Genetic Engineering of Phenylpropanoid Pathway in Leucaena leucocephala

Trees are reservoirs of many economically and biotechnologically significant products. Wood, one such gifts of nature, consists of lignin, hemicelluloses, and cellulose. Biochemistry of lignin, which being one of the most abundant biopolymers on earth, has been studied extensively, partly due to the significance, and interest of such knowledge from industrial point of view (Boerjan et al., 2003). Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism, a major route for synthesis of lignin in plants, a globally important part of plant chemistry.


Introduction
Trees are reservoirs of many economically and biotechnologically significant products. Wood, one such gifts of nature, consists of lignin, hemicelluloses, and cellulose. Biochemistry of lignin, which being one of the most abundant biopolymers on earth, has been studied extensively, partly due to the significance, and interest of such knowledge from industrial point of view . Lignin has far reaching impacts on agriculture, industry and the environment, making phenylpropanoid metabolism, a major route for synthesis of lignin in plants, a globally important part of plant chemistry.
Besides its critical role in normal plant health and development, high levels of lignin are problematic in the agro-industrial exploitation of various plant species. It is considered an undesirable component in paper manufacture due to the cost, energy consumption, and pollutant generating processes required for its removal Boerjan, 2005;Chiang, 2002). Thus, making it essential to provide designer plant species with altered lignin content, and hence, to diminish the pressure on the domestication of natural forest resources in the future. Considerable scientific interest has been focused on the development of trees with improved wood quality through modification of different genes involved in lignin biosynthesis, which could be important for the improved end use of wood material (Chiang, 2006;Higuchi, 2006). Leucaena leucocephala, one of the most versatile fast growing commercially important trees for paper and pulp industry in India, contributes nearly a quarter of the total raw material. The wider use of this tree species in the pulp industry is due to its high rate of biomass production and ability to adapt to a variety of soils and climatic conditions. Every step towards the development of this tree variety in terms of increased biomass and reduced lignin content would be of great help to pulp and paper industry as it will decrease cost and release of hazardous chemicals during the production of paper pulp. This chapter will briefly cover, the chemistry of lignin deposition in plants, role of different monolignol biosynthesis pathway genes, followed by studies concentrated on genetic engineering of phenylpropanoid pathway in Leucaena leucocephala as tool for altering its lignin composition, thereby its application in pulp and paper industries.

Lignin: Occurrence, structure and function
Lignin (from Latin lignum meaning wood), is one of the most abundant natural organic polymer next only to cellulose . It is a vital cell wall component of all vascular plants and represents on an average of 25 % of the terrestrial biomass. It plays crucial role in structural integrity of cell wall & stiffness and strength of stem (Chabannes et al., 2001;Jones et al., 2001). Lignin is primarily synthesized and deposited in the secondary cell wall of specialized cells such as xylem vessels, tracheids and fibers. It is also deposited in minor amounts in the periderm where association with suberin provides a protective role against pathogens. In addition, lignin waterproofs the cell wall; enabling transport of water and solutes through the vascular system (Sarkanen & Ludwig, 1971). Lignins are complex racemic aromatic heteropolymers synthesized from the dehydrogenative polymerization of monolignols, namely coumaryl, coniferyl and sinapyl alcohol monomers differing in their degree of methoxylation (Freudenberg & Neish, 1968). These monolignols produce respectively, p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) phenylpropanoid units when incorporated into the lignin polymer (Fig. 1). The amount and composition of lignins vary among taxa, cell types and individual cell wall layer, and are influenced by developmental and environmental cues (Campbell & Sederoff, 1996). Lignin content is higher in softwoods (27-33%) than in hardwood (18-25%) and grasses (17-24%). The highest amounts of lignin (35-40%) occur in compression wood on the lower part of branches and leaning stems (Sarkanen & Ludwig, 1971).

