A Review on the Improvement of Stevia

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 Scientific 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: 1Á27. 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. L’ame ´ lioration ge ´ ne ´ tique du ste ´ via [Stevia rebaudiana (Bertoni)], tour d’horizon. Can. J. Plant Sci. 91: 1Á27. 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 d’autres 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 n’est pas une plante autogame et la pollinisation s’effectue gra ` des insectes. De tous les glycosides que produisent les feuilles du ste ´ via, le re ´ baudioside-A pre ´ sente un ˆ ce a inte ´ re ´ est le plus de ´ sirable, le ste ´ vioside laissant un arrie ` re-gou ´ ation de ˆ t particulier, car son profil de sapidite ˆ 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 accroıˆ tre la production de ces e ´ dulcorants naturels et en favoriser l’utilisation. Un seul ge ` ne additif, co-se ´ gre ´ gant, commande la proportion de re ´ baudioside-A et C produite, ce qui laisse supposer l’intervention du me ` se. Le ste ´ vioside et le re ´ baudioside-A sont ne ´ gativement corre ´ le ´ s, alors qu’il ˆ 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 d’ame ´ lioration ge ´ ne ´ tique comme la se ´ lection et l’hybridation 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, Can. J. Plant Sci. Downloaded from pubs.aic.ca by 186.147.198.168 on 10/13/13 For personal use only. 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: 1Á27 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 l’he ´ te ´ rosis existante, en raison du fort degre ´ naturel de croisement exte ´ rieur et de l’absence d’un syste ` me efficace re ´ gulant la pollinisation. Des varie ´ te ´ s synthe ´ tiques et compose ´ es comme AC Black Bird et PTA-444 ont d’ailleurs de ´ ja ` e ´ te ´ re ´ alise ´ es. La polyploı¨ die entraıˆ ne une meilleure capacite ´ d’adaptation des plantes, tout en engendrant de plus gros organes et cellules. Les te ´ traploı¨ des se caracte ´ risent par des feuilles plus grandes et e ´ paisses, dont la biomasse et le rendement pourraient de ´ passer ceux des souches diploı¨ des. L’ame ´ lioration par mutation pourrait aussi donner lieu a ` une ame ´ lioration des caracte ` res recherche ´ s, au sein d’une population peu variable. Le recours a ` certaines me ´ thodes biotechnologiques comme la culture tissulaire pour la multiplication massive des meilleurs ge ´ notypes, la culture d’anthe ` res pour le de ´ veloppement d’homozygotes purs a ` double haploı¨ die et l’usage de marqueurs mole ´ culaires pour identifier l’emplacement des locus associe ´s a ` la synthe ` se du re ´ baudioside-A pourrait offrir d’autres possibilite ´ s aux ame ´ liorateurs. Enfin, comprendre le me ´ canisme et les voies de la biosynthe ` se des glycosides du ste ´ viol permettrait d’en 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 150Á200 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 Can. J. Plant Sci. Downloaded from pubs.aic.ca by 186.147.198.168 on 10/13/13 For personal use only. borders of rivers and dry valleys (Robinson 1930).168 on 10/13/13 For personal use only. five in number. CLASSIFICATION OF STEVIA Stevia rebaudiana is one of the 950 genera of the Asteraceae family (Soejarto et al. Stevia satureiaefilia. Korea. and used it as sweetener for their green herbal tea ‘‘mate’’. Stevia rhombifolia Á Kunth. 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. open forests. Shock 1982. with an average of 238C. southward to Central Argentina (King and Robinson 1987). including Brazil. (2004) . Stevia commixta. Canada and India (Lee et al. It also occurs from non-Amazonian South America. Tanzania. Its first botanical description is attributed to M. Plant Sci. Stevia serrata Á sawtooth stevia. with temperatures ranging from 6 to 438C. the first seeds were exported to the United Kingdom where it could not be brought under cultivation. J. Stevia origanoides and Stevia triflora. In Brazil. rebaudiana the pollen viability was 65%. It is a perennial herb with an extensive root system and brittle stems. Fors 1995). In the native state it grows on the edges of marshes or in grassland communities on soils with shallow water tables (Shock 1982). Stevia nepetifolia. The pollen can be highly allergic. Stevia viscida Á viscid stevia. Brandle and Rosa 1992. between latitudes 238 and 248S. but also for the medicinal effects that were attributed to it (Bertoni 1905). in honour of Rebaudi. Stevia consists of a group of annual and perennial herbs. Goenadi 1983. 1) and produce all its flowers (Taiariol 2004. Its name was later changed to the current one. which differs from the results of Oliveira et al. the United States of America. Anther. 2005). Although there are about 230 species in the genus. Stevia leptophylla. Pollen and Stigma Anthers are small. Downloaded from pubs. only S. Stevia plummerae var. 1983. A systematic study of the North and Central American species of Stevia was done by Grashoff (1972). 1982. BOTANICAL DESCRIPTION Floral Biology Flower Structure The inflorescence is loosely paniculate with the heads appearing opposite the bracts in irregular sympodial cymes. 1983). borne in small corymbs of two to six florets (Goettemoeller and Ching 1999). Frederico et al. The climate can be considered as semi-humid subtropical. S. elliptical leaves. The Guarani Indians called the plant ‘‘kaa he-he’’. 1998.ca by 186. Jeppensen et al. By now. 1996). subshurbs and shrubs that occur in mountain regions. The cultivated plants are reported to be more vigorous (Shock 1982. Stevia oligophylla. In 1943. 36 species have been found. Stevia lemmonii Á Lemmon’s stevia. In 1968 it was exported to Japan. Stevia micrantha Á annual stevia. The plant was first called Eupatorium rebaudianum Bert. Indonesia. Dwivedi 1999) with pale purple throat corollas. 2006). while other species contain other biochemicals of interest (Frederico et al. the crop has been introduced to many countries. Bertoni. Stevia ovata var. Srimaroeng et al. Monteiro (1980) observed that in some diploid individuals of S. Saxena and Ming 1988. Stevia salicifolia Á willow-leaf dtevia. Melis 1999. 2003. and it was recommended not only for food production. 2002. southward through Mexico and Central America. The plant can initiate flowering after a minimum of four true leaves have formed. the first chemist to study the chemical characteristics of the substances extracted. Using the acetocarmine technique. Donalisio et al. producing small. * A REVIEW ON IMPROVEMENT OF STEVIA 3 Can. 1983). Lester 1999). 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. Under some environmental conditions and management situations it behaves as an annual or a mixture of plants of both types. It is indigenous to the Rio Monday Valley of the Amambay moutain region at altitudes between 200 and 500 m. 1996).aic.147.198. sh et al. texana Á roundleaf candyleaf. and as a flavour enhancer (Soejarto et al. rebaudiana gave the sweetest essence (Soejarto et al. Stevia ophryphylla. Stevia rebaudiana originated in the highland regions of northeastern Paraguay (on the Brazilian border). 1996). The flowers are small (15Á17 mm) and white (Marsolais et al. The tiny white florets are perfect (hermaphrodite) having both male and female organs. Mexico. Stevia plummerae Á Plummer’s stevia. and rainfall ranging from 1500 to 1800 mm per annum. and from there awareness of and cultivation of the plant spread throughout the world (Lewis 1992). They are arranged in indeterminate heads. The plant takes more than a month to pass through the various developmental flower stages (Fig. Stevia selloi. 1979. Stevia myriadenia. which translates as ‘‘sweet herb’’. distributed mainly in southern and central regions (Frederico et al.YADAV ET AL. 1998. alba. . (a) Stevia anthers.4 CANADIAN JOURNAL OF PLANT SCIENCE Fig. b) (Felippe 1978. Downloaded from pubs.aic. whereas infertile seeds are usually pale or clear (Fig. ranging from 8 h to 14 h (Valio and Rocha 1977.7 to 68. Different stages of flower opening. J. Each achene has about 20 persistent pappus bristles (Goettemoeller and Ching 1999). Zaidan et al. Zaidan et al. a b c d Fig. (b) germinated pollen with pollen tube. Maiti and Purohit 2008). Seeds have a very small endosperm and are dispersed in the wind via hairy pappus. Stigma is bi-lobed/ bifurcated from the middle and style is surrounded by anthers (Fig. Agamospermy. It can be induced to flower from the four-leaf-pairs stage onwards. 1980. The variability for photoperiod sensitivity is large. 1986. rebaudiana produce normal and malformed pollen. (1997) and Lester (1999) reported a poor and highly variable percentage of viable seeds. Duke (1993). Reproduction and Pollination Behaviour Stevia is self-incompatible (Miyagawa et al.198. Seed Seeds are contained in slender achenes about 3 mm in length. could explain the reproductive capacity of this species.7%. 1997) and probably insect pollinated (Oddone 1997). 1999. Seed production in 1 ha would be enough for leaf production in 200 ha (Lester 1999). a minimum of two inductive short-day cycles are necessary for flowering induction. but plants remained vegetative under an 8-h photoperiod with interrupted night (Valio and Rocha 1977).1 kg ha1 have been recorded. 1976. Flowering under short-day conditions should occur 54Á104 d following transplanting. Chalapathi 1997). but it is common to achieve less than 50% germination. Brandle et al. i. (c) stigma and (d) stigma coming out of anthers. 2. seed collected from an individual plant would represent a half-sib family. respectively. Seed yields of up to 8. Fertile seeds are usually dark coloured.3Á1. Sexual and apomictic plants of S.ca by 186. Goettemoeller and Ching (1999) revealed that some active manipulation of the blossoms is necessary to achieve pollination.5%. According to Monteiro (1980). even if one takes into account the total lack of viable pollen observed.168 on 10/13/13 For personal use only. Since stevia is selfincompatible. Carneiro et al. the asexual formation of seeds (Lumaret 1988). rebaudiana shown by embryological studies may be related to specific physiological and/or ecological factors. reaching a size suitable for transplanting to the field at 45Á60 d (Colombus 1997. Shock (1982). (1980) identified three photoperiod classes based on the daylength. Plant Sci. 2). Monteiro 1980.e. Analysis of sporogenesis allows the detection of irregularities that can lead to the formation of inviable gametes.0 g) and as a result seedlings are slow to develop. while outcrossing ranged from 0. 