Limitations, progress and prospects of application of biotechnological tools in improvement of bamboo—a plant with extraordinary qualities

Abstract

Bamboos (family Poaceae) are the most beautiful and useful plants on the Earth, mainly found in the tropical and sub-tropical regions of the world. Bamboos are fast growing and early maturing, but lack of proper management of bamboo resources is leading to rapid reduction of the existing bamboosetum. Bamboo propagation through seeds is limited due to long flowering cycle of upto 120 years, seed sterility and short seed viability. Infrequent and unpredictable flowering events coupled with peculiar monocarpic behaviour i.e. flowering once before culm death, and extensive genome polyploidization are additional challenges for this woody group. Similarly, vegetative propagation by cuttings, offsets and rhizomes are also inadequate to cope up with the demand of planting stock due to large propagule size, limited availability, seasonal dependence, low multiplication rate and rooting percentage. Therefore, attempts have been made to propagate bamboos through in vitro techniques. In vitro flowering has also been achieved successfully in some bamboo species. Classification systems proposed to date need further support, as taxonomic delineation at lower levels is still lacking sufficient resolution. Tremendous advancement in molecular markers holds the promise to address the needs of bamboo taxonomy (systematics and identification) and diversity studies. Successful application of molecular marker techniques has been achieved in several bamboo species although, more studies are required to understand the population structure and genetic diversity of bamboos in a better way. In addition, some efforts have also been made to clone important genes from bamboos and also for genetic transformation using Agrobacterium and particle bombardment methods. An overview of the recent developments made in improvement of bamboos through in vitro propagation, molecular marker technologies, cloning, and transformation and transgenics has been presented. The future potential of improvement of bamboos using modern biotechnological tools has also been discussed.

Introduction

Bamboos are the most unique, fascinating and versatile group of plants known to mankind which are commonly called “Green Gold” or “Poor Man’s Timber” and are unique with complex branching patterns, woody culms and gregarious, monocarpic flowering. It is one of the largest members of the grass subfamily Bambusoideae of family Poaceae (Gramineae), which includes ~1,575 species distributed mainly in tropical and subtropical countries of the world. Major species richness is found in Asia Pacific (China: 626, India: 102, Japan: 84, Myanmar: 75, Malaysia: 50 and few others) and South America (Brazil: 134, Venezuela: 68, Colombia: 56 and few others) while least (5) is found in Africa (Bystriakova et al. 2003, 2004). The herbaceous bamboos with ~110 species are mostly concentrated in the Neotropics of Brazil, Paraguay, Mexico, Argentina and West Indies (Judziewicz et al. 1999). Brazil is the most prominent place representing 89 % of the genera and 65 % of the species that are reported from the New World. The largest natural bamboo forests, known as ‘tabocais’ in Brazil and ‘pacales’ in Peru, cover approximately 600,000 ha across Brazil, Peru and Bolivia (Filgueiras and Goncalves 2004).

Conventionally, bamboos are propagated through seeds, offsets and culm cuttings. However, propagation through seeds is beset with several problems like long flowering cycle (upto 120 years), monocarpic nature of plant, poor seed set, short seed viability, highly heterogeneous seedling populations and consumption of seeds by birds, rodents and wild animals. Similarly, bulkiness and limited availability of propagules, difficulties in transportation over long distances, seasonal dependence, low survival rate and limited rooting of the propagules are the major constraints in bamboo propagation through vegetative methods. Shortage of bamboo planting material is expected to become a bottle neck in the reforestation process due to inefficacy of the conventional propagation techniques like seed propagation, clump division, rhizome and culm cuttings etc.

Pests and diseases also play an important role in the success or failure of the establishment of nursery and plantation of bamboo stands. Tar spot caused by Phyllachora shiriana complex and leaf rust caused by Phakopsora louditiae are the most common diseases of B. blumeana, Bambusa sp. and D. latiflorus. However, mite (Schizotetranycus floresi) was the most prevalent pest observed (Dayan 1988). Similarly, it is susceptive to the Bamboo mosaic virus (BaMV), a potexvirus which infects 13 species of bamboo and is considered as an important limiting factor in the production of B. edulis in Taiwan. The virus reduces the quality and yield of bamboo by over 50 %. No chemicals effectively control or eliminate BaMV from infected plants (Hsu et al. 2000). Development of disease-resistant bamboo may be a solution to this problem. However, it is difficult to obtain virus-resistant bamboos using traditional breeding methods (Chang and Ho 1997; John and Nadgauda 1999).

The conventional methods of taxonomical classification are based on the morphological and flowering features of any plant species. However, in bamboos, taxonomic delineation has been done predominantly on the basis of various morphological features due to erratic and long flowering cycles, which severely restricts the study of reproductive features. Hence, the identification keys are mostly dependent on various vegetative features that need further refinement and re-investigation. In particular, the taxonomic demarcation of woody bamboos at lower ranks, such as genera and species, is not well resolved and requires additional efforts.

Peculiar flowering habits have made it almost impossible to undertake breeding programs for superior traits in woody bamboos. In addition, the characteristic death of bamboo clumps after flowering makes the study of bamboo flowering quite difficult. Tissue culture has been used for rescue of hybrid seeds produced by conventional breeding methods. Alexander and Rao (1968) were the first to report aseptic germination of seeds of hybrid bamboo (Bambusa x Saccharum) on a sucrose enriched medium, heralding the start of tissue culture of bamboos. However, no breakthrough has been achieved in bamboo breeding using conventional methods.

In spite of enormous volume of research work undertaken in bamboos, no compilation of data is available which can provide a ready reference to the various aspects of biotechnological improvements being undertaken in bamboo, therefore this review has been compiled to encompass most of the available literature on bamboo biotechnology.

Progress made using modern biotechnological tools

A number of successful reports documenting propagation of bamboos through in vitro techniques have been published during the last three decades. An attempt has been made to summarize the available information regarding micropropagation of bamboos through tissue culture. Many groups have also attempted to induce flowering in vitro in bamboos to study the floral details. In addition, an overview of the information available on use of various molecular markers in diversity analysis, phylogenetic and taxonomic studies; attempts being made to develop transgenics in bamboos using various transformation techniques and cloning of genes has been presented. Figure 1 summarizes the uses, limitations and the possible areas wherein biotechnological interventions can be made for improvement of bamboos.

Fig. 1.

Uses, limitations, applications of in vitro techniques and future prospects of biotechnological interventions in bamboos

Micropropagation

Micropropagation is a valuable technique for rapid multiplication of difficult-to-propagate plants, both for commercial production and germplasm conservation. Micropropagation using tissue culture techniques offers substantial advantage over largely insufficient and inefficient classical techniques used for mass scale propagation of bamboos. Two distinct patterns of in vitro micropropagation used for bamboos are organogenesis and somatic embryogenesis (Tables 1, 2, 3).

Table 1.

Micropropagation of bamboos through enhanced axillary branching using juvenile and mature explants

