Introduction

1

Bamboo is a versatile material. Its versatility can be gauged by the fact that

Thomas Edison successfully used a carbonized bamboo filament in his experiment

with the first light bulb. This light bulb still burns today in the Smithsonian Museum

in Washington, DC.

Bamboos are tall arborescent perennial grasses belonging to Bambusoideae,

family Graminae (Poaceae). Bambusoideae consists 75 genera and 1250 species

throughout the world (Soderstrum and Ellis, 1988). India is second after China in

genetic resources of bamboo, consisting of 125 species in 23 genera and 10 exotic

species (Anonymous, 2003). Wide gene pool of bamboos provide scope for selection

of economically and industrially important species for various agro-climatic regions

for plantation and afforestation programmes. Bamboo contributes about 12.8 % of

total forest area in 25 states and union territories in 8.96 million hectares area in the

country (Tiwari, 1992).

Its fast growth, short rotation period, adaptability in variety of soils and agro-

climatic conditions, requirement of low input for maintenance and more importantly

increasing demand in indigenous and global markets, provide scope for cultivation in

agro-forestry, farm forestry, social forestry and degraded land for short term and long

term benefits. A large number of cottage industries are dependent on bamboo for raw

material.

Bamboos are among renewable natural resource with a great impact on global

economy and ecology. The combined value of internal and commercial consumption

of bamboos in the world is to tune of US $ 10 billion and expected to reach about $ 20

billion by 2015 and considered to be a Timber of the 21st century.

Currently, indigenous demand of bamboo is mostly met from the natural

forests. Over exploitation, death after gregarious flowering and short seed viability

lead to poor natural regeneration. A consistent supply of quality bamboo is key to the

growth and development of bamboo based industrial plantation sector as well as

bamboo bio-resource dependent rural communities.

Bamboo has more than 1500 documented uses. Major uses are; 1) Industrial:

pulp, paper and ryan. 2) Agriculture: bamboo baskets, stacking material and

Introduction

2

agricultural implements. 3) Construction: scaffolding 4) Housing: bamboo houses,

disaster resistant bamboo buildings, walling, roofing and structural material 5)

Handicrafts: furniture, cottage industries, bamboo mat, agarbathi and Joss sticks 6)

Fisheries 7) Sericulture 8) Activated charcoal 9) Bamboo shoots, pickle, candy,

chutney, curry, vinegar and food nutrient (mineral and vitamins) 10) Medicinal 11)

Fodder 12) Food (bamboo seeds and shoots) 13) Panels as a substitute of traditional

timber species, plywood, particle board, hard board and medium density board and

14) Other important uses: carbon sequestration, checking soil erosion, water

conservation, wind barrier, biofencing and restoration of degraded land (Tewari,

1992).

Its fast growth, easy to cultivate, wide adaptation, excellent soil binding

capacity, short rotation, multifarious uses are incomparable with other plant species.

Demand of bamboo is estimated to 26.6 million ton and supply is only 13.47 million

ton/year (Anonymous, 2003). New uses of bamboo, particularly as a substitute of

wood, housing sector and value added products will increase further demand of

bamboo. Currently, India is importing timber worth Rs.10,000 crores, annually, which

can be partly prevented by the use of bamboo as a substitute of commercial timber.

Current world market of bamboo is estimated to the tune of US $ 10

billion/year, which will be $ 20 billion by 2015 (Anonymous, 2003). Indian market

size of the domestic bamboo economy is estimated at Rs. 2,043 crores by planning

Commission. The market potential is estimated at Rs.4,463 crores, which is expected

to grow to Rs.26, 000 crores by 2015 (Madhab, 2003)

To meet the indigenous demand, bamboo is extracted mainly from the forests,

which is depleting gradually and will not be sustainable. Plantation outside the forest,

particularly in agroforestry, farm forestry, social forestry and in waste lands are

envisaged as a long term solution to reduce pressure on forest, and will act as a source

of raw material for various uses and to mitigate ecological, environmental and

generation of employment. Planning Commission Govt. of India has envisaged 6.0

million bamboo plantations during 10th and 11th five-year plan to meet the growing

demand, development of bamboo bio- resource and poverty alleviation (Anonymous,

2003).

