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A review on the improvement of stevia [Stevia rebaudiana (Bertoni)]

Ashok Kumar Yadav, S. Singh, D. Dhyani, and P. S. Ahuja


Institute of Himalayan Bioresource Technology (Council of Scientic and Industrial Research), Palampur-176061, Himachal Pradesh, India. IHBT Publication number 2067. Received 23 April 2010, accepted 10 August 2010.
Yadav, A. K., Singh, S., Dhyani, D. and Ahuja, P. S. 2011. A review on the improvement of Stevia [Stevia rebaudiana (Bertoni)]. Can. J. Plant Sci. 91: 127. Stevia rebaudiana (Bertoni) is a herbaceous perennial plant (2n 22) of genus Stevia Cav., which consists of approximately 230 species of herbaceous, shrub and sub-shrub plants. Leaves of stevia produce diterpene glycosides (stevioside and rebaudiosides), non-nutritive, non-toxic, high-potency sweeteners and may substitute sucrose as well as other synthetic sweetners, being 300 times sweeter than sucrose. In addition to its sweetening property, it has medicinal values and uses. Stevia is self-incompatible plant and the pollination behaviour is entomophilous. Rebaudioside-A is of particular interest among the glycosides produced in the leaves of stevia because of the most desirable flavour profile, while, stevioside is responsible for aftertaste bitterness. Development of new varieties of S. rebaudiana with a higher content of rebaudioside-A and a reduced content of stevioside is the primary aim of plant breeders concerned with the improvement and utilization of this source of natural sweeteners. The proportions of rebaudioside-A and -C are controlled by a single additive gene known to be co-segregating suggesting synthesis by the same enzyme. Stevioside and rebaudioside-A are negatively correlated, while rebaudioside-A and -C are positively correlated. Conventional plant breeding approaches such as selection and intercrossing among various desirable genotypes is the best method for improving quality traits in a highly cross-pollinated crop like stevia. Various plant types with larger amounts of specific glycoside have already been patented, such as RSIT 94-1306, RSIT 94-75, RSIT 95-166-1 through selection and intercrossing. Composites and synthetics can be used to capture part of the available heterosis because of the high degree of natural out-crossing and the absence of an efficient system of pollination control. Synthetics and composites like AC Black Bird and PTA-444 have already been developed. Polyploidy results in better adaptability of individuals and increased organ and cell sizes. Tetraploids have larger leaf size, thickness and have potential use in increasing biomass and yield in comparison with diploid strains. Characters of interest with low variability in the population may be improved through mutation breeding. Use of biotechnological approaches, such as tissue culture for the mass propagation of elite genotypes, anther culture for development of pure homozygous doubled haploid and molecular marker technology for identification of marker loci linked to rebaudioside-A trait, can create new opportunities for plant breeders. Understanding the mechanism and pathway of biosynthesis of steviol glycosides can help to improve the glycoside profile by up-regulation and down-regulation of genes. Key words: Stevia, diterpene glycoside, rebaudioside A, selection, gibberellic acid pathway, gene cloning Yadav, A. K., Singh, S., Dhyani, D. et Ahuja, P. S. 2011. Lame lioration ge ne tique du ste via [Stevia rebaudiana (Bertoni)], tour dhorizon. Can. J. Plant Sci. 91: 127. Stevia rebaudiana (Bertoni) est une herbace e vivace (2n 22) du genre Stevia Cav., lequel compte environ 230 espe ` ces ve ge tales de type herbace , arbustif ou sous-arbustif. Les feuilles du ste via produisent des glycosides diterpe ` ne (ste vioside et re baudiosides), non nutritifs et non toxiques, mais au tre ` s fort pouvoir sucrant, susceptibles de remplacer le sucrose et dautres e dulcorants artificiels, car ils sont 300 fois plus doux que le sucrose. Outre cette proprie te , le ste via posse ` de des vertus me dicinales. Le ste via nest pas une plante autogame et la pollinisation seffectue gra ` des insectes. De tous les glycosides que produisent les feuilles du ste via, le re baudioside-A pre sente un ce a inte re est le plus de sirable, le ste vioside laissant un arrie ` re-gou ation de t particulier, car son profil de sapidite t amer. La cre nouvelles varie te s de S. rebaudiana plus riches en re baudioside-A et contenant moins de ste vioside est le but principal que recherchent les ame liorateurs, qui souhaitent accro tre la production de ces e dulcorants naturels et en favoriser lutilisation. Un seul ge ` ne additif, co-se gre gant, commande la proportion de re baudioside-A et C produite, ce qui laisse supposer lintervention du me ` se. Le ste vioside et le re baudioside-A sont ne gativement corre le s, alors quil me enzyme dans la synthe existe une corre lation positive entre le re baudioside-A et le re biaudioside-C. La meilleure fac ` res on de renforcer les caracte recherche s dans une espe ` ce a ` pollinisation aussi croise e que le ste via consiste a ` recourir aux me thodes classiques dame lioration ge ne tique comme la se lection et lhybridation des ge notypes inte ressants. On a de ja ` homologue plusieurs cultivars cre e s de cette manie ` re, et produisant une quantite supe rieure de tel ou tel glycoside, notamment RSIT 94-1306,

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Abbreviations: BAP, benzylaminopurine; CDP, ( )-copalyl diphosphate; DMADP, dimethylallyl diphosphate; DXP, 1-deoxyD-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; DXS , 1-deoxy-D-xylulose-5-phosphate synthase; EST, expressed sequence tag; GGDP, geranylgeranyl diphosphat; IBA , indolebutyric acid; IDP , isopentenyl diphosphate; NAA, napthalene acetic acid Can. J. Plant Sci. (2011) 91: 127 doi:10.4141/CJPS10086 1

2 CANADIAN JOURNAL OF PLANT SCIENCE RSIT 94-75 et RSIT 95-166-1. On peut aussi recourir a ` des varie te s compose es ou synthe tiques pour saisir une partie de lhe te rosis existante, en raison du fort degre naturel de croisement exte rieur et de labsence dun syste ` me efficace re gulant la pollinisation. Des varie te s synthe tiques et compose es comme AC Black Bird et PTA-444 ont dailleurs de ja ` e te re alise es. La polyplo die entra ne une meilleure capacite dadaptation des plantes, tout en engendrant de plus gros organes et cellules. Les te traplo des se caracte risent par des feuilles plus grandes et e paisses, dont la biomasse et le rendement pourraient de passer ceux des souches diplo des. Lame lioration par mutation pourrait aussi donner lieu a ` une ame lioration des caracte ` res recherche s, au sein dune population peu variable. Le recours a ` certaines me thodes biotechnologiques comme la culture tissulaire pour la multiplication massive des meilleurs ge notypes, la culture danthe ` res pour le de veloppement dhomozygotes purs a ` double haplo die et lusage de marqueurs mole culaires pour identifier lemplacement des locus associe s a ` la synthe ` se du re baudioside-A pourrait offrir dautres possibilite s aux ame liorateurs. Enfin, comprendre le me canisme et les voies de la biosynthe ` se des glycosides du ste viol permettrait den bonifier le profil par une re gulation en amont ou en aval des ge ` nes. s: Stevia, glycosides diterpe Mots cle ne, re baudioside A, se lection

Stevia rebaudiana (Bertoni) is a herbaceous perennial plant of the Asteraceae family, native to Paraguay (South America). Stevioside, the major sweetener present in leaf and stem tissues of stevia, was first seriously considered as a sugar substitute in the early 1970s by a Japanese consortium formed for the purpose of commercializing stevioside and stevia extracts (Kinghorn and Soejarto 1985). Diterpene glycosides produced by stevia leaves are many times sweeter than sucrose. They can be utilized as a substitute to sucrose (Robinson 1930; Soejarto et al. 1982, 1983; Lyakhoukin et al. 1993; Matsui 1996; Megeji et al. 2005; Sekaran et al. 2007); they are natural sources of non-caloric sweetener and alternatives to the synthetic sweetening agents that are now available to the diet conscious consumers. Randi (1980) reviewed the potential uses of Stevia rebaudiana, which produces stevioside, a non-caloric sweetener that does not metabolize in the human body. The sweet compounds pass through the digestive process without chemically breaking down, making stevia safe for those who need to control their blood sugar level (Strauss 1995). This is more important, especially in the context of the current social movement towards more natural foods (Brandle and Rosa 1992; Kamalakannan et al. 2007). With the increased incidence of diabetes in India and abroad, and growing concern over the safety of some chemical sweeteners, there is a need for a natural non-caloric sweetener with acceptable taste and health properties. In addition to its non-caloric sweetening properties, it has many therapeutic values: it is as antihyperglycaemic, anticancerous (Jeppensen et al. 2002, 2003), and antihypersensitive (Chan et al. 1998; Jeppensen et al. 2002), it has contraceptive properties (Melis 1999), and prevents dental caries (Fujita and Edahira 1979). It can also inhibit bacterial and fungal growth (Rojas and Miranda 2002). Commercial exploitation of stevia increased when Japanese researchers developed a series of processes for the extraction and refinement of sweeteners from its leaves. Research has also made possible simpler, water-only extraction processes (similar to sugar processing) in place of the older solvent extraction technology. The main producers of stevia are Japan, China, Taiwan, Thailand, Korea,

Brazil, Malaysia and Paraguay. Currently, Stevia is consumed in Japan, Brazil, Korea, Israel, the United States of America, Argentina, China, Canada, Paraguay and Indonesia (Crammer and Ikan 1986; Singh and Rao 2005) and to date there have been no reports of adverse effects from its use (Kinghorn and Soejarto 1985; Brandle and Rosa 1992). In the past, the main commercial constraint for the stevia industry was the ban on its use in food products as a food additive in the United States of America, although its use as a dietary supplement was approved by the Food and Drug Administration in 1995 (Bespalhok-Filho and Hattori 1997). In India, the plant was introduced at the University of Agricultural Sciences, Bangalore, during the late 1990s, and studies on its adaptability were initiated. Research focused on cultivation rather than crop improvement. Later, the Institute of Himalayan Bioresource Technology (CSIR), Palampur, introduced two accessions for domestication and cultivation in Himachal Pradesh. As well as cultivation, research has now been aimed at crop improvement through conventional breeding and biotechnological approaches. Products like stevia sweetener will increasingly be in demand due to consumer interest in natural products. Such demand will need to be supported by varieties of stevia improved for agronomical traits as well as for higher quantities and quality of iterpene glycosides, such as rebaudioside-A, which does not have an bitter aftertaste. The purpose of this review is to summarize the existing literature for the improvement of stevia through conventional plant breeding and selection and modern biotechnological approaches to provide a baseline for further improvement. ORIGIN AND ANTIQUITY The genus Stevia Cav. consists of approximately 150200 species of herbaceous, shrub and sub-shrub plants (Gentry 1996) and is one of the most distinctive genera within the tribe Eupatorieae, mainly because of the morphological uniformity of its flowers and capitula, which consist of five tubular flowers and five involucral bracts (King and Robinson 1987); it is distributed from the southwestern United States

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YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 3

