**3.2 Polymer composition of rice biomass**

Cellulose is the utmost abundant organic compound available on earth. It is a linear polymer of repeating units of cellobiose molecule. Cellobiose, a β (1–4)-linked residue, is produced when two glucose molecules (one in 180 deg. rotation) are in proximity to yield a β (1–4)-linkage. These cellulose fibres impart a greater rigidity and strength to the cell wall and hence, enabling plants to exhibit a wide spectrum resistance to various biotic and abiotic factors [12]. The non-cellulosic polysaccharides further enhance the rigidity and strength of plant cell walls by cross-linking with cellulose and lignin. Various reports have suggested this cellular constituent as a mixture of various monosaccharides such as xylose, arabinose, glucose, galactose and rhamnose as well as certain acids [44, 45]. This complex nature of non-cellulosic polysaccharides as well as their involvement in cross linking with cellulose, possesses a major setback in the efficient enzymatic degradation of cellulose to produce biofuel.

Lignin, the second most abundant biopolymer after cellulose, is polymerised with three main types of monolignols namely, Syringyl alcohol (S), Coniferyl alcohol (H) and p-Coumaryl alcohol (H) [46, 47]. As a complex phenolic compound, it improves cell wall rigidity and strength, imparts resistance to a wide array of microbes [48], fosters transporting of minerals through vascular bundles [49], involves in resistance against lodging as well as abiotic anomalies [50–52].

Cellulose and hemicellulose in rice straw can be subjected to fermentation for production of biofuels. However, their efficient conversion into fermentable sugars is hindered by presence of higher amount of lignin (5–24%), ash (10–17%), silica (75% of ash) and potassium [53].

In rice, silica comprises 74.67% of the stem ash content. Both high ash and high silica (SiO2) silica content of ash negatively affect biochemical conversion of lignocellulosic feedstock [11, 53]. High silica content reduces the availability of cellulose to enzymatic digestion and thus, reducing saccharification efficiency. Besides this, high silica accumulation in the cell walls disrupts the cellulosic microfibrils and such aberration hinders overall sugar release and ultimately, ethanol yields in subsequent stages. Therefore, considerable efforts are required to engineer silica content along with lignin and non-carbohydrate polysaccharides content to develop rice genotypes, amenable to greater enzymatic digestibility.

Although, different enzymatic and chemical pre-treatment methods are being employed for the disruption of this complex network but these procedures are energy intensive, costly and harmful to the environment. Hence, genetically modifying the cell wall architecture by employing conventional and modern breeding methods are beneficial for sustainable biofuel production [11, 54].

#### **3.3 Modifications in polymer composition for elevating cellulose utilisation**

As discussed earlier, lignin serves as a key element in cross linking of cellulose and hemicellulosic polysaccharides. This feature is beneficial to the rice plant as it helps it to counteract biotic and abiotic stress but it is a limiting feature for biofuel production. The cross linking creates a barrier for the cellulose degrading enzymes to freely access cellulose for conversion. So, efforts are being made towards reducing the degree of lignification and cross linking through various approaches so as to enhance the efficiency of cellulose degrading enzymes.

#### *3.3.1 Modification of lignin*

Some noticeable work has been done to alter the plant cell wall architecture with the help of biotechnology in model dicot plants such as Arabidopsis and tobacco (**Table 1**). The purpose behind these experiments has been the downregulation of key genes involved in monolignol biosynthesis, as well as the essential enzymes involving in polymerisation thereof. Nearly 40% reduction in total lignin content was achieved by downregulating laccases and peroxidases in the Arabidopsis [61] and tobacco mutants [62], respectively. So, these available reports documenting successful reduction of lignin composition in model dicot plants can be judiciously used by the researchers to favourably alter the cell wall architecture of the less exploited prospective biofuel crops such as rice. The *japonica* rice 'Nipponbare' harbouring an Arabidopsis TF (SHN), was found to be deficient in total lignin content. Expression of essential genes such as *CAD* (cinnamyl alcohol dehydrogenase) and 4-CL (4- coumarate- CoA ligase) were reported to be repressed, which might have contributed in producing lower lignin content [9].

Alternatively, there is another way of altering the plant cell wall architecture, by curbing the expression of essential genes involved in lignin monomers synthesis (**Figure 3**). In rice, flexible culm (*fc1*) mutant with repressed *CAD* gene, a cinnamyl alcohol dehydrogenase gene, was reported to synthesise reduced level of H and G lignin monomers [7]. Zhang and co-workers, [60] were able to produce some transgenics with improved saccharification efficiency as compared to wildtype by targeting same *OsCAD2* gene in rice. Apart from these genes, few other genes including caffeoyl-CoA- methyl transferase (*CCoAOMT*) and caffeic acid o-methyl transferase (*COMT*) were genetically engineered in different species such as alfalfa, canola, maize, poplar, tobacco and sugarcane, to alter the lignin monomers composition [63–66, 70–74]. Several reports enumerating the modifications of some key TFs such as *OsMYB103L* are also available for improved plant architecture in rice [58, 59].


#### *Breeding Rice for Sustainable Bioenergy Production DOI: http://dx.doi.org/10.5772/intechopen.98572*

#### **Table 1.**

*Candidate genes for preferable altering the cell wall polymers (cellulose, hemicellulose and lignin) in plant system.*

## *3.3.2 Modification of hemicellulose*

A general trade-off has been discovered between saccharification efficiency and ferulic acid [75, 76]. Bartley and co-workers, [68] reported the possible role of OsAt10, a BAHD acetyltransferase gene in achieving higher sugar release by favourably modifying glucuronoarabinoxylan (GAX) in rice. Young leaf tissues of the genetically engineered plants were found to be deficient in ferulic acid (FA). The possible role of other genes such as OsXAX1 and OsIRX10 were known to reciprocate similar results in rice [67, 68].

