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17
CHAPTER 2
LITERATURE – a review
Rice is an important cereal crop that that has long been thought cultivated
6500 years ago in southern Asia. It is primarily a high calorie food which is
cultivated mainly for its nutritious grain because of its protein and carbohydrate
content. It is the only cereal that can withstand flood and produce food for
sustaining larger number of people per unit of land than any other cereals (Chang,
1984). About 90 per cent of rice grown in the world is produced and consumed in
the Asian region (Rothschild, 1998).
Taxonomy of genus Oryza has been a topic of discussion since Linneaus
described the taxon. Roschevicz (1931) published a comprehensive study of 20
species which provided a basis for later taxonomic studies in the genus.
Chatterjee (1948) listed 23 species and Sampath (1962) presented a revised list,
enumerating 23 species of Oryza. Vaughan (1994) recognized 22 species to be
valid, in which two are cultivated, Oryza sativa L. and Oryza glaberrima Steud.
Khush (1997) reported two cultivated and twenty-one wild species in the genus
Oryza. Brar and Khush (2003) and Deepak et al., (2006) reported 23 wild species
and 2 cultivated species of Oryza viz. O.sativa and O.glaberrima.
More than hundred names have been proposed for the Oryza species at
various times, including 19 for Oryza sativa alone (Oka, 1988; Lu, 2004). Many
phenotypic differences are obvious between Oryza sativa and its wild relatives
(Xiao et al., 1998; Xiong et al., 1999; Bres-Patry et al., 2001; Cai and Morishima,
2002; Thomson et al., 2003; Uga et al., 2003; Li et al., 2006). Numerous traits
separate wild and domesticated rices including changes in pericarp colour,
18
dormancy, shattering, panicle architecture, tiller number, mating type and number
and size of seeds (Sweeney and Mc Couch, 2007).
2.1. Origin and evolution of domesticated rice germplasm
Origin of rice has been a matter of debate for rice researchers. Ancientness
of genus Oryza dates back to cretaceous period of about 130 million years ago
(Melville, 1966; Chang, 1985). Rice became an important crop in India since 2500
BC before the time of Greeks and Vavilov (1951) considered India and Myanmar
as the centre of origin of domesticated rice.
Koldihwa site in Belan valley in Uttar Pradesh reported the oldest rice
remains in India as carbonized kernels proving existence of rice cultivation in
Northern India in olden time (Sharma et al., 1980). Rice chaffs and shell knives of
6000BC - 4000BC has been excavated from Khok Phanon Di near the Gulf of
Thailand (Higham, 1989). Rice remains obtained from Yangtze basin in China
also show antiquity of rice (Chang, 1983).
The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome
wild relatives of rice in Asia. However, the AA genome wild relatives involve both
annual and perennial forms (Yamanaka et al., 2003).The cultivated species
originated from a common ancestor with AA genome. According to Morishima et
al., (1963) wild progenitor of Oryza sativa is Oryza rufipogon or the Asian form of
Oryza perennis complex, and that of Oryza glaberrima is Oryza breviligulata. A
divergent array of wild species was proposed by different workers as the putative
ancestor of O. sativa (Chang, 1976a). The common rice, O. sativa and the African
rice Oryza glaberrima are thought to be an example of parallel evolution in crop
plants (Khush, 1997). Morishima et al., (1963) also reported parallel evolution of
Oryza sativa and Oryza glaberrima. However the ultimate common progenitor of
19
African and Asian cultivated rice is considered to be Oryza perennis by few
workers (Chang, 1976b).
Regarding rice domestication, it is agreed that rice was domesticated from
wild Asian species belonging to A-genome group of genus Oryza (Chang, 1976b;
Second, 1982; Oka, 1988; Khush, 1997; Ge et al., 1999). It has long been
debated which species or ecotype is the direct progenitor of cultivated rice (Oka,
1988; Morishima, 2001). Hypothesis of origin from Oryza nivara was based on
phenotypic similarity between Oryza nivara and Oryza sativa, including annuality,
self fertilization and high reproductive allocation (Chang, 1976a; Khush, 1997;
Sharma et al., 2000).
Second (1982, 1986) treated the perennial and annual form of Oryza
rufipogon as a single species which was considered to be the wild progenitor of
Oryza sativa as they do not have genetic difference between them and shared
abundant sequence polymorphism (Zhu and Ge, 2005; Zhu et al., 2007). Close
relationship between Oryza sativa and Oryza rufipogon was easily demonstrated
by geographic ranges throughout Asia, lack of major reproductive barriers
between them and also by genetic studies using molecular markers.
Wild species of perennial and annual form of Oryza rufipogon have been
found in South east Asia viz. East and West Malaysia, Indonesia, Taiwan, New
Guinea and Hainan Islands (Chang, 1988). According to Chang (1985) and Oka
(1988), annual and perennial forms were recognized as two distinct species,
Oryza nivara and Oryza rufipogon,in course of time developed into two ecogenetic
groups, indica and japonica. Asian mainland is considered as the centre of origin
of Javanica, a junior form of Oryza sativa, (Chang, 1988) and a third ecogenetic
group which is thought to be evolved from japonica (Randhawa et al., 2006).
20
According to Khush (1997), annual types of Oryza nivara were domesticated
to become Oryza sativa. Many workers have considered that the annual and
perennial wild relatives of Oryza sativa should be considered as separate species
(Vaughan et al., 2003).
A hundred years back, there were about 100,000 traditional rice varieties in
India. With the advent of high yielding rice varieties in 1960s, these local cultivars
have been progressively depleted from cultivation in preference to the high
yielding modern varieties. These traditional types are the genetic wealth of the
country as they provide the basic raw material for the improvement of rice crop in
future. Intensified efforts were made to collect and conserve these genetic
resources before they are lost forever.
Appreciable efforts have been made to collect, evaluate and utilize
germplasm for genetic improvement of rice. Considering the need for future
preservation of rice diversity in the Asian realm, exploration and collection of rice
germplasm had been carried out from unexplored areas between 1983 and 1985
(Vaughan, 1990). Indian rice germplasm collection, from primary as well as
secondary diversity, is continuously providing genetic resources for a wide range
of traits in the breeding programmes in India as well as other Asian rice growing
countries (Singh and Singh, 2003).
2.2. Rice Germplasm Characterization in India
2.2.1. Morphology
Morphological characterization is the first step in the classification and
evaluation of the germplasm (Smith and Smith, 1989). It is an indispensable tool
for selecting varieties or lines based on agronomical, morphological, genetic or
physiological characters (Ndour, 1998).Qualitative characters are important for
21
plant description (Kurlovich, 1998) and germplasm characterization can be at
species, variety and cultivar levels.
Plant morpho-physiological characterization is used to evaluate the
phenotypic diversity through agro-morphological traits (Bajracharya et al., 2006).
Reports on the genetic diversity using agro-morphological characterization
identified the phenotypic variability in rice (Ogunbayo et al., 2005; Bajracharya et
al., 2006; Barry et al., 2007; Parikh et al., 2012).Identifying promising morpho-
physiological traits associated with quality and yield play an important role in
varietal development programs (Ashrafuzzaman et al., 2009).
Grain dimensions have formed the basis of systems of classification mainly
on account of their constancy in deciding commercial grading. The physical
properties of rice grain are determined by the dimensions, shape and weight of
the grain and the hardness of the endosperm. In rice, considerable variation in
grain size ranging from long bold to short bold was noticed (Anon, 1971).
Preferences for grain shape vary from one group of consumers to another.
In general, long grains are preferred in the Indian subcontinent, but in South-East
Asia, the demand is for medium to long rices. There was a strong demand for long
grain rice in the international market. Webb et al., (1979, 1985) noticed a
particular trend of grain cooking quality characters related to grain shape.