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southward through Mexico and Central America. It also occurs from non-Amazonian South America, southward to Central Argentina (King and Robinson 1987). In Brazil, 36 species have been found, distributed mainly in southern and central regions (Frederico et al. 1996). Stevia rebaudiana originated in the highland regions of northeastern Paraguay (on the Brazilian border), between latitudes 238 and 248S, where the unique sweetening power of its leaves and its medicinal properties have been known by the local Guarani Indians many hundreds of years (Chan et al. 1998; Melis 1999; Jeppensen et al. 2002, 2003; Srimaroeng et al. 2005). The Guarani Indians called the plant kaa he-he, which translates as sweet herb, and used it as sweetener for their green herbal tea mate, and as a flavour enhancer (Soejarto et al. 1983). In the native state it grows on the edges of marshes or in grassland communities on soils with shallow water tables (Shock 1982). It is indigenous to the Rio Monday Valley of the Amambay moutain region at altitudes between 200 and 500 m. The climate can be considered as semi-humid subtropical, with temperatures ranging from 6 to 438C, with an average of 238C, and rainfall ranging from 1500 to 1800 mm per annum. In 1943, the first seeds were exported to the United Kingdom where it could not be brought under cultivation. In 1968 it was exported to Japan, and from there awareness of and cultivation of the plant spread throughout the world (Lewis 1992). By now, the crop has been introduced to many countries, including Brazil, Korea, Mexico, the United States of America, Indonesia, Tanzania, Canada and India (Lee et al. 1979; Donalisio et al. 1982; Shock 1982; Goenadi 1983; Saxena and Ming 1988; Brandle and Rosa 1992; Fors 1995). CLASSIFICATION OF STEVIA Stevia rebaudiana is one of the 950 genera of the Asteraceae family (Soejarto et al. 1983; Lester 1999). A systematic study of the North and Central American species of Stevia was done by Grashoff (1972). Stevia consists of a group of annual and perennial herbs, subshurbs and shrubs that occur in mountain regions, open forests, borders of rivers and dry valleys (Robinson 1930). Its first botanical description is attributed to M. S. Bertoni. The plant was first called Eupatorium rebaudianum Bert. in honour of Rebaudi, the first chemist to study the chemical characteristics of the substances extracted. Its name was later changed to the current one, and it was recommended not only for food production, but also for the medicinal effects that were attributed to it (Bertoni 1905). Although there are about 230 species in the genus, only S. rebaudiana gave the sweetest essence (Soejarto et al. 1983), while other species contain other biochemicals of interest (Frederico et al. 1996). It is a perennial herb with an extensive root system and brittle stems, producing small, elliptical leaves. Under some environmental conditions and management situations it behaves as an annual or

a mixture of plants of both types. The cultivated plants are reported to be more vigorous (Shock 1982; Frederico et al. 1996). Kingdom Subkingdom Superdivision Division Class Subclass Group Order Family Plantae Tracheobionta Spermatophyta Magnoliophyta Magnoliopsida Asteridae Monochlamydae Asterales Asteraceae (Compositae formerly) Subfamily Asteroideae Tribe Eupatorieae Genus Stevia Species rebaudiana

Some other related species of Stevia rebaudiana are Stevia eupatoria, Stevia lemmonii Lemmons stevia, Stevia micrantha annual stevia, Stevia ovata var. texana roundleaf candyleaf, Stevia plummerae Plummers stevia, Stevia plummerae var. alba, Stevia rhombifolia Kunth, Stevia salicifolia willow-leaf dtevia, Stevia serrata sawtooth stevia, Stevia viscida viscid stevia, Stevia commixta, Stevia satureiaefilia, Stevia leptophylla, Stevia myriadenia, Stevia ophryphylla, Stevia selloi, Stevia nepetifolia, Stevia oligophylla, Stevia origanoides and Stevia triflora. BOTANICAL DESCRIPTION Floral Biology

Flower Structure The inflorescence is loosely paniculate with the heads appearing opposite the bracts in irregular sympodial cymes. They are arranged in indeterminate heads. The flowers are small (1517 mm) and white (Marsolais et al. 1998; Dwivedi 1999) with pale purple throat corollas. The tiny white florets are perfect (hermaphrodite) having both male and female organs, borne in small corymbs of two to six florets (Goettemoeller and Ching 1999). The plant can initiate flowering after a minimum of four true leaves have formed. The plant takes more than a month to pass through the various developmental flower stages (Fig. 1) and produce all its flowers (Taiariol 2004; sh et al. 2006). Anther, Pollen and Stigma Anthers are small, five in number. The pollen can be highly allergic. Using the acetocarmine technique, Monteiro (1980) observed that in some diploid individuals of S. rebaudiana the pollen viability was 65%, which differs from the results of Oliveira et al. (2004)

4 CANADIAN JOURNAL OF PLANT SCIENCE

Fig. 1. Different stages of ower opening.

with no viable pollen grains. Stigma is bi-lobed/ bifurcated from the middle and style is surrounded by anthers (Fig. 2).

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Reproduction and Pollination Behaviour Stevia is self-incompatible (Miyagawa et al. 1986; Chalapathi et al. 1997) and probably insect pollinated (Oddone 1997). It has been reported that the amount of selfing ranged from 0 to 0.5%, while outcrossing ranged from 0.7 to 68.7%, indicating that the self-incompatibility system is operating (Katayama et al. 1976; Maiti and Purohit 2008). Since stevia is selfincompatible, seed collected from an individual plant would represent a half-sib family. Grashoff (1974) and Monteiro (1980) reported agamospermy in certain genotypes of S. rebaudiana. Sexual and apomictic plants of S. rebaudiana produce normal and malformed pollen, respectively. According to Monteiro (1980), the presence of apomixis in S. rebaudiana shown by embryological studies may be related to specific physiological and/or ecological factors. Analysis of sporogenesis allows the detection of irregularities that can lead to the formation of inviable gametes. Photoperiod and Flowering Time Stevia is a short-day plant that flowers from January to March in the southern hemisphere and from September to December in the northern hemisphere. Flowering under short-day conditions should occur 54104 d following transplanting, depending on the daylength sensitivity of the cultivar. The variability for photoperiod sensitivity is large, ranging from 8 h to 14 h (Valio and Rocha 1977; Zaidan et al. 1980; Chalapathi 1997).

Zaidan et al. (1980) identified three photoperiod classes based on the daylength. According to Valio and Rocha (1977), a minimum of two inductive short-day cycles are necessary for flowering induction. It can be induced to flower from the four-leaf-pairs stage onwards. Flowering is more precocious in the 8-h photoperiod, but plants remained vegetative under an 8-h photoperiod with interrupted night (Valio and Rocha 1977). Seed Seeds are contained in slender achenes about 3 mm in length. Each achene has about 20 persistent pappus bristles (Goettemoeller and Ching 1999). Seeds have a very small endosperm and are dispersed in the wind via hairy pappus. Shock (1982), Duke (1993), Carneiro et al. (1997) and Lester (1999) reported a poor and highly variable percentage of viable seeds. Fertile seeds are usually dark coloured, whereas infertile seeds are usually pale or clear (Fig. 3a, b) (Felippe 1978; Monteiro 1980; Oddone 1997, 1999; Goettemoeller and Ching 1999). Seeds are very small (1000 seeds weigh 0.31.0 g) and as a result seedlings are slow to develop, reaching a size suitable for transplanting to the field at 4560 d (Colombus 1997; Brandle et al. 1998a). Goettemoeller and Ching (1999) revealed that some active manipulation of the blossoms is necessary to achieve pollination. Seed yields of up to 8.1 kg ha1 have been recorded, but it is common to achieve less than 50% germination. Seed production in 1 ha would be enough for leaf production in 200 ha (Lester 1999). Agamospermy, i.e., the asexual formation of seeds (Lumaret 1988), could explain the reproductive capacity of this species, even if one takes into account the total lack of viable pollen observed.

Fig. 2. (a) Stevia anthers, (b) germinated pollen with pollen tube, (c) stigma and (d) stigma coming out of anthers.

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 5

Fig. 3. Stevia seeds (a) infertile seeds are usually pale or clear and (b) fertile seeds are dark coloured.

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Leaf Stevia has an alternate leaf arrangement and herbacious growth habit (Singh and Rao 2005). Leaves are small, sessile, lanceolate to oblanceolate, oblong, serate above the middle and somewhat folded upwards. Trichomes on the leaf surface are of two distinct sizes, large (45 mm) and small (2.5 mm) (Shaffert and Chebotar 1994). 9 stevia, the leaf area index (LAI) 80 d after sowing was 4.83 (Fronza and Folegatti 2003). Stevia leaves vary widely in quality due to many environmental factors, including soil conditions, irrigation methods, sunlight, air purity, farming practices, sanitation, processing and storage. The leaf has a pleasantly sweet, refereshing taste that can linger in the mouth for hours. The material contains the sweet components, surrounded by the bitter components in the veins (Maiti and Purohit 2008). PROPAGATION a) Vegetative Propagation

Shoot Cuttings Propagation of stevia is usually done by stem cuttings, which root easily, but require high labour inputs. Some plant varieties/selections produce virtually no viable seed and vegetative propagation is the only way of multiplication. Shock (1982) opined that stem cuttings were the prime means for the propagation of stevia. The direct planting of stem cuttings in the field was found to have a limited success (Chalapathi et al. 1999a). In general, a vigorous branch is cut at the base with a sharp blade and planting in the field, keeping two to three nodes above the soil. The cut portion of the branch is dipped in neem oil or any other fungicide (Maiti and Purohit 2008). Cuttings of new stems and shoots can be propagated successfully (Lee et al. 1979; Shock 1982; Gvasaliya et al. 1990; Nishiyama et al. 1991). Gvasaliya et al. (1990) reported that nearly 98100% rooting can be obtained, when the current years cuttings are taken from the leaf axils. Further, the location on the plant from which cuttings are taken can also affect growth and rooting. Cuttings from the top part of the main shoot with four internodes generally gave the best results (Tirtoboma 1988). However, the pair of leaves in the cuttings as well as the season also acts as

determinants for the rooting percentage and growth. Cuttings with four pairs of leaves rooted poorly, especially in February. Cuttings with two pairs of leaves rooted best in February and those with three pairs of leaves in April (Zubenko et al. 1991a; Ramesh et al. 2006). Cuttings taken in late winter rooted better than those taken at other times (Carvalho and Zaidan 1995). The sprouting percentage and shoot growth is also affected by the length of the cuttings. The sprouting percentage and shoot growth of sprouted cuttings were significantly higher with 15-cm-long cuttings compared with 7.5-cm-long cuttings (Chalapathi et al. 2001). The superior performance of the 15-cm-long cuttings may be because they have double the quantity of food reserves compared to 7.5-cm-long cuttings. Stolz (1968) indicated the necessity of higher quantities of starch, carbohydrates, sugar and phenolic compounds for root initiation. Rooting of cuttings can sometimes be stimulated by the use of growth regulators (Zubenko et al. 1991b; Carvalho and Zaidan 1995; Kornienko and Parfenov 1996; Kornilova and Kalashnikova 1996; Bondarev et al. 1998). Some growth regulators can sometimes influence (increase) the concentration of steviosides in the leaves (Chen and Li 1993; Acuna et al. 1997). Sprouting, however, was not influenced by NAA or IBA treatment at 500 ppm. IBA or NAA at a higher concentration of 1000 ppm, however, caused complete inhibition of rooting and sprouting of cuttings, which may be due to the injury caused to the callus tissue. Shin and Lee (1979) also reported that higher concentrations of IBA inhibited the rooting and sprouting of chrysanthemum cuttings due to the injury caused to the tissues. Zubenko et al. (1991b) found better rooting and sprouting of stevia cuttings by prolonged dipping of cuttings in 50 ppm IBA solution. Pre-treatment of cuttings with paclobutrazol at 50 or 100 ppm was found to be an effective growth regulator treatment for the induction of roots and sprouts from stem cuttings. Shoot vigour index is an important indication of vigorous growth of sprouted cuttings. Paclobutrazolinduced sprouts were found to be more vigorous than IBA- or NAA-treated cuttings (Chalapathi et al. 2001).

