review of literature -...

What we learn about is not nature itself, but nature exposed to our

methods of questioning.

— Werner Heisenberg

Chapter 2

REVIEW OF LITERATURE

Cytogenetical studies in Delphinium malabaricum (Huth) Munz. : A potential ornamental plant 0

Laboratory of Cytogenetics and Plant Breeding

Chapter 2: Review of Literature

2. REVIEW OF LITERATURE

2.1. THE GENUS DELPHINIUM

The genus Delphinium of the family Ranunculaceae is an important genetic

resource for cut flower cultivars, comprising some 370 species. They are

concentrated in the northern hemisphere temperate zone, with a few scattered in the

high altitudes of Africa (Wilde, 1931). Delphinium includes some horticulturally

important species such as D. elatum L., D. cheilanthum Fisch., D. formosum Boiss.

and Heut. and their interspecific hybrids (Anon, 1949). This genus shows an

interesting diversity in shapes and colors of the flowers, as well as different growth

habits. They have handsome irregular flowers, resembling somewhat the fanciful

figures of the dolphin or the spurs of larks and are commonly known as larkspurs.

The blossoms of the delphiniums are very showy and in some sorts they are even

extremely rich and magnificent.

The genus Delphinium has a bipolar diversity pattern in the Mediterranean

area, with both western and eastern regions rich in endemic species or species

groups (Blanche´, 1991). The Himalayan region in India is home to several

Delphinium species that are adapted from subtropical to temperate climatic

conditions (Polunin and Stainton, 1984 and Chowdhery and Wadhwa, 1984).

Although the genus comprises 370 species, no more than 3 species are commonly

grown in floriculture, namely D. ajacis, D. elatum and D. nudicaule. Besides these

two hybrid species, viz. D. belladonna and D. ruysii are of some importance. By far

the most important however is D. elatum, from which some years ago a list of 4,000

named cultivars, from 211 different breeders and growers was published (Anon,

1949) and this number is increasing every year. This large number of cultivars may

give the idea that there is a wide variation in D. elatum, the hardy perennial, which

has been used so many years as a garden plant. Indeed there is, but although there

are all kinds of color shades and tints and color combinations of the calyx and the

corolla (the so-called bee) no other hues than white, blue and violet are present.

Yellow, orange and red are missing. Yet these hues are present in the genus. D. zalil

from Pakistan and Persia is of a brilliant Sulphur- yellow, D. nudicaule (California)

has more or less orange and D. cardinale (California) scarlet florets. During the last

Cytogenetical studies in Delphinium malabaricum (Huth) Munz. : A potential ornamental plant Page 21



half century investigators and breeders set themselves to introduce the missing hues

into the Delphinium elatum assortment by crossing the above species as well as

others with latter one. Since these results were very poor until 1953, soon after

Legro (1961) started to study the hybridization possibilities in the genus Delphinium

at the Wageningen Horticultural laboratory and succeeded in producing red

flowering Delphiniums by interspecific hybridization of red flowering D. nudicaule

and D. cardinale with D. elatum.

Humans have bred plants with an eye to aesthetics for centuries; flowers are

selected for colorful blossoms or luxuriant foliage. Aesthetic appeal may have

played a role in the domestication of plants, but the rise of pure ornamentals, that is,

plants cultivated only for their aesthetic characterstics, is a much later development.

Despite the relatively late emergence of cultivated ornamental plants in human

history, flowering plants have become very important for displaying and in

commerce. How and why wild ornamental species have taken into cultivation

describes the history of product development in floriculture.

Selection of domesticated crops over the millennia has affected their genetic

makeup. Harlan (1992) suggests that automatic selection, the unintentional

improvement of plants, was routinely practiced during early domestication of crop

plants. For example, seed dormancy and non-synchronous flowering are associated

with wild plants and rapid germination and synchronous flowering with

domestication. Automatic selection continues to improve the germination of

ornamental species in cultivation, as cultivation genetically modifies the

morphology, as well as the physiology of flowering plants (Stebbins, 1974).

Systematic selection, the conscious practice of domesticating wild plants and

improving specific traits, began only 200 years ago in a few technically advanced

countries (Hancock, 1992).

Selection breeding and propagation of native herbaceous perennials or other

perennials discovered in botanical gardens are required to benefit commercially

from the potential diversity and ornamental qualities of these plants. New Guinea

Impatiens, for example, was collected in 1970 in the wild (Mikkelsen, 1987). They

have become a major potted crop only after extensive breeding and propagation.




Clerodendrum ugandense was located in a botanical garden and considered as a

flowering potted plant. In addition to its propagation requirements, growth

regulation and postproduction performance have been investigated which led to its

introduction as a commercial product (Andersen et al., 1993).

2.2. SEED GERMINATION STUDIES

Over the last three decades, a number of works have dealt with general

aspects of the environmental control of germination and dormancy (Koller, 1972;

Roberts, 1972; Heydecker, 1973; Bewley and Black, 1994; Mayer and Poljakoff-

Mayber, 1989 and Kigel and Galili, 1995). In addition there have been several

contributions that have specifically addressed ecological aspects of germination

behaviour (Harper, 1977; Angevine and Chabot, 1979; Grime, 1979; Thompson,

1981 and Fenner, 1992) and recently Baskin and Baskin (1998) have assembled the

most comprehensive review of seed germination and dormancy covering over 3500

wild plant species. Other more focused reviews have also been published during this

time. For example, Mott and Groves (1981) gave an account of germination

strategies in Australian ecosystems; Egley and Duke (1985) concentrated on

germination and dormancy in agricultural weeds; Gutterman (1993) described seed

germination in desert plants; and Simpson (1990) has produced a comprehensive

account of germination and dormancy within a single plant family – the grasses.

While the importance of temperature is recognized in all of these publications, in the

last 40 years only a few reviews have dealt exclusively with the role of temperature

(Hegarty, 1973; Thompson, 1974; Simon, 1979; Roberts 1988 and Probert 1992).

Angevine and Chabot (1979) pointed out that, when a seed germinates under natural

conditions, the individual has, in a sense, „bet its life‟ on the favorability of

environmental conditions for seedling establishment. Consequently, selection favors

environmental cueing mechanisms that decrease the probability of encountering

unacceptable growth conditions following germination. Angevine and Chabot

(1979) recognized a number of so-called germination syndromes, according to the

physical and biotic stresses of the environment that influence seedling establishment.

Seed responses to temperature play a pivotal role in several of these germination

syndromes and it is therefore arguably the most important environmental variable




responsible for the synchronization of germination with conditions suitable for

seedling establishment.

Additional evidence about factors controlling seed germination derives from

studies of taxonomically related species. According to recent studies, it is reasonable

to expect that, within a family or a genus, seed germination could be affected by

phylogenetic constraints and developmental allometries that limit segregation

(Baskin et al., 1993; Baskin and Baskin, 2004; Figueroa and Armesto, 2001;

Nikolaeva, 1999 and Smith-Ramı´rez et al., 1998). These examples lend support to

the hypothesis that germination strategies can be stable evolutionary traits, thus

constraining interspecific variation in germination behavior.

2.3. PHENOLOGICAL STUDIES

Flower is an actively metabolizing system and carries out all its metabolic

activities at the expense of stored food in the form of carbohydrates, proteins and

fats (Nowak and Rudnicki, 1990; Singh et al., 2001 and Bhattacharjee and De,

2003). Besides, high level of turgidity and sensitivity towards ethylene contribute to

potential vase life of the flowers (Bhattacharjee, 1999; Singh et al., 2002 and

Srivastava et al., 2005). Cut flowers detiorate very quickly and hence, to maintain

freshness of flowers, they have to be handled with utmost care. While petal

senescence is clearly a degenerative process, the up regulation of new genes and

synthesis of new proteins appear to be necessary for this process (Suttle and Kende,

1980; Borochov and Woodson, 1989 and Arora and Singh, 2004). Senescence is

considered to be internally programmed, because it is specific and orderly in terms

of when, where and how it occurs (Nooden and Leopold, 1978).

