6 Genetic Diversity in : Conservation and Improvement for Productivity Ajay Thakur, Santan Barthwal and H.S. Ginwal

1. Introduction Bamboos are interesting in their growth, morphogenesis, , distribution, ecology, reproduction as well as diversity. Bamboos belong to the subfamily Bambusoideae of grass family, . Woody bamboos mostly belong to Bambuseae tribe which is further divided into nine subtribes and 67 genera (Ramanayake et al., 2007). An estimated 1,400 species are distributed across the globe from 51oN latitude in Island of Sakhalin (Japan) to 47oS latitude in South Argentina. The bamboos can grow in an altitudinal range which extends from just above the sea level up to 4,000 m (Behari, 2006). The major species richness is found in Asia-Pacific region followed by South America, whereas the least number of species are found in Africa (Bystriakova et al., 2003). Bamboos can thrive in hot, humid rainforests to cold resilient forests. They can tolerate as well as grow in extreme low temperature of about - 20oC and precipitation ranging from 800 mm to 1,300 mm annual rainfall (Goyal et al., 2012). Asia alone is estimated to have more than 6.3 Mkm2 of bamboo forests, with most densities indicated from southern China to northeastern India and through Sumatra to Borneo. The maximum species richness (144 spp km-2) was estimated in forests of south China, including Hainan Island (Bystriakova et al., 2003). It is the fastest growing perennial and its morphogenesis includes functioning of intercalary meristem which supports rapid growth of internodes and their elongation to form the erect stem axis supported by considerable amount of lignin. Such magnificent growth habit shows its potential to suffice the demand of wood biomass despite that bamboos are still considered as poor man's timber. Traditionally, most commercially used bamboos are 38 species belonging to nine genera that comprise only a small portion of bamboo resources and those were 132 Ajay Thakur, Santan Barthwal and H.S. Ginwal

explicitly narrowed down for genetic improvement studies (Williams and Rao, 1994; Rao et al., 1998). Genetic improvement of bamboos were discussed during late nineties and it was advocated to promote work on genetic analysis and conservation (Rao et al., 1998). Studies on genetic variation started in early twentieth century but halted for some time and again restarted during later part of that century. DNA based techniques made understanding of evolutionary trends in bamboos along with inter- and intra-specific relationships easy and now being used in population and conservation genetics. There is a need to understand genetic variation of woody bamboos on morphological, cytological and molecular basis vis-à-vis relationship between them. This chapter reviews the genetic diversity and improvement of bamboo species using classical as well as molecular genetics approach.

2. Bamboos: Genetic Variation and Evolution of Species

2.1.Phenotypic Variation Genetic diversity is the main building block for evolution and speciation. Bamboos are such a diverse group and provide many good examples to analyze and discuss the morphological and genetic concepts of species. A wide spectrum of variability is noticed within species of bamboos in natural distribution. Phenotypic variability exists with respect to flowering of bamboos, morphological traits like internodal length, culm wall thickness, sheath size and their cytology, variation in chromosome number, pollen grain size, fertility and germination. Variation also exists with respect to morphological and anatomical features. Kochhar et al. (1990) studies on populations of pallida, B. tulda and hamiltonii and at West Siang (Arunachal Pradesh) and North Lakhimpur showed little interspecific and more intraspecific variation for seven clump management and five clump morphological traits in the base population. These characterization of traits paved way for improvement through selection of plus bamboo clumps from polymorphic populations based on individual traits. Spontaneous mutants with morphological variations were detected in colour, shape and structure in B. bambos, B. vulgaris, Guadua angustifolia, Oxytenanthera abyssinica and Phyllostachys edulis (Venkatesh, 1984). Genetic variations are recorded in certain species of bamboo most likely due to putative outcrossing, segregation and recombination. Inter- and intra- species variability in end-use linked chemical composition of five commercially important bamboo species, namely, B. balcooa, B. nutans, B. pallida, B. tulda and D. hamiltonii was analysed. High range of lignin content in B. nutans (25.64-29.46%) and that of holocelulose content in B. tulda (70.7-75.0 %) (Thakur et al., 2014). Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 133