Micro-propagation Many different parts of the plant viz., leaves, auxiliary shoots, root-neck sprouts, shoot primordia, internodal explants etc., can be used successfully for tissue culture propagation (Handro et al. 1977; Yang and Chang 1979; Miyagawa et al. 1986; Ferreira and Handro 1987a; Bespalhok-Filho et al. 1992, 1993; Swanson et al. 1992; Akita et al. 1994; Kornilova and Kalashnikova 1996; Constantinovici and Cachita 1997). In vitro multiplication has frequently been used to multiply individually selected or bred clones and successful procedures have been documented (Handro et al. 1977; Ferreira and Handro 1988; Kornilova and Kalashnikova

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Shade can reduce total growth, delay flowering time and reduce the rate of flowering (Slamet and Tahardi 1988). Direct seeding to the field is not practiced, but may be a requirement for large-scale commercial production. Lee et al. (1979) found that plants from seeds were less productive in the first year than those from cuttings. SEED VIABILITY AND GERMINATION Two types of seeds are found in stevia, black and tancoloured, with black seeds being heavier than tan seeds. Goettemoeller and Ching (1999) investigated the low seed germination of stevia and tested the viability of seeds based on tetrazolium chloride, finding that the viability of black seeds was much higher than that of tan-coloured seeds, i.e., 76.7 vs. 8.3%, respectively. Cross-pollination and self-pollination was accomplished by transferring pollen with a bumble bee (Bombus impatiens) thorax on the end of a toothpick. The influence of pollination treatments as well as the effect of light and darkness during germination were also evaluated. Light increases the germination of black seed but not of tan seed. This suggests that tan seeds represent inviable seed that are produced without fertilization. The germination study of seeds obtained from five pollination treatments: cross-pollination by bumble bees (78.3%); cross-pollination by hand (92.0%); crosspollination by wind (68.3%); self-pollination by hand (93.3%) and control (36.3%) suggested that incompatibility is not a factor in these clones. However, all pollination treatments increase seed germination of black seed over the control, suggesting that some active manipulation of the blossoms is necessary to achieve pollination (Goettemoeller and Ching 1999). Germination rates of stevia seeds vary greatly. It can take 46 d to reach two-thirds of the final germination of 6290% at 258C (Shock 1982; Carneiro and Guedes 1992; Chen and Shu 1995; Takahashi et al. 1996). Germination requires at least 208C and often more than 258C; light generally increases germination. Increased temperature (408C on moist paper) for less than 24 h can accelerate germination, but reduce total germination (Tanaka 1985). In Japan, seed remained viable for up to 3 yr when stored under low humidity and in darkness (Kawatani et al. 1977). This contrasts with claims of optimum storage at 08C still producing a 50% loss of viability after 3 yr (Brandle et al. 1998a). In one experiment, seeds collected in the field and greenhouse were compared for germination. The greenhousecollected seeds had a 90% germination rate, whereas field collected seeds gave only 34% germination (Carneiro 1996). Total quantities of viable seed produced in 1 yr are uncertain. Seed yield up to 8 kg ha1 with 50% germination would be sufficient for 200 ha of crop (Lester 1999).

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Fig. 4. Micro-propagation of stevia from nodal explants.

1996; Carneiro et al. 1997; Bondarev et al. 1998; Nepovim 1998). Explants from leaf, nodal (Fig. 4) and inter-nodal segments were cultured on MS medium containing 2,4-D at 2.0, 3.0, 4.0 and 5.0 mg L 1 for callus induction. Inter-nodal segments initiated callus formation earlier than node and leaf. The greatest amount of callus was produced in MS medium with 3.0 mg L 1 2,4-D, whereas, MS medium with 5.0 mg L 1 2,4-D gave the poorest callus (Salim-Uddin et al. 2006). The plant growth and stevioside content in the leaves of the plants grown from stem cuttings or tissue culture were more uniform than the plant grown from seeds. The number of roots, shoot biomass and stevioside content were greater in the vegetatively grown plants (Truong and Valicek 1999). Seed Propagation Reproduction in the wild is mainly by seed, but germination and establishment from seed are often poor and sometimes unsuccessful (Shaffert and Chebotar 1994). Seeds germinate within 710 d after sowing. Propagation through seeds is not a common method of propagation owing to the problem of low seed production and poor germination capacity. Using seed to establish crops of stevia is more successful in tropical climates, where there is no climatic restriction on the length of the growing season. In northern climates the shorter growing season necessitates seedling establishment in a glasshouse/greenhouse prior to the growing season. Stevia flowers need to be fertilized by pollen from another plant to produce viable seed. A high density of bees (three to four hives per hectare) is recommended for good seed production (Oddone 1999). Harvesting of immature seed may also contribute to poor germination (Colombus 1997). Seed production and fertility studies suggest that high germination rates are possible from selected lines (Carneiro and Guedes 1992). Timing of flowering, seed harvest and pollination methods play an important role in seed production (Melis and Sainati 1991; Strauss 1995). Rain at flowering can also reduce seed setting.

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 7

GLYCOSIDES Eight diterpene glycosides with sweetening properties have been identified in leaf tissues of stevia. These are synthesized, at least in the initial stages, using the same pathway as gibberellic acid, an important plant hormone (Singh and Rao 2005). The four major sweeteners are stevioside, rebaudioside-A, rebaudioside-C and dulcoside-A. According to Kinghorn (1987) the sweetness of these compounds relative to sucrose are 210, 242, 30 and 30 times, respectively. The two main glycosides are stevioside, traditionally 510% of the dry weight of the leaves, and rebaudioside-A (Reb-A), 24%; these are the sweetest compounds. There are also other related compounds including minor glycosides, such as rebaudioside-B, rebaudioside-C (12%), rebaudiosideD, rebaudioside-E, rebaudioside-F, dulcoside-A, dulcoside-C and steviolbioside, as well as flavonoid glycosides, coumarins, cinnamic acids, phenylpropanoids and some essential oils (Erik et al. 1956; Erich et al. 1961; Harry et al. 1956; Hiroshi et al. 1976; Masur et al. 1977; Yohei and Masataka 1978; Rajbhandari and Roberts 1983; Makapugay et al. 1984; Crammer and Ikan 1986; Kinghorn 1987; Tsanava et al. 1989; Shaffert and Chebotar 1994; Putieva and Saatov 1997; Dzyuba 1998; Dacome et al. 2005; Sekaran et al. 2007). Among the components of stevia, one, called rebaudioside-A, is of particular interest because it has the most desirable flavour profile (DuBois 2000). Stevioside traditionally makes up the majority of the sweetener (6070% of the total glycosides content) and is assessed as being 110270 times sweeter than sugar. It is also responsible for the bitter aftertaste, sometimes reported as a licorice taste. As well as sweetness, stevioside may have a lingering effect or certain degree of pungency, which is not appreciated by the majority of people, and which reduces its acceptability. Rebaudioside-A is usually present as 3040% of total sweetener and has the sweetest taste, assessed as 180400 times sweeter than sugar with no bitter aftertaste (licorice taste or lingering effect). The ratio of rebaudioside-A to stevioside is the accepted measure of sweetness quality; the more rebaudioside-A the better. If rebaudioside-A is present in equal quantities to stevioside, it appears that the aftertaste is eliminated. The minor glycosides are considered to be less sweet, 3080 times sweeter than sugar (Crammer and Ikan 1986; Brandle 1999; Oddone 1999). The sweetening effect of these compounds is purely taste; they are undigested and no part of the chemical is absorbed by the body. They are therefore of no nutritional value (Hutapea 1997). The yield of sweetening compounds in leaf tissue can vary according to method of propagation (Tamura et al. 1984a), daylength (Metivier and Viana 1979) and agronomic practices (Shock 1982). Unlike many low-calorie sweeteners, stevioside is stable at high temperatures (1008C) and over a range of pH values (Kinghorn and Soejarto

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1985). It is also non-calorific, non-fermentable and does not darken upon cooking (Crammer and Ikan 1986). There are reports of stevioside content (total glycosides) ranging between 4 and 20% on a dry weight basis, depending on the cultivar and growing conditions (Kennely 2002; Starrat et al. 2002). The sweetening potency (sucrose 1) of stevioside, rebaudioside-A, rebaudioside-B, rebaudioside-C, rebaudioside-D, rebaudioside-E, dulcoside-A and steviolbioside are 250300, 350450, 300350, 50120, 200300, 520300, 50120 and 100125, respectively (Crammer and Ikan 1986). The essential oil composition of the aerial parts of five different Stevia rebaudiana genotypes cultivated in on the Tuscan coast (Italy) was examined by CC and GC/MS. Forty different components were identified and the main constituents in all studying samples were spathulenol (13.440.9%), caryophyllene oxide (1.318.7%), beta-caryophyllene (2.116.0%) and betapinene (5.521.5%) (Cioni et al. 2006). In general, Paraguan leaves contain the highest concentration (913%) of the sweet steviosides/rebaudiosides molecules, Chinese stevia contains only 56%, and Indian stevia is midway between these. Under Indian conditions stevioside concentration was about 9.08% of the dry weight of leaves (Ashwini 1996; Chalapathi 1996). The genotypes under study at the Institute of Himalayan Bioresource Technology show considerable morphological variation. Stevioside content is higher in Accession 1 compared with Accession 2 (Megeji et al. 2005). Biosynthesis of Steviol Glycosides The steviol glycoside and gibberellin pathways diverge at kaurene. In stevia, kaurene is converted to steviol, the backbone of the sweet glycosides, then glucosylated or rhaminosylated to form the principle sweeteners. The precursor compounds are synthesized in the chloroplast, and from there are transported to the endoplasmic reticulum, Golgi apparatus and then vacuolated. The purpose of these compounds in the stevia plant is not yet clear, but their high concentration in the leaf and the conservation of the pathway within the species indicate that, at some point in evoluntionary time, their presence conferred significant advantage upon those individuals that possessed them. Some researchers feel that they act to repel certain insects and others speculate that it is an elaborate means of controlling levels of gibberellic acid (Smith and Van-Stadin 1992). Expressed sequence tags (EST) are a powerful tool that has emerged from genomics research. Expressed sequence tag collections can reveal gene expression patterns, gene regulation and sequence diversity. Now, that enriched libraries and efficient high-throughput sequencing is widely available, ESTs have also become an effective means of gene discovery in focused metabolic situations (Sterky et al. 1998; Ohlrogge and Benning 2000). This concept was first applied to the isolation of oleate hydroxylase from castor (Van de Loo et al. 1995). Since then, ESTs have been used to find

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new genes from 1-deoxy-D-xylulose 5-phosphate (DXP) pathway of different crops. It is now clear that transcriptome analysis can be used to identify highly expressed genes that are involved in many different metabolic events (Brandle et al. 2002). Stevia rebaudiana leaves can accumulate high concentrations (up to 30%) of seven different glycosides derived from the tetracyclic diterpene steviol (Brandle et al. 1998a). Their intense sweetness and close structural relationship to gibberellic acid, coupled with the highly active nature of the pathway have fostered interest in steviol glycoside biosynthesis and metabolism (Richman et al. 1999; Totte et al. 2000). In S. rebaudiana, these compounds are synthesized exclusively in the mesophyll cells of leaves and are undetectable in the roots. Although the adaptive value of steviol glycosides is not known, it is clear that S. rebaudiana has committed a very large portion of total metabolism to their synthesis, making S. rebaudiana a good candidate for an EST-based gene discovery effort. Despite this amazing metabolic capability, only a few genes involved in the biosynthesis of steviol glycosides have been isolated and characterized (Richman et al. 1999; Brandle et al. 2002). Recent experiments have shown that the early steps in steviol biosynthesis involve the plastid localized DXP pathway and not the mevalonate pathway (Totte et al. 2000). Therefore, the first step in the steviol glycoside biosynthetic pathway is the formation of DXP from pyruvate and glyceraldehyde 3-phosphate by thiamine phosphate-dependent DXP synthase (Lange et al. 1998; Eisenreich et al. 2001). It has also been found that dimethylallyl diphosphate (DMADP) is not necessarily the committed precursor of isopentenyl diphosphate (IDP) and that IDP and DMADP may arise from separate syntheses (Arigoni et al. 1999; RodriguezConcepcio n et al. 2000; Brandle et al. 2002). Isopentenyl diphosphate and DMADP are converted to geranylgeranyl diphosphate (GGDP) by GGDP synthase via three successive condensation reactions (McGarvey and Croteau 1995). Like all diterpenes, steviol is synthesized from GGDP, first by protonationinitiated cyclization to ()-copalyl diphosphate (CDP) by CDP synthase (Richman et al. 1999; Hedden and Phillips 2000). Next, ()-kaurene is produced from CDP by an ionization-dependent cyclization catalyzed by ( )-kaurene synthase (Richman et al. 1999). ()-Kaurene is then oxidized at the C-19 position to ()-kaurenoic acid, by a novel P450 mono-oxygenase (Helliwell et al. 1999). Steviol is produced by the hydroxylation of ()-kaurenoic acid at the C-13 position, but the gene for this FAD-dependent monoxygenase has not yet been isolated (Kim et al. 1996). The two oxygenated functional groups of steviol, the C-19 carboxylate and the C-13 alcohol, provide attachment points for the sugar side chains that determine the identity of the different glycosides. The C-13 alcohol is successively glucosylated, first yielding steviolmonoside then steviol-bioside, next the C-19 carboxylate is

