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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.