Flower provides an excellent system for the study of senescence. Different

flower parts senesce at different rates. In the commercial use of cut flowers, it is

usually the life span of the petals, an ornamental part of the flower which determines

its effective life. Therefore, the study of petals senescence should provide insight

into the methods to improve the post harvest longevity of cut flowers and insight

into the mechanisms involved in the control of plant senescence. For cut flower

industry, one could first distinguish between the two distinct stages in the

physiology of the flower. The first growth would be flower growth and development




of the plant to full opening. The second stage would be of maturation, senescence

and wilting (Kende and Baumgartner, 1974). On the other hand, Elgar et al., (2003)

defines senescence in second stage which is also the final phase in the ontogeny of

the organ, in which a series of normally irreversible events is initiated that it is the

end of flower life (Chooruut and Kanlayanarat, 2002 and Ting et al., 2008). The

most important barrier in the marketing and commercialization of many cut flowers

is their short vase life and their inability to withstand stresses during storage or

transit (Halevy and Mayak, 1981 and Nowak and Rudnicki, 1990). Harvesting

maturity, in the context of commercial maturity is concerned with the timing of

harvest to meet particular market requirements (Wills et al., 1998). The correct stage

of maturity at harvest is one of the key factors to be considered in order to preserve

and prolong the postharvest life of cut foliage (Dole and Wikins, 1999).

2.3.1. ETHYLENE AND POSTHARVEST LIFE

Ornamental flowers have a short vase life after harvest. The most important

parameter responsible for this is their sensitivity to ethylene. Most ornamentals are

non-climacteric, but the climacteric plants produce an ethylene and respiratory peak.

There is a difference in the response to ethylene. Some flowers such as non-

climacteric delphinium are very sensitive to ethylene, while climacteric carnation is

relatively tolerant. Ethylene induces the start of abscission, but the short postharvest

life of cut flowers is limited by carbohydrate reserves and a rapid rate of metabolism

(Wills et al., 1998).

The length of vase life is one of the most important factors for quality of cut

flowers. The vase life varied among various cultivars in carnation (Wu et al., 1991

and Onozaki et al., 2001), Eustoma (Shimizu and Ichimura, 2002) and gerbera

(Wernett et al., 1996). Ethylene is involved in flower senescence in many potted

plants (Woltering, 1987). Delphiniums that are sold not only as cut flowers, but also

as potted plants exhibit a peak of ethylene production before the sepals abscise; the

sepals have a high sensitivity to exogenous ethylene (Ichimura et al., 2000).

Application of silver thiosulfate complex (STS), an inhibitor of ethylene action,

reduced ethylene- induced abscission of flowers and/or flower buds (Dostal et al.,

1991 and Reid et al., 2002). In cut sweet pea flowers, vase life is extended when




sucrose is added to vase water together with STS (Mor et al., 1984). Ichimura and

Hiraya (1999) previously reported that pulse treatment with sucrose alone extended

the vase life of florets of cut sweet peas, accompanied by the inhibition of ethylene

production.

Several methods to increase the vase life of cut flowers and keep their

freshness for longer periods have been reported. Cut flowers should be free of any

deterioration, as this is one of the principal entry points for decay organisms

(Hardenburg, 1968). A major form of deterioration in cut flowers is the blockage of

xylem vessels by air and microorganisms that cause xylem occlusion (Hardenburg,

1968). The vase life of many flowers can be extended by the application of different

chemicals. Ketsa and Narkbua (2001) reported that cut roses held in solutions

containing 5% mM aminooxyacetic acid (AOA) prolonged the vase life of cut roses.

Ketsa et al., (1995) who opined that AgNO3 prevented microbial occlusion of xylem

vessels in Dendrobium, thereby enhancing water uptake and increasing longevity of

flowers. Awad et al., (1986) also attributed the beneficial effect of AgNO3 in the

vase-water to the production of Ag+

ions, which might inhibit the rise of ethylene

precursor, thereby enhancing the longevity of cut flowers. The germicide 8-

hydroxyquinoline sulfate (8-HQS) is one of the very important preservatives used in

floral industry (Nowak and Rudnicki, 1990). Treatment of carnation flowers with

sucrose in combination with 8-hydroxyquinoline sulphate (HQS) or HQS alone

extends the vase life of cut flowers (Ichimura et al., 1999). Sucrose is widely used in

floral preservatives, which acts as a food source or respiratory substrate and delays

the degradation of proteins and improves the water balance of cut flowers. Steinitz

(1982) opined that addition of sucrose to the solution increased the mechanical

rigidity of the stem by inducing cell wall thickening and lignification of vascular

tissues. Sucrose antagonizes the effect of ABA, which promotes senescence (Halevy

and Mayak, 1979).

2.4. SCENARIO OF CYTOLOGICAL STUDIES

Chromosomal studies dealing with somatic chromosome number

determination and its ploidy level are of fundamental consideration for any given

species in understanding the basic structure of the genetic complement. It has also




been known that chromosomal or cytological studies help in determining the

path of evolution of new species. Over the past two decades, cytogenetic studies

progressed through the information generated by classical methods, allowing to

establish the first cytogenetic models in species such as tomato, wheat and rice. At

the end of the last century cytogenetic studies showed a significant improvement by

implementing new techniques for the analysis of chromosomes, somatic and

meiotic, including molecular cytogenetic techniques (Guerra, 2008). The karyotype

is the final result of many forces that act in the genome at structural, organizational

and functional levels. The use of karyological data in taxonomy, traditionally

referred to as cytotaxonomy or karyosystematics, contributes to evaluate the genetic

relationships among species or populations and to a better understanding of the way

they diverged from each other (Guerra, 2008).

The field of plant cytogenetics was heavily influenced by Barbara

McClintock‟s pioneering work on maize (Zea mays). Her method for unequivocal

identification of individual chromosomes permitted major discoveries regarding the

structure and dynamic behavior of the maize genome (Creighton and McClintock,

1931 and McClintock, 1929, 1932, 1938, 1941 and 1984). Using carmine-based

chromatin staining procedures, McClintock showed that all of the individual

chromosomes could be uniquely identified from a single meiotic nucleus with a

combination of two metrics, the relative lengths and arm ratios of the chromosomes

(McClintock, 1929). This approach proved useful for cytogenetic map development

in other plant species, including rice (Oryza sativa) (Misra and Shastry, 1967),

sorghum (Sorghum propinquum) (Magoon and Shambulinguppa, 1961) and tomato

(Lycopersicon esculentum) (Ramanna and Parkken, 1967).

In literature, studies on cytogenetics, chromosome structure, behaviour

and manipulation in plants are well documented (Karpenchenko, 1925; Sarbhoy,

1977a; Okoli and Olorode, 1983 and Obute, 2001). The usefulness of information

from such studies in the understanding of phylogenetic relationships, genetic

mapping and breeding studies has been very significant (Hartwell et al., 2000 and

Kurata et al., 2002). Chromosome research has made extensive contributions

particularly in the elucidation of the systematic relationships of many closely related

species (Bocher et al., 1955; Rahn, 1957; Cartier, 1973; Zemskova, 1977 and Roy,




1988) karyomorphology of various plants (Geitler and Ischermak-woess, 1962;

Govindarajan and Subramanian, 1983 and Roy, 1988) and cytology of species of

different phytogeographic regions (Gregor, 1939; Runermark, 1969; Briggs, 1973

and Pramanik and Raychaudhuri, 1997).

2.4.1. CYTOLOGICAL STATUS OF DELPHINIUM

Available literature on the basic and applied chromosome features of

Delphinium species showed that the genus Delphinium is cytogenetically studied in

terms of chromosome numbers, karyotypes and meiotic behavior. The chromosome

complement of Delphinium has been studied by a number of workers (Tjebbes,

1927; Tischler, 1927; Langlet, 1927, 1932; Beckman, 1928; Lewitzky, 1931;

Lawrence, 1936; Propach, 1939, 1940; Gregory, 1941; Mehlquist et al., 1943,

Lewis, 1947; Lewis et al., 1951 and Hocquette, 1992). The studied species of the

genus Delphinium reported the presence of 2n=16 (diploid), 2n=24 (triploid), 2n=32

(tetraploid) and 2n=48 (hexaploid) levels for the genus (Langlet, 1927; Lewitzky,

1931; Lawrence, 1936; Gregory, 1941; Lewis, et al., 1951; Mehra and Ramanandan,

1972; Al- Kelidar and Richards, 1981; Love, 1981, 1984 and Subramanian, 1985).