glucosylated, which forms stevioside (Shibata et al. 1991, 1995). The pathway terminates with the glucosylation of stevioside, which forms rebaudioside A. Rhamnosylated glycosides can also be formed by the addition of a UDP rhamnose moiety to steviolmonoside (Richman et al. 1999). The enzymes involved in steviol glucosylation have been partially characterized and there is an understanding of the inheritance of certain glycoside patterns; however, corresponding genes have not been isolated (Shibata et al. 1991; Richman et al. 1999). In an effort to create a resource for gene discovery and to understand the synthesis of steviol glycosides, Brandle et al. (2002) sequenced 5548 random cDNAs from a S. rebaudiana leaf library. With electronic probes, database searches and differential representation, candidate genes for 70% of the steps in the steviol glycoside biosynthetic pathway have been searched. The average GC content of the ESTs was 42.5%, similar to what has been found with Arabidopsis ESTs (Asamizu et al. 2000). Of the 278 ESTs classified into the secondary metabolism category, 62 were candidates for diterpene glycoside synthesis. No members of the mevalonic acid pathway were identified among the S. rebaudiana leaf ESTs. This supports the work of Totte et al. (2000), and further proves that the DXP pathway is used to synthesize IDP for conversion to steviol. Candidates were identified for 70% of the steps in the pathway from pyruvate and glyceraldehydes 3-phosphate to rebaudioside A. The most highly represented pathway EST was an orthologue of the Arabidopsis GA3 gene, which is known to be involved in the three step oxidation of kaurene to kaurenoic acid (Helliwell et al. 1999). The synthesis of kaurenoic acid is a key step in the synthesis of both steviol and gibberellic acid (Brandle et al. 2002). The ent-kaurene skeleton of chloroplast diterpene glycosides, which are produced in large quantities in the leaves of Stevia rebaudiana, is formed via the recently discovered 2-C-methyl-D-erythritol 4-phosphate pathway. The enzymes catalyzing the first two steps of this pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), were characterized. The cDNAderived amino acid sequences for DXS and DXR contain 716 and 474 residues, encoding polypeptides of about 76.6 and 51 kDa, respectively. DXS and DXR from Stevia both contain an N-terminal plastid targeting sequence and show high homology to other known plant DXS and DXR enzymes (Totte et al. 2003). Glycoside Content in Different Plant Parts Plant organs contain different amounts of the sweet glycosides, which decline in the following order: leaves, flowers, stem, seeds and roots. Roots are the only organs that do not contain stevioside. The sweetness in the leaves is two times higher than that in inflorescence (Dwivedi 1999). Sekaran et al. (2007) reported that individual tissues of stevia appear to differ significantly,

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YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 9

with the stevioside content declining in a different order: leaves shoots roots flowers. The fact that the highest stevioside content is found in the leaves suggests that they serve as the main tissue for both synthesis and primary accumulation of stevioside compounds. The largest amount of stevioside was found in the upper young, actively growing shoot sections, whereas the lowest senescent shoot sections exhibited the smallest amount of such compounds. During ontogeny, a gradual increase in the stevioside concentration was observed in both mature leaves and stems, and this process lasted to the budding phase at the onset of flowering (Bondarev et al. 2003). According to Bondarev et al. (2003), the lower, mature leaves of stevia have fewer glands per leaf surface area than the upper, younger leaves, i.e., there is a positive correlation between gland distribution density and steviol glycoside content. This argues in favor of possible increased accumulation, by 30 to 170% of glycosides, in upper young leaves as compared with lower senescent ones. Depending on the clone, the portion of the rebaudioside-A in the total glycosides content appeared to be increased as well. During ontogeny, little variation in the glycoside content is also found in roots. In these organs, from the vegetative phase to flowering, a gradual decrease in the glycoside content was observed. During the fruit development stage the levels of glycosides were found to revert to the initial level. However, in roots the total glycosides content never exceed 0.1% (Bondarev et al. 2003). Kang and Lee (1981) demonstrated that the maximal content of stevioside in leaves is achieved during the formation of flower buds and it then gradually declines. All this information may indicate that the steviol glycosides are transported to generative organs. Similar results were obtained for ecdisteroids in Rhaponticum carthamoides, Ajuga reptans and Serratula coronata (Vereskovskii et al. 1983; Revina et al. 1986; Tomas et al. 1993; Anufrieva et al. 1998). At the whole-plant level, steviol glycosides tend to accumulate in tissues as they age, so that older lower leaves contain more sweetener than younger upper leaves. Since, chloroplasts are important in precursor synthesis, those tissues devoid of chlorolphyll, such as roots and lower stems, contain no or trace amounts of glycosides. Once flowering is initiated, glycoside concentrations in the leaves start declining (Singh and Rao 2005). Stems of stevia plants contain little or no sweeteners, although it is suggested that they may contain some flavour enhancers, odourisers and other agents of potential use for improving foodstuffs or alcoholic beverages (Singh and Rao 2005). As stems mature and lose colour, any steviosides present dissipate. The structure, development and chemical content of stevia roots have also received attention, often associated with culturing procedures (Yamazaki and Flores 1989; Yamazaki et al. 1991; Zubenko et al. 1995).

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Environmental Effect The growth and flowering of stevia are affected by radiation, daylength, temperature, soil moisture, and wind (sh et al. 2006). Stevia is grown as a perennial crop in subtropical regions, including parts of the United States, and as an annual crop in mid to high latitude regions (Goettemoeller and Ching 1999). The results indicate that yield depends mainly on the genetic characters of the plant, the phenotypic expression of which is influenced by climatic and environmental factors (Metivier and Viana 1979; Ermakov and Kotechetov 1996). Moreover, synthesis of terpenes is affected by climatic and environmental factors (Langston and Leopold 1954). Chen et al. (1978) studied the seasonal variation in stevioside content. Tateo et al. (1998) opined that environmental and agronomic factors have more influence on stevioside production. The ideal climate for stevia is a semi-humid subtropical with temperatures ranging from 6.0 to 438C with an average of 238C (Brandle and Rosa 1992). Research conducted in Egypt revealed that climatic conditions, such as temperature, and length and intensity of photoperiod, greatly affect stevia production and quality, as evident from the remarkable increase in yield during the summer compared with winter (Allam et al. 2001). Long-day conditions, as compared with short days, increase internode length, leaf area, and dry weight, and reduce the interval between the appearances of successive leaf pairs in S. rebaudiana. Total soluble leaf sugars, protein, and stevioside content are also augmented in both absolute and relative terms and the biosynthesis of steviol, the aglucone present in stevioside, is increased by 45%. The concentration of glycoside in the leaves of stevia increases when the plants are grown under long days. Since glycoside synthesis is reduced at or just before flowering, delaying flowering with long days allows more time for glycoside accumulation (Metivier and Viana 1979, 2005; Singh and Rao 2005). Sekaran et al. (2007) have shown a rebaudioside A/stevioside ratio of 1.65 in ex vitro green leaves and 0.91 in in vitro shoots. Only severe Ca deficiency caused reduction in the glycoside concentration (DeLima et al. 1997). The chemical content of the last five fully expanded leaf pairs showed the plant nutritional status (Utumi et al. 1999). CYTOLOGY The genus stevia shows great variation in chromosome number. The chromosome number of Stevia rebaudiana (2n 22), previously reported by Frederico et al. (1996) and Monteiro (1980, 1982), has been confirmed (Fig. 5) for various strains. However, strains with 2n 33 and 2n 44 (representing triploid and tetraploid cytotypes) also occur, which show a high degree of male sterility owing to the chromosomal abnormalities during gamete formation. Cytological studies on many species of genus Stevia, performed by Grashoff et al. (1972), concluded that in North America, all the shrubby species have a gametic chromosome number of n 12, while,

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Fig. 5. Chromosome count of diploid stevia plant.

herbaceous species with flower heads in a lax paniculate cluster have n 11. Further, although most reports indicate that n 11 (2n 22), values of 2n 24, 33, 34, 44, 48, 66, 70 have also been observed (Darlington and Wylie 1955; Bolkhoviskikh et al. 1969; Moore 1973, 1974, 1977; Goldblatt 1981, 1984, 1985, 1988; Goldblatt and Johnson 1990, 1991, 1996, 1998; Oliveira et al. 2004). Galiano (1987) considered Stevia as a multibasic genus, with x 11, x 12 and x 17, while, Frederico et al. (1996) considered its basic chromosome number as x 11. Accordingly, this variation reflects numerical (aneuploidy and polyploidy) and possibly structural changes, mainly pericentric inversions (Frederico et al. 1996). Aneuploidy also occurs in Picris babylonica, a plant that belongs to the same family as Stevia (Malallah et al. 2001). All South American species of stevia studied are diploid with the exception of hexaploid S. elatior (2n 66) from Colombia (Jansen et al. 1984). There is a predominance of the basic chromosome number x 11 among stevia species from South America, with only three species (S. lucida from Colombia, one population of S. jujuyensis from Argentina, and S. organensis from Brazil) having x 12 (Coleman 1968; Galiano 1987; Galiano and Hunziker 1987). These three species may have originated by ascending aneuploidy from species with x 11. The main mechanism in the evolution of the South American species of stevia is probably chromosome inversions, with a small amount of aneuploidy and polyploidy. The chromosomal morphology of six Brazilian species of Stevia, including S. rebaudiana, was studied by Frederico et al. (1996). They observed that karyotypes were very similar in chromosome number (2n 22) and size (1.02.4 mm). On the other hand, the comparative analysis of arm ratios of each karyotype, revealed that every species has a difference in arm ratios for at least one chromosome pair. Even in species with the same karyotypic formula, the sm pairs were located in

different positions in the karyotypes. The distinct pattern strongly suggests the occurance of pericentric inversions as the rule in the divergence of Brazilian species of stevia. Most of the chromosomes were metacentric, with a variable number of submedian ones. Only S. ophryophylla and S. rebaudiana had a pair with a subterminal centromere (Frederico et al. 1996). These results of the chromosome size and centromere position were confirmed by Oliveira et al. (2004). Chromosome lengths were similar in other strains with 2n 22 or 2n 44. The presence of a nucleolus organizing region on the short arm of the third major chromosome pair was also confirmed for S. rebaudiana. In the Brazilian species of stevia, the main mechanism of chromosomal evolution is also by pericentric inversion. Thus, Frederico et al. (1996) concluded that, in addition to the numeric changes, structural rearrangements have played an important role in the chromosomal evolution of the tribe Eupatorieae. Based on pollen viability, normal meiosis can be inferred. Although pairing at diakinesis was normal, with the formation of bivalents in all diploids, in none of the S. rebaudiana strains studied was the pollen viable. High rates of tetrad normality (93%) were also observed in the diploid strains. The lowest tetrad normality rates were those of the tetraploid (80%) and triploid (64.5%) strains. Because of irregularities that produce unbalanced gametes during meiosis, polyploids with an odd number of chromosome sets have a high level of sterility (Lawrence 1980). The low tetrad normality rates and the lack of viable pollen in the triploid and tetraploid cytotypes may be related to the pairing of multivalents (tetravalents and trivalents) at diakinesis. Other meiotic abnormalities, such as irregular chromosomal disjunction in anaphase, could also explain these results. However, as with the diploid cytotypes, the lack of viable pollen cannot necessarily be linked with regular pairing at diakinesis (bivalents) and high rates of tetrad normality. Oliveira et al. (2004) studied pairing at diakinesis of diploid, triploid and tetraploid cytotypes with 2n 22, 2n 33 and 2n 44, respectively. For diploid strains, pairing at diakinesis was n 11II, which agrees with the literature, While, pairing at diakinesis for the triploid and tetraploid strains was n 11III and n 11IV, respectively. This multivalent formation suggests that these strains may have an autopolyploid origin. Unlike the above cytotypes, which occur spontaneously in nature, the Stevia cytotypes analyzed here were obtained artificially by inducing polyploidy (Valois 1992). Multivalents at diakinesis would be expected in autopolyploids, since the latter are derived from a single genome and result in three or four homologous sets of chromosomes in triploid and tetraploids, respectively (Stace 1980).