Introduction

3

Based on the property and assessment of inherent characteristics matching

with end uses, the National Mission on Bamboo Application (NMBA) has selected 15

commercially important bamboo species, viz;

1) Bambusa affins 2) B. bambos 3) B. balcooa 4) B. nutans 5) B. polymorpha

6) B. tulda 7) Dendrocalamus asper 8) D. brandisii 9) D. giganteus 10) D. hamiltonii

11) D. strictus 12) Melaconna baccifera 13) Ochlandra travancorica 14)

Dendrocalamus stocksii (Pseudoxytenanthera stocksii) and 15) Phyllostachys

pubescens.

Pecculiar flowering habit in bamboo has made almost impossible to breed

artificially for superior traits, particularly in woody bamboos (Janjen, 1976). Most of

the bamboo species have long flowering cycle (30-120 years), which is a limiting

factor for the planting programme (Mandal and Subramanian, 1992). Sporadic

flowering is also uncertain and short seed viability period further restrict availability

of seeds as and when required. Bamboos flower at long intervals and in many

instances the plants die after flowering. The length of flowereing cycle varies with the

species. It ranges from three years (Schizosttacchyum elegantiaaium) to 60 years

(Bambusa polymorpha), but for most species it lies between 30-35 years. Genetic

diversity between and within bamboo species provides scope for selection of superior

genotypes, propagation, improvement and cultivation for improved productivity,

quality of product, development of bioresource and sustainable utilization. In the

absence of flowers, certain bamboo species are classified according to their vegetative

characteristics.

Fortunately, most of the bamboos are natural polyploids (Darlington and

Wylie, 1955, Janaki Ammal, 1959) and natural variability occurs in seedling and

natural population in tropical and temperate bamboos (Banik, 1995). Characteristics

and properties are quite variable, which provide scope for the selection of the best

genotypes for planting programmes. Clonal forestry based on one or more elite

selected genotypes if propagated through clonal propagation, allows a considerable

genetic improvement as compared to natural populations (Geilis et.al. 2002).

Maintenance of broad genetic base allows further low risk and better scope of further

improvement (Geilis and Oprins, 1998).

Introduction

4

In India, productivity of bamboo forest is far below (0.3 ton/ha/yr) then

potential (Tewari, 1992). Similarly, productivity of bamboo plantation is low and

varies with the species (3.2 – 10.0 ton/ha/yr), which is far below its potential (20-40

ton/ha/yr). In China, productivity of bamboo plantation and bamboo shoots has

reached to 37 ton/ha/yr and 15 ton/ha/yr, respectively, which is mainly attributed to

the selection of right species, high quality planting material, genotype matching with

the site quality and proper management.

Genetic improvement work on bamboo species is at beginning stage in India.

Selection of Candidate Plus Clumps of Bambusa bambos, Dendrocalamus strictus

and Dendrocalamus stocksii have been carried out in Karnataka by the Sirsi Forestry

College (University of Agricultural Sciences, Dharwad). Tamilnadu Agricultural

University, Coimbatore has also selected candidate plus clumps of Bambusa bamboos

and D. strictus. Arunachal Pradesh Forest Research Institute, Itanagar and Rain Forest

Research Institute, Jorhat are pioneer in the selection of candidate plus clumps of

various bamboo species including Bambusa nutans and establishment of germplasm

bank.

For bamboo, different propagation techniques are used, such as seed based

propagation, offset cuttings, rhizome and culm cuttings (Banik, 1994; Hasan, 1977).