2.2.Genetic Variation DNA based molecular markers paved way for fast and reliable estimates of genetic diversity and distance. Considering complexities in taxonomic status of bamboo; mostly due to long flowering interval, initial studies have been focused on molecular phylogeny and taxonomy and a few were on intra specific diversity of bamboo. Molecular markers like random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter simple sequence repeat (ISSR), simple sequence repeat (SSR), expression sequence tag (EST), chloroplast (cp) genome, single nucleotide polymorphic (SNP) markers, etc. are being used for phylogeny, inter- and intra-specific studies. Universally marker based techniques like AFLP, ISSR and cp genome are effective for phylogenetic studies, whereas RAPD and SSRs are co- dominant markers and have high rate of mutation, hence effective in studies related to genetic diversity. AFLP marker technique has been widely used to assess genetic diversity within and among the population of different bamboo species (Ghosh et al., 2011). Four genera of tropical bamboos: Bambusa, Dendrocalamus, Gigantochloa and Thyrsostachys are being reported to be differentiated by AFLP markers and with unique AFLPs for 13 out of 15 species (Loh et al., 2000). Bambusa (six species) is separated into two clusters, Gigantochloa (six species) has formed a discrete cluster diverging Bambusa clusters, while Thyrsostachys is less similar to the Bambusa clusters. Among Dendrocalamus, two species were reported to be different than D. brandisii, clustering within one of the Bambusa clusters. Interspecific diversity studies between nine species of tropical bamboos: Arundinaria hindsii, Bambusa atra, B. bambos, B. ventricosa, B. vulgaris 'Striata', D. asper, D. giganteus, D. longispathus and Gigantochloa atroviolacea using RAPD reveals that contrary to distinguished morphological dissimilarity between B. ventricosa and B. vulgaris, their genetic distance was only 0.143 (Ramanayake et al., 2007). Genetic distance between B. atra from B. vulgaris, B. ventricosa and B. bambos are greater as compared to G. atroviolacea. A. hindsii is most genetically distant from all other species and not related to any of them. A high level of genetic diversity was recorded in molecular analysis of random collections of industrially important reed bamboos (Ochlandra travancorica) using RAPD and AFLP marker technique. Microsatellite or SSR markers are valuable tool for genetic diversity analysis. SSR developed for cereal crops has been cross amplified for other bamboo species and successfully used for genetic diversity studies (Barkley et al., 2005; Dong et al., 2011; Zhang et al., 2011). Polymorphic EST-SSR markers derived from major cereal crops were used to assess the genetic diversity of the USDA temperate bamboo collection consisting of 92 accessions classified into 11 separate genera and 44 species (Barkley et al., 2005). The resulting dendrograms have two distinct clusters of main 134 Ajay Thakur, Santan Barthwal and H.S. Ginwal

clades, which correspond to accessions into either clumping (sympodial) or running (monopodial) bamboos. Similarly, cross amplification of SSR markers of rice and sugarcane were successfully used for analysis of genetic diversity of 23 bamboo species (Sharma et al., 2008). In 20 accessions of D. hamiltonii, genetic diversity was 0.25. SSR markers were identified and characterized from B. bambos and tested for cross amplification in other 18 bamboo species (Nayak et al., 2003). Many novel microsatellite markers have also been developed in some bamboo species using method based on microsatellite enriched genomic library. Availability of draft genome sequence of P. heterocycla var. pubescens (Peng et al., 2013), opens another possibility to use its SSR markers in other bamboo species for assessment of genetic diversity and population genetics.

2.3.Molecular Phylogenomics Understanding of evolution of species is of prime importance for genetic studies and can be applied for improvement and conservation practices. Molecular phylogenomics based on whole cp genome can be used to resolve major relationships within and between subfamilies. Divergence of Bambusoideae from Poaceae is important to study because former is the only subfamily of Poaceae that contains woody members. Three subfamilies of Poaceae, i.e., Bambusoideae, Ehrhartoideae and Pooideae, formed the BEP clade, yet the internal relationships of this clade are controversial. Phylogeny construction of Poaceae from 24 complete chloroplast (cp) genomes including 21 grass species shows difficulty in resolving the diversification among three BEP clade, though it appears that there is a sister relationship between Bambusoideae and Pooideae. It suggests that these lineages may have diverged very rapidly and complete nucleotide sequences of six woody bamboo cp genomes are similar to those of other grasses and rather conservative in evolution (Zhang et al., 2011). The repeats in the cp genome could provide phylogenetic information and caution is needed while using indels based on few selected genes. In North American temperate bamboo species, A. gigantea species complex: A. appalachiana, A. gigantean and A. tecta studies using AFLPs and cpDNA sequences support the recognition of three species and also demonstrate that A. appalachiana and A. tecta are sister species, forming a clade that is significantly divergent from A. gigantea (Triplett et al., 2010). The first major divergence in Arundinaria occurred around 2.3 to 3.2 M yr ago and that A. appalachiana and A. tecta diverged from their common ancestor around 0.57 to 0.82 M yr back (Burke et al., 2014). Paleoclimatic events, including an early Pliocene warming, subsequent cooling, and North American glaciations appear to play an important role in divergence. Though phylogenomic and divergence analyses between A. gigantea and Crytpochloa strictiflora, suggested that former diverged from within Arundinarieae between 1.94 to 3.92 M yr and that later diverged Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 135

as the sister to tropical woody species between 24.83 and 40.22 M yr. These two New World bamboos show unique plastome features accumulated and maintained in biogeographic isolation from Old World taxa.