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. Oddone 1997. According to Valio and Rocha (1977). Grashoff (1974) and Monteiro (1980) reported agamospermy in certain genotypes of S. with no viable pollen grains. It has been reported that the amount of selfing ranged from 0 to 0. the presence of apomixis in S. rebaudiana. Goettemoeller and Ching 1999).147. Chalapathi et al. 1. 1998a). Flowering is more precocious in the 8-h photoperiod. depending on the daylength sensitivity of the cultivar. 3a.. Seeds are very small (1000 seeds weigh 0. Can. indicating that the self-incompatibility system is operating (Katayama et al. Carvalho and Zaidan 1995.5 mm) (Shaffert and Chebotar 1994). The superior performance of the 15-cm-long cuttings may be because they have double the quantity of food reserves compared to 7. root-neck sprouts. the pair of leaves in the cuttings as well as the season also acts as determinants for the rooting percentage and growth. 2006).aic. 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. 1994. sugar and phenolic compounds for root initiation. sanitation. The material contains the sweet components. 9 stevia. Yang and Chang 1979. farming practices. lanceolate to oblanceolate. 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. (1991b) found better rooting and sprouting of stevia cuttings by prolonged dipping of cuttings in 50 ppm IBA solution. 1998). Some growth regulators can sometimes influence (increase) the concentration of steviosides in the leaves (Chen and Li 1993.147. 1990. Cuttings of new stems and shoots can be propagated successfully (Lee et al. (1990) reported that nearly 98Á100% rooting can be obtained. which root easily. including soil conditions. carbohydrates. 3. Rooting of cuttings can sometimes be stimulated by the use of growth regulators (Zubenko et al. can be used successfully for tissue culture propagation (Handro et al. Gvasaliya et al. shoot primordia. Can. PROPAGATION a) Vegetative Propagation Shoot Cuttings Propagation of stevia is usually done by stem cuttings. the leaf area index (LAI) 80 d after sowing was 4. 1999a). keeping two to three nodes above the soil. when the current year’s cuttings are taken from the leaf axils. Leaves are small. Ramesh et al. Swanson et al.198. Nishiyama et al. The cut portion of the branch is dipped in neem oil or any other fungicide (Maiti and Purohit 2008).YADAV ET AL. especially in February. Stevia seeds (a) infertile seeds are usually pale or clear and (b) fertile seeds are dark coloured. Shoot vigour index is an important indication of vigorous growth of sprouted cuttings. 1986. * A REVIEW ON IMPROVEMENT OF STEVIA 5 a b Fig. processing and storage. Bespalhok-Filho et al. refereshing taste that can linger in the mouth for hours. Trichomes on the leaf surface are of two distinct sizes. Constantinovici and Cachita 1997). Akita et al. caused complete inhibition of rooting and sprouting of cuttings. 1991a. 1991). Kornilova and Kalashnikova 1996. however. The sprouting percentage and shoot growth is also affected by the length of the cuttings. Cuttings with two pairs of leaves rooted best in February and those with three pairs of leaves in April (Zubenko et al. Miyagawa et al. Cuttings with four pairs of leaves rooted poorly. Shock 1982. internodal explants etc. but require high labour inputs. Zubenko et al. irrigation methods. Leaf Stevia has an alternate leaf arrangement and herbacious growth habit (Singh and Rao 2005). a vigorous branch is cut at the base with a sharp blade and planting in the field.. Kornilova and Kalashnikova 1996. 1979. Plant Sci.5-cm-long cuttings. Some plant varieties/selections produce virtually no viable seed and vegetative propagation is the only way of multiplication. large (4Á5 mm) and small (2. Sprouting. however. 1977. serate above the middle and somewhat folded upwards. 1992. Micro-propagation Many different parts of the plant viz. the location on the plant from which cuttings are taken can also affect growth and rooting.5-cm-long cuttings (Chalapathi et al. Bondarev et al. In vitro multiplication has frequently been used to multiply individually selected or bred clones and successful procedures have been documented (Handro et al. was not influenced by NAA or IBA treatment at 500 ppm. 1992.ca by 186. The leaf has a pleasantly sweet. Kornilova and Kalashnikova . J. 1977. sessile. Shock (1982) opined that stem cuttings were the prime means for the propagation of stevia. Acuna et al. Ferreira and Handro 1988. which may be due to the injury caused to the callus tissue. oblong. Stolz (1968) indicated the necessity of higher quantities of starch. leaves. The sprouting percentage and shoot growth of sprouted cuttings were significantly higher with 15-cm-long cuttings compared with 7. surrounded by the bitter components in the veins (Maiti and Purohit 2008). 1991b. However. IBA or NAA at a higher concentration of 1000 ppm. air purity. 2001).168 on 10/13/13 For personal use only. Stevia leaves vary widely in quality due to many environmental factors. Kornienko and Parfenov 1996. Cuttings from the top part of the main shoot with four internodes generally gave the best results (Tirtoboma 1988).. The direct planting of stem cuttings in the field was found to have a limited success (Chalapathi et al. Gvasaliya et al. 1997). Ferreira and Handro 1987a. 1993. Further. Paclobutrazolinduced sprouts were found to be more vigorous than IBA.83 (Fronza and Folegatti 2003). In general. Cuttings taken in late winter rooted better than those taken at other times (Carvalho and Zaidan 1995).or NAA-treated cuttings (Chalapathi et al. sunlight. 2001). auxiliary shoots. Downloaded from pubs. Takahashi et al. 3.0 mg L 1 2. Lee et al. whereas field collected seeds gave only 34% germination (Carneiro 1996).168 on 10/13/13 For personal use only. Fig. The germination study of seeds obtained from five pollination treatments: cross-pollination by bumble bees (78. nodal (Fig. shoot biomass and stevioside content were greater in the vegetatively grown plants (Truong and Valicek 1999). crosspollination by wind (68.0 mg L 1 2.7 vs. Using seed to establish crops of stevia is more successful in tropical climates. Timing of flowering. seeds collected in the field and greenhouse were compared for germination. 1996). The number of roots. However. but germination and establishment from seed are often poor and sometimes unsuccessful (Shaffert and Chebotar 1994).0 mg L 1 for callus induction.e. 4) and inter-nodal segments were cultured on MS medium containing 2. cross-pollination by hand (92. Bondarev et al. 4. self-pollination by hand (93. 2006). Nepovim 1998). Seed yield up to 8 kg ha1 with 50% germination would be sufficient for 200 ha of crop (Lester 1999). 1977). Strauss 1995).3%). Carneiro et al. 1998a). Goettemoeller and Ching (1999) investigated the low seed germination of stevia and tested the viability of seeds based on tetrazolium chloride. 8. SEED VIABILITY AND GERMINATION Two types of seeds are found in stevia.4-D at 2.4-D. Carneiro and Guedes 1992. Inter-nodal segments initiated callus formation earlier than node and leaf. (1979) found that plants from seeds were less productive in the first year than those from cuttings. Rain at flowering can also reduce seed setting. Stevia flowers need to be fertilized by pollen from another plant to produce viable seed. 1998. Increased temperature (408C on moist paper) for less than 24 h can accelerate germination.3%) suggested that incompatibility is not a factor in these clones. 76. finding that the viability of black seeds was much higher than that of tan-coloured seeds.ca by 186. but reduce total germination (Tanaka 1985). Harvesting of immature seed may also contribute to poor germination (Colombus 1997).aic. In Japan. Can. respectively. The greatest amount of callus was produced in MS medium with 3. seed harvest and pollination methods play an important role in seed production (Melis and Sainati 1991.6 CANADIAN JOURNAL OF PLANT SCIENCE Shade can reduce total growth.4-D gave the poorest callus (Salim-Uddin et al. 4.147. In one experiment. The greenhousecollected seeds had a 90% germination rate. where there is no climatic restriction on the length of the growing season. Seed production and fertility studies suggest that high germination rates are possible from selected lines (Carneiro and Guedes 1992). Downloaded from pubs. with black seeds being heavier than tan seeds. 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. light generally increases germination. Germination rates of stevia seeds vary greatly. 1996. J. i.0%). This suggests that tan seeds represent inviable seed that are produced without fertilization. This contrasts with claims of optimum storage at 08C still producing a 50% loss of viability after 3 yr (Brandle et al. Germination requires at least 208C and often more than 258C. 1997. but may be a requirement for large-scale commercial production.. Total quantities of viable seed produced in 1 yr are uncertain. In northern climates the shorter growing season necessitates seedling establishment in a glasshouse/greenhouse prior to the growing season. Plant Sci. Cross-pollination and self-pollination was accomplished by transferring pollen with a bumble bee (Bombus impatiens) thorax on the end of a toothpick. Light increases the germination of black seed but not of tan seed. all pollination treatments increase seed germination of black seed over the control.198. black and tancoloured. The influence of pollination treatments as well as the effect of light and darkness during germination were also evaluated. whereas. Propagation through seeds is not a common method of propagation owing to the problem of low seed production and poor germination capacity. . suggesting that some active manipulation of the blossoms is necessary to achieve pollination (Goettemoeller and Ching 1999). A high density of bees (three to four hives per hectare) is recommended for good seed production (Oddone 1999).0. Chen and Shu 1995.0. Seeds germinate within 7Á10 d after sowing. seed remained viable for up to 3 yr when stored under low humidity and in darkness (Kawatani et al.3%). delay flowering time and reduce the rate of flowering (Slamet and Tahardi 1988). Direct seeding to the field is not practiced. Explants from leaf. Seed Propagation Reproduction in the wild is mainly by seed.3%) and control (36.0 and 5. It can take 4Á6 d to reach two-thirds of the final germination of 62Á90% at 258C (Shock 1982. MS medium with 5.3%. Micro-propagation of stevia from nodal explants. 1961. 2007). their presence conferred significant advantage upon those individuals that possessed them. and rebaudioside-A (Reb-A). rebaudioside-A.198. non-fermentable and does not darken upon cooking (Crammer and Ikan 1986). 