But these methods suffer from serious drawback for large scale propagation. Most of

the classical techniques for clonal propagation are useful for the small scale

production (up to 10,000 plants /yr). For mass scale propagation (>50000 plants /yr)

classical techniques are largely insufficient and inefficient and tissue culture is the

only reliable method (Geilis et al., 2002).

In India, macropropagation of bamboo is through offset cutting, rhizome

splitting, air layering, culm cuttings and culms branch cutting. Culm cutting and

branch cutting are comparatively better methods and varying success have been

reported in various bamboo species viz; B. balcooa (Seethalakshmi et.al., 1983), B.

nutans (Singh et.al., 2002), B. pallida (Nath et. al., 1986), B. tulda (Ahlawat and

Singh, 2000), B. vulgaris (Agnihotri and Ansari, 2000), Ochlandra travancorica and

O. scriptoria (Seethalakshmi et al., 1988), Dendrocalamus stocksii (Oxytenanthera

stocksii), (Yellappa Reddy and Yekanthappa, 1989; Reddy and Devar, 2004).

Introduction

5

An integrated approach of biotechnology has played a key role in the fast

development in the improvement programme of forestry species, including bamboos.

Tissue culture based propagation through axillary shoot proliferation and somatic

embryogenesis have potential for mass production and improvement (Thorpe et al.,

1992). Micropropagation techniques have been employed for propagation of various

bamboo species (Chambers et al. 1991; Prutpongse and Gavinlertvatana, 1992;

Woods et al., 1995).

For large-scale production, efficiency of the propagation methods is important

at the same time genetic stability also has prime concerned. Axillary shoot

proliferation method is safe for production of true to type plants with lesser risk of

aberration and basic research allows identifying endogenous and exogenous factors

for rapid shoot induction, multiplication and rooting (Geilis and Oprins, 1998).

Commercially feasible micropropagation for large-scale propagation is in

practice for species of Arundunaria, Chimonobambusa, Fragesia, Pleiobshibataes

and Yushania (Geilis and Oprins, 1998).

Delhi University, Delhi and NCL, Pune are the pioneers in the bamboo tissue

culture work in the country. Plant tissue culture techniques have been employed for

the micropropagation of bamboo species like; B. bambos, B. tulda and D. strictus for

the mass production of planting material from the seedling explants and to establish

field trials (Rao et. al. 1990b; Saxena, 1990; Saxena and Dhawan, 1999).

Micropropagation has been carried out from the seedling material and very

few reports deal with field grown plant materials either through axillary shoot

proliferation in D. asper (Arya., 1997; Arya et al., 1999), B. tulda (Saxena., 1990), D.

hamiltonii (Chambers et al., 1991), D. strictus (Shirgurkar et al., 1996) P. stocksii

(Sanjaya et al., 2005), B. vulgaris (Aliou Ndiaye et al., 2006), B. nutans (Negi and

Saxena, 2011). In vitro propagation of bamboo through somatic embryogenesis have

been successful in D. strictus (Rao et al., 1985; Mukunthakumar and Mathur, 1992;

Rout and Das, 1994), D. giganteus (Rout and Das, 1994), D. hamiltonii (Godbole et

al. 2002), B. balcooa (Gillis et al. 2007), D. hamiltonii (Zhang et al., 2010) and B.

nutans (Mehta et al., 2010).

Introduction

6

Most of the earliar reports on bamboo micropropagation described below are

based on seedling explants, either through axillary shoot proliferation or by somatic

embryogenesis (Chambers et al., 1991; Woods et al., 1995; Chang, 1995). Very few

studies deal with in vitro cloning from explants of field grown/ mature culm (Hassan

and Debergh, 1987; Huang et al., 1989; Prutpongse and Gavinlertvatana, 1992; Lin

and Chang, 1998). Indeed, technically the propagation of adult plants via axillary

branching is much more difficult than seedling of tropical bamboos. Problem

associated with adult bamboos are:

1. Endogenous contamination,

2. Hyperhydricity and instability of multiplication rate at initial stage,

3. Initially poor rejuvenation and low rate of rooting (Geilis et al., 2002)

The use of starting material (seed or adult plant) and the choice of the

propagation methods are crucial. Disadvantages of seedling material are:

1. Insufficient or no knowledge of genetic background

2. Restricted availability and rapid loss of germination capacity

Somatic embryogenesis can be described as the process by which haploid and

diploid somatic cells develop into structure that resemble zygotic embryos (i.e. bi

polar structure without any vascular connection with parental tissue), through an

orderly series of characteristic embryological stages without the fusion of gametes

(Emons, 1994; Raemakers et al., 1995). One striking characteristic of the somatic

embryo is that it continuous growth phase, resulting from the absence of

developmental arrest (Faure et al., 1998).

Somatic embryogenesis is a very valuable tool to achieving a wide range of

objectives, from the basic biochemical, physiological and morphological studies to the

development of technologies with a high degree of practical application.

Cell dedifferentiation and organ formation in plants precedes critical

biochemical changes, which are manifested through expression/ elevation of enzymes.

They in turn regulate different biochemical pathways and reflect flow of energy and

incorporation of carbon along the different metabolic pathways (Tsala et al., 1996).

The role of certain enzymes in cellular differentiation and organ formation has been

pointed out in the literature. Somatic embryogenesis including organogenesis

Introduction

7

precedes a cascade of rapid biochemical changes such as emergence of new enzymes

and metabolites that are directly or indirectly involved in the process.

Glutamine synthetase is iron sulphur flavoprotein, which regulates nitrogen

input in the biological system through ammonia assimilation (Hirel and Gadal, 1980).

It is expected to play a vital role in growth and development of various organs,

including somatic embyos, shoots and roots.

Nitrate reductase is a metalloflavoprotein i.e. Mo-protein and is a key enzyme

in nitrogen metabolism, regulating first step of reduction of inorganic nitrogen for its

subsequent incorporation into organic forms. The enzyme is considered to be a

limiting factor for higher plant growth (Srivastava, 1990; Lea, 1997), development

and protein production. Nitrate reductase is known to change during organ formation

and plant development (Kenis et al., 1992) as do other enzymes involved in nitrate

assimilation pathway (de la Haba et al., 1988).

Plant peroxidases are glycoproteins characterized by the presence of

oligosaccharide chain linked to protein moiety (Hu and van Huystee, 1989).

Peroxidases occur in multiple forms and act as marker of growth/ organogenesis

possibly due to their ability to catalyze oxidation of many substrates using H2O2. The

enzyme is involved in auxin metabolism and formation of cross links between cell

wall components (Cella and Carbonera, 1997).

The dedifferentiation is also characterized by an increase in phenols, but

decrease in soluble sugars (Thorpe et al., 1978; Tsala et al., 1996). Phenols are

aromatic compounds with hydroxyl groups, which act as stimulator for rooting. The

action of phenolic compounds on plant growth is frequently attributed to their

interaction with IAA-oxidase, thus, regulating IAA levels in vivo (Schneider and

Wightman, 1974). O- dihydroxy phenolics inhibit IAA oxidation and so stimulate

growth.

The soluble sugars are important to the development of plants in several ways.

They are source of metabolic energy that is converted from light and important

constituents of supporting tissues that enable plant ot achieve erect growth. They also

provide carbon skeletons for vital organic compounds that make up the plant.

Introduction

8

Biochemical studies in embryogenic and non-embryogenic callus and plant

regeneration. The genotype, plays a vary important role in the establishment, growth

and subsequent differentiation of callus cultures.