3. Bamboo Genetic Improvement Bamboos are established as one of the fastest biomass producers that are being used as an alternative to wood, which leads to increase in their consumption and subsequently exerts pressure on their genetic resource. The number of species, their geographic range of distribution, species and ecosystem diversity are important to determine in situ conservation programme and selection of appropriate species from good populations for ex situ conservation. International funding is being focused on a relatively small set of commercially important and widely distributed 38 priority species of bamboo (Williams and Rao, 1994; Rao et al., 1998). The focus on such a narrow range of species is paving way for genetic improvement for productivity enhancement which can be achieved by comprehensive intraspecific studies on flowering and breeding behavior of bamboos, hybridization, cytogenetics, selection of desirable population and individuals, etc. and their application for productivity enhancement (Williams, 1998). Lately, traits specific molecular and genetic information is also being used for genetic improvement.

3.1.Flowering and Breeding Behaviour Floral biology and breeding behaviour of bamboos have perplexed researchers for long time. Bamboos are primarily a cross pollinated species though adelphogamy (sib pollination) cannot be ruled out (Venkatesh, 1984; Nadgauda et al., 1993a, b; Banik, 1995). Flowering in bamboos is a strange reproduction behaviour among plants; they flowers gregariously like annuals but only after completing a lifecycle of several years. Gregarious flowering in bamboo species is termed for a phenomenon when a particular species flowers simultaneously at one location followed by death of flowering clumps and is thought to be under genetic control (John and Nadagauda, 1999; Bhattacharya et al., 2009). Two AP1/SQUA-like MADS-box genes; PpMADS1 and PpMADS2 from P. praecox characterized during floral transition showed involvement in floral transition. PpMADS2 might play more important roles than PpMADS1 in floral development of P. praecox (Lin et al., 2010). RNA sequencing analysis flowering tissues in bamboos suggests a potential connection between drought-responsive and flowering genes (Peng et al., 2013). In vitro conversion of vegetative meristem to floral meristem is achieved in D. hamiltonii and subsequent protein profiling of floral meristem suggests that metabolism related proteins affect this conversion through nutrient resources 136 Ajay Thakur, Santan Barthwal and H.S. Ginwal

though interactive effect of other proteins in profile cannot be ruled out (Kaur et al., 2014). Sudden death of B. bambos after flowering is explained in the perspective of programmed cell death (PCD) where two mRNA-transcripts for selected bamboo PCD-specific ESTs, namely V2Ba48 (aldehyde dehydrogenase 2) and V2Ba19 (glycogen phosphorylase) were detected. Differential expression kinetics of the aforementioned genes was confirmed during the progress of PCD after setting of seeds (Rai and Dey, 2012). Intraspecific variations of flowering are reported but to a limited extent. Bamboo species flower gregariously at one location but may not flower at adjacent location showing distinct flowering populations which subsequently die (Banik, 1995). Populations flowered during one year may not be likely to mate with populations flowered another year and this temporal reproductive isolation may likely to have direct genetic consequences such as increase of genetic distance between populations and subsequently sub-populations may develop. One possible long term impact may be small effective population size and low genetic diversity of population which, subsequently, increases inbreeding depression. Also these populations struggle to survive in adaptive stress and impact of random genetic drift is likely to be severe. One of the solutions may be sampling and collection of adequate representative germplasm from all possible location which flowered in the different years and grown randomly in either in situ or ex situ future gene bank for genetic diverse population.

3.2.Hybridization Depending on the evolutionary tendencies, hybridization is possible between closely or widely related species or even among different genera. Earliest report of intergeneric hybrid is between Sacharrum and Bambusa. Sacharrum officinarum (mother) and the two seedlings derived through pollination with B. bambos, one of the seedlings was much thicker than the mother, more vigorous and taller and had a somatic chromosome number of 116. The other was much thinner with a somatic chromosome number of 86. Thick seedling had probably come into being through the fusion of an unreduced egg of S. officinarum (2n=80) with a normal sperm of B. bambos (2n=72). The thin seedling had presumably been formed by the union of an S. officinarum egg having neither the haploid nor the diploid number, with the sperm of bamboo (Raghvan, 1952). In bamboos, there is likelihood of natural inter specific hybridization if two species are flowering simultaneously. Interspecific hybrids were obtained between Bambusa and Dendrocalamus spp., Phyllostachys and Dendrocalamus and others. Superior bamboo hybrids with good vitality, reproductive potential and adaptability are being cultivated in China covering more than 600 ha. Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 137

In vitro induction of flowering is considered as an opportunity to increase hybridization which can be achieved by a shift in auxin-cytokinin equilibrium or by a cytokinin only. In vitro and in vivo flowering of B. bambos has shown in vitro-induced florets fairly comparable to normal florets. Though reduced pollen fertility and some impairment in pollen wall development are hindrance to achieve good in vitro flowering. Biochemical studies suggest minimal peroxidase activity before rhizogenesis and induction of in vitro flowering during somatic embryogenesis (John and Nadagauda, 1999).

