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The assessment of intraspecific variability existing in the germplasm of a

species is of great interest for conservation of genetic resources as well as for

broadening of genetic base of species to be exploited by plant breeders. The aim of

the present study was to find out whether phenotypic variations in the plants collected

from different locations in North India were merely epigenetic or genetic. Further, if

the morphovariants have any bearing on their medicinally active principles. So a set

of 34 accessions of E. alba belonging to three different morphotypes were assembled

from different locations in North India. Presently, all the 34 accessions of E. alba

were maintained at Botanic Gardens, Punjabi University, Patiala. Investigations were

carried out to characterize and evaluate the collected germplasm of E. alba for

morphological, cytological, biochemical and molecular parameters.

5.1 . MORPHOLOGICAL ANALYSIS

Morphology has been used to distinguish subspecies and varieties.

Morphological variation and geographical isolation among populations are also

prerequisite to the formation of subspecies and species (Losos and Glor, 2003). The

survey of wild plants revealed the existance of intraspecific morphological variations.

Intraspecific morphological variations are considered as one of the important factor in

the process of evolution. Intraspecific morphovariants within a species have been

considered the incipient species at various stages of development of new species

(Fosberg, 1942). Intraspecific variations of morphological characters are quite

common in flowering plants. The morphology of Eclipta genotypes varied, as has

been previously reported by Gupta (1977). The variability can be attributed to some

genetic reasons as the variants within a species posess same chromosome number.

Morphological variations have been reported in medicinal plants like Lasiurus

scindicus (Yadav and Krishna, 1986; Arshad et al., 2009; Nisar et al., 2010),

Hedysarum boreale (Johnson et al., 1989), Eragrostis species (Tefera et al., 1992),

Cymbopogon jawarancusa (Arshad et al., 1995), Sporobolus iocladus (Arshad et al.,

1999), Dicanthium annulatum (Agarwal et al., 1999), Cenchrus ciliaris (Mansoor et

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130

al., 2002; Arshad et al., 2007), Zizania palustris var. palustris (Lu et al., 2005) and

Panicum antidotale (Sohail et al., 2010).

Taxonomically there exist many synonyms of Eclipta alba as Eclipta

prostrata (L.) L., Eclipta erecta L., Eclipta puncatata L., Verbesina alba L.,

Verbesina prostrata L. In various Floras, Eclipta alba (L.) Hassk., and Eclipta erecta

L., are accepted as synonyms and Eclipta prostrata (L.) L. is considered as a distinct

species. Boissier (1875) described Eclipta marginata Hochst. and Steud. ex Boiss.,

characterizing its cypselas winged differing from Eclipta alba (L.) Hassk., which has

wingless cypselas. He cited Eclipta prostrata under the synonymy of Eclipta

marginata and Eclipta alba. But Vassilczenko (1959) has considered Eclipta alba and

Eclipta prostrata as two distinct species. He kept Eclipta marginata under the

synonymy of Eclipta prostrata and Eclipta erecta under Eclipta alba. He

characterized Eclipta prostrata by having cypselas smooth and fringed and Eclipta

alba with cypselas tuberculate along the ribs or over whole surface. Rechinger (1977)

has considered Eclipta prostrata and Eclipta alba as two valid species but with some

modification in characters different from Vassilczenko. He characterizes Eclipta

prostrata by having cypselas marginate and tuberculate or smooth and Eclipta alba

with cypselas not marginate and smooth. He placed Eclipta marginata under the

synonymy of Eclipta prostrata as suggeated by Vassilczenko (1959). Abedin and

Tajuddin (2009) examined a large number of specimens and found that Eclipta

prostrata (L.) L., is the only distinct species and the various names mentioned above

are all synonyms. Actually the cypselas of the ray florets are smooth and winged

while of disc florets are tuberculose and winged completely or basally in young

condition. It is these incomplete specimens that have resulted in 2-3 different

species. In the light of Article 57 of the code (International Code of Botanical

Nomenclature, 1978) Eclipta alba (Linn.). Hassk. should be followed. Eclipta alba is

commonly known as safed bhangra (Hindi) when in flower and as Kalobhangro when

in fruit. Pila bhangra is the name given to closely related plant Wedelia chinensis

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131

(Wealth of India, 1952; Bhargava and Seshadri, 1972). Wedelia chinensis is often

confused with Eclipta alba and thus considered as its adulterant.

Eclipta alba is monospecific, but its evolution under natural selection in a

wide range of agro-climatic areas has resulted in wide phenotypic diversity. Thus it is

evolutionary dynamic genera. It is erect, partly erect or prostrate and is described as

variable species by taxonomists. There is intraspecific variation in this polymorphic

species. It is very variable in habit, hairiness, size, color and shape of leaves and stem

and size of flower head. Among North Indian populations, three morphotypes of

Eclipta alba can easily be identified i.e. erect, semierect and prostrate (Table 5.1). The

erect plants are tall and upright while the prostrate types have all creeping branches.

The semierect type plants have lower parts of branches creeping and tips are

ascending. The prostrate type of accessions show highest incidence (Fig.VI).

Table 5.1: Habit variation among different accessions of Eclipta alba germplasm

Morphotype Accessions

Erect EPBP1, EPBG1, EPBG2, EPBL1, EPBJ1, EPBF1, EJKJ1

Semierect SPBP1, SPBG1, SPBG2, SPBG3, SPBA1, SPBT1, SPBT2, SHRA1,SUPM1

Prostrate PPBP1, PPBG1, PPBG2, PPBA1, PPBA2, PPBA3, PPBA4, PPBJ1,PPBN1, PPBR1, PPBM1, PRJS1, PJKJ1, PUTC1, PHRP1, PHRP2,PHPS1, PUPM1

Fig. VI: Frequency distribution of three morphotypes on the basis of plant habit

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132

As far as the color of the leaves is concerned the present study revealed two

types of colour variants in Eclipta accessions viz. green and green dark. The different

colour variants were distributed among all the three morphotypes (Table 5.2).

Maximum accessions were of green type (70.6 %) as given in Fig. VII.

Table 5.2: Different color variants of Eclipta alba

AccessionsColourVariants Erect Semierect Prostrate

Green EPBL1, EPBJ1,EPBF1, EJKJ1

SPBP1, SPBG1,SPBG3, SPBA1,SPBT2, SHRA1

PPBP1, PPBG1, PPBA1, PPBA2,PPBJ1, PPBN1, PPBR1, PRJS1,PJKJ1, PUTC1, PHRP1, PHRP2,PHPS1, PUPM1

DarkGreen

EPBP1, EPBG1,EPBG2

SPBG2, SPBT1,SUPM1

PPBG2, PPBA3, PPBA4, PPBM1

Fig. VII: Frequency of various colour variants in Eclipta alba

Morphological variations within the same species are a common phenomenon.

The morphological variation has been detected among various accessions of Eclipta

alba maintained at Punjabi University, Patiala. Since all these accessions have been

grown in the same environmental and soil conditions, the presently observed

variability can be related to their genetic makeup. It is very variable in habit,

hairiness, size, color and shape of leaves and stem and size of flower head. The three

morphovariants viz. erect, semierect and prostrate have been studied for

70.6%

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height/spread, leaf length, number of nodes, branches and inflorescences diameter

(Table 5.3). Among the three types of morphotypes, semierect morphotypes have

highest values of morphological characters as compared to erect morphotypes and the

prostrate morphotypes are the smallest.

Table 5.3: Mean values of various morphological characters of Eclipta alba

Erect type Semierect type Prostrate typeCharacters

Range Mean Range Mean Range Mean

Plant height/Spread (cm)

38.2 – 54.7 45.4 32.6 – 56.8 46.2 21.5 – 38.9 29.3

Nodes/plant 9 -15 11.2 9 - 16 12.4 9 - 13 10.9

Branches/plant 5 -9 7.0 6 - 13 9.4 8 - 11 8.8

Leaf length (cm) 4.2 - 5.8 4.9 3.2 - 5.3 4.5 1.8 - 3.8 2.6

Inflorescence(mm)

5 - 7 5.7 4 - 7 5.4 3 - 5 4.0

5.2 REPRODUCTIVE STUDIES

Studies in the field of reproductive biology can provide important paradigms

for biodiversity conservation, reclamation and restoration. Such studies in

reproductive biology will also help in developing strategies to preserve the genetic

potential of rare species and are crucial for restoration and reintroduction. The

phenological studies are useful in planning out for cultivation of the species on large

scale (Schemske et al., 1994; Delanoe et al., 1996; Wafai et al., 1996; Bernardello, et

al., 2001). There is always a buzz all over the globe to unravel the basic and detailed

information about the reproductive biology of important plant species preferably for

their genetic improvement and cultivation. Phenology in general and reproductive

phenology in particular is a critical and important trait of a plant because it determines

the growth, developmental pattern and number of potential mates thus providing a

mechanism for reproductive isolation and speciation over time (Rathcke, 1983;

Bronstein et al., 1990).