An early biochemical marker for the identification of embryogenic potency

would be of great help for efficient plant regeneration. Reports have been published

on biochemical differences between embryogenic and non-embryogenic callus

cultures with respect to antigens (Khavkin et al., 1977), polypeptide pattern (Chen

and Luthe1987; Strin and Jacobsen, 1987), ethylene production (Wann et al., 1987)

and the amount of tripsin inhibitor (Carlberg et al., 1987). However, these

biochemical systems are either time consuming or insufficiently specific to identify

subsequent stages of development. As an alternative, a simple and rapid identification

of embryogenic callus might be established by isozymes. Isozymes are easily

detectable and their variation is often associated with genetic differences and

developmental stages (Scandalios, 1974).

The application of isozymes as markers in embryogenic culture has been

reported in several studies (Wochok and Burleson, 1974; Negrutiu et al., 1979;

Everett et al., 1985; Key and Basile, 1987; Chawla, 1988). Using starch gel

electrophoresis, Everett et al. (1985) analysed the zymograms of glutamate

dehydrogenase, alcohol dehydrogenase, β, glucoronidase esterase and glutamate

dehydrogenase to distinguish between embryogenic and non-embryogenic cultures.

The initiation and maintenance of embryogenic cultures has been demonstrated in a

great number of plants including many member of poaceae family (Williams and

Maheswaram, 1986).

Embryogenic culture grow slowly and form plants by somatic embryogenesis

while, non-embryogenic calli grow fast, in a disorganized way and form shoots or

root by organogenesis. Both types of calli can be visually differentiated by

morphology but, little is known about the biochemical and molecular event that take

place when somatic cells become competent to produce somatic embryos. A better

knowledge of these culture aspect will have useful application in biochemistry and

molecular identification and characterization of different stages of somatic

embryogenesis and in artificial seed technology.

Introduction

9

Comparing micropropagation through axillary shoot proliferation and somatic

embryogenesis, later method has high potential for rapid and mass production of

planting material. The mass propagation of plants through multiplication of

embryogenic propagules is the most commercially attractive application of somatic

embryogenesis (Merkle et al., 1990). In addition to the clonal propagation, production

of artificial seeds, genetic transformation and conservation of genetic resources are

the other applications of somatic embryogenesis.

There is no published report on studies on somatic embryogenesis from

mature clump vis-a-vis endogenous biochemical changes in embryogenic and non-

embryogenic callus and genetic fidelity of in vitro raised plantlets through somatic

embryogenesis in B. nutans.

Evaluation of genetic fidelity of the micropropagated plants is essential before

large scale/operational planting of micropropagated plants to ensure genetic stability.

Lack of such study may lead to great economic loss due to late visibility of genetic

variation due to long gestation period.

The ultimate success of micropropagation depends upon the ability to transfer

plants out of culture condition on a large scale at low cost and with high survival

rates. Tissue culture conditions in addition to promotion of rapid growth and

multiplication of shoots, results in the formation of structurally and physiologically

abnormal plants. The heterotrophic mode of nutrition and poor mechanism to control

water loss render micropropagated plants vulnerable to transplantation shocks.

Although, considerable efforts have been directed to optimize the conditions for the in

vitro stages of micropropagation, scant attention has been paid to understand the

process of acclimatization of micropropagated plants to the soil environment.

Consequently the hardaning stage continues to be a major bottleneck in the

micropropagation of many plants (Conner and Thomas, 1981; Ziv, 1986).

The genetic stability of in vitro regenerated plants is an essential pre-requisite

for large scale clonal forestry and somaclonal variation is of special relevance in

perennial plants (Skirvin et al., 1994) and long generation forest trees, since

occasional mutations can some times only be noticed at a very late developmental

stages, or even in their offsprings. The tissue culture environment may cause general

Introduction

10

disruption in cellular controls, leading to numerous genomic changes in the tissue

culture derived progeny (Phillips et al., 1994). The occurrence of somaclonal

variation is a potential drawback, when the propagation of mature trees is intended,

where clonal fidelity is required to maintain the advantages of desired elite genotypes

(superior growth, wood properties, disease resistance and other quality traits).

