Chapter 2

REVIEW OF LITERATURE

2.1. Importance of Bamboo

Bamboo belongs to the Gramineae family and has about 90 genera with over
1200 species. Bamboo is naturally distributed in the tropical and subtropical belt
between approximately 46° North and 47° South latitude, and is commonly found in
Africa, Asia and Central and South America. Some species may also grow successfully
in mild temperate zones in Europe, North East Asia and North America. Bamboo is an
extremely diverse plant, which easily adapts to different climatic and soil conditions.
Bamboo flowers rarely and in irregular cycles, which is not yet clearly understood.
Thus taxonomists do not always agree on the identification of bamboo species and
genera, but modern genetic analysis may shed new light on bamboo taxonomy. Dwarf
bamboo species grow to only a few centimetres, while medium-sized bamboo species
may reach a few metres and giant bamboo species grow to about 30 m, with a diameter
of up to 30 cm.
Bamboo stems are generally hard and vigorous, and the plant can survive and
recover after severe calamities, catastrophes and damage. Young bamboo shoots were
the first sign of new plant life after the nuclear bombing of Hiroshima and Nagasaki.
Bamboo shoots and culms grow from the dense root rhizome system. There are two
main categories of rhizomes: monopodial and sympodial. Monopodial rhizomes grow
horizontally, often at a surprising rate, and thus their nickname of ‘runners’. The
rhizome buds develop either upward, generating a culm, or horizontally, with a new
tract of the rhizomal net. Monopodial bamboos generate an open clump with culms
distant from each other and can be invasive. They are usually found in temperate
regions and include the genera Phyllostachys and Pleioblastus. Sympodial rhizomes are
short and thick, and the culms above ground are close together in a compact clump,
which expands evenly around its circumference.
Bamboo has received increasing attention over the last two decades for its
economic and environmental values.Bamboo is often cultivated outside the forest in

17
farms, where it is more easily managed. In Africa, Asia and Latin America, it is closely
associated with indigenous culture andknowledge and is widely used for housing,
forestry, agroforestry, agricultural activities and utensils. Incountries undergoing
economic development, traditional bamboo culture gradually disappears.
However,industrial development of bamboo is offering a new opportunity to younger
generations to retain andcontinue developing cultural traditions related to the
cultivation, harvesting and use of bamboo.Bamboo can meet industrial and rural
requirements, check erosion and conserve soil. Because of its extensive rhizome and
root system, bamboo is useful for soil erosion control, and road and steam embankment
stabilization. Bamboo can be extremely important in providing vegetative cover to
deforested areas. It produces leafy mulch on the soil surface, its foliage provides shade
and protections against rains, and its habit of producing new culms from rhizomes
enables the culms to be harvested without disturbing the soil (Soderstrom and
Calderon, 1979).
The physical and environmental properties of bamboo make it an exceptional
economic resource for awide range of uses and for poverty reduction. It grows quickly
and can be harvested annually withoutdepletion and deterioration of the soil. Bamboo
can grow on marginal land, not suitable for agricultureor forestry, or as an agroforestry
crop. It is relatively light weight because of hollow culms, and unlike wood can be
easily harvested and transported without specialized equipment or vehicles. It
splitseasily for weaving and is thus easy to handle. Processing normally does not
require highly skilledlabour or special qualifications and can be started by rural poor
communities at a minimal cost. For thesame reason, it offer income-earning
opportunities to handicapped people.Bamboo use and trade have been growing rapidly
in recent years.

2.1.2 Utilization of bamboo

Bamboo is an enduring, versatile and highly renewable resource in the Indian
socio-economic- cultural- ecological-climatic functional context with 1.500 recorded
uses. Young and tender bamboo shoots are used as seasonal vegetables in both rural
and urban areas. In Manipur, about 2.11 lakh tonnes succulent sticks are consumed
annually for the processing of bamboo vegetables (Borah et al., 2008). Trade of
Bamboo pickle in tourist spot on Nongpoh (Meghalay) is estimated to be 1,170 kg to
2,210kg annually (Singh, 2002). While screening bamboo species for pickle
preparation, Bhagwati and Deka (2004) reported B. balcooa better among the species

18
studied. The species of bamboo commonly used as food are Dendrocalamus asper, D.
hamiltonii, D. brandisii, D strictus, Bambusa blumenna, B balcooa, B. polymorpha,
Thyrsostachys siamensis, Thyrsostachys oliveri, Gigantochloa albociliata, Melocanna
baccifera etc.
In India, usage of bamboo in scaffoldings is high. But there is virtually no
value addition in the raw bamboo used for scaffolding purposes. Of the 13.47 million
tonnes of bamboo, 3.4 million tonnes are currently being consumed for scaffolding
alone all over India (Anon., 2003). Bamboo can be fashioned artistically to make doors
and windows. Bamboo mat composites can be effectively used for manufacturing panel
and flush doors in combination with plantation grown timbers. The most expensive
holiday resort in the world is made of bamboo. Use of bamboo grids market size
estimated to grow at 25 percent per year with increased penetration in the total market
and greater acceptability of the product.
Bamboo is regarded as substitute of wood due to its physical and mechanical
properties. The woody material of bamboo has vascular bundles, which remains
separate in the long internodes, giving bamboo an easy splitting characteristic and great
flexibility. Bamboos are endowed with another feature –long fibre length, which makes
bamboo pulps suitable for papermaking. It also contains relatively low proportion of
lignin. In the tropics, therefore, bamboo are perhaps the best alternative to softwood
(Rao et al., 1990) Although bamboo is reported to be susceptible to fire, borer, termites
etc. It is now a days increasingly used as wood substitute in some industrial products
like bamboo plywood, bamboo mat, roofing, door shutters, bamboo mat board, bamboo
flooring, bamboo laminates, particle composite etc. after value addition. Various
industries are looking for suitable substitute for wood in production of various items
like pencil, match boxes, match sticks etc. Melocanna baccifera (muli bamboo) makes
excellent matchsticks splints. One kg of splints equals 160 matchsticks packets, each
containing 50 sticks. The annual consumption of wood by the matchsticks industry in
India is 2.6 million cubic feet (Anon., 2003)
Million tonnes of bamboo are utilized in different types of cottage industries
such as agarbatti, kites, and cracker industry, ice cream industry and match
industry.Tribal Development Agencies have also adopted bamboo resources for cottage
industry. There are more than 10,000 units in the handmade sectors of match industry.
Agarbatti is another important industry using bamboo. Bamboo from Tripura and other
North-eastern states is transported all the way to Mysore and Banglore for production
of agarbatti (Kulkarni and Rao, 2002). The current value of bamboo used in agarbatti

19
market is approximately Rs. 135 crore. Besides the agarbatti industry, bamboos are
used in some other miscellaneous items such as lathics and fishing rods and are valued
at Rs 186 crore (Anon., 2003)
Bamboo craft sector is another important area where it generates about 250
million days work by employing mainly women from the rural and tribal area and
generating incomes of Rs.15 billion a year. Various craft products made of bamboo are;
mats, flooring material , purses, bags, satchels, tea packing, different kinds of furniture,
floor tiles, boards, general household products, utensils etc. Crafts products are to be
developed differently, marketed and promoted in innovative and survive in
international market.
General consumption pattern in India indicates that 35 percent bamboo is being
consumed by pulp and paper industries, while the rest 65 percent is used by local
cottage, construction, furniture implements industries etc. (Kulkarni and Rao, 2002). It
is reported that one tonne of pulp is produced from four tonnes of bamboo which is
utilized in production of different paper and boards. Presently, 1.4 million tonnes
bamboo per annum is used by 10-15 paper mills (Kulkarni and Rao, 2002) Total
production of paper and paper board is estimated to be about 3.1 million tones annum-1.
The total important bill during 2000-01 was Rs. 3,300 crores making an increase of 10
percent over the previous year (Anon., 2003).

2.2. Occurrence and distribution of bamboo on farmer’s field

Bamboos are usually distributed naturally in the forest areas; however, farmers
maintain bamboo clumps in their homegarden or farm boundary to meet various
cultural, ecological and economic requirements. Bamboos are often divided into
cultivated or village bamboos and native or forest bamboos (McNeely, 1995). Bamboo
seems to have followed many ancient maritime spice routes between China, Indonesia,
Sri Lanka and India, sometimes making it difficult to establish the centre of origin for
some species (Soderstrom ad Calderon, 1979).

Nair and Sreedharan (1986) have mentioned that farmer’s prefered bamboo
along with other tree species as perennial component in their homestead in Kerala.
Kumar et al. (2005) claimed that Bambusa bambos (L.) is most important bamboo
species in rural areas of Kerala. Singh et al. (2010) have reported that Bambusa tulda is
traditionally grown around the house as live fence, wind break and is used in rituals,
ceremony and for household purpose. It was also reported that, people have prioritized

20
Bambusa cacharensis in their homegardens and bamboo groves because of its desirable
growth architecture (few branches and a straight culm) and multiple uses (Nath et al.,
2004). Tree diversity values in comparable with tropical rain forest was observed for
the homegardens of Central Western Ghats (Shastri et al., 2002). Ried et al. (1991)
observed that culms under the understory of evergreen and deciduous forest produced
thicker and taller culms.Further, taller and thicker culms were also observedunder
mixed evergreen and deciduous c a no p y than under clear-cut canopies (Donald et al.,
1991).

2.3. Dendrocalamus stocksii

Dendrocalamus stocksii is endemic to Central Western Ghats and is found in

Karnataka, Goa, Kerala and Maharashtra. Its natural distribution is mostly confined to
the banks of streams as it requires well drained deep soil. D.stocksii is cultivated in
the coastal belts of Karnataka (Seethalakshmi and Mukteshkumar, 1998) and is
primarily planted around the arecanut gardens and paddy fields (Devar, 2000).
Studies on reproductive biology (Beena, 2012) have hinted the possibility of
anemophilous mode of pollination. Anthers emerge at 6 AM in the morning and
dehisce by 11 AM. It is observed that only 15 percent of anthers dehisce
longitudinally and others dry out without shedding pollens. Seed formation is not
known in this species probably due to dichogamy and protogyny (Beena, 2012).The
gynoecium matures 3-4 days before androecium which effectively prevents self-
pollination. Very low pollen viability and low germination have been observed
in this species. Macro and micro propagation assumes importance in this species
since seed setting is very poor.
Macro propagation techniques have been standardized at Institute of Wood
Science and Technology (IWST), Bangalore and micro propagation through axillary
and somatic embryogenesis have also proved successful in this species. Propagation
through culm cuttings has been the practical and time tested method of propagation of
this species for State Forest Departments (Viswanath et al., 2013). This species can
be macro propagated through double and triple noded culm cuttings containing
dormant buds. These are collected from 1-2 year old culms of ahealthy clump
preferably from Candidate Plus Clumps (CPCs) in February-March. Cuttings from
entire culm can be made from basal region to top, after removing the side branches
with secateur. Later these cuttings are dipped in 0.25 per cent Bavistin for 10-15

21
 minutes for prophylactic measure and treated with 2500 ppm IBA in powder form (99
g talcum powder + 750 mg Bavistin + 250 mg IBA + acetone to create a mixture) or
in solution (2500 mg IBA dissolved in 5-10ml ethanol and made upto 1 L by adding
water) as pulse treatment for 30 minutes and laid horizontally in 1x5m sandbeds,
covered with 2 cm of sand layer and watered regularly. The beds are covered with
shade net (50 per cent) to avoid drying of the cuttings. Ambient conditions are
provided for the cuttings to remain green as sprouting will be hampered if it turns
yellow. Sprouting can be noticed in 10-15 days and rooting completes in 45-60 days
depending on ambient temperature. The rooted plants are scooped from the culm and
then transferred in poly bags, hardened and planted thereafter.
Comparative growth performance of D. stocksii in various locations indicate
that this species performs well in tropical humid, sub-humid and also reasonably well
under managed conditions in semi arid regions (Viswanath et al., 2013). Species is
suitable for agroforestry. Preliminary observations revealed that there was a
significant improvement in culm emergence, culm collar diameter and diameter at
fifth internode of emerging culms after intercropping (Bhave et al., 2011). Based on
strength properties (MOR), D. stocksii is comparable with Dendrocalamus strictus
and is better than B.nutans. Hence this species can be recommended for uses like
walking sticks, umberalla handles, roofing for huts, tent poles, baskets, javelin etc
(Rao et al., 2004). Lastly National Bamboo Mission has prioritized this species for
mass scale cultivation in Maharashtra and Karnataka (Haridasan and Tiwari, 2008).