Lignin biosynthesis
Although lignin has been studied for over a century, many aspects of its biosynthesis still remain unresolved. The monolignol biosynthetic pathway has been redrawn many times, yet still remains a matter of debate. During the last two decades, significant headway has been made in isolating and characterizing a number of genes pertaining to monolignol biosynthesis from different plants. Several reviews on the advancements of monolignol biosynthesis pathways are also available Whetten et al., 1998).
In plants, lignins are synthesized by the polymerization of monolignols, namely p-coumaryl, coniferyl and sinapyl alcohol monomers differing in their degree of methoxylation (Higuchi, 1985;Sederoff & Chang, 1991) via phenylpropanoid biosynthetic pathway (Gross, 1985). This pathway comprises a complex series of branching biochemical reactions responsible for synthesis of a variety of products like lignin, flavonoids and hydroxycinnamic acid conjugates. Many intermediates and end products of this pathway play important role in plant such as phytoalexins, antiherbivory compounds, antioxidants, ultra violet (UV) protectants, pigments and aroma compounds. Finally, the diverse functions of lignin and related products in resistance to biotic and abiotic stresses make this pathway vital to the health and survival of plants.
The synthesis of lignin represents one of the most energy demanding biosynthetic pathways in plants, requiring large quantities of carbon skeletons. Deposition of lignin in plants proceeds via the following steps: 1. The biosynthesis of monolignols 2. Transport of monolignols from the site of synthesis to the site of polymerization 3. Dehydrogenation & Polymerization of monolignols.

Biosynthesis of monolignols
The biosynthesis of monolignols proceeds through the phenylpropanoid pathway starting with deamination of phenylalanine to produce cinnamic acid and involves successive hydroxylation reactions of the aromatic ring, followed by phenolic o-methylation and conversion of the side chain carboxyl to an alcohol group (Fig. 2). Immense amount of work has been done in characterizing the monolignol biosynthesis pathway in past two decades. It is a complex pathway comprising of enzymes with functions like methyltransferase, hydroxylase, reductase and dehydrogenase. Some of the important enzymes involved in monolignol biosynthesis have been discussed below.
The enzyme phenylalanine ammonia-lyase (PAL; EC: 4.3.1.5) that catalyzes the conversion of phenylalanine to transcinnamic acid, is the initial step towards monolignol biosynthesis and other phenolic secondary plant metabolites. Genes encoding PAL have been studied in Populus species (Kao et al. 2002;Osakabe et al., 1995), loblolly pine and other plant species (Bate et al., 1994;Hatton et al., 1995;Jones, 1984;Kumar & Ellis, 2001;Leyva et al., 1992;Ohl et al., 1990). PAL exists as a multiple member gene family and the individual members can be involved in different metabolic pathways as suggested by their expression patterns in association with certain secondary compounds accumulated in specific tissue or www.intechopen.com  developmental stage. The biochemical activity of all known PALs is verified as specific deamination of phenylalanine, but genetic and physiological function may vary among different PAL members. The expression of PAL genetic function is controlled by various genetic circuits and signaling pathways.

Cinnamate 4-Hydroxylase (C4H)
C4H (EC: 1.14.13.11) constitutes the CYP73 family of the large group of Cyt P450 monooxygenases. It catalyzes the 4-hydroxylation of trans-cinnamate, the central step in the generation of phenylalanine-derived substrates for the many branches of phenylpropanoid metabolism. The first and the last enzymes of this short sequence of closely related reactions, termed the general phenylpropanoid metabolism, are PAL and 4CL, respectively. A second metabolic link couples C4H to the membrane-localized Cyt P450 Reductase (CPR). The expression patterns of all three C4H-linked enzymes, PAL, 4CL, and CPR, and of the corresponding mRNAs have been analyzed in cell-suspension cultures and various intact tissues of parsley (Logemann et al., 1995) and Arabidopsis (Mizutani and Ohta, 1997). A reduction in PAL levels leads to an increase in the S/G ratio, whereas reduced C4H activity leads to a decrease in the S/G ratio. These observations support the existence of some sort of metabolic channeling between the enzymes of the central phenylpropanoid pathway and those of monolignol biosynthesis and also provide a basis for the development of new strategies for modified or reduced lignin content.
Similar to PAL, C4H is thought to be involved in a number of secondary metabolism pathways in addition to monolignol biosynthesis as p-coumarate is an intermediate for biosynthesis of many secondary compounds (Croteau et al., 2000). Multiple C4H gene members are identified in many plant species, however, only one C4H is known in the Arabidopsis genome (Raes et al., 2003). The expression study of two C4H members in quaking aspen indicated that one is strongly expressed in developing xylem tissues and the other is more active in leaf and young shoot tissues (Shanfa et al., 2006). In other species, C4H gene is expressed in a variety of tissues and the expression is induced by wounding, light, pathogen attacks and other biotic & abiotic stimuli (Bell-Lelong et al., 1997;Raes et al., 2003). The mechanisms that regulate the genetic function of C4H gene and its family members are yet unknown.