The varietal characteristics such as shape, size and density affect the quality
of rice grain (Juliano, 1990). Market quality was determined by the physical
appearance of the grain, test weight of the grain, percentage of head rice (whole
kernels) and broken rice (Chang, 1997). Grain morphology is among the first to be
a visible character for selection and quality marking (Siddiqui et al., 2007).
22
Among the morphological characters grain length, length/breadth ratio, grain
pubescence and size and color of sterile lemmas appeared to be quite stable
characters and could, therefore be used as primary diagnostic characters in the
classification of paddy varieties. Other morphological characters viz. grain colour,
colour of sterile lemma, awns, were also found to be useful and paddy varieties
could be grouped into distinct classes on the basis of each of these
characteristics. However, they may be altered by external factors. These
characters, therefore, should be used as secondary diagnostic characters (Gupta
and Agarwal, 1988).
Grain length is the strongest determinant of grain size (Takeda, 1986).
Increasing grain size has been proposed as one of the means to increase not only
paddy yield but also the milling yield of rice (Venkateswarlu et al., 1986). Hence
increasing number of large grains on a panicle could result in raising the yield
plateau of rice (Vergara et al., 1990). Grain size has a direct effect on the
marketability and the commercial success of rice varieties (Redona and Mackill,
1998). There is no international standard for grain length and grain shape but the
International Rice Research Institute (IRRI, 2002) developed a scale for
germplasm evaluation. In rice, grain size, aroma, yield and yield related
parameters are important economic traits (Ashikari et al., 2005).
Node base colour, awning, distribution of awns, anthocyanin colouration of
stem nodes and internodes and stigma colour were of less importance for
identification of rice cultivars (Mehla and Kumar, 2008) They also reported that
awn length, panicle length, leaf blade colour and leaf sheath colour can be used
for identification of rice cultivars.
23
Seedling vigor is an important agronomic trait in plant establishment
(Chauhan et al., 1985; Teng et al., 1992) where seed potential plays a major role
especially in direct seeded rice. Seed size and weight are important
characteristics associated with seedling vigor and quality, good crop stand and
good grain yield (Malik et al., 1989)
Large amounts of starch accumulate before heading in the culm and sheath
which was believed to contribute to stiffness of the shoot (Sato, 1959). Physically,
lodging can be examined in terms of the bending movement and the breaking
strength of the culm and sheath (Chang, 1964). Based on culm length, rice plant
could be classified into dwarf, short, medium, tall and very tall plant types
(Richharia and Govindaswami, 1990). The structural strength of rice culms can be
partly indicated by the slenderness ratio (l/r) which is quotient between the length
of the culm and the radius of the culm at Basal Iinternode 1(BI1). Indica varieties
with slenderness ratios greater than 600 were liable to bend or lodge
(Anon, 1964).
Leaf length was the primary factor affecting leaf size (Tanaka et al., 1966).
Leaf length increases as leaf number advances. In most varieties, the second or
third leaf from the last is the largest and the last leaf is called the flag leaf
(Yoshida, 1981). Thakur (1981) reported leaf angle as erect, horizontal or droopy
and was largely influenced by leaf length. The wider the angle, the more the
spread of leaves for light interception, especially in the lower leaves.
Slow leaf senescence is a desirable trait in nitrogen responsive plant types
(Beachell, 1964). Rice breeders from experience considered that small, dark
green and erect leaves were desirable in varieties to be grown under high nitrogen
conditions (Tsunoda, 1965; Beachell and Jennings, 1965).
24
Flowering duration of rice is influenced by several environmental factors, the
chief of them being the time of sowing the seeds (Ramiah, 1933). Environmental
factors especially temperature affect the rice flowering dates and maturity
(McKenzie et al., 1987). Days to flowering varied from 45 to 150 days in rice
varieties of Kerala (Leenakumari and Nair, 1996). Apiculous color at heading
ranged from pink to dark purple for which several iso alleles had been described
(Oka, 1991).
Studies conducted on twenty cultivars of rice to know various morphological
characters responsible for identification of rice cultivars revealed that plant
morphological characters viz, awn length, panicle length, leaf blade colour and
leaf sheath colour were the most important morphological characters for varietal
identification. On the other hand, leaf pigmentation, flag leaf angle, colour of
lemma and palea, leaf hairiness, secondary branching of panicles and altitude of
panicle branching were not helpful in distinguishing the rice cultivars (Mehla and
Kumar, 2008).
Dhulappanwar and Mensinkai (1967) classified the Indian rice varieties
broadly into four maturing groups namely very early (110 days or less), early (110
-140 days), late (150-170 days) and very late (180 days). The achievements of
high yields by growing early maturing varieties are a desirable goal because it
would result in a more efficient daily production of carbohydrates and more
efficient utilization of land (Anon, 1971). Early maturing varieties increased grain
production per day, increased water use efficiency, required closed spacings to
achieve yield potential, while late maturing varieties were adapted to low fertility
conditions (Yoshida, 1977).
25
The early maturing varieties were adapted to a wider range of sowing
(Ghose et al., 1960). Cultivars varied considerably in maturity when grown in
different regions (McKenzie et al., 1987). Rao (1988) reported that early varieties
showed higher percentage of high density grains among primary tillers while no
difference was observed in late cultivars between primary and tertiary tillers.
Number of panicles per unit area is the most important component of yield and
contributes 89% of the variations in yield.
Decreased pollen production and pollen reception at high temperature cause
decrease in spikelet fertility and cultivar difference (Takeoka et al., 1992). Lower
spikelet fertility at elevated temperature resulted in fewer filled grains, lower grain
weight per panicle and decreased harvest index. Spikelet fertility is thus an
important component of yield that is sensitive to high temperature
(Prasad et al., 2006). The shedding or shattering of grain from the ear at the time
of harvest is one of the important factors contributing to loss in yield of rice
(Ghose et al., 1960).
Semi dwarf plants showed increased tillering ability and responsiveness to
nitrogen fertilization and both contributed directly to high yield (Mc Kenzie et al.,
1987). Positive correlation between plant height and grain yield was observed by
Jangalee et al., (1987) and Deosarkar et al., (1989).However, negative correlation
between them was reported by De and Rao (1988). Plant height had positive and
direct effect on grain yield (Reuben and Katuli, 1989; Ramakrishnayya et al.,
1991; Kumar, 1992).
Kalaimani and Kadamavanasundaram (1988) reported positive correlation
between panicle length and grain yield. However, days to panicle emergence was
26
found to be negatively correlated with 100 grain weight (Saini and Gagneja 1975),
plant height, number of productive tillers and panicle length (Dhanraj et al., 1987).
Significant correlation between panicle length and number of grains per
panicle was also reported by Saini and Gagneja (1975), Nagesha (1976) and
Natarajamoorthy (1979). Rao and Jagdish (1987), Manuel and Palaniswamy
(1989) observed positive correlation between days to panicle emergence and
grain yield. However, Brar and Saini (1976), Dhanraj et al., (1987), Kalaimani and
Kadambavanasundaram (1988) reported negative association between them.
Larger panicles with more florets per panicle, larger grain size and a
reduction in floret sterility had a positive effect on yield (McKenzie et al., 1987).
The number of panicles per hill and plant height is reported to have negative direct
effects on yield (Prasad et al., 2001 and Zahid et al., 2006) but 1000-grain weight
have high and positive direct effects on rice yield (Yolanda and Das, 1995; Surek
and Beser, 2003; Zahid et al., 2006).
