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: 127. Stevia rebaudiana (Bertoni) is a herbaceous perennial plant (2n22) of genus SteviaCav., which consists of approximately 230 species of herbaceous, shrub and sub-shrub plants. Leaves of stevia producediterpene glycosides (stevioside and rebaudiosides), non-nutritive, non-toxic, high-potency sweeteners and may substitutesucrose as well as other synthetic sweetners, being 300 times sweeter than sucrose. In addition to its sweetening property, ithas 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 desirableflavour profile, while, stevioside is responsible for aftertaste bitterness. Development of new varieties of S. rebaudiana witha higher content of rebaudioside-A and a reduced content of stevioside is the primary aim of plant breeders concerned withthe improvement and utilization of this source of natural sweeteners. The proportions of rebaudioside-A and -C arecontrolled by a single additive gene known to be co-segregating suggesting synthesis by the same enzyme. Stevioside andrebaudioside-A are negatively correlated, while rebaudioside-A and -C are positively correlated. Conventional plantbreeding approaches such as selection and intercrossing among various desirable genotypes is the best method forimproving quality traits in a highly cross-pollinated crop like stevia. Various plant types with larger amounts of specificglycoside have already been patented, such as RSIT 94-1306, RSIT 94-75, RSIT 95-166-1 through selection andintercrossing. Composites and synthetics can be used to capture part of the available heterosis because of the high degree ofnatural out-crossing and the absence of an efficient system of pollination control. Synthetics and composites like ACBlack Bird and PTA-444 have already been developed. Polyploidy results in better adaptability of individualsand increased organ and cell sizes. Tetraploids have larger leaf size, thickness and have potential use in increasing biomassand yield in comparison with diploid strains. Characters of interest with low variability in the population may be improvedthrough mutation breeding. Use of biotechnological approaches, such as tissue culture for the mass propagation of elitegenotypes, anther culture for development of pure homozygous doubled haploid and molecular marker technology foridentification 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 glycosideprofile 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. Lamelioration genetique du stevia [Stevia rebaudiana (Bertoni)],tour dhorizon. Can. J. Plant Sci. 91: 127. Stevia rebaudiana (Bertoni) est une herbacee vivace (2n22) du genre SteviaCav., lequel compte environ 230 espe`ces vegetales de type herbace, arbustif ou sous-arbustif. Les feuilles du steviaproduisent des glycosides diterpe`ne (stevioside et rebaudiosides), non nutritifs et non toxiques, mais au tre`s fort pouvoirsucrant, susceptibles de remplacer le sucrose et dautres edulcorants artificiels, car ils sont 300 fois plus doux que le sucrose.Outre cette propriete, le stevia posse`de des vertus medicinales. Le stevia nest pas une plante autogame et la pollinisationseffectue grace a` des insectes. De tous les glycosides que produisent les feuilles du stevia, le rebaudioside-A presente uninteret particulier, car son profil de sapidite est le plus desirable, le stevioside laissant un arrie`re-gout amer. La creation denouvelles varietes de S. rebaudiana plus riches en rebaudioside-A et contenant moins de stevioside est le but principal querecherchent les ameliorateurs, qui souhaitent accrotre la production de ces edulcorants naturels et en favoriser lutilisation.Un seul ge`ne additif, co-segregant, commande la proportion de rebaudioside-A et C produite, ce qui laisse supposerlintervention du meme enzyme dans la synthe`se. Le stevioside et le rebaudioside-A sont negativement correles, alors quilexiste une correlation positive entre le rebaudioside-A et le rebiaudioside-C. La meilleure facon de renforcer les caracte`resrecherches dans une espe`ce a` pollinisation aussi croisee que le stevia consiste a` recourir aux methodes classiquesdamelioration genetique comme la selection et lhybridation des genotypes interessants. On a deja` homologue plusieurscultivars crees de cette manie`re, et produisant une quantite superieure de tel ou tel glycoside, notamment RSIT 94-1306,

Abbreviations: BAP, benzylaminopurine; CDP, ()-copalyldiphosphate; DMADP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose-5-phosphatereductoisomerase; DXS, 1-deoxy-D-xylulose-5-phosphatesynthase; EST, expressed sequence tag; GGDP, geranylgeranyldiphosphat; IBA, indolebutyric acid; IDP, isopentenyldiphosphate; NAA, napthalene acetic acid