2.4. Morphological Variations

2.4.1. Variation among populations
Geographical variation is very common in a species having wide distribution
and is usually present at varying levels between and within the populations.
Precipitation affects distribution and limits growth of bamboo more than any other
component of climate (Biswas, 1988). Variation arises from combined effects of gene
mutation and migration, drift, selection, and historical factors, including population
bottlenecks, and rate of population expansion following a bottleneck or founding event
(Cwynar and MacDonald, 1987). Variation in morphological parameters has been
reported in various species across the gradients of altitude, rainfall, latitude, longitude
and region. Within populations, intraspecific variation in physiological and
morphological traits can lead to certain individuals exhibiting superior growth. Large
variations of bud burst were observed between populations of sessile oak (Quercus

22
petraea) and these variations are linked to altitude, latitude and spring frost tolerance
(Ducousso et al. 1996). It was also observed that variation exhibited a striking
geographical pattern among Q. petraea populations, with little within populations,
indicating substantial isolation of and genetic drift within many populations in the
Southern half of the species distribution in Italy.
A significant variation in height and diameter of the populations was reported
in an open pollinated species of Swietenia macrophylla in Costa Rica (Navarro 2004).
Walter and Epperson (2001) observed a low genetic diversity in natural populations of
red pine, Pinus resinosa, a species that has a vast range across North-Eastern North
America. They observed that cone traits showed clinal variation with latitude and
altitude, the biggest cones being collected in high altitude southern populations.
Genetic diversity and population genetic structure of Shorea leprosula was investigated
using seven natural populations distributed throughout Peninsula Malaysia and one
natural population from Borneo (Lee, 1989). Wild beet (Beta vulgaris L.) accessions
from Sicily were evaluated morphometrically and they were divided into coastal and
inland populations based on the geographical distribution pattern (Letschert and Frese,
1993). Significant variation was observed for their petiole length, leaf length and
biomass. Geographical variations in Phyllostachys heterocycla were observed in China
for more than 70 indices including shoot growth and it was observed that number of
spring shoots in the provenances located in South was greater than those in North, but
reverse was true for the diameter and height (Cunji et al. 2002). A significant
intraspecific variations was observed for thirteen morphological and anatomical traits
of seed in nine native populations of Pinus pinaster distributed along mountain and low
land areas of Morocco (Ahida et al. 2006). Vitasse et al. (2009) found significant
differences in phenology and growth among provenances of common garden tree
species and evidences stating that differences in phenology were related to the annual
temperature. Significant altitudinal clines for growth were also revealed and finally it
was observed that despite the proximity of the populations in their natural area,
differences in altitude led to genetic differentiation in their phenology and growth.
In the practice of forestry, even small differences in intraspecific relative
growth rate could compound through time, resulting in large differences in stand
productivity (Atwood et al., 2002, Martin and Shiver, 2002). High levels of variation
for vegetative characters were observed in natural stands of Bambusa tulda surveyed at
17 eco-geographical locations in different districts of West Bengal, India (Battacharya
et al., 2006). Twenty-eight key vegetative characters as well as reproductive

23
morphology were studied. Statistical analyses of quantitative vegetative characters
revealed that a significantly high variability existed between populations, but not within
populations. Ravikanth et al. (2008) examined the population genetic variability of
Dendrocalamus strictus and Bambusa bambos. These two species exhibited strikingly
contrasting patterns in the distribution of genetic variability. While the diversity of D.
strictus was higher in the northern latitudes, the diversity of B. bambos was higher in
the southern latitudes. Yang et al. (2009) used three year old culms of eight
provenances of Bambusa chungii from Guangdong, Guangxi and Hainan Provinces and
evaluated indexes of wood properties, such as fiber dimensions and chemical
composition. Analysis of variance and correlation coefficient used to reveal geographic
genetics. The results showed significant differences between fiber length, fibrin and 1
percent NaOH extraction contents of B. chungii provenances. Finally, three B. chungii
provenances with high qualities and culm yield, i.e. Huaiji, Xinyi and Guilin, were
selected as superior sources based on principal component analysis.
2.4.2. Variation in clump parameters

Number of old culms influences culm recruitment among the clumps and is
indicative that culm density mediates culm recruitment (Taylor and Zisheg, 1993).
Jha and Das (2007) reported that, of the total culms, 21 percent were represented by
current year shoots and 55 percent by old shoots in a seven year old plantation of
Dendrocalamus strictus in Bihar. Among the total culms, 15 percent were represented
by current year shoots and 54-55 percent by old shoots while the new recruits
contributed 5864 culms ha-1. They also reported that production of new culm was
linearly related with number of old culms and the majority of new culms were produced
by 1-10 years old rhizomes. Singh and Singh (1999) also reported that production of
new culm was linearly related with number of old culms and majority of new culms
were produced by rhizomes of 1-10 years old. So also, mean number of new shoots
emerging and surviving per plot was positively correlated with the proportion of adult
shoots carrying new (one year old) leaves of Phyllostachys pubescens (Li et al., 1998).
The annual production of new shoots of Phyllostachys pubescens was not significantly
correlated with the annual values of logging intensity of bamboo poles, rainfall or
temperature, however, survival rate of new shoots was constant over years. However,
severe pests (e.g., plague of locusts), extreme drought or heavy overlogging of adult
shoots with new leaves can result in low production of new shoots during the following
spring.

24
 Fargesia robusta and Bashania fangiana, studied over seven years in south
west China and wa s o bser ved t hat culm numbers remained relatively constant (±
20 percent) in most plots, but culm numbers increased (>20 percent) on some sites
(Taylor and Zisheng, 1993). S o a ls o , s preading clones had greater recruitment
response to density than compact clones, but predation and climate may have
diminished or amplified the observed demographic patterns. Culm production
capacity is usually governed by the age of the clump. Das and Chaturvedi (2006)
observed culm recruitment, standing biomass, net production, litterfall, floor litter mass
and carbon flux at the age of 3, 4 and 5 years in a Bambusa bambos plantation in the
northwest alluvial plains of Bihar, India. The number of culms produced per clump
varied from 22 to 31. The recruitment to culm population varied between 16 and 21
percent and shoot mortality between 3 and 5 percent per year. Short-lived components
(leaves and roots) contributed about 20 percent of net production of bamboo. The
relationships of initial culm number per clump, spatial distribution of culm and culm
number per clump with culm morphological variation of Bambusa ventricosa were
studied (Chen et al., 2008). The results showed that abnormal rate of new culm
decreased with increasing initial culm number per clump and time. The number of new
bamboo in outer and inner clump was correlated negatively and positively respectively
with culm number per clump after shooting.

2.4.3. Variation in culm parameters

Selection of particular species for particular use has been practiced for
millennia, contributing to the development of many cultures and livelihoods.
Nowadays, there is an increasing demand for bamboos with specific characteristics.
Several authors have attempted to identify superior genotypes among bamboo species
by ascertaining variation in morphological characters. Accessions of bamboo species
vary in growth rate and biomass accumulation, growth habit, culm thickness and
internode length, and also the fibre length and silica content. Tools such as INBAR’s
species-to-site matching software have helped greater international germplasm
exchange, and the establishment of collections and commercial bamboo forests alike.
Selection and mass multiplication of elite clones are now often practiced.
Quick production gains can be achieved by selection through faster
evaluation and multiplication of superior germplasm and by utilizing them directly in
plantations. In this regard, some of the following parameters may be considered for the
selection work. The following characters have been used world over to select superior

25
Germplasm of bamboo following parameters are usually evaluated (Banik, 1993)
Clump nature: Thorny or non-thorny, erect, erect with pendulous tips, broadly arched to
clambering, branching through out preferably only at top or up to mid part of culm, leaf
size, evergreen or deciduous, culm colour, clump congested or open and disease
infection (resistant /susceptible). Along with it, the growth habit and productivity,
culm height, diameter and wall thickness are also considered. Culm emergence time,
juvenile mortality, culm production per clump per year, clump expansion rate, edible
shoot production, shoot taste and palatability, canning ability may also be considered
(Banik and Rao, 1995). Hybridization of different species has also shown the potential
of new hybrids to break into existing commercial markets. However, further studies are
needed to select best candidates for hybridization, and more work is needed on inter-
and intra-specific and -generic compatibility (Hoogendoorn et al., 2013).
Pioneering work in improving bamboo was initiated by Beniwal and Singh
(1988). They studied eight important species of bamboos and selected plus bamboos
from the bamboo grown in the villages and also from natural forests. Offsets were
purchased from the villagers and bamboo germ-plasm banks were established during
1984 over 6 hectares at Chessa and Namsai (Arunachal Pradesh). Among the plus
bamboos, BB/CH-4, BT/NS-13, BT/CH-7, BP/CH-3, BP/CH-7, BN/CH-I, Nangal CH-
2 and CH-4 had shown superiority over other plus bamboos.A total of 113 sample
culms of Bambusa blumeana studied in Philippines for growth characters showed
variations for merchantable lengths (6.7 to 15.6 m) and basal diameter (6.6 to 12.3 cm)
and tapering were observed among all the culms (Taudug and Torres, 1985). Culm
anatomy shows substantial variation, but it is difficult to define character states
objectively (Jeffery, 1995). The study of genetic diversity of Dendrocalamus strictus
Nees for increasing the productivity of Bamboo plantation in Vindhyan Region of Uttar
Pradesh was confined to diameter and volume of culm, which are the major traits for
better quality of Bamboo. The rhizomes of one year old D. strictus culms were
collected from the different CPCs (Candidate plus Clumps) from Renukoot, Mirzapur
and Obra Forest Divisions. The collected rhizomes were raised at Research Nursery,
Kotwa and Mirzapur for ex-situ conservation. Significant variations were observed in
diameter of the culm and volume along with culm height among all the 12 CPCs. Good
selection of CPCs were made for cultivation at Mirzapur and adjoining places for
improvement and multiplication of good quality bamboo vis-a-vis enhancing the yield
(Niwedita et al., 2001). Forty eight candidate clumps of Dendrocalamus stocksii from
five different forest ranges of Banvasi, Hulekal, Janamane, Siddapur and Sirsi were

26
analysed for phenotypic variation (Ravi Shankar, 2004). Wide variations were observed
for almost all characters studied, clump characters showed higher variation except for
clump height. The altitude and temperature had a positive effect on growth and yield of
bamboo. Culm diameter varied among the forest ranges from 3.58 cm (Siddapur) to
5.90 cm (Hulekal), while nodal length ranged between 22.7 cm (Sirsi) to 27.7 cm
(Siddapur).