Venkateswarlu et al., (1986) suggested that increasing the proportion of
high-density grains would increase grain yield by as much as 30%. Positive
correlation of grain yield with number of productive tillers, and number of grains
per panicle are also reported (Ramakrishnan et al., 2006)
An analysis of the interrelationships amongst all grain characteristics
suggested wide variation was found in grain size and shape in Pakistan local rice
genotypes.Genetic diversity analysis based on agromorphological traits grouped
45 indigenous aromatic rice germplasm into 10 different clusters (Siddiqui et al.,
2007).
27
Eventhough morphological characterization studies have been focused on
different cultivars and landraces in different parts of India, the same for the
medicinal rice germplasm in Kerala are meager and are reviewed independently.
2.2.2. Anatomy
Anatomical traits in rice plant were utilized in connection with the
classification of different varieties and selection of disease resistant and non-
lodging type. Anatomical traits are of great value for some plants for application at
generic and sub-generic levels (Jones and Luchsinger, 1987). Cheadle (1942)
emphasized the importance of anatomy in taxonomic and phylogenetic studies of
monocots.
Stem anatomy of rice plant has been studied in detail by different authors
(Hector, 1936, Juliano and Aldama, 1937; Roy, 1963). It has been reported that
mechanical system in wild species of rice is stronger than that of the cultivated
ones (Hedayetullah and Chakravarty, 1941).
Vascular bundles are arranged roughly in two rings in the stem of Oryza
sativa (Chaudhary et al., 1971; Joarder and Eunus, 1981). However three rows of
vascular bundles have been also observed in rice varieties by Hector (1936) and
Wada (1956). But slight scattered arrangement of vascular bundles in the stem of
dwarf mutant has also been reported (Roy and Richharia, 1967).
The number of sclerenchymatous tissue constituting the hypodermis is
variable in rice (Chaudhary et al., 1971; Joarder and Eunus, 1981). Modern
cultivars are reported to have higher number of sclerenchymatous cells in
hypodermis than traditional cultivars (Sarwar and Prodhan, 2000).
But Joarder and Eunus (1981) reported that different mechanical cells in the
lodging susceptible varieties were found to be significantly larger than those of the
28
lodging resistant varieties. Higher number of vascular bundles in the inner circle
probably gives lodging resistance to modern cultivars (Sarwar and Prodhan,
2000). However major part of the culm is occupied by hollow pith in traditional
cultivars (Mia, 1978; Sarwar and Prodhan, 2000).
Lodging resistance is positively correlated with the culm diameter and wall
thickness of the basal internodes, both in wheat (Li et al., 2000; Tripathi et al.,
2003; Wang et al., 2006) and barley (Dunn and Briggs, 1989). Moreover, aside
from the thick culm, the culm vascular bundle number in rice also contributes to
lodging resistance (Xu et al., 1996; Duan et al., 2004). Apart from improving
lodging resistance, a thick culm may also act as a carbohydrate store for high
yield in rice (Hirose et al., 2006). Cultivars with large culms, therefore, may be
ideo types for super rice breeding because the characteristics of semi-dwarfism,
lodging resistance and heavy panicles have been considered to be important traits
for super rice breeding (Khush, 2000; Duan et al., 2004; Ma et al., 2004).
Chaudhary et al., (1971) reported that the number of inner vascular bundle
was greater than outer vascular bundles. However equal number of inner and
outer vascular bundles has been reported in different rice varieties by Joarder and
Eunus (1981).The difference in number between inner and outer vascular bundle
was found to be greater in modern cultivars compared to traditional ones (Sarwar
and Prodhan, 2000)
Leaf anatomy has proved to be a good phylogenetic tool for
grass systematics. Many researchers have succeeded using leaf anatomy to
define species and infer phylogenies (Breakwell, 1914; Brown, 1958, 1975, 1977;
Cerros-Tlatilpa, 1999; Columbus, 1996; Ellis, 1987; Fisher, 1939; Morden and
Hatch, 1987; Sanchez, 1971).
29
The number of vascular bundles in the midrib of lamina varied in different
species of Oryza. The members belonging to Oryza sativa had maximum number
ranging from 8 to 13 bundles and those belonging to Oryza granulata had the
minimum number, with 1 to 4 bundles, whereas those belonging to Oryza
officinalis had a intermediate number with 2 to 6 bundles (Roy, 1963).
Many of the anatomical characters including epidermal characters of leaf
play an important role in distinguishing the modern and traditional cultivars of rice
(Metcalfe, 1960; Shimizu and Mamum, 1975; Ellis, 1979). Epidermal cells of the
leaf blade are rectangular in surface view with wavy margin. They develop
cuticular papillae, the size and the arrangement of which differ in different species
of Oryza (Roy,1966).The size, shape and arrangement of epidermal long and
short cells could be varietal characteristics, and the size and shape of the cells
differed accordingly to the position of the cells in the same leaf (Shimizu and
Mamum, 1975). The leaf epidermis of the rice plant is differentiated into long and
short cells, stomatal apparatuses and dermal appendages like papillae, macro
hair and prickle hair (Sarwar and Ali, 2002). The arrangement of different
epidermal cells and appendages with their fine structure has been also studied by
many researchers (Kaufman et al., 1973; Takeoka et al., 1983; Wilkins and Cutter,
1987).
The foregoing review on anatomical studies in different rice species and
cultivars presented the fact that most of the studies in this dimension are focused
on assessment of lodging resistance, some studies on identification of rice
species and others on differentiating traditional and modern cultivars. As far as the
rice landraces of Kerala in general and Navara complex in particular are
30
concerned, no attempt has been made to analyze the anatomical features or to
utilize it in differentiating the different morphoforms in Navara complex.
2.2.3. Palynology
Micromorphological characters such as pollen traits are widely accepted as
reliable taxonomic characters and are frequently used to complement
macromorphological and molecular data for discriminating taxa (Datta and
Chaturvedi, 2004).
Pollen morphological characters can be used in studies of plant taxonomy as
pollen traits are influenced by pollination, dispersal, and germination (Erdtmann,
1952; Nowicke and Skvarla, 1979; Stuessy, 1990; Moore et al., 1991). Botanists
and ecologists have been able to reconstruct past assemblages of plants and
identify periods of environmental change by identifying the plants from their pollen
(Faegri and Iversen, 1989; Moore et al., 1991).
The pollen of the cereals and a few wild grasses, such as Agropyron,
Ammophila, Elyniis and Glyceria, are distinguished from other grass pollen by
their larger size and annulus diameter. Their surface sculpture has been studied
by optical means and by the transmission electron microscope (Andersen and
Bertelsen, 1972). Damblon (1975) adds to the fine sculpture of some grass pollen
by demonstrating the advantage of the sputtering method. Chaturvedi et al.,
(1994) observed variation in pollen exine surface pattern among the species of the
genus Sorghum by SEM and identified seven exine surface patterns that were
taxonomically informative at the section, subsection and series level.
A number of authors studied the pollen morphology of cereals and millets
which forms the most important group of family Poaceae (Wodehouse, 1935;
Firbas, 1937; Jones and Newell, 1948; Sampath and Ramanathan, 1951;
31
Ethirajan, 1953; Rowley, 1960; Bourreil and Reyre, 1968; Bourreil et al., 1970; De
Lisle, 1970; Bonnefille, 1972; Gornall, 1977; Siddiqui and Quiser, 1988; Kohler &
Lange, 1979; Chaturvedi et al.,1994; Chaturvedi et al., 1998). Pollen morphology
of 49 species of family Gramineae from Venezuelan mountain have been
examined by Salgado-Labouriau and Rinaldi (1990, 1993).