520Á300. ESTs have also become an effective means of gene discovery in focused metabolic situations (Sterky et al. 1995). * 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. rebaudioside-C (1Á2%). It is also responsible for the bitter aftertaste. 2Á4%. 2005. There are also other related compounds including minor glycosides. As well as sweetness. Harry et al. that enriched libraries and efficient high-throughput sequencing is widely available. The two main glycosides are stevioside. stevioside may have a lingering effect or certain degree of pungency. rebaudioside-C and dulcoside-A.08% of the dry weight of leaves (Ashwini 1996. rebaudiosideD. Stevioside content is higher in Accession 1 compared with Accession 2 (Megeji et al. Among the components of stevia. 2006). Rebaudioside-A is usually present as 30Á40% of total sweetener and has the sweetest taste. 50Á120. 30 and 30 times. 1977. 1985). Under Indian conditions stevioside concentration was about 9. but their high concentration in the leaf and the conservation of the pathway within the species indicate that. sometimes reported as a ‘‘licorice’’ taste. The purpose of these compounds in the stevia plant is not yet clear.7%).0%) and betapinene (5.168 on 10/13/13 For personal use only. one. There are reports of stevioside content (total glycosides) ranging between 4 and 20% on a dry weight basis. Expressed sequence tags (EST) are a powerful tool that has emerged from genomics research. daylength (Metivier and Viana 1979) and agronomic practices (Shock 1982). an important plant hormone (Singh and Rao 2005). This concept was first applied to the isolation of oleate hydroxylase from castor (Van de Loo et al. Brandle 1999. The minor glycosides are considered to be less sweet. dulcoside-C and steviolbioside. Yohei and Masataka 1978. 2005). They are therefore of no nutritional value (Hutapea 1997). Hiroshi et al. Oddone 1999). rebaudioside-B.3Á18. 2002). 1984a). If rebaudioside-A is present in equal quantities to stevioside. gene regulation and sequence diversity. cinnamic acids. Makapugay et al. 1956. traditionally 5Á10% of the dry weight of the leaves. rebaudioside-C. Paraguan leaves contain the highest concentration (9Á13%) of the sweet steviosides/rebaudiosides molecules. The ratio of rebaudioside-A to stevioside is the accepted measure of sweetness quality. Starrat et al. 50Á120 and 100Á125. they are undigested and no part of the chemical is absorbed by the body. the more rebaudioside-A the better. Putieva and Saatov 1997. 200Á300. 1998. Expressed sequence tag collections can reveal gene expression patterns.ca by 186. ESTs have been used to find . rebaudioside-F. rebaudioside-E. 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. The genotypes under study at the Institute of Himalayan Bioresource Technology show considerable morphological variation. Plant Sci. then glucosylated or rhaminosylated to form the principle sweeteners. 300Á350. The yield of sweetening compounds in leaf tissue can vary according to method of propagation (Tamura et al. 1976. Biosynthesis of Steviol Glycosides The steviol glycoside and gibberellin pathways diverge at kaurene. It is also non-calorific. at some point in evoluntionary time.147. Sekaran et al. Dzyuba 1998.5Á21. at least in the initial stages. According to Kinghorn (1987) the sweetness of these compounds relative to sucrose are 210. 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). beta-caryophyllene (2. 350Á450. coumarins. assessed as 180Á400 times sweeter than sugar with no bitter aftertaste (licorice taste or lingering effect). respectively. In general. such as rebaudioside-B. these are the sweetest compounds. which is not appreciated by the majority of people. In stevia. The sweetening potency (sucrose 1) of stevioside. 1984. Downloaded from pubs. caryophyllene oxide (1. 1956. 1989. Tsanava et al. Crammer and Ikan 1986. and Indian stevia is midway between these. as well as flavonoid glycosides. Dacome et al. dulcoside-A and steviolbioside are 250Á300. The four major sweeteners are stevioside. kaurene is converted to steviol. The precursor compounds are synthesized in the chloroplast. stevioside is stable at high temperatures (1008C) and over a range of pH values (Kinghorn and Soejarto Can. Chalapathi 1996).YADAV ET AL. 242. respectively (Crammer and Ikan 1986). Chinese stevia contains only 5Á6%.9%). it appears that the aftertaste is eliminated. The sweetening effect of these compounds is purely taste.aic. Masur et al. depending on the cultivar and growing conditions (Kennely 2002. Rajbhandari and Roberts 1983. using the same pathway as gibberellic acid. called rebaudioside-A. Kinghorn 1987.5%) (Cioni et al. and from there are transported to the endoplasmic reticulum. Shaffert and Chebotar 1994.1Á16. rebaudioside-A. Ohlrogge and Benning 2000). Now. dulcoside-A. the ‘‘backbone’’ of the sweet glycosides. Golgi apparatus and then vacuolated. Erich et al. phenylpropanoids and some essential oils (Erik et al. rebaudioside-D.4Á40. 30Á80 times sweeter than sugar (Crammer and Ikan 1986. rebaudioside-E. Since then. Forty different components were identified and the main constituents in all studying samples were spathulenol (13. Unlike many low-calorie sweeteners. and which reduces its acceptability. Stevioside traditionally makes up the majority of the sweetener (60Á70% of the total glycosides content) and is assessed as being 110Á270 times sweeter than sugar. is of particular interest because it has the most desirable flavour profile (DuBois 2000). J. The average GC content of the ESTs was 42. 62 were candidates for diterpene glycoside synthesis. In S. Glycoside Content in Different Plant Parts Plant organs contain different amounts of the sweet glycosides.aic. similar to what has been found with Arabidopsis ESTs (Asamizu et al. 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. The sweetness in the leaves is two times higher than that in inflorescence (Dwivedi 1999). which is known to be involved in the three step oxidation of kaurene to kaurenoic acid (Helliwell et al. 1999). 1999). stem. Therefore. 2003). by a novel P450 mono-oxygenase (Helliwell et al. 2002). steviol is synthesized from GGDP. ()-Kaurene is then oxidized at the C-19 position to ()-kaurenoic acid. Of the 278 ESTs classified into the ‘‘secondary metabolism’’ category. and further proves that the DXP pathway is used to synthesize IDP for conversion to steviol.147. Rhamnosylated glycosides can also be formed by the addition of a UDP rhamnose moiety to steviolmonoside (Richman et al. which forms stevioside (Shibata et al. The enzymes involved in steviol glucosylation have been partially characterized and there is an understanding of the inheritance of certain glycoside patterns. RodriguezConcepcio ´ n et al. next the C-19 carboxylate is glucosylated. Isopentenyl diphosphate and DMADP are converted to geranylgeranyl diphosphate (GGDP) by GGDP synthase via three successive condensation reactions (McGarvey and Croteau 1995). The enzymes catalyzing the first two steps of this pathway. 1991. Sekaran et al. Plant Sci. which decline in the following order: leaves. seeds and roots. The ent-kaurene skeleton of chloroplast diterpene glycosides. 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. provide attachment points for the sugar side chains that determine the identity of the different glycosides. 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. Eisenreich et al. Next. (2000). flowers. encoding polypeptides of about 76. Totte ´ et al. rebaudiana leaf ESTs. The synthesis of kaurenoic acid is a key step in the synthesis of both steviol and gibberellic acid (Brandle et al. rebaudiana a good candidate for an EST-based gene discovery effort. The C-13 alcohol is successively glucosylated. corresponding genes have not been isolated (Shibata et al. In an effort to create a resource for gene discovery and to understand the synthesis of steviol glycosides. Steviol is produced by the hydroxylation of ()-kaurenoic acid at the C-13 position. 1991.6 and 51 kDa. 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). Richman et al. Downloaded from pubs. With electronic probes. The most highly represented pathway EST was an orthologue of the Arabidopsis GA3 gene. Brandle et al. 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. but the gene for this FAD-dependent monoxygenase has not yet been isolated (Kim et al. The cDNAderived amino acid sequences for DXS and DXR contain 716 and 474 residues. first yielding steviolmonoside then steviol-bioside. 1998a). Stevia rebaudiana leaves can accumulate high concentrations (up to 30%) of seven different glycosides derived from the tetracyclic diterpene steviol (Brandle et al.168 on 10/13/13 For personal use only. 1995). 1999). 2000.5%. first by protonationinitiated cyclization to ()-copalyl diphosphate (CDP) by CDP synthase (Richman et al. these compounds are synthesized exclusively in the mesophyll cells of leaves and are undetectable in the roots. ()-kaurene is produced from CDP by an ionization-dependent cyclization catalyzed by ( )-kaurene synthase (Richman et al. 1999). rebaudiana. The pathway terminates with the glucosylation of stevioside. Like all diterpenes. 2000). The two oxygenated functional groups of steviol. 2000). No members of the mevalonic acid pathway were identified among the S. is formed via the recently discovered 2-C-methyl-D-erythritol 4-phosphate pathway. which forms rebaudioside A. database searches and differential representation. (2007) reported that individual tissues of stevia appear to differ significantly. 2001). Brandle et al. respectively. 1999.198. 1999). 1996).8 CANADIAN JOURNAL OF PLANT SCIENCE new genes from 1-deoxy-D-xylulose 5-phosphate (DXP) pathway of different crops. Although the adaptive value of steviol glycosides is not known. Roots are the only organs that do not contain stevioside. Their intense sweetness and close structural relationship to gibberellic acid. This supports the work of Totte et al. candidate genes for 70% of the steps in the steviol glycoside biosynthetic pathway have been searched. the C-19 carboxylate and the C-13 alcohol. rebaudiana has committed a very large portion of total metabolism to their synthesis. however. 2002). 1998. Hedden and Phillips 2000). 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.ca by 186. J. Candidates were identified for 70% of the steps in the pathway from pyruvate and glyceraldehydes 3-phosphate to rebaudioside A. Can. were characterized. 2002). Brandle et al. . 2000). (2002) sequenced 5548 random cDNAs from a S. 1999. making S. only a few genes involved in the biosynthesis of steviol glycosides have been isolated and characterized (Richman et al. coupled with the highly active nature of the pathway have fostered interest in steviol glycoside biosynthesis and metabolism (Richman et al. Despite this amazing metabolic capability. rebaudiana leaf library. 