In the genus Delphinium there is clearly a polyploidy series with a basic

chromosome number x=8 (Darlington and Janaki-Ammal, 1945). Numbers of

chromosomes published so far in Delphinium are listed in the Table 2.1. In

Delphinium species diploid species are numerous, tetraploids are occasional, and

hexploids are known only in cultivated forms.

Table 2.1 : Previous reports of somatic chromosome number in Delphinium species.

Taxon Chromosome number Author and Year

n 2n

D. ajacis L. 8 16 Langlet, 1927; Gregory,

1941

D. alabamicum 8 16 Warnock, 1995

D. altissimum Wallich. 8 16 Mehra and Kaur, 1963

Continued…………




D .andersoni Gray 8 16 Lewis et al., 1951

D. belladonna 12, 24 24, 48 Langlet, 1927 and

Subramanian, 1985

D. brunonianum Royle. 8, 16 16, 32 Lewitzky, 1931, Al-Kelidar

and Richards, 1981

D. californicum T.& G. 8 16 Lewis et al., 1951

D. candelabrum Ostenf.

Var.monanthum

8 16 Yang and Wu, 1993

D. cardeopetallum DC. 8 16 Lewitzky, 1931

D. cardinale Hook. 8 16 Mehlquist et al., 1943

D. carolinianum Walt 16 32 Gregory, 1941

D. cashmerianum Royle. 8, 16 16,32 Love, 1981 and Al-Kelidar

and Richards, 1981

D. decorum Fisch. and

Mey.ssp.Tracyi Ewan

8 16 Lewis et al., 1951

D. denudatum Wallich. 8, 10, 16 16,20,32 Mehra and Ramanandan,

1972, Sarkar et al., 1982,

Al-Kelidar and Richards,

1981

D. elatum L. 16 32 Lawrence, 1936

D. formosum Boiss.& Huet 16 32 Gregory, 1941

D. glaucum Wats. 8 16 Lewis et al., 1951

D. gracilentum Greene 8 16 Lewis et al., 1951

D. grandiflorum L. 8 16 Propach, 1940

D. gypsophilum Ewan 8, 16 16,32 Lewis et al., 1951

D. hanseni Greene 8, 16 16, 32 Lewis et al., 1951

D. hesperium Gray 8 16 Lewis et al., 1951

D. hesperium var.

cuyamacae (Abrams) Jeps.

8 16 Lewis et al., 1951





D. inopinum (Jepson)

Lewis and Epling

8 16 Lewis et al., 1951

D. lamartinii (Hybrid) 24 48 Lawrence, 1936

D. malabaricum (Huth)

Munz.

8 16 Pai et al., 2007

D. nudicaule T.& G. 8 16 Gregory, 1941

D. nuttallianum Pritz. 8 16 Lewis et al., 1951

D. occidentale 8 16 Ward and Spellenberg,

1982

D. parryi Gray 8 16 Lewis et al., 1951

D. parryi var.blochmanae

(Greene) Jeps.

8 16 Lewis et al., 1951

D. parishii Gray 8 16 Lewis et al., 1951

D. patens Benth. 8 16 Lewis et al., 1951

D. polycladon Eastw. 8 16 Lewis et al., 1951

D. purpusii Brandg. 8 16 Lewis et al., 1951

D. recurvatum Greene 8 16 Lewis et al., 1951

D. scabriflorum D. 8 16 Love, 1984

D. staphisagria L. 8 16 Hocquette, 1922; Langlet,

1927; Lewitzky, 1931;

Gregory, 1941

D. tricorne Michx. 8 16 Gregory, 1941

D. trolliifolium Gray 8 16 Lewis et al., 1951

D. uliginosum Curran 8 16 Lewis et al., 1951

D. umbraculorum Lewis

and Epling

8 16 Lewis et al., 1951

D. variegatum T. and G. 8, 16 16, 32 Lewis et al., 1951

D. vestitum Wallich. 8 16 Mehra and Kaur, 1963

D. zalil Aitch. and Hemsl. 8 16 Gage, 1953




Meiotic studies of Delphinium have been largely confined to the garden

forms, the major exception being the work reported by Lewis et al., (1951) on the

California species, where both diploids and tetraploids were analyzed. For the

diploid species, metaphase bivalents fall into several easily recognized types, since

terminalization regularly occurs and chiasma frequency for a given pair shows little

variation. The large chromosomes have usually two chiasmata at metaphase (there

may be three or rarely four at diplotene and diakinesis) and others have regularly

one chiasma in the longer arm (Mehlquist et al., 1943 and Lewis et al., 1951).

2.5. PHYTOCHEMICAL SCREENING OF PLANTS

Phytochemicals are chemical compounds formed during the plants normal

metabolic processes. Plant produces these chemicals to protect itself but recent

research demonstrates that many phytochemicals can protect humans against

diseases. These chemicals are often referred to as “secondary metabolities” of which

there are several classes including alkaloids, flavonoids, coumarins, glycosides,

gums, polysaccharides, phenols, tannins, terpenes and terpenoids (Harborne, 1973

and Okwu, 2004). Plants produce near about 50,000 types of secondary metabolites,

in which 12000 types of alkaloids, 60 types of cynogenic glycosides, 15000 types of

terpenes, 4000 types of flavonoids and 100 types of glucosinolates have already

identified (Croteau et al., 2000). These phytochemicals have been used as drugs for

millennia. For example, Hippocrates may have prescribed willow tree leaves to

abate fever. Salicin, having anti-inflammatory and pain-relieving properties, was

originally extracted from the bark of the white willow tree and later synthetically

produced became the staple over-the-counter drug called Aspirin. An important

cancer drug, Taxol (paclitaxel), is a phytochemical initially extracted and purified

from the Pacific yew tree.

Phytochemicals are produced by specific biochemical pathways, which occur

inside the plant cells. The phytochemicals can range from medicinally useful agents

to deadly poisons. The pharmacological and other beneficial effects of

antinutritional factors in plants have been reviewed by Soetan (2008). The presence

of these secondary metabolites in plants probably explains the various uses of plants

for traditional medicine.




2.5.1. ALKALOIDS

Alkaloids are naturally occurring organic substances, predominantly found in

plant sources including marine algae and rarely in animals (e.g. in the toxic

secretions of fire ants, ladybugs and toads). They occur mostly in seed-bearing

plants mainly in berries, bark, fruits, roots and leaves. These are basic in nature and

so referred the term alkaloid (alkali-like). Alkaloids rank among the most efficient

and therapeutically significant plant substances (Okwu, 2005). Some 5,500 alkaloids

are known and they comprise the largest single class of secondary plant substances,

which contain one or more Nitrogen atoms, usually in combination as part of a

cyclic structure (Harborne, 1973). They exhibit marked physiological activity when

administered to animals (Okwu and Okwu, 2004). Furthermore, alkaloids are often

toxic to man and many have dramatic physiological activities, hence their wide use

in medicine for the development of drugs (Harborne, 1973 and Okwu, 2005). Many

drugs used by man for both medical and non medical purposes are produced in

nature in the form of alkaloids e.g. atropine, strychnine, caffeine, nicotine,

morphine, codeine, cocaine etc. Naturally occurring receptors for many alkaloids

have also been identified in human and other animals, suggesting an evolutionary

role for the alkaloids in physiological processes.