Grohne (1957) and Anderson (1979) also made attempts to distinguish
between pollen grains of members of the Poaceae, but all have met with limited
success (Fageri and Iversen, 1989). According to these researchers, size of the
pollen grains varied between cultivated cereals and wild grasses and the cereal
crops produced large pollen grains on average.
Erdtman and Praglowski (1959) reproduced phase contrast micrographs of
Zea, Avena, Triticum, Elimus and Hordeum. Grohne (1957) and Faegri and
Iversen (1964) observed its characteristics with phase contrast for the separation
of various cereals and wild grasses. Various authors disagree about the
identification of cereal pollen based on phase contrast studies. Pollen of the
common cereals and some wild grasses of a similar size range were examined
with a scanning electron microscope. Rowley et al., (1959) and Rowley (1960)
studied thin sections, etched exines and surface replicas of various grasses with
the transmission electron microscope.
Pollen grains of Poaceae are morphologically very similar to each other
with smooth or slightly sculptured exine (Wodehouse, 1935, Faegri and Iversen,
1989) under the light microscope (LM). Several attempts were carried out to
differentiate wild grass from cereal pollen by light microscopy (Firbas, 1937;
Grohne, 1957). But recently it has been reported that pollen grains of tropical
grasses typically show rather little variation in size (Joly et al., 2007)
32
The homogenous morphological feature observed throughout the family
makes pollen morphological differentiation in the graminaceous taxa very difficult
(Chaturvedi et al., 1998). General description of gramineae pollen grains has been
explained by Perveen (2006) as apolar, medium, rarely large sized, spheroidal,
mono-diporate, rarely triporate, operculate to non-operculate, annulate to non-
annulate or with reduced annulus, generally sexine as thick as nexine, often
thicker or some time thinner than nexine.
Fine structural details of exine surface are difficult to discern with the light
microscope, hence the scope of distinguishing pollen exine surface patterns
broadened with examination under SEM by subsequent studies (Andersen and
Bertelsen, 1972; Watson and Bell, 1975; Page, 1978; Kohler and Lange, 1979)
and differences at generic or higher taxonomic levels were revealed. The study of
pollen exine surface patterns under SEM has considerably broadened the scope
of application of this pollen feature at a number of taxonomic levels in grasses
(Chaturvedi et al., 1994).
Attempts were made to identify poaceous taxa by studying pollen exine
sculpture with phase contrast microscopy and other optical means (Erdtman and
Praglowski, 1959; Rowley, 1960; Sorsa, 1968). The study of SEM of some cereal
grasses has shown the way for a possible separation of the different cereal grass
species (Rowley, 1960).
Chaturvedi et al.,(1998) identified eleven exine surface patterns viz.
granulose-punctate, densley- granulose, sparsely-granulose, grouped-granulose,
mixed –granulose, sparsely-spinulose, grouped spinulose, insulae formed by
spaced granules(Type-1), insuale formed by compact granules(Type-2), mixed
insular patern(Type-3) distributed worldwide amongst both wild and cultivated
33
Oryza species. The pollen exine surface pattern of indica, japonica and javanica
races was found to be distinct The race Indica has an Insular Type-3 pattern ,
japonica has an Insular Type-1 pattern and javanica has a sparsely-granulose
pattern (Chaturvedi et al., 1998). Muasya et al., (2002) reported microechinate
ornamentation as a highly informative character to split certain genera that are
most likely non-monophyletic. Variations in the exine pattern of Cyperaceae
revealed extremes of evolutionary changes with finely granulate being more
primitive (Nagels et al., 2009). Five distinct pollen types were identified based on
exine ornamentation among 20 species belonging to 14 genera of the family
Gramineae (Perveen, 2006).
Pollen morphological studies using LM and SEM have been carried out on
six cultivars of Basmati – a variety of cultivated species, Oryza sativa race indica
(Datta and Chaturvedi, 2004). This study revealed distinct variations in pollen
exine surface patterns, in relation to the arrangements of fine surface
excrescences (spinules or granules) and their clustering patterns. Three distinctly
different insular patterns were reported in cultivars Basmati-370, Karnal local and
Type-9. Cultivar Bakul Joha is characterized by free spinules. Mixed type i.e. both
free and fused excrescences were observed in cultivars Bengali Joha and Bhog
Maniki but can be differentiated on the basis of the dimensions of the
excrescences (Datta and Chaturvedi, 2004)
The exploration on the literature on pollen traits of Oryza revealed that no
attempt has been made to study the pollen morphology of rice landraces in
general in Kerala. As far as Navara complex is concerned the results obtained in
the present study is the pioneer attempt and has been reported (Shiny and Nair,
2011).
34
2.2.4. Biomass accumulation
Biomass accumulation is a good measure of competitive success, because it
reflects resource capture under the interference of neighbours (Roush and
Radosevich, 1985; Gaudet and Keddy, 1988; Fernando et al., 2006). Yang and
Hwa (2008) reported that the leaf traits such as leaf thickness, size and shape,
leaf number and orientation are key factors influencing biomass formation.
In the study on the distribution of dry matter in a plant, Brouwer (1962)
reported significant influence of the stage of plant development on the allocation
of dry matter between roots and shoots. However, he also observed that the
biomass allocation pattern may be affected by environmental conditions. The
availability of nutrients, moisture regime of the soil, weather conditions and
photosynthetic activities of the vegetative parts of the plant largely determine plant
biomass accumulation, distribution and plant structure (Cackett and Wall, 1971;
Brady, 1990; White, 1999). Nitrogen is a vital component of plant proteins, nucleic
acids, chlorophyll and a host of other important compounds of living plant cells.
Together with photosynthetic carbohydrates, these complex organic substances
constitute the greater bulk of plant biomass (Kumakov, 1988).
In rice, seedling height ranged from 14 to 30 cm. and was not necessarily
correlated with plant height at harvest (Anon, 1968). But rapid seedling growth is
considered as a desirable trait of tropical rice varieties particularly when combined
with early maturity. It enables the young plants to become fully established before
weeds become a problem. Faster early growth suppressed weeds and is essential
for early maturing varieties (Yoshida, 1977). A high rate of growth can lead to a
considerable increase in final biomass (Richards, 1987). According to Ensminger
35
et al. (2006) photosynthetic performance and biomass accumulation are tightly
related.
Tillering is directly linked to the number of panicles formed, which is an
important determinant of grain yield (Miller et al., 1991; Li et al., 2003; Yang et al.,
2006). Tillering plays an important role in biomass accumulation as intercepted
radiation is increased with the greater leaf area associated with tillering. However,
excessive tillering can lead to high tiller abortion, poor grain set and small panicle
size, which cause reduced grain yield. In adverse environmental conditions when
water for transpiration is limited, low tillering is expected to allow more efficient
use of available water. It was observed that the leaf area, panicle grain number,
fertility percentage and grain yield of tillers were higher in a low - tillering cultivar
than in a high-tillering cultivar (Kariali and Mohapatra, 2007).
Significant relationships between grain yield and biomass at anthesis or
biomass during grain filling have been reported in bread wheat (Tanno et al.,
1985; Turner, 1997) and barley (Ramos et al., 1985). According to Penrose and
Payne (1996) wheat plant biomass accumulation and distribution is directly linked
to yield levels. In the review it has been found that studies on biomass
accumulation and its relation with yield dependent parameters in rice landraces
were missing. It revealed a lacuna in knowledge base especially with regard to the
medicinal rice germplasm in Kerala.
2.2.5. Salinity tolerance
Salinity stress is one of the major constrains faced by the farmers of India
especially at the saline soils and in areas irrigated with brackish water. Reports
show that 20.24 million ha of land in India is affected by salinity (Akbar and
36
Ponnamperuma, 1982). Sensitivity to salinity varies with the growth stage for
many plants, particularly cereal crops.