Coumarate Coenzyme-A ligase (4CL)
Genetic and biochemical functions of 4-Coumarate Coenzyme A ligase (4CL; EC: 6.2.1.12) genes have been clearly demonstrated in association with monolignol biosynthesis (Lewis and Yamamoto, 1990;Lee et al., 1997;Hu et al., 1998Hu et al., , 1999Harding et al., 2002). 4CL genes usually exist as a multi-gene family. Four 4CL genes were detected in the Arabidopsis genome and the expression of each member was regulated differentially in tissues and development stages (Raes et al., 2003). In aspen trees, two 4CL genes were cloned and their expression were clearly distinct, with one in epidermal & leaf tissue and the other specifically in developing xylem tissue Hu et al., 1998). Furthermore, the enzymatic activities of 4CL members from aspen, loblolly pine, tobacco, soybean, Arabidopsis, and many other species were found to have distinct substrate specificities (Hu et al., 1998;Lindermayr et al., 2003;Voo et al., 1995). Whether the substrate specificity of the 4CL members relates to different metabolic pathways is unknown. As the 4CL catalytic kinetics vary among species, it is also likely that the mainstream pathway mediated by 4CL may not be exactly the same in all plant species or tissues. Nevertheless, monolignol biosynthesis is tightly controlled by 4CL.

Cinnamoyl CoA reductase (CCR)
The reduction of cinnamoyl CoA esters to cinnamaldehydes is the first metabolic step committed to monolignol formation (Lacombe & Hawkins, 1997). This first reductive step in www.intechopen.com lignin biosynthetic pathway is performed by Cinnamoyl CoA reductase (EC: 1.2.1.44) and it controls the over-all carbon flux towards lignin. CCR activity is found to be generally low in plants so it is hypothesized that it may play a crucial role as a rate limiting step in regulation of lignin biosynthesis (Ma & Tian, 2005). CCR is apparently encoded by a single gene per haploid genome in Eucalyptus (Lacombe & Hawkins, 1997), poplar (Leple et al., 1998), ryegrass (Larsen, 2004;McInnes et al., 2002), Triticum (Ma, 2007) and tobacco ) and by two genes in maize (Pichon, Courbou et al., 1998), and Arabidopsis (Lauvergeat & Lacomme, 2001). The CCR genes in various species appear as a multiple member family. In the Populus genome, there exist 8 CCR-homolog or CCR-like gene sequences (Li & Cheng, 2005). Triticum (Ma & Tian, 2005;Ma, 2007), maize (Pichon & Courbou, 1998), switchgrass (Escamilla-Trevino & Shen, 2010), Medicago (Zhou & Jackson, 2010) and Arabidopsis (Lauvergeat & Lacomme, 2001) have been shown to possess two or more than two isoforms (CCR1 and CCR2) which are involved in mutually exclusive or redundant functions like, constitutive lignifications and defense. Several other CCR gene sequences have been deposited in the GenBank database, but their functions have still not been demonstrated. It is proposed that all CCR enzymes have a similar catalyzing mechanism for converting the CoA esters to aldehydes in monolignol biosynthesis.