Alkaloids have traditionally been of great interest to humans because of their

pronounced physiological and medicinal properties. From the beginning of

civilization, alkaloid-containing plant extracts have been used in all cultures as

medicines and poisons. The physiological effects of alkaloids have made them

important compounds in medicine. They are used as a remedy for painkillers,

stimulants, muscle relaxants, tranquilizers, anaesthetics, antimalarial, antimicrobial,

antidiabetic, anticancerous, anti-HIV, antioxidants etc.

Proaporphines and crotsparine isolated from Cocculus sparciflorus showed

significant hypotensive and anticancer activity (Bhakuni et al., 1969).

Homoerythrine derived alkaloids isolated from stem of Galipea bracteata (Viera

and Kubo, 1990) showed molluscicidal activity. In modern times, the stimulants

caffeine in coffee, tea and cacao and nicotine in cigarettes are consumed worldwide.

Alkaloids with hallucinogenic, narcotic or analgesic properties have found

applications in medicine e.g. morphine, atropine and quinine.




The first medicinally useful example of an alkaloid was morphine, isolated in

1805 from the Opium poppy Papaver somniferum (Fessenden and Fessenden, 1982).

After that more than ten thousand alkaloids have been discovered from different

sources (Evans, 2006). Alkaloids are commonly found in the orders Centrospermae,

Magnoliales, Ranunculales, Papaverales, Rosales, Rutales, Gentiales, Tubiflorae

and Campanulales. Diterpenoid alkaloids, commonly isolated from the plants of the

Ranunculaceae (Jones and Luchsinger, 1986 and Atta-ur-Rahman and Choudhary,

1995) are commonly found to have antimicrobial properties (Omulokoli and

Chhabra, 1997). Solamargine, a glycoalkaloid from the berries of Solanum

khasianum and other alkaloids may be useful against HIV infection (McMohan et

al., 1995 and Sethi, 1979) as well as intestinal infections associated with AIDS

(McDevitt et al., 1996). While alkaloids have been found to have microbial effects

including against Giardia and Entamoeba species (Ghoshal et al., 1996), the major

antidiarrheal effect is probably due to their effects on transit time in the small

intestine. Berberine is an important representative of the alkaloid group. It is

potentially effective against trypanosomes (Freiburghaus et al., 1996) and plasmodia

(Omulokoli et al., 1997). The mechanism of action of highly aromatic planar

quaternary alkaloids such as berberine and harmane (Hopp et al., 1976) is attributed

to their ability to intercalate with DNA (Phillipson and O‟ Neill, 1987).

DELPHINIUM ALKALOIDS

Plant species of the genera Aconitum, Delphinium and Consolida

(Ranunculaceae) are known sources of C19-norditerpenoid and C20-diterpenoid

alkaloids (NDAs and DAs, respectively) of pharmacological (anti-inflammatory,

analgesic, anti-arrythmia, antifungal and cytotoxicactions) and economic importance

(Atta-ur -Rah-man and Choudhary, 1995). Delphinium plants, long known to be

insecticidal, are a rich source of C-19 norditerpene alkaloids (Jennings et al., 1986;

Kukel and Jennings, 1994; Manners et al., 1995). These alkaloids have been

investigated in invertebrate (Jennings et al., 1986 and Satelle et al., 1989) and

vertebrate (Macallan et al., 1988 and Namby Aiyar et al., 1979) isolated tissue

preparations and found to act as potent nicotinic receptor antagonists. Furthermore,

certain C-19 alkaloids could be candidates for insecticide development due to their




potency and selectivity as ligands of the insect nicotinic receptor (Kukel and

Jennings, 1994).

Alkaloids of Aconitum and Delphinium plant species have been applied in

eastern folk medicine owing to their wide spectrum of therapeutic action including

antiarrhythmic, analgesics, neurological etc. Early pharmacological studies

suggested that some of diterpenoid alkaloids occurring naturally in Aconitum and

Delphinium sp. are curare like activity and therefore, act at neuronal nicotinic

acetylcholine receptors (nAChRs) and exhibit potent N-cholinolytic activity (Benn,

1966). The nAChRs are prototypes for Cys-loop receptor family of pentameric

ligand-gated ion channels. They are considered as an important drug targets since

neuronal nAChRs are involved in high brain function and neurodegenerative

pathologies (Hogg et al., 2003). In particular, dysfunction of human heteromeric

α4β2 and homomeric α7nAChRs (most abundant subtypes in the central nervous

system, CNS) have been associated with a number of human diseases such as

schizophrenia, Alzheimer‟s and Parkinson‟s diseases, epilepsy, anxiety and

depression. At present, several nicotinic receptor ligands are being clinically

investigated (Arneric et al., 2007 and Cassels et al., 2005).

Traditionally, plants from the genus Delphinium have been used as poisons

and insecticides and for medicinal purposes. These activities are linked to the

alkaloids present in these plants (Aiyar et al., 1979). The work so far done on the

alkaloids of Delphinium species has been presented in Table 2.2.

Table 2.2 : Various alkaloids in different Delphinium species.

Species Alkaloids Reference

D. ajacis L. Ajadelphine, ajadelphinine, delcosine,

delsoline, deltaline, gigactonine,

methoxygadesine, delphisine.

Pelletier et al.,

1992

D. barbeyi Huth. Methyllycaconitine, Barbine Aniszewski, 2007

D. cardinale Hook. Brownine, dehydrobrownine, hetisine,

dehydrohetisine

Benn, 1966





D. cardiopetalum DC Cardiopine, Cardiopinine,

Cardiopimine, Cardiopidine, Cardiodine

Reina et al., 1996

D. corymbosum

Regel.

Delcorine, Delsonine Aniszewski, 2007

D. denudatum Wall. 8- Acetylheterophyllisine Atta-ur-Rahman

et al., 1997

D. elatum L. Elasine, isodelpheline, eladine,

delpheline, deltaline,

methyllycaconitine, nudicauline, 14-

deacetylnudicauline, lycoctonine and

elatine.

Pelletier et al.,

1989

D. fangshanense

W.T.Wang

16-demethyldelsoline,

tetrahydrobenzylisoquinoline, O-

methylroefractine N-oxide,

methyllycaconitine, nudicauline,

delavaine A, delavaine B, and

magnoflorine

Zhang et al., 1999

D. formosum Boiss. &

A. Huet.

Lycoctonine, delsemine Tanker and

Ozden 1975

D. giraldii Diels. Giraldines A, Giraldines B, Giraldines

C, dihydrogadesine, tatseinsine,

siwanine A

Zhou et al., 2003

D. glaucescens Rydb. Lycoctonine, dictyocarpine, brownine,

14-dehydrobrowniine,

methyllycaconitine, delcosine,

dictyocarpinine, deltaline, glaucenine,

glaucerine, glaucephine and glaucedine

Pelletier et al.,

1981

D. grandiflorum L. 7-0-Acetylgrandine,

Delgrandine

Pelletier et al.,

2001

D. linearilobum N.

Busch.

Ajaconine, hetisine, acochlearine,

isotalatizidine, cammaconine,

Suzgec et al.,

2009





winkleridine, deltatsine and

condelphine

D. linearilobum N.

Busch.

Linearilobin, linearilin, lycoctonine, 14-

acetyltalatizamine, browniine,

cammaconine, talatizamine,

cochlearenine.

Kolak et al., 2006

D. occidentale S.

Watson.

Methyllycaconitine, Barbine Aniszewski, 2007

D. pentagynum Lam. 2-dehydrodeacetylheterophylloidine,

14-demethyl-14-

isobutyrylanhweidelphinine, 14-

demethyl-14-acetylanhweidelphinine,

14-deacetylnudicauline,

methyllycaconitine, 14-deacetyl-14-

isobutyrylnudicauline, 14-

acetylbrowniine, browniine, delcosine,

lycoctonine, 18-methoxygadesine,

neoline, karakoline, magnoflorine.

Diaz et al., 2004

D. poltoratzkii Rupr. Ajacine, Anthranoyllycoctonine,

Condelphine, Delphyrine, Delpoline,

Delsonine, Karacoline, lycoaconitine

Aniszewski, 2007

D. staphisagria L. Delphisine, Neoline, Chasmanine,

and Homochasmanine

Pelletier et al.,

1975

D. tiantaishanense W.

J. Zhang et G. H.

Chen.

tiantaishansine, tiantaishannine,

tiantaishanmine, and tiantaishandine.