Asana and Kale (1965) reported that under saline conditions germination
ability of seeds differ from one crop to another and even a significant variation is
observed among the different varieties of the same crop. Khan et al., (1997)
observed that rice varieties showed a great variation in germination due to salinity
effect. A decrease in germination at various level of salinity was reported in
perennial halophytic grasses (Khan and Ghulzar, 2003). Speed of germination,
final germination percentage, plumule and radicle length and dry weight were
reported to decrease in rice as the salinity level increased (Hakim et al., 2010).
Similar results have been reported in Zea mays L. by Khodarahmpour (2012).
Rice crop is rated as a salt sensitive crop (Shannon et al., 1998) which show
a range of tolerance to environmental factors like availability of water, salt
concentration and variation in temperature.
In rice seeds, germination occurs as a consequence of very active metabolic
changes during the activation stage (Takahashi, 1965). Reduction in germination
under salinity was reported in rice by many workers (Varghese and Thampi, 1966;
Bhattacharya, 1967; Panchaksharaiah and Mahadevappa, 1971). Varietal
difference in tolerance to salinity at germination was reported by Maliwal and
Paliwal (1971) and Paliwal and Mehta (1973). Approximately 50% reduction in
germination at 0.6% NaCl was reported by Balasubrahmaniam (1965) and
Bhumbla et al., (1968).
Adverse effect of high level of CI- on nitrate and phosphate uptake under
saline environment was reported in rice seedlings (Akbar, 1975). Rice is tolerant
during germination, becomes sensitive during early seedling growth, and then
37
becomes more tolerant as it matures (Pearson and Ayer, 1960; Hakim et al.,
2010). Rahman et al., (2012) reported that the growth of plumule as well as the
radicle decreased significantly with increasing arsenic and NaCl concentration.
Salinity affects all stages of the growth and development of rice plant and
the crop responses to salinity varies with growth stages, concentration and
duration of exposure to salt. In the most commonly cultivated rice cultivars, young
seedlings were very sensitive to salinity (Flowers and Yeo, 1981; Lutts et al.,
1995). There are other reports where grain yield is much more depressed by salt
than the vegetative growth. Yield is a very complex character which comprise of
many components are related to final grain yield which are also severely affected
by salinity. Panicle length, spikelets per panicle and 1000-grain weight is
significantly affected by salinity (Khatun et al., 1995; Khatun and Flowers, 1995).
Large amount of soluble sodium ions accumulates in soil and water because
of the combined effects of natural and human factors and this seriously affects
plant growth and yield (Sahin et al., 2002). Salt stress is becoming one of the key
factors that restrict agricultural productivity, especially in irrigated areas and in
rainfed coastal areas (Neue, 1991; Castillo et al., 2000). The seedling stage is one
of the most sensitive stages to salt stress in rice, and studies on salt tolerance
during this stage could probably provide insights for enhancing tolerance
throughout the plant life cycle (Munns and Tester, 2008). Moreover, the relation
between sodium concentration in plant tissue and growth and yield were observed
to be negative, and with greater effects on shoot growth than on root growth
(Eschie et al., 2002). Percentage survival of transplanted seedlings correlated
positively with the dry weight of seedling at transplanting, as well as with biomass
accumulation during stress (Maiale et al., 2004).
38
The harmful effect of salinity occurs due to osmotic stress and specific ion
toxicity. Almansouri et al., (2001) reported that seed germination and seedling
establishment are inhibited or delayed by salt and osmotic stresses. It has been
reported that under saline conditions, germination ability of seeds differ from one
crop to another and even a significant variation is observed among the different
varieties of the same crop (Maas and Hoffman, 1977; Hakim et al., 2010).
Germination and seedling characteristics are the most viable criteria used for
selecting salt tolerance in plants. Thus germination and seedling development is
very important for early establishment of plants under stress condition. Selecting
cultivars for rapid and uniform germination under saline conditions can contribute
towards early seedling establishment (Kazemi and Eskandari, 2011).
VTL 5, released in September 1996 is a rice variety which is reported to
have multiple tolerances to abiotic stresses such as salinity, acidity and
submergence (Shylaraj and Sasidharan, 2005). Pokkali land race of Kerala is
reported to be the global donor for most of the salinity tolerance breeding
programs in rice. ‗Kuthiru‘ and ‗Orkayama‘ are reported to be saline tolerant from
Kaipad tracts of Kerala. Using these two tolerant varieties, saline tolerant, non-
lodging and high yielding varieties, Ezhome-1 and Ezhome -2 have been released
(Vanaja and Mammootty, 2010). Kuttusan, Orthadiyan and Chovverian are
reported as other native saline tolerant cultivars from Kaipad area in North Kerala
(Chandramohanan and Mohanan, 2012).
The studies on salinity tolerance of different medicinal rices in Kerala have
not been attempted so far and the studies in this dimension may prove the
possibility of cultivating these races in areas affected with increased salinity.
39
2.2.6. Primary metabolites
Rice has been considered as the most nutritious food as it is rich in
carbohydrate, and contains vitamins, iron, phosphorous and protein in adequate
amounts. Juliano et al., (1964) stated that nutritional composition of rice differs
with variety, nature of soil, environmental conditions and fertilizers applied.
Various groups of nutritional components are not uniformly distributed in different
parts of the kernel.
Carbohydrate of rice mainly comprises of 85-90% starch (Houston and
Kohler, 1970) along with small portions of hemicelluloses, pentosans and sugars.
The difference in starch content of the cultivars is attributed to their variation in
photosynthetic activities of leaves as well as translocation and utilization of
photosynthates (Ahmed et al., 1998). Most important nutritional property of total
carbohydrate is their easy digestibility in the small intestine (Devindra and
Longvah, 2011).
Starch accumulates in the rice grain during the grain filling period (Ishimaru
et al., 2003; Fitzgerald, 2004). Rice starch comprises of amylose and amylopectin
which is major component of carbohydrate. In common rice, 12 - 35% of total
starch consists of amylose whereas waxy rice contains less or no amylose
(Juliano et al., 1964). Amylose content is considered to be the single most
important characteristic for predicting rice cooking and processing behaviors
(Juliano, 1979a, 1979b; Webb, 1985). Rice varieties are grouped on the basis
of their amylose content into waxy (0-2%); very low (3-9%), low (10-19%),
intermediate (20-25%) and high (>25%). Intermediate amylose rices are the
preferred types in most of the rice growing areas of the world except where low-
amylose Japonicas are grown (Shobarani et al., 2006).
40
Rice protein is considered as indicator of nutritional quality (Juliano, 1978). It
is one of the most important sources of protein and has highest biological value
among all the cereals. The proteins of rice are mainly composed of glutilin while
prolamin is in lower quantity, making rice kernels rich in high quality proteins
(Mullins, 1999). Chaudhary and Tran (2001) reported higher lysine content in rice
which makes it more nutritious than any other cereals. About 4-5% lysine content
is reported in rice which is more than that of wheat, corn and sorghum (James and
Mc Caskill, 1983; Janick, 2002).
Chemical compositions of cereals are characterized by protein content
(Lasztity, 1999). Protein content in rice is influenced by variety and environment,
crop season, planting date (Cagampang et al., 1966) and nitrogen fertilization
(Sturgis et al., 1952; Kik and Hall, 1961; Juliano et al., 1964). But most protein is
the alkali-soluble (glutelin) called Oryzenin in rice. Rice glutelin is composed of
alpha and beta subunits, which were separated into alpha-1 (39 kDa), alpha-2 (38
kDa), alpha-3 (37.5 kDa and 37 kDa) and alpha-4 band (34 and 33 kDa) for alpha
subunit and beta-1 (23 kDa), beta-2 (22.5 kDa) and beta-3 (22 kDa) bands for
beta subunit, respectively (Jahan et al., 2005).