Can. J. Plant Sci. (2011) 91: 127 doi:10.4141/CJPS10086 1

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RSIT 94-75 et RSIT 95-166-1. On peut aussi recourir a` des varietes composees ou synthetiques pour saisir une partie delheterosis existante, en raison du fort degre naturel de croisement exterieur et de labsence dun syste`me efficace regulant lapollinisation. Des varietes synthetiques et composees comme AC Black Bird et PTA-444 ont dailleurs deja` ete realisees. Lapolyplodie entrane une meilleure capacite dadaptation des plantes, tout en engendrant de plus gros organes et cellules.Les tetraplodes se caracterisent par des feuilles plus grandes et epaisses, dont la biomasse et le rendement pourraientdepasser ceux des souches diplodes. Lamelioration par mutation pourrait aussi donner lieu a` une amelioration descaracte`res recherches, au sein dune population peu variable. Le recours a` certaines methodes biotechnologiques comme laculture tissulaire pour la multiplication massive des meilleurs genotypes, la culture danthe`res pour le developpementdhomozygotes purs a` double haplodie et lusage de marqueurs moleculaires pour identifier lemplacement des locusassocies a` la synthe`se du rebaudioside-A pourrait offrir dautres possibilites aux ameliorateurs. Enfin, comprendre lemecanisme et les voies de la biosynthe`se des glycosides du steviol permettrait den bonifier le profil par une regulation enamont ou en aval des ge`nes.

Mots cles: Stevia, glycosides diterpene, rebaudioside A, selection

Stevia rebaudiana (Bertoni) is a herbaceous perennialplant of the Asteraceae family, native to Paraguay(South America). Stevioside, the major sweetener pre-sent in leaf and stem tissues of stevia, was first seriouslyconsidered as a sugar substitute in the early 1970s by aJapanese consortium formed for the purpose of com-mercializing stevioside and stevia extracts (Kinghornand Soejarto 1985). Diterpene glycosides produced bystevia leaves are many times sweeter than sucrose. Theycan be utilized as a substitute to sucrose (Robinson1930; 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 andalternatives to the synthetic sweetening agents that arenow available to the diet conscious consumers. Randi(1980) reviewed the potential uses of Stevia rebaudiana,which produces stevioside, a non-caloric sweetener thatdoes not metabolize in the human body. The sweetcompounds pass through the digestive process withoutchemically breaking down, making stevia safe for thosewho need to control their blood sugar level (Strauss1995). This is more important, especially in the contextof the current social movement towards more naturalfoods (Brandle and Rosa 1992; Kamalakannan et al.2007). With the increased incidence of diabetes in Indiaand abroad, and growing concern over the safety ofsome chemical sweeteners, there is a need for a naturalnon-caloric sweetener with acceptable taste and healthproperties. In addition to its non-caloric sweeteningproperties, it has many therapeutic values: it is asantihyperglycaemic, 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 andEdahira 1979). It can also inhibit bacterial and fungalgrowth (Rojas and Miranda 2002). Commercial exploi-tation of stevia increased when Japanese researchersdeveloped a series of processes for the extraction andrefinement of sweeteners from its leaves. Research hasalso made possible simpler, water-only extraction pro-cesses (similar to sugar processing) in place of the oldersolvent extraction technology. The main producers ofstevia are Japan, China, Taiwan, Thailand, Korea,

Brazil, Malaysia and Paraguay. Currently, Steviais consumed in Japan, Brazil, Korea, Israel, theUnited States of America, Argentina, China, Canada,Paraguay and Indonesia (Crammer and Ikan 1986;Singh and Rao 2005) and to date there have been noreports of adverse effects from its use (Kinghorn andSoejarto 1985; Brandle and Rosa 1992).In the past, the main commercial constraint for

the stevia industry was the ban on its use in foodproducts as a food additive in the United States ofAmerica, although its use as a dietary supplement wasapproved by the Food and Drug Administration in1995 (Bespalhok-Filho and Hattori 1997). In India, theplant was introduced at the University of AgriculturalSciences, Bangalore, during the late 1990s, and studieson its adaptability were initiated. Research focusedon cultivation rather than crop improvement. Later,the Institute of Himalayan Bioresource Technology(CSIR), Palampur, introduced two accessions for do-mestication and cultivation in Himachal Pradesh. Aswell as cultivation, research has now been aimed at cropimprovement through conventional breeding and bio-technological approaches. Products like stevia sweetenerwill increasingly be in demand due to consumer interestin natural products. Such demand will need to besupported by varieties of stevia improved for agrono-mical traits as well as for higher quantities and quality ofiterpene glycosides, such as rebaudioside-A, which doesnot have an bitter aftertaste. The purpose of this reviewis to summarize the existing literature for the improve-ment of stevia through conventional plant breeding andselection and modern biotechnological approaches toprovide a baseline for further improvement.

