Present invention relates to a genetically modified sweet sorghum plant with reduced lignin content. Also, the present invention relates to reduction of lignin content in sorghum by down regulation of Caffeic acid O-methyl transferase (COMT) and Caffeoyl CoA-O-methyltransferase (CCoAOMT) activity through antisense approach. Particularly, the invention relates to different anti-sense construct incorporated in a binary vector. More particularly, the present invention relates to a method of Agrobacterium mediated gene transfer in sweet sorghum plant.
Background of the present invention
Optimal realization of the benefits of various plant products as biological resources for human welfare is a challenging proposition. However, some compounds present in plant that are essential for plant growth, development and defense, do sometimes hinder in realization of the desired benefits of the plant product. One such component is lignin. Presence of high level of lignin often limits the extraction of beneficial components from plants and use of biomass of plants. Its removal often become cumbersome and when removed they generate large amount of chemical wastes, unfriendly to the environment. One possibly way to tackle this situation is to reduce the in vivo lignin content without seriously impeding the benefit of its presence for the plants. Attempts in the past to genetically engineer the quality and quantity of lignin in several commercially important plant species have yielded interesting results (Feuillet et al, 1993; Ni et al, 1994; Dwivedi et al, 1994; Antanassova et al, 1995; Doorsellare et al, 1995; Mackay et al, 1995; Jouanin et al, 2000 and Chabannes et al, 2001). It has bee observed that inhibition of some of the enzyme activity involved in lignin biosynthetic pathway could alter lignin content without affecting normal physiology of the plant. The scientific rational on which these studies were undertaken was based on the pioneering studies carried out in the past on brown-midrib mutant of maize. It was found that the mutant character was associated with reduced cinnamaldehyde alcohol dehydrogenase (CAD) and cacffeic acid O-methyl transferase (COMT) activities. It was further observed that inactivation of CAD generates a pool of cinnamaldehyde, which is in turn incorporated into lignin polymer instead of cinnamyl alcohol, and thereby cross-link with cellulose decreases, albeit the total lignin content remained the same. This situation is however expected to facilitate easy removal of the component.

Recently, Chabannes et al (2001) demonstrated that the simultaneous down regulation of cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase causes drastic reduction of lignin content in plants. It was further demonstrated that down regulation of caffeic acid O-methyltransferase activity could result dramataic decrease in syringyl lignin biosynthesis but with little effect on the synthesis of guaiacyl lignin (Atanassova et.al, 1995; Van doorsselaere et.al, 1995; Tsai et.al, 1998; Jouanin et.al, 2000). The unequivocal demonstration of the essential role of caffeoyl CoA-O-methyltransferase (CCoAOMT) in lignin biosynthesis came from the result of biochemical, immunolocalization studies in Zinnia (Ye et.al. 1997) and also from transgenic studies in tobacco, Zhong et.al.(1998) showed that down regulation of CCoAOMT in tobacco plants not only reduce total lignin content but also dramatically decreased both guaiacyl and syringyl lignin units. Similar results were also observed in transgenic poplar with down-regulated CCoAOMT gene (Meyermans et. al., in press).
Sweet sorghum, Sorghum bicolor (L) Moench, is the only crop that provides grain and stem that can be used for the production of alcohol, sugar, syrup, fuel etc. But the major problem with this plant is the presence of lignin in cell wall that adversely affects the process of extraction of beneficial materials. Reduction of lignin content is likely to improve the quality of the plant. All conventional breeding programmes undertaken to generate cultivars with reduced lignin content met with limited success. Thus, possibility to develop cultivars with altered lignin content through genetic engineering figured as a distinct possibility. Keeping this in view, the present proposal is aimed at manipulating the function of caffeoyl CoA-O-methyltransferase, the pivotal enzyme of lignin biosynthetic pathway, in Sorghum through transgenic approach.
Objectives of the present invention:
1. Preparation of cDNA library of Sorghum.
2. Isolation of CCoAOMT gene by screening of cDNA library.
3. Characterization of CCoAOMT gene by sequencing.
4. Molecular construction of double strand mediated antisense construct.
5. Standardization of transformation method for sorghum following Agrobacterium
mediated gene transfer.
6. Transformation of antisense construct into sorghum tissue.
7. Selection and regeneration of primary transformants.
8. Analysis of primary transformants for low lignin content.