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The present work summarizes the salient features of the reproductive

strategies of the herb Eclipta alba and this information may be of immense use in

planning its cultivation and long term conservation. The genus has considerable

phenotypic plasticity and has morphological variations viz. erect, semierect and

prostrate. Many aspects of reproductive behavior of plants are plastic and can vary

greatly among individuals of the same species living in different habitats. As all the

morphotypes of Eclipta alba are grown under identical conditions. Considerable

diversity was observed among the three morphotypes which includes number of ray

and disc florets, number of seeds produced per capitulum, pollen fertility and seed

germination. Taylor (1987) stated that intra-population morphological variation was

as great as or greater than inter-population variation and therefore, the morphological

variation was largely due to phenotypic plasticity. The phenotypic variability in E.

alba increases its ability to colonize a wide range of habitats. In cool or dry weather,

or in closely mown lawns, the leaves usually spread flat against the surface of the

ground to form an almost prostrate rosette (Longyear, 1918; Lovell and Rowan,

1991). In warmer weather or in areas where it is crowded by taller vegetation, the

leaves stand in more or less erect tufts (Longyear, 1918). The rosette enables it to

survive mowing, grazing and competition with grasses. Also its ability to reproduce

by sexual and vegetative means accounts for the species colonizing and stabilizing

fast in new habitats. This probably accounts for the rapid spread of Eclipta alba.

Vegetative reproduction allows it to occupy a temporary site quickly while light seeds

produced by sexual reproduction allow distance dispersal to new sites.

5.2.1 Floral Morphology

Studies on biology of the species revealed that the taxon is hermaphrodite. The

Eclipta alba is propagated by seeds and rarely through vegetative cuttings. E. alba is

annual to biennial plant that flowers throughout year. The flowers are showy and are

borne in aggregates of capitular heads for pollinator attraction. Several workers

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135

contended that the heads of composites represent single flowers (Good, 1931; Leppik,

1960). However, this is true only in terms of pollinator attraction. Presentation of

many single ovuled flowers for pollination over a great period of time results in much

higher time results in much higher rates of out crossing than in many ovuled single

flowers (Brutt, 1961). Onset and duration of flowering, relative maturation of male

and female sex organs and the number and arrangement of flowers in a plant

profoundly influence the pollinator visitation pattern in the taxa under reference

which has a direct bearing on the success of their sexual cycle (Siddique, 1991).

Different reproductive characters of Eclipta alba were quantitatively assessed.

The total number of ray and disc florets in a flower head found to be variable for the

three accessions i.e. in erect (81.34) followed by semierect (79.66) and least in

prostrate (66.67) type. Occasional occurrence of twin capitulum has been observed in

some accessions is also observed in other members of Asteraceae. Ray florets were

larger than disc florets. However, their ovary size and bifid stigma length were almost

of equal size. A very thick mat of dense hairs was present on both upper and lower

surfaces of stigma in disc floret while dense hairs were present only on the upper

stigma surface in ray floret. In the disc floret stigma protruded through the

syngenesious column of anther. Protandry was the common phenomenon. The

characteristics of reproductive apparatus influenced the reproductive fitness. The ray

florets open before the disc florets to enhance the chances of cross pollination. The

flower opening is asynchronous which ensures pollen availability for a longer time to

affect pollination and possibilities of higher fruit and seed set. In Asteraceae the

florets are protandrous when hermaphrodite, and among the species in which the level

and type of self-incompatibility has been measured, the vast majority are

sporophytically self-incompatible (Nettancourt, 1977; Patil et al., 1979; Stuessy et al.,

1986). Consequently vector mediated crossing is extremely important.

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136

5.2.2 Flowering Phenology and Anthesis

Phenological studies play an important role in planning of conservational

strategies and finalizing the protocol for large scale cultivation of plants (Schemske et

al., 1994; Delano et al., 1996; Wafai et al., 1996; Bernardello et al., 2001; Wani et al.,

2006).

According to Reich (1995), the phenology of vegetative phase is important as

cycles of leaf flush and leaf fall are intimately related to growth, plant water retention

and gas exchange. Floral initiation, development, and anthesis of Asteraceae are

generally described as occurring in an acropetal or centripetal direction on the

capitulum (Cronquist, 1955; Sattler, 1973; Reese and Hilger, 1984). In E. alba,

anthesis proceeds gradually towards the centre of a head. Ray florets present in the

periphery opens as the capitulum start opening. The anthesis starts at morning and

florets remained open till the stigma shrivels. The outer ray florets opens first so as to

enhance the chances of cross pollination from pollens of disc florets of other flowers

than the pollens from disc florets of the same capitulum. Presently, it has been

observed that anthesis in E. alba is asynchronous from floret to floret as well as

capitulum to capitulum. The protracted asynchronous mode of pollen presentation by

the species guarantees the long term availability of the pollen to ensure effective

pollination (Wyatt, 1982). The present study confirms the earlier reports of

asynchronous anthesis in Inula racemosa (Wani et al., 2006)

5.2.3 Pollen Fertility

Pollen represents a critical stage in the life cycle of plants as viable pollen is

crucial for successful plant reproduction. Plant fertility can influence many aspects of

population ecology and evolution. The quantity and quality of pollen produced by a

plant is an important component of reproductive success.

Pollen analysis revealed that Eclipta alba has high pollen fertility. Pollen

fertility was studied throughout the year from January to December to study the effect

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137

of seasonal variations. The erect morphotypes have maximum percentage of viable

pollens than the semierect and the prostrate morphotypes (Fig. VIII). Pollen fertility

was highest in the month of November in the erect morphotypes (99.34%) In the

semierect plants, it was highest in the month of September (98.41%) and in prostrate

it is highest in the month of May (97.80%). The Eclipta plants did not show any heat

stress as far as pollen fertility was concerned. In erect morphotypes, pollen fertility is

lowest in the month of September (80.04%). In semierect and prostrate morphotypes,

the pollen fertility was lowest in the month of January i.e. 74.04% and 71.96%,

respectively. The pollen is spheroidal, tricolporate and echinate. Polar diameter was

22.45 μm (20-25 μm) and equatorial diameter was 22.5 μm (20 - 25 μm). P/E ratio is

0.99 μm. The length of spine was 2.5 μm (1.5- 4 μm). Exine thickness was 7.4 μm (1 -

2 μm (Zafar et al., 2007).

Pollen fertility depends on the efficiency of the meiotic process. In the

presently analyzed accessions meiotic abnormalities have been found to be

responsible for pollen sterility which could have an important effect on fitness of the

population. However, if meiosis was regular, for example, chromosomes pair and

segregate normally, sterility of the pollen grain was not expected to occur because of

cytological reasons (Boff and Schifino-Wittmann, 2002). There are many non-genetic

causes of decline in pollen viability, including pollen age and exposure to

environmental stresses such as temperature and humidity (Stone et al., 1995; Kelly et

al., 2002). Variations in temperature, humidity, and cloud cover can influence pollen

production and viability (Artschwager and McGuire, 1949; Brooking, 1979; McLaren

and Wehner, 1992). Cold temperature stress prior to flowering appears to reduce

pollen viability during anthesis by disrupting meiosis during early stages of

microsporogenesis (Brooking, 1979). Environmental factors like high temperature,

drought conditions and soil factors may seriously affect pollen performance (Zamir

and Gaddish, 1987). Assessment of pollen viability is very important in understanding

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138

the effect of environmental stress during blooming period (Thomson et al., 1994).

Environmental variation can affect pollen fertility in Sorghum as temperature below

13 °C during sensitive stages can induce male sterility (Downes and Marshall, 1971;

Brooking, 1976) and high temperature appear to have similar effect (Dhopte, 1984).

Fig. VIII: Pollen fertility of three morphotypes of E. alba vs Temperature

Fig IX: Minimum and maximum temperature and Average temperature during2009

According to Reed and Tuinstra (2005) the mean maximum temperature,

maximum and minimum relative humidity and precipitation before anthesis were all

highly correlated with pollen viability. High temperature just prior or during

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139

microsporogenesis significantly reduced the pollen fertility. According to Sato et al.

