Proc. Indian Acad. Sci. ( Sci.), Vol. 93, No. 3, July 1984, pp. 253-274 Printed in India.

Physiology of bud growth and opening

H Y MOHAN RAM and I V RAMANUJA RAO Department of Botany, University of Delhi, Delhi 110007, India Al~traet. Flower growth and opening are commonplaceevents, but physiologicallyintrieate and inadequately explained. In .this review, we have brou8ht together and evaluated informaª on this subject to for attention on the dynamicfacets of flowerdevelopment. In particular, the physiolo8icalbasis of flowerbud dormancy,nature of cleistogamy,mechanism of flowerbud growth and turgor maintenanceand role of in corolla growth have been examined. The regulation of flower movementsand openingby temperature and light, and circadian rhythmsin floweropening have beendiscuss~ alongwith a considerationof the role of the epidermis in light pereeption. It is emphasized that studies on flower physiologyneed to be intensified in view of the lacunae in our basic knowledgeas wellas to providea sound basis for improvingyields of both agricultural and hortieultural crops. Keywords. a-amylase; blossom showers; coroUa;flower bud dormaney;flower bud growth; flower opening; flower movements; flower physiology;invertase; stamens; coffee;gladiolus; Kalanchoe; Lamiwn; Pharbitiy, Ruellia; Turnera.

1. Introduction

Angiosperms constitute the dominant and most ubiquitous group of vascular and occupy a wide range of ecological habitats. The presence of a flower that bears ovules concealed in a hollow ovary makes them unique. De¡ what a flower is and how it carne to be established in the long course of evolutionary history have engaged the interest of generations of botanists. Lack of fossil evidence and the deep gaps between what is presently considered as the most primitive flower and the most highly evolved anthostrobilus of fossil ancestors are serious handicaps in traeing the origin of the flower. Several widely divergent viewpoints have been expressed in interpreting the fundamdntal nature of the flower, which has a varied structure and organization. Although angiosperms have breeding systems other than sexual reproduction, the main functions of the flower are: formation and release of microspores, reception of pollen, guidance of male gametophytes, gametic union, formation of seeds and associated dispersal structures and auxillary functions for the attraction or exclusion of animal vectors (Pijl 1978). display various adaptive mechanisms to speed up evolutionary responses to meet the selective pressures by promoting genetic recombi- nation and gene migration. On account of their economic, aesthetic and scientific value, flowers have captivated the attention of professional and amateur botanists alike. Naturalists have also been drawn to the study of flowers because of the close interrelationship between the latter and mammals, birds and insects. For a student of developmental botany the transition from the vegetative to the reproductive phase is a remarkable event. Despite the impressive amount of literature on flower initiation, our present knowledge about flower development, involving specific changes in gene function for the differentiation of floral organs in a close

253 254 H Y Mohan Ram and 1 V Ramanuja Rao succession on the floral axis is quite incomplete. The understanding of the inherent genetic mechanisms and their modification by the environmental and physiological factors are challenging questions for the plant scientist. A glimpse into the work of earlier scientists such as Pfeffer (1897), Goldsmith and Hafenrichter (1932), Ball (1933, 1936) and Bª (1929) on flowers displays originality of approach, a thoroughness in establishing facts anda penetration in understanding complex phenomena about flower structure, growth, movements and functions. The optical and scanning electron microscopic facilities, analytical tools, photographic appliances and the tissue culture technique available to the modern plant scientist ate so refined that dynamic phenomena like flower growth and development can be studied not only in depth but also in a multidisciplinary perspective. On account of limitation of space, only a few sr aspr of flower growth ate dealt with. Floral morphogenesis as understood through the application of in vitro culture (Johri and Ganapathy 1967; Mohan Ram and Jaiswal 1974; Konar and Kitchlue 1982) and experimental modification of flower sex have been covered elsewhere (Heslop- Har¡ 1972; Mohan Raro 1980; Durand and Durand 1984). As corolla generally constitutes the most conspicuous part of the flower, corolla expansion and divergence are considered synonymous with flower growth. In the account that follows, material from classical works that have remained remarkably novel are drawn to illustrate poignant points and advances made in the recent years are discussed at appropriate places.

2. Genetic and environmental regulation of bud dormancy, corolla growth and opening

2.1 Bud dormancy

2.la Low temperature induction ofgrowth: In most plants flower buds, once initiated, follow a continuous course ofdevelopment leading to anthesis and fruit formation. In some perennial plants especially the temperate deciduous trees, flower buds undergo a pr ofdormancy. Bud dormancy is broken by exposure to low temperatures. Under natural conditions chilling (1 -10 ~C) in winter fulfils this requirement. Ir the winters ate mild and do not extend over a sufficient length of time, the buds rail to biossom and set fruit. In sour cherry, bud growth is arrested after the and petal primordia have been formed, anthers areat the stage of sporogenous cells and ovule primordia have not yet been differentiated (Felker et al 1983). In sour cherry, flower bud chilling is accompanied by meiosis, first in the anthers and then in the ovule. Importantly, starch accumulation takes place with the progression of chilling. Ultrastructural studies indicate that du¡ dormancy, carbohydrate meta- bolism plays a more crucial role than nuclear events (Felker et al 1983). However, in Rhododendron flower buds, starch accumulation occurs before the rest pe¡ and its utilization is postponed until the last weeks of cell expansion prior to anthesis (Schneider 1972). Temperature is an important factor affecting flower initiation and development in bulbous plants (Hartsema 19£ Low temperature promotes the translocation of gibberellin-like substances from the bulb scales of iris to the shoot apex (Rodrigu› Pereira 1964). Whereas the concentration ofgibberellins in the bulb scales does not vary Flower bud growth and opening 255 much, a larger bulb would contain more total gibberellins than a smaller one. Work on narcissus, tulip and hyacinth (see Rees 1972) has shown that flower initiation and differentiation takes place in warm temperatures, but flower maturation and anthesis are rapid only when the plants become exposed to low temperatures during of after flower differentiation. Although the precise role of gibberellins is not known, it has been suggested that in iris they are involved in flower initiation and flower stem development and in tulip in flower extension. In narcissus flower primordia may be laid down one year before anthesis. Thus at the time of planting, a bulb contains already formed flower buds. In Zephyranthes, flower initiation alternates with formation throughout the growing period. Thus tiny immature buds and large buds approaching the point of anthesis occur in a dormant state in the bulb. In Z. rosca flower growth and opening are promoted when the temperature is lowered in the presence of water (Kerling 1949). It is also important that the period oflow temperature exposure exceeds 10 hr. Kerling (1949) was of the opinion that low temperature caused auxin release which promoted pedicel growth. Holdsworth (1961) working with Z. citrina, Z. tubispatha and with two species of Pancratium showed that increased soil moisture after rains could be the factor trigge¡ gregarious flowering. The situation is thus reminiscent of that in coffee.

