stable.

Stage-Specific and Tissue-Specific Expression of Cellulases: Because the enzymes to be expressed by the transformed plant hosts hydrolyze components of the plant cell wall, high levels of expression might have a deleterious effect on the plant host. Therefore, targeting of the expressed enzyme to particular subcellular compartments may be preferred. Targeting of the expressed enzyme may also be preferred to avoid expression of the enzyme in sub-cellular compartments where proteolytic activity is high. Targeting of the expressed enzyme may also be preferred if the exogenous cellulase activity interferes with the normal cellular metabolism of certain compartments.

Example:

Identification of cellulose degrading enzymes (glycolases) and construction of super binary vector harboring the identified glycolases

Plant lignocellulosic biomass is a complex matrix of polymers composing of polysachharides cellulose and hemicellulose and a polyphenolic complex lignin as the major structural components. Cellulose, the most abundant biopolymer on the earth, is a simple, linear polymer of glucose. Nature has developed effective cellulose hydrolytic machinery for recycling of carbon from plant biomass in the environment, without it the global carbon cycle would not function. To date many cellulase genes have been cloned and sequenced from a wide variety of fungi, bacteria, protozoans and plants. Cellulase refers to a class of enzymes that catalyze the hydrolysis of cellulose (cellulolysis). Several different kinds of cellulases are known, which differ structurally and mechanistically. Cellulose is degraded through the synergistic action of two genearal types of cellulase enzymes. Enzymes that cleave the cellulose chain are referred to as endo-1,4 β-D-glucanases (endoglucanase, EG; EC 3.2.1.4) and serve to provide new reducing and non- reducing chain termini on which exo-1,4- β-D glucanases (cellobiohydrolase, CBH; EC 3.2.1.91) can operate. A third activity, β-D Glucosidase (EC 3.2.1.21) is required to cleave cellobiose and other oligomers to glucose. The glucosidase activity is required at 100-1000 times lower concerntration than the cellulases.

Most natural cellulose is closely associated with hemicellulose. β-l,4-Xylans are major constituents of hemicellulose. The gram-positive anaerobic thermophilic bacteria Clostridium thermocellum exhibits a highly active and thermostable (optima 7O ⁰ C) xylanase activity when cells are grown on cellobiose. Xylanase (Endo β-1, 4- xylanase, XynZ; EC 3.2.1.8) enzymes secreted by during growth on cellobiose may make cellulose accessible to cellulolytic enzymes. Based on this criterion T. reesei xylanase SEQ ID No 4 was chosen. Thus the objective of this work is to enable the high level expression of the aforesaid genes in tissue specific manner through vacuolar targeting in pearl millet in a system in which the cost of production could potentially meet the cost target for the enzymes in the biomass to ethanol industry. T. reesei produces at least five endoglucanases (EI, EII, EIU, EIV, and EV), two exoglucanases (CBHI and CBHII), and two β-glucosidases (BGLI and BGLII). The need for five endoglucanase species in the T. reesei cellulase system has not been clearly explained, particularly considering that endoglucanases (with EI and EH as major species) represent less than 20% of the total cellulase protein of T. reesei. Synergism between endoglucanases and cellobiohydrolases has been shown for EI, EII, and EHI. However, synergism between endoglucanases has not been clearly demonstrated. Part of the problem may be that natural cellulosic substrates are not used for laboratory experiments due to their heterogeneous nature and the true functions of the different endoglucanases may not be observed on purified cellulose. It is noteworthy that endoglucanase, such as EI, have broad substrate specificity (e.g., xylanase activity). T. reesei produces β-glucosidases at low levels compared to other fungi such as Aspergillus species. Furthermore, the β-glucosidases of T. reesei are subject to product (glucose) inhibition whereas those of Aspergillus niger are more glucose tolerant. The levels of T. reesei β-glucosidase are presumably sufficient for growth on cellulose, but not sufficient for extensive in vitro saccharification of cellulose. T. reesei cellulase preparations, supplemented with Aspergillus β-glucosidase, are considered most often for cellulose saccharification on an industrial scale. Using this criteria A. naeslundii β-glucosidase SEQ ID No. 8 was chosen. As regards, choosing enzymes for these experiments, the following criteria were considered: (i) The enzyme should show exhibit synergistic activity on lignocellulosic substrates; (ii) Enzyme should be thermostable to at least 45-5O ⁰ C, and (iii) Enzyme should have compatible pH optima. CBHI and CBHII are the principal components of the T. reesei cellulase system, representing 60 and 20%, respectively, of the total cellulase protein produced by the fungus on a mass basis. Using these criteria we chose El endoglucanase from Acidothermus cellulyticus SEQ ID. No 2 and CBHI from T. reesei SEQ TD No 6. El and CBHI exhibit synergistic activity on lignocellulosic substrates. Although EI has optimal activity at 81 ⁰ C; it is still highly active at 45-5O ⁰ C, compatible with the CBHI enzyme, which exhibits optimal activity at temperature up to 50 ⁰ C. These two enzymes have compatible pH optima at pH 5-6. The actual enzymes chosen for this invention are key to demonstrating this production system technology. Therefore following genes were chosen to express in pearl millet as a first step towards developing the self -glucogenic pearl millet system for lignocellulosic degradation.