Detailed description of the present invention:
Accordingly, the present invention relates to an antisense construct, said construct incorporated in a binary vector, said vector in Agrobacterium and Agrobacterium mediated transformant thereof. The invention also relates to a process of preparing the above said. Following is a flow chart showing various components of the invention.
Isolated total RNA from sorghum stem: Total RNA was isolated from Sorghum stem using not phenol method. mRNA was purified form total RNA using oligo-dT cellulose matrix.
Synthesis of first strand cDNA: First strand cDNA was synthesized using random hexamer.
Isolation of partial COMT gene: Partial COMT gene of 790bp in length was amplified from cDNA synthesized through RT-PCR using gene specific 51 & 3! primers designed from published sequences of sorghum COMT gene. Sequences of the primers are: 51-attcgcggatccatggggtcgacggcggaggacg-31 and 5'-tatcccgagctcttacttgatgaactcgatgg-3Cloning and characterized of the amplified COMT fragment: Amplified COMT gene fragment was cloned in pUC18 vector using restriction sites BamHI and Sacl incorporated in the primers. Clones were characterized by sequencing.
Construction of antisense COMT gene cassettes in binary vector, The COMT gene fragment was dropped out by BamHI and Sacl digestion, ligated with BamHI and Bglll digested linker in sense and antisense oriented and incorporated in pUC18 digested with Sacl. The double stranded antisense construct was then digested out with Sacl and incorporated into binary vector (BREF 121) under ubiquitin promoter (Hindlll/Sacl) and nos terminator (Sacl/EcoRI) through tetrapartite ligation to make complete cassettes for plant transformation.


Isolation of partial CCoAOMT gene: Partial CCoAOMT gene was amplified from cDNA synthesized through RT-PCR using degenerated 51 & 3' primers designed from corresponding to the consensus amino acid sequence regions of CCoAOMT enzyme of heterologous plants. The sequences of the primers are: 5J-gaattcggatccac(t)caa(g) gaa(g)gta(c/t/g)gga(c/t/g)cac(t)aa-3' and 5'-ccgggtaccg(a)ttccaa(c/t/g)aga(c/t/g)gt g(a)ttg(a)tcg(a)ta-3'
Cloning and characterization of the amplified CCoAOMT fragment: Amplified CCoAOMT fragment was cloned in pUC18 vector using restriction sites BamHI and Kpnl incorporated in the primers. Clones were characterized by sequencing.


Construction of antisense CCoAOMT gene cassettes in binary vector the CCoAOMT gene fragment was dropped out by BamHI and Kpnl digestion, ligated with BamHI and Bglll digested linker in sense and antisense orientation and incorporated in pUC18 digested with Kpnl. This double stranded antisense contruct was digested out with Kpnl and incorporated into a binary vector (BREF 121) under ubiquitin promoter (Hindlll/Kpll) and nos terminator (Kpnl/EcoRI) through tetrapartite ligation to make complete cassettes for plant transformation.
Isolation of 3' end CCoAOMT gene by 3' RACE: First strand cDNA was synthesized from mRNA isolated from stem of sorghum plant using oligo-dT primer. Then it was amplified with RACE-AMP primer and gene specific primer constructed on the basis of sequences analysis of partial CCoAOMT gene fragment.
Amplified product was fun on a gel, blotted onto membrane and checked the presence of CCoAOMT gene through hybridization using partial CCoAOMT gene as probe. Amplified product was digested with BamHI and Kpnl and cloned into pUC 18 vector. Colonies were hybridized to identity the positive clones and characterized by further hybridization of digested recombinant plasmid DNA isolated from the suspected clones.

Agrobacterium mediated transformation was carried out to transfer the construct in sweet sorghum plant and the genetically modified plant shows less lignin content. The process is given in the form of a flow chart.

In the present invention the medium composition used for the above said transformant is given below. Also given the results of

We Claim:
1. A purified preparation of an antisense nucleic acid, which inhibits expression of said
COMT gene and wherein said antisense nucleic acid is further selected from the group
consisting of a synthetic oligonucleotide, a single-stranded oligonucleotide, a linear
oligonucleotide and combinations thereof.
2. A purified preparation of an antisense nucleic acid, which inhibits expression of said
CCoAOMT gene and wherein said antisense nucleic acid is further selected from the
group consisting of a synthetic oligonucleotide, a single-stranded oligonucleotide, a
linear oligonucleotide and combinations thereof.
3. A method of decreasing lignin content in sweet sorghum plant, said method
comprising: introducing into a cell by conventional method to said cell an antisense
construct of claim 1 and/or 2 in an amount sufficient to produce in the cell a population
of antisense nucleic acids which specifically hybridize to mRNA expressed by a
gene(s) COMT and/or CCoAOMT encoding lignin under physiological conditions and
wherein inhibition of expression of said COMT and/or CCoAOMT genes results in a
statistically significant decrease in the cellular lignin production to a control population
of cells.
4. An expression vector comprising an antisense nucleic acid molecule which specifically
hybridizes under physiological conditions to mRNA transcribed by CCoAOMT gene.
5. An expression vector comprising an antisense nucleic acid molecule which specifically
hybridizes under physiological conditions to mRNA transcribed by COMT gene.
6. Agrobactehum tumefaciens comprising an antisense nucleic acid molecule of claim 1.
7. Agrobactehum tumefaciens comprising an antisense nucleic acid molecule of claim 2.
8. Primers useful in partial CCoAOMT gene amplification, represented by SEQ ID NO 1
and 2.
9. Primers useful in partial COMT gene amplification, represented by SEQ ID NO 3 and
4.


Dated this on 18th Day of August, 2006