(2002), the most sensitive period for heat stress was found to be 7-15 days before

anthesis. Presently, the pollen fertility has been studied in various morphotypes of E.

alba to see the seasonal variation and it was found that average temperature has no

correlation with pollen fertility (Table 5.4). However, there was positive and

significant correlation between maximum temperature and pollen fertility in the three

morphotypes of Eclipta. The minimum, maximum and average temperature

throughout the year is shown in Fig. IX.

Table 5.4: Correlation coefficient between Pollen fertility in E. alba and Averagemonthly temperature during 2009

MorphotypePollen fertility vs

Avg.temperature

Pollen fertility vsAvg. min.

temperature

Pollen fertility vs Avg.max. temperature

Erect 0.117454 0.555268** 0.540597**

Semierect 0.16533 0.602594** 0.511173**

Prostrate 0.178289 0.178289 0.624462**

** Significant at 0.01

5.2.4 Pollination system

In Asteraceae the basic attraction unit for pollinators is the inflorescence

(capitulum) composed of ray florets, which are petal analogues (Mani and Saravanan,

1999). The presence of ray florets is positively associated with plant fitness when

pollinators are abundant (Stuessy et al., 1986, Nielsen et al., 2002). Flowers

essentially act as a food source for pollinators and other visitors thereby giving the

study of flowering phenology both ecological and evolutionary significance.

Purseglove (1968) postulated that flowers with white corolla, strong perfume,

Discussion

140

abundant nectar and sticky pollen are entomophilous and are visited by bees and

thrips. However, our study showed that white flowers of E. alba were not visited by

many insects except thrips and ants and these may carry out the pollination suggesting

entomophilous pollination. Small Thrips tabaci insects were often seen visiting all

florets of a head one by one going down to the corolla tube and comes out to visit the

next one. Earlier studies have shown that Thrips tabaci and Thrips palmi of order

Thysanoptera are hosts of Eclipta (Salas, 2003).

5.2.5. Seed production and Fertility

The total number of seeds produced per capitulum in the three morphotypes of

Patiala populations of E. alba was also variable i.e. erect morphotypes (83±6.55)

followed by semierect (72±7.54) and least in prostrate (59±3.0). Bagging experiment

revealed that E. alba is self as well as cross compatible which is evident from high

seed set in open pollinated flowers and low seed set in bagged flowers. The seed

production was enhanced by insect pollination. E. alba showed protandrous condition

which favors outbreeding. The species was cross compatible. Open pollinated heads

produced large quantity of seeds while those bagged to exclude the foreign pollen

produced less number of seeds. Self and cross pollination has been recorded in several

species of Compositae. Self pollen can germinate as revealed by the bagging

experiments. Low fruit and seed set in bagged flowers may be due to the existence of

protandry, which tends to prevent self pollination and encourages cross pollination.

There is seasonal variation on seed germination in E. alba as observed in the months

of January and July. It was observed that in the month of January seed germination

was very low in all the three morphotypes and was very high during July in all the

three morphotypes.

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141

5.3 CYTOLOGICAL STUDIES

The data on chromosome number and meiotic analysis are prerequisites to

overall understanding of a genome and its genetics. Presently detailed cytological

studies have been carried out in different accessions of Eclipta alba (L.) Hassk. All

these accessions have same chromosome number n=11 and is in accordance with

earlier records for the species (Keil and Stuessy 1975; Koul et al., 1976a; Pinkava and

keil, 1977; Powell and Powell, 1978; Sidhu, 1979; Robinson et al., 1981; Morton,

1981; Kumar and Trivedi, 1983; Trivedi and Rajesh, 1984; Razaq et al., 1988; Gill

and Omoigui, 1988; Mathew and Mathew, 1988; Gupta and Gill, 1989; Trivedi and

Trivedi, 1992; Hunziker et al., 1989; Mohamed, 1997). Most of the previous reports

from India and outside also reported the same chromosome number. However Kumar

et al. (1990), Singh (1993) and Kumar and Singh (1989) reported aneuploid cytotypes

(2n=20,22) in the species. Mohan et al. (1962) found somatic variability i.e. 2n =18;

Silvestre (1980) investigated n=11 and 2n=18 and Renard et al. (1983) found 2n=18

for E. alba. Husaini and Iwo (1990) reported its haploid number n=12.

Meiotic studies carried out in various accessions of Eclipta alba revealed that

the majority of the collections showed normal meiotic behavior with 11 bivalents at

metaphase-I and regular chromosome segregation at anaphase-I and II. At diakinesis

one or two bivalents were seen to be associated with darkly stained nucleolus. Ring

and rod bivalents were observed at diakinesis, ring shaped were more frequent than

rod shaped bivalents.

In the life cycle of an organism the meiosis is genetically the most significant

activity consisting of highly coordinated physiological, biochemical, cytogenetic and

phenotypic events which lead to gene recombination, chromosome reduction and

gamete formation (Kaul and Singh, 1991). Mutation in any of the genes governing the

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142

events disrupts meiosis and culminates in gametic sterility and other abnormalities.

Basic data on meiotic behavior and pollen viability estimations are important for the

germplasm characterization, to determine the genetic variability and evolution

processes of the species analyzed.

The observed precocious chromosome migration to the poles or early

disjunction in accession PHPS1 may have resulted from precocious chiasma

terminalization at diakinesis or metaphase-I. Late disjunction of chromosomes as

observed in accession PHPS1 was generally attributed to the interlocking of

chiasmata. The presence of bridges with or without fragment reflected structural

changes such as heterozygous inversions, chromatid exchanges, deletions and

duplications. The observed subchromatic or side arm bridges could be originated from

an abnormal function at the places where the chiasmata are formed (Brandham, 1969;

Brandham, 1970).

Abnormalities in microsporogenesis become evident by occurrence of dyads,

triads, polyads, tetrads with micronuclei, etc. Dyads, triads with three microspores of

different sizes suggest that restitution nuclei may also occur. Dyads were formed

when there was failure of one meiotic division. Formation of triads could be due to

asynchrony in the second division where one nucleus fails to divide while the other

enters telophase-II. Usually in a triad one 2n microspore and two n microspores are

formed. The consequences of 2n are jumbo pollen formation.

The micronuclei at dyad or tetrad stage of PMC’s might have resulted due to

non-orientation of chromosomes and laggards. Micronuclei lead to the formation of

micro pollen which indicates the loss of genetic material. Their presence, therefore,

suggests that the resultant product of meiotic division is deficient in one or the other

chromosome. This usually leads to the formation of sterile pollen grains. According to

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143

Singhal et al. (2010) the large sized pollen grains, almost double size, may be of ‘2n’

constitution and play an important role in the origin of intraspecific polyploidy in the

species. The small sized fertile pollen grains which are the products of hypoploid

PMCs could lead to the origin of aneuploids. Presently, different sized pollen grains

have been observed in different accessions E. alba but the cytological status could not

be ascertained. This aberrant meiotic behavior has an influence on the low viability of

pollen. The presence of laggards, bridges, unoriented chromosomes during anaphase-

I/telophase-II and consequently micronuclei and microcytes and polyads during

meiosis was reported in many species such as Capsicum annuum L. (Raja Rao et al.,

1987), Vicia faba and Pisum sativum (Souguir et al., 2008) and Brachiaria hybrid

(Mendes-Bonato et al., 2006) etc.

Chiasmata Frequency Analysis

Estimation of chiasma frequency has been regarded as most easy and direct

method of scoring the total number of crossing over events in the genome. Chiasma

corresponds to the points of physical exchange between homologous non-sister

chromatids (Tease and Jones, 1978) and their frequency is regarded as a good

estimate of the level of genetic recombination in the species (Colombo, 1992). Studies

conducted on a variety of plants revealed the existence of intraspecific variability in

chiasma frequency among different accessions (Sanhez-Moran et al., 2002). It is well

known that chiasma frequency varies in different environments (Barber, 1940; Fedak,

1973; Dowrick, 1957). Rees and Dale (1974) have considered the intraspecific

variability as a mean of generating new forms of recombination which influence

variability within natural populations in an adaptive way.