#### **3.4 Role of cellulose synthase genes**

Cellulose synthase enzymes are pivotal for cellulose synthesis. These proteins organise to form a hexameric 'rosette' structure approx. 25–30 nm diameter [77]. The plant cellulose synthase (*Ces A*) genes were first identified during random sequencing of cotton ESTs [78] and its role in cellulose synthesis was first reported in Arabidopsis Ces *A* mutants [10, 79]. The Ces *A* gene family was also identified in rice, maize, barely and poplar [57, 80–92].

Tanaka and co-workers [10] generated four different introgressed lines, showing brittle culm phenotypes by suitably introgressing Tos17, a retroposons in the genetic background of rice wildtype. They identified three cellulose synthase genes namely, *OsCesA4*, *OsCesA7* and *OsCesA9* on three different chromosomes. The mutant *Osfc16* with a mutation on *CesA9*- conserved sequence was found with altered cellulose crystallinity (crt1), which possibly enhanced the saccharification efficiency [93]. In a similar experiment, conserved site of another potential cellulose synthase CesA4 is mutated to alter cellulose crystallinity (crt1) for enhanced cellulose synthesis in *fc17* mutants [57]. Considerable efforts have been made to alter various structural properties of cell wall constituents including cellulose crystallinity (crt1) and degree of polymerisation (DP) which usually negatively affect the saccharification potential. In this regard, some noticeable work has been done to identify and characterise few genes of glycoside hydrolase family (*OsGH9B 1, 3 and 16*), promising candidate genes for favourably modifying structural properties of cell wall polymers as well as cellulose synthesis in rice [55, 56]. Beside cellulose synthase genes, other genes including KORRIGN [94–96], COBRA-like protein [7] and KOBITO [80] need to be explored properly to develop energy efficient elite cultigens in rice.

#### **3.5 Genomics and QTL identification for biomass traits**

Correlation between biomass traits and grain yield in rice is negative. Breeding varieties for high grain yield usually involves designing the varieties for medium plant height with short erect flag leaves which in turn affect the total biomass yield. This can be addressed to some extent by crossing rice cultivars, showing high polymorphism for grain yield potential as well as biomass traits and identify the candidate genes or QTLs involved. After the successful mapping of genes or, QTLs, the linked markers can be used for marker assisted selection (MAS) as well as can be used to screen the existing wild types or landraces for dual characteristics. As we have discussed earlier, cell wall polymers i.e., cellulose, hemicellulose and lignin composition can be altered for improved saccharification traits, hence, it is essential to search for the genetic link between cell-wall polymer composition and grain yield in order to breed dual purpose rice cultigens [97–103]. Gui- Fu and co-workers [97] identified few major QTLs associated with three plant traits namely, total biomass yield, straw yield and grain yield by developing suitable doubled haploid population. A QTL co-associated with both cell wall polymer composition and heading

*Breeding Rice for Sustainable Bioenergy Production DOI: http://dx.doi.org/10.5772/intechopen.98572*

date (HD17) has also been identified by crossing parents with considerable polymorphism for the dual characters [102]. Recently, Genome wide association survey (GWAS) involving high throughput molecular markers (SNPs) were employed to identify the genomic regions exhibiting significant association between markers and phenotypic trait and characterise the candidate genes involved [101]. Dissecting the genomic fragments involving lignin and cellulose biosynthesis is possible now with the application of GWAS technique [100].

#### **3.6 Plant breeding strategies for improving biomass traits**

Pre- existence of variability is of paramount importance in any crop improvement programme. Selection, being the core stone of plant breeding activities, is employed to harness the existing variability present in various germplasms including wild types and landraces, before creating additional variability by mutation.

A preferred high HI for good yield reduces the vegetative biomass of the rice as a whole including reduction in plant height. There is a trade-off for plant height vs. biomass yield. Hence, the role of long- culm rice cultivars in breeding high energy efficient varieties has been given due consideration [104–108]. However, an increase in culm length may increase the risk of plant lodging, which is a major factor influencing rice grain yield stability especially in direct-seeded rice. A thick culm with tolerable lignin content in cell wall will decrease the risk of plant lodging. So, there should be a balance between the cell wall constitution and morphological characters. Hence, judicious selection of genotypes for increased plant height with thick culm along with high grain yield can address the negative impact exerted by short culm height on overall biomass production. Few researches have enumerated the importance of selecting certain traits such as stem girth, plant height, leaf, sheath and stem weight for higher biomass yield in rice [40, 108, 109].

Another way for breeding dual purpose cultigens is to incorporate 'stay green' traits in cultivated type [110, 111]. Varieties possessing these traits are able to maintain higher photosynthetic activity at post-flowering stage, increasing yield thereof. At the same time, higher decomposability of these traits could serve the dual objectives as discussed above. Hence, there is a possible opportunity to exploit this stay green character in developing dual purpose rice genotypes as it has been exploited in other biofuel/bioenergy crops. Also, this character genetically enhances the photosynthetic efficiency, there may be no need to apply extra N inputs. Nevertheless, more research is required in this aspect. As of till date there are no reports of this strategy being exploited in breeding rice varieties for dual purpose.

Next it is important to identify the genetic loci (QTL) associated with these stay green trait and the markers flanking those regions [112–115]. Various biotechnological tools can then be employed for their successful integration into the plant genome or alternatively, marker aided selection (MAS) can be employed for varietal improvement. Also, heterosis breeding can be used to exploit the possible heterotic gene combinations in remodelling the plant architecture for higher biomass yield in rice, as it has been done in sorghum which possesses similar architectural traits [116–119].