Thus candidate genes glycolases for lignocellulosic degradation are Endoglucanase (El) SEQ ID No. 2, Cellobiohydrolase (CBHl) SEQ ID No. 6, Beta Glucosidase (BGL) SEQ ID No. 8, Xylanase (XynZ) SEQ ID No. 4; and GAD gene SEQ ID No. for stress tolerance:

The four glycolases identified from different sources have been codon optimized for their expression in plant system (Pearl millet). YWERLFV Fraction score closer to 0.4 indicates thermostability. Thus glycolases genes for lignocellulosic degradation used for plant transformation after codon optimization for efficient expression in the pearl millet system are Endoglucanase (El) SEQ ID No. 3, Cellobiohydrolase (CBHl) SEQ ID No. 7, Beta Glucosidase (BGL) SEQ ID No. 9, Xylanase (XynZ) SEQ ID No. 5; and GAD gene SEQ ID No. 1 for stress tolerance, which code for the cellulose degrading enzymes Endoglucanase (El) SEQ ID No. 11, Cellobiohydrolase (CBHl) SEQ ID No. 13, Beta Glucosidase (BGL) SEQ ID No. 14, Xylanase (XynZ) SEQ ID No. 12; and GAD gene SEQ ID No. 10. Each of the enzymes were first cloned into individual expression cassettes as shown in Fig 1 and then cloned into the single binary vector within the same T-DNA region as shown in Fig 2.

Selection of suitable genotype of pearl millet with high biomass

Three parental inbred lines of pearl millet (P. glaucum L.) viz. ABI- 13B, ABI-56B and 843B were procured from Atash Seeds Private Ltd. Hyderabad. They were initially screened for their agronomic performance in green house at Avesthagen. Selection of elite cultivar was based on the criteria like seed germination percentage and higher biomass yield. Seed germination percentage was highest (97%) in ABI- 13B followed by 843B (68 %) and ABI-56B (58 %) (Fig-3A). However, biomass yield was highest in ABI 56 B (82.7 g) followed by ABI13 B (76 g) and 843 B (41 g) respectively (Fig-3 B & C). DW/FW ratio was 0.43 in ABI13B and 0.4 in ABI 56B, however in 843B this was 0.32 only. Seed germination and dry biomass yield is highest in ABI 13B. On this basis of over all productivity ABI 13B genotype was selected for further tissue culture studies (Fig 3).

Plant transformation with the binary vector harboring the four different glycolases

Optimization of regeneration protocol in pearl millet

An efficient and reproducible tissue culture regeneration protocol is a pre-requisite for successful development of transgenic plants. Therefore attempts are being made to evolve a proficient regeneration protocol in pearl millet.

Surface Sterilization

Surface sterilization of the explant is a very decisive step in initiating a tissue culture experiment, as the sole success depends upon an efficient decontamination method, if sterilization is not up to the mark, the entire exercise may be futile due to microbial contamination. Even over-sterilization may also limit the success of culture initiation due to the death of the explants. Therefore a simple, cost effective and less time consuming sterilization protocol was optimized. The optimized protocol is as follows: wash the seeds (mature or immature) with Teepol (Tween 20). Rinse 3-4 times with distilled water. Soak the seeds in 0.1% HgCl ₂ for 5 minute. Again rinse 3-4 times with sterile distilled water, and use the sterile seeds for inoculation.

