Chapter 11

REVIEW OF LITERATURE

Section 1. CassJ8 mphera L.

A. Seed dormancy and Germination

1. Seed dormancy

Dormnncy may be defined as a state in which growth is temporarily

suspended. Seed physiologists have distinguished Dormancy from Quiescence.

Quiescence is the condition of a seed when it is unable to germinate only because

the external conditions normally required are not present; and dormancy is the

condition of a seed when it fails to germinate because of internal conditions, even

though external conditions (e.g., temperature, moisture and atmosphere) are suitable.

A number of concepts and theories of seed dormancy have emerged through the

years and some notable reviews in this subject are : Crocker and Barton, 1957;

Toole, 1959; Chouard, 1960; Evenari, 1961, 1965 a, 1965 b; Vegies, 1964; Barton,

1965 a, 1965 b; Stocks, 1965; Wareing, 1965 a, 1965 b; Amen, 1968; Viers , 1972;

Khan, 1977; Nikolaeva, 1977; Roberts and Smith, 1977; Taylorson and Hendricks,

1977; Rolston, 1978; Bewiey and Black, 1982; Mayer and Poljakoff-Mayber, 1982,

1989.

A widespread cause of seed dormancy is the presence of a hard seed coat,

which occurs in many plant families. According to Quinlivan (1971), the seed

dormancy in leguminosae family is due to hard seededness. The impermeability of

the seed coat is related to changes occllrring in the fine structure of the hilum

(Poptsov, 1976). Different types of legume seeds which produce hard seed coats

show considerable variability depending on the species, the degree of maturity, the

ripening conditions and storage time. Low air humidity during fruit ripening results

in a considerable increase in seed hardiness. For instance, the number of hard seeds

in Casda from mid-Asia varied from 10 to 30 percent whereas those from other

places (BaQuni) showed 2 to 3 percent variation (Nikolaeva, 1977). Rolston (1978)

showed that impermeability of seed coat to water was the most common feature in

Leguminosae family. According to him, the seed coats of many members of

Leguminosae are hard, resistant to abrasion, covered with a wax-like layer and such

seed coats appear to be entirely impermeable to water. In some seeds, most of the

water enters through chlazal region as the micropylar region is apparently

impregnated with materials which have low permeability to water (Janerette, 1979).

According to Dell (1980) the impermeability of the seed coat is controlled by a small

opening present in the seed coat called the strophiolar cleft. The surrounding tissues

in the strophiole are impregnated with pectins, suberins, cutins and mucilages which

controlled the entry of water into the seed (Werker, 1980). Datta and Bas11 (1982)

have reported the dormancy in Cessia sophem seeds are exogenous and coat

imposed. These authors tried to break the dormancy of the above seeds by treating

them with chemical substances like Thiourea, Potassium nitrate, Potassir~m

thiocyanate and Magnesium nllphate. But the seeds did not germinate. Some

possible causes and reasons for coat imposed dormancy have been discussed by

Bewley and Black (1982). However, Bewley and Black (1982) and Mayer and

18

Pobakoff-Mayber (1989) have also mentioned the controlling factors which

established coat imposed dormancy and some methods to overcome it. A general

discussion of coat imposed dormancy has been put forward by Tran and Cavanagh

(1985).

In nature, most of the seeds with coat imposed dormancy become permeable

to water when the seed coat is broken down or punctured in various ways. This

includes mechanical abrasion, microbial attack, passage through the digestive tract

of animals and exposure to high and low temperatures (the exposure of seeds to

varying temperatures possibly causes crack in the seed coat due to expansion and

contraction). Under laboratory conditions and in field trials, various means are

adopted to render the seed coat permeable. These include either mechanical

treatment or chemical treatment. Mechanical treatments l i e scratching or shaking

the seeds with some abrasives or cutting the seed coats or thermal treatment with

boiling water improve the permeability of the seed (Rolston, 1978; Shirai and Kagei,

1985; Kohli and Kumari, 1986; Muir and Pitman, 1987; Singer and Pitman, 1988).

Chemical treatment is mainly of two kinds : removal of the waxy layer of the

seed coat by suitable solvent such as alcohol (Barton and Crocker, 1948; Koller,

1954; Rolston, 1978; Kohli and Kumari, 1986) or treatment with strong acids. There

are reports of sulphuxic acid scarification being an efficient means to break the coat

imposed dormancy of seeds and to achieve maximum germination (Aganval and

Vyas, 1970; Sdva-Barbosa et nl., 1971; Shaybany and Rouhani, 1976; Stibbumm~~ and

Sridhar, 1977; Hutchinson and Ashton, 1979; Johnston et al., 1979; Kohli and

K~unari, 1986; Menon and Kulkarni, 1987; Sangai, 1988; Thak~lr, 1989; S igh and

Singh, 1990; Maithnni, 1991; Zodape, 1991). It is evident from the above reports that

the dormancy breaking action of mechanical abrasion and alcohol or sulphtuic acid

scarification of the seed could be directly related to an increase in the permeability

of water leading to germination.