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YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 11

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GENETIC INHERITANCE The magnitude of heritable variation is of utmost importance since it has relevance to selection response. Stevia produces steviol glycoside sweeteners in its leaves that are up to 240 times sweeter than sugar. Understanding the genetic basis of glycoside proportions will aid in their manipulation through plant breeding. The experiments conducted by Brandle (1999) were focused on the genetic control of the proportions of two of these glycosides, rebaudioside-A and rebaudioside-C. The study was conducted using F2 population from crosses between two sets of parents with divergent glycoside profiles. Segregation in the first set of F2s showed that the presence/absence of rebaudioside-A is controlled by a single dominant gene, but that the actual proportions of rebaudioside-A may be controlled by multiple loci or alleles. In a second cross, proportions of rebaudioside-A and rebaudioside-C were found to co-segregate and were shown to be controlled by a single additive gene. This result suggests that both rebaudioside-A and -C are synthesized by the same enzyme. The results were used to propose a model for glycosylation of steviol glycosides (Brandle 1999). The presence of significant heritability (h2) for three economically important characters, i.e., leaf yield, leaf:stem ratio and stevioside (62.1, 78.8 and 76.6, respectively) clearly suggested that genetic improvement of stevia is possible (Brandle and Rosa 1992). These high heritabilities enable selection and breeding programs, aimed at higher yield, to achieve substantial gains. Linkage Map To lay a foundation for molecular breeding efforts, the first genetic linkage map for S. rebaudiana was constructed by Yao et al. (1999) based on RAPD markers. This information will be useful to those interested in developing marker-assisted selection procedures and quantitative trait analysis as well as provide a starting point for those interested in genome organization in stevia. Despite the fact that the Compositae is one of the largest and most diverse families of flowering plants, there has been little research involving molecular markers, largely because the family possesses very few major crop species (Kesseli and Michelmore 1996). This work represents the first detailed genetic study ever conducted in this genus and one of only a few conducted among the members of the Compositae. Yao et al. (1999) constructed the genetic linkage map for S. rebaudiana using segregation data from a pseudo test-cross F1 population. A total of 183 randomly amplified polymorphic DNA (RAPD) markers were analysed and assembled into 21 linkage groups covering a total distance of 1389 cM, with an average distance between markers of 7.6 cM. The 11 largest linkage groups consisted of 419 loci, ranging in length from 56 to 174 cM, and accounting for 75% of the total map distance. Of the primers that showed amplification products, 35.5% detected polymorphic loci and 62.5%

of those marker loci segregated in a 1:1 ratio, indicating a high level of genetic diversity in stevia. Most of the RAPD markers segregated in normal Mendelian fashion. Similar findings have been demonstrated in other cross-pollinated species (Grattapaglia and Sederoff 1994). This may enable stevia breeders to conduct marker-assisted selection with genes that are found closely linked to the markers in the map. In many linkage maps, loci are often found to be concentrated in certain areas (hot spots) of a few linkage groups (Paterson 1996; Keim et al. 1997). In comparison, the marker loci in the stevia linkage map were distributed more evenly and may better represent of the whole genome (Yao et al. 1999). Genetic diversity detected with RAPDs varies with species and crosses (Beaumont et al. 1996). It appears that the limited breeding efforts undertaken to date have not significantly reduced levels of genetic diversity among the stevia breeding lines. This may be partly because stevia has not undergone a great deal of selection (Yao et al. 1999). Construction of the stevia genetic linkage map has laid a foundation on which to conduct marker-assisted selection in stevia. The next step is to associate genes involved with economically significant traits to the RAPD markers and to convert those markers into easily scorable PCR-based markers such as SCARs. Mapping newly cloned cDNAs from stevia genes involved in glycoside synthesis, such as copalyl diphosphate synthase, can be addressed using cleaved amplified polymorphic sequences markers and will lead to a better understanding of the genomic organization of secondary metabolism (Richman et al. 1998; Konieczny and Ausubel 1993). Further, marker development (e.g., using AFLP or the unmapped RAPD markers segregating 3:1) that will allow the resolution of the stevia map into 11 linkage groups is also an important goal for the future. Character Associations Information and understanding of the interrelationships among characters are important to aid selection and set limits of each economic character that a breeder can choose without adversely affecting another important character. Several authors have studied the dependence of yield on various growth parameters as well as stevioside content (Buana and Goenadi 1985; Shu and Wang 1988; Buana 1989; Nishiyama et al. 1991; Brandle and Rosa, 1992; Shyu 1994; Chalapathi et al. 1998, 1999b; Truong et al. 1999; Utumi et al. 1999). Some interesting correlations have been found which can assist selection programmes (Table 1). Plant height and leaf number at the second and fourth week after planting were positively correlated with biomass production in a greenhouse experiment conducted by Buana and Goenadi (1985). In another study, Buana (1989) reported that plant height had no significant correlation with production, leaf number, or branch number in the

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Table 1. List of some important correlations Characters Plant height, Leaf number Plant height Stevioside content Dry leaf yield Rebaudioside-A Dry yield Leaf dry weight per plant Stevioside content Chemical content of last fully expanded leaf pair Stevioside Plant leaf yield Total stevioside content Leaf thickness Stevioside content at seedling stage Rebaudioside-A content Dulcoside-A Rebaudioside-A Stevioside Dulcoside Correlation ve Uncorrelated ve Uncorrelated ve ve ve ve ve ve Uncorrelated ve ve (not always) ve ve Uncorrelated ve ve ve ve ve Characters Biomass production Production, leaf number, branch number Total soluble carbohydrates Yield, leaf:stem ratio Leaf size and thickness Leaf thickness Plant height, number of branches, leaves per plant and dry matter Yield. Leaf surface, number of roots Plant nutrient status Yield, leaf:stem ratio Branch number, leaf number Plant height Leaf/stem ratio Rebaudioside-A/Stevioside ratio Stevioside content at maturity Leaf area, net photosynthetic rate, chlorophyll and protein content Stevioside Rebaudioside-C Rebaudioside-A Rebaudioside-C References Buana and Goenadi (1985) Buana (1989) Nishiyama et al. (1991) Brandle and Rosa (1992) Shyu (1994) Shyu (1994) Chalapathi et al. (1998) Shu and Wang (1988) Truong et al. (1999) Utumi et al. (1999) Brandle and Rosa (1992) Buana (1989); Buana and Goenadi (1985); Shu and Wang (1988) Tateo et al. (1998) Shyu (1994) Weng et al. (1996) Weng et al. (1996) Nakamura Nakamura Nakamura Nakamura and and and and Tamura Tamura Tamura Tamura (1985) (1985) (1985) (1985)

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first 4 wk. A positive correlation between total soluble carbohydrates and stevioside content was established by Nishiyama et al. (1991). Stevioside content was uncorrelated with yield or leaf:stem ratio (Brandle and Rosa 1992). Further, dry leaf yield was correlated with leaf size and thickness and content of rebaudioside-A was highly correlated with leaf thickness (Shyu 1994). The dry yield of stevia was positively correlated with plant height, number of branches, leaves per plant and dry matter accumulation. About 96.88% of the total variation in dry leaf yield was explained by a linear function of these four characters (Chalapathi et al. 1998). Shu and Wang (1988) showed that leaf dry weight per plant had the greatest influence on yield. Stevioside content is influenced by both leaf surface and number of roots; however, the leaf surface has more influence on stevioside content than the number of roots (Truong et al. 1999). The chemical content of the last fully expanded leaf pair was well correlated with plant nutrient status (Utumi et al. 1999). Furthermore, stevioside concentrations were uncorrelated with yield or leaf:stem ratio indicating that concurrent improvement of agronomic and chemical characteristics is possible (Brandle and Rosa 1992). The observed correlation suggests that the carbohydrate reserve in the leaves of stevia is found mainly in the form of diterpenic glycosides of the stevioside type. The parameters obtained from this correlation allowed the establishment of a simple method to determine the stevioside content in dry stevia leaves (Nishiyama

et al. 1991). Plant leaf yield is proportional to branch number, leaf number and (not always) plant height (Buana and Goenadi 1985; Shu and Wang 1988; Buana 1989). Total stevioside content is positively correlated with leaf:stem ratio (Tateo et al. 1998). Leaf thickness is positively correlated with rebaudioside-A:stevioside ratio (Shyu 1994). The total stevioside content of leaves at the seedling stage and when mature is not correlated, making plant selection at the seedling stage ineffective. High rebaudioside-A content is also linked to large leaf area, high net photosynthetic rate, high chlorophyll and protein content (Weng et al. 1996). Nakamura and Tamura (1985) reported that the levels of dulcosideA and stevioside and rebaudioside-A, and -C are positively correlated with each other, while stevioside and rebaudioside-A, and dulcoside and rebaudioside-C are negatively correlated with each other. PHENOTYPIC VARIABILITY In the wild populations of Stevia rebaudiana, there is great variation in phenotype and leaf analysis. The collections made as part of the various breeding and selection research programs have invariably included a range of genotypes and selections of plants with distinct levels of steviosides in their leaves. Shock (1982) planted 200 lines for survival testing and screened 17 lines for productivity. The stevioside content of leaves can vary substantially (416%) between individual plants, even after a selection program has been continued for some time (Bian 1981; Nakamura and Tamura 1985).

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 13

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This natural variability could be partially due to the largely out-crossing nature of the species (Handro et al. 1993). Monteiro (1980) studied the phenotypic differences present in the population and was unable to separate them into a valid taxonomic variety. There are also reports of irregular quantitative and qualitative production of the sweetening molecules from cultivated S. rebaudiana. The phenotypic variability within the population is linked to the open pollination behavior of the species (Tateo et al. 1998). Nurhaimi and Toruan (1995) showed somaclonal variations in DNA fingerprints between six groups of plantlets. It has been reported from China, that, in a sample of plants from one clone, stevioside varied from 1.48 to 6.98% and rebaudioside-A from 4.5 to 12.1%, with total glycoside varying from 10.26 to 19.57% (Huang et al. 1995). In another report from China, total sweet glycoside concentration in some lines has been reported to be as high as 20.5%, and in separate cultivar rebaudioside-A: stevioside ratios of 9:1 have been disclosed (Morita 1987; Shizhen 1995). Such variability in the raw material would permit the use of conventional extraction methods to produce a stevia sweetener with more than 85% rebaudioside-A, without the need to recrystalize individual glycosides. Much of the morphological variability has been observed in the population of S. rebaudiana under study at the Institute of Himalayan Bioresource Technology, Palampur. It also exhibits considerable variability for stevioside content, which may vary from 2 to 10% (Megeji et al. 2005). The genetic improvement of stevia is only possible through the characterization of the available variability at the morphological, chemical and biochemical, cytogenetic and molecular levels, in order to utilize the information to develop an ideal plant type. Stevia grown at the Delhi Research Station (Ontario, Canada) had 1.22 times as much leaf dry weight as stem dry weight, this same ratio was about 0.67 in California (Brandle and Rosa 1992). DISEASE RESISTANCE Stevia is known to be free from attacks by insects, which may be due to its inherent sweetness acting as a repellent. Therefore, insecticides are not required at an essential basis as in other crops, which helps in producing organic Stevia. The fungal diseases Septoria leaf spot (Septoria steviae), Alterneria leaf spot (Alternaria alternata), stem rot (Sclerotium dephinii Welch.), root rot (Sclerotium rolfsii), powdery mildew (Erysiphe cichoracearum DC), damping-off (Rhizoctonia solani Kuehn.) and Sclerotinia sclerotoirum have been reported (Ishiba et al. 1982; Lovering and Reeleeder 1996; Chang et al. 1997; Thomas 2000; Megeji et al. 2005; Kamalakannan et al. 2007) (Table 2). There is a need to develop and identify resistant sources to develop varieties resistant to or tolerant of these diseases.