Culm diameter, height, internode length and leaf area of 12 candidate plus
culms of Dendrocalamus strictus varied between 2.35-2.86 cm, 333.3 – 423.3 cm,
13.17-16.00 cm and 51.11 – 103.71 cm2 respectively in dry deciduous forest of Uttar
Pradesh (Singh et al., 2006). Further the morphometric measurements revealed that the
clumps from natural origin had larger diameter, height and leaf area and smaller
internode length compared to the clumps from plantations.High levels of variation for
vegetative characters were observed between 17 populations of Bambusa tulda in West
Bengal (Battacharya et al., 2006). Extensive morphological variation in B. vulgaris in
Ghana was observed in relation with the wide range of geographical and ecological
niches and climatic regimes. This may be because seed setting in B. vulgaris is not
known, even though flowering may occur sporadically in rare instances (Ofori et al.,
2006). Morphological characteristics of 2 and 4 year old managed natural stands of
Gigantochloa scortechinii in Malaysia varied depending on age and height level of
culms (Razak et al., 2007).

Pan et al. (2009)investigated the morphological characteristics, aboveground

biomass and its allocation of Fargesia angustissima (Mitford) along an altitudinal
gradient ranging from 1200 MASL (the lowest distribution boundary of F.
angustissima) to 1810 MASL (the uppermost distribution limit) in the Wolong Nature
Reserve, south western China. Except for a nearly linear increase in internode number
and a linear decrease in shoot biomass with increasing elevation, other parameters
studied changed non-linearly with increasing elevation. These results may imply that
the local-environmental conditions do not change linearly with altitude. It also indicates
that, growth of F. angustissima, as a low-altitude species, may be impacted by
precipitation rather than by temperature.

A solid stemmed province germplasms of wheat were collected from

lower altitude comprising of higher temperatures and shorter growing season in Turkey
(Damania et al., 1997). Nearly 53 accessions of white clover stolons (Trifolium

27
repens)were collected from naturalized populations from Argentina and studied for
morphological parameters. There were significant correlations among seven pairs of
characters and these accessions were clustered into agglomerative hierarchical
clustering procedure with squared Euclidean distance using the statistical analysis
system (Rosso and Pagano, 2001). It was observed thatLatitude negatively influenced
seed size of Aleppo pine in Morocco (Boulli et al., 2001).

High heritability with high genetic gain was observed for parameters like
diameter of culms, height of culms and number of internodes by Singh et al. (2004) in
Dendrocalamus strictus. Heterozygosity varied among populations, ranging from
0.326 to 0.340, with the highest values found in the populations from Central
Peninsular Malaysia and the ecological variables (longitude, latitude and annual
rainfall) were not responsible for the observed genetic differences among populations.
Lewis et al. (2010) observed that there was a high genotypic coefficient of variation for
biomass, number of new sprouts, number of culms, leaf breadth, leaf length, height and
diameter at third node of 15 bamboo species of India. They also observed, high
estimates of heritability for leaf breadth, biomass, leaf length, culm height, and
internode length along with a high genetic advance estimates (as percent of mean) for
biomass, diameter at 5th internodes and leaf length. High heritability coupled with high
genetic advance indicated that such characters are controlled by additive gene action
and will respond effectively on phenotypic selection.Highgeneticadvance estimate (as
percentof mean) wasobservedforbiomass, diameteratbreastheightand leaflength of
most of the bamboo species (Lewis et al., 2010). Generally, characters showing
highheritabilityalso possess high value of genetic advance. Highheritability coupled
with high genetic advance indicate that such characters are controlled by additive gene
action. Usually, environment affects polygenic trait (Wright et al., 2007).

2.4.4. Demarcation of populations

Clustering of accessions into populations based on location attributes

have been attempted by many researchers. New Brunswick area in Canada was divided
into 11 regions using principle component analysis based on the data of precipitation,
temperature, elevation, latitude and longitude to undertake a first division forest
classification (Groenewoud, 1984). It was observed that the distribution of plant
community was controlled by climatic or geologic parameters.Various environmental
attributes like rainfall, types of vegetation and geomorphology of the region

28
discriminate species into populations (Bottini, et al., 2000). Ying & Yanchuk (2006)
claimed that geographical ranges of a species can be delimited based on latitude,
longitude and elevation within which a seed source can be used for research. Twenty
nine experimental sites evaluating black spruce (Picea mariana) showed differential
correlations for growth and environmental attributes. These experiments were divided
into eight regional groups or clusters, with adjacent clusters sharing more of the same
provenances than distant clusters. This arrangement was checked by cluster analysis
using the Ward and Average Linkage (SAS Institute Inc.) and led to the modification in
grouping (Mullin and Morgenstern, 1990). Vitousek (1998) divided Metrosideros
polymorpha into five populations based on elevation gradient and studied physiological
and morphological variations in Hawaii. It was indicated that large phenotypic variation
of this species was determined by genetic differences. A study revealed that aridity
indices, longitude, and latitude together explained 61 percent of the variance in PC1
(Principal Component 1) and 39 percent of the variance in PC2 (Eckert et al., 2010).
Geographic variables viz. latitude, longitude and elevation predicted 52 percent of the
genetic variation in the growth parameters of Aspen (Hamann et al., 2010). Rehfeldt
(1983) introduced the floating principle into seed transfer in a same tree species. The
floating principle recognizes that similar genotypes tend to occur in similar
environments. For example, similar genotypes occur at different elevations as
geographic distances in latitude and longitude change.

2.4.5. Selection of superior phenotypes based on multiple traits

Hierarchical cluster analysis can readily be used to asses relatedness and

distance of any type of samples characterized by any type of descriptors. Therefore, it
may be used to assess genetic similarity and dissimilarity in germplasm collections, and
the technique could also have applications for the selection of parental lines for which
varying degrees of segregation are sought. Peeters and Martinelli (1989) suggested that
hierarchical cluster analysis is the best to classify entries from a germplasm collection
according to their degree of similarity. A high degree of accuracy is observed when the
collection is described by many characters. Sokal (1986) and Rolf and Wooten (1988)
agree that the UPGMA clustering method (or group average or average-linkage cluster
analysis) generally yields results which are the most accurate for classification
purposes.Lin et al. (1986) have shown the advantage of using hierarchial cluster
analysis in stability analysis over a range of environments, particularly by the inclusion

29
of reference cultivars. Murphy et al. (1986) have used cluster analysis using
coefficients of parentage for Red Winter Wheat to determine overall patterns of
relationship between existing cultivars in the United States. Wahid et al. (2006) has
used Principal Component Analysis to understand the variation for 13 morphological
characters among 9 native populations of Pinus piaster in Morocco. Similarly,
UPGMA dendrogram based on morphological similarity values with a cophentic value
of 0.67 and produced two groups for 10 accessions of Dendrocalamus hamiltonii from
Chandel and Impal areas of Assam (Waikhom et al. 2012).

2.4.6. Diversity in clonal and non-seeding species

The variation in bamboo ramet population of Fargesia nitida for population

structure, biomass allocation, and morphological characteristics growth along the
succession of coniferous forest was studied in Wolong Nature Reserve, China
(Xiaohong et al., 2006). It was observed that clonal ramets, constituting bamboo
population, tended to become short and small with succession. The ramets changed
towards having a greater mass investment in leaves, branches and underground roots
and rhizomes rather than in the culm. The results demonstrate that the ramet population
structure of F. nitida is unstable and its growth performance is inhibited by succession.
In clumping clonal plants, the often discrete nature of clumps may be enhanced by
competition among genets. Franklin et al. (2008) examined the genetic composition of
clumps of Bambusa arnhemica F. Muell., a bamboo from northern Australia, by
analysis of microsatellites. Three of the ten clumps were demonstrably multiclonal,
containing a minimum of two, four and five genets, respectively. This raises intriguing
questions about the development of clumps and suggests that intergenet competition
may at times be overwhelmed by the benefits of coloniality in B. arnhemica. It was also
observed that, Non-seeding behavior of a bamboo species does not have any influence
on intraspecific diversity of a species, as was observed in Bambusa vulgaris, which
showed a clear pattern of genetic dissimilarity index among the individuals of
evergreen and semi-deciduous populations (Ofori et al., 2006).

2.4.7. Variation in Aboveground Biomass

Biomass is one of the productivity indicators of any species and carbon storage
in plants is an indication of reduction of atmospheric carbon dioxide to minimize the
potential danger of global warming. Bamboo biomass varies between and within the

30
species along the geographical gradients. A detailed study on biomass and productivity
of four important bamboo species was done by Upadhyaya et al. (2008). Bambusa
balcooa, B. pallida, Phillostachys bambusoides, Arundinaria racemosa growing at
different altitudinal regimes in Arunachal Pradesh were studied for biomass. The
biomass of respective bamboo components per culm increased with age, except in case
of P. bambusoides. Among the species, the total aboveground biomass was in the
sequence of B. balcooa > B. pallid > P. bambusoides > A. racemosa.Further, Jha and
Das (2007) observed that the total energy stored as biomass by a 5 year old plantation
of Dendrocalamus strictus was 2375 TJ ha -1 and that by 7 year old plantation was 3579
TJ ha-1. The total biomass was 118.23 MG ha-1 in the 5 year old and 176.31 MG ha-1 in
the 7 year old plantations. The recruitment to culm population varied between 20 and
21 percent and shoot mortality between 4 and 5 percent per year.

Aboveground biomass varies among the various culm components.The above

ground biomass of Phyllostachys pubescens was estimated as 116. 5 t ha-1 for culms,
15.5.t ha-1 for branches, 5.9 t ha-1 for leaves and 137.9 t ha-1 in total in China (Isagi et
al. 1997).Das and Chaturvedi (2006) reported that a 5 year of Bambusa bambos
plantation in North-West alluvial plains of Bihar produced 257.3 Mg ha-1 of above
ground biomass of which 65 percent, 21 percent and 4 percent biomass was contributed
respectively by culms, branches and leaves. It was observed that live culms constituted
82 percent of the total biomass.Variation in biomass of above ground components viz.
culm (0.952 – 0.843 kg), branch (0.311 – 0.248 kg culm-1) and leaves (0.249- 0.265 kg
culm-1) was observed among 12 candidate plus clumps of Dendrocalamus strictus in
dry deciduous forest of Uttar Pradesh (Singh et al., 2006). The relative contribution of
various components to the standing state of biomass for the species was in the order;
bamboo culms (1.862 kg culm-1) > branches (0.255 kg culm-1) > inflorescence (0.235
kg culm-1) > leaves (0.034 kg culm-1) in three year old Melocanna baccifera located in
natural forests of Mizoram (Pynskhem et al., 2010). The bamboo Fargesia denudata
was studied for biomass allocation and environmental conditions most conducive for
growth and survival in South Western subalpine coniferous forest of China (Xie et al.,
2010). Above ground biomass of four bamboo species viz. Bambusa balcooa, B.
pallida, Phillostachys bambusiodes and Arundinaria racemosa growing at different
altitudinal regimes was carried out in Andhra Pradesh by using linear equations
(Upadhyaya et al., 2008). It was observed that maximum biomass allocation was

31
observed in culms (64-81 percent) and minimum in leaves (5-15 percent). So also, the
foliage biomass in the lower altitude was lower compared to the higher altitude.

Above ground biomass production of Fargesia yunnanensis was evaluated in

three provenances of Yunnan, China and it was observed that above ground biomass
was highest in Jizhushan Provenance (7.88 kg culm-1) because of high organic matter
and nitrogen status in these soils (Wang et al., 2009). The biomass, height, and basal
diameter of ramets and biomass of modules were highest in the small gap canopy
condition and it was also observd that with the increase of canopy cover, there were
increase in biomass allocation to leaves, specific leaf area and percentage of branching
and decrease in biomass allocation to roots and number of roots.