2. Germination

The seed occupies a unique position in plant science. It has fimdamemtal

importance as a tool in plant physiology because most of the known physiological

processes are concentrated in the growth and development of this smaU unit (Chinoy

et d., 1969). The reaction between activation of essential enzymes, sequential release

of hormones and the energy relations of the process duuing the germination of seed

are still unknown. It is here that plant physiologists, biochemists and biophysicists

need to work together (Sen, 1984). There is a voltrminous data on seed germhation

which has been adequately dealt with in a number of excellent reviews. (Crocker,

1938; Toole, 1939; Evenari, 1956,1961; Toole etal., 1956; Crocker and Barton, 1957;

Barton, 1961; Koller, 1961; Koller et d., 1962; Mayer and Poljakoff-Mayber, 1963,

1975,1982, 1989; Wareing, 1963;Vegies, 1964; Bonner and Vamer, 1965; Borthwick,

1965; Brown, 1965, 1972; Lang, 1965; Kozlowski, 1972; Khan, 1977; Bewley and

Black, 1978, 1982; Oaks, 1983; Murray, 1985).

Plant growth substances are known to influence germination of seeds. The

effect of these substances have been studied primarily with a view to improving

germination under normal as well as unfavourable conditions. The externally applied

plant growth substnnces affect various metabolic pathways as well as developmental

stages such as regulation of precocious germination. It is generally assumed that

endogenous hormones and those applied exogenously act in a similar fashion

although this assumption is by no means justified (Mayer and Poljnkoff-Mayber.

1989). A great deal of information is available on the effect of exogenously applied

20

compounds on the germination of seeds. Some of the notable reports in this context

are : (Paleg, 1965; Weaver, 1972; Krishnamoorthy, 1975; Varner and Ho, 1976;

Khan, 1977, 1982; Letham et nl., 1978; Crozier and Hillman, 1984; Jacobsen and

Beach, 1985; Murray, 1985; Chadwick and Garrod, 1986; Davies, 1987; Mayer and

Poljakoff-Mayber. 1989). There are a few reports available on the effect of growth

regulators on seed germination of certain species of Cassi'a (Singh and Mu@,

1987 a, 1987 b; Sigh, 1989; Thakur, 1989).

The importance of seed treatments at a high temperature on seed

germination has been emphasised by several workers (Lang, 1965; Koller, 1972;

Heydecker, 1977; Menon and Kulkarni, 1987; Mayer and Poljakoff-Mayber, 1989).

According to them, different seeds have different temperature ranges for their

germination. The temperature optima of the germination of seeds may vary from

species to species or even in different cultivars of the same species. The optimal

temperature for the germination of a seed is that at which maximum percentage of

germination is obtained in the shortest time. For example, in Cucrunis sativo, the

maximal temperature at which germination occurs may be as high as 48OC (Knapp,

1967). The seeds of Solmum nigm progressively lose their ability to germinate

within 48 hr. at 50°C or within 6 hr. at 5S°C (Givelberg et al., 1984). In many desert

seeds, storage at 50°C promotes germination while storage at normal temperature

lowers the rate of germination. Thus, there is usually an optimal temperature below

and above which germination is delayed but not prevented (Mayer and Poljakoff-

Mayber, 1989). An interesting interaction between light, temperature and high

humidity resulting in the promotion of germination is characteristic of Oldenhdia

corymbosa which grows in tropical areas. Its seeds can germinate only in the

21

presence of light and at a high temperature of 35 to 40°C. (Mayer and Poljakoff-

Mayber, 1989).

Reports on the cumulative effect of seed treatments with high temperature

and phytohormones are limited. Holm and Miller (1972) found that either

Gibberellic acid or any one of the various treatments which raised the temperature

to 50-llO°C caused certain weed seeds to germinate. Singh et d. (1974) succeeded

in germinating Datrm inoxia seeds by treating with aqueous thiourea or ascorbic

acid (two reducing agents) and also with X-rays, electric shocks or high temperature

shocks. They have suggested that all these can operate by inactivating 'an interfering

enzyme or hormone. Hendricks and Taylorson (1976) have reported that, many

light- temperature- and hormone responses could have a common focus in some

lecithin and / or phosphatidyl ethanolamine molecules in membranes. A cumulative

effect of seed treatment with high temperature (40°C) and growth substances in the

germination of Desmodirun gmgetirun has been observed in this laboratory

(Abraham and Thomas, 1993). It was therefore conch~ded that even the range of

high temperature at which germination could occur was not absolute and could be

enhanced by the exogenous application of plant growth substances.

B. Metabolic changes during the juvenile phase:

The metabolic activity of dry seeds begins with the imbibition of water and

gradrlally increases as germination marches on. The storage materials in the seed are

broken down and part of the breakdown products is transported from the cotyledon

or endospenn to the growing embryo. The metabolic changes in the composition of

seeds during germination have been investigated in a number of plant species (Oota

et aL, 1953; Ingle et al, 1964, 1965; Ching, 1966; Chinoy et aL, 1969; Palmiano and

22

Juliano, 1972). The series of events during seed germination are well documented by

many authors (Ching, 1972; Mayer and Shain, 1974; Mayer and Poljakoff-Mayber,

1975, 1982, 1989; Ashton, 1976; Khan, 1977, 1982; Bewley and Black, 1978). Smith

(1974) carried out some interesting work on the reserve hydrolysis in legumes and

this study of 500 legume species has revealed that there are 8 basic pattems of

hydrolysis of reserves from the cotyledons. According to him, in Cmia species, the

mobilization of reserves begins on the abaxial side of the cotyledons and the pattems

of mobilization is not associated with the vascular strands. Major changes during the

juvenile phase of seedling growth are carbohydrate metabolism, protein metabolism

and enzyme activities.