Eclipta alba plants flower throughout the year. The recombination processes

of the three morphotypes of the species have been studied to know if there is any

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144

fluctuation in the process in different months of the year. Natural variation in

recombination frequency in Eclipta alba has been assessed by analyzing variation in

chiasma frequency among three morphotypes. There was no significant variation in

mean chiasma frequency between plants within accessions, but there was significant

variation between accessions. The different prostrate type of accessions of E. alba

were found to have higher recombination values than the values shown by plants of

other morphotypes except during the months of March and September when the erect

type of plants show higher recombination values (Fig. X). In all the accessions

belonging to the three morphotypes of the species a common trend was observed i.e.

in each morphotype the average chiasma frequency and recombination index were

highest during the months of July and lowest during the months of Janauary.

The chiasma frequencies of large number of plant chromosomes have been

determined in the last centaury (Nilson et al., 1993). Studies conducted on a variety of

plants revealed the existence of intraspecific variability in chiasma frequency among

different accessions (Sanhez-Moran et al., 2002). It is well known that chiasma

frequency varies in different environments (Barber, 1940; Fedak, 1973; Dowrick,

1957).

According to Fedak (1973) chiasma frequency varies in different

environments. The seasonal variation in chiasma frequencies has been reported in a

number of animals (Cobror et al., 1986). There is considerable literature documenting

the influence of temperature (Dowrick, 1957), mineral nutrients (Law, 1963; Bennett

and Rees, 1970), chemicals (Barber, 1940) and ionizing radiations (Lawrence, 1961)

on chiasma formation. Since specific environmental factor such as temperature might

have contributed to seasonal variation in the chiasma frequency in the present case an

attempt has been made to correlate minimum temperature with chiasma frequencies in

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145

the three morphotypes (Fig. XI). One cannot conclude that the average minimum

temperature during the first week of alternate months is more important than the day

temperature in causing changes in pairing and chiasma formation in chromosomes.

Maximum and minimum temperature is not sufficiently accurate criteria of daily

fluctuation in temperature. Nevertheless, the present study has shown the decrease in

the chiasma frequency with increase in minimum night temperature. In all the

morphotypes of E. alba minimum chiasma frequency was observed during the months

of July when average night temperature was 26.9 ºC. However, Dowrick (1957)

recorded increased chiasma frequencies in Tradescantia bracteata and Uvularia

perfoliata as a result of increase in temperature. Similar observations have been made

by Hovin (1958) in amphihaploid of Poa annua. Genetic as well as environmental

factors have generally been considered the reason for differences in chromosome

pairing.

Variation in chiasma frequency and localization has also been shown to be

genetically controlled (Quick, 1993). The existence of significant interaccession

variation for chiasma frequency implies that the accessions concerned differ for

genetic factors or elements which effects chiasma frequency (Sanchez-Moran et al.,

2002). The presently observed cyclic variation in average chiasma frequencies,

recombination index and excess chiasma in the three morphotypes of Eclipta alba

clearly depict the influence of environmental factors on process of recombination.

Discussion

146

Fig. X: Distribution of Recombination index and the number of ExcessChiasmata per bivalent during different months in various morphotypes ofE. alba

Fig. XI: Relationship of mean Chiasma Frequency and mean minimum nighttemperature

Discussion

147

5.4 MOLECULAR ANALYSIS

An enormous amount of genetic diversity is characteristic of natural

populations. The random genetic drift, restricted gene flow and differential selection

pressures jointly influence genetic structure of natural populations. These effects lead

to low within and comparatively high among population genetic variation in species

consisting of small and isolated populations (Nevo, 1983; Holderegger and Schneller,

1994). DNA based molecular markers are versatile tool for the plant genome analysis

and for differentiating different genotypes. A number of molecular techniques like

Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length

Polymorphic DNA (AFLP), Restricted Length Polymorphic DNA (RFLP) and Inter

Simple Sequence repeat (ISSR) etc are now being successfully used for genetic

analysis of medicinal plants (Joshi et al., 2004). DNA markers are highly stable and

specific. It has immense applications in the standardization of medicinal plants and its

products. Molecular analysis techniques allow the estimation of genetic variability

and divergence between species and populations, and thus they can be used for

phylogenetic studies, conservation programs and control of the genetic origin of

products.

In general, genetic diversity can be measured at any functional level from

blueprint (DNA) to phenotype. Presently, possible genetic variation among E. alba

genotypes collected from different locations was analyzed using analysis to

discriminate molecular variability. Random Amplified Polymorphic DNA markers

were chosen since they are an effective and relatively inexpensive technique and do

not require any prior sequence information (Jones et al., 1997; Chenuil, 2006). These

dominant neutral markers have been applied to a wide range of plant species like

Azadirachta (Farooqui et al., 1998), Juniperus communis (Adams and Pandey, 2003),

Codonopsis pilsula (Fu et al., 1999), Allium schoenoprasum (Friesean et al., 1999),

etc.

Discussion

148

Presently, RAPD based cluster analysis revealed genetic variability in

different populations of E. alba. Accession PHPS1 collected from district Sirmaur

(Himachal Pradesh) was found to be significantly different from the rest of accessions

displaying a similarity coefficient of 0.59 with rest of the accessions. RAPD based

analysis revealed genetic variability among different accessions belonging to three

morphotypes of E. alba. Closeness among prostrate accessions collected from district

Amritsar (PPBA1 and PPBA2), Patiala and Jalandhar (PPBP1 and PPBJ1) and

Panchkula (PHRP1 and PHRP2) was quite evident. Similarly genetic closeness in

semierect accessions SPBP1 and SPBG3 collected from Patiala and Gurdaspur has

been demonstrated. The association between genotypes from contiguous regions may

be the result of similar agro climatic conditions or due to seed movement and gene

flow, however the closeness between distantly located genotypes may be attributed to

the unique and broad genetic base of the species. Thus the accessions collected from

different localities on molecular analysis revealed high variation within the species

indicating their adaptive potential.

Genetic diversity within a species has always enthused evolutionary and

conservation biologists. The capability of a species to adapt to environmental changes

depends upon genetic diversity in the species (Neel and Ellstrand, 2003). The species

with depletion in gene pool and reduced genetic diversity pose challenges in the

selection pressure brought in by the environmental changes (Caro and Laurenson,

1994). The evolution of plants in distinct agro-climatic zones demonstrates significant

levels of variations in response to the selection pressure in the zones (Singh et al.,

1998).

Although the data presented here are not conclusive to infer the genetic

relationship between the various accessions but they reflect the utility of RAPD in the

analysis of genetic variability distribution within this important medicinal herb. The

Discussion

149

present study and similar studies on lotus, sweet potato, oil palm and potato (Campos

et al., 1994; Connoly et al., 1994; Shah et al., 1994; Demeke et al., 1996) suggest that

RAPD is more appropriate for analysis of genetic variability in closely-related

genotypes. Ray and Bhattacharya (2010) used the RAPD technique for confirmation

of genetic fidelity of synseed generated plantlets of Eclipta alba. Molecular analysis

of intraspecific variation in particular may find application in resolving disputes of

taxonomic identities, relations and authentication of the species, developing a

comprehensive database of genetic variability in the species for future reference.

5.5 BIOCHEMICAL ANALYSIS

The curative activity of plant based drugs is attributed to the chemicals present

in them and majority of which are their secondary metabolites which help the plant to

adapt to biotic and abiotic stress. Most of the Ayurvedic herbs are collected from

nature without bothering about their genetic makeup which is bound to affect their

chemical constitution. The major challenge for conservation and improvement of

genetic resources of medicinal plants is the identification of better chemotypes which

have both good quality and quantity of active principle. Plant secondary metabolites

(PMS) play an important role in the survival of the plants in their habitat and are

involved in resistance against pest and diseases, the attraction of pollinators, the

attraction with symbiotic microorganisms, etc (Dixon, 2001; Harborne, 2001). PMS

are also of interest because they determine the quality of food (color, taste and aroma)

and ornamental plants (flower color, smell).

The chemical composition of the Eclipta alba plants has been investigated by

many workers. The herb Eclipta alba contains mainly coumestans i.e. wedelolactone

and demethylwedelolactone, polypeptides, polyacetylenes, thiophene-derivatives,

steroids, triterpenes and flavonoids. Qualitative analysis of the phytochemicals of

aqueous extracts revealed the presence of carbohydrates, saponins, phytosterols,

Discussion

150

phenols, flavonoids and tannins in E. alba plants (Khanna and Kannabiran, 2007).

Study of chemical constituents of E. alba has been carried out by a number of workers

(Govindachari et al., 1956; Bhargava et al., 1972; Karrer, 1977; Wagner et al., 1986;

Singh et al., 1988; Nguyen and Doan, 1989; Han et al., 1998).