1999. it is clear that S. which are produced in large quantities in the leaves of Stevia rebaudiana. 2002). 1999. Since. Ajuga reptans and Serratula coronata (Vereskovskii et al.ca by 186.1% (Bondarev et al. Long-day conditions. has been confirmed (Fig. During the fruit development stage the levels of glycosides were found to revert to the initial level. all the shrubby species have a gametic chromosome number of n 12.YADAV ET AL. As stems mature and lose colour. At the whole-plant level. i. Cytological studies on many species of genus Stevia. although it is suggested that they may contain some flavour enhancers. in upper young leaves as compared with lower senescent ones. The concentration of glycoside in the leaves of stevia increases when the plants are grown under long days. According to Bondarev et al. (1972). Only severe Ca deficiency caused reduction in the glycoside concentration (DeLima et al. Tomas et al. 1995). development and chemical content of stevia roots have also received attention. Plant Sci. Chen et al. 1999). 1983. little variation in the glycoside content is also found in roots. the phenotypic expression of which is influenced by climatic and environmental factors (Metivier and Viana 1979. increase internode length. Ermakov and Kotechetov 1996). Stevia is grown as a perennial crop in subtropical regions. strains with 2n 33 and 2n 44 (representing triploid and tetraploid cytotypes) also occur. performed by Grashoff et al. The largest amount of stevioside was found in the upper young. 2005. In these organs. 2006). synthesis of terpenes is affected by climatic and environmental factors (Langston and Leopold 1954). while. any steviosides present dissipate.147. 5) for various strains. 1998). often associated with culturing procedures (Yamazaki and Flores 1989. including parts of the United States. This argues in favor of possible increased accumulation. there is a positive correlation between gland distribution density and steviol glycoside content. Similar results were obtained for ecdisteroids in Rhaponticum carthamoides. delaying flowering with long days allows more time for glycoside accumulation (Metivier and Viana 1979. Sekaran et al. (1998) opined that environmental and agronomic factors have more influence on stevioside production. and reduce the interval between the appearances of successive leaf pairs in S. odourisers and other agents of potential use for improving foodstuffs or alcoholic beverages (Singh and Rao 2005). rebaudiana. 1997). which show a high degree of male sterility owing to the chromosomal abnormalities during gamete formation. leaf area. whereas the lowest senescent shoot sections exhibited the smallest amount of such compounds. 1991. those tissues devoid of chlorolphyll. and wind (sh et al. the portion of the rebaudioside-A in the total glycosides content appeared to be increased as well. Anufrieva et al. from the vegetative phase to flowering. Total soluble leaf sugars. The results indicate that yield depends mainly on the genetic characters of the plant. . such as roots and lower stems. Moreover. Stems of stevia plants contain little or no sweeteners. steviol glycosides tend to accumulate in tissues as they age. concluded that in North America. Once flowering is initiated. * A REVIEW ON IMPROVEMENT OF STEVIA 9 with the stevioside content declining in a different order: leaves shoots roots flowers. and this process lasted to the budding phase at the onset of flowering (Bondarev et al. The chromosome number of Stevia rebaudiana (2n 22).e. Tateo et al. in roots the total glycosides content never exceed 0. Since glycoside synthesis is reduced at or just before flowering. However.168 on 10/13/13 For personal use only.91 in in vitro shoots. mature leaves of stevia have fewer glands per leaf surface area than the upper. chloroplasts are important in precursor synthesis. soil moisture. a gradual decrease in the glycoside content was observed. However. a gradual increase in the stevioside concentration was observed in both mature leaves and stems. The ideal climate for stevia is a semi-humid subtropical with temperatures ranging from 6. the lower. and stevioside content are also augmented in both absolute and relative terms and the biosynthesis of steviol. younger leaves. J. temperature. Singh and Rao 2005). Revina et al. 2001). (1978) studied the seasonal variation in stevioside content. 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. 1982).65 in ex vitro green leaves and 0. CYTOLOGY The genus stevia shows great variation in chromosome number. contain no or trace amounts of glycosides. Environmental Effect The growth and flowering of stevia are affected by radiation. 1993. daylength. by 30 to 170% of glycosides. such as temperature. protein. Depending on the clone.0 to 438C with an average of 238C (Brandle and Rosa 1992). so that older lower leaves contain more sweetener than younger upper leaves. and length and intensity of photoperiod. 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. All this information may indicate that the steviol glycosides are transported to generative organs. actively growing shoot sections. Downloaded from pubs. During ontogeny. The structure.aic. 1986. (2003). and dry weight. Research conducted in Egypt revealed that climatic conditions. glycoside concentrations in the leaves start declining (Singh and Rao 2005). the aglucone present in stevioside. During ontogeny. (1996) and Monteiro (1980. greatly affect stevia production and quality. Yamazaki et al. Can. and as an annual crop in mid to high latitude regions (Goettemoeller and Ching 1999). previously reported by Frederico et al..198. 2003). 2003). (2007) have shown a rebaudioside A/stevioside ratio of 1. Zubenko et al. The chemical content of the last five fully expanded leaf pairs showed the plant nutritional status (Utumi et al. as evident from the remarkable increase in yield during the summer compared with winter (Allam et al. is increased by 45%. as compared with short days. which occur spontaneously in nature. 34. For diploid strains. (1996) concluded that. 1985. Even in species with the same karyotypic formula. such as irregular chromosomal disjunction in anaphase. However. The main mechanism in the evolution of the South American species of stevia is probably chromosome inversions. organensis from Brazil) having x 12 (Coleman 1968. in none of the S. the lack of viable pollen cannot necessarily be linked with regular pairing at diakinesis (bivalents) and high rates of tetrad normality. This multivalent formation suggests that these strains may have an autopolyploid origin. Unlike the above cytotypes. was studied by Frederico et al.aic. with a variable number of submedian ones. Most of the chromosomes were metacentric. pairing at diakinesis for the triploid and tetraploid strains was n 11III and n 11IV. with only three species (S. jujuyensis from Argentina. polyploids with an odd number of chromosome sets have a high level of sterility (Lawrence 1980). The chromosomal morphology of six Brazilian species of Stevia. Accordingly. 1984). the sm pairs were located in different positions in the karyotypes. (1996). revealed that every species has a difference in arm ratios for at least one chromosome pair. respectively (Stace 1980). rebaudiana. respectively. rebaudiana strains studied was the pollen viable. which agrees with the literature. structural rearrangements have played an important role in the chromosomal evolution of the tribe Eupatorieae. while. They observed that karyotypes were very similar in chromosome number (2n 22) and size (1. Although pairing at diakinesis was normal. the main mechanism of chromosomal evolution is also by pericentric inversion. and S. as with the diploid cytotypes. Based on pollen viability. Goldblatt 1981. 1974. rebaudiana had a pair with a subterminal centromere (Frederico et al.147. respectively. Frederico et al. Thus. although most reports indicate that n 11 (2n 22). . Other meiotic abnormalities. Goldblatt and Johnson 1990. (2004). including S. 33. rebaudiana. The presence of a nucleolus organizing region on the short arm of the third major chromosome pair was also confirmed for S. The lowest tetrad normality rates were those of the tetraploid (80%) and triploid (64. All South American species of stevia studied are diploid with the exception of hexaploid S. Bolkhoviskikh et al. herbaceous species with flower heads in a lax paniculate cluster have n 11. this variation reflects numerical (aneuploidy and polyploidy) and possibly structural changes.168 on 10/13/13 For personal use only. 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. normal meiosis can be inferred. (2004) studied pairing at diakinesis of diploid. with x 11. J. 1996). Galiano and Hunziker 1987). since the latter are derived from a single genome and result in three or four homologous sets of chromosomes in triploid and tetraploids. triploid and tetraploid cytotypes with 2n 22. These three species may have originated by ascending aneuploidy from species with x 11. Frederico et al. the Stevia cytotypes analyzed here were obtained artificially by inducing polyploidy (Valois 1992). 1988. In the Brazilian species of stevia. Multivalents at diakinesis would be expected in autopolyploids. While. 2004). lucida from Colombia. 48. Chromosome lengths were similar in other strains with 2n 22 or 2n 44. 1991. Moore 1973. Galiano 1987. Oliveira et al. x 12 and x 17. 5. The distinct pattern strongly suggests the occurance of pericentric inversions as the rule in the divergence of Brazilian species of stevia. 1984. High rates of tetrad normality (93%) were also observed in the diploid strains. Because of irregularities that produce unbalanced gametes during meiosis. Can. with a small amount of aneuploidy and polyploidy. 70 have also been observed (Darlington and Wylie 1955.4 mm). These results of the chromosome size and centromere position were confirmed by Oliveira et al. 1977. Chromosome count of diploid stevia plant. There is a predominance of the basic chromosome number x 11 among stevia species from South America. 1996). (1996) considered its basic chromosome number as x 11.10 CANADIAN JOURNAL OF PLANT SCIENCE Fig. elatior (2n 66) from Colombia (Jansen et al. a plant that belongs to the same family as Stevia (Malallah et al. Further.5%) strains. 44. On the other hand. one population of S. pairing at diakinesis was n 11II. 2n 33 and 2n 44.0Á2. 66. Oliveira et al. 1998. 1969. Plant Sci. the comparative analysis of arm ratios of each karyotype. with the formation of bivalents in all diploids.ca by 186. Galiano (1987) considered Stevia as a multibasic genus. Downloaded from pubs. mainly pericentric inversions (Frederico et al. Aneuploidy also occurs in Picris babylonica. 