1. Carbohydrate metabolism

a Starch content

Starch is a polysaccharide composed of a-1,4 glucan with a-1,6 glucan side

chains and serves as a nutrient source of germinating seeds. According to

Abrahamsen (1964) and, Abrahamsen and Sudia (1966) in many seeds storage

carbohydrates are the principal substrates during germination. During germination,

starch is normally broken down by a or R amylases. Swain and Dekker (1966) have

shown a pathway in Peas during germination.

a amylase R amylase Starch -------------- + soluble oligosaccharides -------------- -,

a glucosidase ~ a l t ~ ~ ~ --------------- :, Glucose

It has been reported that the starch content in the endosperm of many

monocotyledonous seeds decreases with the advance in germination (Saxena et d.,

1970, Vora et al., 1974 a, 1975). This suggests thnt the mobilization and utilization

of starch increases with the advance in the germination process of seeds. In legume

seeds also, the starch content depletes and the amylase activity increases during the

early periods of germination (Juliano and Vamer, 1969; Yomo and Vamer, 1973;

Tarrago and Nicolas, 1976; Bewley and Black, 1978; Sharma and Pant, 1979). On the

contrary, in soya beans, the starch content increases during seed imbibition and

germination (Adams et al., 1980; Kamaladevi et al., 1990). Savitd and Desikachar

(1990) showed an increase in starch content in soya bean seedlings and a starch

depletion in pigeon pea seedlings in 24, 48 and 72 hours after the commencement

of germination.

b. Sugar content

Sucrose is often present in dty seeds in small amounts or is formed as a

result of breakdown of raffinose. During germination of seeds the degradation of

starch results in the formation of glucose and eventually sucrose is synthesised.

Sucrose is formed by a complex mechanism. Mayer and Poljakoff-Mayber (1989)

have depicted this as follows : Glucose is phosphorylated in the presence of ATP.

Part of glucose-6-phosphate formed is converted to fructose-6-phosphate (F-6-P)

and part to Glucose-1-phosphate (G-1-P). The G-1-P, in presence of uridine

triphosphate (UTP), is converted to uridine diphospho glucose (UDPG). Sucrose is

then formed by the condensation of UDPG and F-6-P. Finally sucrose is syr~thesised

24

andthe breakdown products are transported to the developing embryo. It is reported

that in seedling embryos, the sucrose synthase pathway and acid invertase are active

and sucrose is broken down by the sucrose synthase pathway. This pathway is

dependent upon UDP and Pyrophosphate (PPi), adequate quantities of which are

produced by a cyclic series of reactions (Huber and Akazawa, 1986; Sung et d.,

1986; Xu et d., 1986; Black et d., 1987). When sucrose is broken down by this

pathway the hexoses from sucrose can enter glycolysis. In addition, Xu et d. (1989)

proposed that in the plant cell cytoplasm, alternative enzymes are present at various

steps in glycolysis and gluconeogenesis for the interconversion of sucrose and

pyruvate. They have further reported the sucrose metabolism of developing and

germinating lima bean seeds with a view to characterising the fundamental portion

of cellular carbon nutrition.

It is generally reported that the reducing sugar content of seeds increases

during the early hours of germination (Vonohlen, 1931; Sasaki, 1933; Tada and

Kawamura, 1963; Abrahamsen and Sudia, 1966; Saxena et d., 1970; Sugavanam,

1992). The soluble sugar present in the cotyledons of different varieties of pigeon

pea increases to a maximum on the 2nd day of germination and decreases slowly

upto the 4th day followed by a rapid decline (Sharma and Pant, 1979). In the

seedlings of Sorghum vulgare, the total sugar content increases upto 72 hours of

germination and then falls (Vora et d., 1974 a). Vora et nl. (1975) have reported a

gradual increase in the reducing sugars from 24 to 72 hours of germination period

and a decline at 96 hours. Kamaladevi et d.(1990) have evaluated the changes in

glucose, fructose, sucrose, maltose, raffinose, stachyose and verbascose contents of

25

wingcd bean (Psophoctup~~s tetmgonolobr~s) seedlings. Savitri and Desikachar (1990)

have reported an increase in mono- and &-saccharides during the germination of

soya bean and pigeon pea seeds.