The present study conducted on various accessions of Eclipta alba revealed

the presence of inter-accession variation in various phytochemicals. The data is

presented in Tables 4.10 and 4.11.

5.5.1 Carbohydrates

Anthrone assay (Chitra et al., 2009) revealed that the extract has

0.11µg/100mg and 1.37µg/100mg of carbohydrates on dry basis in root and leaf

sample, respectively. Anitha (2002) reported 232 mg/g of carbohydrates in E. alba.

Among various accessions of E. alba maintained at Patiala the carbohydrate content

ranged from 16.13 mg/g to 18.69 mg/g with a mean value of 17.45±0.701 mg/g. The

semierect plants have overall highest carbohydrate content.

5.5.2 Proteins

The protein content determined by Chitra et al. (2009) by using Lowry’s

method was 1.31µg/100mg and 0.74µg/100mg for leaf and root samples, respectively.

Anitha (2002) reported 0.1 mg/g of proteins in E. alba. Rao et al. (2009) reported 14.5

mg/g total protein content in E. prostrata in wet sample. In the present study, the

protein content varied between 12.21 mg/g to 14.85 mg/g with mean value of

13.69±0.678 mg/g. Protein content was highest among erect plants.

5.5.3 Phenols

These nitrogen-containing compounds protect plants from a variety of

herbivorous animals, and many possess pharmacologically important activity. So far

as plant phenolics constitute one of the major groups of compounds acting as primary

antioxidants or free radical terminators, it was reasonable to determine their total

Discussion

151

amount in the selected plant extracts (Cook and Samman, 1996). A recent study by

Chitra et al. (2009) reported 0.12 µg/100 mg of phenolic compounds in leaf samples

and 0.06 µg/100mg of phenolic compounds in root samples. In the fresh leaves of

Eclipta alba Rao et al. (2009) observed phenol content as 19.5±0.458 GAE units in

fresh E. alba. According to another study the phenol content in the methanolic extract

was 30.4 mg/g of dry mass in Eclipta alba (Veeru et al., 2009). In the presently

studied accessions of Eclipta alba, the phenol content varied between 17.33 mg/g to

20.97 mg/g with a mean value of 18.83±1.088 mg/g. Among three morphotypes, the

minimum phenol content was observed in prostrate accessions.

5.5.4 Saponins

Saponins are amphipathic glycosides grouped phenomenologically by the

soap-like foaming they produce when shaken in aqueous solutions. Saponins, a

diverse group of secondary metabolites characterized by their structure containing a

steroidal or triterpene aglycone and one or more sugar chains (Hostettmann and

Marston, 1995; Osbourn, 2003; Guclu and Mazza, 2007), occur constitutively in

many plants and, everting various biological activities, may be considered as part of

plant defense system (Potter and Kimmerer, 1989; Osbourn, 1996; Agrell et al.,

2003).

Present study has revealed the variation in saponins content in the leaves of

various accessions. Saponins content ranged between 28.3 mg/g to 48.5 mg/g with

overall mean of 39.4±4.89 mg/g. Crude saponins was the major phytochemical

constituent present in highest percentage in E. prostrata (4.5%). Other

phytochemicals estimated in this plant were present only in very low concentrations

(Khanna and Kannabiran, 2007).

Discussion

152

5.5.5 Wedelolactone

Wedelolactone is a naturally occurring coumestan isolated from aerial parts of

Eclipta alba. The E. alba plants contain wedelolactone (1.6%) as principle coumestan

compound due to which it is valued as hepatic drug. The coumestan Wedelolactone is

found to be present in leaf and stem of natural herb but absent in roots.

Chemoprofiling using HPLC have a great role in quality control of medicinal plants

(raw drugs) and in the finished herbal drugs.

The present HPLC estimation revealed the considerable phytochemical

(wedelolactone) variation in the studied accessions of Eclipta alba. The

phytochemical diversity measured as quantitative difference in the accumulated

wedelolactone ranged from 0.111 mg/g to 1.43 mg/g with a mean value of

0.512±0.385. Plant specific marker compound (wedelolactone) showed quantitative

variations among the genotypes, which could not be correlated with allelic variation

but the active principle content when measured under uniform growing conditions,

variations so observed presumably had a genetic basis.

Variation in morphological characters and active principle constituents of

Eclipta prostrata Linn. under different seasonal and soil conditions has been

determined by Gupta (1977). The concenteration of wedelolactone as estimated by

UV spectrophotometric method at 351 nm is 0.0078±0.000081% in leaf and

0.0042±0.000081% in stem (Zafar and Sagar, 1999). HPLC analysis of Eclipta alba

demonstrated the presence of major proportion of wedelolactone in case of petroleum

ether extract (1.9±0.2% w/w) and very small quantity of around 0.2±0.01% w/w in

case of ethanolic extract (Roy et al., 2008). According to Wagner et al. (1986)

extracts of vegetative tissue of E. alba plants cultivated in India produce 15.9%

Discussion

153

wedelolactone. Methanol extracts of E. alba contain 1.6% wedelolactone (Jayathirtha

and Mishra, 2004).

5.5.6 Phytate Content

Elements are necessary for the healthy development of man, animals and

plants. Their concentrations in the plant differ from part to part and from plant to

plant, depending on the elements level in the soil and presence of allelochemicals.

Phytate is one of the allelochemicals found in plants (Aletor, 1993). It is the most

important antinutritional factor because more than 50% of phosphorus is present in

the form of phytate in plant foods and organic soils serving as phosphorus depot in the

body, which is broken down by phytate to myo-inositol. Myo-inositol increased the

oxygen transporting capacity of hemoglobin in red blood cells, improves and

regulates cellular metabolism. Phytate has been shown to play a role in preventing

colorectal carcinoma, hypercholesterolaemia and renal calculi (Marounek et. al.,

2000). The aquatic weed samples were found to be high in phytate. It is likely that

high phytate intake interferes with mineral absorption. Even though some plants have

a substantial quantity of the minerals, it is felt that a large portion of these minerals

may not be available due to the presence of phytate (Abulude, 2005).

In the present study phytate contents in three morphotypes of E. alba has been

compared and it was found that phytate was highest in the erect type i.e. EPBP1

(467.26 mg/100g) followed by semierect i.e. SPBP1 (463.80 mg/100g) and least in

the prostrate type i.e. PPBP1 (449.96 mg/g). Earlier, Abulude (2005) reported 440

mg/100g DM of phytate in E. alba. The present results suggest that the weed is not

suitable as diet as it has high phytate content as the bioavailability of minerals of the

weed would be low.

Discussion

154

5.6 CORRELATION ANALYSIS

The better understanding of genetic variation at the intraspecific level help in

identifying superior genotypes for crop improvement as well as to evolve strategies

for the effective in situ and ex situ conservation programmes, although such empirical

determination of genetic diversity can be obtained by evaluating morphological and

biochemical traits. The aim of the present study was to find out whether phenotypic

variations in the genotypes grown in different locations are merely epigenetic or

genetic. Furthermore, if the same are genetically different, then their medicinally

active principles have to be estimated to find out possible quantitative variation,

which accord for the variation in medicinal activity of the plants collected from

different locations.

The morphological and biochemical characteristics of the different accessions

of E. alba have been studied. The correlation coefficients among the major

characteristics showed that these are positively associated with each other. The

present correlation study revealed that the various phytochemicals in the leaves are

positively correlated with morphological characters such as plant height and leaf size.

The plant height was found positively and very significantly correlated with

biochemicals i.e. carbohydrates (0.96), proteins (0.86), phenols (0.96), saponins (0.93)

and wedelolactone (0.45). The correlation coefficients among different characteristics

are given in Table 5.5. Medicinal plants are valued for the biochemicals present in the

leaves. The results clearly indicate that the selection of Eclipta alba plants with good

yield depends upon large plants with big sized leaves. Thus the data of morphological

characters showed usefulness of selecting the genotypes with good yield for

commercial cultivation.