2.1b Role of moisture in breaking bud dormancy in coffee: In certain coffee growing arcas, flower buds undergo a period of rest prior to meiosis after attaining a certain stage of development. Bud opening occurs only after rer a rainfall--appropriately termed "blossom shower" (Went 1917; van der Meulen 1939; Mathew and Chokkanna 1961). In Chickmagalur arca in Karnataka, flower buds ate initiated in October and remain dormant until April the following year. Failure of blossom showers can cause a se¡ set back in coffee production. Leliveld (1939) had noted that meiosis in C. canephora was dependent on the occurrence of rain. Mes (1957a) showed that flower bud dormancy could be broken and meiosis triggered by submerging the bud-bearing branches of C. arabica in water of supplying water by other mcans. Water treatment of twigs bearing buds has now become a standard method for studying meiosis in Coffea spp (Sreenivasan 1983). According to Alvim (1958) the moisture effect is also dependent on temperature, higher temperatures being more productive. Whereas Mes (1957b) believed that dormancy was caused by water stress in the young flower buds, Alvim (1960) observed that water stress was not the cause of dormancy but a condition necessary for overcoming it. It was only after experiencing a period of water stress that coffee plants become receptive to water, resulting in resumption of flower bud growth. Continuous growth under saturated conditions does not promote flower bud growth. The dormant coffee flower buds are covered with a gum-like substance (Gopal et al 1975). The coffee planters in India believe that this gum-like substance has some tole in flowering and that it has to first dissolve in the blossom showers before the buds can resume active growth. Chemical analysis of this gummy material has shown the presenc• of growth-promoting as well as inhibiting substances (Gopal et al 1975). Browning (1973a, b) has proposed that the resumption of flower growth may depend on the levels of endogenous abscisic acid, gibberellins and cytokinins in the flower bud. Gopal and Venkataramanan (1976) have observed that after sprinkler irrigation and during the enlargement of the flower buds, there is a marked reduction in the total 256 H Y Mohan Ram and I V Ramanuja Rao content of growth-inhibiting substances with simultaneous increase in growth- promoting substances. Spraying of gibberellic acid (Alvim 1958) was effective in promoting the opening of flower buds. Alvim (1958) has suggested that the effect of moisture supply is through syntbesis or activation of hormones (probably gibberellins).

2.2 Cleistogamy In a majo¡ of flowers the corolla opr to expose the stamens and the pistil (chasmogamy). There are some flowers in which the corolla remains permanently closed (cleistogamy). Production of both thesr floral forms is noticed in 287 spr br to 56 families (Lord 1981). A marked reduction in the size of the corolla and stamens distinguishes the cleistogamous flowers. Cleistogamy #ves rise to offsp¡ genetically very similar to the parents. However, the advantage of cleistogamy is that genes responsible for a local adaptive pr will be brought to a high localized frequency (Stebbins 1970; Williams 1975). Cleistogamy functions to slow or moderate evolutionary response (Thompson 1976). The role of chasmogamy is to speed up evolutionary responses to changing selection pressures by promoting both gene migration and reassortment (Thompson 1976). Many spr of Viola produce large quantities of seed by means of obligately cleistogamous (sr flowers (Clausen et al 1964; Beattie 1978). Gopinathan and Babu (1984) have reported the occurrence of a population of Viona minima the wild progenitor of rice bean--which bears obligate subterranean cleistogamous flowers. A genetic basis for cleistogamy has been suggested for Salvia cleistogama (Burck 1906), Con~elina spp (Uphof 1934), Carthamus tinctorius (Dille and Knowles 1975) and sinuata (Lee et al 1976) although its expression is sensitive to environmental factors (Uphof 1938; Khoshoo et al 1969; Lee et al 1976). In a study on Ruellia Khoshoo et al (1969) showed that during winter, the plants bear only cleistogamous flowers. The pr0geny of a cross between R. tweediana and R. tuberosa yielded a hybrid which was completely sterile and totally cleistogamous (Khoshoo et al 1969). Interestingly, gamma ray irradiation of the hybrid resulted in induction of chasmogamy (Raghuvanshi et al 1981). Hybrids formed between three tropical American spr of Ruellia ( R. tuberosa, R. nudiflora and R. brittoniana) were also completely cleistogamous but partly fertile (Long 1977). Whether the closed corolla of cleistogamous flowers is a consequence ora cause of cleist0gamy was debated by early workers. In Lamium amplexicaule Lindman (1908) observed that precocious maturation of antbers and early pollination could retard corolla expansion. Rr162 Lee et al (1978) proposed that a burst of ethylene production in the cleistogamous flowers of at pollination was responsible for arrested corolla expansion. However, Lord (198 I, 1982) showed that the destiny of the two floral forms is determined much earlier, with the corolla shape divergence occurring subsequent to antber divergente. Cleistogamous flowers produce a smaller amount of pollen and tbeir germination invariably occurs within the anthers (Staedtler 1923). Lord (1982) has re~ently noted that in L. amplexicaule, cleistogamous and chasmogamous flower primordia are morphologically and histologically indistinguishable prior to meiosis. With the onset of meiosis and by the time the microspore mother cells are in prophase I, the two floral Flower bud growth and opening 257 types manifest their difference as evidenced by the reduced number of microspore mother cells in the cleistogamous anthers. Although clearly noticeable by the beginning of meiosis, differentiation of the two forms must have occurred even earlier, since the microspore mother cells are reduced in number in the cleistogamous anthers. The cause of this earlier differentiation is still unknown. Lord (1982) has indicated that this developmental divergence in terms of cell division activity can now be narro wed down to a short phase between the beginning of anther differentiation and prophase I of meiosis. If lack of anthesis is excluded, the principal differences between the two floral forms is a slight quantitative modification in anther and corolla growth with calyx and remaining unaltered. Lord (1981) believes that some mechanism that dissociates development in the gynoecium/calyx with that of the androecium/corolla operates in cleistogamous flowers. It is now possible to modify development and expression in cleistogamous/chasmo- gamous flowers by hormone application. In cleistogamous L. amplexicaule (Lord 1980), Ruellia hybrid (R. tweedianax R. tuberosa: Raghuvanshi et al 1981) and Collimia orandiflora (Minter and Lord 1983), gibberellic acid (GAa) application induces chasmogamous flowers, without altering anther morphology. Ir is interesting to recall that water stress had been shown to increase cleistogamous flower production in Stipa leucotricha (Brown 1952) and Bromus unioloides (Langer and Wilson 1965). Recently, Minter and Lord (1983) have shown that both water stress and application of abscisic acid (ABA) induce cleistogamous flowers, causing a reduction in both total flower number and chasmogamy. These authors have proposed that anthers are the target organ for hormonal control whose size is determined by the relative levels ofABA and GA s in the floral . This contention disagrees with the earlier observation (Lord 1980; Raghuvanshi et al 1981; Minter and Lord 1983) that anther morphology is unaltered. The decrease in corolla size in cleistogamous flowers is attributed to reduction in anther tissue (Lord 19~/9) since gibberellin application results in normal corolla expansion (Lord 1980; Raghuvanshi et al 1981). There is evidence to suggest that gibberellins produced in anthers are involved in corolla expansion (Murakami 1975; Rajan Bala 1982). It may be inferred that the gibberellin levels in the small anthers of cleistogamous flowers are too low to permit anthesis (Lord 1981). There is no expe¡ evidence to believe that anthesis is dependent on gibberellin levels. Once normal corolla growth is attained through enhanced availability of gibberellins, anthesis should follow asa normal consequence. Among hydrophytes, cleistogamy has been recorded in several submerged and floating-leaved species (see Sculthorpe 1967 and references cited therein). In these the closed and submerged flower buds undergo self-pollination. Ottelia ovalifolia bears normal aerial flowers in summer and small, submerged and cleistogamous flowers in sp¡ or autumn or when the plants are overcrowded (Ernst-Schwarzenbach 1956). Autogamy occurs and the resulting fruits bear fewer seeds than the normal fruits. Whether or not photoperiod regulates the formation of the two types of flowers in this plant is not clear. In some other submerged hydrophytes such as Subularia aquatica, Blyxa alternifolia and Ottelia alismoides, self-poUination occurs within the closed flower buds, but the buds subsequently open (see Sculthorpe 1967). It was pointed out earlier that in many land plants cleistogamy results from water stress. The occurrence of cleistogamy in submerged water plants needs to be eXplained in physiological terms. 258 H Y Mohan Raro and I V Ramanuja Rao