Veblen et al. (1980) also reported that the above ground biomass of Chusquea
culeon in Chile as 10.5 tonnes ha-1 yr-1. In Thailand, Suwannapinut (1983) reported that
the mean biomass of Thyrsostachys siamensis was 4.3 tonnes ha-1 yr-1. The mean above
ground biomass of stands was 32 t ha-1. Isagi et al. (1993) reported that the above
ground biomass of Phyllostachys bambusoides in central Japan (350N) was 24.6 tonnes
ha-1 yr-1 over a 6-year period. In southern India, Shanmughavel and Francis (1996)
reported that the above ground biomass of Bambusa bambos was 47 t ha-1 y-1after
fertilization and irrigation. Lin (2002) reported that the mean above ground biomass of
Phyllostachys heterocycla was 35.45 t ha-1 and the maximum biomass was 49.48 t ha-1
according to 3,046 stand ha-1 density in Sha, Fujiang, China. Li et al. (2007) also
reported that the above ground biomass of P. heterocycla was 26.66 t ha-1 according to
the average 1805 stands ha-1 and 14.77 kg stand-1 in Jiangxi, China. An et al. (2009)
reported that different management patterns have affected the biomass of Sinocalamue
oldhami in Pingyang, Zhejiang, China.

2.4.7.1. Predication of Aboveground Biomass

Equations that predict the biomass of a tree from dendrometrical characteristics

such as diameter or height that are easier to measure should be developed (Ebuy et al.,
2011). Species specific biomass equations have been developed for temperate forests
(Zianis et al., 2005). In highly diverse ecosystems such as tropical rainforests, either
specific, local equations with good precision but narrow range of application (Araujo et
al. 1999, Basuki et al., 2009) or general multispecies pantropical equations (Brown,
1997, Chave et al., 2005) have been developed. Aboveground biomass stock estimate

32
was made in nine hedgerow raised Bambusa bambos clumps through destructive
sampling in Kerala (Kumar et al., 2005) by developing allometric relationships linking
clump biomass and culm number. Oli and Kandel (2005) predicted biomass in
Bambusa nutans growing is Eastern Terai by using a liner regression W = a + b (D2L)
with a R2 value of 90, 82 and 73 percent respectively for culm, branch and foliage.
They also observed that 48 percent of dry matter content was found in the branches,
while culm and foliage contained 44 and 37 percent respectively. Allometric equations
were built to predict biomass by DBH for Phyllostachys makinoi in Taiwan (Yen et al.,
2010) and it was observed that foliage, branches and culms respectively constituted 73-
78 percent, 13-17 percent and 6-9 percent of the total above ground biomass.

Most researchers (Virtucio et al., 1994; Othaman, 1992; Li et al., 1999; Singha,
2006) adopted two measurable growth parameters of individual culms i.e., dbh and
culm height. Although, inclusion of height in the regression equations can improve the
r2 values, measuring the culm height is extremely difficult as the culm tops are not
properly visible in the stands (Singha, 2006). R2 values depicting quality of allometric
equation vary for various above ground biomass components in bamboo and usually
culm biomass is strongly predicted (Oli and Kandel, 2005). It is also argued that the
prediction of leaf yield form biomass equations is less accurate and more site specific
than that of stem component (Satoo and Madgwick, 1982). Regression analysis was
used to create equations for biomass calculations of a tropical bamboo, Guadua
weberbaueri in Brazil revealed that bamboo culm diameter and height can be used to
calculate biomass (Torezan and Silveira, 2000). Biomass equations were developed for
three tree species in Africa using linear regression equation by using DBH, height and
specific gravity as independent variable (Ebuy et al., 2011) and it was observed that all
the equations were within the 95 percent confidence interval.

The above ground biomass of different stands of Phyllostachys heteroclada was

studied by allometric method. It was observed that the total above ground biomass
decreased with increasing age (Tieren et al., 1985).Above ground biomass of
Schizostachyum lumanpo was investigated in Philippines and logarithmic based
allometric equations were also developed (Suzuki and Jacalne, 1986). They reported
that the above ground biomass constituted about 146.8 t ha-1 and composed of 8.8 ton
ha-1 foliage, 22.2 t ha-1 of branch and 115.8 t ha-1 of culm. Avaro and Montagnini
(2006) computed equations for predictions of above ground biomass by using
logarithmic linear equation for DBH and height in Calophyllum brasiliense and it was

33
observed that 91.4 percent of above ground biomass composed of stem, 24.5 percent of
branches and 14.5 percent of foliage.

Qiu et al.,1992 had reported for P. pubescencs in China, a highly significant

relationship between annual precipitations and annul timber yield. Huang et al., 1993
also showed that Phyllostachys pubescens located in areas with little available soil
moisture, less biomass was accumulated and was mostly partitioned below ground.
Riano et al.,2002 observed that optimum growth in Columbia for Guadua angustifolia
was achieved at 500 to 1500 m elevation because factors like rainfall, temperature and
relative humidity which govern the culm production were more favorable for growth of
this species.

2.5. Variation in Soil Nutrients between various locations

Soil nutrient status varies among the bamboo growing sites. Soil physical
factors such as slope of land, texture, bulk density, moisture-holding capacity,
temperature are the nonchemical properties of soil which influence bamboo
productivity (Kleinhenz and Midmore, 2001), moreover, high bulk density of clay soils
negatively affects bamboo growth (Kleinhenz, et al., 2003). The contents of available
nutrients in the soil are positively related to yield and explain much of the variation in
yield across bamboo growing sites and regions in China (Hong, 1994). Hence, bamboo
growth and biomass is positively related to soil organic matter, which is a primary
source of nutrients in bamboo cultivation sites (Chung and Ramm, 1990). Nutrient
availability is the most important soil chemical property governing bamboo growth and
yield but other factors such as soil pH and soil salinity are also important (Kleinhenz
and Midmore, 2001). Soil pH and salinity are factors which affect bamboo in both
natural stands and cultivated plantations. There is a positive trend towards better
growth of bamboo under higher soil pH (Jin and Chong, 1982). Cai and Wang (1985)
measured best growth of P. fimbriligula at a soil pH of about 5.2 and suboptimal
growth below and above this value. This implies that, in contrast to other crops,
bamboos are comparably less affected by acid soils, making them suitable for
cultivation on degraded land in sub/tropical regions where soils are frequently highly
weathered and therefore, low in pH.

Usually, Leaf litter from above and below ground organic matter components,
increase the soil organic carbon content (Jose, 2009). Litter biomass contributes
significantly to soil organic matter and supplies bamboos with nutrients in natural

34
stands and cultivated plantations (Kleinhenz and Midmore 2001). Soil nutrient
properties varied among the three bamboo growing sites in Yunnan province of China
with soil of Jizushan site being more fertile with higher N (39.06 mg 100g-1) and
organic matter status (10.20 percent), which positively influenced the growth and
biomass production in that region (Wang et al., 2009). The spatial variability was also
observed for available nitrogen and organic carbon at various soil depths of Moso
bamboo forest under different management types in Jiangxi Province, China (Shunbao
et al., 2010). Liu et al. (2011) observed N2O fluxes were 0.230, 0.102 and 0.093 mg
N2Om-2 h-1 from the intensively managed bamboo forest, conventionally managed
bamboo forest, and broadleaf evergreen forest, respectively, and calculated annual
cumulative N2O flux and soil water content. Results showed that with increased
plantation years, SOM (Soil Organic Matter) decreased slightly at the beginning (1-5
years), and then rose up steadily. Based on the average of the three locations, the
highest SOM content of 75.82 g kg-1 was the surface layer (0-10 cm) of the 15 years.
As plantation year increased, the variation of SOM in the surface layer (0-10 cm) was
represented by a parabolic shape, and in the second layer (10-20 cm), it was a similar
mode, but less vigorous. Soil organic carbon (SOC) storage significantly increased
during 5 to 15 years after it reached full production, and the calculated annual SOC
increment in 0-40 cm soil profile was about 6.3 Mg Cha-1 year-1.
Carbon stock in forest soils is the greatest carbon stock in terrestrial ecosystem,
and its effect occupies an important place in carbon emission and carbon sequestration
in terrestrial ecosystems. Organic carbon in the soils is the greatest carbon stock in
bamboo Forest system. Organic Carbon in the soils usually occupies 54-67percent of
the total Carbon stock. There is a great difference in soil organic contents among
different bamboo forest ecosystems. For example, organic content in 0-40 cm of soil
depth in forest ranged between 20 -30 g kg-1 (Li et al., 2003). Carbon stocks in the soil
under the bamboo forest ecosystems depend on organic content in the soils. Carbon
stock in the soils layers of bamboo forests decrease with soil depth. Carbon stock in the
soil layer of bamboo forest mainly distributed in 0-20 cm of soil layer, which
accounting for about 40 percent of total CS (Carbon Stock) in 0-60 cm soil layer (Zhou
& Jiang, 2004; Li et al., 2003; Wang et al., 2009). Carbon stock in 0-60 cm of soil layer
accounted for 58.2 percent of total Carbon stock in the soil layer, whereas Carbon stock
in 0-160 cm of soil layer accounted for 89.6 percent of total Carbon stock in the soil
layer (Wang et al., 2009) the content of organic matter in the soils under Phyllostachys
praecox forest increased time or cultivation years. The contents of organic matter in the

35
soils in 1999, 2002 and 2007 were 28.9, 44.4 and 55.6 g kg-1, respectively, the content
of organic matter in 0-10 cm of soil depth under Phyllostachyspraecox forest with 0-,1-
,5-,10-, and 15 years cultivation history were 30.96, 25.55, 26.35, 33.07 and 79.24 g/kg,
respectively (Jiang et al., 2009).

2.6. Physiological variations

There can be wide environmental variation within the natural range of a species
(Court, 1974). Adaptation of a species to geographic variation in the environment may
often depend on genetic variation among populations (Berry and Bjorkman 1980).
Several studies have documented genotypic variation in the gas exchange, water
relations and gross morphology of tree species (Parker and Pallardy, 1985; Bongarten
and Teskey, 1986; Abrams 1988) and in some cases such genetic variation has been
found to have a relationship with the habitat from which the plants originated (Parker
and Pallardy, 1985; Bongarten and Teskey, 1986). Bamboo shows variation in
physiological parameters like photosynthetic rates between species, different ages of
culm, season and location (Volker and Midmore, 2001). Studies conducted by Koyama
and Uchimura (1995) with P. bambusoidesshowed that net photosynthesis increased
until air temperature was 270C, but decreased rapidly thereafter. In a similar study a
significant variation in physiological properties was found between Zostera marina
genotypes (Hughes et al., 2009).
Significant provenance variations in net photosynthesis, stomatal conductance,
leaf area and whole plant dry weight of neem seedlings (Kundu & Tigerstedt 1998)
indicated possibility of choosing the best provenances for a breeding program.
Similarly, Mebrahtu and Hanover (1991) for black locust (Robinia pseudoacacia L.)
and Jhonsen and Major (1995) for black spruce (Picea mariana Mill.) have reported
variations. A considerable genotypic variation in physiological responses to drought
between low and upland sources of Acer rubrum L was observed (Abrams and
Kubiske, 1990).Cordell et al. (1997) divided Metrosideros polymorpha into five
populations based on elevation gradient and studied physiological variations in Hawaii.
It was observed that site and elevation influenced net CO2 assimilation rates.

Hall et al. (2005) suggested that single genes or clusters of genes may have an
influence on the expression of physiologically related traits controlling water-use and
photosynthesis. So also increasing crop yield requires an increase in photosynthesis is

36
now recognized, and genetic manipulation of photosynthetic processes has become a
key target for crop improvement (Mann, 1999a; Mann, 1999b). However, there is no
direct control of genes on photosynthesis but the connection between different parts of
the photosynthetic process is to be considered together with the influence of
environmental factors, development and acclimation, and metabolic regulation (Horton,
2000). Dwarf bamboos are an important understory component of the lowland and
montane forests in the subtropical regions of Asia and South America, yet little is
known about their physiology and phenotypic plasticity in response to changing light
environments.