3.3.Ploidy Level and Genetic Variation Apart from hybridization, polyploidy and somatic mutation are an important tools to achieve genetic improvement quickly. Just in one generation, the polyploids become separate and distinct from the diploid species. In polyploids, sexual reproduction system is modified by inclusion of strong asexual reproduction system; the common examples are bamboos and grasses. Most of the woody bamboos so far studied are polyploids, and diploids are rarely found in any of them. P. heterocycla var. pubescens which is a tetra-ploid and is considered to have undergone whole-genome duplication 7-12 M yr ago (Peng et al., 2013). Chromosome number in bamboo varies sometimes according to genera as well as climatic conditions of its distribution range. Four chromosome, numbers: 48, 54, 64 and 72 were mostly reported for bamboos. Among temperate bamboo species of genera Arundinaria, Chinomobambusa, Himalayacalamus, Phyllostachis, Pleioblastus, Sinobambusa, chromosome number is 48 (2n). Most species of tropical bamboo genera like Bambusa, Cephalostychum, Dendrocalamus, Gigantochloa, Melocanna, are reported to have chromosome number 72 (2n). Chromosome number of bamboo species appear to vary with variation in native climatic zones, from temperate, sub-tropical to tropical zones. The number ranges between 72 and 48, which decreases gradually from the tropical zone to the temperate zone (72- 64 - 48). In earlier studies, basic chromosome number of Bambuseae was 6, as most chromosome numbers reported were 48, 54 and 72 which were multiples of 6 (Uchikawa, 1935). Later studies suggested that basic chromosome number of most woody bamboos is 12 (x = 12) which is also a multiple of 6 (Clark et al., 1995). It is now hypothesized that two different polyploidy groups are present in woody bamboos: tropical woody bamboos mostly hexaploid (2n = 6x = 72) and temperate woody bamboos mostly tetraploid (2n = 4x = 48). This seems to be consolidated by the reports of large genomic DNA content of tropical woody bamboos as compare to temperate woody bamboos (Gielis et al., 1997). Molecular markers studies in bamboos reported 9.62 alleles per locus in Asian bamboo species and 8.44 alleles per locus in American bamboo species, indicating a high level of polyploidy (Barkley et 138 Ajay Thakur, Santan Barthwal and H.S. Ginwal al., 2005; Sharma et al., 2008). Though there are some exceptions in chromosome numbers for some species of Bambusa and Dendrocalamus (Ruiyang, 2003). Also, New World bamboos (bamboos of American continents) show exceptions, such as Gaudua has chromosome number 46. Chromosome number of some bamboos is given in Table 3.3.1.

Table 3.3.1. Chromosome number of important bamboo species

Bamboo species Chromosome Bamboo species Chromosome number number Arundinaria fortunez 48 Cephalostchyum pachystachis 48 A. fargesii C. pergracii 72 syn Bashania fargesii 48 A. pygmaea 54 Chinomobambusa falcata 48 A. falconeri syn Himalayacalamus falconeri 48 Dendrocalamus asper 72 A. iwatekensis D. giganteus 72 syn Sasa hidaensis 48 D. hamiltonii 72 A. gigantea 48 D. latiflorus 48, 64, 72 A. racemosa 48 D. longispathus 72 A. simonii 48 D. minor 72 Bambusa balcooa 72 D. strictus 72 B. bamboos 72 D. brandisii 72 B. bicicatricata 64, 72 B. emeiensis Gigantochloa macrostachya 72 syn. Sinocalamus affinis 70 Guadua capitaya 46 B. floribunda 72 G. chacoensis 46 B. multiplex 72 G. paraguayana 46 B. nana 72 B. nutans 72 Indocalamus wightianus 48 B. pervariabilis 56, 64 Melocanna baccifera 72 B. changii syn. Lingnania changii 52, 64 Phyllostachis aurea 48 B. desemulate 64 P. bambusoides 48 B. guabgxi 64 P. flexuosa 48 B. lepida 72 P. iexuosa 54 B. rutila 64 P. pubescens 48 B. sinospinosa 64 P. marliacia 48, 72 B. textilis 72 P. straita 48 B. tulda 72 Pleioblastus 24, 48 B. vario-striates 84, 96 Pseudosasa japonica 48 B. polymorpha 72 Sasa spp. 24, 48 B. tuldoides Munro syn A.angulata Munro 48, 64, 72 Sinobambusa spp. 48 Cephalostchyum Yushania pantlingii syn mormorea 48 Arundinaria pantlingii 48 Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 139

Knowledge of ploidy level and chromosome segregation during mitosis or meiosis of bamboos is vital for genetic improvement via polyploidy. Autopolyploids and allopolyploids are identified by studying and matching chromosomes at metaphase in mitosis and these details are wanting for bamboos. Seeds of B. bambos and D. strictus responded well when treated with colchicine showing increase in size and greater vigour (Tewari, 1992). However, limited studies are available in application of polyploidy for genetic improvement.