2. Protein metabolism

The proteins which are metabolically inactive are regarded as storage proteins

and are usually located in protein bodies. Oota et al. (1953) have concluded that the

proteins which are a frequent reserve material in the seed are often broken down

during germination with concomitant rise in arninoacids and amides followed by de

novo protein synthesis in the growing part of the embryo. Thus, the first observable

change in germination is a change in protein to soluble nitrogen (Klein, 1955). He

showed clearly that there was an increase in soluble nitrogen only in the seeds of

lettuce which germinated and not in those which remained dormant. Cheny (1963)

showed that during the germination of peanut seed, over 60 percent of the dry

weight of cotyledon and 70 percent of the protein are depleted. Varner (1965)

suggested that during germination, seed proteins are hydrolysed in the endosperm

or cotyfedon into peptidases and aminoacids which are translocated to the growing

axis. The maximum rate of hydrolysis of storage proteins coincides with the

maximum rate of growth of the seedling. The early protein degradation might be due

to the combined activity of preformed soluble peptidases present in dry seeds whose

activity rapidly declines over the first few days after imbibition (Chrispeels and

Varner, 1967) and an insoluble membrane bound proteinase which appears to be

synthesised de novowithin 48 hours of initial imbibition (Taiz and Jones, 1970). The

26

biochemical aspects of legume seed proteins have been reviewed by Millerd (1975).

The storage proteins of seeds generally have a high nitrogen and proline content and

have low lysine, tryptophan and methionine content (Mayer and Poljakoff-Mayber,

1989).

3. Enzymic activities

a Catalase activity

The catalase enzyme contains iron in its porphyrin nucleos. Some workers

reported a direct correlation of catalase activity and seedling growth (Nanda, 1950;

Chikasne, 1953; Vema and VanHuystee, 1970) whiie others showed an inverse

correlation (Galston, 1951; Halevy, 1964; Prathapasenan et al., 1969). Yokoyarna

(1956) studied the relation of catalase activity to mitochondrial respiration and

pointed out that catalase directly affected the oxidation and reduction of cytochrome

C oxidase system. A rise in the enzyme catalase has been reported, as germination

progresses (Papov, 1965; Hendricks and Taylorson, 1974). On the other hand, low

moisture content due to water stress reduced catalase activity in Wheat, Sesamum

and Eleusine as and when germination advanced (Achslya, 1968). Vora et al.

(1974 b) have also observed that the low moisture level of seedlings can depress the

enzyme activity. Esashi et al. (1979) have studied the involvement of catalase on the

regulation of germination of cocklebur seeds. Eising (1989) has determined the

catalase synthesis and turn over during peroxisome transition in surfflower

cotyledons.

27

Plant hormones play a pivotal role in the regulation of enzyme synthesis and

activity in plant cells. Stimulation of the enzyrnic system and an enhanced growth by

the exogenous application of certain growth substances like Ascorbic acid (Chiioy

et al., 1969) and GA3 (Prathapasenan et d, 1969; Sangeeta and Varshney, 1991)

have been reported.

b. Peroxidase activity

Peroxidase (donor: H,O, oxido reductase) catalyses the oxidation of diverse

hydrogen donors and peroxidase activity is implicated in many biological events in

plants depending on the nature of the donor. This enzyme is known to fulfill many

functions in plants. Peroxidase activity is reported to be associated with active

differentiation (Galston, 1951; Halevy, 1964; Saxena, 1979). Siege1 (1955) has pointed

out a broad hydrogen donor specificity to plant peroxidases. The enzyrne has been

implicated in respiration (Chance, 1954; Hackett, 1963), as a key component in IAA

oxidase system (Ray, 1958; McCune, l961), and in the breakdown of IAA (Goldacre,

1961). It can act as a mixed function oxidase (Buhler and Mason, 1961; Nicholls,

1962) and as an agent in the oxidation of metabolites by means of H, 0, by product

(Fruton and S i o n d s , 1963). It has also been put forward as a constituent of

terminal oxidation (Alexander, 1964) and in the biosynthesis of auxin (Riddle and

Mazelis, 1964). An increase in peroxidase activity during gemination has been

reported (Papov, 1965; Hendricks and Taylorson, 1974). Fridovich (1976) suggested

that the peroxidase removes all noxious products in plant cells and according

to Gasper et d. (1982) it is a natural source of scavenger in seeds. Dendsay

28

and Sachar (1982) observed a 30-fold stimulation of peroxidase activity in mung

bean seedlings after 4 days of germination. Balasirnha (1982) reviewed the regulatory

role of peroxidase in higher plants. Bhattacharjee and Gupta (1984) have reported

that higher peroxidase activity indicates higher plant potential. Bhatia and S H X ~ I I ~

(1991) observed a direct correlation between enzyme activity and seedling growth.

The regulation of peroxidase activity by growth i~omones in many plant

species has been reported by various authors (Lavee and Galston, 1968; Stuber and

Levings, 1969; Birecka and Galston, 1970; Ritzert and Turin, 1970; Lee, 1972). It is

well established that high concentrations of auxin promotes the release of ethylene.

In cotton plants, auxin induced evolution of ethylene coincides with the

enhancement of peroxidase activity (Sakai and Imaseki, 1971; Fowler and Morgan,

1972). Wolter and Gordon (1975) have also reported such enhancement in

peroxidase activity in Aspen callus cultures. It is well known that plant pemxidases

play a central role in metabolism and growth through the biosynthesis of essential

constituents, in the action of hormones and in defence mechanism. According to

Batra and Kuhn (1975) peroxidases are involved in disease resistance in plants.