To understand how bamboo species adapt to different light intensities, leaf

morphological, anatomical, and physiological differentiation of Sinarundinaria nitida
(Mitford) Nakai, a subtropical woody dwarf bamboo was observed, growing in open
and shaded natural habitats in the Ailao Mountains, SW China was observed (Yang et
al., 2014). Compared with leaves in open areas, leaves in shaded areas had higher
values in leaf size, specific leaf area, leaf nitrogen, and chlorophyll concentrations per
unit area but lower values in leaf thickness, vein density, stomatal density, leaf carbon
concentration, and total soluble sugar concentration. However, stomatal size and leaf
phosphorus concentration per unit mass remained relatively constant regardless of light
regime. Leaves in the open habitat exhibited a higher light-saturated net photosynthetic
rate, dark respiration rate, non-photochemical quenching, and electron transport rate
than those in the shaded habitat. It was finally revealed that the bamboo species
exhibited a high plasticity of its leaf structural and functional traits in response to
different irradiances. The combination of high plasticity in leaf morphological,
anatomical, and physiological traits allows this bamboo species to grow in
heterogeneous habitats.

Cordell et al. (1998) claimed that intraspecific differentiation may result also
from phenotypic plasticity among any of a number of morphological and physiological
characters (e.g., water and nitrogen use efficiency, leaf spectral characteristics, leaf size
and orientation).There are conflicting reports concerning the adaptive features of tree
populations originating from cold, high-altitude environments. It is always
hypothesized that such trees will possess adaptive features that will be demonstrated in
a common environment, such as elevated rates of net CO2 exchange, elevated needle
nitrogen concentration and high proportional biomass allocation to roots (Oleksyn et al.

37
1998). Driever et al. (2014) using gas-exchange analysis and protein analysis examined
natural variation in photosynthetic capacity in a diverse panel of 64 elite wheat
cultivars grown in the field in relation to growth traits, including biomass and harvest
index. Significant variations in photosynthetic capacity, biomass, and yield were
observed, although no consistent correlation was found between photosynthetic
capacity of the flag leaf and grain yield when all cultivars were compared.Guillermo et
al. (2011) compared two new pineapple somaclones (P3R5 and Dwarf) derived from in
vitro culture of the donor cv. Red Spanish Pinaras for various physiological
parametersviz. Photosynthetic rate, the transpiration rate, the water use efficiency, the
internal leaf CO2 concentration, and the chlorophyll pigment contents. It was observed
that small changes in the pineapple DNA may result in relevant phenotypic
modifications.Significantvariations in netphotosynthetic rate,transpiration rate,stomatal
resistant,stomatal conductance, dark respiration and mesophyll efficiency (mesophyll
capacity to fix CO2) were observed among these productive branches among two
mango groups of cultivars viz. alternate year and regular flowering behaviour (Singh et
al., 2011).So also, Variation between strawberry cultivars in plant canopy and growth
and transpiration efficiency was not strongly influenced by environment, genotypic
variation and these variables could be utilised in a breeding programme for improved
drought tolerance in strawberry (Grant, 2010).

2.6.1. Variation in growth of seedlings

Pot culture experiments have been conducted under open field conditions to
understand variation among genotypes in various species (Hussain et al., 1999; Liu et
al., 2010; Krishnamurthy et al., 2007; Chandrasekar, 2010). Qiu et al. (1992) and
Biswas (1988) showed how dramatically bamboo responds to increasing availability of
water. Given that temperatures are sufficiently high for sympodial bamboo growth,
bamboo could produce new shoots and culms year-round, with no distinct shoot phase
if water demand is satisfied by rainfall (Farrelly, 1984) or irrigation. Lin (1995) has
also said that irrigation promotes shoot and culm growth when rainfall is insufficient.
Many authors have noted the positive effects of greater availability of water on bamboo
production and how lack of water limits its productivity (Kleinhenz and Midmore,
2001). Differences in seedling height growth were reported among populations of
Populus davidiana (Zhang et al. 2004) and Pinus harwegii (Viveros-Viveros et al.,
2009). Water supply had a major effect on bamboo growth and shoot yield of

38
Phyllostachys pubescens established in South-East Queensland, Australia (Kleinhenz et
al., 2003). Without irrigation and only little rainfall (80 – 140 mm) prior to and during
the shoot season in the first 2 years of the study, shoot numbers were greater in plots
closer to a supply of temporal pond water. With irrigation in the following years,
bamboo shoot numbers and individual shoot weights were much greater at the higher
rate of irrigation.

2.6.2. Variation in leaf photosynthesis rate

Photosynthesis is influenced by three main processes; a photochemical process
involving the utilization of light energy for photosynthesis, a diffusion process
associated with the transport of CO2 to the photosynthetic reaction site, and
biochemical processes involved with the fixation and chemical reduction of CO2
(Gaastra, 1962; Bierhuizen and Slatyer, 1964). The magnitude of the three processes
can be influenced by many factors, and the interaction of such factors will ultimately
affect the photosynthetic rate. The relationship between photosynthesis and radiant
energy can best be defined by a hyperbolic shaped curve. At low light flux densities the
relationship is essentially linear but as light flux density is increased, photosynthetic
rate increases less rapidly until light saturation is reached in many species. There are
four major features of the light response curve which need further explanation; the
linear portion, the curvilinear portion, the light saturation point, and the light
compensation point (Criswell, 1970).The efficiency of conversion of intercept radiation
to biomass is affected mainly by the density of plants, as well as by altitude, position in
the canopy and the time and season of measurement. A clear diurnal variation of light
use efficiency at different times of each measuring day, light use efficiency was
generally higher in the summer than in winter and in the afternoon than in the morning
(Cao et al., 2011).
Diurnal variation in photosynthesis of Calamus nambariensis var. alpinus at
seedling stage was observed with an obvious double peaks curve of diurnal variation
both in net photosynthesis rate (Pn) and transpiration rate (Tr) with obvious midday
depression at 12 and the peak value appeared at 10 to 14 (Fengying et al., 2010).The
photosynthetic rate of Phyllostachys edulis and related factors were studied in different
time scales and the results showed that the curves of diurnal variation of net
photosynthetic rate (Pn) were different in four seasons, demonstrating bimodality in
summer and autumn and single peak curve in winter and spring. Wen et al. (2011)
studied temporal and spatial dynamics of carbon fixation by Moso Bamboo

39
(Phyllostachys pubescens) in subtropical China, using a LI-6400 portable
photosynthesis system. The results showed that the capability of carbon fixation of P.
pubescens leaves had obvious temporal and spatial dynamic variations. It was revealed
that there were two peak periods and two low periods in the seasonal variation of
carbon fixation capability. Data also revealed that the capability of carbon fixation by
five-year-old P. pubescens was more than that of one-year-old and three-year-old.
Results showed two peaks of carbon sequestration within a year, from May to June,
October, and two depressions, in the high temperature period of July to August and in
the low temperature period and leaf displacement period. However, Huang et al. (1989)
found that the peak of carbon sequestration appeared in the summer. They also
observed that Daily changes and seasonal changes of carbon sequestration were
positively correlated with mean temperature, precipitation, rainy days, monthly
sunshine hours, leaf temperature, relative humidity of chamber and light saturation
point.
Kobayashi et al. (2000) compared photosynthetic light utilization efficiency for
Sasa kurilensis beneath the closed canopy of Fagus crenata. They observed that
photosynthetic light utilization efficiencyat relatively low light was greater, and the
dark respiration rate was smaller in the leaves of seedlings (10 cm in height) beneath
the closed canopy than in the leaves of saplings at the sunny forest edge. The
culm/foliage (C/F) ratio of F. crenata seedlings at the shady site was small, suggesting
effective matter-production beneath the Beech canopy. Because the shoot density of S.
kurilensis was smaller beneath the beech canopy than in the gap, light availability at the
bottom of S. kurilensis layer was greater beneath the beech canopy. These results
suggest that the photosynthetic productivity of the F. crenata seedlings would be
enough for the seedlings to survive in the understory with a low density of S. kurilensis
shoots beneath the closed beech canopy.
Intraspecific variation in the rate of photosynthesis has been reported in many
species (Kumar, 1982; Hutmacher and Kreig, 1983; Collins et al., 1988). Genotypic
variation in photosynthesis and plant water relations during drought and the leaf and
seedling morphology were examined in greenhouse-grown Fraxinus pennsylvanica
from five populations located along an east-west transect from New York State to
South Dakota (Abrams et al., 1990). During a 17 day drought, South Dakota seedlings,
from the most xeric habitat, maintained the highest net photosynthesis and leaf
conductance, and New York seedlings, from the most mesic habitat, exhibited the
lowest net photosynthesis and leaf conductance. Photosynthesis differed significantly

40
among sweet potato genotypes (Bhagsari, 1990). Single leaf net photosynthesis varied
among sweet potato genotypes and ranged between 0.61 to 1.09 mg CO2 m-2 sec-1 and
the stomatal conductance ranged from 0.68 to 1.34 cm s-1 and these parameters peaked
in August period.Koyama and Uchimura (1995) reported photosynthesis value of 12 µ
mol cm-2 sec-1 in Phyllostachys bambusoides. Differences in net photosynthesis along
the genotypic and phenotypic lines have been observed (Victoria and Abrams, 1994).
Phyllostachys edulis seedlings responded differently to the elevated CO2
concentration in four seasons leading to variation in growth under same light intensity
(Shi et al., 2007).Photosynthetic activities of different bamboo species (Phyllostachys
viridi-glaucescens, Phyllostachys pubescens, Phyllostachys bambusoides and Bambusa
ventricosa) growing at the Botanical Garden of Rome werestudied(Gratani et al., 2008).
The highest net photosynthetic rates (NP) of the considered species were measured in
early autumn, while the lowest ones in spring (30percentof the maximum in the genus
Phyllostachys), in the period of vegetative activity, and in winter (10percent of the
maximum in B. ventricosa). The correlation between NP and leaf temperature (LT)
indicated that the favourable temperature enabling 50–100 percent of NP was in the
range 2.2–32.1 and 16.2–36.3 °C for the genus Phyllostachys and B. ventricosa,
respectively. According to their origin, the species of the genus Phyllostachys,
originating in a temperate climate had a higher sensibility to high air temperatures than
B. ventricosa, originating in a tropical and subtropical climate, and having a lower
sensibility to low air temperatures. Owing to the great potential for biomass production,
bamboos could be a significant net sink for CO2 carbon sequestration with the mean
maximum yearly assimilation rate (9.5±4.5 μmol m−2 s−1), P. pubescens contributed in
major role to carbon sequestration (14±0.6 kg CO2 year−1 per culm) compared with the
other considered species (on the average 3.0±1.6 kg CO2 year−1, mean value.
A significant variation was established for net photosynthesis (8.14 to 15.13
µmol m-2 s-1) and stomatal conductance (0.37 to 0.59µmolm-2 s-1) among the
provenances of Neem (Kundu and Tigerstedt, 1998).Net photosynthetic rate (Pn) increased
in bamboo (Phyllostachy pubescens) with increased atmospheric CO2 concentrations (Cao et
al., 2011). Average maximum Pn was 17.76, 16.66 and 16.77 (µmol-1m-2s-1) in lower, middle
and upper layers canopy respectively at CO2saturation point. Light use efficiency of leaves
ranged from 0.15 percent to 2.25 percent on different days and at different times of year
measured by LP-6400 at 900 m altitude in the Tiannmu Mountain Natural Reserve (TMNR) in
Northwest Zhejiang Province, China.