Rice proteins are valuable because these are colorless and have a bland
taste. Moreover they are non allergenic and possess cholesterol reduction
properties (Chrastil, 1992). The brown rice protein content of 17,587 cultivars
maintained at International Rice Research Institute (IRRI) ranged from 4.3 to 18.2
per cent with a mean of 9.5 per cent.
Zhang and Tang (1986) reported the free amino acid pool in rice which was
dominated by serine, alanine, aspartate and glutamate during the embryo
differentiation stage. In the maturation stage, serine, alanine, arginine and lysine
41
were the main components. These amino acids play an important role in
regulating the availability of the whole amino acid pool. The free amino acid
concentration of rice is becoming an increasingly important grain quality factor
because of its apparent influence on the organoleptic acceptability of cooked rice
(Kamara et al., 2010).
Proteins and amino acids are saturated in outer layers of milled rice and are
reduced at centre (Primo et al., 1963; Houston et al., 1964, 1968). Similarly,
albumin and globulin are found in outer layers of bran, but glutelin have a reverse
distribution (Houston et al., 1968).
2.2.7. Molecular characterization
Molecular markers have become fundamental tools for finger printing,
establishing phylogenetics, tagging desirable genes, characterization of
transformants and study of genome organization (Rafalski et al., 1996). It gives
information that helps in deciding the distinctiveness of species and their ranking
according to the number of close relatives and phylogenetic position
(Rahman et al., 2007). The use of molecular markers has proven its value for a
variety of purposes in molecular biology and it helps in genetic analysis in
understanding the structure and behavior of genomes in cereals (Korzun, 2002). It
is a useful tool for assessing genetic variations and resolving cultivar identities
(Malik et al., 2008).
Morphological and biochemical markers may be affected by environmental
factors and growth practices (Higgins, 1984; Xiao et al., 1996; Ovesna et al.,
2002), but DNA markers portray genome sequence composition, thus enabling to
detect differences in the genetic information carried by different individuals (Saker
et al., 2005).
42
Molecular markers based on DNA sequence are found to be more reliable
(Ragunathachari et al., 2000) than selection of plant varieties based on
morphological characters because major characters of interest possess low
heritability and are genetically complex (Rahman et al., 2007). Molecular markers
are prominent tool in augmenting conventional breeding methods for crop
improvement in various cultivated species (Mohan et al., 1997) Broad range of
molecular markers have become available which are being used in a variety of
ways not only to supplement and expedite the conventional methods for
improvement, but also for characterization and maintenance of plant genetic
resources that are so vital for crop improvement programmes (Gupta et al., 2002).
Genetic markers are used for clonal identification, linkage mapping,
population diversity, taxonomy and germplasm conservation in rice (Parsons et
al., 1997; Virk et al., 2000; Qian et al., 2001; He et al., 2004; Jeung et al., 2005).
Comparison of effectiveness among different classes of PCR-based markers were
carried out for genotype identification in different plants (McGregor et al., 2000;
Corazza-Munes et al., 2002; Ferdinandez and Coulman, 2002; Palombi and
Damiano, 2002; Archak et al., 2003).
Development of PCR reaction technology has introduced a considerable use
of molecular markers viz. SSRs (Levinson and Gutman, 1987; Cregan, 1992),
RAPDs (Welsch and McClelland, 1990; Williams et al., 1990; Tingey and
Deltufo,1993), RFLPs (Paran and Michelmore,1993; Becker et al., 1995), AFLPs
(Vos et al., 1995; Zhu et al.,1998), ISSRs (Albani and Wilkinson,1998) and SNPs
(Vieux et al., 2002) to assess the variability and diversity at molecular level (Joshi
et al.,2000).
43
2.2.7.1. Random Amplified Polymorphic DNA
Random Amplified Polymorphic DNA (RAPD) finger printing method was first
described by Welsh and Mc Clelland (1990) and William et al., (1990).It has
proven useful in genotype identification and gene mapping (Robert et al., 1999). It
does not require any prior knowledge of DNA sequence, but still revealed a high
level of polymorphism (Williams et al., 1990; Hadrys et al., 1992; Karp et al.,
1997).
Genetic variations of nine upland and four lowland rice cultivars was
investigated at the DNA level using RAPD method via polymerase chain reaction
by Yu and Ngyuyen (1994). They also reported that RAPD analysis is useful in
determining genetic variation at DNA level among rice cultivars and the technique
is sensitive and powerful.
RAPD markers have been used successfully to detect genetic variation
among low land and upland rice cultivars, the genetic characterization and
classification of japonica cultivars into temperate and tropical groups and for
analysis of genetic variability in wild rice populations (Baishya et al., 2000).
Nadarajan et al., (1999) analyzed the genetic relationship between seven
Japonica, two Indica and one tropical japonica rice varieties using RAPD method.
Jeung et al., (2005) had employed RAPD markers for fingerprinting temperate
japonica and tropical indica rice genotypes. Significant polymorphism was
detected among the genotypes.
Monna et al., (1994) mapped 102 RAPD markers on all twelve
chromosomes of rice using DNAs of cultivars Nipponbare (japonica) and Kasalath
(indica) and of F2 population generated by a single cross of these parents and
44
they were able to detect polymorphisms appearing in the range from less than
100bp to 2000bp.
Yamamoto (1994) analysed 35 rice cultivars with 13 RAPD primers and
obtained 54 scorable polymorphic bands to calculate genetic distance. Ko et al.,
(1994) confirmed that commercial Australian and USA lines and their relatives are
closely related with similarity indices of 88-97% using RAPD markers. Parsons et
al., (1997) studied genetic variation between samples of Oryza sativa from 19
localities in Bengladesh and Bhutan using 14 decamer primers. 94 reproducible
bands were obtained out of which 50% were polymorphic. Ravi et al., (2003)
assessed the genetic diversity among 40 cultivated varieties and wild relatives of
rice with RAPD markers. They observed 90% polymorphism from 499 RAPD
markers. Pervaiz et al., (2010) reported the use of RAPD primers of OPA, OPB,
OPC, OPJ and OPK series in assessing genetic variability of aromatic and non-
aromatic rice germplasm.
RAPD analysis using OPC, OPF and OPK series in scented rice revealed
rice varieties under cultivation with similar names are genetically quite different
(Raghunathachari et al., 2000). Genetic diversity among traditional and improved
cultivars of rice was analyzed using RAPD primers of OPA and OPB series
(Rabbani et al., 2008).
RAPD markers have been used to identify and tag the important genes for
Basmati quality traits like aroma (Jin et al., 1995; Trangoonrung et al., 1996),
cooked kernel elongation, amylose content and length/breadth ratio (Ram et al.,
1998).
Genetic diversity in 23 Bora rice (Glutinous rice) landraces of Assam at DNA
level was analyzed using RAPD markers (Sarma and Bahar, 2005). The genetic
45
diversity analysis of traditional Sali rice germplasm of Assam through RAPD
markers was carried out by Barooah and Sarma (2004) using 51 Sali rice
accessions and 72 RAPD primers. The result indicated high level of diversity and
emphasized the potentiality of using molecular markers in rice germplasm
management of Assam rice collection.
2.2.7.2. Microsatellites
Microsatellite markers also known as Simple Sequence Repeats (SSRs) are
highly informative and are easily detectable with PCR. They occur frequently in
plant genomes showing an extensive variation in different individuals and
accessions (Akkaya et al., 1992; Senor and Heun 1993; Wu and Tanksley, 1993).