Shannon (1976) reported a decreased peroxidase activity in GAS- treated PhaseJors

ndgms. Auxin, ethylene and GA, differently activated the growth and activity in

mung bean seedlings (Dendsey and Sachar, 1982) and in the seedlings of cauliflower

(Bhatia and Saxena, 1991). According to Barendse (1983), hormones apart from

influencing the synthesis of enzyme proteins, directly or indirectly influence the

activity of synthetic and degradative enzymes. It also plays a major role in modifying

the conformation of enzyme proteins e.g., RNA polymerase. Sangeeta and Varshney

29

(1991) reported a suppression of peroxidase activity in Avena sativnseedlings by the

action of GAS

C. Presowing seed treatments

Kiddand West (1918,1919) exarninedthe scientific aspects of seed treatment

with chemicals and concluded that, factors which influence the plant during the early

stagesof development may also profoundly influence its subsequent life history. They

considered the size of the seed, degree of its maturity, soaking seeds in water and

in solutions of various chemicals before sowing and the temperature as important

factors. The addition of ions such as calcium caused no significant amelioration of

the injurious effects of distilled water in beans Pailey, 1933). According to Barton

(1950), exclusion of oxygen during the presoaking period may prevent germination.

Martyanova eta/. (1962) claimed that alternate soaking and drying of seeds before

planting doubled the yield under drought conditions. Henckel (1964) and Chinoy

(1968) have reviewed the importance of presowing treatment of seed for drought

resistance, salt stress and in crop production. Sashidhar et a/. (1977) suggested mat

the favourable effects of presowing seed treatments may be due to the ability of

plants from such treated seeds to produce and accumulate proline under stress. An

important adaptation found in many organisms subjected to water stress is the

accumulation of certain organic compounds such as sucrose, the aminoacid - proline

and several other compounds which lower the osrnotic potential (Salisbury and Ross,

1986). Such compounds appear in plant cells as and when water stress increases, and

the resultant drop in the osmotic potential is known as osmotic adjustment or

30

osmoregulation (Morgan, 1984). The embtyonic axis damage can be reduced by

exposing the seed to a limited amount of water (Presowing seed treatment), which

enables all parts of the seed to become hydrated uniformly before planting (Noggle

and Fritz, 1986). However, Basu and Sur (1988) reported that the presowing

treatments in general gave a beneficial effect on crop growth by inducing tolerance

to stress.

The beneficial effect of presowing treatment of seeds with growth regulators

during seed germination and growth has been reported by a number of workers

(Gupta, 1956; Nanda et d., 1959; Henckel, 1961; Denisova, 1962; Mohanty and

Mishra, 1962; May et n1.,1962; Komeev, 1963; Dawson, 1965; Chinoy et d.,1969,

1970; Abraham, 1970; Jani et d.,1970; Saxena, 1974; Kumar and Aka, 1978; Vora

and Patel, 1979; Sahai et nl., 1980; Singh et a1.,1985; Singh and Saxena, 1991). Thus

the presowing seed treatment with growtl-I regulators has been a matter of interest

for plant physiologists for a long time. However, negative results due to presowing

seed treatments have also been reported (Henckel, 1964; Salim and Todd, 1968;

Heydecker, 1973; Saxena, 1974, 1979). Presowing seed treatments with bioregulants

have shown to stimulate many physiological and biochemical processes which

ultimately result in a high rate of seedling growth as well as enhanced vegetative

growth and high productivity (Abraham et d., 1968; Chinoy 1968; Abraham, 1970).

Incorporation of growth regulators through presowing seed treatment is found

beneficial in enhancing growth and yield of a large number of cereals (Chinoy et d.,

1970; Chinoy and Saxena, 1978; Saxena, 1985). Wittwer (1978) has also observed the

importance of chemical regulators in crop production.

3 1

D. Growth and Metabolism

In an experiment with pea plants (Pisrun sativum) Padma (1980) observed

more leaves and higher dry matter production due to the presowing seed treatment

with 100 ppm GA3 solution as compared to the control. Saxena et a1. (1987)

investigated the pretreatment effect of pea seeds with IAA, GA3 and Kinetin during

growth and found that the pretreatments increased plant dry weight and yield

characters. Singh and Saxena (1991) reported that the presowing seed treatment with

growth substances like IAA, GAS and kinetin increased plant height, leaf number, dry

weight of leaves, stem and roots as well as the total dry matter production.

During plant growth, the rise in the carbohydrate level is a cause or

consequence of the shift from vegetative phase to reproductive phase. Ito and Saito

(1961) reported that the accumulation of carbohydrates in the leaves of cucumber

is correlated with the accumulation of flower forming substances. Gurumurti et al.

(1969) found a higher sugar content in the spikelet of wheat when compared to the

earlier stages of shoot apex differentiation. It showed a steep fall at the time of

anther and carpel formation, again rose in the seed setting stage and declined during

maturity. These authors have also reported that starch appears in wheat at the time

of newly formed grain stage and increases as maturation advances. According to

Randhawa and Singh (1972), a sufficiently high carbohydrate level is essential for the

initiation of flowers in CI~cumis melo. Saimbhi and Nandpuri (1981) observed a pre-

flowering rise in the leaf carbohydrate content of Cr~crlmis melo. Yadav and

Goswami (1990) estimated the starch and total sugars of tender, medium matured

32

and matured leaves of some plants in the Lauraceae family. According to them, the

starch content has an increasing trend with age and maturity. The sugar content in

the medium aged leaves was found to be higher than in the tender and mature

leaves. Patil et al.(1991 a) investigated the carbohydrate content of Alphonso mango

during the fruit bud differentiation period. In this study they found a higher content

of reducing sugars, low amount of non reducing sugars and total sugars in the buds

which were going to differentiate into fruit buds. The starch content showed no

variation during fruit bud differentiation period. nevi and Tyagi (1991) suggested

that the leaves of flowered shoots of mango contain higher amount of non-reducing

sugars and total soluble sugars where as the leaves from the non-flowered shoots

contain a higher amount of reducing sugars and starch.