ORIGIN AND ANTIQUITYThe genus Stevia Cav. consists of approximately150200 species of herbaceous, shrub and sub-shrubplants (Gentry 1996) and is one of the most distinctivegenera within the tribe Eupatorieae, mainly becauseof the morphological uniformity of its flowers andcapitula, which consist of five tubular flowers andfive involucral bracts (King and Robinson 1987); itis distributed from the southwestern United States

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southward through Mexico and Central America. It alsooccurs from non-Amazonian South America, southwardto Central Argentina (King and Robinson 1987). InBrazil, 36 species have been found, distributed mainly insouthern and central regions (Frederico et al. 1996).Stevia rebaudiana originated in the highland regions ofnortheastern Paraguay (on the Brazilian border), be-tween latitudes 238 and 248S, where the unique sweet-ening power of its leaves and its medicinal propertieshave been known by the local Guarani Indians manyhundreds of years (Chan et al. 1998; Melis 1999;Jeppensen et al. 2002, 2003; Srimaroeng et al. 2005).The Guarani Indians called the plant kaa he-he,which translates as sweet herb, and used it assweetener for their green herbal tea mate, and as aflavour enhancer (Soejarto et al. 1983). In the nativestate it grows on the edges of marshes or in grasslandcommunities on soils with shallow water tables (Shock1982). It is indigenous to the Rio Monday Valley of theAmambay moutain region at altitudes between 200 and500 m. The climate can be considered as semi-humidsubtropical, with temperatures ranging from 6 to438C, with an average of 238C, and rainfall rangingfrom 1500 to 1800 mm per annum. In 1943, the firstseeds were exported to the United Kingdom where itcould not be brought under cultivation. In 1968 it wasexported to Japan, and from there awareness of andcultivation of the plant spread throughout the world(Lewis 1992). By now, the crop has been introduced tomany countries, including Brazil, Korea, Mexico, theUnited States of America, Indonesia, Tanzania, Canadaand India (Lee et al. 1979; Donalisio et al. 1982; Shock1982; Goenadi 1983; Saxena and Ming 1988; Brandleand Rosa 1992; Fors 1995).

CLASSIFICATION OF STEVIAStevia rebaudiana is one of the 950 genera of theAsteraceae family (Soejarto et al. 1983; Lester 1999). Asystematic study of the North and Central Americanspecies of Stevia was done by Grashoff (1972). Steviaconsists of a group of annual and perennial herbs,subshurbs and shrubs that occur in mountain regions,open forests, borders of rivers and dry valleys (Robinson1930). Its first botanical description is attributed toM. S. Bertoni. The plant was first called Eupatoriumrebaudianum Bert. in honour of Rebaudi, the firstchemist to study the chemical characteristics of thesubstances extracted. Its name was later changed to thecurrent one, and it was recommended not only for foodproduction, but also for the medicinal effects that wereattributed to it (Bertoni 1905). Although there are about230 species in the genus, only S. rebaudiana gave thesweetest essence (Soejarto et al. 1983), while otherspecies contain other biochemicals of interest (Fredericoet al. 1996). It is a perennial herb with an extensiveroot system and brittle stems, producing small, ellipti-cal leaves. Under some environmental conditionsand management situations it behaves as an annual or

a mixture of plants of both types. The cultivatedplants are reported to be more vigorous (Shock 1982;Frederico et al. 1996).

Kingdom PlantaeSubkingdom TracheobiontaSuperdivision SpermatophytaDivision MagnoliophytaClass MagnoliopsidaSubclass AsteridaeGroup MonochlamydaeOrder AsteralesFamily Asteraceae

(Compositaeformerly)

Subfamily AsteroideaeTribe EupatorieaeGenus SteviaSpecies rebaudiana

Some other related species of Stevia rebaudiana areStevia eupatoria, Stevia lemmonii Lemmons stevia,Stevia micrantha annual stevia, Stevia ovata var.texana roundleaf candyleaf, Stevia plummerae Plummers stevia, Stevia plummerae var. alba, Steviarhombifolia Kunth, Stevia salicifolia willow-leafdtevia, Stevia serrata sawtooth stevia, Stevia viscida viscid stevia, Stevia commixta, Stevia satureiaefilia,Stevia leptophylla, Stevia myriadenia, Stevia ophryphylla,Stevia selloi, Stevia nepetifolia, Stevia oligophylla, Steviaoriganoides and Stevia triflora.

BOTANICAL DESCRIPTION

Floral Biology

Flower StructureThe inflorescence is loosely paniculate with the headsappearing opposite the bracts in irregular sympodialcymes. They are arranged in indeterminate heads. Theflowers are small (1517 mm) and white (Marsolais et al.1998; Dwivedi 1999) with pale purple throat corollas.The tiny white florets are perfect (hermaphrodite)having both male and female organs, borne in smallcorymbs of two to six florets (Goettemoeller and Ching1999). The plant can initiate flowering after a minimumof four true leaves have formed. The plant takes morethan a month to pass through the various developmentalflower stages (Fig. 1) and produce all its flowers(Taiariol 2004; sh et al. 2006).

