In this article we will discuss about the transgenic improvement of trees.

Improvement of tree species through biotechnology is one of the potential goals in plant science. Genetic improvement of trees is a tedious endeavour because of their long reproductive cycle. Several advancement has been made in the development of tissue culture protocols for the propagation of conifer and pine and these are in fourth generation.

In order to speed up the tree improvement cycle, genetic engineering offers novel method to transfer novel genes into different tree species and also direct regeneration of transgenic trees through tissue culture to eliminate the need to produce seeds.

The first report on the transgenic conifer regeneration was appeared in 1993, where gene gun mediated DNA delivery system was employed in the production of transgenic white spruce. Presently, several transgenic approaches have been implicated in the modification of lignin biosynthesis, cellulose accumulation and rapid growth of the certain tree species.

Examples of Transgenic Improvement of Trees:

Lignin Engineering:

Lignification is an important process for the plant due to its importance of rigidity for cell wall and adaptation to water conduction and more importantly in abiotic and biotic stress toler­ance. The lignified secondary wall results from the expression of several classes of genes. The assembly and deposition of polysaccharide wall and lignin involves coardinated expression of the numerous genes (Fig. 26.1). Thus, modification of wood quality is indispensable in view of human utilization and environmental significance.

Pathway for the Production of Lignin

Wood is the chief source for fiber, chemical and energy. But processing of wood for indus­trial benefit is costly and economically not viable. This is because secondary xylem (wood) of trees, from which pulp is derived, is composed of cellulose, lignin and hemicellulose in approxi­mate proportion of 2:1:1. Although cellulose and lignin make a collaborative effort in maintain­ing tensile strength and rigidity to the cell wall and act as important component during growth, lignin becomes main hurdles to wood processing (pulping) in timber industries.

Pulping process in which cellulose separate from lignin requires enormous amount of energy, chemical and pose environmental impact. As a consequence, persistent endeavour has been made to produce transgenic trees that accumulate less lignin or more extractable lignin to facilitate pulping.

Since lignin reductase has positive implications, some of the earliest attempts to re­duce lignin biosynthesis were carried out by down regulation of certain genes encoding caffeate- o-methyl transferase or cinnamyl alcohol dehydrogenase (Fig. 26.1). But success was limited to the modifications of lignin structure in transgenic tobacco plants.

This was accomplished by supporting phenyl alanine ammonialyase in transgenic tobacco. This enzyme catalyse initial step in phenyl propanoid pathway upstream of lignin biosynthesis. The down regulation of this enzyme restricts overall phenyl propanoid biosynthesis resulting in several abnormal features as well as reduced lignin in transgenic tobacco.

Another classic example on lignin reduction have also been accomplished in transgenic Arabidopsis and tobacco by down regulating 4Cl (cinnamoyl-coenzyme A reductase), but growth was retarded and collapse of cell wall were reported in transgenic plant where lignin reduction was more than 40%. However, these anoma­lies were not noticed in transgenic aspen (Populus tremuloidesmicax) tree in which expression or lignin biosynthetic gene pt 4Cl, encoding 4-coumarate: coenzyme A ligase (4Cl) has been down regulated by antisense inhibition.

The 4Cl genes, pt 4Cl, pt Cl2 encodes protein which catalyze the CoA ligation of hydroxycinnamic acid, provides enough phenolic precursors for lignin biosynthesis. While pt 4cl2 expression is restricted to flavanoids production, pt 4cl is committed for lignin biosynthesis in developing xylem tissue. Transgenic aspen exhibit upto 45% reduction in lignin.

In the same line of work, an interesting outcome has been observed about increase in cellulose content upto 15% which maintains structural integrity in the plant and accomplished growth in transgenic trees. The probable reason for cellulose elevation is due to increased hydroxycinnamic acid and its incorporation into the non-nuclear cell wall by glucosyltransferase pathway and hence, direct substrate after 4Cl activity was suppressed.

It has also been proposed that cellulose synthesis is substrate limited and that reduces flow of carbon into the lignin pathway and pt CI, step increases the availability of carbon for cellulose deposition. This would even probably meet the higher carbon demand for woody than herbaceous plants. Reduction of lignin upto 50% without compromising in growth and development have also been witnessed in french bean by down regulating peroxidase.

By employing antisense technology it has been possible to modify structure of lignin with altering its content by blocking the expression of several enzymes involved in lignin biosynthetic path­ways. The transgenic aspen and poplar tree plants expressing antisense gene for caffeic acid/5- hydroxy-ferulic acid-o-methyl transferase (COMT), resulted in its reduced activity and varia­tion in the lignin composition without altering basic level of lignin content.

It is possible to increase lignin content in transgenic trees by modification of transcrip­tion factors expression and employed two myb factors EgMyb1 and EgMyb2 expressed in xylem tissue have been cloned from eucalyptus cDNA library. The recombinant proteins (Egmyb1 and Egmyb2) recognises specific myb binding sites in the promoter region of both hydroxy cinnamyl CoA reductase (CCA), cinnamyl alcohol dehydrogenase (CAD) genes. The Myb2 acts as activator of lignification genes. The overexpression of Myb2 increased lignin content and thicker cell wall in transgenic tobacco.