An increase in protein content takes place during certain stages of cell

growth especially flowering (Pearsall and Billimoria, 1938; Kursanov and Rryushkova,

1940). The increased protein content is correlated with increased metabolic activity

(Brown etnl., 1952). Semenenko (1963)observed adecrease in the protein, RNA and

DNA contents towards the end of maturation period. Josef et al. (1966) have shown

an increased protein synthesis towards the maturity of Phaseolus vufgaris. During the

ripening of wheat and pea seeds the content of nucleic acids and proteins increased

at the same rate initially but the production of protein quickly surpassed that of

RNA and DNA (Chinoy et al., 1969). In TrigoneUa, a higher level of protein wns

observed in 2-week-old seedlings as compared to 8-week-old seedlings (Jain and

Agrawal, 1987). The total soluble prolein content in the roots of cowpea was

minimum during the initial stage (55 days after sowing), then there was an upsurge

33

at 70 days after sowing and it declined at 85 days after sowing of the seeds (Dayal

andBharti, 1989). The total protein content in mango increased in the post frlit bud

differentiation stage and showed an increasing trend in the differentiate81 buds

whereas the undifferentiated buds maintained the same concentration of protein

which was observed in the buds during fmit bud differentiation (Patil et d., 1990).

A rise in the catalase and pemxidase activities in the reproductive shoots of

grape vine has been noticed by Srinivasnn and Rao (1971). Vora and Vyas (1971)

suggested that where there is intense cellular activity involving cell divis on and

differentiation, the activities of catalase and peroxidase are increased. The catalase

activity of rice leaves decreased during senescence (Kar and Mishra, 1976). Vora

(1978) reported that the catalase of oats leaves was enhanced by long dly (LD)

treatment in vegetative and transforming apex as well as in the spikelet and the

activity lowered in stamens and carpels. The peroxidase activity of transforming apex,

stamens and milky grains was also enhanced by LD treatment. He further suggested

that the catalase and peroxidase activities are greater during the transformation stage

which actually mark the initiation of flowering. On the other hand, the pc roxidase

activity of Cicer adentinurn decreases in the flowering stage and at maturity (Jaiwal

and Sigh, 1989). In this respect, Parish (1968) reported that, the peroxidas: activity

has been found to be well connected with senescence. Patil et al. (1991 b) reported

that the peroxidase activity in mango increased significantly during the ons~:t of fruit

bud differentiation and decreased thereafter. They further correlated th~! specific

activity of peroxidase with reducing sugar content during fruit bud differentiation.

34

E. Phenols in Plant growth and Metabolism

There is still considerable uncertainty as to whether phenolic compounds

have a physiological role in plant growth and metabolism. The phenols are known

to affect the biosynthesis of ethylene. It is known that a p-coumaric acid ester was

anecessary co-factor for ethylene biosynthesis from methionine in cauliflower florets

(Mapson, 1970). Many phenols are able to exert considerable effect on growth

processes when applied exogenously to plant tissue at physiological concentrations

(Kefeli and Kadyrov, 1971; Kefeli and Kutacek, 1977; Kefeli, 1978; Ray, 1983). It

may be noted that, tannins have generally been shown in plant systems to have an

antagonistic effect on GAS activity (Corcoran et al., 1972). Nanda et al. (1976)

studied the effect of GA, and some phenolic compounds on the flowering of

Impatiens bdsanuha Harborne (1980) has reported that, in vitro, flavonoids with a

catechol R- ring have a sparing effect on IAA by inhibiting IAA-oxidase activity and

theoretically they have a stimulating effect on plant growth. By contrast, flavonoids

with a monohydroxy phenol 13- ring uniformly augment enzymic activity and thus

have a potentially inhibiting effect on growth (Stenlid, 1976 a). Related hydroxy

cinnamic acids also have similar effects. The in. ntro effects of phenolics on the

pathway of auxin biosynthesis from anthranilic acid and tryptophan have also been

demonstrated, but in vivo significance of such interactions have yet to be established

(Kefeli and Kutacek, 1977). Turetskaya et al. (1977) have made attention to study

the effect of phenolic compounds on the IAA-oxidase enzyme causing auxin

destruction. There are evidences that dihydroconiferyl alcohol in lettuce has a

35

synergistic effect on GAS-stimulated elongation of hypocotyl (Kamisaka and Shibata,

1977). By contrast, substitution of diconiferyl alcohol by any one of several common

hydroxy cinnnmic acids reverses this effect. IIowcvcr, informntion rcgnrding the

endogenous level of plant phenols due to the effect of exogenous application of

plant growth substances is limited. There are reports that the plant phenols may

affect growth by interaction withphytohomones such as auxins (Kefeli and Kutacek,

1977). Ray and Laloraya (1984) have suggested that the biosynthesis of phenolic

compounds can be activated in the presence of light. It has also been reported that

there is relation between the endogenous production of total phenols and the effect

of phytohormones like GAS (Raysand Laloraya, 1984; Jain and Agrawal, 1987).