Anther, Pollen and StigmaAnthers are small, five in number. The pollen canbe highly allergic. Using the acetocarmine technique,Monteiro (1980) observed that in some diploid indivi-duals of S. rebaudiana the pollen viability was 65%,which differs from the results of Oliveira et al. (2004)

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

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

Reproduction and Pollination BehaviourStevia is self-incompatible (Miyagawa et al. 1986;Chalapathi et al. 1997) and probably insect polli-nated (Oddone 1997). It has been reported that theamount of selfing ranged from 0 to 0.5%, while out-crossing ranged from 0.7 to 68.7%, indicating that theself-incompatibility system is operating (Katayama et al.1976; Maiti and Purohit 2008). Since stevia is self-incompatible, seed collected from an individual plantwould represent a half-sib family.Grashoff (1974) and Monteiro (1980) reported

agamospermy in certain genotypes of S. rebaudiana.Sexual and apomictic plants of S. rebaudiana producenormal and malformed pollen, respectively. Accordingto Monteiro (1980), the presence of apomixis inS. rebaudiana shown by embryological studies may berelated to specific physiological and/or ecological fac-tors. Analysis of sporogenesis allows the detection ofirregularities that can lead to the formation of inviablegametes.

Photoperiod and Flowering TimeStevia is a short-day plant that flowers from January toMarch in the southern hemisphere and from Septemberto December in the northern hemisphere. Floweringunder short-day conditions should occur 54104 dfollowing transplanting, depending on the daylengthsensitivity of the cultivar. The variability for photoper-iod sensitivity is large, ranging from 8 h to 14 h (Valioand Rocha 1977; Zaidan et al. 1980; Chalapathi 1997).

Zaidan et al. (1980) identified three photoperiod classesbased on the daylength. According to Valio and Rocha(1977), a minimum of two inductive short-day cycles arenecessary for flowering induction. It can be induced toflower from the four-leaf-pairs stage onwards. Flower-ing is more precocious in the 8-h photoperiod, butplants remained vegetative under an 8-h photoperiodwith interrupted night (Valio and Rocha 1977).

SeedSeeds are contained in slender achenes about 3 mm inlength. Each achene has about 20 persistent pappusbristles (Goettemoeller and Ching 1999). Seeds have avery small endosperm and are dispersed in the wind viahairy pappus. Shock (1982), Duke (1993), Carneiro et al.(1997) and Lester (1999) reported a poor and highlyvariable percentage of viable seeds. Fertile seeds areusually dark coloured, whereas infertile seeds are usuallypale or clear (Fig. 3a, b) (Felippe 1978; Monteiro 1980;Oddone 1997, 1999; Goettemoeller and Ching 1999).Seeds are very small (1000 seeds weigh 0.31.0 g) and asa result seedlings are slow to develop, reaching a sizesuitable for transplanting to the field at 4560 d(Colombus 1997; Brandle et al. 1998a). Goettemoellerand Ching (1999) revealed that some active manipula-tion of the blossoms is necessary to achieve pollination.Seed yields of up to 8.1 kg ha1 have been recorded, butit is common to achieve less than 50% germination.Seed production in 1 ha would be enough for leafproduction in 200 ha (Lester 1999). Agamospermy, i.e.,the asexual formation of seeds (Lumaret 1988), couldexplain the reproductive capacity of this species, even ifone takes into account the total lack of viable pollenobserved.

Fig. 1. Different stages of ower opening.

a b c d

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


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LeafStevia has an alternate leaf arrangement and herbaciousgrowth habit (Singh and Rao 2005). Leaves are small,sessile, lanceolate to oblanceolate, oblong, serate abovethe middle and somewhat folded upwards. Trichomeson the leaf surface are of two distinct sizes, large(45 mm) and small (2.5 mm) (Shaffert and Chebotar1994). 9 stevia, the leaf area index (LAI) 80 d aftersowing was 4.83 (Fronza and Folegatti 2003). Stevialeaves vary widely in quality due to many environmentalfactors, including soil conditions, irrigation methods,sunlight, air purity, farming practices, sanitation, pro-cessing and storage. The leaf has a pleasantly sweet,refereshing taste that can linger in the mouth for hours.The material contains the sweet components, sur-rounded by the bitter components in the veins (Maitiand Purohit 2008).