BREEDING OBJECTIVES Breeding programmes for stevia should be aimed at improving total glycoside content and rebaudiosideA:stevioside ratio with higher leaf yield. So far, plant breeding efforts with stevia have been largely focused on improving leaf yield and rebaudioside-A concentration in the leaves. High leaf:stem ratios are desirable in cultivated stevia because of the low stevioside concentrations (B5 mg g1) in stem tissue. Cultivar descriptions indicate that sufficient genetic variability exists to make significant genetic gains in leaf yield, rebaudioside-A content and the rebaudiosideA:stevioside ratio (Lee et al. 1982; Morita 1987; Brandle and Rosa 1992; Shizhen 1995) (Table 3). The native rebaudioside-A:stevioside ratio in Stevia rebaudiana leaves is usually about 0.5 or less. The predominance of stevioside gives a characteristic bitter aftertaste to the crude extract. Conversely, the most valuable extracts are those that have rebaudioside-A as the major component, because of its organoleptic and physicochemical features, i.e., it has the best profile relative to all other glycosides, and is more soluble in water (Ahmed and Dobberstein 1982; Crammer and Ikan 1986; Huang et al. 1995), allowing a greater variety of formulations. Therefore, the development and phytochemical characterization of new varieties of S. rebaudiana with higher levels of rebaudioside-A is a primary aim of plant breeders concerned with the improvement and utilisation of this source of natural sweeteners (Yao et al. 1999; Dacome et al. 2005; Sekaran et al. 2007). With the high level of natural variability due to constant out-crossing, breeders are able to improve the level of sweeteners in the leaves and alter the rebaudioside-A:stevioside ratio (Shu 1989; Huang et al. 1995). BREEDING METHODS Germplasm Introduction, Collection and Conservation Germplasm is a very important material for the improvement of crops. Introduction of germplasm from one area to another continues to be an important activity for breeding, particularly in developing countries. It is generally used as source of superior genes and increasing genetic diversity in the germplasm for breeding programmes. Introductions can be used directly as commercial cultivars. Adapting exotic germplasm is, however, a long-term programme. Intermating should be carried out for several generations and selection pressure applied gradually for desirable gene combinations. Institutions around the world that have undertaken research and/or appraisal studies on stevia have collected seed and plant material from Paraguay in its wild, natural environment (Grashoff 1972; Bian 1981; Shock 1982; Soejarto et al. 1983; Suhendi 1989; Tateo et al. 1998). The rationale behind seed collection is to conserve genes and not genotypes, since, in stevia, due to heterozygosity, no genotype is true breeding. Two

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Table 2. Various diseases reported in stevia Disease Septoria leaf spot Alterneria leaf spot Root rot disease Causal organism Septoria steviae Alternaria alternate Sclerotium rolfsii Symptoms Depressed, angular, shiny olive grey foliar lesions are formed that rapidly coalesced and often surrounded by a chlorotic halo. Leaves quickly become necrotic and often drop off the plant. Initially appears as small circular spots, light brown in colour. Later, many became irregular and dark brown to grey, while others remain circular with concentric rings or zones. Yellowing and drooping of leaves, with wilting of plants and white cottony mycelial growth at the collar region takes place. The mycelial growth spread to the stem and roots, with associated tissue rotting. Brown sclerotia appear on the diseased areas. Brown lesions on the stem, near the soil line are formed, followed by wilting and eventually by the complete collapse of affected individuals.

Stem rot

Sclerotinia sclerotiorum

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genotypes of S. rebaudiana, Accessions I and II, which are morphologically diverse with respect to their growth habit and sweetness are being maintained and multiplied at the Institute of Himalayan Bioresource Technology. Further selections for desirable plant types are being performed in segregating progenies of individual selections. Selection The success of stevia breeding depends on the choice of parents, making crosses, raising adequate population and further selections. In the wild, the total glycoside
Table 3. Characters for improvement/utilization through breeding Sr. no. Steviol 1 2 3 Characters glycoside characters Total glycosides content Rebaudioside-A content Rebaudioside-A: stevioside ratio Reference Sys et al. (1998) Marsolais et al. (1998) Brandle (2001), Morita and Yucheng (1998) Brandle and Rosa (1992)

Yield contributing characters 4 Leaf:stem ratio 5 Leaf size/area index 6 Leaf thickness 7 Number of leaves/plant or branch 8 Number of branches/plant 9 Internode length 10 Stalk weight and thickness 11 Leaf angle 12 Photoperiod sensitivity/short day plant 13 Plant vigour/growth rate 14 15 16 17 18 19 20 21 22 Seed germination percent Seed viability for short duration Self-incompatibility Lodging susceptibility Drought tolerance Sensitive to water logging Asynchronous seed maturity and seed dispersal Disease resistance Poor tolerant to high soil pH

Lester (1999), Valio and Rocha (1977) Borie (2000), Andolfi et al. (2002) Barathi (2003), Carneiro et al. (1997), Duke (1993), Shock (1982) Marcavillaca (1985) Chalapathi et al. (1997) Jia (1984)

Shock (1982)

concentration in stevia leaves typically varies from 2 to 10% on a dry weight basis. Nearly three decades of breeding and selection have increased glycoside concentration in stevia leaves by as much as 20% (Huang et al. 1995). However, this improvement was based on phenotypic selection for total glycoside concentration in stevia leaves, which is heavily influenced by environmental conditions, such as soil and weather. More importantly, it requires selection relatively late in the growing season. Selection at the early seedling stage is least effective, because seedlings are so influenced by the environment that only 2030% of the variability is genetic, and measurements are based on expensive and tedious high performance liquid chromatography (HPLC) procedures (Brandle and Rosa 1992). As a result, selection for plants producing high amounts of glycoside is expensive, time consuming, and relatively inefficient (Yao et al. 1999). Countries that have been researching stevia for some time, especially Japan, China, Korea, Taiwan and Russia, have all reported success in their breeding/ selection programmes and have released new varieties with improved glycoside content and higher yields (Table 4). Most breeding programs are based on cross breeding and selection. Vegetative propagation and cloning have frequently been used to multiply individually selected plants. Some of these selections, although very high yielding, are self-incompatible and can only be reproduced vegetatively (Lee et al. 1982). This limits their commercial use, although they may be useful for breeding new hybrids. Attempts made elsewhere in the world have resulted in patents for the superior plant types (Table 5). A cultivar with a rebaudioside-A:stevioside ratio of 0.96:1, compared with 0.36:1 in the starting material, was developed with total glycosides of 22.4% (Lee et al. 1982). Other plants have been developed that exhibited rebaudiosideA:stevioside ratios as high as 9.1:1, but total steviol glycosides were 10.1% (Morita 1987). Again, due to self incompatibility, the cultivar could not be reproduced using a seed-based production system. The costs associated with clonal propagation limit the general applicability for large-scale production of stevia plants.

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 15


Table 4. Some varieties/cultivar selections and releases Year 1979 1982 1989 1995 1994 1994 1996 1996 2000 2000 Country Korea Korea China China Taiwan Indonesia China Russia India India Reference Lee et al. (1978) Lee et al. (1982) Shu (1989) Shu (1995) Shyu (1994) Suhendi (1989) Weng et al. (1996) Kornienko and Parfenov (1996) IHBT (CSIR) annual report IHBT (CSIR) annual report Variety Suweon 2 Suweon 11 Yunri, Yunbing Zongping K1, K2, K3. BPP72 SM4 Ramonskaya Slastena Madhuguna Madhuguni Features High yield and steviosides Thick leaves, high Reb% Highest Rebaudioside and stevioside High yield, better Rebaudioside:stevioside ratio High yield and Rebaudioside:stevioside ratio High yield and total glycoside content High yield and total glycoside content

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Development of Stevia Plant RSIT 94-1306 and RSIT 94-751 Sys et al. (1998) and Marsolais et al. (1998) set out to develop stevia plants with high concentrations of individual steviol glycosides that could be extracted and recombined in ratios suitable for specific product uses. Landrace stevia has a combination of steviol glycosides that is not optimal for all product applications. In 1989, seed of a landrace variety from China was introduced at Agriculture and Agri-Food Canada, Delhi Research Station, Ontario, Canada. Superior plants were selected from this population and designated SR1 to SR15 (breeding procedure shown in Fig. 6). These plants were inter-crossed and half-sib seed was collected from each parent plant. The half-sib families were evaluated in a genetic heritability trial (Brandle and Rosa 1992). RSIT 94-1306 was selected from the SR2 half-sib population and RSIT 94-751 was selected from the SR13 half-sib population on the basis of agronomic traits and steviol glycoside profile, based on replicated trials. RSIT
Table 5. List of patents Title Stevia rebaudiana with altered steviol glycoside composition Stevia rebaudiana with altered steviol glycoside composition Variety of Stevia rebaudiana Bertoni Stevia plant named RSIT 94-751 Stevia plant named RSIT 94-1306 Stevia plant named RSIT 95-166-13 Extraction of sweet compounds from Stevia rebaudiana Bertoni Method of cultivating hybrid new variety via systematically breeding stevia rebaudiana clone parental plant. Country USA USA USA USA USA USA USA

94-1306 and RSIT 94-751 were vegetatively propagated by shoot-tip and stem cuttings.

Development of RSIT 95-166-13 Brandle et al. (1998b) developed RSIT 95-166-13 as a unique combination of characteristics, which distinguished it from its parents and all other stevia varieties for a high rebaudioside-C:stevioside ratio. Different seed germplasm accessions of stevia from China were grown for evaluation at the Delhi Research Station, Agriculture and Agri-Food Canada. Plant samples RSIT 94-1838, RSIT 94-1833, and RSIT 94-1560 were retained as a result of the higher than average concentrations of rebaudioside-C in their leaves on a dry weight basis. A plant (RSIT 94-1829) selected from the variety Brazil Zairai was also retained, because its leaves had higher than average concentrations of rebaudiosideC. Brazil Zairai is an open-pollinated landrace variety of stevia obtained from the Japanese National Germplasm Depository. These four clones were used as parents and intercrossed. Half-sib seeds collected from clone RSIT

Inventer Brandle, J. Brandle, J. Morita, T. and Yucheng, B. Marsolais, A. A.; Brandle, J. and Sys, E. A. Sys, E. A.; Marsolais, A. A. and Brandle, J. Brandle, J.; Sys, E. A. and Marsolais, A. A. Wang, Q

Year 2001 1999 1998 1998 1998 1998 1999 2006 1984a, b 1985 1986 1987 1988 1985 1990

Patent no. 6,255,557 PCT-WO99/49724 6031157 PP10,564 PP10,562 PP10,563 5,972,120 CN1985575 JP-59034848; JP-59034826 JP-60160823 JP-61202667 JP-62096025 JP-63173531 EPA0154235 JP-2242622

Japan Japan Japan Japan Japan Europe Japan

Morita Morita Morita Nakazato, T Nakazato, T Stevia co. Inc. Sanyo Kokusaku Pulp Co.

New triploid of Stevia Rebaudiana Bertoni- contains sweet diterpenoid.

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In 1989, seeds of landrace var. from china In summer 1990

1000 plants grown


15 Plants selected

SR1 - SR15
Crown dug-out & grown in green house in winter 1990-91

In summer 1991

Intercrossed & half-sib seeds collected

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These half-sib families were evaluated in genetic heritability trial


In summer 1994

evaluated visually, and superior rows are identified. The selected rows are harvested separately. Equal quantities of reserve seed from selected single plants, based on progeny performance, is composited. The first recurrent selection cycle starts when the new composite is grown in an isolated plot where random mating among the plants is allowed. A bulk seed sample is harvested from the remaining plants of each composite for use in replicated yield trials to determine the response to selection in each recurrent selection cycle for the characteristics under improvement. Recurrent selection is continued until a reasonable response to selection is achieved. Each cycle of recurrent selection produces a new population that may be a potential cultivar. The improved population with increased frequency of desirable alleles can be used as a cultivar per se or as a source to identify superior individual genotypes. Development of Synthetics and Composites There is a need to develop a stevia cultivar that is enriched in rebaudioside-A and has a high steviol glycoside content that can be produced using a relatively low-cost method based on transplants produced from seed. Therefore, a synthetic cultivar produced by intercrossing clones or sibbed lines obtained from a breeding population during cycles of recurrent selection is required. In order to develop a synthetic variety more than one line is required and the lines or clones are typically tested for combining ability, preserved for future synthesis of the synthetic cultivar, as well as combined by random crossing. Such synthetic cultivars are intended for use in crop production systems. Because of the high degree of natural out-crossing and the absence of an efficient system of pollination control, composites and synthetics are used to capture part of the available heterosis. This is the most practical and effective breeding method, and there is a large effort aimed at establishing stevia as a crop in Japan as well as a number of other countries based on developing synthetic cultivar.

SR 2

SR 13

RSIT 94-1306
Cuttings evaluated

RSIT 94-751
Cuttings evaluated

Stevioside (%) Reb-A

= 17.25 = 0.0

Stevioside (%) Reb-A

= 4.88 = 11.82

Total glycosides = 18.37

Total glycosides = 18.08

Fig. 6. Breeding procedure followed by Sys et al. (1998) and Marsolais et al. (1998).