It is also evident Chat phenolics may have indirect effect on physiological

processes of plant growth. Many phenols are capable of inhibiting ATP synthesis in

mitochondria, uncoupling respiration and of inhibiting ion absorption in roots

(Stenlid, 1970). The biosynthesis of phenol in plants is linked with glucose and its

breakdown. In higher plants the production of phenols is mediated through acetate-

mevalonate pathway and also through shikimic acid pathway. All these components

are derived from glucose breakdown (Hess, 1975). There are reports that flavonoids

affect the polar transport of auxins (Stenlid 1976 b) and the protoplasmic streaming

in root hairs (Popovici and Re*, 1976). Certain phenolics particularly caffeic acid

esters and flavonoids occur in plant chloroplasts (Saunders and Mc Clure, 1976) in

small amounts and they may have a possible function in photosynthesis or on the

effect of light on plant processes. The phenolic constituents are capable of uv

absorption thus providing protection from uv radiation.

36

The peroxidase and polyphenol oxidase are involved in disease mistance in

plants and are known to degrade phenols to quinones which are reported to be more

fungitoxic than phenols (Le Tourneau et d., 1957; Batra and Kuhn, 1975). It has

been reported by many authors that the phenolic compounds play an important role

in defence mechanism of plants against various diseases (Swain, 1977; Friend, 1979;

Bell, 1981; Nicholson and Hammerschmidt, 1992). The role of polyphenol oxidase

and phenols in plants has been reviewed by Mayer (1987).

Section 2. Spilanthes ciliata H.B.K.

F. Hormonal and thermal regulation on the rooting of stem cuttings

1. Role of Growth regulators

There are a number of reports to show that root initiation and regeneration

of stem cuttings of various plant species are differently aflected by various growth

regulators (Nanda et d., 1972; Gupta et d., 1975; Abraham and Fapohunda 1981;

AJ'Kinany, 1981; Gupta et d., 1984; Iliev et al., 1984; Singh and Paliwal, 1985;

Trivedi and Sigh, 1988; Panda and Das, 1989; Sharnet and Dhiman, 1991). Nanda

(1975, 1979) reviewed the seasonal rooting response of varying concentrations of

different auxins and other plant growth substances in different plant species with a

view to understanding the physiology of adventitious root formation. Baadsmand

(1983) made an extensive survey on the application of rooting hormones.

It is generally accepted that auxin has a central role in the initiation and

development of adventitious roots. Auxins have attracted the attention of many

37

workers because of their marked influence on the root formation of cuttings. It was

originally shown by Went (1934) andThirnann and Went (1934) that auxins stimulate

adventitious root formation instern cuttings. Later, Thimann (1935) and Zimmerman

and Wilcoxon (1935) reported that NAA and IBA are also active in promoting

adventitious root formation of cuttings. It is reported that the synthetic auxins like

IBA and NAA are more effective than IAA in rooting. (Hitchcock and Zimrnennan,

1940; Evans, 1953; Vanonsem, 1953; Fernqnst, 1966; Kawase and Matsui, 1980;

Geneve and Heuser, 1982). The above findings have been extensively substantiated

by many others. Some workers have suggested that the rapid root stimulation by

exogenous application of auxins is due to some favourable biochemical changes

taking place in plant cells (Nanda and Anand, 1970; Nanda, 1975; Gupta et af.,

1975). The maximum rooting of cuttings occurs when high concentrations of auxin

are given soon after cuttings are made (Shibaoka, 1971; J a ~ s et Ill., 1983) and

symptoms of toxicity have been induced just below such concentrations (Jackson and

Hamey, 1970; Middelton, 1977, Middleton et a/., 1980). The treatment of cuttings

with low concentrations of supplied auxin results in the retention of 'sensitivity' to

high doses of auxin (Shibaoka, 1971). This represents an example of auxin binding

activity being induced by the exogenous supply of auxins has been found in other

tissues (Trewavas, 1980). Auxins comprise the only group of chemicals which

consistently enhance root formation innaturally responsive or so called easy-to-root

cuttings (Haissig, 1974, 1982). In such cuttings, auxins invariably induce the

formation of a greater number of roots per cuttings than other chemicals. There are

reports that the exogenously supplied IAA does not persist at high concentrntions

38

in the region of regeneration through out the entire period required for the

formation of root primordium (Brunner, 1978; Kefeli, 1978). It has also been

reported that high concentrations of auxin, even when applied for a period as short

as 30 minutes initiates cell division and controls the organisation of primordim

(Kantharaj et d., 1979). Non-woody cuttings usually show high response to supplied

auxins (Hartmann and Kester, 1983; Haissig, 1986). A progressive decline in the

response of cuttings to exogenous auxin is evident if they are kept in water for

longer periods prior to treatment with high dosage of auxin. The supply of awin at

a very low concentration partially overcomes the effect of such declining response

(Jarvis et d., 1983). Jawis (1986) reported that the basis of such declining response

is unknown. Furthermore, excessively high concentrations of auxins inhibit growth

of primordia or even root initiation itself (Jarvis, 1986).