3. Energy reqnirement and turgor mainten-nce dnring flower bud growth

The magnitude ofcorolla growth va¡ with the type of flower and its ultimate size. In gladiolus, for example, the corolla adds nearly 16 times its fresh weight and over seven times its dry weight between the time the outer bract separates out until the corolla attains its full expansion (Rao 1979; Rajan Bala 1982). Ah entire mechanism has to be built up and sustained in the corolla to support the massive inflow of materials, since it is largely a non-photosynthetic organ. There are several reports which suggest that flowers are strong sinks for assimilates (Harris and Jeffcoat 1972). In gladiolus the constitutes the main sink until anthesis (Ginzburg 1974; Robinson et al 1980; Shillo and Halevy 1981). Thus spikes harvested one week before the first floral bud opens out need exogenous supply of sugars for subsequent flower growth and opening (Rao and Mohan Raro 1981). Shillo and Halevy (1975) have shown that short days and reduced light intensity enhance flower abortion in gladiolus. Low light also impedes the development of inflorescence by increasing the competing ability of the corra for available assimilates. Flower bud abortion or 'blasting" occurs asa result ofcompetition for nutrients. For example in Easter lilies, Einert and Box (1967) showed that with increase in flower number per inflorescence, increased flower bud abortion took place. Smith and Langhans (1961) traced bud abortion to sudden moisture deficits. However, Mastalerz 0965) demonstrated that a reduction in the translocation of carbohydrate to the inflorescence caused by cooling the bud pedicel or bud removal promoted abortion. A direct dependence of flower bud abortion on the duration of darkness was also recorded. Working with lilies, Schenk and Boontyes (1970) noted that enclosure in the dark for one week caused bud blast and bud abscission in 27 and 72 ~ of the cases respectively. In tulips and narcissus a rapid mobilization of reserves from the bulb to the shoot (amounting to three quarters of the bulb weight) occurs from sprouting of the bulb to flower opening (see Rees 1972). Early flowedng stages in several plants such as bougainvillea (Hackett and Sachs 1966), iris (Mae and Vonk 1974), tomato (Kinet 1977) and tose (Mor and Halevy 1980) ate sensitive to light, and buds abort under low light conditions. Whether abortion of flower buds under low light intensities is caused by lower supply of photoassimilates or by a weakening of flowers as sinks because of hormonal of other factors leading to reduction in competing ability, has not been resolved satisfactorily. In carnations Harris and Scott (1969) showed that low light caused decreased rates of flower growth, but the rate of flower development was independent of the levels of assimilates available. These workers suggested that the processes controlling flower development regulate the partitioning of assimilates I~etween the flower and the test of the shoot system. Mae and Vonk (1974) concluded that bud abortion under low illumination cannot be attributed to lack of photosynthates. According to them hormone imbalance plays a key role in the assimilate distribution pattern and consequently in bud abortion, since application of hormones increases flowering. Kinet (1977) working with tomato also noted that light conditions were critical from the time of appearance (macroscopically visible) of the inflorescence onwards. He showed that the develop- ment of the inflorescence could be stimulated by direct application of benzylamino- purine and gibberellins under low light conditions. Hormone treatment favoured assimilate distribution to the inflorescence at the expense of young . The work of Goldschmidt and Huberman (1974) has shown that wounding or application of Flower bud growth and openin# 259 ct-naphthaleneacetic acid to a single petal of citrus causes an increase in accumulation of ~4C-sucrose. Wounding of one petal causes accumulation in the remaining , often at the expense of the stamens. Light has a promotory effect on the translocation of assimilates to reproductive sinks. For example in roses the terminal flower bud degenerates ifit is enclosed in dark. Young rose shoots (2-3 cm long after ftower initiation) are most sensitive to dark treatment (Mor and Halevy 1980; Mor et al 1980). It has been suggested that this occurs under conditions of low photosynthate availability, since the developing inflorescence is a weak sink that cannot compete effectively with vegetative sinks. It has been proposed that the rate ofcarbohydrate flow from source to sink is related to rates of activities of enzymes such as amylase and invertase (Hofmann et al 1963; Wª 1974; EI-Fouly and Garas 1974). Fawzi and EI-Fouly (1979) reported that following morphological initiation of floral primordia, amylase and invertase activities as well as total soluble sugar content increased in the shoot apex of carnation plants. In tomato also, the concentration of reducing sugars increased with floral development (Russell and Morris 1982). This was accompanied by an increase in acid invertase activity which rose to a maximum before anthesis. Flower bud abortion was correlated with reduction in the rate ofdry matter accumulation, reducing sugars/sucrose ratios and in acid invertase levels. These workers suggested the operation of an invertase-mediated unloading mechanism for transported sucrose in the flower buds and noted that the transport of sucrose to the inflorescence might depend on the production of invertase rather than rice-versa. Limitations in invertase production under conditions of low photoassimilate availability would limit the import ofcarbohydrates and thus lead to flower bud abortion. Ho and Nichols (1977) reported that the growing corolla in roses continues to import dry matter throughout its development and that reducing sugars (sucrose does not accumulate) and starch account for 50 ~ of its dry weight. According to these workers, hydrolysis of stored starch is probably associated with flower opening in so far as decrease in water