3.4.Genetic Improvement Classical genetic improvement programme reported on bamboos is very limited because of their unique habit and long flowering cycles. They are a perennial monocot, hence strategies and breeding techniques employed on trees or even cereal crops may not be applicable, like, since they have long gestation period similar to tree species but habit of cereals to die immediate after flowering. Hence, it is difficult to create breeding programme or even develop breeding populations for long term genetic gain. Most of the reported work on genetic improvement is for short term genetic gain, viz., natural intraspecific variation in desirable morphological traits is studied, individual clumps with desirable traits are selected and conserved ex situ. This is complemented with a fairly fast propagation method to multiply individuals with desirable traits. Selection methods presently being used include enumeration of phenotypic traits of clumps then each trait is assigned some weightage and clumps having high scores are selected. These clumps are called as plus clumps and their selection is based mostly on quantitative traits like, height, girth or diameter at midpoint of 5th internode, 5th internodal length, number of culms/clump, culm wall thickness, lumen size, etc. (Subramaniam, 1995). The concept behind such selection is to collect a pool of germplasm having most of the desirable characters. Genetic studies in bamboos improvement were started for the first time in India during 1980 in Arunachal Pradesh under the aegis of All-India Coordinated Research Programme on underutilized and under-exploited plants. Plus clumps of B. balcooa, B. nutans, B. pallida, B. tulda, Bambusa spp. (nal and nangal), D. giganteus, D. hamiltonii and D. strictus were selected (Beniwal and Singh, 1990). Similar study was conducted at Rain Forest Research Institute, Jorhat during late nineties where desirable germplasms of B. balcooa, B. nutans, B. pallida, B. tulda, D. hamiltonii and M. baccifera were selected, assessed at multilocation sites and some of them tested for anatomical properties and pulp. Further, these selected individual are being multiplied (Tripathi et al., 2011). Though selection on scoring based on weighted phenotypic traits is a good approach to select individuals with average desirable morphological characters. 140 Ajay Thakur, Santan Barthwal and H.S. Ginwal

Perhaps it may be likely to ignore those individuals having one or two extremely good phenotypic traits. In fact, various bamboo utilization have specific requirements and hence require raw material accordingly, e.g., kite and agarbatti industry requires bamboos with long internodes; bamboos in structural uses requires solid and for architectural purpose requires a specific ratio between wall thickness and collar diameter; energy sector require bamboos with high calorific values and low ash content. KONBAC industry manufactures designer bamboo furniture and they require bamboos with at least 7.5 cm culm diameter and 1.2 cm minimum wall thickness and ratio between wall thicknesses to diameter in a range of 1:3 to 1:8. According to industry, there are bamboos but there is a dearth of suitable raw material. Preference and choice of the user group is an important aspect for selection of bamboo species, e.g., the Javanese like to use culms of Gigantochloa apus for handicrafts which grow on the slopes rather than in the valley or the river banks. In Bali, people do not like to eat betung biasa but prefer betung manis, because betung manis is sweeter than betung biasa (Widjaja, 1998). A traits specific genetic improvement approach is being suggested which comprises, first characterization and intraspecific diversity study of each trait, followed by linkages studies between traits. Selection of clumps should be based on desirable industry specific traits and ex situ conservation of best individuals possessing those traits would have been suggested as slightly better tools for germplasm collection and creation of base population. Kochhar et al. (1990) studied populations of B. pallida, B. tulda, and D. hamiltonii at West Siang (Arunachal Pradesh) and North Lakhimpur showed little inter- and more intra-specific variation for seven clump management and five clump morphological traits in the base population. These traits paved way for improvement through selection for individual traits vis-à-vis single plus bamboo clumps from polymorphic populations. Estimation of genetic variation on geographical scale through provenance trial is also a quick, easy and cheaper way for genetic improvement of bamboos though not many reported. Genetic improvement through plus clump selection from provenance trial of D. strictus at Forest Research Institute, Dehradun is going on. Selected plus clumps are being cloned through vegetative propagation and tissue culture. Heritability studies of important traits give a fair idea of genetic control of that trait though it is difficult to estimate in bamboo because of long flowering cycle. It is pertinent to start seed based progeny trial and genetic improvement programme whenever flowering happens. Seed based selection of desirable germplasm in D. hamiltonii was reported, where early good performing seedlings were tested in field for growth traits (Sood et al., 2002). Selection of superior provenances and even individuals within provenances is needed. These selections should include local knowledge and choice of desirable character of bamboos. Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 141