Species	Explant	Medium + PGRs		References
		Induction	Rooting
Juvenile explants
54 Bamboo species	Node	MS + BAP	MS + NAA	Prutpongse and Gavintertvatana 1992
Bambusa balcooa, B. bambos	Node	MS + BAP + NAA	MS + IBA/NAA	Rathore et al. 2009
B. bambos	Node	MS + BAP	MS + NAA	Arya and Sharma 1998
	Embryonic axis of caryopsis	MS + BAP	MS + BAP + GA3 + NAA	Kapoor and Rao 2006
B. nutans	Node	MS + BAP	MS + IBA	Yashoda et al. 1997
B. oldhamii	Shoot apices	MS + TDZ	MS + NAA	Lin et al. 2007a
	Node	MS + BAP + AdS	MS + IBA + NAA	Thiruvengadam et al. 2011
B. tulda	Shoot apices	MS + BAP + Kn	½ MS + IAA + Cou	Saxena 1990
B. ventricosa	Node	MS + BAP + NAA + AC	MS + BAP + NAA + AC	Dekkers and Rao 1989
	Shoot apices	MS + BAP	MS + BAP + NAA	Huang and Huang 1995
Dendrocalamus asper	Seed	MS + BAP	MS + IBA + NAA	Arya and Arya 1996; Arya et al. 1999, 2002a
D. brandisii	Seed	MS + BAP + CW	MS + IBA	Nadgauda et al. 1990
D. giganteus	Node	MS + BAP + Kn	½ MS + IBA + Cou	Ramanayake and Yakandawala 1997
	Node	MS + BAP + NAA	MS + IBA	Agnihotri et al. 2009
	Node	MS + BAP	MS + IBA/IAA/NAA + Cou	Sood et al. 2002
D. membranaceus	Node	MS + BAP	MS + IBA	Yashoda et al. 1997
D. strictus	Shoot apices	MS + BAP + CW	MS + IBA	Nadgir et al. 1984
	Node, Coleoptile	½ MS + BAP	MS	Shirgurkar et al. 1996
	Node	MS + GA3 + Kn	MS + GA3 + Kn	Maity and Ghosh 1997
	Shoot apices	MS + BAP + triacontanol	MS + NAA + rice bran extract	Mishra et al. 2001
	Shoot apices, node	½ MS + TDZ	½ MS + IBA	Singh et al. 2001
D. strictus, D. giganteus	Node	MS + BAP + AdS	½ MS + IBA	Das and Rout 1991
Oxytenanthera abyssinica	Node	MS + BAP + NAA	MS + IBA	Diab and Mohamed 2008
Phyllostachys meyeri	Node	½ MS	½ MS	Ogita et al. 2008
Thamnocalamus spathiflorus	Zygotic embryos	MS + BAP + IBA	MS + IBA	Bag et al. 2000
Mature explants
Bambusa balcooa	Node	MS + BAP	MS + BAP + NAA	Mudoi and Borthakur 2009
	Node	MS + BAP + Kn	½ MS + IBA	Das and Pal 2005a
B. balcooa, B. nutans, B. salarkhanii, B. vulgaris	Node	MS + BAP	½ MS + NAA + IBA	Nurul Islam and Rahman 2005
B. bambos	Node	MS + BAP	MS + NAA	Arya and Sharma 1998
B. edulis	Inflorescence	MS + NAA + IBA + 2,4- D	–	Lin et al. 2005
B. glaucescens	Node	MS + BA + AC	MS + BA + NAA + AC	Banik and Alam 1987
	Node	MS + BAP + Kn	MS + IBA	Shirin and Rana 2007
B. nutans	Node	MS + BAP	MS + IBA	Yashoda et al. 2007
	Node	MS + BAP + Kn	MS + IBA + IAA + NAA	Negi and Saxena 2011
B. oldhamii	Node	MS + BAP	MS + IBA + NAA	Thiruvengadam et al. 2011
B. polymorpha	Node	MS + BAP	MS + NAA	Arya et al. 2005
B. tulda	Node	MS + Glut + IAA + BAP	MS + Cou	Mishra et al. 2008
B. ventricosa	Node	MS + BAP	MS + NAA	Arya et al. 2002b
B. vulgaris, B. arundinacea	Node	MS + BAP + Kn + CW	½ MS + IBA	Nadgir et al. 1984
B. vulgaris	Node	MS + BAP + AdS	MS + BAP + IBA	Das and Rout 1994
	Node	MS + BAP	MS + IBA	Ramanayake et al. 2006
B. wamin	Node	MS + BAP + Kn	½ MS + IBA	Arshad et al. 2005
Dendrocalamus asper	Node	MS + BAP	MS + IBA + NAA	Arya and Arya 1996; Arya et al. 1999, 2002a
	Node	MS + BAP	MS + IBA	Banerjee et al. 2011
	Node	MS + BAP + AdS	MS + IBA + NAA	Singh et al. 2011
D. giganteus	Node	MS + BAP + Kn + CW	½ MS + IBA + Cou	Ramanayake and Yakandawala 1997
	Node	MS + BAP	–	Ramanayake et al. 2001
	Node	MS + BAP	MS + IBA + NAA	Arya et al. 2006
D. hamiltonii	Node	MS + BAP + 2,4-D	½ MS + IBA + NAA	Sood et al. 1994
	Node	MS + BAP + NAA	MS + IBA	Agnihotri and Nandi 2009
	Node	MS + BAP + NAA	MS + IBA	Agnihotri et al. 2009
	Node	MS + TDZ + AA	MS + IBA + CC	Singh et al. 2012a
D. longispathus	Node	MS + BAP + Kn	½ MS + IBA + Cou	Saxena and Bhojwani 1993
D. strictus	Node	MS + IAA + AdS	MS + IBA + NAA + Phloroglucinol	Chaturvedi et al. 1993
	Node	MS + BAP + Kn		Ravikumar et al. 1998
D. strictus	Node	MS + BAP + Kn + CW	½ MS + IBA	Nadgir et al. 1984
Guadua angustifolia	Node	MS + BAP	MS + BAP	Jimenez et al. 2006
Pleioblastus pygmaeus	Node	MS + BAP	MS	Watanable et al. 2000
Pseudoxytenanthera stocksii	Node	MS + BAP + NAA + AA + Cyst + Glut	½ MS + BAP + IBA + AA + Cyst + Glut	Sanjaya et al. 2005
Thamnocalamus spathiflorus	Node	MS + BAP + IBA	MS + IBA	Bag et al. 2000
Thyrsostachys oliveri	Node	MS + BAP	½ MS + NAA + IBA	Nurul Islam and Rahman 2005

AA ascorbic acid, AC activated charcoal, AdS adenine sulphate, BAP 6-benzylaminopurine, CC choline chloride, CW coconut water (milk), Cou coumarin, Cyst cysteine, 2, 4-D 2, 4 Dichlorophenoxy acetic acid, GA₃ gibberellic acid, Glu glutamine, IAA indole-3-acetic acid, IBA indole-3-butyric acid, Kn kinetin, NAA α-napthaleneacetic acid, PGR plant growth regulator, PVP polyvinylpyrolidone, TDZ Thidiazuron

Table 2.

Callogenesis and indirect organogenesis in bamboos

Species	Explants	Medium + PGRs		References
		Callus formation	Organogenesis
54 Bamboo species	Shoot tips, leaf, inflorescence	MS + 2,4-D + CW	–	Prutpongse and Gavintertvatana 1992
Bambusa glaucescens	Young leaves	MS + CH + CW + 2,4-D + PVP	–	Jullien and Van 1994
B. multiplex	Shoot tip	MS + 2,4-D	–	Huang and Murashige 1983
B. nutans	In vitro shoots	MS + 2,4-D + BAP + ABA	MS + 2,4-D + BAP	Kalia et al. 2004
B. oldhamii, B. multiplex	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
B. vetricosa	Internode, sheath base	MS + 2,4-D	MS + 2,4-D	Dekkers and Rao 1989
Dendrocalamus farinosus	Seed embryo, young shoots	MS + 2,4,5-T + Kn + IBA	MS + Kn + IAA	Hu et al. 2011
D. giganteus	Shoots, spikelets, roots	MS + 2,4-D + NAA	MS + 2,4-D + NAA	Ramanayake and Wanniarachchi 2003
D. hamiltonii	Node	MS + BAP + 2,4-D + GA3	MS + IBA	Sood et al. 1994
D. latiflorus	Internodes	MS + 2,4-D	MS + 2,4-D + BA	Zamora et al. 1989
	Inflorescence	MS + 2,4-D + Kn + CW + PVP	MS + TDZ	Lin et al. 2007b
Phyllostachys aurea	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
P. nigra	Shoots	½ MS + 2,4-D	½ MS + 2,4-D	Ogita 2005
Sasa pygmaea	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
Schizostachyum brachycladum, Thyrsostachys sinensis	Internode, sheath base	MS + 2,4-D	MS + 2,4-D	Dekkers and Rao 1989

ABA Abscisic acid, BAP 6-benzylaminopurine, CH casein hydrolysate, CW coconut water (milk), 2, 4-D 2, 4 Dichlorophenoxy acetic acid, GA₃ Gibberellic acid, IAA indole-3-acetic acid, IBA indole-3-butyric acid, Kn kinetin, NAA α-Napthaleneacetic acid, PGR plant growth regulator, PVP polyvinylpyrolidone, TDZ thidiazuron, 2, 4, 5-T 2, 4, 5-Trichlorophenoxyacetic acid

Table 3.