Coniferaldehyde 5-hydroxylase (CAld5H)
CAld5H enzyme like C4H belongs to cytochrome P450 monoxygenase family. The hydroxylation reaction in the biosynthesis of S-unit (syringyl) was first considered to occur at the ferulate level (Grand, 1984), and hence, the enzyme was called Ferulate 5-hydroxylase (F5H). However, studies later have revealed that F5H can also function at later steps in the pathway, mainly at the coniferyl aldehyde or coniferyl alcohol level (Humphreys et al., 1999;Li et al., 2000). This enzyme was therefore alternatively renamed coniferaldehyde-5hydroxylase (CAld5H) (Osakabe et al., 1999). F5H/CAld5H is unusual in that it is a multifunctional plant P450 with three physiologically relevant substrates. The Km for the substrates such as coniferaldehyde, coniferyl alcohol and for the ferulic acid are 1 μM, 3 μM and 1000 μM respectively. This study demonstrates that the coniferaldehyde is the most preferred substrate for the enzymes (Humphrey et al., 1999). Considerable evidence is now available that shows that in angiosperm trees, the syringyl monolignol pathway branches out from guaiacyl pathway through coniferaldehyde and is regulated in sequence by three genes encoding coniferaldehyde 5-hydroxylase (CAld5H), 5-hydroxyconiferaldehyde O-methyltransferase (COMT) and sinapyl alcohol dehydrogenase (SAD).

Cinnamyl/Sinapyl alcohol dehydrogenases (CAD/SAD)
In gymnosperm wood, coniferyl alcohol is the major monolignol units while both coniferyl and sinapyl alcohols are present in angiosperm wood. CAD (E.C: 1.1.1.195), depicts a class of NADPH dependent oxidoreductase, suggested to catalyze multiple cinnamyl alcohol formations from their corresponding cinnamaldehydes (Lewis & Yamamoto, 1990;Whetten et al., 1998). This reduction of aldehydes to corresponding alcohols has been considered to be an indicator of lignin biosynthesis because of its specific role at the end of the monolignol biosynthesis pathway (Baucher et al., 1996). When the Populus tree was studied for monolignol biosynthesis in wood forming tissue, in addition to CAD, it was found in aspen that another gene, its sequence similar to but distinct from CAD, is also associated with lignin biosynthesis (Li et al., 2001). The biochemical characterization of the recombinant protein encoded by this gene indicated that the www.intechopen.com enzymatic activity has specific affinity toward sinapaldehyde, therefore it was named SAD. Compared with SAD enzyme kinetics, CAD showed a catalytic specificity towards coniferaldehyde instead. The catalytic specificities of the two enzymes have been further verified in protein structure analysis (Bomati & Noel, 2005). Furthermore, it was demonstrated that the expression of CAD is associated with G-lignin accumulation while SAD was associated with S-lignin formation during xylem differentiation (Li et al., 2001). The evidence from molecular, biochemical and cellular characterizations strongly suggest that CAD is involved in G-monolignol biosynthesis and SAD in S-monolignol biosynthesis in aspen wood formation.

Transport of monolignols
After the synthesis, the lignin precursors or monolignols are transported to the cell wall where they are oxidized and polymerized. The monolignols formed are insoluble and toxic to the plant cell and hence are converted to their respective glucosides by the action of UDPglycosyltransferases (UDP-GT). This conversion renders the monolignols, soluble and less toxic to the plant cells, which can be stored in plant vacuoles, and transported to the cell wall as the need arises. It has been hypothesized that these monolignol glucosides are storage or transport forms of the monolignols (Steeves et al., 2001).

Dehydrogenation and polymerization
After transport of the monolignols to the cell wall, lignin is formed through dehydrogenative polymerization of the monolignols (Christensen et al., 2000). The dehydrogenation of monolignol radicals has been attributed to different class of enzymes, such as peroxidases (POX), laccases (LAC), polyphenol oxidases, and coniferyl alcohol oxidase. Lignin is a hydrophobic and optically inactive polymer, which is highly complex and heterogeneous in nature. Lignin polymerization is a radical coupling reaction, where the monolignols are first activated into phenoxy radicals in an enzyme catalyzed dehydrogenation reaction. These radicals couple to form dimers, oligomers and eventually the lignin polymer (Freudenberg, 1968). Peroxidases are heme-containing oxidoreductases that use H 2 O 2 as the ultimate electron acceptor. The natural electron donor molecules in a peroxidase catalyzed reaction vary and include, monolignols, hydroxycinnamic acids (Zimmerlin et al., 1994), tyrosine residues in extensions (Brownleader et al., 1995) and auxin (Hinman & Lang, 1965). Several reports on peroxidase activity or gene expression in ligninforming tissues have appeared, but only a few isoenzymes or genes have been specifically associated with lignification (Christensen et al., 2001;Marjamaa et al., 2006;Quiroga et al., 2000;Sato et al., 1993).