PROPAGATION

a) Vegetative Propagation

Shoot CuttingsPropagation of stevia is usually done by stem cuttings,which root easily, but require high labour inputs. Someplant varieties/selections produce virtually no viableseed and vegetative propagation is the only way ofmultiplication. Shock (1982) opined that stem cuttingswere the prime means for the propagation of stevia. Thedirect planting of stem cuttings in the field was found tohave a limited success (Chalapathi et al. 1999a). Ingeneral, a vigorous branch is cut at the base with a sharpblade and planting in the field, keeping two to threenodes above the soil. The cut portion of the branch isdipped in neem oil or any other fungicide (Maiti andPurohit 2008). Cuttings of new stems and shoots can bepropagated successfully (Lee et al. 1979; Shock 1982;Gvasaliya et al. 1990; Nishiyama et al. 1991). Gvasaliyaet al. (1990) reported that nearly 98100% rooting canbe obtained, when the current years cuttings are takenfrom the leaf axils. Further, the location on the plantfrom which cuttings are taken can also affect growthand rooting. Cuttings from the top part of the mainshoot with four internodes generally gave the bestresults (Tirtoboma 1988). However, the pair of leavesin the cuttings as well as the season also acts as

determinants for the rooting percentage and growth.Cuttings with four pairs of leaves rooted poorly,especially in February. Cuttings with two pairs of leavesrooted best in February and those with three pairs ofleaves in April (Zubenko et al. 1991a; Ramesh et al.2006). Cuttings taken in late winter rooted better thanthose taken at other times (Carvalho and Zaidan 1995).The sprouting percentage and shoot growth is also

affected by the length of the cuttings. The sproutingpercentage and shoot growth of sprouted cuttings weresignificantly higher with 15-cm-long cuttings comparedwith 7.5-cm-long cuttings (Chalapathi et al. 2001).The superior performance of the 15-cm-long cuttingsmay be because they have double the quantity of foodreserves compared to 7.5-cm-long cuttings. Stolz (1968)indicated the necessity of higher quantities of starch,carbohydrates, sugar and phenolic compounds for rootinitiation.Rooting of cuttings can sometimes be stimulated by

the use of growth regulators (Zubenko et al. 1991b;Carvalho and Zaidan 1995; Kornienko and Parfenov1996; Kornilova and Kalashnikova 1996; Bondarevet al. 1998). Some growth regulators can sometimesinfluence (increase) the concentration of steviosides inthe leaves (Chen and Li 1993; Acuna et al. 1997).Sprouting, however, was not influenced by NAA orIBA treatment at 500 ppm. IBA or NAA at a higherconcentration of 1000 ppm, however, caused completeinhibition of rooting and sprouting of cuttings, whichmay be due to the injury caused to the callus tissue. Shinand Lee (1979) also reported that higher concentrationsof IBA inhibited the rooting and sprouting of chry-santhemum cuttings due to the injury caused to thetissues. Zubenko et al. (1991b) found better rootingand sprouting of stevia cuttings by prolonged dippingof cuttings in 50 ppm IBA solution. Pre-treatmentof cuttings with paclobutrazol at 50 or 100 ppm wasfound to be an effective growth regulator treatmentfor the induction of roots and sprouts from stemcuttings. Shoot vigour index is an important indicationof vigorous growth of sprouted cuttings. Paclobutrazol-induced sprouts were found to be more vigorous thanIBA- or NAA-treated cuttings (Chalapathi et al. 2001).

Micro-propagationMany different parts of the plant viz., leaves, auxiliaryshoots, root-neck sprouts, shoot primordia, internodalexplants etc., can be used successfully for tissue culturepropagation (Handro et al. 1977; Yang and Chang 1979;Miyagawa et al. 1986; Ferreira and Handro 1987a;Bespalhok-Filho et al. 1992, 1993; Swanson et al.1992; Akita et al. 1994; Kornilova and Kalashnikova1996; Constantinovici and Cachita 1997). In vitromultiplication has frequently been used to multiplyindividually selected or bred clones and successfulprocedures have been documented (Handro et al. 1977;Ferreira and Handro 1988; Kornilova and Kalashnikova

ba

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


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1996; Carneiro et al. 1997; Bondarev et al. 1998;Nepovim 1998). Explants from leaf, nodal (Fig. 4) andinter-nodal segments were cultured on MS mediumcontaining 2,4-D at 2.0, 3.0, 4.0 and 5.0 mg L1 forcallus induction. Inter-nodal segments initiated callusformation earlier than node and leaf. The greatestamount of callus was produced in MS medium with 3.0mg L1 2,4-D, whereas, MS medium with 5.0 mg L1

2,4-D gave the poorest callus (Salim-Uddin et al. 2006).The plant growth and stevioside content in the leaves ofthe plants grown from stem cuttings or tissue culturewere more uniform than the plant grown from seeds. Thenumber of roots, shoot biomass and stevioside contentwere greater in the vegetatively grown plants (Truongand Valicek 1999).