Somatic embryogenesis in bamboos

Species	Explants	Medium + PGRs		References
		Embryogenesis	Germination
Bambusa arundinacea	Embryonal axis	N6 + BAP + 2,4-D + PVP	N6 + BA + 2,4-D + PVP	Mehta et al. 1982
B. balcooa	Pseudo-spikelet	MS + BAP	MS + BAP	Gillis et al. 2007
B. beecheyana	Inflorescence	MS + 2,4-D + Kn	MS + 2,4-D + Kn	Yeh and Chang 1986b
	Roots	MS + Kn + 2,4-D	MS + Kn + 2,4-D	Chang and Lan 1995
B. edulis	Node, internode	MS + Kn + 2,4-D + CW	MS + TDZ + NAA	Lin et al. 2004
B. glaucescens	Leaves	MS + BAP + 2,4-D	–	Jullien and Van 1994
B. oldhamii	Inflorescence	MS + 2,4-D + Kn	MS + 2,4-D + Kn	Yeh and Chang 1986a
	Floral tissue	MS + BAP + NAA	MS + 2,4-D + Kn	Ho and Chang 1998
B. oldhamii, B. multiplex	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
B. ventricosa	Stem segment	MS + BAP + IBA	MS + BAP	Cheah and Chaille 2011
B. vulgaris	Node, zygotic embryos	MS + 2,4-D + Kn + AdS	MS + 2,4-D + Kn + AdS	Rout and Das 1994
Dendrocalamus asper	Seed	MS + 2,4-D	MS + 2,4-D	Kanyaratt 1991
	In vitro shoots	MS + BAP + 2,4-D + IAA	MS + BAP + IAA	Arya et al. 2008
	Roots, leaves, node	MS + 2,4-D	MS + BAP	Ojha et al. 2009
D. giganteus, D. strictus	Node, zygotic embryos	MS + 2,4-D + Kn + AdS	MS + 2,4-D + Kn + AdS	Rout and Das 1994
D. hamiltonii	Node	MS + BAP + 2,4-D	MS	Godbole et al. 2002
	Axillary bud	MS + BAP + 2,4-D	½ MS + IBA	Bag et al. 2012
D. latiflorus	Meristems	MS + 2,4-D + BAP	MS + 2,4-D + BAP	Zamora et al. 1989
D. longispathus	Internode	B5/MS + 2,4,5-T + 2,4-D	–	Saxena and Bhojwani 1993
D. membranaceus	Node	MS + BAP + 2,4-D	–	Vongvijitra 1988
D. strictus	Zygotic embryo	B5 + 2,4-D	½ B5 + CW	Zamora and Gruezo 1990
	Seed	MS + 2,4-D + BAP + PVP	½ MS + NAA + IBA	Saxena and Dhawan 1999
	Seed	B5 + 2,4-D	B5 + IBA + NAA	Rao et al. 1985
	Seed	MS + 2,4-D + CW	MS	Dekkers and Rao 1989
	Seed	MS + 2,4-D + Kn	MS + 2,4-D + Kn	Kumar and Mathur 1992
Otatea acuminata	Seed	MS + 2,4-D + BAP	MS + 2,4-D + BAP	Woods et al. (1992)
Phyllostachys aurea	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
P. bambusoides	Node	MS + picloram	–	Komatsu et al. 2011
Sasa pygmaea	Shoot apices	MS + BAP + NAA	MS + NAA	Huang et al. 1989
Sinocalamus latiflora	Zygotic embryos	MS + 2,4-D + Kn + PVP	MS + 2,4-D + Kn + PVP	Yeh and Chang 1987
	Anthers	N6 + 2,4-D + BAP + AC	N6 + 2,4-D + BAP + AC	Tsay et al. 1990

AC activated charcoal, AdS adenine sulphate, BAP 6-benzylaminopurine, CW coconut water (milk), 2, 4-D 2, 4 Dichlorophenoxy acetic acid, GA₃ gibberellic acid, IAA indole-3-acetic acid, IBA indole-3-butyric acid, Kn kinetin, NAA α-Napthaleneacetic acid, PGR plant growth regulator, PVP polyvinylpyrolidone, TDZ thidiazuron, 2, 4, 5-T 2, 4, 5-Trichlorophenoxyacetic acid

Organogenesis

Clonal propagation via organogenesis is a two-staged process involving the proliferation (axillary meristems) or induction (adventitious meristems) of unipolar shoots on explants followed by shoot excision and induction of root meristems. It is generally agreed that plants regenerated from shoot tips or nodal buds are genetically stable and free from somaclonal variations associated with plants differentiated from callus. Therefore, a lot of studies are available wherein enhanced axillary branching has been utilized for micropropagation of bamboo species using juvenile and mature tissues (Table 1) and only few reports document indirect organogenesis (Table 2).

Factors controlling organogenesis

Explant

Success in micropropagation of bamboos was obtained using both juvenile and mature explants (Table 1). Sprouting of nodal buds into shoots is primarily determined by genotype, physiological state of the tissue, and time of the year when the explants are collected and cultured (Saxena and Dhawan 1994; Ramanayake et al. 1995; Ramanayake and Yakandawala 1997; Singh et al. 2011, 2012a). Saxena and Bhojwani (1993) reported that bud-break frequency in Dendrocalamus longispathus was strongly influenced by the juvenility of lateral shoots, position of axillary bud on the branch, and the season in which cultures were initiated. Explants collected during spring (February-April) gave better response in terms of decreased contamination, early shoot initiation and increased percent bud break with higher number of shoots in D. asper (Singh et al. 2011), while early summer (April–June) was best for explant collection and establishment of D. hamiltonii (Singh et al. 2012a).

Medium

The nutritional requirements for optimum growth of a tissue in vitro may vary with species. In bamboos, mostly MS medium has been used for both direct as well as indirect organogenesis. Singh et al. (2011, 2012a) compared four media viz. MS (Murashige and Skoog 1962), SH (Schenk and Hildebrandt 1972), B₅ (Gamborg et al. 1968) and NN (Nitsch and Nitsch 1969) during axillary bud break and found better response in MS medium. Similarly, half strength rather than full strength MS was reported better for axillary shoot formation and organogenesis in some bamboo species (Shirgurkar et al. 1996; Singh et al. 2001; Ogita et al. 2008) (Tables 1, 2).

In general, agar or gellan gum solidified medium is used for tissue culture of plants, however, several workers have reported higher rates of shoot multiplication and improved growth in liquid medium in comparison to semi-solid medium (Saxena and Bhojwani 1993; Sood et al. 2002; Das and Pal 2005a; Arya et al. 2006; Shirin and Rana 2007; Ogita et al. 2008). The slower growth or poor shoot multiplication on semi-solid medium vis-a-vis liquid medium may be attributed to the fact that solubilized agar binds water, absorbs nutrients and PGRs resulting in reduced uptake of nutrients, PGRs and other essential constituents by cultured tissues, however, in some instances vitrification of shoots leading to reduced multiplication rates have also been reported in liquid medium.

Growth regulators

The frequency of bud break on PGR free basal medium is usually very low (Arya et al. 2006; Singh et al. 2011). The variable endogenous levels of growth regulators are known to be the cause of varied responses of species and genotypes to growth regulator supplemented media. Therefore, detailed information regarding the requirement of plant growth regulators (PGRs) is necessary before we can exploit plant tissue culture on commercial scale. The level and kind of PGRs included in the culture medium largely determine the success of tissue culture protocol. Incorporation of BAP into the medium improved the axillary bud proliferation (Nadgir et al. 1984; Dekkers and Rao 1989; Hirimburegama and Gamage 1995; Arya and Arya 1996; Arya et al. 2006), while Kinetin (Kn) alone was found to be less effective (Ramanayake and Yakandawala 1997; Arya et al. 2006; Singh et al. 2011). Synergistic effect of the two cytokinins BA and Kn was reported best for shoot multiplication in D. giganteus (Arya et al. 2006) and B. glaucescens (Shirin and Rana 2007). TDZ has been used during axillary shoot propliferation in B. oldhamii (Lin et al. 2007a), D. strictus (Singh et al. 2001) and D. hamiltonii (Singh et al. 2012a) while gibberellic acid (GA₃) was used during in vitro propagation of D. strictus (Maity and Ghosh 1997).

In addition to cytokinins and auxins, other additives like adenine sulphate, activated charcoal and amino acids have also been included in the proliferation medium. The lethal browning or blackening of cultures due to phenolic compounds has been controlled using polyphenol adsorbents or antioxidants. Significant control of browning with enhanced shoot multiplication was achieved using ascorbic acid in D. hamiltonii while PVP and activated charcoal were ineffective in doing so (Singh et al. 2012a). On the contrary PVP improved shoot health in D. strictus cultures (Saxena and Dhawan 1999).

Medium pH

The hydrogen ion concentration of the media effect growth of the tissue by altering pH of cells. Higher ‘H’ ion concentration induced precipitation of phosphates, gelatinization of agar and destruction of vitamins and growth regulators. Though majority of plant tissues have optimum pH from 5.0 to 5.5 (Butenko et al. 1984) yet the pH range is variable for individual plant tissues. In an investigation, Arya et al. (2006) found that shoot growth was well in the pH range of 4.5 to 5.8 in D. giganteus, however, best shoot multiplication rate was obtained in the medium with pH of 4.5.