Gibberellic acid has been widely reported to inhibit root formation in cuttings

of a number of plant species (Brian and Randley, 1955; Kato, 1958; Brian et d.,

1960; Fernqvist, 1966; Fabijan et d., 1981). Such inhibition is particularly evident

when gibberellii is supplied before or soon after cuttings are made (Brian et d.,

1960). There are reports that Gibberellin antagonists or inhibitors of gibberellin

synthesis (viz., EL. 531, CCC and AM0 1618) have enhanced the adventitious root

formation in cuttings (Libbert and Krelle, 1966; Kefford, 1973; Fabijan et d., 1981).

However, root formation may also be stimulated by the application of GA, (Nanda

et d., 1967; Altman and Wareing, 1975; Hansen, 1975). A very low concentration of

GAS has also been found to promote the root initiation of cuttings (Eriksen, 1972;

Hansen, 1976). Such a situation is dependent upon the conditions of irradiance

39

during the growth of the stock plant from which cuttings are taken. In the leaf

cuttings of Phaseolus vulgaris, root formation is stimulated by the treatment of

primary leaves with GA, prior to the excision of the cuttings, but application of

TIBA below the pulvinus inhibits the effect of GA3 (Varga and IIurnphries, 1974).

In cuttings of pea, the rooting has been stimulated in response of GA3 application >

under relatively low irradiance, which itself is beneficial to root formation (Hansen,

1975, 1976).

However, reports on the rooting response of cuttings with the application of

ascorbic acid are scanty. Bove (1957) noted the beneficial effect of orange juice with

NAA and aneurin for the rooting in Cifn~s cuttings. Cailahjan arid Nekrasova (1958)

induced the rooting in lemon cuttings with NAA and heteroauxin plus ascorbic acid.

Abraham and Fapohunda (1981) reported an enhancement of rooting in cuttings due

to the application of 100 ppm ascorbic acid in Onion, Cassava and Bougainvillea.

2. Temperature effects

The rate of many plant metabolic processes is controlled by temperature and

hence it is not surprising that temperature influences the root stimulation in cuttings.

A decrease of hydrolysing enzyme activity by lowering of temperature has been

reported (Alexander 1938; Nanda and Anand, 1970; Gupta et al., 1975). Wassink

(3953) reported that an increase in temperature modified the hydrolytic enzyme

activity to a maximum at a temperature of 40°C. Heide (1964) tested a range of

temperatures for the stock plants and for cuttings. He found that generally longer

rodts are formed with higher propagation temperature. Heide (1965) also reported

that, two weeks of low temperature (lB°C) followed by a longer period with high

temperature (27OC) increased root length (but not root number) as well as the

number of adventitious buds in Begonin leaf cuttings. In the cotyledons of

Sjnapsjs albn, Moore et d. (1975) found a higher root initiation at 25 to 30°C, an

inhibition below 25OC and elimination above 35OC. These authors suggested that the

temperature might influence the translocation of supportive and inhibitory factors

and also stimulate the mitosis in the rooting zone. Dykeman (1976) tested the

rooting of Chtysanthemluo and Forsythia cutting at 25 and 30°C. More rapid rooting

and more roots per cuttings were obtained at 30°C, but root elongation, root

diameter and root hair development are found superior at 25OC. Thus he suggested

that higher temperatures favour primordium initiation where as lower temperatures

favour root developxnent. Anderson et d. (1975) and Andersen (1986) hnve also

suggested that the optimal temperature for rooting were considerably higher for the

cuttings taken from high temperature stock plants.

The carbohydrate content and catabolism of cuttings are influenced by

temperature and these factors have important implications for rooting. Ooishi et d.

(1978) found that the effect of temperature on cuttings might be mediated through

carbohydrate metabolism and that rooting occurred in direct relation to temDerature.

i.e., 16, 36 and 87 percent of cuttings rooted at 17, 23, and 30°C respectively. The

respiration rate at the rooting zone was also positively associated in the above work.

They further reported that the peak respiration rate during rooting occurred earlier

and became higher with increasing temperature. Agnirl Veierskov and Andetsen

41

(1982) reported that the carbohydrate metabolism might be one of the supporting

factors within pea cuttings and it is found to be dependent on temperature. Hence

the above authors concluded that the beneficial effect of higher temperature on root

initiation might be due to the related increase in respiration and the catabolism of

simple sugars that would have been stored at lower temperatures.

There are reports to show that the carbohydmte concentrations may also be

influenced by treatment with auxin. Treatment wlth auxin can enhcuice mobilization

of carbohydrate from the leaves and upper portion of the stem by an increased

transport to the rooting zone (Nanda and Dhaliwal, 1974; Weyland, 1978; Orton,

1979; Hyndman et al., 1982; Gislerod, 1983; French, 1983). However reports on the

cumulative effect of high temperature and its interaction with growth regulators are

scanty (Heide, 1965).