potential favours influx of water into the corolla tissue and promotes cell enlargement. In Lilium corceum the concentration of reducing sugars in the flower bud also increases until anthesis, accounting for over 30 Y/oof the dry weight of the corolla (Combes 1936). Reducing and non-reducing sugars account for 40-60 % of the total dry weight of the ray florets in chrysanthemum (Pardha Saradhi, unpublished work). Sacalis and Durkin (1972) have also shown that sugars fed to cut roses and carnations accumulate largely in the petals, although probably only a small proportion is used metabolicaUy. The function of sugar which is not directly involved in metabolism is unknown. Other than the obvious osmotic function in maintaining the turgidity of the petals, a part of the accumulated sugar in the petals is secreted as nectar. In gladiolus this occurs on anthesis and is also related to senescence at a later stage (Rao unpublished work). In gladiolus r and acid invertase activities increase with the progression of corolla development and have a positive correlation with the amount of carbohydrates (Rajan Bala 1982). The decline in starch in late developmental stages can be correlated with the sharp rise in ct-amylase activity and reducing sugar content. It may be inferred that the reducing sugars being osmotically active cause an influx of water, causing turgescence of expanded corolla (Winkenbach 1970; Rao and Mohan Ram 1980). Similar correlations between reducing sugar content, invertase activity and growth have been reported in the petals of Ipomoea purpurea (Winkenbach and Matile 1970) 260 H Y Mohan Ram and I V Ramanuja Rao carnations (Hawker et al 1976), rose (Ho and Nichols 1977) and in tomato inflorescence (RusseU and Morris 1982). In gladiolus, a-amylase is formed exclusively in the petal epidermis on perception of light. The enzyme is transported to the ground parenchyma where it hydrolyzes the extensive starch reserves (Rao and Mohan Raro 1980). In this respect it is comparable to the barley grain system. In barley, a-amylase synthesized in the aleurone layer causes the breakdown of starch stored in the endosperm. Whereas the barley grain represents a fulty differentiated but quiescent system activated by gibberellin emanating from the embryo asa result of hydration, the gladiolus petal is a dynamic system triggered by light. The production of a-amylase is also regulated by the sequential basipetal differentiation of the epidermal cells (Rao 1982). In gladiolus, the system of overlapping outer bracts which completely enclose the flower bud and their gradual separation represents a mechanism programmed for sequential exposure of successive buds to light and stimulation of a-amylase to ensure ah orderly development of the buds. The outer bract has been shown to actas a natural qualitative light filter and regulates the production of~t-amylase and petal growth by a red/far-red control (Rao 1982; Rao and Mohan Raro unpublished work). A critical stage in flower bud growth in the spike ofGladiolus which is initiated by gibbr acid and sustained by sucrose has also been identified (Rao and Mohan Raro 1982). This corresponds to the stage at which separation of the outer bract occurs, leading to light induction ofct-amylase (Rao and Mohan Raro 1980). Buds not induced by light were shown to respond more significantly to GA 3 and sucrose than those induced by light. Since the separation of the outer bract results in light-induced ~t-amylase production and starch hydrolysis leading to petal growth, it has been proposed that growth promotion by GA 3 is related to light-induced petal growth at this specific stage (Rao and Mohan Ram 1982). The demonstration in gladiolus (Rao 1982) that the growth of the petals at different developmental stages is a function of the levels of r,-amylase in those stages, strengthens the conclusion that the sugars released through ~-amylase activity are directly involved in petal growth. Light-mediated a- amylase production could thus be an important step in the formation of an active sink to draw materials from the rest of the plant. One important role of continued and sequential basipetal starch hydrolysis in the gladiolus petal (Rao and Mohan Ram 1980) could be to maintain a constant osmotic as well as sink potential in the growing arca of the petal, in spite of water uptake. Investigations on cell and organ expansion have been largely confined to vegetative parts. Information on cell expansion in floral organs is scanty. This may be traced to lack of good experimental systems rather than to absence of interest. In fact, production of larger flowers has been an important concern of flower growers, which would require the understanding of the basic mechanisms involved. Ah elegant method has been developed in this laboratory which involves the floating of ray florets (9 to 9.5 mm) of Chrysanthemum morifolium var 'Jyotsna', removed from the outermost whorl of young capitula in petri plates containing 30 mi of the test solution (Pardha Saradhi and Mohan Ram 1982). Using this technique it has been shown that KCI causes up to 33 % increase in elongation. The values for GA 3 and sucrose when used individually are 39.8 and 28.9 % respectively. Maximal growth response (82.8 ~) is recorded in KCI + GA 3 + sucrose. It is inferred that the increased turgor resulting from sucrose-promoted potassium uptake along with GA3-caused tissue extensibility accounts for the enhanced floret growth. Fiower bud growth and opening 261

N,N'-dicyclohexyl carbodiimide (DCCD), a potent inhibitor of membrane-bound ATPase strongly inhibits the growth of ray florets of Chrysanthemum even under in vivo conditions, showing the importante of membrane-bound ATPases in ray floret expansion (Pardha Saradhi, unpublished work). Ray floret expansion is also retarded by (2-chloroethyl) trimethyl ammonium chloride (CCC), an inhibitor of gibbereUin biosyntbesis (Pardha Saradhi unpublished work) implying that endogenous gib- br are involved in ray floret growth. The CCC effect can be overcome by simultaneous application of GA 3.