Yang et al., (1994) stated that the greater resolving power of SSR assay can
provide more informative data than other techniques. SSR markers which
generate Simple Sequence Length Polymorphisms (SSLPs) have been found to
discriminate between closely related accessions and genotypes with a narrow
genetic base (Yang et al., 1994; Olufowote et al., 1997). These second generation
markers are somatically stable and inherited in a co-dominant Mendelian manner
and can, therefore, distinguish between heterozygotes and homozygotes
(Morgante and Olivieri, 1993; Thomas and Scott, 1993).
Microsatellites demonstrate a high degree of transferability between
species, as PCR primers designed to an SSR within one species frequently
amplify a corresponding locus in related species, enabling comparative genetic
and genomic analysis (Duran et al., 2009). Among PCR- based markers, SSR
markers are becoming popular, both for genetic diversity and breeding research
as they are robust markers compared to RAPD and AFLP markers in
discriminating even closely related individuals (Vanaja et al., 2010).
46
In rice, about 50% of the genome consists of repetitive DNA sequences
(Deshpande and Ranjekar, 1980). The presence of approximately 5,700 to 10,000
SSRs out of which 2740 SSRs are identified and mapped on the 12 rice
chromosomes with an average distance of one SSR per 157 kb (McCouch et al.,
1997, 2002). SSRs are more popular in rice because they are highly informative,
mostly monolocus, co-dominant, easily analyzed and cost effective (Chambers
and Avoy, 2000).
SSR analysis using 19 microsatellites followed by clustering based on
UPGMA method on different samples of ‗Echizen‘, an old Japanese landrace
along with other advanced cultivars and landraces showed that old Echizen was a
diverse landrace (Kobayashi et al., 2006). SSR markers were used to analyze
genetic diversity among rice accessions including landraces, cultivars and wild
relatives. The study revealed that genetic diversity was high for wild relatives, low
for cultivars and moderate for landraces (Ram et al., 2007).
Mapping of rice glabrous gene using different markers viz. SSR, RAPD and
AFLP have shown that highest level of polymorphism was obtained with SSR
marker followed by RAPD and AFLP (Dong et al., 2009). Microsatellite (SSR)
markers were used for mapping of Minute (Mi) genes, a major gene for grain size
on rice chromosome 3 (Fraker et al., 2004). The genetic diversity of Jumli
Marshiwas, the most common traditional rice variety of Assam was assessed by
agro-morphological variability and microsatellite marker polymorphism which
showed low morphological diversity (Bajracharya, et al., 2006). SSR analysis
using 164 markers on 24 rice cultivars carrying good quality traits revealed higher
genetic diversity among them (Lapitan et al., 2007). Seetharam et al., (2009)
estimated genetic diversity in 30 rice genotypes adapted to coastal environments
47
using 35 primers of SSR markers and morphological characters and classified into
five groups.
The review focus light on preference of SSR markers for the effective
characterization of the rice germplasm to reveal the identity due to high degree of
polymorphism than RAPD and AFLP markers.
2.3. Research progress in Navara
The efforts for collection, conservation and documentation of this unique rice
landrace are meager. Most of the studies were aimed in collection of Navara types
from different parts of the state. The study includes morphological evaluation,
isozyme and RAPD analysis for the characterization of ecotypes of this cultivar
and identification of markers for distinguishing Navara ecotypes from other rice
cultivars. Navara rice landrace has become a matter of study in recent years and
has been examined at its morphological, molecular (Sreejayan et al., 2005) and
physico-chemical (Deepa et al., 2009) dimensions.
2.3.1. Morphological delineation of Navara germplasm
Morphological evaluation of Navara samples based on 6 quantitative and 15
qualitative characters grouped germplasm into three morphotypes viz., tall golden
yellow, dwarf golden yellow and dwarf black (Sreejayan et al., 2003). However
Sreejayan et al., (2003) grouped Navara into four groups based on culm length
and sterile glume color. They classified Navara cultivars into long yellow
(Kuttadan), short yellow (Palakkadan), intermediate yellow (Vadakkan) and short
black (Wynadan). Leenakumari (2004) classified Navara germplasm into two
broad groups based on grain color as golden yellow glumed and black glumed
types. Leenakumari (2004) also described Navara genotypes as tall, lanky plants
with erect and glabrous pale green to green leaves, green basal leaf sheath, white
48
and two cleft ligules with pale green collar and auricle. Characterization of Navara
germplasm based on qualitative morphological characters revealed three different
morphotypes in cluster analysis (Joseph et al., 2007).
Kumar et al., (2010) recorded that among the qualitative characters apiculus
colour, lemma and palea colour and seed coat colour showed great variability in
Navara and hence, can be considered as markers for the identification of Navara
types.
2.3.2. Pollen studies in Navara
LM and SEM studies of Oryza sativa cv. Navara, indigenous to Kerala,
revealed the existence of a comparatively primitive third morphotype Navara
Punja, in addition to the already reported black and golden yellow morphotypes.
The analysis also revealed variation in exine ornamentation, size of the aperture,
annular thickening, operculum and pollen volume in different morphoforms (Shiny
and Nair, 2011)
2.3.3. Isozyme analysis
The alcohol dehydrogenase isozyme expression has been explored for
Navara accessions collected from different parts of Kerala along with two local
check varieties to demonstrate the utility of alcohol dehydrogenase as a marker
for grouping Navara ecotypes (Kumar et al., 2010). This study revealed two
bands, ADH-3 and ADH-4 as common for all the genotypes analyzed. Germinated
seed sample expressed more alcohol dehydrogenase banding pattern and nine
bands were resolved for germinated seed samples. The Navara ecotypes showed
two unique bands viz., ADH-4 and ADH-8 (Kumar et al., 2010).
49
2.3.4. Estimation of genetic diversity in Navara
The genetic relationship between Navara and a larger set of other varieties
(including 19 traditional and 6 improved varieties) was assessed using data
generated from five microsatellite markers. The 40 Navara genotypes were
separated into a distinct cluster in the dendrogram showing three distinct varietal
types of Navara (Sreejayan et al., 2005). Kumar et al., (2008) reported unique
bands for Navara ecotypes with RAPD primers OPE 6, OPP6 and OPP11.
Genetic variations and some of the physico-chemical properties of Navara
were studied using microsatellite markers and compared with those of non
medicinal rice varieties: Jyothi and IR 64. Navara showed 11 unique positive and
36 unique negative markers to differentiate it from Jyothi and IR 64 (Deepa et al.,
2009).
RAPD analysis for characterization of Navara ecotypes and identifying
markers for distinguishing Navara ecotypes from other rice cultivars have also
been reported by Kumar et al., (2010). Studies including morphological evaluation
and SSR markers were carried out to characterize and analyze the genetic
relationship of Navara germplasm with other traditional rice landraces. SSR study
also revealed that Navara is genetically distinct from other traditional rice
landraces despite sympatric cultivation over several centuries (Jose et al., 2010).
Characterization of the genetic resources of Navara using 24 morphological
traits and 664 amplified fragment length polymorphic (AFLP) markers revealed
that Navara germplasm represents a composite of highly homozygous genetically
isolated units. The distinctness of Navara accessions in the AFLP dendrogram in
relation to other traditional rice strains further demonstrates that the genotypes are
50
nevertheless genetically cohesive and perpetuated with minimum genetic
admixing (Sreejayan et al., 2011).
Similar works using RAPD primer OPB-05 and OPF-01 distinguished Navara
accessions from other rice landraces in Kerala. Primer, OPB-05 produced unique
product with a size of less than 1.0 kb and OPF-01 produced product at size of
less than 0.5kb unique for the Navara accessions (Reshmi et al., 2011).