Carbon source

Sucrose is the most widely used carbon source in various plant tissue culture media, but its concentration varies from 2 to 6 %. The most commonly used sucrose concentration in bamboos is 3 %. Saxena (1990) found that 2 % sucrose was ideal for shoot multiplication in B. tulda. A high concentration (~6 %) of sucrose was used in medium for callus initiation and proliferation (Yeh and Chang 1986a, b, 1987; Tsay et al. 1990; Lin et al. 2004). Replacement of sucrose with less expensive table sugar had negligible effect on rate of shoot multiplication in D. asper and D. hamiltonii but reduced the cost of plant production considerably, however, the use of glucose showed deleterious effects on shoot multiplication (Singh et al. 2011, 2012a).

Propagule size

Single shoots usually do not survive under in vitro conditions. A propagule of three to five shoots has been reported best for multiplication of shoots in Bambusa tulda (Saxena 1990), Dendrocalamus longispathus (Saxena and Bhojwani 1993) and D. hamiltonii (Agnihotri and Nandi 2009; Agnihotri et al. 2009) in comparison to lesser or more shoots (Nadgir et al. 1984; Arya et al. 1999). Agnihotri and Nandi (2009) and Agnihotri et al. (2009) reported 20-fold shoot multiplication rate with propagule size of 3–5 shoots in D. hamiltonii. A propagule of 7–10 shoots was found optimum (supporting 5–6 fold multiplication rate) for large scale propagation of D. asper and D. hamiltonii (Singh et al. 2011, 2012a).

Culture duration

Sub-culturing of shoots is usually done at periodic interval of 3–4 weeks so as to maintain healthy cultures. Longer sub-culture durations usually lead to longer and pale shoots which gradually turn brown to black instead of enhancing the multiplication rate further (Mudoi and Borthakur 2009; Bisht et al. 2010; Singh et al. 2012a). The available nutrients in the culture medium become a limiting factor hampering the health of shoots.

Rooting of shoots

Induction of roots in excised shoots and subsequent survival of plantlets in the soil are the most crucial steps for success of any micropropagation protocol. Shirgurkar et al. (1996) and Watanable et al. (2000) reported rooting of Pleioblastus pygmaeus shoots in MS basal medium without growth regulators. The role of auxins in root development is well established and has been reviewed by Scott (1972) and Torrey (1976). Different auxins differ in their physiological activities depending upon the extent to which they move through tissues, remains bound inside the cells, or gets metabolized. Usually there is sufficient residual cytokinin in the shoots, thus little or no cytokinin is required for root induction. IBA alone or in combination with NAA are the most commonly used growth regulators for rooting in bamboos. Although, some reports are available where cytokinins (BAP or Kn) in combination with auxins were used for root induction in bamboos (Table 1). Addition of choline chloride along with IBA enhanced the rooting response in D. hamiltonii upto 89 % (Singh et al. 2012a) in comparison to the earlier report of Sood et al. (2002) where rooting response was not consistent and only 25–30 % of the shoots developed into plantlets. On the other hand, Saxena (1990) and Ramanayake and Yakandawala (1997) accomplished rooting of B. tulda and D. giganteus shoots respectively in the presence of coumarin. Usually a single step procedure is used for rooting of shoots in bamboos, however, a two step procedure has also been used for rooting of D. hamiltonii shoots (Agnihotri and Nandi 2009; Agnihotri et al. 2009) and a very high rooting rate (>90 %) was reported when the propagules were cultured on IBA supplemented medium for a week followed by transfer to IBA-free medium. Similarly, Singh et al. (2011) and Bag et al. (2012) also observed very high rooting efficiency (100 %) in D. asper and D. hamiltonii, respectively, while Singh et al. (2012a) reported 89 % rooting in D. hamiltonii. Shoots cultured on ½ MS medium gave better response among the four strengths tested (¼, ½, ¾ and full) in D. asper and D. hamiltonii (Singh et al. 2011, 2012a). This was attributed to reduction in total nitrogen required for rooting (Ajithkumar and Seeni 1998).

Acclimatization and field transfer

Lab to land transfer remains the major bottleneck in commercialization of tissue culture technique. This is mainly because of the shock which the in vitro raised plantlets experience when they are transferred from in vitro culture environment with low irradiance and high humidity to natural environment with high irradiance and low humidity. The in vitro raised plants usually have leaves with poor or no development of cuticular wax, impaired stomatal mechanism, low photosynthetic pigments, biochemicals e.g. carbohydrates, proteins, proline and phenols, poor photosynthetic activity, poor vascular development and connections, etc. Therefore, an efficient hardening and acclimatization technique is necessary to ensure better survival of in vitro raised plantlets in the field. Gradual reduction in the supply of nutrients and humidity during these procedures forces the plant to strengthen its own photosynthetic and defense mechanisms, and prepare them to grow under in vivo conditions. The healthy rooted plantlets are usually transferred to seedling trays or polybags containing different types of potting mix like soil, sand, soilrite, perlite, vermiculite, compost or farm yard manure either alone or in various ratios (Mishra et al. 2011; Singh et al. 2011) and maintained under high humidity. Initial application of reduced MS minerals to the plantlets has been found essential for their better acclimatization. After 2 to 3 weeks growth in mist chamber, the plants are transferred to net house for hardening for another 2 to 3 weeks. Addition of vermi-compost to the sand was found to improve the survival of plants probably due to increased porosity of sand and better aeration of roots (Singh et al. 2011). Verma and Arya (1998) studied the effect of arbuscular mycorrhizal fungal isolates and organic manure on growth and mycorrhization of micropropagated D. asper plantlets and spore production in their rhizosphere. Finally the acclimatized and hardened plants (1 to 2 ft height) are transferred to the field under natural conditions. Season of field transfer has also been found to influence the survival rate and growth of the plantlets (Mishra et al. 2011; Singh et al. 2011). Plantlets transferred in the months of July to August showed higher survival rate with sprouting of more new shoots than other months.

Macroproliferation, a method of plant multiplication by separating the rooted tillers has been used by many workers for enhancing the rate of multiplication of in vitro raised plants and for continuous supply of plantlets. Splitting of rooted tillers could double the production of D. asper plants (Singh et al. 2011), while three-fold increase was achieved in B. tulda (Mishra et al. 2011) and B. balcooa (Mudoi and Borthakur 2009).

Micropropagation has been widely used for rapid mass multiplication of bamboos, however, its application on commercial scale is restricted often due to high rate of plant loss when transferred to natural or ex vitro conditions. Only few reports are available regarding successful field transfer of micropropagated bamboos. Arya et al. (1999) reported 95 % field survival of D. asper and transferred 6,000 plants raised through seed tissue culture to the field. Sood et al. (2002) and Agnihotri et al. (2009) reported a survival percentage of 70 % in the field for the plants of D. hamiltonii. Mishra et al. (2011) reported 91 % survival of the plants of B. tulda in the green house. Negi and Saxena (2011) have successfully produced 2,500 plantlets with 95.83 % hardening rate up to nursery stage and transferred 12 plants with 100 % success in the field. Singh et al. (2011, 2012a) transferred 25,000 and 3,000 plants of D. asper and D. hamiltonii respectively to the Forest Department land in Yamunanagar, Haryana under the DBT’s Bamboo Mission. They reported a success rate of 92.34 % and 100 % for D. asper and D. hamiltonii in the green house, while 79.76 % and 85 % success was achieved in the field. Morphological growth variations were not observed among these plants over a period of 1–2 years. Few other reports have also documented good field performance of the tissue culture raised plantlets (Nadgir et al. 1984; Saxena 1990; Mudoi and Borthakur 2009; Agnihotri et al. 2009). Besides evaluating the morphological parameters, physiological parameters like photosynthesis, transpiration, water use efficiency, etc. have also been compared with mother plants in D. hamiltonii (Agnihotri and Nandi 2009; Agnihotri et al. 2009). The rate of photosynthesis increased from 3.55 CO₂ l mol m⁻² s⁻¹ (hardened plants, ready for field transfer) to 5.44 l mol CO₂ m⁻² s⁻¹ (6 months of field transfer); after a year of plantation, the rate of net photosynthesis was 14.0 l mol CO₂ m⁻² s⁻¹, while after 1.5 years it was 12.76 CO₂ l mol m⁻² s⁻¹. These values are comparable to those observed for the mother bush. Transpiration rate also increased simultaneously with the age of the plant. Water used efficiency also showed a similar pattern like net photosynthesis. A similar trend was also observed for Ci/Ca ratio for 18-month-old field transferred plants and the mother bush with values of 0.497 and 0.617, respectively. Similarly, Bag et al. (2012) compared 18 months old filed transferred in vitro propagated plants of the two age groups, and the corresponding mother plants (MPs) of D. hamiltonii in respect of gas and water vapour exchange rates, related parameters, morphological features and leaf anatomy. The rate of photosynthesis was significantly influenced by the age of the MPs and was found to be higher in the tissue culture (TC)-raised plants; plants (both TC-raised and MPs) of the younger age group performed better than the corresponding plants of the older age group. The same trend was found when the water-use efficiency was taken into consideration. Many groups have tested the genetic fidelity of the tissue culture raised plants using molecular markers also. The same will be discussed in the section on achievements made using molecular approaches.