94-1560 were designated half-sib population RSIT 95166. Plants from half-sib population RSIT 95-166 were evaluated in the field and one plant, RSIT 95-166-13, was selected on the basis of its novel steviol glycoside traits high in rebaudioside-C content. RSIT 95-16613 was vegetatively propagated at the Delhi Research Station by shoot-tip and stem cuttings. Population Improvement

Recurrent Selection Recurrent Selection is useful for improving quantitatively inherited characters in cross-pollinated species. In S. rebaudiana, where self-incompatibility ensures a high degree of heterogeneity, recurrent selection is the most effective method of increasing foliage yield as well as total glycoside content. In population improvement, plant breeders aim to increase the frequency of desirable alleles through the selection of superior recombinants. Recurrent selection typically starts with harvesting individual open-pollinated plants from the source population. A portion of the seed of these plants is saved as a reserve. The agronomic characters of progeny rows are

Synthetic Cultivar AC Black Bird Brandle (2001) developed the synthetic cultivar AC Black Bird (breeding procedure shown in Fig. 7), which is characterized by exhibiting a high level of total glycosides (at least 14%), and a high ratio of rebaudioside-A to stevioside (at least 9.1:1). In order to create parents for the synthetic cultivar, crosses were made among a number of single plants and a large number of progeny were planted out to the field, and selections were made among those progeny. Leaves sampled from those selected plants were analyzed for glycoside concentration as well as composition, and selections that were high in glycosides, with rebaudioside-A to stevioside ratios of at least about 9.3:1 (denoted as A to D), were inter-crossed in the greenhouse and seeds were collected from the maternal parents. At the same time, cuttings were taken from

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 17


In order to create parents for the synthetic cultivar, crosses were made among a no. of single plants and a large no. of progeny planted out in field and selections were made among those progeny

OxO OxO

OxO OxO

OxO OxO

OxO OxO
Crossing among no. of selected plants

Seeds collected
Large no. of progeny planted in field

IIIIIIIIIIIIIIIIIIIIIIIIIII
1...12
Selection among these families

60 plants in each family

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20 plants from each family selected for HPLC analysis


Selected high in glycosides and reb-A/stevioside = 9.3:1

4 plants selected (9.3:1) (A,B,C,D)

IIII
ABCD Sexual inter-crossing Cuttings to duplicate the synthetic as required

Isolated from field in green-house, trimmed inside 1.3x1m insect case screened with 10mm mesh. A hive of bumble bees was used for inter-crossing

AC Black bird (Synthetic cultivar)

Parental clones, Selections, half-sib families of selections and synthetic cultivar evaluated in replicated field trial Stevioside (%) = 1.27 Reb-A Total Reb-A/Stev = 12.49 = 15.06 = 9.96

Fig. 7. Breeding procedure followed by Brandle (2001) for development of synthetic cultivar AC Black Bird.

the plants so that they could be used to duplicate the synthetic as required. Seed from the maternal parents was retained as half-sib families and a portion bulked to create a synthetic cultivar AC Blackbird. The half-sib families, the bulked sample and the parental clones were evaluated in a replicated field trial. The plants so obtained are characterized as exhibiting high levels of total steviol glycosides and being enriched in rebaudioside-A. It is preferred that the cultivar seed be produced from at least two intermating genotypes.

Variety of Stevia ATCC Accession No. PTA-444 A variety of S. rebaudiana developed by Morita and Yucheng (1998), which contained 2.56 times or more rebaudioside-A than stevioside and is capable of being cultivated by seed propagation, is produced from SF-6 seed, having ATCC Accession No. PTA-444, or progeny thereof. Morita and Yucheng (1998) carried out stevia breeding by repetitive crossing and selection to obtain a variety with a high content ratio of rebaudioside-A to stevioside. Hills (SF3 grade) containing 2.56 times or

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more rebaudioside-A than stevioside were selected from seeds formed by crossing between SF3s described in Japanese patent publication no. 61-202667. Further, crossing and selection were repeated, and as a result of crossing between both hills containing 2.56 times or more rebaudioside-A than stevioside, SF5-1 (designated No. 103), which has excellent resistance to Septoria, and SF5-2 (designated No. 109), which is excellent in sweetening components, were finally selected. More than 80% of the resulting hills (SF6-I and SF6-II) obtained from the crossing between No. 103 and No. 109 contained 2.56 times or more rebaudioside-A than stevioside. However, crossing between SF3 and No. 103 or No. 109 (SF6-III) did not give hills containing a stable component proportion of rebaudioside-A.

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Mutation Breeding Mutagenesis is a potential tool to broaden variability and to isolate desirable economic traits in a shorter period compared with conventional breeding procedures. The variability found in nature is due to the accumulation of natural mutations that have occurred during the evolution of the plant. With the discovery of mutagenic agents, both physical and chemical, plant breeders have the ability to induce variability and use it in their breeding programmes. Several mutagenic agents, such as X-rays, g-rays, fast neutrons, thermal neutrons and chemicals such as EMS, DES, MNUA, ENUA, MNU, ENU, can be used to produce useful mutations. Irradiation with Cobalt-60 gamma rays has been used to induce variation in breeding lines (ToruanMathius et al. 1995). Characters of interest can only be improved through mutation breeding if the population shows less variability for the character concerned. Because leaves are the economically important part of this crop, mutation breeding can play a very important role in the improvement of stevia. Gamma irradiation of the seeds of stevia does not affect germination, but at higher doses suppresses root development. Polyploid Breeding The induction of polyploidy to improve agronomic yields is a process commonly used in plants of economic interest (Allard 1960) and has been applied to other species, such as Nicandra physaloides (Gupta and Roy 1986), coffee (Cruz et al. 1993), Clitoria ternatea (Gandhi and Patil 1997) and orange (Romero-Aranda et al. 1997). Better adaptability of individuals and increased organ (Fig. 8) and cell sizes are usually associated with polyploidy (Guerra 1988). Crossbreeding between individuals with different numbers of chromosomes usually leads to sterile progeny. According to Allard (1960), in various agricultural plants with diploid and tetraploid individuals as parents, the progeny are partially or completely sterile triploids that do not bear seeds. Valois (1992) developed polyploid strains by soaking the seeds of S. rebaudiana in nine treatments of

Fig. 8. Comparison for leaf size of (a) polyploid and (b) diploid plant.

colchicine (an antimitotic agent) at concentration ranging from 0.001% to 0.5% for 18 h. Polyploidization was confirmed in only two treatments, which corresponded to the lowest colchicine concentrations. Specifically, triploid plants of S. rebaudiana can be produced by mating tetraploid female and diploid male parents. Triploid plants of S. rebaudiana contain a large amount of rebaudioside-A, a very sweet diterpenoid (Sanyo 1990; Shuichi et al. 2001). The glycoside quality of stevia is improved by using the polyploids. Triploid and tetraploid plants had a lower tetrad normality rate than the diploids. All of the strains had inviable pollen (Oliveira et al. 2004). Thus, the higher the ploidy number, the greater the size of the pollen and the stomata, and the lower their number per unit area. The triploid strain produced the shortest plants and the lowest number of inflorescences, whereas the tetraploid strain had the largest leaves. Analysis of variance revealed highly significant differences among the strains, with a positive correlation between the level of ploidy and all of the morphological features examined (Oliveira et al. 2004). Breeding of triploid plants of stevia was conducted by Shuichi et al. (2001), and 42 triploid plants, grouped into eight cultivars, were obtained. The chromosome number of these plants, obtained by counting the chromosome number of root tip cells and by flow cytometry analyses, showed that the plants were triploid (2n 33). In these triploid plants, the leaves, flowers and guard cells were larger than that in the plants classified as diploid. The leaf shape of the triploid plants was categorized as needle-like or wide needle-like. In thin-layer chromatography and HPLC analyses, seven kinds of triploid

YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 19

plants, T1 to T7, obtained from crossing tetraploid SMX1W with a high rebaudioside-A content with seven kinds of diploid cultivars with a high rebaudioside-A content, showed high rebaudioside-A contents with a ratio (rebaudioside-A:stevioside) of more than 0.63. On the other hand, T-8, obtained from crossing tetraploid KSW with a high stevioside content with diploid SMX6 with a high RA content, showed high STV contents (rebaudioside-A ratio 0.23). The seasonal changes in the total content of the sweeteners and the RA ratio of the triploid plants showed that total content increased until August, whereas the RA ratio remained constant (Shuichi et al. 2001).

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Heterosis Breeding Hybrids offer an opportunity to mobilize greater genetic variation and heterotic response. Success in developing hybrids in any crop depends on the availability of heterotic response for economic yield and economic feasibility in terms of cost involved in seed production. Sun (2001) suggested a method for breeding hybridized seed of S. rebaudiana that involves hybridizing between female plants produced from cuttings and male plants produced from seed and collecting seeds from plants produced from cuttings. Its advantages are low cost, a high rebaudioside-A content and high output of stevioside. Wang (2006) claims a method of cultivating a new hybrid variety by systematically breeding a S. rebaudiana clone parental plant, which systematically breeds good single plants, selects and matches good combinations, propagates clone parental plants, hybridizes the producing seeds, and mixes the collected seeds. Asexual propagation of the parental plant is adopted to fix its good properties and make the parental plant clone to process group hybridization, which has simple programme and more rapid yielding efficiency than traditional breeding method; the new variety of S. rebaudiana bred according to this method has strong resistance, high leaf output, and a high content of total glycoside in the leaves. BIOTECHNOLOGICAL APPROACHES Tissue Culture Seeds of stevia show a very low germination percentage (Felippe and Lucas 1971; Felippe et al. 1971; Monteiro 1980; Toffler and Orio 1981) and vegetative propagation through cuttings is limited by the small number of individuals (Sakaguchi and Kan 1982). Tissue culture is the only rapid process for the mass propagation of stevia, and there have been a few reports of in vitro growth of stevia (Miyagawa et al. 1986) and in vitro micropropagation from shoot tips and leaves (Akita et al. 1994; Kornilova and Kalashnikova 1996; Constantinovici and Cachita 1997; Sivaram and Mukundan 2003; SalimUddin et al. 2006; Ahmed et al. 2007). Cell and tissue culture techniques have been widely used to study the

growth, metabolism, etc., of dicotyledonous plants (Yuang-ling and King-In 1978; Cheng et al. 1980; Facciotti et al. 1985; Barwale et al. 1986; Christou et al. 1987; Fametaer et al. 1990; Dhir et al. 1991a, b; Ahsan et al. 2000). Plant regeneration from in vitro culture can be achieved by embryogenesis or organogenesis. In stevia, regeneration has been achieved by organogenesis from different explants, such as leaves (Yang and Chang 1979; Ferreira and Handro 1987a, b), axillary shoots (Bespalhok-Filho et al. 1992), stem tips (Tamura et al. 1984b), suspension cultures (Ferreira and Handro 1988) and anthers (Flachsland et al. 1996). Somatic embryogenesis has previously been reported from leaves (Wada et al. 1981; Bespalhok-Filho et al. 1993) and stems (Miyagawa et al. 1984; Bespalhok-Filho and Hattori 1997). Somatic embryogenesis can also be obtained from floret explants of S. rebaudiana cultured on MS medium supplemented with 2,4-D (9.05 and 18.10 mM) and kinetin (0 to 9.29 mM). On 9.05 mM 2,4-D supplemented medium maximum embryogenic callus formation occurred in medium without kinetin. On 18.10 mM 2,4-D supplemented medium the best treatment was 2.32 mM kinetin. Embryogenic callus started at the base of the corolla and ovary (BespalhokFilho and Hattori 1997). The embryogenic callus is usually characterized by a light green or light yellow colour, compact structure and the presence of globular somatic embryos on its surface. The embryogenic callus formed first on the base of the ovary and/or corolla, and then proliferated throughout the whole explant. Nonembryogenic callus may also be present, characterized by a white colour and a hyperhydric appearance (Bespalhok-Filho and Hattori 1997). A synthetic auxin such as 2,4-D or picloram is usually used for the induction of somatic embryogenesis (Merkle et al. 1990). Auxin is needed to cause de-differentiation and to elicit totipotency (Terzi and Loschiavo 1990; Bespalhok-Filho and Hattori 1997). Anther Culture Anther culture is usually used to obtain haploid plants from which doubled haploids/homozygous plants can be developed through colchicine treatment in a short time and also in crops where self-incompatibility is the limiting factor for the development of homozygous plants or inbred lines. In this method, immature anthers from a differentiating population are grown or cultured on substrate. Anther culture is usually carried out at the beginning of a breeding programme. Once the populations of plants homozygous for a particular trait are available, new varieties are developed by hybridization and genetic studies can be carried out efficiently. Flachsland et al. (1996) regenerated plants from anthers of S. rebaudiana cultured in vitro under defined conditions. Anthers (containing uninucleate microspores) were induced to form callus when aseptically cultured on Murashige and Skoog liquid medium