4. Role of other floral organs in corolla growth

In most investigations on flower growth, emphasis has been on the growth ofcorolla as it generally constitutes the most conspicuous part of a flower and since the changes occurring in it are dramatic. By and large, the role played by other floral organs in bud opening has been overlooked. For example, unfil recently, bracts were considered as organs meant for the protection of the immature floral buds. The discovery of the crucial role of the outer bract as a light filter in the trigge¡ of corolla growth in gladiolus (Rao and Mohan Raro 1980) demonstrated the close interaction between the different components of the flower bud. Histo¡ however, the stamens were the first floral organs shown to influence corolla growth (Lang 1961). DevelopmentaUy there is a correlation among the various floral organs. Factors which favour the development of the ovary also favour the and those promoting development also stimulate corolla expansion. In Glechoma hederacea, the hermaphrodite flowers have larger corollas than the female flowers (Plack 1957). Exposure to long days or application of ~t-naphthaleneacetic acid to Silene pendula plants caused male sterility and reduction in corolla growth, but promoted ovary development markedly (Heslop-Harrison and Heslop-Harrison 1958). In Bryophyllum and Kalanchoe low light intensity causes abortion of the stamens and reduction in corolla development while not affecting ovary growth (Resende 1949/1951). Whereas very little is known about the relationship of the sepals and the ovary, it can be concluded that the stamens have a strong influence on coroUa growth. The exact nature of this influence has not been defined so far. Thus the influence of ah important event like meiosis on corolla development has still not been understood. Observations on natural anther abortion and male sterility as weU as from experimental emasculation indicate that functional anthers are necessary for the normal growth and development of many flowers (Lang 1961). Male sterility and reduced petal growth are sometimes correlated (Michaelis 1954). Application of male gametocidal compounds such as mendok and dalapon cause not only abortion of the sporogenous tissue in the anthers but also result in poor growth and opening of the corolla in Linum (Rustagi and Mohan Raro 1971). Flowers of treated plants become progressively smaller and cleistogamy increases with increasing concentrations of the compounds. Kinoshita (1971) is of the opinion that a good deal of similarity exists in the mode of pollen abortion in chemically induced and genetically controlled male ste¡ plants. The tapetal cells generally become hypertrophied or radially elongated, occlude the anther locule and crush the microspores. In Tulipa gesneriana, spontaneous atrophy of stamens leads to a marked decline in mobilization of starch into the bud and is followed by the decay of the entire bud 262 H Y Mohan Raro and I V Ramanuja Rao

(Marr› 1946). Interestingly in many Euphorbiaceae, the male flowers havr petals whereas the female flowers are naked. It is important to recall a few exceptional cases such as banana and papaya in which the functionally female flowers bear larger pe¡ than the male flowers. Baker (1957) first pointed out the difference in size of pistillate and bermaphrodite flowers in gynodioecious and gynomonoecious plants. In Glechoma hederacea the female flowers on predominantly hermaphrodite plants and those on purely female plants ate smaller than the hermaphrodite flowers (Plack 1957). Sbe found that occasional flowers which have one, two and threr aborted anthers ate intermediate in size between the female and complete bermaphrodite flowers. Emasculation of the hermaphrodite flowers in bud condition reduces the size of the corollas in proportion of the number of anthers removexl, with the corolla of the flower in which all four anthers had been removed being similar in size to the normal female flower. Ir was proposed that a hormone was releasexi post-meiotically from the anthers since the critical stage for treatment in Glechoma hederacea was a 7 mm bud whereas meiosis is completed when the bud is 1 mm long. Wittwer (1943) had also found that the production of 'growth hormone' in staminate flowers occurs only after the onset of meiosis. According to Marr› (1946) the effect ofantbers on the growth ofother parts of the flower ce,ases wben the anther reaches matu¡ and the tapetum is used up. The ma~imal auxin production by the ovary of Papaver occurs when there is mei0sis in the megaspore motber cells and mitosis in the surrounding tissues (Katunskij 1936). In Soldanella mŸ the development of flower buds grown at 15~ of above became arrested at the stage of microspore mother cells in anthers (Lona 1968). This could be overcome by low temperature which not only promoted microsporogenesis but also subsequent bud growth and opening. Thus, microsporogenesis in this plant must be considered as the phase initiated only at low temperature. Application of gibberellic acid could substitute for the low temperature requirement of flower buds. Lona (1965) detected gibberellin-like activity in methanolic extracts of Corylus avellana pollen and that of other plants after cold treatment. However, the efficiency of GA3 in promoting flower bud growth till full opening in plants where sucia 'intra-reproductive vernalization' is necessary is not always equal to that obtained in Soldanella, Saxifraga, etc. Thus, the development ofDraba azoides flower buds till anthesis can be induced by gibberellin applications only with great difficulty. Some other plants do not even respond. According to Lona (1965) it is possible that other compounds may also be responsible for the "vernalization process" in these plants and that flower development may be controlled by a sequential complex of morphogenetic substances such that GA alone cannot mimic the cold effect. The promotion of corolla growth by gibberellin was first demonstrated by Plack (1958) in Glechoma hederacea. Application of GA3 to emasculated flowers was able to replace the stamen effect. She proposed that anther-dedved gibberellin may probably be important in normal maturation of some flowers. Indoleacetic acid (Plack 1957, 1958), ~-naphthaleneacetic acid, triiodobenzoic acid and L-proline did not have a similar effect. Lindstrom and Wittwer (1957) also observed an increase in petal size of Pelargonium hortorum treated with GAa. Blake (1966) reported that gibberellins ate involved in the growth of Viscaria candida flowers. Staby et al (1972) showed that 90 9£ of terpene-synthesizing potential of cell-free extract leading to gibberellin synthesis of young tulip buds was derived from stamens. Murakami (1973) also reported that removal of anthers from flower buds reduced the final length of the floral tube in Flower bud orowth and openino 263