Somatic embryogenesis

Micropropagation via somatic embryogenesis offers another easy and reliable method for mass propagation as both the root and the shoot primordia are produced in a single step. It can be used for large scale propagation of bamboo at minimum cost in a relatively shorter time and with lowest labor inputs. Encapsulation of somatic embryos in alginate beads to produce synthetic seeds holds great promise for establishment of bamboo plantations. Intensive research on tissue culture of bamboos related to somatic embryogenesis was initiated by Mehta et al. (1982) with the production of plantlets of Bambusa arundinacea. After that, somatic embryogenesis and plantlet regeneration has been reported in several bamboo species (Table 3). Recently, Bag et al. (2012) reported maximum embryogenesis (93.3 % and 90.0 %) with highest number of somatic embryos (38.7 and 37.3 per callus lump) and regenerated plantlets (11.9 and 11.3) per callus lump from 10- and 45-year-old D. hamiltonii bushes respectively, on MS medium supplemented with 5.0 μM BAP and 7.5 μM 2,4-D.

In general, embryogenic tissue is initiated on a medium containing low concentration of auxins, usually in the form of 2,4-D and NAA, and cytokinins (BA and TDZ). Mostly, MS medium has been used for embryogenesis in bamboos, however, in few studies other media such as B5 and N6 have also been used for somatic embryogenesis. The generation of morphologically developed somatic embryos does not guarantee satisfactory post-embryonic performance. Embryo development in bamboos is initiated by arresting cell proliferation through the removal of auxins and cytokinins and putting them on PGR free medium (Godbole et al. 2002). Although, Rout and Das (1994) reported development, maturation as well as germination of somatic embryos on (MS) basal medium supplemented with Kn, 2, 4-D and AdS. In general, maturation of somatic embryos is achieved on agar solidified media, however, as demonstrated by Hassan and Debergh (1987) somatic embryos can also be obtained in liquid medium. Mature somatic embryos germinate and convert to plantlets in a growth regulator free medium (Godbole et al. 2002). Although in some studies, cytokinin has been found to be an essential component in germination of bamboo somatic embryos. Kn was used to promote the germination of B. oldhamii, B. beecheyana and Sinocalamus latiflora somatic embryos (Yeh and Chang 1986a, b, 1987), while Lin et al. (2004) used TDZ for somatic embryo germination in B. edulis.

Flowering of bamboos in vitro

The most unique feature of bamboos is their monocarpic flowering behaviour (John and Nadgauda 1999). Most bamboos flower (and seed) gregariously at the end of long vegetative growth phases, ranging between 3 and 120 years or more (Janzen 1976; John and Nadgauda 1999) and usually die after flowering. This characteristic makes the study of bamboo flowering quiet difficult. It has been almost impossible to breed woody bamboos for superior traits due to these peculiar flowering habits. The first reports on flowering of bamboos in tissue culture (Nadgauda et al. 1990; Rao et al. 1990) created great excitement among the plant biologists. Since then tissue culture systems have been significantly used to reduce the juvenility stage of bamboo, and many studies have shown that bamboos flowered in vitro in just a few months (Table 4). It opened up the possibility of controlled flowering that can be used for breeding of bamboos. There is no synchrony in the timings of anthesis under in vitro conditions, whereas in nature the timing of anthesis is influenced by environmental conditions and usually takes place in the morning hours. Once in vitro methods are standardized for obtaining flowering comparable to that observed in nature, this technology can be used for attempting hybridization between bamboo species (Nadgauda et al. 1993).

Table 4.

In vitro flowering studies in bamboos

Species	Medium + PGRs	References
Bambusa sp., Cephalostachyum pergacil, Dendeocalamus membranaceus	–	Prutpongse and Gavintertvatana 1992
Bamboo sp.	B5 + BAP + 2,4-D	Rao and Rao 1990
B. arundinacea	MS + BAP + NAA	Ansari et al. 1996
	MS + BAP + CW	Nadgauda et al. 1997
	MS + BAP	Joshi and Nadgauda 1997
B. arundinacea, D. strictus, D. brandisii	MS + BAP + CW	Nadgauda et al. 1990
B. arundinacea, D. strictus, D. brandisii	MS + BAP	John and Nadgauda 1999
B. edulis	MS + TDZ + 2,4-D	Lin and Chang 1998
	MS + TDZ	Lin et al. 2003, 2004
	MS + 2,4-D + IBA + NAA	Lin et al. 2005
B. oldhamii	MS + 2,4-D + Kn	Ho and Chang 1998
B. vulgaris, D. strictus, D. giganteus	MS + IBA + AdS + GA3	Rout and Das 1994
D. asper	MS + BAP	Satsangi et al. 2001
D. giganteus	MS + BAP	Ramanayake et al. 2001
D. hamiltonii	MS + BAP	Chambers et al. 1991
D. latiflorus	MS + 2,4-D	Lin et al. 2007b
D. strictus	½ MS + TDZ	Singh et al. 2000

AdS adenine sulphate, BAP 6-benzylaminopurine, CW coconut water (milk), 2, 4-D 2, 4 Dichlorophenoxy acetic acid, GA₃ gibberellic acid, IBA indole-3-butyric acid, Kn kinetin, NAA α-Napthaleneacetic acid, PGR plant growth regulator, TDZ Thidiazuron

In vitro flowering is an important phase in growth, development and physiological science. It is a difficult phenomenon sensitive to the environment. The conversion from vegetative to reproductive phase in vitro is thought to be regulated by external and internal factors, which include plant growth regulators, auxin-cytokinin equilibrium and genotypic variation, nutrients, pH of the medium and light conditions (Heylen and Vendrig 1988) which interact in complex and erratic ways (Van Tran Thanh 1973; Teixeira da Silva and Nhut 2003). More than 10 years after the first report on in vitro flowering in bamboo some significant results have been obtained, but practical and commercially exploitable results have not been reported yet.

Investigations have revealed that cytokinin is a key factor for in vitro flowering of bamboos (Nadgauda et al. 1990; Chambers et al. 1991; Rout and Das 1994; Lin and Chang 1998). Cytokinins are constituents of floral stimulus transported from phloem sap to the apical part stimulating in vitro flowering (Bernier et al. 1993). These are required to keep up the cell division cycle but might also be involved in promoting the transition from undifferentiated stem cells to differentiation (Werner et al. 2001). Lin et al. (2003) reported that multiple shoots grown from spikelet-derived somatic embryos of Bambusa edulis flowered on MS medium containing TDZ, while NAA was a negative regulator for cytokinin-dependent in vitro flowering. TDZ was also found effective in the induction of flowering in the cultures of D. latiflorus (Lin et al. 2004) and D. strictus (Singh et al. 2000). The cultures behave like natural plants during in vitro flowering as the rate of shoot proliferation gradually increases to over three-fold before flower induction. However, in vitro flowering was not the expression of a species specific mechanism believed to occur during gregarious flowering, as the mother clump did not flower (Ramanayake et al. 2001). In most of the studies, flowering was induced in medium supplemented with BA. Rout and Das (1994) used adenine hemisulphate, IBA and gibberellic acid for flower induction in B. vulgaris, D. giganteus and D. strictus. Many other cytokinins such as AdS, 2iP, Kn and zeatin tested were ineffective and presence of BA in the culture medium was absolutely essential for induction of flowering. Although, 2iP showed synergistic effect in combination with BA, zeatin showed antagonistic effect on induction of flowering (Joshi and Nadgauda 1997). However, Prutpongse and Gavintertvatana (1992) reported that in 8 species of bamboo, flowering was not affected by the culture conditions like light, medium, temperature, etc.

The long and unpredictable flowering cycles, and gregarious flowering is speculated to be genetically programmed. It is now known that genes control flowering in plants and that the expression of these genes is due to endogenous or exogenous signals. Some of these genes control the transition of the meristem from a vegetative to a reproductive state, while others control when to flower (Hempel et al. 1997). However, much needs to be done to understand the precise mechanisms controlling these unique flowering characteristics in bamboos.