Li et al., 2007

D. tongolense Franch. Tongolenine C, Tongolenine D. He et al., 1998

D. tricorne Michx. Lycoctonine, delsemine, tricornine Pelletier and

Bhattacharyya,

1977





D. trifoliolatum Finet

& Gagnep.

Trifoliolasines D, trifoliolasines E,

trifoliolasines F.

Zhou et al., 2005

D. umbrosum Hand.-

Mazz.

Delsemine, delsemine A, delavaine A,

delavaine B, giraldine G, ajacine,

methyliycaconitine, lycoctonine,14-

acetyldecosine, delcosine,delectinine,

umbrosumines A, umbrosumines B,

umbrosumines C.

Chen et al., 2010

D. yunnanense

(Franch.) Franch.

methyllycaconitine,

deacetylnudicauline, anhweidelphinine,

postanisine, delelatine, delbonine,

delsemine A, delsemine B, 14-

dehydrodecosine, deltaline, delcosine,

blackmine, brownine, 14-

dehydrobrownine

Chen et al., 2011

2.5.2. ANTHOCYANINS

Anthocyanins, common plant pigments, are part of the very large and

widespread group of water soluble plant constituents collectively known as

flavonoids and are part of the natural beauty of the plant world (Kuhnau, 1976).

Anthocyanins are the secondary metabolites produced by the plants (Tsuda et al.,

2004) and are responsible for most of the red, blue, purple and other intermediate

colors of many plant tissues (Frank et al ., 2002). Anthocyanins are glycosylated

polyhydroxy and polymethoxy derivatives of 2-phenylbenzopyrylium (flavylium)

salts (Wu et al., 2004). Approximately 400 different anthocyanins are found in

nature (Kong et al., 2003); the most common anthocyanidin aglycons in plants are

cyanidin (Cy), delphinidin (Dp), malvidin (Mv), petunidin (Pt) peonidin (Pn) and

pelargonidin (Pg). The chemical structures of these different aglycons vary only in

the position of the R group that is located at position C-3' or C-5' (Table 2.3).

Aglycons have rarely been found in fresh plant materials (Prior, 2004). The

flavylium ion structures of these six anthocyanidins are shown in Fig. 2.1.




Figure 2.1: Commom anthocyanidin structure.

Anthocyanins are the major set of structural pigments that give flowers their

unique colors. They are synthesized in a branch of the flavonoid biosynthesis in six-

step pathway that begins with three molecules of malonyl-CoA and one molecule of

p-coumaroyl-CoA biosynthesis are well characterized and conserved across the

flowering species that control expression of the anthocyanin biosynthetic genes are

well characterized in a number of model systems including petunia, snapdragon and

Table 2.3 : Different anthocyanidin aglycons (Wu et al., 2004).

Anthocyanidin R1 R2 MW Color

Cyanidin OH H 287 Orange red

Delphinidin OH OH 303 Blue

Malvidin OCH3 OCH3 331 Red

Petunidin OCH3 OH 317 Dark red/Purple

Peonidin OCH3 H 301 Purplish red

Pelargonidin H H 271 Orange




Arabidopsis thaliana. Changes in flower colors depend on the degree of acidity as

well as on the type of anthocyanidin pigments in the cells and the structure of their

substituents (Brouillard and Dangles, 1994). Such modifications are known to be

controlled in the flowers by single gene substitutions (Forkmann, 1994). The study

of the genetics of anthocyanin synthesis began in the 19th

century with Mendel‟s

work on flower color in peas. Since that time, there have been periods of intensive

study into the genetics and biochemistry of pigment production in a number of

different species. In the early studies, genetic loci were correlated with easily

observable color changes. After the structures of anthocyanins and other flavonoids

were determined, it was possible to correlate single genes with particular structural

alterations of anthocyanins or with the presence or absence of particular flavonoids.

Mutations in anthocyanin genes have been studied for many years because they are

easily identified and they generally have no deleterious effect on plant growth and

development. In most cases, mutations affecting different steps of the anthocyanin

biosynthesis pathway were isolated and characterized well before their function was

identified or the corresponding gene was isolated. More recently, many genes

involved in the biosynthesis of anthocyanin pigments have been isolated and

characterized using recombinant DNA technologies (Holton and Cornish, 1995).

DELPHINIUM ANTHOCYANINS

The fascination with Delphinium emanates from its magnificent multi-

flowers and gorgeous cyanic color. Delphiniums are widely cultivated in the world

and have a wide range of flower colors from white, yellow, red, violet to blue.

Analysis of their flower pigments was started in 1915 by Willstatter and Meig, who

isolated a pigment, delphinidin, from reddish purple petals of D. consolida, which is

recently classified in the genus Consolida. Thereafter analysis has been carried out

mainly on garden cultivars and four anthocyanins were isolated: violodelphin from

violet flower of D. hybridum cv. „Black Night‟ (Kondo et al., 1990), cyanodelphin

from blue flower of D. hybridum cv.„Blue Springs‟ (Kondo et al., 1991), delphinidin

3-rutinosoid-7-glucoside from white flower of D. hybridum cv.„Snow White‟ and

„Galahad‟ and delphinidin 3-rutinoside from reddish purple flower of D. hybridum

„Astorat‟ (Toki et al., 1994). On the other hand, the red flowered species such as D.




cardinale Hook. and D. nudicaule Torr. and A. Gray were found to possess

pelargonidin anthocyanins (Werckmeister, 1954).

The flower color and specifically the sepal coloration of cyanic Delphinium

cultivars, depends on the pigmentation with acylated and non-acylated anthocyanins

concomitantly produced and their accumulation affecting the appearance. The search

for acylated anthocyanins was begun by Kondo et al., (1990, 1991). Two pigments,

viodelphin and cyanodelphin, were isolated and reported as delphinidin glycosides

esterified by two and four p-hydroxybenzoic acids from the purple and blue

flowered cultivars, respectively. Furthermore, delphinidin 3-O-rutinosyl-7-O-

gluciside (bisdeacyl-platyconin) and delphinidin 3-O-rutinoside (tulipanin) were

identified as the major anthocyanins in the white and pink flowered cultivars,

respectively (Goto et al., 1983; Brandt et al., 1993 and Ishikura and Hayashida,

1980). However, the flower color is expressed by a blend of the respective major

pigments, acylated and/or non-acylated anthocyanins, which results in the various

color attributes (Hashimoto et al., 2000).

In some cyanic Delphiniums, the coloration of the sepals changes with

anthesis. Such a color change has been extensively discussed with Lathyrus

odoratus (Sakata and Uemoto, 1976 and Sakata and Arisumi, 1977), Impatiens

balsamina (Hagen, 1966), Rosa spp. (Arisumi et al., 1976), Antirrhinum majus

(Toki and Uemoto, 1977) and Campanula isophylla (Justesen et al., 1997). Changes

in the flower color during flowering might be correlated with a shift in the

biosynthesis of anthocyanins. In Delphinium, the pigments of the flowers which

comprise anthocyanins (pigmented) and flavonols (colorless) are synthesized

through the flavonoid pathway. The blue and white color of the flower of D. elatum

is associated with delphinidin derivatives and precursors, respectively, but red or

orange flowers of the two wild Delphinium species, D. nudicaule and D. cardinale

contain pelargonidin derivatives. Generally, delphinidin is dominant to pelargonidin

in various species (Beale, 1941). Legro (1961) and Honda et al., (1999) suggested

that delphinidin is dominant to pelargonidin in Delphinium based on their

crossbreeding results. Thus the flower color of the hybrids between red-flowering

wild species and D. elatum was assumed to be blue or purple due to the presence of

delphinidin derivatives.




2.6. MUTATION BREEDING

Ever since the epoch-making discoveries made by Muller (1927) and Stadler

(1928), that ionizingradiations can induce hereditary alterations and thereby enhance

the frequency of mutations many times over the one occurring spontaneously in

nature, the breeder is no longer limited to the availability of natural mutations.