Callus Induction Since monocots like pearl millet are not amenable to direct regeneration, indirect regeneration through callus/ somatic embryo paves the way to achieve the desired goal. In order to optimize the same different explants viz. seeds, seedling roots, mature and immature embryos were explored for callus initiation. In the entire study basal MS salts with 3% sucrose and 0.8% agar, were used. The pH of the media was adjusted to 5.8 prior to autoclaving. Callus Induction Medium (CIM) was supplemented with different concentrations of 2,4-D (2,4-dichlorophenoxy acetic acid) ranging from 1 to 5 mg/L. All the cultures were initially incubated under dark condition in culture room (BOD incubator) at 26 ⁰ C for callus induction. Seed as explant

After surface sterilization seeds were directly inoculated on CIM. Callus initiation started after 2-3 days of inoculation on MS+ 3mg/L 2,4-D. After 2 weeks callus growth was observed on all the levels of 2,4-D, however on 3 and 5 mg/L it was almost equal. Therefore in the ensuing studies 2,4-D @ 3-mg/L was used for induction and growth of callus. The callus induced initially was of spongy type, which was very fragile (Fig-4B). After 20 days a small solid mass was enclosed by spongy calli could be seen. In order to obtain a healthy rapidly multiplying embryogenic calli, at this stage the spongy calli was gently removed and the remaining callus was further sub-cultured for proliferation on the same medium. Since tissue was so delicate at this stage, more than 50 % calli died due to the mechanical injury. Therefore to minimize this loss, in another experiment, calli were first sub-cultured as such without removing spongy calli. This allowed them to proliferate rapidly and within four weeks of subculture (total 7 weeks of inoculation), about 20% of embryogenic calli were obtained.

Mature embryo as explant

After soaking of sterilized seeds in water for 48 hrs, mature embryos were excised and inoculated on CIM. It was very difficult to dissect out the mature embryo and therefore about 60-70% seeds were damaged during that. Although, the growth of calli from mature embryo was very rapid and proliferated very well, but most of the calli were spongy in texture.

Immature embryo as explant

After complete anthesis (10-12 days after the onset of first flush of pollination), the fascicles were manually separated from the rachis of the pearl millet spike, then young green seeds were cleaned and as surface sterilized as mature seeds. Then immature embryos (2 mm in size) were aseptically excised and inoculated on CIM. Immature embryos were excised from young seeds at around 90% efficiency. A rapid callus induction and growth thereafter was recorded on CIM. About 40% of the callus obtained from these embryos was embryogenic as seen in Fig-4A.

Seedling roots as explants

After removal of callus from seeds, the remaining roots were excised and were also cultured to test their callogenic potential. Callus obtained from roots was highly spongy and was of less use for regeneration. Hence on the basis of these observations, it was inferred that in pearl millet immature embryos is the most preferred explant, followed by seeds, for in vitro studies and the preferred right stage for immature embryo collection is 10-12 days after first pollination.

Shoot Regeneration from callus

Embryogenic callus distinguishable by its compact and globular texture was transferred to Shoot Regeneration Medium (SRM) containing 1, 2, 3 mg/L of Benzyl amino purine (BAP), Kinetin (Kn) zeatin (1, 2, 3 mg/L) alone or in combination and with (0.2%) or without activated charcoal. In a separate experiment BAP and Kn were also supplemented with low concentrations (0.1 mg/L) of IAA, IBA and NAA. In all the experiments, media was supplemented with 10 mg/L AgNO ₃ as ethylene inhibitor. All the cultures were incubated under 16h photoperiod in culture room. Of all the SRM tested so far, multiple shoot regeneration from embryogenic calli was recorded only in media containing 3 mg/L BAP with 0.2% activated charcoal and 10 mg/L AgNO ₃ after 4 weeks of transfer (Fig- 4C), however growth of shoots in this media was observed to be very slow. The same media devoid of activated charcoal favored extensive growth of green roots (Fig- 4D). Figure- 4E and F show the different stages of shoot development from callus. These shoots have further been subcultured for shoot elongation. BAP is most supportive for shoot induction while charcoal in SRM favors shoot regeneration and suppress extensive root induction.

Xylose sensitivity assay

The use of selection and reporter gene has been vital for developing strategies for the production of transgenic plants and to increase their frequency. Currently the most widely used selectable marker genes confer resistance to antibiotics or herbicides, enabling the transformed cells to be selected by growth on a media containing the corresponding compound. But there have been associated a number of possible, but as yet unfounded food safety concerns with the use of antibiotic resistance marker in genetically modified organisms (GMO) used for food production. They may derive either from the marker gene itself or from its secondary gene product or from both. Public concerns have been paying attention on the practice of antibiotic resistance markers, which encompass: i) Inherent toxicity of the marker gene product; ii) Transfer and expression of the marker gene in gut microorganism and subsequent transfer to pathogen; iii) Toxicity or allergenicity of the gene product; iv) Intake of genetically modified plant products might compromise orally administrated antibiotics.