Seed PropagationReproduction in the wild is mainly by seed,but germination and establishment from seed areoften poor and sometimes unsuccessful (Shaffert andChebotar 1994). Seeds germinate within 710 d aftersowing. Propagation through seeds is not a commonmethod of propagation owing to the problem of lowseed production and poor germination capacity. Usingseed to establish crops of stevia is more successfulin tropical climates, where there is no climatic restrictionon the length of the growing season. In northernclimates the shorter growing season necessitates seedlingestablishment in a glasshouse/greenhouse prior to thegrowing season. Stevia flowers need to be fertilizedby pollen from another plant to produce viable seed.A high density of bees (three to four hives per hectare) isrecommended for good seed production (Oddone 1999).Harvesting of immature seed may also contribute topoor germination (Colombus 1997).Seed production and fertility studies suggest that

high germination rates are possible from selected lines(Carneiro and Guedes 1992). Timing of flowering, seedharvest and pollination methods play an important rolein seed production (Melis and Sainati 1991; Strauss1995). Rain at flowering can also reduce seed setting.

Shade can reduce total growth, delay flowering time andreduce the rate of flowering (Slamet and Tahardi 1988).Direct seeding to the field is not practiced, but may be arequirement for large-scale commercial production. Leeet al. (1979) found that plants from seeds were lessproductive in the first year than those from cuttings.

SEED VIABILITY AND GERMINATIONTwo types of seeds are found in stevia, black and tan-coloured, with black seeds being heavier than tan seeds.Goettemoeller and Ching (1999) investigated the lowseed germination of stevia and tested the viability ofseeds based on tetrazolium chloride, finding that theviability of black seeds was much higher than that oftan-coloured seeds, i.e., 76.7 vs. 8.3%, respectively.Cross-pollination and self-pollination was accomplishedby transferring pollen with a bumble bee (Bombusimpatiens) thorax on the end of a toothpick. Theinfluence of pollination treatments as well as the effectof light and darkness during germination were alsoevaluated. Light increases the germination of black seedbut not of tan seed. This suggests that tan seedsrepresent inviable seed that are produced withoutfertilization.The germination study of seeds obtained from five

pollination treatments: cross-pollination by bumble bees(78.3%); cross-pollination by hand (92.0%); cross-pollination by wind (68.3%); self-pollination by hand(93.3%) and control (36.3%) suggested that incompat-ibility is not a factor in these clones. However, allpollination treatments increase seed germination ofblack seed over the control, suggesting that some activemanipulation of the blossoms is necessary to achievepollination (Goettemoeller and Ching 1999).Germination rates of stevia seeds vary greatly. It can

take 46 d to reach two-thirds of the final germinationof 6290% at 258C (Shock 1982; Carneiro and Guedes1992; Chen and Shu 1995; Takahashi et al. 1996).Germination requires at least 208C and often morethan 258C; light generally increases germination. In-creased temperature (408C on moist paper) for less than24 h can accelerate germination, but reduce totalgermination (Tanaka 1985). In Japan, seed remainedviable for up to 3 yr when stored under low humidityand in darkness (Kawatani et al. 1977). This contrastswith claims of optimum storage at 08C still producing a50% loss of viability after 3 yr (Brandle et al. 1998a). Inone experiment, seeds collected in the field and green-house were compared for germination. The greenhouse-collected seeds had a 90% germination rate, whereasfield collected seeds gave only 34% germination(Carneiro 1996). Total quantities of viable seed pro-duced in 1 yr are uncertain. Seed yield up to 8 kg ha1

with 50% germination would be sufficient for 200 ha ofcrop (Lester 1999).

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


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GLYCOSIDESEight diterpene glycosides with sweetening proper-ties have been identified in leaf tissues of stevia. Theseare synthesized, at least in the initial stages, using thesame pathway as gibberellic acid, an important planthormone (Singh and Rao 2005). The four major sweet-eners are stevioside, rebaudioside-A, rebaudioside-C anddulcoside-A. According to Kinghorn (1987) the sweet-ness of these compounds relative to sucrose are 210, 242,30 and 30 times, respectively. The two main glycosidesare stevioside, traditionally 510% of the dry weight ofthe leaves, and rebaudioside-A (Reb-A), 24%; theseare the sweetest compounds. There are also other re-lated compounds including minor glycosides, such asrebaudioside-B, rebaudioside-C (12%), rebaudioside-D, rebaudioside-E, rebaudioside-F, dulcoside-A,dulcoside-C and steviolbioside, as well as flavonoidglycosides, coumarins, cinnamic acids, phenylpropa-noids and some essential oils (Erik et al. 1956; Erichet al. 1961; Harry et al. 1956; Hiroshi et al. 1976; Masuret al. 1977; Yohei and Masataka 1978; Rajbhandari andRoberts 1983; Makapugay et al. 1984; Crammer andIkan 1986; Kinghorn 1987; Tsanava et al. 1989; Shaffertand Chebotar 1994; Putieva and Saatov 1997; Dzyuba1998; Dacome et al. 2005; Sekaran et al. 2007).Among the components of stevia, one, called