They drew the conclusion that induced mutations were similar to spontaneous

mutations forming the basis for natural selection and evolution. Initial attempts to

induce mutations in plants mostly used X-rays, but soon other types of irradiations

such as gamma-rays (acute and chronic), neutrons (fast and thermal), electrons,

protons, α-rays from radon, β-rays from Phosphorus 32 and Sulphur 35 were used.

Thereafter in 1946, Auerbach and Robson showed that nitrogen mustards produced

mutations in Drosophila. Subsequently, a number of chemicals with mutagenic

action were described. The successful application of induced mutation in plant

breeding was first achieved by Gustafsson in Sweden during the World War I. He

produced the first mutant cultivar “Jutta” in barley by using X-rays during the

1950s. Since then efforts on mutagenesis have yielded many commercial varieties of

crops like rice, barley, castor, french bean and tomato (Swaminathan, 1972). Now

with this advent the genotypes and phenotypes of plants are under human control

and can be changed at any time according to desired niches and needs. This creation

of mutations at will and their utilization for the production of new crop varieties is

known as mutation breeding. In recent years, it has become a common trend among

all the plant breeders, not only in India but all over the world, to use either mutation

breeding or polyploidy breeding as a magic tool for creating new varieties because

by their application entirely new and original characters are produced in a variety as

against hybridization and other methods where merely the already present characters

are combined together into form of a new variety.

Mutation breeding has been widely used for the improvement of plant

characters in various crops. It is a powerful and effective tool in the hands of plant

breeders especially for autogamous crops having narrow genetic base (Micke, 1988).

Many reports are available for the successful use of mutation breeding in the

production of new cultivars in many crops (Micke et al., 1985; Anonymous 1987).

During the past seventy years, more than 2,252 mutant cultivars from 175 plant




species including cereals, oilseeds, pulses, vegetables, fruits, fibers and ornamentals

have been officially released in 50 countries all over the world (Maluszynski et al.,

2000 and Chopra, 2005). Of these, 60% were released from 1985 onwards. Most

mutant varieties were released in China (26.8%), India (11.5%), USSR and Russia

(9.3%), the Netherlands (7.8%), USA (5.7%) and Japan (5.3%). Many induced

mutants were released directly as new varieties; others were used as parents to

derive new varieties. For example, of the 2,252 varieties, 1,585 (70%) were released

as direct mutants, i.e. from direct multiplication of a selected mutant and its

subsequent release as a new variety. Of the 2,252 accessions, 75% are in crops and

25% in ornamental and decorative plants. Most crop mutant varieties (1,603) were

released in seed-propagated species, which include 1,072 cereal and 311 legumes

(Maluszynski et al., 2000). Mutation induction with radiation was the most

frequently used method to develop direct mutant varieties (89%). The use of

chemical mutagens was relatively infrequent. A great majority of mutant varieties

(64%) were developed by the use of gamma rays (Ahloowalia et al., 2004). Among

the chemical mutagens, EMS is reported to be the most effective and powerful

mutagen (Minocha and Arnason, 1962 and Hajra, 1979). Several attempts in these

regards have been made to evolve the desirable plants by using physical and

chemical mutagens.

2.7. ROLE OF MUTATION IN ORNAMENTAL PLANTS

Ornamental plants are ideal for the application of mutation induction

techniques because many economically important traits e.g. flower characteristics or

growth habit, are easily monitored after the mutagenic treatment. Not surprisingly,

for centuries breeders have made use of spontaneously occurring sports, which have

contributed extensively to the generation of diversity in ornamental species. For

example, the origin of the moss rose was first observed in 1696 as a mutant of Rosa

centifolia (Hurst and Breeze, 1922). In azaleas and chrysanthemums, approximately

50% of cultivars have been derived from natural sports or induced mutations

(Heursel, 1980 and Preil, 1986). Since the 1930s mutation induction has been

applied to ornamental plants. The first commercial officially released mutant was in

tulip, cultivar Faraday, with altered flower color resulting from irradiation of cv.




Fantasy by De Mol in 1936 (Broertjes and van Harten, 1988). Broertjes and van

Harten (1988) comprehensively reviewed and listed about 100 species which have

been subjected to experimental mutation induction. According to the available

information, about 500 mutant cultivars in 29 ornamental species have been

officially released (Table 2.4) (Broertjes and Van Harten, 1988; Bhatia, 1991; Kawai

and Amano, 1991; Wang, 1991; Maluszynski et al., 1992 and Anonymous, 1994,

1996).

Table 2.4 : Number of officially released mutant varieties in ornamental

plant species.

Latin name Common name Mutagen No. of varieties

Abelia sp. Abelia Gamma rays 01

Alstroemeria sp. Alstroemeria X rays, gamma rays 35

Antirrhinum sp. Snapdragon Cross, X rays 05

Achimenes sp. Achimenes X rays, Fn 08

Begonia sp. Begonia X rays, gamma rays 25

Bougainvillea Bougainvillea Gamma rays 12

Calathea crocata Calathea X rays 01

Canna indica Canna lilies Gamma rays 04

Chrysanthemum sp. Chrysanthemum X rays, gamma rays 232

Dahlia sp. Dahlia X rays, gamma rays 36

Dianthus sp. Carnation X rays, gamma

rays, EMS

18

Euphorbia fulgens Euphorbia X rays 01

Ficus benjamina Ficus X rays 02

Gerbera jamesonii Gerbera gamma rays 01

Gladiolus sp. Gladiolus X rays, gamma rays 05





Guzmania peacockii Guzmania gamma rays 01

Hibiscus sp. Hibiscus gamma rays 07

Hoya carnosa Hoya Radiation 04

Hyacinthus sp. Hyacinth X rays 01

Kalanchoe sp. Kalanchoe X rays 03

Lagerstroemia indica Crapemyrtle EMS 02

Lantana depressa Wild sage gamma rays 03

Lilium sp. Lily X rays 02

Malus sp. Apple

(ornamental)

X rays 01

Pelargonium

grandiflorum

Geranium X rays 01

Polyanthus tuberose Tuberose Gamma rays 02

Portulaca grandiflora Portulaca Gamma rays 11

Rhododendron Azalea X rays, gamma rays 15

Rosa sp. Rose X rays, gamma

rays, EMS

61

Saintpaulia sp. African violet Gamma rays 01

Streptocarpus sp. Streptocarpus X rays 30

Tulip sp. Tulip Chemical, X rays 09

Weigela sp. Weigela Gamma rays 03

FAO/IAEA Mutant Varieties Database (2009)

Many mutants in ornamentals, e.g. Achimenes, Chrysanthemum, Carnation,

Roses and Streptocarpus, were obtained by irradiating rooted stem cuttings,

detached leaves and dormant plants (Broertjes and van Harten, 1988). The altered

flower color and shape, growth habit (dwarf or trailing) and other novel phenotypes




of commercial value were selected (Table 2.5). According to the FAO/IAEA

Database (2009), of the 552 mutant cultivars of floricultural plants, most were in

Chrysanthemum (232), followed by Rose (61), Dahlia (36), Alstroemeria (35),

Streptocarpus (30), Begonia (25), Carnation (18), Azalea (15), Bougainvillea (12)

and Achimenes (8) (Maluszynski et al., 2000).

Table 2.5 : Different mutated traits among officially released mutant varieties

of ornamental and decorative plants.

(Jain and Spencer, 2006)

In several ornamental crops, successful attempts have been made to induce a

wide range of flower color variations (Table 2.6). The most prominent example is

Chrysanthemum (Dendranthema x Grandiflorum), in which hundreds of new

cultivars have been developed by mutation induction throughout the world.

Sr. No. Mutated traits Number of mutants

1. Flower color 417

2. Flower morphology 31

3. Plant architecture 25

4. Leaf color 13

5. Variegated leaves 09

6. Ornamental type 09

7. Leaf morphology 07

8. Earliness 06

9. Compact growth 05

10. Dwarf 04

11. Flower type 03

12. Others 27




Table 2.6 : Induced Mutations in flower color.