2001). 1996. ophryophylla and S. values of 2n 24. could also explain these results.198. in addition to the numeric changes. Only S. the marker loci in the stevia linkage map were distributed more evenly and may better represent of the whole genome (Yao et al. Shu and Wang 1988. (1999) based on RAPD markers. 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. 1996). indicating a high level of genetic diversity in stevia. The study was conducted using F2 population from crosses between two sets of parents with divergent glycoside profiles.YADAV ET AL. Despite the fact that the Compositae is one of the largest and most diverse families of flowering plants. * A REVIEW ON IMPROVEMENT OF STEVIA 11 Can.g. 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). i. In a second cross.8 and 76. Yao et al. In many linkage maps. leaf:stem ratio and stevioside (62. 1999). leaf yield.. J. Utumi et al. such as copalyl diphosphate synthase. leaf number. Genetic diversity detected with RAPDs varies with species and crosses (Beaumont et al. rebaudiana using segregation data from a pseudo test-cross F1 population. Mapping newly cloned cDNAs from stevia genes involved in glycoside synthesis. 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. 1998. 1999). aimed at higher yield.6 cM. It appears that the limited breeding efforts undertaken to date have not significantly reduced levels of genetic diversity among the stevia breeding lines. 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. (1999) constructed the genetic linkage map for S. This may enable stevia breeders to conduct marker-assisted selection with genes that are found closely linked to the markers in the map. The results were used to propose a model for glycosylation of steviol glycosides (Brandle 1999). GENETIC INHERITANCE The magnitude of heritable variation is of utmost importance since it has relevance to selection response. ranging in length from 56 to 174 cM. 1992. This result suggests that both rebaudioside-A and -C are synthesized by the same enzyme. Further. In another study. proportions of rebaudioside-A and rebaudioside-C were found to co-segregate and were shown to be controlled by a single additive gene. 1998.. Konieczny and Ausubel 1993). 1997). Downloaded from pubs.ca by 186. 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. with an average distance between markers of 7. The experiments conducted by Brandle (1999) were focused on the genetic control of the proportions of two of these glycosides. Understanding the genetic basis of glycoside proportions will aid in their manipulation through plant breeding. 35. there has been little research involving molecular markers. 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. Similar findings have been demonstrated in other cross-pollinated species (Grattapaglia and Sederoff 1994). The presence of significant heritability (h2) for three economically important characters. 1999).147. 1999. The 11 largest linkage groups consisted of 4Á19 loci.5% of those marker loci segregated in a 1:1 ratio. 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. rebaudiana was constructed by Yao et al.e. Shyu 1994.168 on 10/13/13 For personal use only. Nishiyama et al. the first genetic linkage map for S. Several authors have studied the dependence of yield on various growth parameters as well as stevioside content (Buana and Goenadi 1985.1. Truong et al. 1991. Brandle and Rosa. Chalapathi et al. 1999b. Linkage Map To lay a foundation for molecular breeding efforts.aic. Segregation in the first set of F2s showed that the presence/absence of rebaudioside-A is controlled by a single dominant gene. respectively) clearly suggested that genetic improvement of stevia is possible (Brandle and Rosa 1992). largely because the family possesses very few major crop species (Kesseli and Michelmore 1996). Plant Sci.5% detected polymorphic loci and 62. rebaudioside-A and rebaudioside-C. 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. marker development (e.198. Some interesting correlations have been found which can assist selection programmes (Table 1). Stevia produces steviol glycoside sweeteners in its leaves that are up to 240 times sweeter than sugar. but that the actual proportions of rebaudioside-A may be controlled by multiple loci or alleles. Buana 1989. This may be partly because stevia has not undergone a great deal of selection (Yao et al. Of the primers that showed amplification products.6. loci are often found to be concentrated in certain areas (hot spots) of a few linkage groups (Paterson 1996. or branch number in the . and accounting for 75% of the total map distance. These high heritabilities enable selection and breeding programs. Keim et al. Buana (1989) reported that plant height had no significant correlation with production. 78. In comparison. to achieve substantial gains. Most of the RAPD markers segregated in normal Mendelian fashion. Construction of the stevia genetic linkage map has laid a foundation on which to conduct marker-assisted selection in stevia. Leaf surface. Shu and Wang (1988) showed that leaf dry weight per plant had the greatest influence on yield. 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. 1991).ca by 186. Total stevioside content is positively correlated with leaf:stem ratio (Tateo et al. 1998). Buana and Goenadi (1985). Plant Sci. while stevioside and rebaudioside-A. (1991). 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. High rebaudioside-A content is also linked to large leaf area. Downloaded from pubs. The stevioside content of leaves can vary substantially (4Á16%) between individual plants. dry leaf yield was correlated with leaf size and thickness and content of rebaudioside-A was highly correlated with leaf thickness (Shyu 1994). the leaf surface has more influence on stevioside content than the number of roots (Truong et al. (1996) Weng et al. Shock (1982) planted 200 lines for survival testing and screened 17 lines for productivity. .168 on 10/13/13 For personal use only. 1996). there is great variation in phenotype and leaf analysis. net photosynthetic rate. J. Stevioside content is influenced by both leaf surface and number of roots. (1998) Shu and Wang (1988) Truong et al. Plant leaf yield is proportional to branch number. 1999). (1996) Nakamura Nakamura Nakamura Nakamura and and and and Tamura Tamura Tamura Tamura (1985) (1985) (1985) (1985) Can. (1999) Utumi et al. About 96. number of branches. leaf number and (not always) plant height (Buana and Goenadi 1985. (1998) Shyu (1994) Weng et al. high chlorophyll and protein content (Weng et al. 1998).12 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Buana 1989). high net photosynthetic rate. Stevioside content was uncorrelated with yield or leaf:stem ratio (Brandle and Rosa 1992). Nakamura and Tamura 1985). 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 dry yield of stevia was positively correlated with plant height. List of some important correlations Characters Plant height.aic. Shu and Wang 1988. making plant selection at the seedling stage ineffective. Further. branch number Total soluble carbohydrates Yield. 1999). The chemical content of the last fully expanded leaf pair was well correlated with plant nutrient status (Utumi et al. chlorophyll and protein content Stevioside Rebaudioside-C Rebaudioside-A Rebaudioside-C References Buana and Goenadi (1985) Buana (1989) Nishiyama et al. number of branches. A positive correlation between total soluble carbohydrates and stevioside content was established by Nishiyama et al. first 4 wk. Leaf thickness is positively correlated with rebaudioside-A:stevioside ratio (Shyu 1994). leaf number Plant height Leaf/stem ratio Rebaudioside-A/Stevioside ratio Stevioside content at maturity Leaf area. however. The total stevioside content of leaves at the seedling stage and when mature is not correlated. leaf number. leaves per plant and dry matter accumulation. number of roots Plant nutrient status Yield. even after a selection program has been continued for some time (Bian 1981. 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. leaves per plant and dry matter Yield.147. leaf:stem ratio Leaf size and thickness Leaf thickness Plant height. Shu and Wang (1988) Tateo et al. (1999) Brandle and Rosa (1992) Buana (1989). Nakamura and Tamura (1985) reported that the levels of dulcosideA and stevioside and rebaudioside-A. leaf:stem ratio Branch number.198.88% of the total variation in dry leaf yield was explained by a linear function of these four characters (Chalapathi et al. 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. and dulcoside and rebaudioside-C are negatively correlated with each other. Furthermore. PHENOTYPIC VARIABILITY In the wild populations of Stevia rebaudiana. (1991) Brandle and Rosa (1992) Shyu (1994) Shyu (1994) Chalapathi et al. and -C are positively correlated with each other. Two . Soejarto et al. The fungal diseases Septoria leaf spot (Septoria steviae). Much of the morphological variability has been observed in the population of S. stevioside varied from 1. In another report from China. Huang et al. Megeji et al.) and Sclerotinia sclerotoirum have been reported (Ishiba et al. Huang et al. no genotype is true breeding.. BREEDING METHODS Germplasm Introduction. this same ratio was about 0.98% and rebaudioside-A from 4. 1995). Brandle and Rosa 1992. root rot (Sclerotium rolfsii). however. Intermating should be carried out for several generations and selection pressure applied gradually for desirable gene combinations. Lovering and Reeleeder 1996.22 times as much leaf dry weight as stem dry weight. It has been reported from China. So far. 1999.5 or less. natural environment (Grashoff 1972. stem rot (Sclerotium dephinii Welch. Palampur. Therefore. that.5 to 12. Plant Sci. rebaudiana under study at the Institute of Himalayan Bioresource Technology. 1997. damping-off (Rhizoctonia solani Kuehn. 1995). It is generally used as source of superior genes and increasing genetic diversity in the germplasm for breeding programmes. High leaf:stem ratios are desirable in cultivated stevia because of the low stevioside concentrations (B5 mg g1) in stem tissue. and is more soluble in water (Ahmed and Dobberstein 1982.26 to 19. Cultivar descriptions indicate that sufficient genetic variability exists to make significant genetic gains in leaf yield.e. Introductions can be used directly as commercial cultivars. in order to utilize the information to develop an ideal plant type.ca by 186. plant breeding efforts with stevia have been largely focused on improving leaf yield and rebaudioside-A concentration in the leaves. total sweet glycoside concentration in some lines has been reported to be as high as 20.168 on 10/13/13 For personal use only. Nurhaimi and Toruan (1995) showed somaclonal variations in DNA fingerprints between six groups of plantlets. 