Regulation of monolignol biosynthesis
Developmental program of lignification associated with certain types of plant cells, such as xylem and fibers, require coordinated regulation of different lignin biosynthesis genes, as well as with genes controlling other aspects of plant growth and development. Different transcription factors such as R2R3-MYB, KNOX, MADS, LIM have been found to be regulating lignin biosynthesis in many plants, although the understanding of the molecular mechanism of pathway regulation is still limiting (Campbell & Rogers, 2004;Zhou et al., 2006Zhou et al., , 2008. Lignification can be modified in a more efficient and precise way by www.intechopen.com understanding the regulation of these pathways via altering the expression of relevant transcription factors.

Lignin as barrier for paper production
From an agro-economical point of view, lignin is considered to have a negative impact because it affects the paper manufacture and limits digestibility of forage crops. High quantity and low Syringyl (S) to Guaiacyl (G) lignin ratio plays a detrimental role in economy and ecology of paper production. Every unit increase in S/G ratio decreases the cost of paper production by two and half times. Both lignin content and composition are known to have impact on pulp & paper because residual lignin in the wood fibers causes a discoloration and a low brightness level of the pulp (Chaing et al., 1988). Consequently, for the production of high quality paper, lignin has to be removed from cellulose during the pulping process without damaging the polysaccharide component of wood. During chemical (Kraft) pulping, a large amount of Sodium hydroxide (NaOH) and Sodium sulfide (Na 2 S) are required to extract lignin from the pulp (Axegard et al., 1992). Subsequently, the residual lignin is further removed with bleaching agents, such as Chlorine dioxide (ClO 2 ), Hydrogen peroxide (H 2 O 2 ), Sodium hypochlorite(NaOCl), Oxygen(O 2 ), or Ozone (O 3 ) (Axegard et al.,1992;Biermann, 1993;Christensen et al., 2000).
These lignin extraction & bleaching procedures can partly degrade cellulose and consequently, reduce pulp quality and paper strength. Lignin extraction consumes large quantities of chemicals and energy leading to poor environmental image for these industries (Biermann, 1996;Higuchi, 1985;Odendahl, 1994). For this reason, engineering of plants with cell wall structures that are more susceptible to the krafting, and thus, more amenable to hydrolysis, or are sufficiently altered so as to shunt the above processes is an attractive approach to improve pulping efficiency and potentially alleviate some of the negative environmental impacts of the paper making industry. Apart from the great deal of work in the lignin field for improving the pulping process, many examples can be found based on research aimed at altering the lignin content for improving bio-fuel production (Chen, 2006;Chen & Dixion, 2007;Davision, 2006;Franke et al., 2002), as well as for improving forage crop digestibility (Table 1).

Genetic engineering of phenylpropanoid biosynthetic pathway
Despite the extensive literature on genetic modifications of lignin biosynthesis in a variety of plants, only a few studies have reported the impact of modified lignin content and composition on pulping and bleaching processes. Nevertheless, significant progress has been made in this field, as summarized in Table 1.
Emerging genetic engineering strategies in planta including manipulation of lignin biosynthesis at regulatory level, controlling monolignol polymerization enzymes, and modifications of lignin polymer structure, together with exploration of lignin degrading enzymes from other organisms provide us the necessary tools for producing designer plant species with reduced/altered lignin traits, so as to meet the needs of paper, livestock industries, etc. However, traditional genetic engineering strategies such as upregulation and downregulation of monolignol biosynthetic genes are still applied and have been successful in facilitating lignin decomposition by altering both lignin content and composition. One such example based on genetic modifications in Leucaena is described here, providing insights into the reactions, and regulation of genes involved in lignin biosynthesis, and its impact on determining lignin quality for paper industries.

Leucaena leucocephala as a source of pulp
A great deal of knowledge on the molecular biology and regulation of phenylpropanoid biosynthesis has been derived from investigations in plants such as Arabidopsis, Alfalfa etc.