Genus Reference

Alstroemeria Broertjes and van Harten, 1988; MBNL, 1992

Amaryllis Broertjes and van Harten, 1988

Begonia Broertjes and van Harten; Soedjono, 1988

Calathea Broertjes and van Harten, 1988

Canna Broertjes and van Harten, 1988; Khalaburdin, 1991;

MBNL; 1992

Dahlia Broertjes and van Harten, 1988

Dendranthema Broertjes and van Harten, 1988; Nikaido and

Onozawa, 1989; Matsumoto and Onozawa, 1989;

Jerzy, 1990; Antonyuk, 1991; Nagatomi, 1991;

MBNL, 1992; Ahloowallia, 1992; Nagatomi et al.,

1993; Jerzy and Zalewska, 1996; Tulmann Neto and

Latado, 1996

Dianthus Broertjes and van Harten, 1988; Silvy and Mitteau,

1986; MBNL, 1992; Simard et al., 1992; Cassells et

al., 1993

Euphorbia (fulgens) Broertjes and van Harten, 1988

Eustoma Nagatomi et al., 1996

Forsythia Broertjes and van Harten, 1988; van de Werken, 1988

Geranium MBNL, 1992

Gerbera Walther and Sauer, 1986b; Laneri et al., 1990; Jerzy

and Zalewsta, 1996

Gladiolus Broertjes and van Harten, 1988; Raghava et al., 1988;

Sedelnikova, 1988; MBNL 1992





Hibiscus Broertjes and van Harten, 1988; MBNL 1992

Hyacinthus Broertjes and van Harten, 1988; MBNL 1992

Iris Broertjes and van Harten, 1988

Kalanchoe Broertjes and van Harten, 1988; Schwaiger, 1992

Lantana Datta, 1995

Lilium Broertjes and van Harten, 1988; Grassotti et al., 1987

Muscaria Broertjes and van Harten, 1988

Pelargonium Broertjes and van Harten, 1988

Petunia Padmaja and Sudhakar, 1987

Phlox Antonyuk, 1991

Portulaca Broertjes and van Harten, 1988

Rhipsalidopsis Broertjes and van Harten, 1988

Rhododendron Broertjes and van Harten, 1988; MBNL 1992

Rosa Broertjes and van Harten, 1988; Zykov and

Klimenko, 1989; Antonyuk, 1991; MBNL 1992;

MBNL 1996

Saintpaulia Broertjes and van Harten, 1988

Streptocarpus Broertjes and van Harten, 1988; MBNL 1992

Tulipa Broertjes and van Harten, 1988

Zinnia Venkatachalam and Jayabalan, 1991, 1994

2.8. CYTOLOGICAL ABERRATIONS

Chromosomal aberrations have been used as a measure of reproductive

success in plants for many years and have been correlated with morphological and

taxonomical changes, fertility-sterility relationships, mutations and other

characteristics. Cytological aberrations in plants serve as an excellent monitoring

system for the detection of effect of mutagens at chromosomal level. Many

researchers have compared the mutagenic efficiencies of different mutagens on




different crops. Their results seem to be entirely specific for particular species and

even varieties. While many researchers like Rao and Rao (1983), Kumar and Dubey

(1998), Dhanayanth and Reddy (2000) and Bhat et al., (2005) found chemical

mutagens to be more effective than physical ones, many others like Tarar and

Dnyansagar (1980a), Zeerak (1991) and Singh (2003) found physical mutagens to be

more effective than the chemical ones.

Auerbach and Robson (1942) presented first elaborate report that mustard

gas could induce mutations as well as chromosomal aberrations in Drosophila.

Mutagen Urethane was reported to produce chromosomal breaks in Oenothera by

Ochlker (1946). Formalin was also reported to have mutagenic effect when fed to

Drosophila (Rapoport, 1946). Sodium azide was found to be very effective mutagen

under certain treatment conditions (Kleinhofs et al., 1974), it made possible to

obtain high mutation frequency, mostly gene mutations, with negligible frequency of

chromosomal mutations.

Gamma rays, Maleic hydrazide (MH) and gamma rays+ Maleic hydrazide

(MH) treatments show disturbed mitotic behavior which was noticed by Grover and

Tejpaul (1982) in Vigna radiata. The sticky chromosomes, fragments and ring

chromosomes at metaphase while the laggards and bridges at anaphase were noticed

by these workers. The chromosomal aberrations were found to be significantly co-

related with the dose. The combined treatment enhanced chromosomal aberrations.

Similarly, the meiotic process was also affected. The quadrivalents presumably due

to translocations, were occasionally encountered in metaphase-I. Irregular

disjunction of chromosome at anaphase-I, accompanied by laggards was also

observed (Grover and Tejpaul, 1982).

Gates (1908) observed delicate threads of cytoplasm connecting adjacent

pollen mother cells in Oenothera. Gates (1911) subsequently suggested that these

connections must form an important avenue of exchange between PMCs and

described the transfer of nuclear material through them from one meiocyte to

another, calling the process cytomixis. According to Heslop – Harrison (1966) and

Risueno et al., (1969), the role of cytoplasmic channels is related to the transport of

nutrients between meiocytes. Investigations in angiosperms have provided evidence

that massive protoplasmic connections are formed among microsporocytes.




Cytomixis involving transfer of chromatin material is reported in a large number of

plants (Shnaider, 1975; Singhal and Gill, 1985; Bedi, 1990; Lattoo et al., 2006 and

Singhal and Kumar 2008 a, b). The phenomenon is better known to exist in

genetically imbalanced plants like, hybrids, mutants, aneuploids and polyploids

(Premachandran et al., 1988, Peng et al., 2003, Zhou, 2003; Sheidai and Attaei;

2005). In various crops, the abnormal spindles have been reported (Harlan and De-

Wet, 1975 and Veilleux, 1985). The spindle apparatus is normally bipolar and acts

as a single unit, playing a crucial role in the alignment of metaphase chromosomes

and their pole ward movement during anaphase. Distortion in meiotic spindles may

be responsible for unreduced gamete formation. The formation of unreduced

gametes has been investigated in studies of evolution (Harlan and De-Wet, 1975)

and in breeding programmes (Veilleux, 1985). It was reported that meiotic

abnormalities cause male sterility (Goyal and Khan, 2009). Chromatin bridges and

micronuclei were described for the first time in interspecific hybrids of Glycine max

x Glycine soja by Ahmad et al., (1977), who found that the extent of abnormalities

was influenced by environmental conditions. Studies on different plant species have

shown that the decline in seed production is correlated with meiotic irregularities

(La Fleur and Jalal, 1972; Dewald and Jalal, 1974; Moraes-Fernandes, 1982; Smith

and Murphy, 1986; Pagliarini and Pereria, 1992; Pagliarini et al., 1993; Consolaro et

al., 1996 and Khazanehdari and Jones, 1997). In most of the mungbean varieties,

pollen fertility showed a close relationship with meiotic abnormalities (Khan, 1990).

The least mutation frequency at higher doses may be attributed to chromosomal

aberration or saturation in the mutational events which may result in the elimination

of mutant cells during growth (Blixt and Gottschalk, 1975).

2.9. ADVANCEMENT OF MOLECULAR PROFILING

The preservation of genetic diversity in endangered species is a main goal in

conservation planning, since long-term species survival depends on the maintenance

of sufficient genetic variability within and among populations to accommodate the

selection pressures brought about by environmental change (Barrett and Kohn,

1991). Although some authors have questioned the importance of genetic studies

with regard to demographic approaches (Lande, 1988 and Schemske et al.,1994),




many others think that assessing genetic diversity and understanding how diversity

in structure is not only a prerequisite in designing suitable conservation strategies

(Falk and Holsinger, 1991 and Avise, 1995), but, furthermore, this knowledge helps

to resolve taxonomic (Van Buren et al., 1994 and Cole and Kuchenreuther, 2001),

phylogenetic (Smith and Pham, 1996), demographic and ecological (Cruzan, 1998)

questions of great relevance for conservation.