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. and in separate cultivar rebaudioside-A: stevioside ratios of 9:1 have been disclosed (Morita 1987. 2005). 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. i. Morita 1987.).5%. Stevia grown at the Delhi Research Station (Ontario. Conversely. Tateo et al. Shizhen 1995). * A REVIEW ON IMPROVEMENT OF STEVIA 13 Can. it has the best profile relative to all other glycosides. Dacome et al.YADAV ET AL. rebaudioside-A content and the rebaudiosideA:stevioside ratio (Lee et al. particularly in developing countries. 2005.57% (Huang et al.198. due to heterozygosity. which helps in producing organic Stevia. With the high level of natural variability due to constant out-crossing. Kamalakannan et al. BREEDING OBJECTIVES Breeding programmes for stevia should be aimed at improving total glycoside content and rebaudiosideA:stevioside ratio with higher leaf yield. in stevia. Crammer and Ikan 1986. since. the most valuable extracts are those that have rebaudioside-A as the major component. which may be due to its inherent sweetness acting as a repellent. with total glycoside varying from 10.aic. 1998). 1998). 2007). Monteiro (1980) studied the phenotypic differences present in the population and was unable to separate them into a valid taxonomic variety. Canada) had 1. 1982. There is a need to develop and identify resistant sources to develop varieties resistant to or tolerant of these diseases. It also exhibits considerable variability for stevioside content. rebaudiana.1%. Alterneria leaf spot (Alternaria alternata). DISEASE RESISTANCE Stevia is known to be free from attacks by insects. in a sample of plants from one clone. 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. Introduction of germplasm from one area to another continues to be an important activity for breeding. The predominance of stevioside gives a characteristic bitter aftertaste to the crude extract.48 to 6. which may vary from 2 to 10% (Megeji et al. 1983. powdery mildew (Erysiphe cichoracearum DC). insecticides are not required at an essential basis as in other crops. the development and phytochemical characterization of new varieties of S. 1995). Bian 1981. cytogenetic and molecular levels. The rationale behind seed collection is to conserve genes and not genotypes. This natural variability could be partially due to the largely out-crossing nature of the species (Handro et al. breeders are able to improve the level of sweeteners in the leaves and alter the rebaudioside-A:stevioside ratio (Shu 1989.67 in California (Brandle and Rosa 1992). Sekaran et al. Therefore. 2005. The genetic improvement of stevia is only possible through the characterization of the available variability at the morphological. because of its organoleptic and physicochemical features. without the need to recrystalize individual glycosides. allowing a greater variety of formulations. J. chemical and biochemical. 1982. Shock 1982. Downloaded from pubs. a long-term programme. Adapting exotic germplasm is. The native rebaudioside-A:stevioside ratio in Stevia rebaudiana leaves is usually about 0. Thomas 2000. Suhendi 1989. 1993).147. There are also reports of irregular quantitative and qualitative production of the sweetening molecules from cultivated S. The phenotypic variability within the population is linked to the open pollination behavior of the species (Tateo et al. Shizhen 1995) (Table 3). Chang et al. Collection and Conservation Germplasm is a very important material for the improvement of crops. 2007) (Table 2). Shock (1982) Marcavillaca (1985) Chalapathi et al. it requires selection relatively late in the growing season. In the wild. The costs associated with clonal propagation limit the general applicability for large-scale production of stevia plants. More importantly. 1995). Valio and Rocha (1977) Borie (2000). Brown lesions on the stem. are self-incompatible and can only be reproduced vegetatively (Lee et al. Stem rot Sclerotinia sclerotiorum Can. Other plants have been developed that exhibited rebaudiosideA:stevioside ratios as high as 9. Initially appears as small circular spots. 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). 1982).147.1:1. 1999). compared with 0. Attempts made elsewhere in the world have resulted in patents for the superior plant types (Table 5).96:1. Characters for improvement/utilization through breeding Sr. Later. 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. This limits their commercial use. . However. angular. Selection The success of stevia breeding depends on the choice of parents. Korea. As a result. (1998) Marsolais et al. and measurements are based on expensive and tedious high performance liquid chromatography (HPLC) procedures (Brandle and Rosa 1992). Plant Sci. time consuming.aic. Nearly three decades of breeding and selection have increased glycoside concentration in stevia leaves by as much as 20% (Huang et al. although they may be useful for breeding new hybrids. due to self incompatibility. Carneiro et al. making crosses. with wilting of plants and white cottony mycelial growth at the collar region takes place. light brown in colour. Most breeding programs are based on cross breeding and selection. near the soil line are formed. Steviol 1 2 3 Characters glycoside characters Total glycosides content Rebaudioside-A content Rebaudioside-A: stevioside ratio Reference Sys et al. (1998) Brandle (2001). Taiwan and Russia. raising adequate population and further selections. the cultivar could not be reproduced using a seed-based production system.1% (Morita 1987).ca by 186. Accessions I and II. such as soil and weather. Yellowing and drooping of leaves. shiny olive grey foliar lesions are formed that rapidly coalesced and often surrounded by a chlorotic halo. Vegetative propagation and cloning have frequently been used to multiply individually selected plants. selection for plants producing high amounts of glycoside is expensive. (1997). with associated tissue rotting. genotypes of S. Again. Leaves quickly become necrotic and often drop off the plant. (1997) Jia (1984) Shock (1982) concentration in stevia leaves typically varies from 2 to 10% on a dry weight basis. China. J.168 on 10/13/13 For personal use only. was developed with total glycosides of 22. Brown sclerotia appear on the diseased areas. because seedlings are so influenced by the environment that only 20Á30% of the variability is genetic. although very high yielding.36:1 in the starting material. and relatively inefficient (Yao et al.14 CANADIAN JOURNAL OF PLANT SCIENCE Table 2. especially Japan. which are morphologically diverse with respect to their growth habit and sweetness are being maintained and multiplied at the Institute of Himalayan Bioresource Technology. have all reported success in their breeding/ selection programmes and have released new varieties with improved glycoside content and higher yields (Table 4).198. Countries that have been researching stevia for some time. A cultivar with a rebaudioside-A:stevioside ratio of 0. this improvement was based on phenotypic selection for total glycoside concentration in stevia leaves. Selection at the early seedling stage is least effective. no. but total steviol glycosides were 10. Andolfi et al. (2002) Barathi (2003). many became irregular and dark brown to grey. Downloaded from pubs. followed by wilting and eventually by the complete collapse of affected individuals. The mycelial growth spread to the stem and roots. which is heavily influenced by environmental conditions. 1982).4% (Lee et al. while others remain circular with concentric rings or zones. Duke (1993). rebaudiana. the total glycoside Table 3. Further selections for desirable plant types are being performed in segregating progenies of individual selections. Some of these selections. . A. (1996) Kornienko and Parfenov (1996) IHBT (CSIR) annual report IHBT (CSIR) annual report Variety Suweon 2 Suweon 11 Yunri. * A REVIEW ON IMPROVEMENT OF STEVIA 15 Table 4. These plants were inter-crossed and half-sib seed was collected from each parent plant. Yunbing Zongping K1. These four clones were used as parents and intercrossed. E. A. 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. A. In 1989. Country USA USA USA USA USA USA USA 94-1306 and RSIT 94-751 were vegetatively propagated by shoot-tip and stem cuttings. A. A. Canada. A plant (RSIT 94-1829) selected from the variety Brazil Zairai was also retained. J. based on replicated trials. which distinguished it from its parents and all other stevia varieties for a high rebaudioside-C:stevioside ratio. Agriculture and Agri-Food Canada. and Sys. (1982) Shu (1989) Shu (1995) Shyu (1994) Suhendi (1989) Weng et al. Half-sib seeds collected from clone RSIT Inventer Brandle. A. Sys. A. T Nakazato. Brandle. (1998) and Marsolais et al. JP-59034826 JP-60160823 JP-61202667 JP-62096025 JP-63173531 EPA0154235 JP-2242622 Japan Japan Japan Japan Japan Europe Japan Morita Morita Morita Nakazato.168 on 10/13/13 For personal use only. A. Superior plants were selected from this population and designated SR1 to SR15 (breeding procedure shown in Fig. 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. and Brandle. T Stevia co.147.120 CN1985575 JP-59034848. Development of Stevia Plant RSIT 94-1306 and RSIT 94-751 Sys et al. A. BPP72 SM4 Ramonskaya Slastena Madhuguna Madhuguni Features High yield and steviosides Thick leaves. E.contains sweet diterpenoid. Development of RSIT 95-166-13 Brandle et al. J. seed of a landrace variety from China was introduced at Agriculture and Agri-Food Canada. Marsolais.562 PP10. Landrace stevia has a combination of steviol glycosides that is not optimal for all product applications.YADAV ET AL. B. Morita. J. Plant samples RSIT 94-1838. Sys. Marsolais.564 PP10.972. Downloaded from pubs. Wang.255. Delhi Research Station.ca by 186. Inc. . Brazil Zairai is an open-pollinated landrace variety of stevia obtained from the Japanese National Germplasm Depository. better Rebaudioside:stevioside ratio High yield and Rebaudioside:stevioside ratio High yield and total glycoside content High yield and total glycoside content Can. and Marsolais. New triploid of Stevia Rebaudiana Bertoni. RSIT Table 5. 