Discussion

155

Table 5.5: Correlation between morphological and biochemical characters indifferent morphotypes of Eclipta alba

Characters X1 X2 X3 X4 X5 X6 X7

Erect plants

X1 Plant height 1

X2 Leaf length 0.76881 1

X3 Carbohydrate 0.80712 0.65082 1

X4 Protein 0.80712 0.72277 0.40075 1

X5 Phenol 0.78923 0.68420 0.69454 0.60356 1

X6 Saponins 0.22869 -0.10455 -0.50396 0.99685 -0.78999 1

X7 Wedelolactone 0.21001 0.52017 0.82516 -0.86703 0.45152 -0.90376 1

Semierect plants

X1 Plant height 1

X2 Leaf length 0.66263 1

X3 Carbohydrate 0.95396 0.71477 1

X4 Protein 0.92332 0.73002 0.97984 1

X5 Phenol 0.97159 0.67557 0.98527 0.95723 1

X6 Saponins 0.95835 0.63983 0.91870 0.87730 0.96840 1

Prostrate plants

X1 Plant height 1

X2 Leaf length 0.10312 1

X3 Carbohydrate 0.91224 0.00926 1

X4 Protein 0.85304 0.19734 0.87237 1

X5 Phenol 0.85249 0.11730 0.89982 0.88941 1

X6 Saponins 0.08757 0.45153 -0.12209 -0.05697 -0.14778 1

X7 Wedelolactone 0.27901 0.204803 0.36016 0.26990 0.47040 0.19241 1

Total accessions

X1 Plant height 1

X2 Leaf length 0.8341 1

X3 Carbohydrate 0.9632 0.8023 1

X4 Protein 0.8539 0.7825 0.8348 1

X5 Phenol 0.9652 0.8129 0.9512 0.8705 1

X6 Saponins 0.9334 0.7676 0.9431 0.8263 0.9349 1

X7 Wedelolactone 0.4584 0.2735 0.3587 0.5177 0.2551 0.3497 1

Discussion

156

5.6.1 Inter Accession variability

Variation among phytoconstituents within and between populations of a

species are not uncommon. Morphological as well as biochemical variability has ben

reported among populations, land races, accessions, etc. in a number of crops (Wilson

et al., 1990; Ouendeba et al., 1995, Zeinali et al., 2004). Such variations have genetic

as well as environmental basis. Variations in medicinal plants is often noticed at

chemical level which is due to synthesis and accumulation of various biochemicals.

Since the presently investigated accessions are growing in same environmental and

edaphic conditions the observed variability may be related to their genetic makeup.

The present study on various accessions of Eclipta alba revealed the presence

of inter-accession variation in various phytochemicals. Analysis of variance

(ANOVA) was carried out in different accessions of Eclipta alba covering various

parameters. The ANOVA revealed significant differences among various accessions

for various characters (Table 5.6). The results indicated the presence of adequate

amount of variability in the germplasm under study.

5.6.2 Phytochemical variability vs Morphotype

Phenotypic variations often give valuable clue to the underlying genetic

variations. The variation of phenotypic characters, especially quantitative ones, differs

greatly among populations rather than within the populations (Schemske et al., 1994).

Variations in the alkaloid content due to differences in the morphological characters

have also been observed in Cymbopogon flaxuosus by Seeni et al. (2003).

Quantitative variation in various phytoconstituents has often been noticed in

medicinal plants. This diversity, may be due to the synthesis and accumulation of

various biochemicals, can be continuous or discrete and may be coded by many or

few genes together with some inputs from the environment (Mallet, 1996).

Discussion

157

Table 5.6: Analysis of variance for various biochemical constituents

Constituents Degree offreedom

S.S. M.S. F - value

Plant HeightError

231

2323.72115.19

1161.8635.97

32.297

Leaf LengthError

231

37.32089.9815

18.66040.3220

57.954

Carbohydrate contentError

231

11.0565.119

5.5280.165

33.48

Protein contentError

231

9.8505.337

4.9250.172

28.61

Phenol contentError

231

24.0714.95

12.030.48

24.95

Saponins contentError

221

320.99229.19

160.4910.91

14.706

Wedelolactone contentError

210

1.4773880.297098

0.7386940.029710

24.86363

The phytochemical variation has been detected among various accessions of Eclipta

alba maintained at Punjabi University, Patiala (Table 5.7). Since these accessions

were growing in the same environmental and edaphic conditions, observed variability

can be related to genetic makeup.

The variation in the quantity of various biochemical constituents has been

studied among the various morphological, colour and cytological variants observed in

Eclipta alba. Among the three morphotypes semierect morphotypes possess higher

amount of various biochemicals i.e. carbohydrates, proteins, phenols and saponins as

compared to erect morphotypes which possess higher amount of wedelolactone and

the prostrate morphotypes possess comparatively least amount of biochemicals.

The two colour variants also showed peculiar trend. The dark green Eclipta

accessions showed higher amount of various constituents as compared with other

Discussion

158

colour variant (Table 5.8). Among the dark green morphotypes of Eclipta alba,

semierect morphotypes possess higher amount of all the biochemicals followed by

erect morphotypes and prostrate morphotypes.

Table 5.7: Mean performance of three morphotypes of Eclipta alba

Constituents ErectMorphotypes

SemierectMorphotypes

ProstrateMorphotypes

Carbohydrate (mg/g) 17.96±0.321 18.12±0.474 16.91±0.399

Protein (mg/g) 14.20±0.217 14.32±0.441 13.19±0.454

Phenol (mg/g) 19.71±0.587 19.73±1.073 18.03±0.466

Saponins (mg/g) 41.5±1.22 44.3±4.07 36.2±3.24

Wedelolactone (mg/g) 1.11±0.304 0.492±0.137 0.292±0.121

Table 5.8: Mean performance of Eclipta alba accessions with green and darkgreen leaves

Green Eclipta Dark Green EcliptaConstituents

Erect Semierect Prostrate Erect Semierect Prostrate

Carbohydrate

(mg/g)

17.80±0.20 18.04±0.45 16.88±0.37 18.19±0.33 18.28±0.57 17.04±0.53

Protein (mg/g) 14.10±0.23 14.19±0.38 13.16±0.47 14.33±0.10 14.49±0.58 13.3±0.40

Phenol (mg/g) 19.33±0.46 19.58±1.04 17.99±0.41 20.21±0.21 20.04±1.28 18.21±0.65

Saponins(mg/g)

40.8±1.41 44.4±4.14 36.04±3.26 42.3±0.353 44.1±4.86 37.2±4.10

Wedelolactone(mg/g)

-- 0.589±0.13 0.245±0.10 1.11±0.30 -- 0.431±0.03

Analysis of variance (one way ANOVA) revealed highly significant

differences among three morphotypes of E. alba for all the characters observed.

(Table 5.9).

Discussion

159

Table 5.9: Analysis of variance for different characters in various accessions ofthree morphotypes of E. alba

Sr.No.

Character Between Groups Within Groups

1. Plant height 1151.525** 35.955*2. Leaf length 20.031* 0.3223. Carbohydrate content 5.555 0.1654. Protein content 4.946 0.1725. Phenol content 12.115* 0.4836. Saponins content 159.390** 10.944*7. Wedelolactone content 0.730 0.031

* Significant at 5% level of significance** Significant at 1% level of significance

5.6.3 Phytochemical variability vs meiotic behaviour

The different accessions of Eclipta alba have been analyzed for meiotic

behavior. Most of the accessions have normal meiotic behavior (85.29%) and only a

few accessions i.e. 14.70% accessions showed abnormal meiotic behaviour. Attempt

has been made to find out if there exists any correlation between cytological

abnormalities with the phytochemical variability. The mean performance of various

types of Eclipta accessions with and without meiotic abnormalities has been given in

Table 5.10. All types of accessions with normal meiotic behavior uniformly showed

the higher quantity of carbohydrates, proteins, phenol, saponins and wedelolactone

content than those possessing meiotic abnormalities.

Table 5.10: Mean performance of Eclipta alba accessions with and withoutmeiotic abnormalities.

Meiotic behaviourMean values

Normal AbnormalCarbohydrate (mg/g) 17.58±0.64 16.69±0.49Protein (mg/g) 13.81±0.61 13.00±0.654Phenol (mg/g) 19.01±1.06 17.77±0.448Saponins content (mg/g)Wedelolactone content (mg/g)

40.59±4.410.642±0.39

35.26±4.650.219±0.11

Discussion

160

Cell cycle is under the control of many genes. A number of genes have been

identified for completion of meiotic cell cycle in plants and animals (Ohta et al.,

1997; Spellman et al., 1998; Doyle et al., 1999). Any disturbance in these genes and

their control system may result in meiotic abnormalities (Kaul and Murthy, 1985;

Caetana-Pereira and Pagliarini, 2001).