The analysis of genetic diversity and relatedness between or within different

populations, species and individuals is a central task for many disciplines of

biological science. Genetic diversity has been conventionally estimated on the basis

of different biometrical techniques (Meteroglyph, D2, divergence analysis, and

principal component analysis) such as phenotypic diversity index (H), or coefficient

of parentage utilizing morphological, agronomical and biochemical data (Matus and

Hayes, 2002; Mohammadi and Prassana, 2003; Jaradat et al., 2004 and Ahmad et

al., 2008). However, evaluation based on these phenotypic data was laborious and

took years to draw a conclusion. The advent of different molecular techniques led

breeders to estimate genetic diversity on the basis of data generated by different

molecular markers, which provided a means of rapid analysis of germplasm and

estimates of genetic diversity, which were often found to corroborate phenotypic

data. These molecular markers are broadly categorized as non-PCR or PCR based.

Restriction fragment length polymorphism (RFLP) belongs to the first category and

polymorphism is restriction site-based and does not require a PCR reaction to

amplify while amplified fragment length polymorphism (AFLP), single sequence

repeats (SSR) and random amplified polymorphic DNA (RAPD) markers belong to

second category which require a PCR reaction and offer several advantages over the

first category i.e. rapid and low cost per analyses, freedom from radio labeling and

high or sometimes comparable polymorphism (Rauf et al., 2010). All these markers

are now being widely used for evaluating genetic relationships of many crop

germplasm (Table 2.7).




Table 2.7 : Molecular assessment of genetic diversity in several crops.

Plant species Family Technique used Reference

Alstroemeria L. Alstroemeriaceae RAPD Aros et al., 2006

Arachis hypogaea L. Fabaceae RAPD Bhagwat et al.,

1997.,Varsha

kumari et al., 2009

Arachis hypogaea L. Fabaceae RAPD, ISSR Raina et al., 2001

Brassica spp. Brassicaceae RAPD Malode et al.,

2010

Chrysanthemum

morifolium L.

Asteraceae RAPD Barakat et al.,

2010

Dendrobium serdang

Sw.

Orchidaceae RAPD Khosravi et al.,

2009

Dendroseris spp. Asteraceae RAPD Esselman, 2000

Eleusine coracana G. Poaceae RAPD Das et al., 2009

Emblica officinalis

Gaertn

Euphorbiaceae RAPD Selvi et al., 2008

Foenicum vulgare

Mill.

Apiaceae RAPD Zahid et al., 2009

Glycine max (L.) Merr. Fabaceae RAPD vs. SSR Doldi et al., 2006

Hippeastrum spp. Amaryllidaceae RAPD Chakrabarty et al.,

2007

Hordeum vulgare L. Poaceae RAPD, ISSR Fernandez et al.,

2002

Ixora spp. Rubiaceae RAPD Rajaseger et al.,

1999

Lachenalia bulbifera J.

Jacq.

Hyacinthaceae RAPD Kleynhans and

Spies, 2000





Lippia spp. Verbenaceae RAPD Viccini et al., 2004

Narcissus tazetta var.

chinensis

Amaryllidaceae AFLP, RAPD Lu et al., 2007

Nierembergia

linariaefolia (Graham)

Solanaceae AFLP, ISSR Escandon et al.,

2007

Ocimum spp. Lamiaceae RAPD, AFLP Carovic – Stanko

et al., 2010

Oryza sativa L. Poaceae RAPD Babaei et al., 2011

Oryza sativa L. Poaceae RAPD vs. AFLP Fuentes et al.,

2005

Oxytropis chankaensis

Jurtz.

Fabaceae RAPD Artyukova et al.,

2004

Pisum sativum L. Fabaceae PCR based

marker vs. RFLP

Lu et al., 1996

Prunus spp. Rosaceae RAPD, SSR Shiran et al., 2007

Ranunculus reptans L. Ranunculaceae RAPD Fischer et al., 2000

Saccharum spp. Poaceae Phenotypic vs.

AFLP

Lima et al., 2002

Stachytarpheta spp. Verbenaceae RAPD Peiris and Godage,

2005

Triticum aestivum L. Poaceae Phenotypic vs.

AFLP

Barrett et al., 1998

Triticum aestivum L. Poaceae SSR, RAPD Tiegu et al., 2007

Vanilla spp. Orchidaceae RAPD Besse et al., 2004

Vigna mungo (L.)

Hepper

Fabaceae RAPD, ISSR Souframanien and

Gopalakrishna,

2004

Zea mays L. Poaceae RFLP, RAPD,

SSR, AFLP

Pejic et al., 1998




Plant breeding systems are a major factor influencing the genetic structure of

populations (Hamrick and Godt, 1990). Detecting genetic variation becomes more

and more important in breeding programmes. Successful cultivar development is

dependent on the availability of an extensive and well documented germplasm

collection. Characterisation and evaluation of wild species accessions is therefore an

important activity in relation to the breeding program. Molecular methods to assess

genetic variation assist the more conventional methods of characterizing accessions.

Determining the genetic variation among species accessions provide information

that is important when choosing the accessions for use in the breeding programme

(Newbury and Ford-Lloyd, 1993 and Williams and St.Clair, 1993).

Mutations have the potential to alter many morphological and physiological

traits (Micke et al., 1990 and Wang et al., 1992). The estimation of genetic diversity

on the basis of morphological traits alone does not determine actual level of genetic

diversity among germplasm because morphological traits are the product of gene

and environmental interactions (Alan, 2007). The degree of gene expression is

highly influenced by the conduciveness of the environment and genetic background

in which gene is present. Therefore, selection based merely on morphological traits

has been often misleading (Astarini et al., 2004 and Asif et al., 2005). Hence in

many instances breeders have been using genetically similar parents in their

breeding programmes, leading to a narrow genetic base (Fouilloux and Bannerot,

1988; Xia et al., 2004 and Rehman et al., 2002). During last ten years, techniques

based on DNA markers along with morphological traits have been used to detect

variation at DNA level to distinguish closely related genotypes (Williams et al.,

1990; Welsh et al., 1991 and Alan, 2007).

However, most mutants deviate from the original variety only in minor

characteristics and may thus be very difficult to distinguish genetically. Correct

identification of new varieties is extremely important to protect plant breeders‟ rights

for commercial exploitation. Accurate identification of plants is also desired for

patent protection of propagated material. Use of present day molecular markers in

addition to the classical methods provides more positive identification of new

cultivars. Restriction fragment length polymorphism (RFLP) and amplified fragment

length polymorphism (AFLP), though used for screening of genetic diversity, are




laborious, usually involves radioactivity and are not suited for routine application of

cultivar identification. Random amplification of polymorphic DNA (RAPD) requires

only small amounts of starting DNA, does not require prior DNA sequence

information, and nor involves radioactivity (Williams et al., 1990), while data can be

generated faster with less labor than other methods like RFLP. The technical

simplicity of the RAPD technique has facilitated its use in the analysis of

phylogenetic relationships in several plant genera, e.g. roses (Debener et al., 1996),

Tuber species (Gandeboeuf et al., 1997), blueberry (Levi and Roland, 1997), barley

(Noli et al., 1997), Passiflora (Fajardo et al., 1998), lentil (Ferguson et al., 1998),

taro (Irwin et al., 1998), Cymbidium (Obara-Okeyo and Kako, 1998) etc.

Furthermore, RAPD markers can be used to detect genetic variation at the intra as

well as interspecific level (Abo-elwafa et al., 1995). There are also some examples

where an RAPD-based finger print technique has been used for mutant

discrimination. Recently, 11 radiomutants from two chrysanthemum cultivars were

characterized by RAPD (Kumar et al., 2006). This technique has also been used to

study genetic variability in radio mutants from the lady group of chrysanthemum

(Ruminska et al., 2004). Similarly, a mutated cherry was differentiated from its

parental by1out of the 40 RAPD primers tested (Stockinger et al., 1996).

review of literature -...

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