Transformation and transgenics

Introduction of foreign genes into plant cells can be achieved by a variety of methods including particle bombardment, electroporation, silicon carbide, polyethylene glycol and Agrobacterium. Table 5 summarizes the reports pertaining to cloning and transgenic development in bamboos. Luciferase genes have been cloned from fireflies (Photinus pyralis) and successfully transferred into D. giganteus using Agrobacterium tumefaciens binary vector (Wiersma 2008). Optimization of the cell culture conditions of target plant cells/tissues is one of the most important factors for transformation studies. Transient expression of the GUS gene in the log phased suspension cells of Phyllostachys nigra indicated that cells having active growth efficiency were ideal targets for transformation using the particle bombardment method (Ogita et al. 2011). In order to construct transgenic bamboo cells with high efficiency, two problems needs to be resolved—establishment of an efficient suspension cell culture system and an improvement of the transformation procedure.

Table 5.

Cloning, transformation and transgenic studies in bamboos

Tools used	Species	Achievements	References
Agrobacterium	Dendrocalamus giganteus	Luciferase genes cloned from fireflies (Photinus pyralis) were successfully transferred into D. giganteus using A. tumefaciens binary vector	Wiersma 2008
Cloning	Bambusa oldhamii	Cloning and characterization of catechol-O-methyltransferase (COMT) gene	Li et al. 2007
	D. latiflorus	A cDNA named DlMADS8 was isolated from the young spikelets of D. latiflorus by rapid amplification of cDNA end (RACE) and transformed into Arabidopsis thaliana. Transgenic plants of DlMADS8 exhibited the phenotypes of curled leaves and early flowering. After bolting, three novel phenotypes related to inflorescence development were observed in different transgenic plants. No obvious homeotic conversions of floral organs were observed in all of the 35S::DlMADS8 transgenic Arabidopsis plants. These results indicated that DlMADS8 probably plays a role in floral meristem determinacy and is involved in controlling the flowering time of D. latiflorus.	Tian et al. 2006
	Neosinocalamus affinis	Cloned 4-coumarate-coenzyme A ligase gene (EU327341)	Hu et al. 2009
Particle bombardment	Phyllostachys nigra	Generation of stable transgenic bamboo cells using constructs expressing hygromycin- phosphotransferase gene and enhanced fluorescent protein genes namely AcGFP1 and mCherry	Ogita et al. 2011

Achievements made using molecular approaches

Polymorphism and phylogenetic relationships

Vegetative and floral characteristics have been used for taxonomic studies and to assess diversity within and between populations in majority of plant species since ancient times. Morphological features are readily available visually and additionally do not require sophisticated equipments for data documentation. However, morphological determinations need to be taken by an expert taxonomist as they are subject to changes due to environmental factors and may vary at different developmental stages (Kalia et al. 2011a). Therefore, taxonomic studies mainly depend on the inflorescence and floral morphology however, flowering has been a major bottleneck in these studies and the basic knowledge of biology and genetics of bamboos is severely lacking (Janzen 1976).

The limitations of morphological markers were complimented with the markers developed at both protein and DNA level. Leaf isozymes were used by Heng et al. (1996) to detect polymorphism among five genera of bamboos. However, the number of isozyme loci that can be scored is limited. To date, only 40–50 reagent systems have been developed that permit the staining of a particular protein. A second drawback of biochemical markers is tissue variability. Therefore, several samplings of the segregating population are necessary to score all the available isozymes. Moreover, protein markers are also influenced by the environmental and developmental changes. Due to these reasons isozymes are not preferred markers for diversity analysis.

Under such circumstances, only molecular approaches are the useful techniques for characterizing the genetic diversity among different cultivars or species, for identifying genes of commercial interest and improvement through genetic transformation technology, and for taxonomic delineation of bamboos especially at lower levels of species and subspecies. Molecular markers are powerful enough to discriminate closely related varieties also. At the nuclear level, several markers viz. RFLP, RAPD, AFLP, SCARs, ISSR, SSRs, EST-SSRs and MITE-TD have been widely applied in genetic variation, systematic classification, and phylogenetic relationships among bamboos, with more or less success. Table 6 summarizes the applications of morphological, biochemical and molecular markers to study polymorphism and phylogenetic relationships in bamboos.

Table 6.

Application of morphological, biochemical and molecular markers to study polymorphism and phylogenetic relationships in bamboos