41
2.6.3. Variation in leaf transpiration rate

Leaf transpiration rate and stomatal conductance also varies among

populations. Transpiration from a stand of Moso Bamboo was estimated with respect to
sap flux and it was observed that there was small individual to individual variation in
sap flux (Kume et al., 2010). In a study, over 70 cultivars were grown in field for over
2 years to determine stomatal density, guard cell length, and potential stomatal
conductance, along with other morphological leaf traits (Tanaka et al., 2010). Large
variation was found in potential stomatal conductance, which was closely associated
with stomatal conductance in the field for representative genotypes.Two cultivars of
strawberry were traditionally grown without irrigation (rain-fed only), one (‘Cambridge
Favourite’) showed very high transpiration rates, while the other (‘Totem’) showed
very low transpiration rates (Grant, 2010).Kullaj et al. (2014) assessed the behaviour of
several apple cultivars under a cultivation regime with high radiation and temperatures.
They observed that due to their vigorous growth, the cultivar Red Delicious and to
some degree also cultivar Golden Delicious showed higher levels of transpiration.
Transpiration Efficiency (TE) and water extraction were assessed in 149 germplasm
entries from sorghum reference set (plus three control cultivars) using a lysimetric
system under terminal water stress and fully irrigated conditions outdoors (Vadex et al.,
2011). A 2-fold range for TE and a 1.25-fold variation for water extraction were
observed under terminal water stress conditions among the germlasms.

2.6.4. Variation in Instantaneous Water Use Efficiency

A first step in breeding for improved water use efficiency is to determine the
extent of adaptation to limited water availability already existing between cultivars.
Variation between the cultivars of strawberry was observed for their Water Use
Efficiencies (Grant et al., 2010). Instantaneous Water Use Efficiency (WUE) observed
among the populations was high in summer season (Kumar and Bera, 2013). So also,
significant genotypic variation was observed for WUE among the cultivars of Triticum
astivum (Ehdaie and Waines, 1993).

2.6.5. Variation in Leaf Nutrients

Higher content of Nitogen promotes development and prevents collapse of
chloroplasts in leaves and is closely correlated with the photosynthetic rate of leaves
(Mengel and Kirkby, 1987). Nitrogen is reported to be the most common nutritional

42
factor limiting photosynthesis and biomass production (De Jong, 1982). Positive
correlations between leaf nitrogen content and photosynthesis have been reported
(Aoki, 1987; Sakai, 1987). Phosphorus may exert a direct effect on photosynthesis by
modifying the energy metabolism and potassium indirectly affects photosynthesis
through its effect on stomatal conductance (Sobhana et al., 1996). Leaf nutrient
accumulation studies in Phyllostachys pubescens in Chinarevealed that Nitrogen,
Phosphorus and Potassium values decreased in older leaves (Huang et al., 1985). Leaf
nitrogen and phosphorus observed in < 1 year old leaves of Bambusa distegia which
were respectively 2.74 percent and 0.21 percent, while, leaf potassium content were
lower than 1.68 percent (Zhou and Wu, 1997).
The antioxidant system in the leaves of two sugarcane cultivars under salt stress
was analysed (Medeiros, 2014). In both salt-stressed cultivars, no alteration in the foliar
nitrogen content was observed; however, there was a reduction in the phosphorus and
potassium levels and an increase in the sodium and chloride concentrations. There was
also a reduction in gas exchange on the third day under salt stress. Although the content
of soluble sugars remained stable in both species, there was a decrease in free amino
acids. Significant differences between accessions of Miscanthus were found for the
nutrient concentration (Lei et al., 2013). In addition, the significant difference conferred
the possibility of achieving a desirable cultivar with significant biomass yield and
relative nutrient removal by harvest.

2.6.6. Leaf chlorophyll content

Chlorophyll content in leaves varies along the season and phenotypes. The
chlorophyll content in leaves vary with the day length, irradiance and radiation quality,
temperature and nutrient status of the soil (Lewandowska and Jarvis, 1977).
Chlorophyll content among tea clones were studied and it was observed that
highestchlorophyll was present in autumn and lowest in summer (moisture stress
period) (Kumar & Bera 2013). So also, significant differences in chlorophyll content
was observed among rice varieties (Djanaguiraman and Ramadass, 2004). Ranunculus
repens populations showed no variation in chlorophyll concentration when grown
under flooded and non-flooded conditions (Lynn and Waldren, 2002).The changes of
leaf area index (LAI), leaf photosynthetic pigments content, photosynthetic rate of
Phyllostachys pubescens in one leaf development cycle (two years) under different
fertilization (Jin et al., 2011). Fertilization significantly promoted the synthesis of

43
chlorophyll. Wu et al. (2002) extracted chlorophyll contents from the epidermis of
moso bamboo by ultrasonic using 80 percent acetone, DMF, and DMSO. The results
demonstrated that, in acetone, the contents of chlorophyll a, b, and total chlorophyll
were 3.23, 1.56. and 4.80 mg g-1, respectively. In contrast, in DMF and DMSO, both
chlorophyll contents were less than that in acetone. The total chlorophyll contents were
only 4.18 and 3.78 mg g-1.

2.6.7. Specific leaf area

Specific leaf area (SLA, the ratio of leaf area to leaf dry mass) and leaf dry
matter content (LDMC, the ratio of leaf dry mass to fresh mass) reflect a fundamental
trade-off in plant functioning between a rapid production of biomass (high SLA, low
LDMC species) and an efficient conservation of nutrients (low SLA, high LDMC
species). It has thus been proposed that these traits – or one of them – be measured
routinely in screening programmes. Valinete et al. (2014) reported a high population-
level differentiation in Specific Leaf Area in Cork Oak. Specific leaf area exhibited
inverse relation with leaf area and leaf dry weight (Nath and Das, 2008) in Bambusa
cacharensis. The lack of clear trends in specific leaf area among such diverse ecotypes
may also indicate that the variation in length and other dimensions of width and
thickness (Tranquillini, 1979) may be largely a phenotypic response.Dijkstra and
Lambers (1989) observed that a fast growing inbred line of Plantago major had higher
specific leaf area than that of the slow growing one. Individual leaf area, leaf specific
weight and dry leaf weight were measured for nine Chinese provenances of
Phyllostachys pubescens in 1996 (Qiu et al., 1999). The provenance with the largest
individual leaf area was from Longhai (Fujian), followed by those from Jianou and
Conghua (Guangdong); the provenance with the smallest leaf area was from Jurong
(Jiangsu) while the specific leaf weight varied through the year in a 'low-high-low'
pattern. A stability of relationship existed between carbon isotope discrimination and

SLA (specific leaf area) in Arachis hypogaea genotypes collected from different
environments suggesting that SLA can be used to screen genotypes for Water use
efficiency (Rao and Wright. 1994).Specific leaf area (SLA) in Sasa palmata, a
temperate bamboo was greater in shade than in the open, irrespective of rhizome
connection, suggesting that individual leaves show morphological plasticity
independently of only physiological integration (Saitoh et al., 2002). Rebetzke et

44
al.,2004 observed large genotypic differences for Specific Leaf Area ranging between
276-335 cm2 g-1 of wheat when evaluated in glasshouse. They further claimed that
genotypic variation in specific leaf area was under additive genetic control with small
to moderate narrow sense heritability (h2=1-51 percent on single plant basis).

2.6.8. Relationship between growth and physiological parameters.

Relationships between most of the growth and physiological parameters have

been reported by various authors. A significant correlation between net photosynthesis
and phytomass was reported by Mahon et al. (1977), who further suggested that total
dry matter weight should be used in gas exchange studies. Net photosynthesis and
stomatal conductance are often related to each other (Salisbury and Ross, 1992). The
photosynthetic capacity of an individual leaf depends upon its nutritional status and
climatic condition (Kleinhenz and Midmore, 2001). Kumar and Bera (2013) also
reported a positive relationship between chlorophyll content and photosynthesis. A
positive relationship between percentage nitrogen and leaf photosynthesis rate can be a
result of direct participation of roughly half of leaf nitrogen in the photo and
biochemical photosynthesis process (Mooney, 1986).

A positive association (P<0.01) was observed between stomatal conductance

and transpiration under irrigated and non-irrigated in 32 public cotton cultivars/bred
lines in two field experiments in both years indicating the prevalence of stomatal
control of transpiration (Ullah et al., 2008).Various physiological parameters were
estimated between two Walnut species J. hopeiensis Hu. (‘Yihe 1’) and J. regia L.
(‘Qingxiang’) and it was significant correlation between PN and Chl content, E and Ci
(Wang et al., 2010). Bamboo species exhibit a high plasticity of its leaf structural and
functional traits in response to different irradiances and the combination of high
plasticity in leaf morphological, anatomical and physiological traits allows this bamboo
species to grow in heterogeneous habitats (Yang et al., 2014). Specific leaf area and
plant growth relationship seem to provide a physiological basis of the variation in
growth of the provenances as observed by Yoshida (1976) and Wang et al. (1995) in
barley (Hordeum vulgare L.) and silver birch (Betula pemdula Roth), respectively. In
evaluating the relationships among specific leaf area, whole plant growth and
photosynthetic parameters, specific leaf area was considered to be an important growth
determinant (Kundu & Tigerstedt 1998).

45
 The combination of elevated CO2 and temperature hardly affected specific leaf
area, chlorophyll content, nitrogen content had positive linear relationship. So also,
variables net photosynthesis rate had positive relationship in all treatments (Li et al.,
2012). Fargesia rufa Yi growing understory in subalpine dark coniferous forest, was
investigated to see the effects of elevated CO2, temperature and their combination, the
dwarf bamboo plantlets were exposed to two CO2 regimes (ambient and double
ambient CO2 concentration) and two temperatures (ambient and +2.2°C) in growth
chambers(Li et al.,2013). Gas exchange, leaf traits and carbohydrates concentration
were measured after the 150-day experiment. Elevated CO2 significantly increased the
net photosynthetic rate (Anet), intrinsic water-use efficiency (WUEi) and decreased
stomatal conductance (gs) and total chlorophyll concentration based on mass (Chlm) and
area (Chla). On the other hand, elevated CO2 decreased specific leaf area (SLA), which
was increased by elevated temperature. Elevated CO2 also increased foliar carbon
concentration based on mass (Cm) and area (Ca), nitrogen concentration based on area
(Na), carbohydrates concentration (i.e. sucrose, sugar, starch and non-structural
carbohydrates) and the slope of the Anet–Na relationship. However, elevated
temperature decreased Cm, Ca and Na. The combination of elevated CO2 and
temperature hardly affected SLA, Cm, Ca, Nm, Na, Chlm and Chla. Variables Anet and Na
had positive linear relationships in all treatments. Results showed that photosynthetic
acclimation did not occur in dwarf bamboo at elevated CO2 and it could adjust
physiology and morphology to enable the capture of more light, to increase WUE and
improve nutritional conditions.

2.7. Genetic Variation

2.7.1. Isolation of DNA

Tender leaves have been extensively used as a source of DNA in various

bamboo species (Mukherjee et al., 2010; Hu et al., 2000; Sahasrabudhe and Deodhar,
2010; Nayak et al., 2010; Ramanayake et al., 2007). Rapid DNA method (Doyle and
Doyle, 1987) was used to extract the DNA from these tender leaves, and it has been
extensively used in DNA extractions in several plant species (Dilworth and Frey, 2000;
Paris and Carter, 2000; Mace et al., 2003). The Doyle and Doyle method along with
slight modifications have been used to obtain good quality total DNA for polymerase
chain reaction (PCR) based downstream applications (Devi et al., 2013). The annealing
temperature maintained for the 15 ISSR primers during thermal cycling was 44.9 °C to

46
57.1 °C. The annealing temperature depends on the GC content of the primer used and
usually ranges from 45 to 65 ◦C (Lian et al., 2001; Ammiraju et al., 2001; Reddy et al.,
2002). The DNA is usually quantified and examined for intactness using the gel
electrophoresis method and is the most suitable and accurate technique available
(Matasyohet al., 2008). Lin et al., (2011) also extracted DNA of Phyllostachys
violascens in similar way and used in ISSR technique.