3.5.Biotechnological Approaches for Genetic Improvement Major research focused also on the clonal propagation of elite genotypes, either juvenile or adult. The literature on this subject, however, is very limited, and this is solely due to lack of success. Technically, the propagation of adult plants via axillary branching is much more difficult than with seedlings in tropical bamboos. When using adult bamboos main problems are: (1) endogenous contamination, (2) hyperhydricity and instability of multiplication rates, and (3) problems with rooting also, though bamboos root readily in nature. Tissue culture can be a good tool for improvement in bamboos. Sodium chloride- tolerant plantlets of D. strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis (Singh et al., 2003). About 39 per cent of mature somatic embryos tolerant to 100 m M NaCl germinated and converted into plantlets in a medium [half-strength MS+2 per cent sucrose+0.02 mg l−1 (0.1 μM) α- naphthaleneacetic acid +0.1 mg l−1 (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl which further give 31 per cent success on transferring into a garden soil and sand (1:1) mixture containing 0.2 per cent (w/w) NaCl. Genetic markers and quantitative trait studies for identifying genetic variation patterns is suggested to use to improve conservation of plant genetic resources. The genomic DNA content of tropical woody bamboos is larger than that of the temperate woody bamboos as estimated by flow cytometric analysis (Gielis et al., 1997). Genome size is assessed in moso bamboo (P. pubescens) which is just more than 2 Gb and similar to maize genome but significantly larger than that of rice genome (Gui et al., 2007; Peng et al., 2013). Moso bamboo genome contains 43.9 per cent GC and 59.0 per cent transposable elements and 31,987 protein coding genes and the average length of protein coding gene is 3,350 bp (Peng et al., 2013). World-wide effort for sequencing of genome and transcriptome of bamboos resulted in deposition of partial genome sequences and expressed sequence tags (ESTs) in database. Characterization of genes, which control economically important characters: lignin and cellulose content, growth, disease resistance, etc., is most sought after for genetic improvement. Gene regulating these characters in plants can be used in genetic transformation. Regulation of Lignin components has impact on pulp-making efficiency and on reduction of pollution in the environment. A comparative analysis of the lignin biosynthesis pathway between P. heterocycla cv. pubescens and rice suggests that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content (Peng et al., 2010). The catalytic subunit of cellulose synthaze complex (BoCesA) in B. oldhamii participates in cellulose synthesis in the primary cell walls of growing bamboo and number of these genes have two or more copies in the bamboo genome, implicates role in rapid growth and also wood forming capacity (Chen et al., 2010). In B. balcooa fibre specific cDNAs among different 142 Ajay Thakur, Santan Barthwal and H.S. Ginwal internodes during bamboo development were studied and for few selected bamboo fibre ESTs namely, V1Bb147 (protein kinase-like protein) and V1Bb88 (myb domain- containing protein) were detected which were accountable for bamboo fibre development (Rai et al., 2011). Further a gene BbKst (Gene bank ID JQ432560) is reported in B. balcooa, which encodes serine-threonine protein kinase and apparently induces higher cellular deposition and enhance fibre qualities like; flexibility coefficient, slenderness ratio and lower runkel ratio (Ghosh et al., 2013). Bamboo flowering can be manipulated and early seed production is possible in P. praecox; where two genes PpMADS1and PpMADS2 reported to be involved in floral transition (Lin et al., 2010). BohLOL1; an LSD1- like gene in B. oldhamii is a nuclear binding protein which plays a role not only in response against pathogen but also in the regulation of growth. The up-regulation of BohLOL1expression occurs in growing bamboos (Yeh et al., 2011).

4. Conclusion Bamboos are widely distributed, fastest biomass producing woody grass which are still important part of livelihood of rural people. Despite this, genetic improvement have been carried out on a very few bamboo species. Molecular diversity of bamboos are fairly better studied than phenotypic diversity. New molecular markers now made studies on evolution more obvious than ever. Gregarious flowering of bamboos is still a mystery, though information generated on flowering cycle is valuable for biologists as well as managers. Flowering is being considered as an opportunity by both. Lately, some genes and proteins responsible for flowering are identified and these studies needs to be validated further. Death of clumps after flowering is being established to have some relation with programmed cell death and studies have revealed that two genes have some significant role. Flowering can be used as an opportunity for genetic improvement work on bamboos to create a broader base population, estimate of genetic variation and genetic control of traits. Hybridization studies are possible if flowering is controlled either in situ or in vitro. Bamboos have become natural polyploid few millions years ago which is substantiated by molecular studies. Temperate bamboos are mostly tetraploid and tropical bamboos are mostly hexaploid, though bamboo of the New World. There is a need to create artificial polypoidy for improvement of quality and quantity of bamboos. Genetic improvement programme is started in some bamboo species mostly based on index selection which is needed to be refined by study on genetic variation of desirable traits and trait specific selection keeping end use into consideration. Genetic improvement programme would have yielded much in short time period if provenance/population studies of specific bamboo had been conducted. Genetic variation in vegetative propagation and multilocation trials of selected clumps of bamboos is key to launch desirable bamboos Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 143 in field. Complete genome studies of bamboos and data mining facilitated determination of gene or protein controlling a desirable character. This may be useful for trait specific genetic modification programme of bamboos.