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.198. E. RSIT 94-1833. T.. Sanyo Kokusaku Pulp Co. Brandle. and Yucheng.557 PCT-WO99/49724 6031157 PP10. high Reb% Highest Rebaudioside and stevioside High yield. K3. b 1985 1986 1987 1988 1985 1990 Patent no. 6). (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. Brandle. Ontario.563 5. (1998b) developed RSIT 95-166-13 as a unique combination of characteristics. J. Different seed germplasm accessions of stevia from China were grown for evaluation at the Delhi Research Station.. because its leaves had higher than average concentrations of rebaudiosideC. 6. J. Plant Sci.aic. K2. J. 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. The half-sib families were evaluated in a genetic heritability trial (Brandle and Rosa 1992). Q Year 2001 1999 1998 1998 1998 1998 1999 2006 1984a. (1978) Lee et al. This is the most practical and effective breeding method.82 Total glycosides = 18. preserved for future synthesis of the synthetic cultivar. and superior rows are identified. 6. is composited. Plant Sci. Equal quantities of reserve seed from selected single plants.198. SR 2 SR 13 RSIT 94-1306 Cuttings evaluated RSIT 94-751 Cuttings evaluated Stevioside (%) Reb-A = 17. were inter-crossed in the greenhouse and seeds were collected from the maternal parents. (1998) and Marsolais et al.147. In order to create parents for the synthetic cultivar. 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.37 Total glycosides = 18. 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. At the same time. Leaves sampled from those selected plants were analyzed for glycoside concentration as well as composition. was selected on the basis of its novel steviol glycoside traits high in rebaudioside-C content. from china In summer 1990 1000 plants grown 15 Plants selected SR1 .16 CANADIAN JOURNAL OF PLANT SCIENCE In 1989. with rebaudioside-A to stevioside ratios of at least about 9. Recurrent selection typically starts with harvesting individual open-pollinated plants from the source population. as well as combined by random crossing. Therefore. composites and synthetics are used to capture part of the available heterosis. J. In S. In population improvement. 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. and selections that were high in glycosides.1:1). Such synthetic cultivars are intended for use in crop production systems. 94-1560 were designated half-sib population RSIT 95166. These half-sib families were evaluated in genetic heritability trial In summer 1994 evaluated visually. plant breeders aim to increase the frequency of desirable alleles through the selection of superior recombinants. and selections were made among those progeny. 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.88 = 11. based on progeny performance. (1998). and a high ratio of rebaudioside-A to stevioside (at least 9. Plants from half-sib population RSIT 95-166 were evaluated in the field and one plant. crosses were made among a number of single plants and a large number of progeny were planted out to the field. Each cycle of recurrent selection produces a new population that may be a potential cultivar. rebaudiana. seeds of landrace var. 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. a synthetic cultivar produced by intercrossing clones or sibbed lines obtained from a breeding population during cycles of recurrent selection is required. Downloaded from pubs.0 Stevioside (%) Reb-A = 4.3:1 (denoted as A to D). Because of the high degree of natural out-crossing and the absence of an efficient system of pollination control. The first recurrent selection cycle starts when the new composite is grown in an isolated plot where random mating among the plants is allowed. which is characterized by exhibiting a high level of total glycosides (at least 14%).168 on 10/13/13 For personal use only. A portion of the seed of these plants is saved as a reserve. The selected rows are harvested separately. Breeding procedure followed by Sys et al. where self-incompatibility ensures a high degree of heterogeneity.aic. Recurrent selection is continued until a reasonable response to selection is achieved. cuttings were taken from . RSIT 95-16613 was vegetatively propagated at the Delhi Research Station by shoot-tip and stem cuttings. recurrent selection is the most effective method of increasing foliage yield as well as total glycoside content. 7).ca by 186. In order to develop a synthetic variety more than one line is required and the lines or clones are typically tested for combining ability.25 = 0.SR15 Crown dug-out & grown in green house in winter 1990-91 In summer 1991 Intercrossed & half-sib seeds collected Can. RSIT 95-166-13. Population Improvement Recurrent Selection Recurrent Selection is useful for improving quantitatively inherited characters in cross-pollinated species.08 Fig. Hills (SF3 grade) containing 2. rebaudiana developed by Morita and Yucheng (1998). 20 plants from each family selected for HPLC analysis Selected high in glycosides and reb-A/stevioside = 9. Selections. Seed from the maternal parents was retained as half-sib families and a portion bulked to create a synthetic cultivar AC Blackbird. of progeny planted out in field and selections were made among those progeny OxO OxO OxO OxO OxO OxO OxO OxO Crossing among no. Variety of Stevia ATCC Accession No..3:1) (A.168 on 10/13/13 For personal use only. J. having ATCC Accession No.49 = 15. PTA-444. Breeding procedure followed by Brandle (2001) for development of synthetic cultivar AC Black Bird. which contained 2. * A REVIEW ON IMPROVEMENT OF STEVIA 17 In order to create parents for the synthetic cultivar. the plants so that they could be used to duplicate the synthetic as required.56 times or .aic.56 times or more rebaudioside-A than stevioside and is capable of being cultivated by seed propagation.ca by 186. crosses were made among a no. Plant Sci.YADAV ET AL.. half-sib families of selections and synthetic cultivar evaluated in replicated field trial Stevioside (%) = 1.B. of single plants and a large no. The plants so obtained are characterized as exhibiting high levels of total steviol glycosides and being enriched in rebaudioside-A. Downloaded from pubs. is produced from SF-6 seed.C. The half-sib families. the bulked sample and the parental clones were evaluated in a replicated field trial.12 Selection among these families 60 plants in each family Can.198.3x1m insect case screened with 10mm mesh. of selected plants Seeds collected Large no. PTA-444 A variety of S.3:1 4 plants selected (9.147.27 Reb-A Total Reb-A/Stev = 12. of progeny planted in field IIIIIIIIIIIIIIIIIIIIIIIIIII 1……………………………………………. trimmed inside 1.D) IIII ABCD Sexual inter-crossing Cuttings to duplicate the synthetic as required Isolated from field in green-house. A hive of bumble bees was used for inter-crossing AC Black bird (Synthetic cultivar) Parental clones. It is preferred that the cultivar seed be produced from at least two intermating genotypes.96 Fig. 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. or progeny thereof.06 = 9. 7. Crossbreeding between individuals with different numbers of chromosomes usually leads to sterile progeny. such as Nicandra physaloides (Gupta and Roy 1986). the progeny are partially or completely sterile triploids that do not bear seeds. 109 contained 2. triploid plants of S. Better adaptability of individuals and increased organ (Fig. the greater the size of the pollen and the stomata. (2001). whereas the tetraploid strain had the largest leaves. Further. Gamma irradiation of the seeds of stevia does not affect germination. g-rays. 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. rebaudiana in nine treatments of Fig. which corresponded to the lowest colchicine concentrations. which has excellent resistance to Septoria.168 on 10/13/13 For personal use only. the higher the ploidy number.56 times or more rebaudioside-A than stevioside. both physical and chemical. In these triploid plants. 109). Analysis of variance revealed highly significant differences among the strains. 2001). 2004). DES. Clitoria ternatea (Gandhi and Patil 1997) and orange (Romero-Aranda et al. Specifically. 2004). the leaves. 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. 1995). Several mutagenic agents. ENU. According to Allard (1960). Characters of interest can only be improved through mutation breeding if the population shows less variability for the character concerned. 103 or No.56 times or more rebaudioside-A than stevioside. can be used to produce useful mutations. were finally selected. SF5-1 (designated No. but at higher doses suppresses root development. With the discovery of mutagenic agents. in various agricultural plants with diploid and tetraploid individuals as parents. crossing between SF3 and No. which is excellent in sweetening components. were obtained. and as a result of crossing between both hills containing 2. Because leaves are the economically important part of this crop. The leaf shape of the triploid plants was categorized as needle-like or wide needle-like. flowers and guard cells were larger than that in the plants classified as diploid. The triploid strain produced the shortest plants and the lowest number of inflorescences. Shuichi et al. Can. thermal neutrons and chemicals such as EMS. Triploid and tetraploid plants had a lower tetrad normality rate than the diploids. colchicine (an antimitotic agent) at concentration ranging from 0. ENUA. coffee (Cruz et al. 1993).147. rebaudiana can be produced by mating tetraploid female and diploid male parents. 109 (SF6-III) did not give hills containing a stable component proportion of rebaudioside-A. rebaudiana contain a large amount of rebaudioside-A. grouped into eight cultivars. Plant Sci. crossing and selection were repeated. and 42 triploid plants. 61-202667.5% for 18 h. MNUA. such as X-rays. Breeding of triploid plants of stevia was conducted by Shuichi et al. J. In thin-layer chromatography and HPLC analyses. All of the strains had inviable pollen (Oliveira et al. with a positive correlation between the level of ploidy and all of the morphological features examined (Oliveira et al. 103). showed that the plants were triploid (2n 33). 103 and No. 8. Downloaded from pubs. 1997).aic. fast neutrons. 