Pharbitis nil. Murakami (1975) further demonstrated that anthers were a source of gibberellins in Mirabilisjalapa. The acetone-extractable gibbereUin-like compounds in petals and stamens of Pharbitis increased rapidly after the petals emerged from the calyx, reaching a maximum 12hr before anthesis and then declining markedly (Murakami 1973). The gibberellin content in stamens was relatively large in these plants and accounted for changes in gibberellins during corolla development. In Impatiens sultani, gibberellin and cytokinin levels increased during the transformation of a bud into a flower and later decreased after stamen abscission (Baltepe 1983). In Gladiolus intact stamens promote the growth of isolated corollas. The increasing indepr of the older stages without stamens in this plant suggests that the influence of stamens decreases as corolla growth advances although it does not stop. The influence of stamens on corolla growth appears to be through the production of gibberellins since the c¡ effect of stamens can be completely replaced by GAa or GAa + sucrose (Rajan Bala 1982). Rr Goldschmidt (1980)demonstrated that the ABA content ofcitrus petals which is initially low, increases gradually with a concurrent decrease in stamens as the bud development proceeded towards anthesis. Ethylene is detrimental for flower growth in bulbous plants. The stage of flower developme~t in tulips that is susceptible to ethylene is at pollen tetrad formation, which is attained by about four weeks after completion of flower differentiation. Perianth growth becomes stunted resulting in the producti0n of 'open' flowers in which the anthers are exposed (see Rees 1972). The smaller flowers are also short-stemmed than normal flowers. Ethylene has been shown to cause anther necrosis after completion of flower differentiation with the perianth frequently remaining green at anthesis. Alternatively the entire flower d¡ up ('blasting'). Even endogenous ethylene production by the anthers of hcalthy flower buds during meiosis (especially at higher temperatures), leads to flower death (see Rees 1972).

5. Regulation of flower movements and opening

As flower opening is an essential requirement for cross pollination, plants have adopted va¡ strategies to attain it at the most appropriate period in a given microenviron- ment. Although it has been recognized since long that flower opening is basically a result of greater expansion of the inner surface of the petals more than the outer, the means by which this is accomplished is diverse. Light and temperature are the most important factors that regulate flower opening, although it is to be expected that many factors may be operating simultaneously or successively. It is important to note that rapid turgor movements so commonly observed in the leaf movements have not been recorded in flower movements and opening. However, Pontecorvo (personal communication 1982) has observed that in some high altitude flowers growing in the Alps, closure of petals occurs almost within a few minutes when the sky above the flowers becomes covered by a passing cloud, causing a reduction in light intensity. As the clouds more away the flowers resume their open position. Light-sensitive turgor changes must account for such rapid flower movements and this aspect needs further study. It has also been observed in the capŸ of certain Compositae such as Helichrysum and Acroclinum that opening is caused by desiccation and closing by treatment with water. Although this is dramatically noticed in the dried capitula, clearcut movements can be observed in the intact capitula also. 264 H Y Mohan Raro and I V Ramanuja Rao

5.1 Sun-tracking flowers and flowers that bend up and down Several flowers follow the sun's movement from east to west. In the sunflower, Shibaoka and Yamaki (1959) showr that the east-west movement was a growth phenomenon of the stem. The movement is probably regulated by the auxin content of the sides of the stem which face or ate away from the sun. Placing the plants in continuous darkness does not terminate the east-west movements for sr days, thereby showing that a circadian rhythm is involved. Whereas the precise mechanism has not br162elucidated so far, in the leaves of the sun-tracking Lavatera cretica, a differential starch deposition in the lamina is thought to actas a "memory" for the le,aves (Fisher and Fisher 1983). Asymmet¡ deposits of starch are produced imm~liately after sunrise each day and these ate believed to be stored until just before the next sundse. The resulting solute asymmetries in the petiole are believed to produce anticipatory movements just before dawn. The up and down movcments of flowers have becn shown to be a function of the differcntial growth of the pcdiccl. In Kalanchoe blossfeldiana (Bª 1953), the movcmcnt of the buds occurs daily till they are opcn. Transferring of the plant to continuous dark docs not terminatc movement for several days. Sincc this is a growth phenomenon of the pcdiccl,the rcgulation of flowcr movcment could be depcndent on the auxin-auxin inhibitor levcls in the pcdicel, as shown by Kaldewcy (1957) in Fritillaria.

5.2 Effect of temperature on flower opening Investigations on the eff~t of tempr on flower opr were initiated during the sr hall of the 19th century. In Crocus and certain va¡ of tulip, flower movement was shown to be dependent on a small change in tempr and was nearly independent of light. Andrews (1929) found that in Crocus increase of only 0.2C was sufficient to initiate the opr movement. For tulips the tempr increase necessary was 1 C (Andrews 1929). A fall in tempr (which is about equal to that necessary for initiating opening) brought about flower closure. B6hner (1934) mentions a critical tempr below which tulip flow•rs have a tendency to close and abovr which they open. It is generaUy agreed that the opening and closing of flowers are not caused by turgor changes but are brought about by differential growth of the two sides of the tepals (Pfeffer 1897; Bª 1929). It was earlier believed that gentle warming of the inner surface of the tepal caused rapid growth, whereas the outer surface did not respond. Bª (1929) demonstrated that warming the outer surface of the tepal while keeping the inner one cool caused growth on the outerside. The closing results from a slower growth response of the inner side at lower tempr162 in comparison to the outerside. Crombie (1962) has written a critical review on thr in flowers. By removing one or both the epidermal layers of petals M~irkert (I 931) showed that the growth reaction (opening of closing) occurred chiefly in the mesophyll region. B6hner (1933, 1934) concluded that whereas the growth of the mosophyll cells was the p¡ mechanism, a controlling effect was exerted by the epidermal layers. There was little change in osmotic pressure anda permanent increase in length was obtained aftr every growth reaction (Bª 1929; B6hnr 1934). Wood (1953) concluded that the two surfaces of a tepal have different optima for growth. She found that the Flower bud growth and opening 265 mesophyll cells near the outer surface of the tepals of tulip hada temperature optimum for growth which was about 10C lower than that of the cells near the inner surface. This was true even when strips of mesophyll and epidermis were taken from the inner and outer sides. When the temperature was raised from 7C to 17C, there was a marked increase in growth of the inner strip whereas the outer strip did not show much response. Lowering the temperature, however, caused the outer strip to grow at ah increased rate while the inner strip showed little change. The reactions of isolated epidermal layers, although similar, were of a smaller magnitude than those of the corresponding mesophyU cells. A much greater growth response was found in complete tepals. Carbon dioxide was shown to have a marked effect on the temperature optimum for growth, especially in the inner surface cells, with a ¡ in CO2 concentration shifting the optimum to a lower temperature. Studies on Turnera elegans (Ball 1936), Cestrum nocturnum (Overland 1960) and S/da rhombifolia (Ugborogho 1980) have indieated that lower temperatures delay and high temperatures hasten flower opening. In contra.st to this general situation, Kaihara and Takimoto (1979) found that lower temperatures at dawn promoted earlier opening in Pharbitis nil and higher temperature delayed it. These authors showed that the temperature to which the buds were subjected during the light period modified the length of the dark period needed to promote rapid flower opening and that lower temperatures eliminated the suppressive effect of light on flower opening, such that at 20C the buds could open rapidly even in continuous light (Kaihara and Takimoto 1981a). It was also found that irrespective of when the low temperature treatment was begun, under continuous light all the buds flowered at about the same time. Kaihara and Takimoto (1981b) also determined that petals are sites of photo- and thermo- perception and that the curvature of the midribs is the primary force in flower opening.