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supplemented with 0.1 to 1 mg L 1 BAP. Regeneration of shoots was readily achieved by transferring pieces of callus to fresh solid medium with the same composition. Shoots were induced to form roots upon transfer to medium with 0.1 mg L1 NAA. Plantlets were successfully potted, but cytological studies of root tips from regenerated plants revealed a normal diploid number of chromosomes (2n 22). The study implies that somatic cells of the anther wall respond to the high BAP concentration in the medium. Marker Assisted Selection The development of molecular marker technology and consequent identification of marker loci linked to important agronomic traits have created exciting new opportunities for plant breeders. Marker-assisted selection provides the potential for improving selection efficiency by allowing for earlier selection and reduced plant population size (Staub et al. 1996). In the past decade the creation of genetic maps has been the foundation of this new plant breeding tool. One of the priorities of plant genome mapping is the identification of genes associated with economically important traits and the use of this information for further improvement of crops. The value of molecular markers for the development of linkage maps and their use in the analysis of economically important traits has been amply demonstrated in both, field crops (Paterson et al. 1991; Yu et al. 1991; Mackill et al. 1993; Paran and Michelmore 1993) and forest trees (Groover et al. 1994; Bradshaw and Stettler 1995). Molecular linkage maps have been constructed for most major crop plants (Staub et al. 1996; Paterson 1996); these maps provide a more direct method for the selection of desirable qualitative and quantitative traits through their linkage to easily detectable genetic markers (Edwards et al. 1987; Paran and Michelmore 1993; Mackill et al. 1993; Yao et al. 1999). FUTURE PROSPECTS Rebaudioside-A is of particular interest among the glycosides produced in the leaves of stevia because it has the most desirable flavour profile, while stevioside is responsible for aftertaste bitterness. Improved genotypes with a high content of rebaudioside-A with respect to other glycosides (like stevioside) need to be developed, as the Food and Drug Administration has approved rebaudioside-A with 95% purity. Further research and development need to be carried out to improve stevias potential as a crop by developing improved varieties with higher yield and quality through plant breeding methods and biotechnological approaches. SUMMARY Stevia rebaudiana is gaining popularity in various developed and developing countries as an important

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crop for the production of nonnutritive, nontoxic, high-potency sweeteners. It has many other curative properties, such as the inhibition of bacterial and fungal growth, and it is an anti-cancerous, antihyperglycaemic, anti-hypersensitive agent, it prevents dental caries and has contraceptive properties, as reported in the literature. In the recent past, research has been conduced around the world on various aspects of crop improvement, the development of new varieties, propagation, seed production, cultivation, disease resistance, improvement of glycosides quality and quantity. The major problem of large-scale cultivation is the lack of quality planting material. Stevia is a selfincompatible plant, and seed-grown plants vary in their growth, quality and quantity of diterpene glycosides and desirable ratio of rebaudioside-A and stevioside, which restrict its cultivation from seed. Generally, plants with desirable characteristics are propagated by stem cuttings and tissue culture practices, which limits the large-scale production of planting material. The emphasis in future research should be on the development of new seed varieties with wider adaptability to different climatic conditions, better germination and viable seed production, better leaf:stem ratio and with a high content of rebaudioside-A compared with other glycosides for successful cropping and higher diterpene glycoside production.
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YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 21 pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosenthesis from 1-deoxyxylulose in higher plants. Proc. Natl. Acad. Sci. USA 96: 13091314. Asamizu, E., Nakamura, Y., Sata, S. and Tabata, S. 2000. A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries. DNA Res. 7: 175180. Ashwini, K. S. 1996. Production of multiple shoots and somatic embryogenesis in Stevia rebaudiana Bertoni through in vitro propagation. M.Sc. (Agri.) thesis, UAS, Bangalore, India. Barathi, N. 2003. Stevia: The calorie free natural sweetner. Natural Product Radiance 2: 120122. Barwale, U. B., Goodman, M. M. and Widholm, J. M. 1986. Somaclonal variation in plants regenerated from culture of soybean. Plant cell rep. 6: 365368. Beaumont, V. H., Mantet, J., Rocheford, T. R. and Widholm, J. M. 1996. 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CANADIAN JOURNAL OF PLANT SCIENCE Dwivedi, R. S. 1999. Unnurtured and untapped super sweet nonsacchariferous plant species in India. Current Sci. (Bangalore) 76: 14541461. Dzyuba, O. O. 1998. Stevia rebaudiana (Bertoni) Hemsley a new source of natural sugar substitute for Russia. RastitelNye Resursy. 34: 8695. [in Russian, English abstract] Edwards, M. D., Stuber, C. W. and Wendel, J. F. 1987. Molecular facilitated investigations of quantitative trait loci in maize. I. Number, genomic distribution and types of gene action. Genetics 116: 113125. Eisenreich, W., Rohdich, F. and Bacher, A. 2001. Deoxyxylulose phosphate pathway to terpenoids. Trends Plant Sci. 6: 678684. Erich, M., Peter, Q., Berlinges, U., Nakes, A., Waters, H. and Helment, V. 1961. Direct correlation of the diterpene alkaloids and hydrocarbons of the phyllocladene group: Interconversion of garryfoline and steviol. J. Am.Chem. Soc. 84: 31633164. Erik, V., Hewritt, G. and Fletcher, J. R. 1956. Stevioside. IV. Evidence that stevioside is a sophoroside. J. Am. Chem. Soc. 78: 47094710. Ermakov, E. I. and Kotechetov, A. A. 1996. Specic features in growth and development of Stevia plants under various light regimes in regulated conditions. Doklady Rossiiskoi Akademii Selskokhozyaistvennykh Nauk 10: 89. Facciotti, D., ONeal, J. K., Lee, S. and Shewmaker, C. K. 1985. Light inducible expression of a chimeric gene in soybean tissue transformed with Agrobacterium. Biotechnology 3: 241246. Fametaer, I., Negrutiu, I., Mouras, A., Vaucheret, H. and Jacobs, M. 1990. Asymmetric hybridization in Nicotiana by Gamma fussion and progeny analysis of self-fertile hybrids. Theor. Appl. Genet. 79: 513520. Felippe, G. M. 1978. Stevia rebaudiana A review. J. Chromatogr. 161: 403405. [in Portuguese, English abstract.] Felippe, G. M. and Lucas, N. M. C. 1971. Estudo da viabilidade dos frutos de Stevia rebaudiana Bert. Hoehnea. 1: 95105. [in Portuguese, English abstract.] Felippe, G. M., Lucas, N. M. C., Behar, L. and Oliveira, M. A. C. 1971. Observacoes a respeito de germinacao de Stevia rebaudiana Bert. Hoehnea. 1: 8193. [in Portuguese, English abstract.] Ferreira, C. M. and Handro, W. 1987a. Micropropagation of Stevia rebaudiana through leaf explants from adult plants. Planta Medica. 54: 157160. Ferreira, C. M. and Handro, W. 1987b. Some morphogenetic responses of leaf explants of Stevia rebaudiana cultured in vitro. Rev. Brasil. Bot. 10: 113116. Ferreira, C. M. and Handro, W. 1988. Production, maintenance and plant regeneration from cell suspension cultures of Stevia rebaudiana (Bert.) Bertoni. Plant Cell Rep. 7: 123126. Flachsland, E., Mroginski, L. and Davina, J. 1996. Regeneration of plants from anthers of Stevia rebaudiana Bertoni (Compositae) cultivated in vitro. Biocell 20: 8790. Fors, A. 1995. A new character in the sweetener scenario. Sugar J. 58: 30. Frederico, A. P., Ruas, P. M., Marin-Morales, M. A., Fuas, C. F. and Nakajima, J. N. 1996. Chromosome studies in some Stevia Cav. (Compositae) species from Southern Brazil. Braz. J. Genet. 19: 605609. Fronza, D. and Folegatti, M. V. 2003. Water consumption of the stevia (Stevia rebaudiana (Bert.) Bertoni) crop estimated through micro-lysimeter. Sci. Agric. 60: 1518.

Chen, K., Chang, T. R. and Chen, S. T. 1978. Studies on the cultivation of stevia and seasonal variation of stevioside. China Gartenbau 24: 3442. Chen, S. and Shu, S. 1995. Study on storage technique of Stevia rebaudiana seed. Acta Agron. Sin. 21: 102105. Chen, S. Y. and Li, Q. R. 1993. Effect of growth substances on the stevioside content of Stevia rebaudiana. Plant Physiol. Commun. 29: 265267. Cheng, T. Y., Saka, H. and Voqui-Ding, T. H. 1980. Plant regeneration form soybean cotyledonary nodes in culture. Plant Sci. Let. 19: 9199. Christou, P., Murphy, J. E. and Swain, W. F. 1987. Stable transformation of soybean by electroporation and root formation from transformed callus. Proc. Nat. Acad. Sci. 84: 39623966. Cioni, P. L., Morelli, L., Andol, L., Macchia, M. and Ceccarini, L. 2006. Qualitative and quantitative analysis of essential oils of ve lines Stevia rebaudiana Bert. genotypes cultivated in Pisa (Italy). J. Essential Oil Res. 18: 7679. Coleman, J. R. 1968. Chromosome numbers in some Brazilian Compositae. Rhodora 70: 228240. Colombus, M. 1997. The Cultivation of stevia, natures sweetener. Ontario Ministry of Agriculture, Food and Rural Affaairs, Toronto, ON. p. 4. Constantinovici, D. and Cachita, C. D. 1997. Aspects of in vitro multiplication in Stevia rebaudiana Bert. Cercetari Agronomic in Moldova. 30: 8086. Crammer, B. and Ikan, R. 1986. Sweet glycosides from the stevia plant. Chem. Britain 22: 915916. Cruz, N. D., Boaventura, M. S., Conagin, C. H. T. M., Dutilh, J. H. A., Forni-Martins, E. R., Medina, D. M., Mendes, A. J. T., Pierozzi, N. I. and Pinto-Maglio, C. A. F. 1993. Cinqu tica Vegetal. s Anos de Pesquisa em Citogene enta e Tre Documents IAC, 27: 60 pp. Dacome, A. S., da Silva, C. C., da Costa, C. E. M., Fontana, J. D., Adelmann, J. and da Costa, S. C. 2005. Sweet diterpenic glycosides balance of a new cultivar of Stevia rebaudiana (Bert) Bertoni: isolation and quantitative distribution by chromatographic, spectroscopic and electrophoretic methods. Process Biochem. 44: 35873594. Darlington, C. and Wylie, A. P. 1955. Chromosome atlas of owering plants. 2nd ed. George Allen and Unwin Ltd., London, UK. 519 pp. DeLima, O. F., Malavolta, E. and Yabico, H. Y. 1997. Inuence of nutritional stress on content and production of stevioside during Stevia rebaudiana development. Pesquisa Agropecuaria Brasileira 32: 489494. Dhir, S. K., Dhir, S. and Widholm, J. M. 1991a. Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.). Plant Cell Rep. 10: 3943. Dhir, S. K., Dhir, S., Sturtevant, A. P. and Widholm, J. M. 1991b. Regeneration of transformed shoots electroporated soybean (Glycine max L.) protoplasts. Plant Cell Rep. 10: 97101. Donalisio, M. G. R., Duarte, F. R., Pinto, A. J. D. A. and Souza, C. J. 1982. Stevia rebaudiana. Agronomico 34: 6568. DuBois, G. E. 2000. Sweeteners: non-nutritive. Pages 2245 2265 in F. J. Francis, ed. Encyclopedia of food science and technology Vol.4. 2nd ed. John Wiley and Sons, Inc., New York, NY. Duke, J. 1993. Stevia rebaudiana. Pages 422424 in J. Duke, ed. CRC handbook of alternative cash crops. CRC Press, Boca Raton, FL.

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