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While these models will continue to serve as platforms for studying lignifications, a number of other plant species, e.g., Leucaena leucocephala have recently been selected for such studies. The paper industry in India mainly uses bamboos, Eucalyptus sp., Casuarina sp. and Leuacaena sp. as a source for paper pulp. Selection of the species depends upon availability, price and acceptability by any given industrial unit. However Leucaena sp. is extensively used in India and nearly a quarter of raw materials for paper and pulp industry comes from this plant (Srivastava et al., 2011). Leucaena is also valued as an excellent source of nutritious forage.
(a) (b) It has been estimated that dried leaves of Leucaena contain nearly 28-35% of protein content of high nutritional quality. Besides this, Leuceana is also an excellent source of firewood, industrial fuel, organic fertilizer, timber and gum (Cottom et al., 1977). A native of Central America, Leucaena has been naturalized pan-tropically, with members of its genera being vigorous, drought tolerant, highly palatable, high yielding & rich in protein and grow in wide range of soils (Hughes, 1998;Jones, 1979). However, these attributes are limited by the occurrence of anti-nutritive factors in the fodder, such as tannins and mimosine (Hammond et al., 1989;Hegarty et al., 1964;Jones, 1979).

Phenylpropanoid biosynthesis genes from Leucaena leucocephala
Different genes involved in phenylpropanoid biosynthesis were studied in detail from Leucaena. Table 2 summarizes the details of such genes isolated from Leucaena along with their GenBank accession numbers.  regeneration of leguminous trees has been reported in few research works. The complete in vitro plantlet regeneration of Leucaena (Cultivar, K-67) from lateral bud explants has been optimized with the maximum shoot multiplication rate of 22 shoots per explants. These regenerated plantlets were transplanted ex vitro with 80% survival rates (Goyal et al., 1985). In addition to the regeneration from lateral bud explants, an alternative (both direct and callus mediated/indirect) plantlet regeneration system has been successfully demonstrated with 100% regeneration frequency using cotyledon explants from 3-4 days old plants (Cultivar,. It is interesting to note that the plantlets regenerated from cotyledonary explants rooted without any requirements of growth regulators on basal media (Saafi et al., 2002). Addition of Thidiazuron (TDZ) to the shoot induction medium has substantially improved the number of in vitro shoots per explants as compared to the basal shoot induction medium with N 6 -Benzyladenine (BA). Liquid pulse treatment of the induced shoots with TDZ resulted in the improvement in the subsequent rooting. The plantlets regenerated in this manner showed more than 90% survival rate ex vitro when grown in coco-peat mixture (Shaik et al., 2009). In order to improve overall in vitro plantlet regeneration efficiency, attempts to propagate elite (cultivar K-8, K-636) and wild type varieties of Leucaena were made by supplementing the basal shoot induction media with puterscin. It has been observed that putriscine (9.3 µM) significantly enhanced the number of regenerated shoots from hypocotyls explants when compared to the induction medium containg only BA (22.2 µM). The incidence of yellowing and leaf abscission was successfully abridged by addition of glutamine (685 µM) or adenine (540 µM) which indirectly added ex vitro survival of the plants. All the regenerated plantlets from hypocotyls explants exhibited 100% in vitro rooting and were subsequently transplanted ex vitro (Sirisha et al., 2008). In vitro regeneration system for some other cultivars of Leucaena (K-8, K-29, K-68 and K-850) from mature trees derived nodal explants as well as seedlings derived cotyledonary node explants have also been reported, where cultivar K-29 gave the best response in vitro. Indirect (through callus phase) somatic embryogenesis of cv. K-29 using 40.28 μM NAA and 12.24 μM were also established. These somatic embryos were further matured in full strength medium (Rastogi et al., 2008).