References Banik, R.L. 1995. Selection criteria and population enhancement of priority bamboos. In: Williams, J.T.; Ramanuja Rao, I.V. and Rao, A.N. Eds. Genetic enhancement of bamboo and rattan. New Delhi, INBAR. pp. 99-110. Barkley, N.A.; Newman M. L.; Wang M. L.; Hotchkiss M.W.; Pederson G. A. 2005. Assessment of the genetic diversity and phylogenetic relationships of a temperate bamboo collection by using transferred EST-SSR markers. Genome, 48(4): 731-737. Behari, B. 2006. Status of bamboo in India. In: India. MoEF. Compilation of papers for preparation of national status report on forests and forestry in India. New Delhi, Survey and Utilization Division, Ministry of Environment and Forests. pp. 109-120. Beniwal, B.S.; Singh, N.B. 1990. Genetic improvement of forest trees in Arunachal Pradesh. Indian Forester, 116 (1): 3-10. Burke, S.V.; Clark, L.G.; Triplett, J.K.; Grennan, C.P.; Duvall, M. R. 2014. Biogeography and phylogenomics of new world Bambusoideae (Poaceae), revisited. American Journal of Botany, 101(5): 886-891. Bystriakova, N.; Kapos, V.; Lysenko, I.; Stapleton, C.M.A. 2003. Distribution and conservation status of forest bamboo biodiversity in the Asia-Pacific Region. Biodiversity and Conservation, 12(9): 1833-1841. Chen, C.Y.; Hseih, M.H.; Yang, C.C.; Lin, C.S.; Wang, A.Y. 2010. Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii. Phytochemistry, 71(11-12): 1270-1279. Clark, L.G.; Zhang, W.; Wendel, J.F. 1995. A phylogeny of the grass family (Poaceae) based on ndhF sequence data. Systematic Botany, 20: 436-460. Dong, Y.R.; Zhang, Z.R.; Yang, H.Q. 2012. Sixteen novel microsatellite markers developed for Dendrocalamus sinicus (Poaceae), the strongest woody bamboo in the World. American Journal of Botany: e347e349. [Available at: http://www.amjbot.org/]. Gielis, J.; Everaert, I.; De Loose, M. 1997. Genetic variability and relationships in Phyllostachys using random amplified polymorphic DNA. In: Chapman, G.P. Ed. The bamboos. Linnaean Society Symposium Series No. 19. London, Academic Press. pp. 107-124. Ghosh, J.S.; Chaudhuri, S.; Dey, N.; Pal, A. 2013. Functional characterization of a serine threonine protein kinase from Bambusa balcooa that implicates in cellulose overproduction and superior quality fibre production. BMC Plant Biology, 13(10):128. 144 Ajay Thakur, Santan Barthwal and H.S. Ginwal

Goyal, A.K.; Ghosh, P.K.; Dubey, P.K.; Sen, A. 2012. Inventorying bamboo biodiversity of North Bengal: A case study. International Journal of Fundamental and Applied Sciences, 1(1): 5-8. Gui, Y.; Wang, S.; Quan, L.; Zhou, C.; Long, S.; Zheng, H.; Jin, L.; Zhang, X.; Ma, N.; Fan, L. 2007.Genome size and sequence composition of moso bamboo: A comparative study. Science China Life Sciences, 50(5): 700-705. John, C.K.; Nadgauda, R.S. 1999. Review in vitro-induced flowering in bamboos. In Vitro Cellular and Developmental Biology - Plant, 35(4): 309-315. Kaur, D.; Dogra V.; Thapa P.; Bhattachrya, A.; Sood, A.; Srinivasulu, Y. 2014. In vitro flowering associated protein changes in Dendrocalamus hamiltonii. Proteomics, 15(7): 1291-1306. doi: 10.1002/pmic. 201400049. Kochhar, S.; Bhag Mal; Chaudhary, R.G. 1990. Population aspect of the phenological behaviour of bamboo germplasm In: International Bamboo Workshop, Cochin 14-18 November 1988. Bamboos - Current research: Proceedings edited by I.V.R. Rao; R. Gnanaharan and C.B. Sastry. Peechi, KFRI. pp. 51-58. Lin, X.C.; Lou, Y.F.; Liu, J.; Peng, J.S.; Liao, G.L.; Fang, W. 2010. Cross breeding of Phyllostachys species (Poaceae) and identification of their hybrids using ISSR markers. Genetics and Molecular Research, 9(3): 1398-1404. Loh, J.P.; Ruth, K.; Set, O.; Gan, L.H.; Gan, Y.Y. 2000. A study of genetic variation and relationships within the bamboo subtribe bambusinae using amplified fragment length polymorphism. Annals of Botany, 85(5): 607-612. Nadgauda, R.S.; John, C.K.; Mascarenhas, A.F. 1993a. Floral biology and breeding behaviour in bamboos Dendrocalamus strictus Nees. Tree Physiology, 13(4): 401-408. Nadgauda, R.S.; John, C.K.; Mascarenhas, A.F. 1993b. Floral biology and breeding behaviour in bamboos Bambusa arundinacea Willd. Tree Physiology, 13(4): 409-416. Nayak, S.; Rout, G.R.; Das, P. 2003. Evaluation of genetic variability in bamboo using RAPD Markers. Plant Soil and Environment, 49(1): 24-28. Peng, Z.; Lu, T.; Li, L.; Liu, X.; Gao, Z.; Hu, T.; Yang, X.; Feng, Q.; Guan, J.; Weng, Q.; Fan, D.; Zhu, C.; Lu, Y.; Han, B.; Jiang, Z. 2010. Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences. BMC Plant Biology, 10: 116. doi: 10.1186/1471-2229-10-116. Peng, Z.; Lu, Y.; Li, L.; Zhao, Q.; Feng,Q. et al. 2013. The draft genome of the fast- growing non-timber forest species moso bamboo (Phyllostachys heterocycla). Nature Genetics, 45: 456-463. doi:10.1038/ng.2569. Raghavan, T.S. 1952. Sugarcane×bamboo hybrids. Nature, 170(4321): 329-330. Rai, V.; Dey, N. 2012. Identification of programmed cell death related genes in bamboo. Gene, 497(2): 243-248. Genetic Diversity in Bamboos: Conservation and Improvement for Productivity 145