The reduced amount of carbohydrate, proteins, phenols and saponins in the

presently studied Eclipta alba plants with abnormal meiosis is an indication that these

plants possess aberrant metabolism as well. The presence of normal meiosis helps

these plants in reproduction and seed setting. The present analysis clearly revealed

that the plants with normal meiotic behavior should be used for medicinal and

commercial purpose.

5.7 BIOACTIVITY OF EXTRACTS

The leaf extracts in three different solvents viz. acetone, methanol and water

were evaluated for antimicrobial activity against five strains of test organisms

obtained from MTCC collection. The results obtained showed that all kind of leaf

extracts of E. alba exhibit bactericidal effects on the test microorganisms. The results

are presented in Fig. XII. Among the three morphotypes, the leaf extracts of erect type

of plants showed maximum antimicrobial activity against all the bacteria except B.

subtilis where the extracts of prostrate type showed maximum activity. The semierect

type of plants also maintained the intermediate position among the three morphotypes.

The most susceptible bacteria was S. aureus followed by E. coli while the most

resistant bacteria were P. aeruginosa followed by K. pneumoniae and B. subtilis.

The antibacterial response of three types of extracts of Eclipta alba showed

good antibiotic activity against both gram positive and gram negative strains except P.

aeruginosa. The reason for the different sensitivity between gram-positive and gram-

negative bacteria could be ascribed to the morphological differences between these

micro organisms, Gram-negative bacteria having an outer phospholipid membrane

carrying the structural lipo-polysaccharide components. This makes the cell wall

Discussion

161

impermeable to lipophilic solutes, while porins constitutes a selective barrier to the

hydrophilic solutes with an exclusion limit of about 600 Da (Nikaido and Vaara,

1985) The Gram-positive bacteria should be more susceptible having only an outer

peptidoglycan layer which is not an effective permeability barrier (Scherer and

Gerhardt, 1971). Phytochemical constituents such as tannins, flavonoids, alkaloids

and several other aromatic compounds are secondary metabolites of plants that serve

as defense mechanisms against predation by many microorganisms, insects and

herbivores (Lutterodt et. al., 1999; Marjorie, 1999). The demonstration of

antibacterial activity of E. alba leaf extracts against both gram positive and gram

negative bacteria may be indicative of the presence of broad spectrum antibiotic

compounds (Srinivasan et. al., 2001). Out of the three solvents used for extraction, the

acetone extracts showed the highest antibiotic activity against the test organisms,

followed by the methanol extracts and water extracts. Different solvents have been

reported to have the capacity to extract different phytoconstituents depending on their

solubility or polarity in the solvent. Acetone extracts in this study might have had

higher solubility for more phytoconstituents, consequently the highest antibacterial

activity.

The biochemical composition of plants is the most common parameter used

for the characterization of plants. The slight variation in biochemical compositions of

the E. alba studied here, however, could be well attributed to genetic rather than

environmental and seasonal factors. Phytochemical screening of the extracts of E.

alba by earlier workers revealed the presence of tannins, coumestans, saponins,

alkaloids, etc. (Dalal et al., 2010; Upadhyay et al., 2001; Wagner et al., 1986; Singh

et al., 2001). Of various phytoconstituents, wedelolactones (coumestan), present E.

alba is believed to be responsible for almost all the antibacterial activities (Dalal et

al., 2009). In addition, other secondary metabolites such as phenolics could be held

partially responsible for some of these biological activities.

Discussion

162

Fig. XII: Histograms showing antibacterial activity of three differentmorphotypes of E. alba against different organisms

Erect

Semierect

Prostrate

Discussion

163

Presently, the high antimicrobial activity observed in the erect type of

morphotypes can be attributed to the presence of higher quantities of wedelolactone

(1.08 mg/g) saponins (42.6 mg/g) and phenols 19.98 mg/g than other morphotypes.

The prostrate type of E. alba plants exhibited minimum antimicrobial activity and had

lower quantities of phytoconstituents (0.374 mg/g, 39.4 mg/g, 18.26mg/g

respectively) too.

5.8 In vitro STUDIES

Plant tissue culture is a fascinating and useful tool which allows the rapid

multiplication of many genetically identical plants using relatively small amounts of

space, supplies and time. In vitro propagation has been proved as a potential

technology for large scale multiplication of medicinal plant species (Lui and Li, 2001;

Wawrosch et al., 2001; Martin, 2002, 2003, Azad et al., 2005; Faisal et al., 2003;

Hassan and Roy, 2005). Therefore, it was important to develop an efficient

micropropagation technique for Eclipta alba to rapidly disseminate superior clones

once they are identified. Micropropagation via shoot culture, often utilized to

maintain clonal fidelity, would be especially appropriate in this respect (Sen and

Sharma, 1991). Micropropagation of E. alba plants was achieved through nodal

culture as well as callus culture. In vitro micropropagation of Eclipta alba from nodal

segment explants was reported earlier by Franca et al., 1995; Borthakar et al., 2000;

Gawde and Partkar, 2004; Dhaka and Kothari, 2005; Baskaran and Jayabalan, 2005;

Hussain and Anis, 2006; Han et al., 2007; Ray and Bhattacharya, 2008. Plantlet

regeneration via callus formation in E. alba is reported by Zafar and Sagar (1999).

5.8.1 Shoot proliferation

Growth regulators especially cytokinins (Kinetin, BA, 2 ip, Zeatin) are one of

the most important factors affecting shoot proliferation (Lane 1979; Stolz 1979;

Bhojwani 1980; Garland and Stoltz, 1981). Studies conducted by different workers

clearly indicated that BA is more effective, reliable and useful cytokinin for shoot

Discussion

164

proliferation in E. alba (Franca et al., 1995; Gawde and Partkar, 2004; Dhaka and

Kothari, 2005; Baskaran and Jayabalan, 2005; Hussain and Anis, 2006; Han et al.,

2007; Ray and Bhattacharya, 2008). However, Borthakur et al. (2000) reported

maximum number of shoots in MS medium supplemented with 0.05 mg/L kinetin.

Presently, nodal explants of Eclipta alba on MS media supplemented with two

concentrations (0.5 mg/L and 1.0mg/L) of BAP alone and BAP with NAA were

cultured for shoot regeneration. A combination of BAP with NAA was not found

satisfactory for shoot multiplication (Table 4.15). This is in contrast to earlier work by

Hassan et al. (2008) who reported 94% of explants forming 18 shoots on the same

medium. In the present study the highest frequency of shoot production was recorded

in MS medium supplemented with 1.0 mg/L BA which confirms the earlier studies by

Franca et al. (1995), Gawde and Paratkar (2004) and Dhaka and Kothari (2005).

5.8.2 Regeneration from Callus

Zafar and Sagar (1999) are probably the first to report of plantlet regeneration

via callus formation in E. alba. They raised plants from totipotent callus of leaf, stem

and root cultured on MS medium supplemented with various growth regulators and

found 2,4-D (4.52µM); 2,4-D (4.52µM ) + 6-BA (4.43); 2,4-D (4.52µM ) + 6-BA

(4.43) + Kin (4.64µM) + IAA (5.7µM) and 2,4-D (22.62µM) to be the most effective

for callus formation.

In the present study, two media were tried i.e. 2,4-D (1.0 mg/L) and 2,4-D (1.0

mg/L) + 6-BA (1.0 mg/L) and callus response was observed in both media as reported

by Zafar and Sagar (1999). The media which showed regeneration from callus are 6-

BA (2.0 mg/L) + IAA (0.5 mg/L), 6-BA (2.0 mg/L) + NAA (0.5 mg/L), Kin (2.0

mg/L) + NAA (0.5 mg/L) and Kin (1.0 mg/L) + IAA (0.2 mg/L). These combinations

of cytokinin and auxin concentrations have not been reported in earlier investigations

in E. alba.

Discussion

165

5.8.3 Rooting

Rooting of microshoots has been reported to be controlled by various factors

like growth regulators in the medium (Bhojwani and Razdan, 1992; Feng et al., 2000;

Abrie and Staden, 2001), basal salt composition (Skirvin and Chu, 1979; Zimmerman

and Broome, 1981; Garland and Stoltz, 1981), genotype (Rines and McCoy, 1981) as

well as cultural conditions (Murashige, 1977). Auxins were found to stimulate root

growth (Thimann and Went, 1934). Use of auxins singly or in combination for rooting

was reported by different authors (Sahoo and Chand, 1998; Ajithkumar and Seeni,

1998; Rai, 2002, Baskaran and Jayabalan, 2005; Sivakumar and Krishnamurthy,

2000; Hassan and Roy, 2005; Rahman et al., 2006; Baksha et al., 2007).