The genetic diversity analysis revealed that the traditional selection
performed by farmers for short maturity coupled with autogamous breeding may
have retained the genetic purity and governed the genetic structure of Navara.
2.3.5. Physico-chemical characterization and bioactive compounds
Pioneer attempts for the physico-chemical characterization of the Navara
ecotypes have begun as early as 1996, but conclusive evidences on the bioactive
compounds responsible for the medicinal traits in Navara is yet to be exposed.
Menon and Potty (1997) reported free amino acid contents of 0.316mg/g and
0.089mg/g respectively in black and golden yellow glumed Navara cultivars grown
under wet land conditions. Black glumed Navara contained amino acids DL-2 –
amino-n-butyric acid and DL-iso–leucine while, golden yellow glumed Navara
contained L-Histidine monochloride, L-ornithine monochloride and DL-isoleucine.
Menon and Potty (1999) also reported the involvement of Mn in the development
of quality components in Navara grain.
Assessment of nutrient composition and physicochemical properties in
Navara, recorded that dehusked Navara rice consisted of 73% carbohydrates,
9.5% protein, 2.5% fat, 1.4% ash and 1628 kJ per 100g of energy. Navara rice
had 16.5% higher protein, and contained higher amounts of thiamine (27-32%),
riboflavin (4-25%) and niacin (2-36%) compared to Jyothi and IR 64. The total
51
dietary fibre content in Navara was found to be 34-44% higher than that of Jyothi
and IR 64. Significantly higher phosphorus, potassium, magnesium, sodium and
calcium levels were found in Navara rice, compared to the other two varieties. The
cooking time of dehusked Jyothi and IR 64 varieties were found to be 30min, while
Navara needed longer time to cook (38min). The cooked rice of Navara was slimy
in nature, probably due to the presence of non-starch polysaccharides (Deepa et
al., 2008).
The study on genetic variations and some of the physicochemical properties
of Navara in comparison with Jyothi and IR 64 revealed that the SSR primers for
protein content and setback viscosity primer (RM 4608) were observed to be
polymorphic in case of Navara (Deepa et al., 2009)
In vitro starch digestibility and glycemic indices of three rice varieties-
`Navara', `Jyothi' and `IR 64' showed that the rate of starch hydrolysis was
maximum (67.3%) in `Navara' rice compared to other two rice varieties. `Navara'
exhibited the lowest kinetic constant (k) indicating inherent resistance to
enzymatic hydrolysis. The glycemic load (GL) and glycemic index (GI) of `Navara'
were similar to `Jyothi' and `IR 64' and concluded that `Navara' is an easily
digestible rice and can be used for baby and geriatric foods (Deepa et al., 2010).
Nutritional value in terms of amino acids and protein was found increasing in
Navara landraces when cultivated in organic condition (Shiny et al., 2010)
Recent phytochemical investigations and spectroscopic studies of the diethyl
ether fraction of methanolic extract of Navara Black (NB) rice bran gave three
important compounds namely, tricin and two rare flavonolignans- tricin 4'-O-
(erythro-β-guaiacylglyceryl) ether and tricin 4'-O-(threo-β-guaiacylglyceryl) ether.
Of the three compounds, tricin and the threo- form of flavonolignan showed anti-
52
inflammatory effect of >65% after 5 h, at 2 mg/kg, in carrageenan-induced, paw
edema experiments in rats. The results of the study corroborate with the
preferential use of Navara in indigenous medicine, over staple varieties (Smitha et
al., 2011)
Structural, electronic and energetic characteristics of tricin, tricin - 4′ - O -
(erythro-β-guaiacylglyceryl) ether (TEGE) and tricin - 4′- O - (threo – β –
guaiacylglyceryl) ether (TTGE), isolated from ―Navara‖ rice bran have been
studied using Density Functional Theory (DFT) to explain their experimentally
determined radical scavenging activity (EC50 values) in comparison with known
standards such as quercetin, myricetin, and catechin (Ajitha et al., 2012).
It has been reported that the extracts of ―Navara‖ (black glumed) contains
significant amounts of oryzanols, phenolic acids, flavonoids, proanthocyanidins
and phytic acid compared with staple varieties. These extracts are potential free
radical scavengers and have anti-inflammatory effect compared with staple
varieties. Higher oryzanol content in Navara is beneficial for lowering plasma
cholesterol, reducing platelet aggregation, nerve imbalances and aortic fatty
streak formation (Smitha et al, 2012).
2.3.6. Activity profiles in Navara
2.3.6.1. Anti-carcinogenic properties
Molecular studies revealed the presence of Bowman-Brik Inhibitor (BBI)
protein in Navara, which is effective especially against breast cancer. This protein,
christened Bowman-Birk Trypsin Inhibitor protein, is also known to possess anti-
inflammatory and anti-allergic properties in animals and is reported to be capable
of imparting resistance to fungal pathogens and pests in crops. There are reports
that the protein had earlier been isolated from a few other crops like soybean,
53
barley and sunflower. But it has not been identified so far in any other rice variety
of the country. The sequenced part of the gene includes 762 base pairs (BP) and
it shows 94 per cent identity with the Bowman-Birk Trypsin Inhibitor protein in
japonica rice in China (Hindu – Dec 21; 2007).
2.3.6.2. Antioxidant, Anti proliferative and radical scavenging properties
Free radical-induced oxidative stress is the root cause for many human
diseases. Naturally occurring antioxidant supplements from plants are vital to
counter the oxidative damage in cells. The crude methanolic extract from Navara
rice bran contains significantly high polyphenolic compounds with superior
antioxidant activity as evidenced by scavenging of free radicals including 1, 1-
Diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO). Navara extracts also
showed highest reducing power activity, anti-proliferative property in C6 glioma
cells. Relatively high total antioxidant activity in the Navara rice bran compared to
the other rice varieties showed a significant correlation with polyphenolic contents
suggesting the importance of polyphenolics as potential antioxidant biomolecules.
Navara had relatively higher reducing power than other samples, indicating a
significantly higher correlation with polyphenolic content (Rao et al., 2010).
Chemical indices, antioxidant and anti-inflammatory activity of extracts of
bran of medicinal rice – Navara ―black glumed‖ type (NB) and its rice (NBr), were
studied in comparison with bran and rice of staple varieties: Sujatha and
Palakkadan Matta. The study revealed that the total oryzanol, phenolic, flavonoid,
proanthocyanidin and phytate contents of Navara bran (1.84 mg/g, 27.16 mg of
gallic acid/g, 4.50 mg of quercetin/g, 0.98 mg of catechin/g and 8.77 mg/g dry
weight of bran, respectively) were higher compared with Navara rice and staple
varieties. The higher oryzanol content, chemical indices, antioxidant and anti-
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inflammatory activity for Navara compared with staple varieties corroborates with
its medicinal use in Ayurveda (Smitha et al., 2012).
Even though reports on characterization of Navara germplasm are available
at morphological, biochemical and molecular level, reports on physiological,
anatomical and palynological studies are very little. Moreover almost all the
studies till date has been under the notion that there are only two morphologically
distinct black and golden yellow forms in the medicinal rice Navara with two
variants having awns, making the morphoforms to four. Many attempts have been
made to study the Navara germplasm in different perspectives and the existence
of Navara Punja has been reported as the results of this study (Shiny and Nair,
2012) but the presence of Cheriya Navara in this complex has not been revealed
till date. In this background, a biosystematic approach in studying the germplasm
using morphology, anatomy, palynology, physiology, biochemistry and molecular
markers has been undertaken to characterize ‗Navara complex‘ and delineate
Navara Punja as a separate entity in Navara germplasm complex.