Remodeling of Cell Wall Composition:

Remodeling of plant cell wall by employing genes of microbial origin is another promising area. For example, decrease in galactose and arabinose was achieved in transgenic potato by the expression of rhamnogalacturonase lyase from Aspergillus. The expression of microbial enzymes like 4-hydroxy cinnamoyl-CoA reductase/lyase in tobacco and Datura stramonium resulted in the enhanced production of benzoic acid derivative glucose conjugate, which had a significant impact on cell wall composition. This is illustrated by the orange-red or red brown colouration of the xylem.

Disease Resistance:

Forest trees are attacked by various disease causing agents such as fungi. Insects can inflict maximum damage which is serious concern in forest ecology and management. In the combating strategy genetic transformation of trees using gene coding for Bt or proteinase inhibitors provides substantial protection and consequently reduces chemical usage.

Some of the tree species which have been modified by transformed pine trees, and hybrid populus. Different crystal toxin gene of Bacillus thuringiensis has been used to generate insect’s resistance, character in populus. Hybrid poplar expressing CrylB gene showed reduced larval feeding and decreased larval weight of cotton leaf beetle.

Similarly, transgenic expression of Cry III A in Populus tremula was found to be toxic to coleoptera insect. Transformation by antifungal and antibacterial gene in tree species is at the early stage. However, introduction of antifungal wheat oxalate oxidase gene in poplar shows limited success.

Herbicide Resistance:

The first herbicide tolerant transgenic trees were reported in 1987 after introduction of aroA gene into poplar. The aroA gene coding for S-enolpyruvyl shikimate synthase that is ac­tive in the synthesis of aromatic amino acids. Transgenic poplar was resistant to glyphosate. In addition, trees transformed with bar gene were also tolerant to herbicide.

Shorter Reproductive Cycle:

Exploration of forest trees for trait analysis is sufficient to the long Juvenile phase. Thus, introduction of genes encodes proteins controlling plant reproductive cycle will be significant for forest tree species. Therefore, it is desirable to introduce gene for early flowering in tree plants.

The gene leafy from Arabidopsis encodes proteins governs early flowering, have been introduced in aspen. Early flowering was noticed after 7 month instead of 8-20 years. Recent studies show that homologous to Lfy gene and PTF L gene from populus is able to induce early flowering in poplar.

Phytoremediation:

The potentials of hybrid poplar tree able to mineralize chlorinated compounds such as trichloroethylene (TCE), carbon tetrachloride (CT) and other halogenated aliphatic hydrocar­bons have been well recorded. Enhancement of TCE metabolism in transgenic poplar has been currently being used by research group at the University of Washington. Active role of expressed cytochrome p450 II El in transgenic tobacco has shown to enhance TCE metabolism.

Flower Sterility:

The manipulation of flowering can provide many benefits. Flower sterility in trees has immense role in tree improvement particularly in productivity and gene flow containment. In tree species most of its energy is utilised for the production of flowers. Generally, tree produces innumerable number of flowers and plant has to invest energy and biomaterial for its produc­tion.

Thus, it is hypothesised that complete elimination or reduction of number of flowers save energy and increase biomass production. By imposing reproductive sterility it may result in ecological concern over the use of transgenic plantations and eliminate unwanted trees. The other potential outcome of the technique is associated with shorter breeding cycle engineering sterility could be accomplished by cell ablation, of reproductive tissues.

In populus tree intro­duction of fused two cytotoxic genes, DTA and barnase resulted in decreased vegetative growth. The promoters from PTP, a poplar homology of the Arabidopsis gene APETALA 3 was used in the expression of reporter and cytotoxic genes in flower tissues of poplar (tree), tobacco and Arabidopsis (model plant).

In the second approach dominant negative mutants (DNM) transgene was expressed using hybrid promoters composed of two copies of the enhancer element from the 35 S promoters attach to ACTH promoter from Arabidopsis. Other options are suppressing flowering by RNA interfering technique using this strategy suppression of PTLF, PTAP1, and PTD are like to give floral sterility.

In addition, blocking the expression of functional genes involved in developmental process of flowering could result in flower formation. For example, several homeotic genes, APETALA 2 (AP2) LEAFY (LFY) involved in the developmental process of flowering have been identified in Arabidopsis and their homologous in populus tree species.

The flowering process can be speed up by accelerating tree breeding program. Therefore, prior knowledge of genes is involved in early flowering to occur in the life cycle of tree is essen­tial to cause flowering early. Constitutive expression of LEY gene resulted in precocious flower development in transgenic aspen.

Overexpression of constitutive expression PTLF gene, homologs of LEY, accelerated flowering times in Arabidopsis. Therefore, understanding flower­ing time genes helpful in the early production of flower in trees in turn reduced breeding cycle, which has a significant contribution in tree improvement programme.

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