Regeneration system of Leucaena leucocephala
Considering all above discussed reports, it can be suggested that our reports (Shaik et al., 2009) of improved method also have produced a consistent regeneration system for Leucaena which will be beneficial for the mass propagation and genetic transformation of Leucaena species. Lignin content in wood pulp adversely influences the quality of paper produced. In Leucaena which is an important paper pulp wood crop, it becomes important to identify and multiply elite clones having naturally low lignin content. In addition to this approach there is a need to develop transgenic plants with altered or reduced lignin content for its efficient and eco-friendly removal from pulp. The above mentioned multiple regeneration pathways are an excellent tool to introducing foreign genes. Out of all these methods shoot regeneration from cotyledonary node explants are more responsive to multiple shoot induction (Hussain et al., 2007). Genetic transformation procedures particularly particle bombardment which is considered as most effective means of gene delivery can be applied to the transformation of these shoot meristems. Cotyledonary explant derived multiple shoots form most suitable tissue for genetic transformation due to their higher regeneration frequency. Therefore, our recent study dealt with multiple shoot induction from the cotyledonary nodes of Leucaena in response to cytokinins, thidiazuron (TDZ) and N6benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots (Fig. 5). The addition of cytokinins to the medium was found essential for multiple shoot induction. Leucaena cotyledonary nodes carried a high potential for rapid multiple shoot regeneration on medium containing lower concentrations of TDZ (0.05 or 0.23 µM) (Shaik et al., 2009). As multiple shoots originated from the mass of closely placed shoot initials of axillary meristems (Fig. 5), this system could be efficiently used for particle bombardment mediated transformation. This efficient and high frequency in vitro regeneration system is highly reproducible and can be used for mass propagation and genetic transformation of Leucaena.

Genetic transformation of Leucaena
The genetic transformation protocols based on Agrobacterium-mediated and/or direct gene transfers by biolistic bombardment have been successfully applied to numerous woody angiosperm species (Merkle & Nairn, 2005), including Populus and Betula. The introduction of transgenes have included both sense and antisense strategies (referring to the orientation of the introduced gene into the plant genome) (Strauss et al., 1995;Baucher et al., 1998) and RNAi technology (Merkle & Nairn, 2005). In the antisense strategy, duplex formation between the antisense transgene and the endogenous gene transcripts is proposed to induce the degradation of duplexes and, correspondingly, lead to suppressed gene expression (Strauss et al., 1995). Regeneration system for Leucaena has already been established in our previous works (Shaik et al., 2009;Sirisha et al., 2008). To exploit this to produce transgenic Leucaena plants for reduced/altered lignin content, various phenylpropanoid pathway genes (C4H, 4CL, CCoAoMT, CCR, CAld5H, CAD and POX) were cloned and used for transformation experiments.  (Shaik et al., 2009) One day old embryo axes without cotyledons were used as explants for transformation. It was carried out by three methods: 1) Particle bombardment; 2) Particle bombardment followed by co-cultivation and 3) Agro-infusion method. The transformation efficiencies with various gene constructs are summarized in Table 3. It can be observed that maximum efficiency (100%) was noted with CAD using particle bombardment followed by cocultivation as a means of transformation. However, CCR with the same procedure gave only 10% efficiency. In general it was concluded that a combination of particle bombardment method followed by co-cultivation was most effective in transforming the shoot meristems of Leucaena.