2.7.2. ISSR analysis

Inter simple sequence repeat (ISSR) technique is a PCR based method, which
involves amplification of 1 DNA segment present at an amplifiable distance in between
two identical microsatellite repeat regions oriented in opposite direction (Reddy et al.,
2002). The technique uses microsatellites, usually 16–25 bp long, as primers in a single
primer PCR reaction targeting multiple genomic loci to amplify mainly the inter- SSR
sequences of different sizes. The microsatellite repeats used as primers can be di-
nucleotide, tri-nucleotide, tetranucleotide or penta-nucleotide. The primers used can be
either unanchored (Meyer et al., 1993; Gupta et al., 1994; Wu et al., 1994) or more
usually anchored at 3’ or 5’ end with 1 to 4 degenerate bases extended into the flanking
sequences (Zietkiewicz et al., 1994). The technique combines most of the benefits of
AFLP and microsatellite analysis with the universality of RAPD. ISSRs have high
reproducibility possibly due to the use of longer primers (16–25 mers) as compared to
RAPD primers (10- mers) which permits the subsequent use of high annealing
temperature (45– 60 ◦C) leading to higher stringency.
The studies on reproducibility show that it is only the faintest bands that are not
reproducible. About 92–95 percent of the scored fragments could be repeated across
DNA samples of the same cultivar and across separate PCR runs when detected using
polyacrylamide (Fang and Roose, 1997; Moreno et al., 1998). The 10 ng template DNA
yielded the same amplification products as did 25 or 50 ng per 20μl PCR reaction. The
annealing temperature depends on the GC content of the primer used and usually
ranges from 45 to 65 ◦C. ISSRs segregate mostly as dominant markers following simple
Mendelian inheritance (Gupta et al., 1994; Tsumura et al., 1996; Ratnaparkhe et al.,
1998; Wang et al., 1998). However, they have also been shown to segregate as co-
dominant markers in some cases thus enabling distinction between homozygotes and
heterozygotes (Wu et al., 1994; Akagi et al., 1996; Wang et al., 1998; Sankar and
Moore, 2001).

47
 ISSRs have been successfully used to estimate the extent of genetic diversity at
inter- and intra-specific level in a wide range of crop species which include rice (Joshi
et al., 2000), wheat (Nagaoka and Ogihara, 1997), fingermillet (Salimath et al., 1995),
Vigna (Ajibade et al., 2000), sweet potato (Huang and Sun, 2000) and Plantago (Wolff
and Morgan-Richards, 1998). Superiority of ISSR-PCR over other marker techniques
has been brought out in such investigations by various workers. Anchored SSR primers
for instance, have been found to be more useful and reproducible than isozymes,
RFLPs and RAPDs in the diversity analysis of trifoliate orange germplasm (Fang et al.,
1997). ISSRs were more useful for the analysis of diversity in the genus Eleusine in
terms of quality and quantity of data output as compared to RFLP and RAPD (Salimath
et al., 1995). Significantly, the efficiency of the technique was evident in
characterization even at the varietal level of a species. For instance, three 5’ anchored
primers together could distinguish 20 cultivars of Brassica napus (Charters et al.,
1996).
ISSR is the marker of choice for assessment of genetic diversity in cocoa
(Charters and Wilkinson, 2000), gymnosperms such as Douglas fir and sugi (Tsumura
et al., 1996) and even fungi (Hantula et al., 1996). In a study on white lupin it has been
demonstrated that among 10 primers used any two were sufficient to distinguish all the
37 accessions studied (Gilbert et al., 1999). Similarly, 4 primers were sufficient to
distinguish 34cultivars of potato (Prevost & Wilkinson, 1999) and 3 primers could
distinguish 16 genotypes of redcurrant (Lanham & Brennan, 1998).Several researchers
have used number of ISSR primers to ascertain genetic diversity viz.Yang, et al. (2012)
observed polmorphic bands in 10 ISSR primers to understand diversity among the
populations of Dendrocalamus membranaceus; Length (2009) used 17 ISSR primers to
study 12 accesions of Melocanna baccifera; Lin et al. (2011) used 15 ISSR primers to
acess the genetic diveristy among cultivars of Phyllostachys violascens; Tian et al.,
(2012) used 7 ISSR primers to ascertain the genetic variation between and among the
populations of Dendrocalamus giganteus.
The use of such highly informative primers lowers the cost, time and labour for
diversity analysis. Various marker techniques have been used inphylogenetic
investigations based on relative similarity. Inspite of their higher efficiency and
reproducibility, ISSR markers have as yet not been used extensively. It has however
been found effective in resolving problems relating to the phylogeny of Asian
cultivated rice Oryza sativa (Joshi et al., 2000), wheat (Nagaoka and Ogihara, 1997),
finger millet (Salimath et al., 1995), Vigna (Ajibade et al., 2000) and Diplotaxis species

48
(Martin & Sanchez-Yelamo, 2000). There is immense scope to use this powerful
technique in resolving species/inter-species status in many a genus and in deciding the
distinctness of different genera within a family. Significantly, genome/species specific
ISSR markers have been reported in four genera Oryza (Joshi et al., 2000), Lolium and
Festuca (Pasakinskiene et al., 2000) and Diplotaxis (Martin and Sanchez-Yelamo,
2000) which are useful in delineating species.
ISSR technology is a rapid and sensitive technique which can be used to
estimate relationship between closely and more distantly accessions (Length 2009).The
use of inter-simple sequence repeats (ISSR) has been reported as being more useful for
assessing genetic diversity for plant populations and inferring genetic relationships
among closely related cultivars compared to standard DNA sequencing methods (Goh
et al., 2010). ISSR markers are proved to be efficient and inexpensive way to provide
molecular data. They have been successfully used in determining genetic relationship
and used for DNA fingerprinting in various bamboo species (Xinc, 2009; Qirong, 2010;
Mukherjee et al., 2010; Yang, 2010; Tian, 2011; Lin et al., 2012).

2.7.3. Polymorphism in primers

Polymorphism among genotypes results from differences in the length of the

tandem repeats, which can be very variable by contraction or expansion of the tandem
repeats (Environment 1995). Relatively large number of polymorphism obtained could
be useful in finding the elite germplasm resources for industrial use (Nag et al., 2013).
Low to moderate levels of polymorphism in few species indicate that diversity of the
species is declining (Godt and Hamrick, 1991). Genetic diversity of Sinocalycanthus
chinensis populations in four different habitats, namely, scrub, evergreen broadleaved
forest, Chinese fir forest and bamboo grove, in China, was analysed by considering 12
primers using polymorphic DNA technique (Zhang et al., 2007). Environmental
conditions (e.g. temperature and precipitation) as well as human activities could affect
genetic diversity (Liu, et al., 2009). It was observed that the percentage of polymorphic
loci of four populations varied from 6.04 to 11.54 percent with an average of 8.93
percent, wherein bamboo grove population was the highest and evergreen broadleaved
forest population was the lowest with similar trend in Shannon's and Nei's indexes were
observed. A total of 209 (136 polymorphic), 222 (152 polymorphic), and 434 (253
polymorphic) bands were detected using 15 ISSR primers, 15 primer combinations of
SRAP, and 15 primer combinations of AFLP, respectively (Yang et al., 2010). Based
on genetic diversity, all the cultivars of P. violascens were divided into four groups,

49
which were reflected by their morphologies. In a report, SSR markers based studies in
Cocos nucifera revealed that all 17 primers showed 100 per cent polymorphism (Rajesh
et al. 2014).

2.7.4. Genetic variation between accessions

Genetic variation among the accessions and populations has been estimated by
various authors in various species by using molecular marker technique. Molecular
markers are commonly used to characterize genetic diversity within and between
populations or groups of individuals because they typically detect high levels of
polymorphism (Kosman and Leonard, 2005). Literature survey reveals that ISSR,
AFLP and RAPD markers are used to understand genetic variation and considerable
genetic variation exists among and between the populations of any bamboo species.
High similarities were observed in Phyllostachys pubescens collected from Ningbo
(East), Jingning (South), Jiangshan (West) and Anji (North) of Zhejiang Province,
China for DNA polymorphism and corresponding physicochemical properties of
bamboo charcoals (Lin et al., 2007). Genetic variability of Dendrocalamus hamiltoni
was studied in Chandel and Imphal East using AFLP molecular markers (Waikhom et
al., 2012). In a study, Waikhom et al. (2012) observed that DICE similarity between 10
accessions of Dendrocalamus hamiltoni ranged from 0.152 to 0.856. On the contrary
high similarity (0.023 to 0.108) was observed among the cultivars of Phyllostachys
pubescens in South China (Xin et al., 2009). UPGMA cluster based groupings were
observed in different bamboo species by several authors (Waikhom et al., 2012;
Length, 2009). The genetic diversity analysis among 35 accessions of Zizania latifolia
Zhejiang Province in China was conducted using 47 pairs of SRAP primers (Chaohang,
2010). It was observed that these accessions clustered into two groups and genetic
distance between single cropping and double cropping bamboo was greater than the
distance within each type, while the accessions with closer geographic origins showed
higher genetic similarity.

Amplified Fragment Length Polymorphism (AFLP) technique was used to

analyze genetic diversity of 26 samples of Iris collected from China. Genomic DNA
was digested with EcoRI and MseI enzymes and amplified with 6 EcoRI+3/MesI+3
primer combinations. A total of 536 scorable bands were produced by AFLP analyses
which were translated into genetic similarity matrix. Genetic similarity, ranging from
0.56 to 0.89, showed abundant genetic diversity. Cluster analysis was made based on

50
the UPGMA method. All the samples were classified into six groups. Different
provenances of Iris belonging to the same species with high genetic similarity were
closely correlated and gathered together (Shaohuao et al., 2011). Amplified fragment
length polymorphism (AFLP) was used to study DNA diversity of 16 species in
Indocalamus and other 5 similar bamboo species (Shaohua et al., 2009). Genomic DNA
was digested with EcoR I and Mse I enzymes and amplified with 9 (EcoR I+3)/(Mse
I+3) primer combinations. In the 21 samples, 9 EcoR I-Mse I AFLP primer
combinations produced 1367 legible bands, of which there were 115 common bands,
273 specific bands and 72 specific missing bands. In the 16 samples of Indocalamus,
1193 legible bands were obtained by using 9 primer combinations, with 190 common
bands, 177 specific bands and 98 specific missing bands. All the specific bands and
specific missing bands would be used as molecular markers to identify these species.

AFLP analysis of DNA samples of Phyllostachys pubescens showed distinct

genets that originated from the previous flowering event and that each genet had its
own flowering time (Isagi et al., 2004). It was revealed further that, different genets are
mixed in a community of a large bamboo species with a leptomorphic rhizome system,
and a community that originated from a single flowering event can have a range of
flowering years. Triplett et al. (2010) made a comparative analysis of amplified
fragment length polymorphisms (AFLPs) and chloroplast DNA sequences representing
diversity within and among three species plus individuals with intermediate or unusual
morphological characteristics (putative hybrids). Molecular results support the
recognition of three species previously defined on the basis of morphology, anatomy,
and ecology, with most of the molecular variance accounted for by among-species
variation. Molecular evidence also demonstrates that A. tecta and A. appalachiana are
sister species, forming a clade that is significantly divergent from A. gigantea.
Phyllostachys were analyzed using Nuclear Restriction Fragment Length
polymorphisms (RFLPs). The RFLP data were used to generate genetic distances
between all pairs of taxa and to examine the degree of genetic variation within and
among bamboo species. The genetic distances were also used to create dendrograms of
accessions and species (Friar and Kochert, 1994).