An alternative approach involving xylose isomerase (XyIA) gene as positive selection marker system is being used for pearl millet. This selection favors the regeneration and growth of transgenic cells, while non-transgenic cells are starved but not killed. XyIA catalyzes the reversible isomerization of D-xylose to D-xylulose as a part of the xylose metabolic pathway in microorganisms. D-xylose is the major component of hydrolysis of hemicellulose from biomass. Generally, Plant cells are not capable of metabolizing D- xylose as such completely. In theory, to make a plant capable of utilizing D-xylose, as a carbohydrate nutrient only one single gene has to be introduced i.e. XyIA. Sensitivity or tolerance of plant cells to the level of a particular compound may be genotype or tissue dependent. Therefore in order to work out the minimum inhibitory level of D-xylose, different concentrations (0.5, 1.0, 2.0, 3.0 and 4.0 %) of xylose were supplemented as sole carbon source in the basal medium. Medium with 3 % sucrose and that devoid of any carbon source served as positive and negative control respectively. Effect of xylose was studied on seed germination, callus induction from seeds as well as callus proliferation. In xylose sensitivity assay in seeds, 2% xylose concentration was regarded as the threshold for seed germination, beyond that xylose was found to inhibit the seed germination (Fig- 5A). However, in case of callus induction from seed and proliferation, all the concentrations 0.5 to 4.0 % were found to inhibit the callus proliferation (Fig- 5B). Callus growth was recorded only in control 3% sucrose. Thus 2% xylose concentration is the preferred concentration to be used for selection of transgenic pearl millet during seed germination. And 0.5% concentration is the preferred concentration to be used for selection of transgenic pearl millet during callus induction and proliferation.

Standardization of Agrobacterium Mediated Transformation in Pearl millet

Agrobacterium mediated transformation (AMT) has become an extensively established mode of producing transgenic plants. There are only a very few reports on genetic transformation in pearl millet but those have been executed only via biolistic method. There is no published report available on AMT in pearl millet so far. Therefore, the current invention has focused towards developing an efficient AMT protocol in pearl millet.

Transformation of pearl millet with binary vector

Pearl millet seeds after surface sterilization were kept for soaking for 48 hrs in sterile water. After 48 hrs, emergence of small embryos can be seen. A. tumefaciens EHA 105 strain harboring pCAMBIA 1305.1 binary vector with GUS reporter gene was grown overnight in LB to obtain the ODgoo 0.5-0.6. The culture was pelleted down at 3000 rpm for 5 min at RT and re-suspended in 50 mL basal MS medium supplemented with 10OmM Acetosyringone. After 10 min Agro-infection, seeds were blot dried and placed on co-cultivation medium (MSO). After two days some of the embryos were checked for transient

GUS Assay. And the remaining seeds were transferred to selection medium (MS+ 2, 4-D + 20 mg/L

Hygromycin). About 20 seeds from those transformed with pi 305.1 vector were checked for transient

GUS assay, and blue staining was seen on the coleoptile part.

A. tumefaciens strains EHA105 and GV 3101 were transformed with pl305.1 vector by electroporation.

Transformed Agrobactrium colonies were screened and confirmed with PCR. These strains were used for further transformation studies. Agrobacterium strains GV3101 and EHA 105 were transformed with p 1305.1 vector, which were confirmed by PCR.

Transformation of immature zygotic embryos of pearl millet with pi 305.1

Immature embryos were excised and infected with GV-3101 (ODβoo 0.5-0.6) and EHA-105 (OD ₆₀₀ 0.25-

0.3) strains A. tumefaciens harboring pl305.1GUS+XI binary vector as seeds were infected earlier. After two days of co-cultivation, embryos were checked for transient GUS assay. And after dis-infection the embryos were transferred to selection medium (0.5% xylose). In a separate experiment calli were also infected. On the basis of transient GUS assay it was observed that, out of the two strains (EHA 105 and GV3101), GV3101 was found to be more efficient and thus the preferred strain for pearl millet transformation. About 80 % of the embryos revealed the transgenic nature through transient GUS staining (Fig- 6). The immature embryos have been transferred to the selection medium for their further growth.