Tools used	Species	Achievements	References
Morphological and biochemical markers
Morphological descriptors	Bamboo sp.	Evaluated phylogenetic relationships among 15 bamboo species using 32 key morphological descriptors	Das et al. 2007
Isozymes	Bamboo sp.	Detected polymorphism in five bamboo species using leaf isozymes	Heng et al. 1996
Molecular markers
RFLP	Phyllostachys sp.	Detected 380 polymorphic bands using 43 probe enzyme combinations in 12 species	Friar and Kochert 1991, 1994
RAPD	Bambusa, Dendrocalamus, Sasa, Dinocloa, Cephalostachyum	Identified genetic relationship between 12 bamboo species belonging to 5 genera	Nayak et al. 2003
	Bambusa sp.	Investigated relationships between samples of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus, by different authors	Sun et al. 2006
	Bamboo sp.	Evaluated phylogenetic relationships among 15 bamboo species	Das et al. 2007
	Dendrocalamus, Bambusa, Gigantochloa, Arundinaria sp.	Reported that genetic distances between genera Bambusa and Gigantochloa are smaller while Dendrocalamus and Arundinaria has greater and greatest distances from other species, respectively	Ramanayake et al. 2007
	Phyllostachys sp.	Assessed phylogenetic relationships among 73 genotypes	Gielis et al. 1997
SCAR	B. balcooa, B. tulda	Generated species-specific SCAR fragments named ‘Balco836’ for B. balcooa and ‘Tuldo609’ for B. tulda	Das et al. 2005
AFLP	Bambusa, Dendrocalamus, Gigantochloa, Thyrsostachys	Examined 15 species from 4 genera and found that 6 species of Bambusa separated into 2 clusters while 6 species of Gigantochloa formed a discrete cluster. Thyrsostachys was less similar to Bambusa while two Dendrocalamus species were very different and required further study	Loh et al. 2000
	Bambusa sp., Dendrocalamus sp.	Studied phylogenetic relationship and genetic variability among 12 edible bamboo species (Bambusa and Dendrocalamus genus) of North-Eastern India using six primer pair combinations.	Ghosh et al. 2011
	Bamboo sp.	Analyzed phylogeny of world bamboos by AFLP of chloroplast DNA	Kobayashi 1997
	Guadua angustifolia	Conducted AFLP analysis of Guadua germplasm in Colombia with emphasis on the coffee region.	Marulanda et al. 2002
	Phyllostachys pubescens	Could clearly identify ten cultivars of P. pubescens that had high similarity and divided them into three groups based on genetic variation and similarity.	Lin et al. 2009
	P. pubescens	Analysis of clonal structure and flowering traits of bamboo species	Isagi et al. 2004
	Phyllostachys sp.	Phylogenetic studies in genus Phyllostachys	Hodkinson et al. 2000
	Sasa senanensis	Studied clonal structure of a dense population of this dwarf bamboo in a 10-ha study plot at Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan	Suyama et al. 2000
SSR	Bamboo sp.	Evaluated the transferability of 98 SSR markers of rice and 20 EST-SSR markers of sugarcane for phylogenetic and genetic diversity analysis in 23 bamboo species.	Sharma et al. 2008
	Bamboo sp.	120 rice SSR markers were assessed for their transferability to 21 different bamboo species. The transferability was 68.3 %. SSR markers located on rice chromosome 7 and 1 showed the highest and lowest transferability, respectively to the bamboo genome.	Chen et al. 2010
	Bambusa arundinacea	Characterized 6 microsatellites, three polymorphic and three monomorphic, in B. arundinacea and tested cross species amplification in 18 other bamboo species.	Nayak and Rout 2005
	Guadua sp.	Demonstrated the usefulness of rice and sugarcane microsatellite sequences to establish the relationships between genotypes, varieties and cultivars of Guadua	Marulanda et al. 2007
	Phyllostachys sp.	Analyzed 1,532 P. pubescens SSR sequences available in public domain DNA databases, and found 3,241 SSR loci comprising repeats of two or more nucleotides in 920 genomic survey sequences (GSSs) and 68 cDNA sequences. SSR PBM014 transferred successfully to six other Phyllostachys species and showed rich polymorphism, therefore could serve as species-specific marker for Phyllostachys interspecies hybrid identification.	Tang et al. 2010
	Phyllostachys pubescens	Studied 176 samples of Phyllostachys in Taiwan and found limited genetic variation. The region around Nantou County consisted of all of the nine identified clones while the remaining regions generally consisted of only one common clone which indicated that center of variation is in Nantou County.	Lai and Hsiao 1997
EST-SSR	Arundinaria, Bambusa, Brachystachyum, Hibanobambusa, Indocalamus, Phyllostachys, Pseudosasa, Sasa, Semiarundinaria, Shibataea, Sinobambusa	Used EST-SSR markers derived from major cereal crops to assess the genetic diversity and phylogenetic relationships of a temperate bamboo collection of USDA consisting of 92 accessions, 11 genera and 44 species	Barkley et al. 2005
	Bambusa edulis, B. oldhamii	Analyzed 3406 publically available ESTs from caespitose bamboo species (B. edulis and B. oldhamii) and found 245 non-redundant SSRs in 205 EST contigs that were used to develop 15 EST-SSR markers. The transferability of markers was 59.6 % among 14 additional caespitose bamboo species. The successfully transferred markers showed 51.4 % polymorphism.	Dong et al. 2011
	B. oldhamii	Selected 10 EST-SSR markers from B. oldhamii public sequence data base and observed their transferability to 25 species of Bambusoideae. Transferability ranged from 30 to 100 %.	Sharma et al. 2009
	Phyllostachys edulis	Development of EST-SSR markers	Zhi-jun et al. 2011
	P. rubromarginata, P. flexuosa, P. glauca	Detection of contamination in a bamboo plot where P. rubromarginata stands were mixed with either P. flexuosa or P. glauca.	Yu et al. 2004
ISSR	Bamboo sp.	Evaluated genetic relationships among 22 taxa of bamboo using 12 ISSR and four EST-based random primers, resulting in amplification of 220 loci.	Mukherjee et al. 2010
	Phyllostachys pubescens	Reported that ten cultivars of P. pubescens having high similarity could be divided into three groups	Lin et al. 2009
	P. violascens	Assessed genetic diversity within different cultivars of P. violascens using 15 ISSR primers and a total of 209 (136 polymorphic) bands were detected. Based on genetic diversity, all the cultivars of P. violascens could be divided into four groups, which are reflected by their morphologies.	Lin et al. 2011
MITEs	B. multiplex	Presence of Ac-like sequences was found	Huttley et al. 1995
	B. vulgaris, Sasa veitchii, Phyllostachys edulis	Isolated partial Ac-like transposon elements	Gielis 1998
	B. vulgaris	Obtained sequence from B. vulgaris that revealed considerable homology to the HAT superfamily of transposons	Keukeleire et al. 2004
	P. pubescens	Observed that 23.28 % of P. pubescens genome is enriched with repeat elements and majority of them (18.89 %) were LTR retrotransposons, mainly Gypsy/DIRS1 and Ty1/Copia type	Jie et al. 2007
	Bamboo sp.	Isolated 79 full-length MLE (Mariner-like elements) transposase genes from 63 bamboo species representing 38 genera in six subtribes mainly found in China. The transposases were highly conserved, mostly uniform in length and contained intact DNA-binding motifs and DD39D catalytic domains with few notable frameshift, indel and nonsense mutations.	Zhou et al. 2011
Cp DNA	Asian bamboos	Examined restriction site mutations of cpDNA for 16 Asian bamboo genera	Watanable et al. 1994
	Bamboo sp.	Utilized rpl 16 intron data to study relationships between 23 species of Chusquea and 15 taxa from Bambusoideae	Kelchner and Clark 1997
	Bamboo sp.	High-throughput sequencing of six bamboo chloroplast genomes	Zhang et al. 2011a
	Bamboo sp.	Studied polymorphism, similarities and relationships among 22 bamboo species using RAPD of chloroplast DNA (RACPD)	Zhang et al. 2011b
ITS Sequences	Arundinaria sp.	Analyzed phylogenetic relationships of Arundinaria and related genera (Pleioblastus, Bashania, Pseudosasa, Oligostachyum, Clavinodum, etc.) using nrDNA ITS sequences and the cpDNAtrnL-F intergenic spacer (IGS)	Qiang et al. 2005
	Phyllostachys sp.	Made a comparison of nrDNA ITS sequences for phylogenetic studies in genus Phyllostachys to review the previous infra-generic classifications	Hodkinson et al. 2000
RT-PCR, cDNA library	B. oldhamii	Four cDNA clones, BoSus1, BoSus2, BoSus3 and BoSus4, were isolated by screening a cDNA library from etiolated bamboo shoots and suggested that, sucrose synthase (SuS) is encoded by at least four genes in bamboo, each with a specific role in providing substrates for the polysaccharide biosynthesis and/or energy production	Chiu et al. 2006
SSH and Microarray analysis	B. edulis	Identified differentially expressed genes in an albino mutant. These genes were not related to photosynthesis	Lin et al. 2006
RT-PCR and microarray analysis	Phyllostachys praecox	Identified several genes related to development of bamboo rhizome bud and cloned six genes, the expression patterns of these genes revealed significant differences in rhizome shoots, rhizome buds, bamboo shoots, leaves, and young florets	Wang et al. 2010

AFLP amplified fragment length polymorphism, Cp DNA chloroplast DNA, EST expressed sequence tag, ISSR inter simple sequence repeat, ITS internal transcribed spacer, MITEs miniature inverted-repeat transposable elements, RAPD randomly amplified polymorphic DNA, RFLP restriction fragment length polymorphism, RT-PCR real time polymerase chain reaction, SCAR sequence characterized amplified regions, SSH suppression subtractive hybridization, SSR simple sequence repeat (microsatellite)

Genetic fidelity testing

In vitro propagation has emerged as a powerful technique for large scale propagation of bamboos. However, culture conditions, explant source, ploidy level and in vitro culture age are known to induce somaclonal variation in vitro. These somaclonal variations may appear due to cell cycle disturbances caused by exogenously applied growth regulators, increased mutation rate per cell-generation over time, and accumulation of mutations over a period of time, alteration in DNA methylation patterns, DNA damage and mutation, and alteration of cell’s ability to repair damaged and mutated DNA (see Singh et al. 2012b). It is therefore extremely important to ascertain the clonal uniformity of the in vitro raised plants. Although, morphological, biochemical, physiological and anatomical parameters such as leaf shape, thickness, leaf mass, chlorophyll and relative water content, photosynthetic parameters and leaf anatomy etc. have been used for the purpose in D. asper and D. hamiltonii (Agnihotri et al. 2009; Singh et al. 2011; Bag et al. 2012), use of more reliable DNA based markers such as RAPD, ISSR, SSR, AFLP, etc., has also been done to test the genetic fidelity of in vitro raised bamboo plants. Amplification of monomorphic bands in tissue culture raised plants and mother bushes confirmed that the former were genetically uniform and true to type to the mother in these studies (Table 7). Use of more than one marker system has been suggested by various workers so as to target a wider region of the genome. Singh et al. (2012b) used a set of four markers namely RAPD, ISSR, SSR and AFLP to access the genetic fidelity of D. asper plants raised through axillary shoot proliferation. Effect of length of culture age on genetic stability was studied in B. balcooa (33 subculture cycles, Negi and Saxena 2010), B. nutans (27 passages, Negi and Saxena 2011) and D. asper (30 subculture passages, Singh et al. 2012b), however, it was not found to affect the genetic stability of the plants raised through enhanced axillary branching.

Table 7.