rebaudioside-A, is of particular interest because it hasthe most desirable flavour profile (DuBois 2000). Stevio-side traditionally makes up the majority of the sweetener(6070% of the total glycosides content) and is assessedas being 110270 times sweeter than sugar. It is alsoresponsible for the bitter aftertaste, sometimes reportedas a licorice taste. As well as sweetness, stevioside mayhave a lingering effect or certain degree of pungency,which is not appreciated by the majority of people, andwhich reduces its acceptability. Rebaudioside-A isusually present as 3040% of total sweetener and hasthe sweetest taste, assessed as 180400 times sweeterthan sugar with no bitter aftertaste (licorice taste orlingering effect).The ratio of rebaudioside-A to stevioside is the

accepted measure of sweetness quality; the morerebaudioside-A the better. If rebaudioside-A is presentin equal quantities to stevioside, it appears that theaftertaste is eliminated. The minor glycosides areconsidered to be less sweet, 3080 times sweeter thansugar (Crammer and Ikan 1986; Brandle 1999; Oddone1999). The sweetening effect of these compounds ispurely taste; they are undigested and no part of thechemical is absorbed by the body. They are therefore ofno nutritional value (Hutapea 1997). The yield ofsweetening compounds in leaf tissue can vary accordingto method of propagation (Tamura et al. 1984a),daylength (Metivier and Viana 1979) and agronomicpractices (Shock 1982). Unlike many low-calorie sweet-eners, stevioside is stable at high temperatures (1008C)and over a range of pH values (Kinghorn and Soejarto

1985). It is also non-calorific, non-fermentable and doesnot darken upon cooking (Crammer and Ikan 1986).There are reports of stevioside content (total glyco-

sides) ranging between 4 and 20% on a dry weightbasis, depending on the cultivar and growing condi-tions (Kennely 2002; Starrat et al. 2002). The sweeteningpotency (sucrose1) of stevioside, rebaudioside-A,rebaudioside-B, rebaudioside-C, rebaudioside-D,rebaudioside-E, dulcoside-A and steviolbioside are250300, 350450, 300350, 50120, 200300, 520300,50120 and 100125, respectively (Crammer and Ikan1986). The essential oil composition of the aerial parts offive different Stevia rebaudiana genotypes cultivated inon the Tuscan coast (Italy) was examined by CCand GC/MS. Forty different components were identifiedand the main constituents in all studying sampleswere spathulenol (13.440.9%), caryophyllene oxide(1.318.7%), beta-caryophyllene (2.116.0%) and beta-pinene (5.521.5%) (Cioni et al. 2006). In general,Paraguan leaves contain the highest concentration(913%) of the sweet steviosides/rebaudiosides mole-cules, Chinese stevia contains only 56%, and Indianstevia is midway between these. Under Indian condi-tions stevioside concentration was about 9.08% of thedry weight of leaves (Ashwini 1996; Chalapathi 1996).The genotypes under study at the Institute of HimalayanBioresource Technology show considerable morpholo-gical variation. Stevioside content is higher in Accession1 compared with Accession 2 (Megeji et al. 2005).

Biosynthesis of Steviol GlycosidesThe steviol glycoside and gibberellin pathways divergeat kaurene. In stevia, kaurene is converted to steviol, thebackbone of the sweet glycosides, then glucosylatedor rhaminosylated to form the principle sweeteners. Theprecursor compounds are synthesized in the chloroplast,and from there are transported to the endoplasmicreticulum, Golgi apparatus and then vacuolated. Thepurpose of these compounds in the stevia plant is not yetclear, but their high concentration in the leaf andthe conservation of the pathway within the speciesindicate that, at some point in evoluntionary time, theirpresence conferred significant advantage upon thoseindividuals that possessed them. Some researchers feelthat they act to repel certain insects and others speculatethat it is an elaborate means of controlling levels ofgibberellic acid (Smith and Van-Stadin 1992).Expressed sequence tags (EST) are a powerful tool

that has emerged from genomics research. Expressedsequence tag collections can reveal gene expressionpatterns, gene regulation and sequence diversity. Now,that enriched libraries and efficient high-throughputsequencing is widely available, ESTs have also becomean effective means of gene discovery in focused meta-bolic situations (Sterky et al. 1998; Ohlrogge andBenning 2000). This concept was first applied to theisolation of oleate hydroxylase from castor (Van deLoo et al. 1995). Since then, ESTs have been used to find