8) and cell sizes are usually associated with polyploidy (Guerra 1988). Valois (1992) developed polyploid strains by soaking the seeds of S. and the lower their number per unit area. The glycoside quality of stevia is improved by using the polyploids. mutation breeding can play a very important role in the improvement of stevia. Thus.001% to 0. obtained by counting the chromosome number of root tip cells and by flow cytometry analyses. However. Irradiation with Cobalt-60 gamma rays has been used to induce variation in breeding lines (ToruanMathius et al. The variability found in nature is due to the accumulation of natural mutations that have occurred during the evolution of the plant.18 CANADIAN JOURNAL OF PLANT SCIENCE more rebaudioside-A than stevioside were selected from seeds formed by crossing between SF3s described in Japanese patent publication no. Polyploidization was confirmed in only two treatments.198. a very sweet diterpenoid (Sanyo 1990. seven kinds of triploid .ca by 186. plant breeders have the ability to induce variability and use it in their breeding programmes. The chromosome number of these plants. Triploid plants of S. Comparison for leaf size of (a) polyploid and (b) diploid plant. and SF5-2 (designated No. More than 80% of the resulting hills (SF6-I and SF6-II) obtained from the crossing between No. MNU. of dicotyledonous plants (Yuang-ling and King-In 1978. 2006. 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. T1 to T7. Christou et al.147.05 mM 2.63. 1994. 1992). Tissue culture is the only rapid process for the mass propagation of stevia. Asexual propagation of the parental plant is adopted to fix its good properties and make the parental plant clone to process group hybridization. Auxin is needed to cause de-differentiation and to elicit totipotency (Terzi and Loschiavo 1990. Flachsland et al. and mixes the collected seeds. propagates clone parental plants. etc. In this method. suspension cultures (Ferreira and Handro 1988) and anthers (Flachsland et al. Kornilova and Kalashnikova 1996. Toffler and Orio 1981) and vegetative propagation through cuttings is limited by the small number of individuals (Sakaguchi and Kan 1982). Plant regeneration from in vitro culture can be achieved by embryogenesis or organogenesis. 1984. T-8. metabolism. b. 1980. 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. Its advantages are low cost. the new variety of S. rebaudiana clone parental plant. showed high STV contents (rebaudioside-A ratio 0. 1990). Sivaram and Mukundan 2003. Somatic embryogenesis can also be obtained from floret explants of S. 1984b). 1985. which has simple programme and more rapid yielding efficiency than traditional breeding method.10 mM 2.4-D supplemented medium the best treatment was 2.29 mM). Bespalhok-Filho et al.4-D supplemented medium maximum embryogenic callus formation occurred in medium without kinetin. and then proliferated throughout the whole explant.4-D or picloram is usually used for the induction of somatic embryogenesis (Merkle et al. Anther culture is usually carried out at the beginning of a breeding programme. On the other hand.10 mM) and kinetin (0 to 9. Bespalhok-Filho and Hattori 1997). Ferreira and Handro 1987a. Somatic embryogenesis has previously been reported from leaves (Wada et al. rebaudiana cultured on MS medium supplemented with 2. Once the populations of plants homozygous for a particular trait are available. Wang (2006) claims a method of cultivating a new hybrid variety by systematically breeding a S. Barwale et al.YADAV ET AL.168 on 10/13/13 For personal use only. Felippe et al. immature anthers from a differentiating population are grown or cultured on substrate. SalimUddin et al. whereas the RA ratio remained constant (Shuichi et al.aic. Dhir et al. J. Fametaer et al. Nonembryogenic callus may also be present. a high rebaudioside-A content and high output of stevioside.32 mM kinetin. Heterosis Breeding Hybrids offer an opportunity to mobilize greater genetic variation and heterotic response. Ahsan et al. 1987. The embryogenic callus is usually characterized by a light green or light yellow colour. The embryogenic callus formed first on the base of the ovary and/or corolla. Facciotti et al. * A REVIEW ON IMPROVEMENT OF STEVIA 19 plants. selects and matches good combinations. In stevia. On 9.198. Sun (2001) suggested a method for breeding hybridized seed of S. 1981. BIOTECHNOLOGICAL APPROACHES Tissue Culture Seeds of stevia show a very low germination percentage (Felippe and Lucas 1971. axillary shoots (Bespalhok-Filho et al. Embryogenic callus started at the base of the corolla and ovary (BespalhokFilho and Hattori 1997). new varieties are developed by hybridization and genetic studies can be carried out efficiently. Can. rebaudiana cultured in vitro under defined conditions. rebaudiana bred according to this method has strong resistance. (1996) regenerated plants from anthers of S. Ahmed et al. 1990. A synthetic auxin such as 2. Plant Sci. obtained from crossing tetraploid SMX1W with a high rebaudioside-A content with seven kinds of diploid cultivars with a high rebaudioside-A content. Anthers (containing uninucleate microspores) were induced to form callus when aseptically cultured on Murashige and Skoog liquid medium . 1993) and stems (Miyagawa et al. Monteiro 1980. stem tips (Tamura et al. and there have been a few reports of in vitro growth of stevia (Miyagawa et al. Cell and tissue culture techniques have been widely used to study the growth. showed high rebaudioside-A contents with a ratio (rebaudioside-A:stevioside) of more than 0.ca by 186. 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. 2007). 2000). 1991a. regeneration has been achieved by organogenesis from different explants. characterized by a white colour and a hyperhydric appearance (Bespalhok-Filho and Hattori 1997).23). 1986. On 18. 2001). Constantinovici and Cachita 1997.05 and 18.4-D (9. 1996). 1971. which systematically breeds good single plants. Bespalhok-Filho and Hattori 1997). b). and a high content of total glycoside in the leaves. such as leaves (Yang and Chang 1979. obtained from crossing tetraploid KSW with a high stevioside content with diploid SMX6 with a high RA content.. Cheng et al. rebaudiana that involves hybridizing between female plants produced from cuttings and male plants produced from seed and collecting seeds from plants produced from cuttings. hybridizes the producing seeds. compact structure and the presence of globular somatic embryos on its surface. 1986) and in vitro micropropagation from shoot tips and leaves (Akita et al. Downloaded from pubs. high leaf output. Agric. 1960.. Allard. Agrario 58: 48Á51. 1999. Biol. Sci. Shigeoka. 1996. better leaf:stem ratio and with a high content of rebaudioside-A compared with other glycosides for successful cropping and higher diterpene glycoside production. I.. J. Nepovim. Anufrieva. Inc. improvement of glycosides quality and quantity. W. V. J. such as the inhibition of bacterial and fungal growth. A. 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. Sultana. Sagner. 1999). research has been conduced around the world on various aspects of crop improvement. Russ.. L.1 to 1 mg L 1 BAP. Paran and Michelmore 1993) and forest trees (Groover et al. and Dobberstein. Paran and Michelmore 1993. as the Food and Drug Administration has approved rebaudioside-A with 95% purity. 1994. better germination and viable seed production. 1991. and Islam. The study implies that somatic cells of the anther wall respond to the high BAP concentration in the medium. Zenk.20 CANADIAN JOURNAL OF PLANT SCIENCE supplemented with 0. and Bacher. Eisenreich. 3: 1985Á1988. Res.. C. 1982.. 2: 121Á125. J. nontoxic. E. L.enal. 2001. M. Razvy. Allam. Arigoni. and Macchia. In the recent past. M. Acuna. high-potency sweeteners. which restrict its cultivation from seed. Salahin.. and seed-grown plants vary in their growth. A. Trop. Bradshaw and Stettler 1995).-Euras. Downloaded from pubs. V.. 1991. which limits the large-scale production of planting material. Further research and development need to be carried out to improve stevia’s potential as a crop by developing improved varieties with higher yield and quality through plant breeding methods and biotechnological approaches.. M..... W. S. while stevioside is responsible for aftertaste bitterness. Tissue culture and breeding of maize (Zea mays L.1 mg L1 NAA. 13: 180Á183. M. Regeneration of shoots was readily achieved by transferring pieces of callus to fresh solid medium with the same composition. Nassar. 2000..) Á A review. Subtrop. crop for the production of nonnutritive. Bioagronomic characteristics of Stevia rebaudiana. seed production. the development of new varieties. M. A. I. rebaudioside A and rebaudioside C.. 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. Radykewicz. as reported in the literature. 1994. A. M. M.147. Improved genotypes with a high content of rebaudioside-A with respect to other glycosides (like stevioside) need to be developed. 1997. cultivation. R. An efficient method for in vitro clonal propagation of a newly introduced sweetener plant (Stevia rebaudiana Bertoni) in Bangladesh. Mehdi. T. P. Mackill et al. S. Mackill et al. Ceccarini. Nosov. and Lafont. A. N. Ahsan. M. M. field crops (Paterson et al. Plant Cell Rep. New York. A. Hossain. Sci. Y. propagation. and it is an anti-cancerous. but cytological studies of root tips from regenerated plants revealed a normal diploid number of chromosomes (2n 22). M. R. Yao et al. Volodin. R. Garcia. Stevia is a selfincompatible plant. M. 1993. and Valicek. Principles of plant beeding. Plantlets were successfully potted. B. Latzel. 2002. Inf. Akita. Koizumi.. R. [Online] Available: Nile. D. Mass propagation of shoots of Stevia rebaudiana using large scale bioreactor. M. Plant Physiol. SUMMARY Stevia rebaudiana is gaining popularity in various developed and developing countries as an important Can. The major problem of large-scale cultivation is the lack of quality planting material. S. High performance liquid chromatographic separation and quantitation of stevioside.. 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. Pak. NY. Plant Sci. Molecular linkage maps have been constructed for most major crop plants (Staub et al. Micropropagation of plants of Stevia rebaudiana in vitrio and content of stevioside in leaves after application of growth regulators under field conditions. Marker-assisted selection provides the potential for improving selection efficiency by allowing for earlier selection and reduced plant population size (Staub et al. Hannan. Andolfi. antihyperglycaemic. Chromatogr... Paterson 1996). 1996). H.sci. plants with desirable characteristics are propagated by stem cuttings and tissue culture practices. Generally. disease resistance. Jpn. 1993. It has many other curative properties. M. T. Y. Ahmed.ca by 186. 1998. M. The content and composition of ecdysteroids in plants and tissue cultures of Serratula caranata.. The emphasis in future research should be on the development of new seed varieties with wider adaptability to different climatic conditions. 485 pp. anti-hypersensitive agent. J.198. H. Shoots were induced to form roots upon transfer to medium with 0. 236: 523Á526. 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