Genetic fidelity testing of in vitro raised bamboos

Tools used	Species	Achievements	References
Morphological, biochemical, physiological and anatomical markers
Morphological descriptors	Dendrocalamus asper	Compared in vitro-raised plants with mother plants and found no variation.	Singh et al. 2011
	D. hamiltonii	Leaf thickness and specific leaf mass of the in vitro raised plants were found comparable to the mother plants.	Bag et al. 2012
Biochemical analysis	D. hamiltonii	Chlorophyll and relative water content of the in vitro raised plants were found comparable to the mother plants.	Bag et al. 2012
Physiological studies	D. hamiltonii	The rate of net photosynthesis and water use efficiency were found comparable to those observed for the mother bush.	Agnihotri et al. 2009
		The rates of photosynthesis and water use efficiency of in vitro propagated and hardened plants were found to be comparable with the corresponding mother plants.	Bag et al. 2012
Anatomical studies	D. hamiltonii	Leaf anatomy of the in vitro propagated and hardened plants was found to be similar with the corresponding mother plants.	Bag et al. 2012
Molecular markers
RAPD	Bambusa balcooa, B. tulda	Confirmed clonal fidelity of in vitro raised plants and advocated the use of axillary meristem culture for true-to-type or clonal propagation.	Das and Pal 2005b
	D. asper	Tested clonal fidelity of in vitro raised shoots uo to 30th passage, hardened plants growing in the ployhouse and randomly selected field transferred plants up to 2 years with mother plant and found no somaclonal variation.	Singh et al. 2012b
	D. hamiltonii	Reported genetic fidelity during various stages of growth and development of in vitro raised plants, up to 1.5 years after field plantation and found no somaclonal variation.	Agnihotri et al. 2009
	Guadua angustifolia	Evaluated clonal fidelity of in vitro raised plants at various stages of subculture along with hardened plants and compared with mother plant.	Nadha et al. 2011
ISSR	B. balcooa	Clonal fidelity testing of in vitro raised plants up to 33rd passage and in vitro raised plants transferred to the field compared with parent plant and found true to type.	Negi and Saxena 2010
	B. nutans	In vitro raised shoots up to 27th cycle of shoot multiplication, hardened plants growing in the ployhouse, plants growing in the field and mother plant were found genetically similar.	Negi and Saxena 2011
	D. asper	Tested clonal fidelity of in vitro raised shoots uo to 30th passage, hardened plants growing in the ployhouse and randomly selected field transferred plants up to 2 years with mother plant and found no somaclonal variation.	Singh et al. 2012b
	G. angustifolia	Evaluated clonal fidelity of in vitro raised plants at various stages of subculture along with hardened plants and compared with mother plant.	Nadha et al. 2011
SSR	D. asper	Tested clonal fidelity of in vitro raised shoots uo to 30th passage, hardened plants growing in the ployhouse and randomly selected field transferred plants up to 2 years with mother plant and found no somaclonal variation.	Singh et al. 2012b
AFLP	B. balcooa	Compared the tissue culture raised plants originating through axillary bud proliferation and somatic embryogenesis and reported that no epigenetic changes could be detected by methylation sensitive AFLP (MSAP)	Gillis et al. 2007
	B. nutans	Assessment of genetic fidelity of tissue culture raised plants at various stages from plant regeneration to field establishment. Reported 98.8 % genetic stability in regenerated plantlets.	Mehta et al. 2011
	D. asper	Tested clonal fidelity of in vitro raised shoots uo to 30th passage, hardened plants growing in the ployhouse and randomly selected field transferred plants up to 2 years with mother plant and found no somaclonal variation.	Singh et al. 2012b

Future prospects of bamboo biotechnology

Bamboo is becoming an increasingly important economic asset in poverty eradication, and economic and environmental development. About 2.5 billion people in the world depend economically on bamboo and the international trade in bamboo amounts to $5–10 billion. With the growth in the demand for environment friendly green products, the world bamboo market is expected to double by 2015, from USD 10 billion to USD 20 billion (Xuhe 2003). Traditionally used as low-cost construction material in developing countries, bamboo is being processed into increasingly sophisticated products that serve consumers in developed countries and high end markets. Nowadays, with new technologies for processing, most products made from wood can be made from bamboos, resulting in the potential for a multi-billion dollar market. In such conditions, it is essential to adopt the techniques like tissue culture for mass multiplication of bamboo to fill the gap of demand and supply. This must be followed by genetic fidelity testing of tissue culture raised plants to ensure their true to type nature. In a similar endeavour, the Department of Biotechnology (DBT), Government of India established a Bamboo Mission, a network project on bamboo resources under which more than 800 ha of land was planted with tissue culture raised plants of eight bamboo species (DBT 2009–10) by more than 10 Institutions located in various parts of India.

Development of genetically engineered plants capable to counter biotic and abiotic stresses is imperative. However, an efficient regeneration protocol must be in place before genetic transformation studies can be initiated. Micropropagation, using mature as well as juvenile explants, through organogenesis and somatic embryogenesis has been attempted in many bamboo species. However, the problems of endogenous contamination, browning of explants or shoots during multiplication, instability of multiplication rate, somatic mutations and somaclones, albinism of plants, low rooting percentage and limited survival of plants during hardening and field transfer needs further attention. Vascular arbuscular mycorrhizal (VAM) fungal isolates may be incorporated in the hardening media to strengthen the growth and mycorrhization of micropropagated bamboo plantlets. This will improve the rhizosphere as well as the field survival rate of tissue culture raised plants.

Commercial scale application of micropropagation technology is still limited in bamboos. To date, somatic embryogenesis has been achieved in only 26 species (~1.5 % of the total species) belonging to six genera, i.e. Bambusa, Dendrocalamus, Phyllostachys, Sasa, Sinocalamus and Otatea. Therefore, additional efforts are required for standardizing the micropropagation techniques for majority of important bamboo species so that they can be used for improvement and clonal forestry programme using bamboos as well as for ex situ conservation and cryopreservation of rare species or populations. Research on haploid culture, callus culture against stress conditions, development of tolerant cell lines, etc. should also be started as a part of the long-term genetic improvement program. The most promising approach for improving the ornamental use of bamboo germplasm involves in vitro manipulations to exploit the somaclonal variations.

The in vitro regeneration techniques need to be improved further so that they can be used for genetic improvement of bamboo through transgenics. Successful introduction of foreign genes into plant cells is primarily governed by two factors, optimization of culture condition of target plant cells/tissues and transformation procedure. In bamboos, only few reports are available regarding transformation and transgenic development. Further inputs are required for establishment of an efficient suspension cell culture system and development of transformation procedure with high efficiency in bamboos.

Monocarpic flowering habit has made it almost impossible to breed for superior traits in woody bamboos. In vitro flowering has opened up the possibility of controlled flowering that can be used for bamboo breeding. Negligible progress has been made in improving the taxon through conventional breeding programs but some traits which need further improvement include yield, growth in all types of soils, wider climatic adaptation, thornlessness, thick walls, disease and pest resistance, and improved palatability, among others. Scientists are trying to develop hybrid bamboo that will be the solution for energy, paper pulp and bamboo charcoal production. Tissue culture technology can also be used for rescue of hybrid seeds produced by conventional breeding methods. Understanding of the precise mechanisms controlling flowering in vitro can lead to new avenues for hybridization between bamboo species. Conventional breeding programs are time, cost and labour intensive therefore marker assisted selection (MAS) must be used for selecting beneficial genetic traits as well as for assessing the genetic potential of specific genotypes prior to phenotypic evaluation. Molecular markers linked with QTL/major genes for traits of interest must be developed. In addition, availability of a broad genetic base is must for initiating breeding programmes in any given crop. The available genetic base can be broadened using modern tools of biotechnology including in vitro selection, mutagenesis and transgenics (Kalia et al. 2011b).

In addition, conservation of agronomically important cultivars through in vitro methods and cryopreservation must be done to defeat the biological and environmental calamities which may threat the germplasm maintained in situ in field genebanks and germplasm gardens. Cryopreserved material (stored as seeds, ovules, embryos, callus, etc.) can be used successfully for breeding in the future.

Taxonomic delineation of species and subspecies is still controversial and needs to be addressed more rigorously. Efforts have been made to classify the genus based on morphological, biochemical and molecular markers but more inputs are required to confirm the phylogeny and taxonomy of the genus. Major challenge associated with any molecular method is to determine the appropriate taxonomic level at which it is most informative and to correlate it with morphologically definable taxonomic groupings. Considerable progress has been made in this area, but much more needs to be done yet. In contrast to the vast majority of studies done to date on bamboo taxonomy and systematics, investigations on genetic diversity at the population level are in its infancy. Therefore, studies are required to better understand the level of population diversity and clonal structure in bamboos.

One of the potentially emerging areas for bamboo biology is the comparative genomic studies, wherein available genomic information of other well characterized cereal crops could be extrapolated to initiate functional genomics in bamboos. The genomic resources have accumulated rapidly for almost all major lineages of grasses except bamboos, which can seriously hamper our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies and for better understanding of gene and genome evolution that underlies phenotypic and ecological divergence of plants. By January 2009, the number of ESTs deposited in the GenBank ranged from 436,535 to 2,018,337 for rice, wheat, maize, barley, sorghum, sugarcane, and switchgrass, but only 3,087 for bamboos. This creates a major missing link in the grass family for comparative genomics. Considering the high economic importance of bamboo in rural economies and industrial applications, genetic and genomic analyses of bamboo need to be significantly advanced (Peng et al. 2010).

Undoubtedly, the relationship between bamboos and man has travelled a long journey and both remain inseparable due to the outstanding capacity of bamboos to improve the human environment and economy. Judicious utilization and conservation of bamboo resources can bring more benefits to mankind throughout the world.