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-

54

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.