Peroxidase (LlPOX): A case study
Numerous reports on peroxidase activity in lignin forming tissues have been reported, but only a few isoenzymes have been specifically associated with lignification (Sato et al.,1993;Quiroga et al., 2000;Christensen et al., 2001;Marjamaa et al., 2006). It is likely that the control of the whole lignification process requires a mechanism for the co-ordinated expression and/or activation of the monolignol biosynthetic genes/enzymes and the radical forming peroxidases. Data from transgenic plants down regulated for peroxidase activity has confirmed the role of some POX isoforms in lignin polymerization in tobacco and Populus sieboldii (Miq.)X Populus grandidentata (Michx.) (Talas-Ogras et al., 2001;Blee et al., 2003;Li et al., 2003b). Both quantitative (up to 50% reduction) and qualitative changes were reported, but no obvious growth phenotypes, other than larger xylem elements were found.
Recombinant pCAMBIA1301 binary vector harboring partial sequence of LlPOX in antisense orientation was used to transform the embryos isolated from aseptically germinated seeds of Leucaena (Fig. 6). Two fragments (one from conserved region and another from nonconserved region) of LlPOX cloned in anti-sense orientation were used for transformation. The Leucaena embryo axes were bombarded with microcarriers coated with recombinant pCAMBIA vectors using PDS-1000/He Biolistic Particle Delivery System. After growing the embryos on regeneration media without selection for one week, these embryos were subjected to three rounds of selections. The plants, which survived were shifted to ½-MS with Cytokinin, 2-ip (2-isopentenyl adenine) 0.5 mg/ L to enhance elongation of transformed shoots. In all the above cases, the bombarded explants were subjected to transient GUS assay 48 hrs after second bombardment and the putative transgenic plants, which survived three rounds of selection, were analyzed for the gene integration into the plant genome.
As an alternative strategy, Leucaena embryo axes were also transformed by a combination of particle bombardment followed by co-cultivation with Agrobacterium (GV2260) harboring www.intechopen.com respective recombinant pCAMBIA vectors. After particle bombardment, the embryo axes were then transferred onto the respective regeneration medium, co-cultivated in the dark at 25 ± 2 °C for 3 days. After co-cultivation, the embryo axes were washed thoroughly with Cefotaxime 250 mg/ L in sterile distilled water and transferred onto the regeneration medium. Higher levels of transient GUS expression confirmed the transformation efficiency. The present study was performed using, two different antisense constructs of Leucaena peroxidase gene. We observed severe stunted or retarded growth in plants when transformed using constructs having conserved domain. These plants were found to grow barely up to 0.5 cm, soon followed by the death of the apical meristem and rise of a fresh axillary bud from its axis, which again dies and this process was found to be repeating. As a www.intechopen.com result, the plant attained a height of 2.5 cm on an average and even failed to produce roots when transferred to rooting medium. When non-conserved AntiPOX construct was used in Leucaena transformation, normal regeneration was noticed but the plants were thin and slow growing compared to the untransformed control plants. Comparative growth pattern of Leucaena are shown in Fig. 7.
LlPOX was immuno-cytolocalized in the transformants generated following the above mentioned protocols. Control and transformed plants of same age group were selected. The control plants showed better growth and bio-metric parameters (height, growth and rooting) over the transformants. POX was immuno-cytolocalized in stem tissues of control untransformed plants (Fig. 8 A, B, C) and putative transformants (Fig. 8 D, E, F), with a view to find whether there exists reduction in peroxidase expression in lignifying tissues (i.e vascular bundle and xylem fibres). It was observed that the transformants showed reduced levels of POX near the sites of lignifications. It was also noted that Leucaena transformed by AntiLlPOX from conserved region resulted in discontinuity in vascular bundle assemblies.

Conclusions
Thanks to years of painstaking research in to the chemistry of lignin, it is now seen as a potential target for genetic engineering of plants, mostly aggravated by its industrial and agricultural applications. However, much of our understanding of lignin biochemistry comes from studies of model plants like Arabidopsis, Tobacco, Poplar, etc. Furthermore, this technology needs to be transferred to other plant species. Leucaena, a multiple utility leguminous tree, is targeted for ongoing research to alter its lignin content due to its importance in paper and pulp industry in India. Keeping this in mind, attempts were made to improve pulp yielding properties by genetically engineering lignin metabolism so as to gratify the demand of such industries. The results presented here highlight the challenges and limitations of lignin down-regulation approaches: it is essential but difficult to find a level of lignin reduction that is sufficient to be advantageous but not so severe as to affect normal growth and development of plants.
These findings may contribute in the development of Leucaena with altered lignin composition/content having higher lignin extractability, making the paper & pulp industry more economic and eco-friendly. The multi-purpose benefits of lignin down regulation in this plant can also be extrapolated to improved saccharification efficiency for biofuel production and forage digestibility, apart from enhanced pulping efficiency. Although genetic engineering promises to increase lignin extraction and degradability during the pulping processes, the potential problems associated with these techniques, like increased pathogen susceptibility, phenotypic abnormalities, undesirable metabolic activities, etc. must be addressed before its large scale application. In order to overcome such barriers, significant progress must be made in understanding lignin metabolism, and its effects on different aspects of plant biology.
Nevertheless, the current genetic engineering technology provides the necessary tools for a comprehensive investigation for understanding lignin chemistry, which were hardly possible using classical breeding methods.