5.3 Effect of li#ht on flower opening

5.3a Day-blooming flowers: One of the earliest and detailed inqui¡ into the mechanism of flower opening was on Turnera ulmifolia L. var. elegans Urb. by BaU (1933, 1936). In this plant the petals of a mature bud emerge from the calyx in the evening and elongate rapidly during the night. Anthesis begins 1 hr after dawn and the ttower is fuUy open in about another hour. The flowers close and wither within 3-4 hours. The closing of the flower is due to active hyponasty and is independent of withe¡ Light interruption of the dark pr inhibits flower opening. Illumination du¡ the second half of the night has a greater effect in inhibiting anthesis than that during the first half (Ball 1933). Buds which were one day younger and scheduled to open the second morning after receiving one night's illumination were affected such that even though the corollas in these buds were contained within the sepals during the night when iUumination was given, the flowers failed to open fully and the petals appeared crumpled at the tip (Ball 1933). He emphasized that in all cases the corolla expanded to its normal size but only its divergence did not take place. However, in Hedera helix Sigmond (1929) observed that flower buds illuminated du¡ the night failed to opr the foUowing morning. BaU (1933) showed that the petals and sepals ofTurnera buds contain abundant starch which largely disappears by anthesis. He attributed the rapid expansion of the petals to increased osmotic pressure resulting from staxch hydrolysis and elevated levels of 266 H Y Mohan Raro and I V Ramanuja Rao reducing sugars. In both petals and sepals, the hydrolysis of starch commenced at the tips and proceeded downwards. Exposure to light at night, which prevents buds from opening, impeded starch hydrolysis and the petals still contained large amounts of starch throughout the mesophyll. Ball (1933) emphasized that starch hydrolysis was more related to anthesis than to corolla growth. He stated "there may well be a causal relationship between the inhibition of starch and the absence of the epinastic expansion of petals" (Ball 1969). The inhibitory effect ofillumination on flower opening (also starch breakdown) was observed in several other plants (Ball 1933). Exceptionally, however, in Cassia occidentalis flower opening was inhibited by nocturnal illumination without affecting starch hydrolysis. Ball (1933) realized that mere hydrolysis of the existing starch reserves would be too insufficient to account for normal growth and opening of the flowers and that sugar movement from the rest of the plant was necessary. Du¡ the period of rapid expansion of the petals quick increments in fresh and dry weight were noted. Starvation of the shoots markedly reduced flower size. Continued starvation caused incomplete o~ning of the flowers. Schmucker (1928) suggested that carbohydrate changes might be responsible for the opening of flowers ofCereus grandiflorus and that the utilization of sugars during anthesis was indicated by a slight rise in flower temperature. Subsequently Ball (1936) observed that red light (650--700 nm) was as effective as white light in preventing flowers from opening, while blue light was ineffective. Red light inhibition was also correlated with lack of starch hydrolysis. Ott (1958) also recognized the importance of red light in causing flowers of Ipomoea purpurea to wilt without opening. Flower buds of Pharbitis nil ordinarily open at dawn. When the photoperiod during the previous day is 10hr or longer, the flower buds open after 10hr of the onset of darkness. But if the photoperiod is 9 hr or shorteri the buds open about 20 hr after the onset of the photoperiod (Kaihara and Takimoto 1979). Exposure to fluorescent light (daylight) in the first 4 hr ofdark period delays opening, but when given later, it has little effect. A 5 min pulse of red light given every 30min delays openin~ Far-red light reverses the action of red light. These observations suggest that the absence of Pfr during the first 4 hr ofdarkness is necessary for opening (Kaihara and Takimoto 1980).

5.3b Night biooming flowers: The last change from night to day appears to be the trigger for flower opening in night-blooming flowers and continuous dark inhibits opening in these flowers. Schmucker (1928) and Rauh and Zehender (1958) working with Cereus, and Arnold (1959) studying two species of Oenothera showed that the timing mechanism was pr0bably located in the buds, as each bud must be exposed to the dark to light transition before it opens. Schmucker (1928) observed that exposure of plants to light at night and keeping them in dark during daytime caused the normal pe¡ to become weaker gradually until it was replao6d by a new one in which the plants opened in the morning. Stomps (1930) working with a hybrid Oenothera, found that plants kept continuously in dark retained the original periodicity for 6-8 days. In Cestrum nocturnum (night queen) also opening and closing in a cyclic manner could be maintained in constant light or darkness. Emission of the strong scent at blooming followed a circadian rhythm (Overland 1960).

5.3c Physiological basis offlower opening: Ordinarily flower buds of Pharbitis nil cut at early developmental stages open slowly and at varying times. When ABA is given to Flower bud growth and opening 267