Rai, V.; Ghosh, J.S.; Amita Pal; Dey, N. 2011. Identification of genes involved in bamboo fibre development. Gene, 478(12): 19-27. Ramanayake, S.M.S.D.; Meemaduma, V.N.; Weerawardene, T.E. 2007. Genetic diversity and relationships between nine species of bamboo in Sri Lanka, using random amplified polymorphic DNA. Plant Systematics and Evolution, 269(1-2): 55-61. Rao, A.N.; Rao, V.R.; Williams, J.T. Eds. 1998. Priority species of bamboo and rattan. Serdang, IPGRI-APO. Ruiyang, C. 2003. Chromosome atlas of major economic plants genome in China (Tomus IV) chromosome atlas of various bamboo species. Beijing, Science Press. 646p. Sharma, R.K.; Gupta, P.; Sharma, V.; Sood, A.; Mohapatra, T.; Ahuja, P.S. 2008. Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo. Genome, 51(2): 91-103. Singh, M.; Jaiswal, U.; Jaiswal V.S. 2003. In vitro selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (Dendrocalamus strictus Nees). In Vitro Cellular and Developmental Biology - Plant, 39(2): 229-233. Sood, A.; Ahuja, P.S.; Sharma, M.; Sharma, O.P.; Godbole, S. 2002. In vitro protocols and field performance of elites of an important bamboo Dendrocalamus hamiltonii Nees et Arn. ex Munro. Plant Cell, Tissue and Organ Culture, 71(1): 55-63. Subramaniam, K.N. 1995. Bamboo genetic resources in India. In: Vivekanandan K.; Rao, A.N. and Ramanatha Rao, V. Eds. Bamboo and rattan genetic resources in certain Asian countries. Serdang, IPGRI-APO. 229p. Tewari, D.N. 1992. A monograph on bamboo. Dehradun, International Book Distributors. 498p. Thakur, A.; Tripathi, Y.C.; Singh, S.P.; Naithani, S. 2014. Chemotyping of some priority bamboo species for functional diversification. In: 13th Silvicultural Conference, Dehradun, 24-28 November 2014. Proceedings. Tripathi, Y.C.; Chand, T.; Thakur, A.; Pathak, K.C. 2011. Genetic improvement and conservation of genetic resources of some economically important bamboo species of northeastern India. Jorhat, Rain Forest Research Institute. Triplett, J.K.; Oltrogge, K.A.; Clark, L.G. 2010. Phylogenetic relationships and natural hybridization among the North American woody bamboos (Poaceae: Bambusoideae: Arundinaria). American Journal of Botany, 97(3): 471-492. Uchikawa I. 1935. Karyological studies in Japanese bamboo. II: Further studies on chromosome numbers. Japanese Journal of Genetics, 11(6), 308-313. 146 Ajay Thakur, Santan Barthwal and H.S. Ginwal

Venkatesh, C.S. 1984. Dichogamy and breeding system in a tropical bamboo, Ochlandra travancorica. Biotropica, 16(4): 309-312. Widjaja, E.A. 1998. Bamboo genetic resources in Indonesia. In: Vivekanandan, K.; Rao, A.N.; Ramanatha Rao, V. Eds. Bamboo and rattan genetic resources in Certain Asian countries. Malaysia, IPGRI-APO. pp. 63-102. Williams, J.T. 1998. Bamboo and rattan research. In: Rao, A.N.; Rao, V.R. and Williams, J.T. Eds. Priority species of bamboo and rattan. Serdang, IPGRI- APO. pp. 3-5. Williams, J.T.; Rao, V.R. 1994. Priority species of bamboo and rattan. INBAR Technical Report No. 1. New Delhi, INBAR. Yeh, S.H.; Lin, C.S.; Wu, F.H.; Wang A.Y. 2011. Analysis of expression of BohLOL1, which encodes LSD1-like zinc finger protein in Bambusa oldhamii. Planta, 234(6): 1179-1189. Zhang, Y.J.; Ma, P.F.; Li, D.Z. 2011. High-throughput sequencing of six bamboo chloroplast genomes: Phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae). PLoS One, 6(5): e20596.