Various strategies have been used to detect variants from micropropagated

plants including phenotypic based on morphological traits (Hwang and Ko, 1987;

Smith, 1988; Paranjothy et al., 1990), cytological analysis for numerical and

structural variation in the chromosomes (D’Amato, 1985; Armstrong and Phillips,

1988), isozyme electrophoresis (Sabir et al., 1992) and use of molecular markers like

RFLP’s for nuclear and organellar genomes for studying single base pair changes,

chromosomal rearrangements or methylation changes (Chaudaury et al., 1994; Natali

et al., 1995).

Isozyme has been used as a major biochemical marker for understanding of

heritable variation within and among plant population (Kephart, 1990). During last

two decades, DNA markers have been employed in the tree improvement programme

of various species (El-Kassaby, 1991; Neale et al., 1992; Dinus and Tuskan, 1997). In

recent years, the development of molecular techniques have gained importance to

assess genetic fidelity of the micropropagated plants (Rani and Raina, 2000; Giri et

al., 2004)

DNA markers (RAPD, RFLP, AFLP, and ISSR) have emerged as important

tools for understanding genetic variability within and between population/clones

varieties, species and genera, evaluation of genetic fidelities of micropropagated

plants and development of genomic libraries (Hela et al., 2000). RAPD has been

chosen widely since, it amplify different regions of the genome, allowing better

analysis of genetic stability/variation of plantlets, as well as simplicity and cost

effectiveness. Molecular markers have been used to assess genetic fidelity of

micropropagated plants of various species viz; Betula pendula (Ryynänen and

Aronen, 2005), Cedrus ibani (Piola et al., 1999), Curcuma longa (Salvi et al., 2002),

Eucalyptus tereticornis and E. camaldulensis (Rani and Raina, 1998), Populus

deltoids (Rani et al., 1995) and Prunus dulcis (Martins et al., 2004).

Introduction

11

Bambusa nutans Wall ex. Munro:

B. nutans is a medium sized bamboo, occurring naturally in Sub-Himalayan

tracts from Yamuna eastwards to Arunachal Pradesh between 600-1500 m altitudes

(Gamble, 1986). It is commonly cultivated in North West India, Orissa and West

Bengal. B. nutans prefers growing on moist hill slopes and flat uplands in well

drained sandy loams and clayey loams. It is resistant to drought, but not to frost

(Jackson, 1987). B. nutans is generally propagated by planting offsets (Gamble,

1986). There are only two reports on micropropagation of B. nutans through axillary

shoot proliferation (Negi and Saxena, 2011) and somatic embryogenesis (Mehta et al.,

2010).

Culms are up to 6- 15 m in height, with a diameter of 5-10 cm and internodal

length of 25-45 cm. Culm is green, smooth, without shining, and slightly white-ringed

below the nodes. Culm sheath is 10-23 cm long; leaves linear lanceollate, 20.3 cm;

inflorescence a panicle with many spikelets, fruit oblong and hairy. B. nutans has

been flowering by sporadic and gregarious manner (30-40years cycle). B. nutans is

one of the commercial species of Thailand and India (Anantachoke 1987). It is

commonly used species in paper and pulp industries (Krishnamachari et al., 1972). B.

nutans is used in construction, preferred for basketry, craft and also used as an

ornamental plant (Anonymous, 2005).

Based on the importance, demand of qality planting material and lack of

efficient methods for large-scale clonal production, lack of studies on biochemical

markers for the identification of embryogenic and non-embryogenic callus and lack of

information on genetic fidelity in B. nutans, studies were taken up to with the

following objectives:

Objectives:

• Development of protocols for rapid and mass production of quality planting

materials of B. nutans through somatic embryogenesis.

• Investigations on biochemical changes at various stages in embryogenic and

non-embryogenic callus.

• Evaluation of genetic fidelity of micropropagated plants raised through

somatic embryogenesis.