the cut buds, opening is rapid and occurs even in continuous light at 25 C (Kaihara and Takimoto 1983). These workers believe that ABA promotes flowr opening by r•setting the timing mechanism in a manner simulating dark treatment. Working with Kalanchoe, Schrempf (1980) proposed that ABA does not directly affect the circadian oscillator which controls opening time but rathr influences the drivr osciUator (pr movemr Thus ABA dampens the amplitudr of the pr movement rhythm but does not affect the period length of the circadian rhythm. Removal of ABA (I0-6M) and giving a light pulse increases the amplitude. Use of a high concentration of ABA (10-SM) results in permanent dampening of petal movemr He suggested that sinc• ABA was taken up in the transpirational stream, the results could be explained in terms of a rhythmic supply of ABA due to rhythmic transpiration (zur Lippr 1957; Schrempf 1980). ABA is supposed to act on ion fluxr (van Steveninck 1976) which correlate strongly with turgor changes leading to pr movement (Schrempf 1980). Interr Goldsr (1980) reported that the levr of ABA in the citrus flower increases in the cour~ of development. (Earlier, Goldschmidt 1968 had indicated that the br of citrus pr might be an auxin- mediated process). The circadian rhythm in pr movement of Kalanchoe blossfeldiana is the best studied flower system (Bª 1953). In this plant, the phasr curve to high temperature pulses resembled the light response curve (Engelmann et al 1974a) and closely approximated those generated by a feedback mathematical model for the circadian osciUator. The prr of lithium ions and deuteration were shown to slow the circadian clock in Kalanchoe petals (Engelmann 1972; Engelmann et al 1974b). In contrast to the proposal of Schrempf (1980) that rhythmic water uptake was the cause for circadian sensitivity in regulation of pr movement of Kalanchoe, Eckhardt and Engr (1984) have recently shown that rhythmic uptake of vanadate (an ATP ase activity inhibitor) is not sufficient to explain its timr162 phase shifting effect. The dose-effect-curvefor vanadate given at a phase when minimal delays are brought about should show larger phase delays at higher concentrations, but the pr is at the same concentration as at a phase where maximal phase delays ate induced. Through the use of vanadate, Eckhardt and Engelmann (1984) have dr that the plasmalemma ATP ase plays a role in the functioning of the circadian rhythm of Kalanchoe. 5.4 The function of the epidermis in light perception It would be pr at this stagr to consider the function of the epidermis in light pr sinc,r it would be the first pr layer to intercr light. The inhibition of opr by light documentr above must occur consequr to light pr by the epidermis followed by transduction of the stimulus through chemical or r mr to the physically responsivr part of the petal. A crucial structural and biochemical role of the epidermis in light pcrception leading to pvtal growth has ber demonstrated in gladiolus. Rao (1979, 1982) has shown that the epidermal cclls of petals of gladiolus possess microlenses. These are formr by the outward growth of the outer radial wall ofthe epidermal cells resulting in the formation of curved structures which are thicker in the middle than at the r These microlenses act to focus light specifically onto the nucleus which is situated in the central zonr of the inner radial wall of the cpidermal cells. P¡ to the developmcnt of this focussing microlens, the nucleus is randomly situatr in the epidermal cell and later

P--8 268 H Y Mohan Raro and I V Ramanuja Rao moves over to occupy the focal point. The development of the microlens is characteristic of differentiated cells; undifferentiated cells have an ordinary flat surface. Importantly, it is only after full differentiation as characterised by formation of the microlens that the epidermal ceUs become competent to be induced by light (Rao 1982). These then start producing ~-amylase which is later transported to the ground parenchyma wbere hydrolysis of the accumulated starch takes place (Rao and Mohan Raro 1980). The shape of the epidermal cells of the petals is useful in the absorption and re- radiation of specific bands of the spectrum (Kay et al 1981). This becomes crucial as the final colouration of the flower is c¡ in terms of insect vision in order to provide adequate guidance for their approach and alighting~ leading to poUination. Petal pigments such as anthocyanins and flavonoids are contained in the vacuoles of the epidermal cells and light is reflected back from an aerenchymatous, unpigmented mesophyll. In certain Ranunculus and Anemone species light is reflected from a specialized layer of starch grains (Kay et al 1981). Haberlandt, the celebrated physiological plant anatomist whose works and ideas were much ahead of his time (1914) had observed that the outer waU of epidermal cells frequently protrudes out in a slightly papillose manner and that in many shade plants, this tendency is accentuated such that large conical epidermal papillae are formed. He initially believed that the epidermal papillae might actas condensing lenses to concentrate the available light upon the chloroplasts of the palisade cells, presumably to increase photosynthesis. Later, he conceptualized that "lens-action" of epidermal papillae is primafily connected with the perception of photic stimuli. Stahl (see Haberlandt 1914) advocated that the conical epidermal papillae of velvety leaves enabled them to absorb oblique rays of light which would have been totally lost ir the outer epidermal cells were of the ordinary fiar type. The cave moss, Schistostega has peculiar epidermal cells in which 'the upper cytoplasm acts asa refractive lens to focus light on specially-o¡ chloroplasts (Richards 1932). Lee and Lowry (1975) observed that the epidermal cells of Selaginella wildenovii which are "egg-shaped" with a convex outer wall and having chloroplasts in a "peculiar position" distal to the leal surface, might also function as lenses similar to that in Schistostega. These workers further reported that the epidermal cell wall surfaces functioned as thin-interference filters causing ah increased reflection of photosyntheti- cally less active light (400-500nm) with increased penetration of the most photo- synthetically active range (600-650 nm). This is an important adaptation because light reaching the floor of rain forests in which these plants occur is extremely limiting and is deficient in photosynthetically active wavelengths. There is a growing recent interest in studying the role of epidermis in flowers. The full impact of the function of this bounding layer of the plant is yet to be fully realized.

6. Conclusions

The process of fiower growth and opening appears simple and commonplace. Yet physiologically it is intricate, involving an interplay of genetic and environmental factors. In adapting to a wide range of bioclimatic conditions, plants have displayed several mr to accomplish flower opening. Plants have also evolved strategir Flower bud growth and opening 269 to produce flowers best suited to the fluctuating seasonal conditions. Cleistogamy is a product of such an effort. The usefulness of studies on flower growth and opening discussed in this paper llave been grossly underestimated. Staple food is all seed and seeds are borne by sur flowers. It is, therefore, critir that a flower bud develops a strong sink capability. The fundamental studies disr by us have demonstrated that light, stamens, petal epidermis, ~-amylase and invertase forro important r in the development of such a capability. In floriculturr the application of scientifir principles and knowledge have yielded unprecedented dividends. Flower buds of some floral crops can now be handled, cold-stored and marketed like any other agricultural commodity. Left to the vagaries of nature, inadequate overcoming of flower bud dormancy could mean the failure of ah entire fruit crop. Can there be a greater attraction for work in the area of flower physiology?

Acknowledgements

The authors thank Dr I Usha Rao, Mrs Asha Juneja, Ms S S Sundari, Mr M Narasimham, Mss K Prema, Vibha Kakkar, Meenakshi Kakkar, Nirmal Tikiya and Dr B Harigopal for their generous help in the preparation of the manuscript.

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