Clonal identification of 176 P. pubescens acessions collected around Nantou

County island in Taiwan was performed using RAPD analysis. Results showed that
genetic variation in the samples was very limited (Lai and Hsiao, 1997). The region
around contained all of the nine identified clones whilst the remaining regions generally

51
consisted of only 1 common clone. This study led to the identification of first area of
successful introduction and that cultivation later spread to other regions. Nineteen
cultivars and 2 affinis species of Phyllostachys praecox were analyzed in China using
10 primers of random amplified polymorphic DNA molecular markers (Wei et al.
2001). Results showed that narrow and broad leaved Lei bamboos are the result of
management practices and the classification of bamboo species based on random
amplified polymorphic DNA markers is somewhat reliable. Twenty random primers
were used to amplify DNA fragments extracted from 56 samples of Dendrocalamus
sinicus from South and South-West of Yunnan Province, China (Lipeng et al., 2004).
The analysis showed that D. sinicus had high level of genetic diversity in same
populations of the 2 variant types 'curved foot' type and ‘straight culm' type. Ten
accessions of different germplasms of moso bamboo , Phyllostachys edulis and 2
outgroups (P. kwangsiensis, P. reticulata) are used to compare viz. AFLP, ISSR,
SRAP, (AFLP+SRAP), (ISSR+SRAP), (AFLP+ISSR), (AFLP+ISSR+SRAP), single-
species and multi-joint identifications method are respectively used for the genetic
similarity cluster analysis and principal coordinates analysis of genetic distance
(Qirong, 2010). This study indicated that the genetic similarity coefficient range of the
10 bamboos was 0.59, -0.86, while the genetic distance was 0.20 -0.677, the sequence
homology 97.40 percent -99.9 percent, the variation rate 0.0-2.06 percent.Ten cultivars
of Phyllostachys pubescens were studied to determine genetic similarity using 16 inter-
simple sequence repeat (ISSR) primers in China (Xin, 2009). Statistical analysis
showed that the genetic similarity matrices obtained from these two sets of molecular
markers had a significant correlation (R=0.959, P=0.013) with genetic distances
ranging from 0.023 to 0.108, and could be divided into three groups based on their
genetic variation and similarity.

Phyllostachys violascens cultivars were assessed for genetic diversity using 15

ISSR (inter-simple sequence repeat) primers (Lin et al., 2012).The mean genetic
similarity of P. violascens was 0.872 and all the cultivars could be divided into four
groups, which are reflected by their morphologies. Genetic relationships among 22 taxa
of bamboo were evaluated using 12 inter simple sequence repeats (ISSR) and four
expressed sequence tag (EST)-based random primers, resulting in amplification of 220
loci (Mukherjee et al., 2010). The grouping of species supports earlier published reports
on molecular phylogenetic studies in bamboos with few deviations.Genetic diversity of
four straight-culmed provenances of Dendrocalamus sinicus was analyzed based on

52
seven inter-simple sequence repeat (ISSR) primers in China (Yang, 2010). A high
degree of genetic differentiation among straight culmed D. sinicus provenances was
observed most likely due to habitat fragmentation and flowering without seed. Inter-
simple sequence repeat (ISSR) markers were used to assess the level and distribution of
genetic diversity in 226 individuals from 15 populations of H. Cordata in China (Wei
et al., 2012). ISSR analysis revealed low genetic variations within populations but high
genetic differentiations among populations.

2.7.5. Genetic variation between and among populations.

The genetic uniformity and non-uniformity is reflected by the genetic distance

value as described by Das and Mukherjee (1997). If the population is more or less
continuous, allele frequencies will fluctuate with distance due to genetic drift and
selection (Sokal et al., 1989). Population with continually small effective population
size will be especially susceptible to the loss and the re-organization of variation by
genetic drift (Barrett and Kohn, 1991; Frankel and Soule, 1981). Yushania
niitakayamensis was studied from nine locations across its distribution range in Taiwan
for genetic differentiation using RAPD (Hsiao and Lee, 1999). The study indicated that
the populations were generally differentiated according to geographical separation and
altitudinal differences that interrupt gene flow and high altitude populations were more
similar genetically. Analysis of molecular variance (AMOVA) revealed that among-
location, between sampling sites within location, and among individuals within
sampling site components accounted for 15.27 percent, 4.80percent and 79.93percent of
the total variance, respectively.Ofori et al. (2006) described within and between-
population generic variation in Bambusa vulgaris using Random Amplified
Polymorphic DNA (RAPD). Six deca-mer primers generated 45 different sized
fragments (loci) across 76 accessions from four populations in Ghana. The percentage
of polymorphic bands ranged from 66.67 to 100 percent with a mean of 89 percent.
Genetic distances among populations ranged from 10 to 27 percent and most of the
genetic variation was found to reside within populations (81.5 percent) with a low
index of populations differentiation (18.5 percent), although significant (P < 0.001).
Cluster analysis produced four major clusters with seven sub-clusters. The evergreen
and semi-deciduous accessions grouped separately and may be classified as two
different management units (MUs). The results suggest that sampling from moist
evergreen in MU1 and dry semi-deciduous zone in MU2 may provide a core collection
that could represent the total gene pool of B. vulgaris in Ghana. Genetic differentiation

53
of eleven Dendrocalamus latiflorus populations naturally distributed in South China
was investigated by using 23 primers through random amplified polymorphic DNA
(RAPD) markers (Xing et al., 2010). The results indicated that sympodial bamboo
exhibited high genetic diversity (amounting to 44percent) with genetic distance ranging
from 0.0387 to 0.4374 and clustered into four groups.
ISSR (Inter Simple Sequence Repeat), SRAP (Sequence Related Amplified
Polymorphism) and AFLP (Amplified Fragment Length Polymorphism) techniques
were used for the first time for assessment of genetic diversity within different cultivars
of Phyllostachys violascens (Lin et al., 2009). Ramanayke et al. (2006) studied genetic
diversity within a population of Dendrocalamus giganteus and Ochlandra stridula in
Sri Lanka. It was concluded that, D. giganteus, introduced as a single plant to Sri Lanka
in 1856, showed a relatively low genetic diversity of 0.092+ 0.027. Although seeding
in this species is rare, seed-raised plants showed the highest genetic distances and
contributed significantly to the genetic diversity. The species is mainly propagated
vegetatively and the presence of groups of individuals with no polymorphism indicated
clones. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic
structure and differentiation of seven populations of Dendrocalamus giganteus (Tian,
2012). Seven ISSR primers generated 140 bands, of which 124 were polymorphic
(88.57 percent). Genetic diversity within populations was relatively low, averaging
11.33 percent polymorphic bands (PPB), while diversity was considerably higher
among populations, with PPB = 88.57percent. Greater genetic differentiation was
detected among populations (GST = 0.8474). We grouped these seven populations into
two clusters within an UPGMA dendrogram-one comprised the Xinping and Shiping
populations from central Yunnan, the other included the remaining five populations.
Mantel tests indicated no significant correlation between genetic and geographic
distances among populations. Breeding system characteristics, genetic drift, and limited
gene flow (Nm = 0.0901) might be important factors for explaining this differentiation.

Genetic diversity of Guadua angustifoliawas evaluated by examining 55

accessions using microsatellite sequences of rice and sugarcane, crops genetically
related to this species (Marulanda et al., 2007). Amplifications were obtained with 27
and six similarity groups were also obtained. Cluster analysis in Melocanna baccifera
revealed that the accessions collected from a relative distance form 30 km were found
in separate groups (Length, 2009).

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2.7.6. Factors influencing genetic variation

Thirty to 70 percent of angiosperm species have polyploidy events in their

evolutionary history (Tiffin and Gaut, 2001), and hence polyploidy events are
extremely common in the evolutionary history of flowering plants. In theory,
allotetraploids should have higher genetic diversity than diploids because they are
formed through hybridization of two divergent genomes. Likewise, auto-polypoidy can
slow the effects of both drift and selection on genetic diversity, potentially resulting in
both greater diversity and different patterns of molecular diversity in autotetraploids
than diploids (Haldane, 1930). Dendrocalamus is most likely an ancient hexaploid
(Yang et al., 2012; Soderstrom, 1981; Li et al., 2001) and polyploidy taxa typically
display a great genetic diversity, both in terms and the number of bands amplified
(Garcia et al.,2009). Results have shown that polyploidy has been one of the major
driving forces in the evolution of woody bamboos (Gielis, 1998). Recent work using
dominant genetic markers has shown that plant polyploids possess a high genetic
diversity (Abbott et al., 2007; Garcia-Verdugo et al., 2009).

Bamboos also undergo somatic mutations that create variation, as bamboos live
for a long period, somatic mutations may occur in their gene pool which may
eventually lead to some variations (Length 2009). A growing body of data indicates
that populations of clonal plants can maintain considerable amounts of genetic diversity
(Eckert and Barrett, 1993; Widen et al., 1994). Psudostaga japonica, aclonally
propagated speciesmaintains relatively high levels of allozyme variation and percentage
of polymorphic loci compared to the average plant species of restricted distribution and
those with both sexual and asexual reproduction (Hong and Man, 2002). Ellstrand and
Roose (1987) in a review on studies of population genetic structure of primarily
obligate clonal plant species concluded that plant species tend to have intermediate
levels of genetic diversity which is consistent with our own results.

Oton et al., (2006) observed genetic structure of B. vulgaris in Ghana under

different forest types and it was observed that the genetic dissimilarity distance among
individuals ranged from 0.87 to 0.00 and clear pattern of differentiation was observed
among evergreen and semi deciduous populations. Low levels of genetic variation were
revealed both at the species level and population in an endemic species,
Monimopetalum chinense (Xie et al., 2004). Some narrow endemics appear to possess
levels of gene diversity within population that are comparable to species with broad

55
ranges (Wright et al., 2007).Yang et al. (2012) also found varying amount of
polymorphism (93-100 percent) among the 12 populations of Dendrocalamus
membranaceus in China. So also when accessions are grown for longer period, they
undergo a process of selection both naturally and artificially within different
populations and these genotypes are adapted to the different environments (Length
2009). Analysis of molecular variance in Sinocalycanthus chinensis populations in four
different habitats, namely, scrub, evergreen broadleaved forest demonstrated that the
genetic differences among population were high. Variation among population occupied
71.91 percent, while that within population occupied only 28.09 percent. The genetic
differences coefficient among populations was 0.672. The gene flow among
populations was very low, N< sub>m</ sub>=0.297 0 (Zhang et al., 2007).

Many clonal plant populations usually can be as diverse as those of non-clonal

plant species (Till and Gaudeul, 2002; Stocklin et al., 2009; Egenter et al., 2009). High
genotypic diversity in clonal accessions of Sasa senanensis were incurred by the
environmental heterogeneity (Suyama et al., 2000). A prevailing assumption is that a
high degree of asexual reproduction is often associated with genetic monomorphism
(Williams, 1975; Harper, 1977). However, Ellstrand and Roose (1987), Hamrick &
Godt (1989) and Widén et al. (1994) concluded that clonal populations may have high
genetic diversity. In China, a high degree of genetic differentiation among straight-
culmed Dendrocalamus sinicus provenances was observed and it was most likely due
to habitat fragmentation and flowering without seed (Yang et al., 2010). Variation in
AFLP banding patterns among clones was partitioned into three potential sources;
clones, stems within-clones and foliage within-stems (Douhovnikoff and Dodd, 2003).
Most of the variation was attributable to clones and then to stems within-clones. Our
method yielded a Jaccard similarity threshold of 0.983 that resulted in a potential
pairwise error rate of 8.1percent putative clone assigned to siblings and 1.5 percent
sibling assigned to clones.

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