According to Dhaka and Kothari (2005) rooting was best achieved on MS

medium supplemented with 1.0 mg/L IBA. Baskaran and Jayabalan (2005) observed

that rooting was highest (94.3%) on full strength MS medium containing 9.8 μM IBA.

Hussain and Anis (2006) demonstrated most efficient rooting on half strength MS

medium augmented with 0.5 µM IBA. Hassan et al. (2008) reported in vitro raised

shoots rooted on half strength MS medium with 0.5 mg/L IBA +0.1 mg/L NAA.

In the present study also IBA has been proved to be more effective among all

other auxins. Maximum root length was achieved in half strength MS medium with

1.0 mg/L IBA which supports earlier investigations by Baskaran and Jayabalan

(2005). However Hussain and Anis (2006) reported maximum root length using 0.5

IBA. This difference may be due to the difference in the genotype of the plant used. In

Eclipta alba, Franca et al. (1995) achieved multiple rooting using MS medium

supplemented with 2.4 µM 2-isopentyladenine (2iP). Gawde and Paratkar (2004)

observed best response of rooting in the low amount of BA (0.44 µM). Cent per cent

shoots developed roots directly from shoot base when transferred to growth regulator-

free MS medium (Ray and Bhattacharya, 2008).

Discussion

166

5.8.4 Hardening

For tissue culture generated plants hardening is the most crucial step. The

tissue culture plants are very soft to face ambient environmental conditions (Bhojwani

and Razdan, 1992). In the present study, rooted plantlets were transferred from culture

bottles to plastic pots containing garden soil: farmyard manure (1:1). Borthakur et al.

(2000) transferred the plantlets to pots containing 1:1 mixture of autoclaved sand and

soil. Earlier Gawde and Paratkar (2004) tried a variety of combinations of the

hardening media like soil, sand: soil (1:1), soil: vermicompost (1:1) and knops

hydroponic solutions and achieved 4-5%, 2%, 95% and 100% survival, respectively.

Baskaran and Jayabalan (2005) suggested a mixture of garden soil, farmyard manure

and sand (2:1:1) as hardening media for E. alba plantlets.

Priming of culture regenerated propagules has been recommended for

obtaining better acclimatized plants (Nowak and Shulaev, 2003; Hazarika, 2003). For

priming E. alba microshoots 6.3 µM of chlorocholine chloride (CCCl) was found

most effective (Ray and Bhattacharya, 2008). The authors observed increased number

of roots, elevation of chlorophyll level in leaves and increase in plant biomass in 30

day old treated shoots of E. alba. The primed micropropagated plants were healthy

with 100% survival in soil as compared to 84% survival in non treated plants.

The technique described here appears to be readily adaptable for large scale

clonal propagation and plantation for sustainable use in the industry. Moreover, by

standardizing the protocols for clonal propagation of selected elite plants, it is

possible to achieve a tenfold increase in the products per unit area of cultivation

(Hassan and Roy, 2005).

5.8.5 Somaclonal Variations

The generation of phenotypic variation (quantitative or qualitative) during in

vitro micropropagation has been described as somaclonal variations. These variations

Discussion

167

are most likely to arise as a result of chromosome structural changes (e.g. small

deletions or duplications), gene mutation (both dominant and recessive have been

recovered as the latter being more common), gene amplification, small chromosome

re-arrangement, transposable elements (these may be induced to transpose due to

stress during in vitro culture) and mitotic crossing over during culture induction.

Chromosomal aberrations like laggards, bridges in meiosis I and meiosis II,

micronuclei and change in ploidy level have been observed in tissue regenerants

(Larkin and Scowcroft, 1981; Gupta, 1998; Pontaroli and Camadro, 2005).

Somaclonal variation can be assessed by analysis of phenotype, chromosome number

and structure, proteins, or direct DNA evaluation of plants (De Klerk, 1990).

Micropropagated plants have exhibited various morphological and

biochemical variations due to mutations believed as somaclonal variations (Larkin

and Scowcroft, 1981). Such variations are the case of changes in morphological

features like plant height and leaf size among micropropagated plants (Ravindran et

al., 2004).

In the present study, attempt has been made to induce genetic variability

among three morphotypes of Eclipta alba. The present results exhibit the occurrence

of variation in morphological and biochemical characteristics when we compare the

mother plant and their tissue culture generated plants. All the accessions showed

increased leaf width, carbohydrate and protein contents. The extent of variation

however, differs from accession to accession. This could be attributed to the

differences in genotypes of various accessions. The clonal plants were shorter in size

with decreased branches as compared to their mother plants (control). However, there

was marginal increase in leaf breadth and quantity of basic constituents carbohydrates

and proteins. Somaclonal variations observed in the present investigations may be

because of hormonal effects as suggested by Yipeng et al. (2005) and Ducos et al.

(2003).

Discussion

168

5.9 CULTIVATION TRIAL

Cultivation of medicinal plants has become almost essential due to unplanned

collection of herbs from wild sources. There is a need for a scientific approach for

propagation of medicinal plants and to collect relevant information regarding agro

technology, genuine planting material, economics of field cultivation, high yielding

varieties, etc. One has to explore wild medicinal plant species and to bring them under

cultivation. There is enormous potentiality for commercial cultivation of useful herbs

and production of safe green medicines by establishing plant based industries tract for

the socio-economic development and conservation of genetic diversity. There is need

to standardize cultivation practices to ensure the sustainable supply of standard plant

material, and to reduce the load on wild forms, there is need to mark out better

chemotypes and to standardize their cultivation practices.

The demand of Indian medicinal plants has increased over the years in the

international market. In current years, E. alba has been harvested in huge quantities

from the wild to meet the pharmaceutical demand. For this purpose raw material from

cultivation is always preferred to the collection from wild, however, systematic

cultivation of Eclipta alba plant is not adopted till date. Though Eclipta alba has

acquired great commercial importance for its medicinal and cosmetic preparations but

information is scarce about agronomic management of this crop.

In the present study, Eclipta shows good response treatment to NPK as

compared to NPK + FYM, FYM treatment and control plants. Carbohydrates and

protein content was highest in the plants treated with NPK (5 g/m2) followed by NPK

(2.5 g/m2) and NPK (5 g/m2) + FYM (2 kg/m2). Combination of NPK and FYM

showed modest productivity. In the absence of FYM there were large interactions

between each of the fertilizer nutrients, but with FYM the interactions were small.

Discussion

169

FYM increased the response to N applied alone, but decreased the N response where

basal P and K fertilizers were also applied (Boyd, 1961).

Effects of fertilizers on production of medicinal plants has been studied in

Rheum habarbarum and Beta vulgaris (Ombidi and Saigusa, 2000), Echinacea

purpurea (El- Gengaihi et al., 1998), Echinacea angustifolia (Berti et al., 2002),

Tanacetum parthenium (Default et al., 2003), etc. Increased amount of active

principle with application of fertilizers has been reported in Datura spp. (Esendal et

al., 2000), Echinacea angustifolia (Berti et al., 2002) Atropa belladonna (Baricevic et

al., 2002) and Artemesia spp. (Usha and Swamy, 2002). The data were analyzed

following ANOVA technique. The increase in plant height was significantly affected

by fertilizer treatment (Table 5.11).

Discussion

170

Table 5.11: Analysis of variance for the various qualitative and quantitativecharacters

S.No

Characters d.f M.S. F - value CD (%)

1 Plant HeightRTE

2816

39.119790109.009107.7246080

5.0614.11

2.776234.80857

2 Nodes/plantRTE

2816

3.59251603.62035102.8842690

11.251.26

NSNS

3 Branches/plantRTE

2816

0.7036404014.0648003.0787120

0.234.57

NS3.03572

4 Leaf lengthRTE

2816

14.54778019.04416015.962360

0.911.19

NSNS

5 Leaf breadthRTE

2816

0.23333020E-010.48333330E-010.64583760E-01

0.360.75

NSNS

6 Inflorescence sizeRTE

2816

0.333328200.749998700.50000060

0.671.50

NSNS

7 CarbohydrateRTE

2816

0.17361110E-0239.7320100.1940190E-02

0.8920478.34

NS0.762080E-01

8 Protein contentRTE

2816

0.9622400E-0117.2125600.26031810E-01

1.14661.21

NS0.279145