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new genes from 1-deoxy-D-xylulose 5-phosphate (DXP)pathway of different crops. It is now clear thattranscriptome analysis can be used to identify highlyexpressed genes that are involved in many differentmetabolic events (Brandle et al. 2002). Stevia rebaudianaleaves can accumulate high concentrations (up to 30%)of seven different glycosides derived from the tetracyclicditerpene steviol (Brandle et al. 1998a). Their intensesweetness and close structural relationship to gibberellicacid, coupled with the highly active nature of thepathway have fostered interest in steviol glycosidebiosynthesis and metabolism (Richman et al. 1999;Totte et al. 2000). In S. rebaudiana, these compoundsare synthesized exclusively in the mesophyll cells ofleaves and are undetectable in the roots. Although theadaptive value of steviol glycosides is not known, it isclear that S. rebaudiana has committed a very largeportion of total metabolism to their synthesis, makingS. rebaudiana a good candidate for an EST-based genediscovery effort. Despite this amazing metabolic cap-ability, only a few genes involved in the biosynthesis ofsteviol glycosides have been isolated and characterized(Richman et al. 1999; Brandle et al. 2002).Recent experiments have shown that the early steps in

steviol biosynthesis involve the plastid localized DXPpathway and not the mevalonate pathway (Totte et al.2000). Therefore, the first step in the steviol glycosidebiosynthetic pathway is the formation of DXP frompyruvate and glyceraldehyde 3-phosphate by thiaminephosphate-dependent DXP synthase (Lange et al. 1998;Eisenreich et al. 2001). It has also been found thatdimethylallyl diphosphate (DMADP) is not necessarilythe committed precursor of isopentenyl diphosphate(IDP) and that IDP and DMADP may arise fromseparate syntheses (Arigoni et al. 1999; Rodriguez-Concepcion et al. 2000; Brandle et al. 2002).Isopentenyl diphosphate and DMADP are con-

verted to geranylgeranyl diphosphate (GGDP) byGGDP synthase via three successive condensation reac-tions (McGarvey and Croteau 1995). Like all diterpenes,steviol is synthesized from GGDP, first by protonation-initiated cyclization to ()-copalyl diphosphate (CDP)by CDP synthase (Richman et al. 1999; Hedden andPhillips 2000). Next, ()-kaurene is produced fromCDP by an ionization-dependent cyclization catalyzedby ()-kaurene synthase (Richman et al. 1999).()-Kaurene is then oxidized at the C-19 position to()-kaurenoic acid, by a novel P450 mono-oxygenase(Helliwell et al. 1999). Steviol is produced by thehydroxylation of ()-kaurenoic acid at the C-13 posi-tion, but the gene for this FAD-dependent mono-xygenase has not yet been isolated (Kim et al. 1996).The two oxygenated functional groups of steviol, theC-19 carboxylate and the C-13 alcohol, provide attach-ment points for the sugar side chains that determinethe identity of the different glycosides. The C-13 alcoholis successively glucosylated, first yielding steviolmono-side then steviol-bioside, next the C-19 carboxylate is

glucosylated, which forms stevioside (Shibata et al. 1991,1995). The pathway terminates with the glucosylation ofstevioside, which forms rebaudioside A. Rhamnosylatedglycosides can also be formed by the addition of a UDPrhamnose moiety to steviolmonoside (Richman et al.1999). The enzymes involved in steviol glucosylationhave been partially characterized and there is an under-standing of the inheritance of certain glycoside patterns;however, corresponding genes have not been isolated(Shibata et al. 1991; Richman et al. 1999). In an effort tocreate a resource for gene discovery and to understandthe synthesis of steviol glycosides, Brandle et al. (2002)sequenced 5548 random cDNAs from a S. rebaudianaleaf library. With electronic probes, database searchesand differential representation, candidate genes for 70%of the steps in the steviol glycoside biosynthetic pathwayhave been searched.The average GC content of the ESTs was 42.5%,

similar to what has been found with Arabidopsis ESTs(Asamizu et al. 2000). Of the 278 ESTs classified into thesecondary metabolism category, 62 were candidatesfor diterpene glycoside synthesis. No members of themevalonic acid pathway were identified among theS. rebaudiana leaf ESTs. This supports the work ofTotte et al. (2000), and further proves that the DXPpathway is used to synthesize IDP for conversion tosteviol. Candidates were identified for 70% of thesteps in the pathway from pyruvate and glyceraldehydes3-phosphate to rebaudioside A. The most highly repre-sented pathway EST was an orthologue of the Arabi-dopsis GA3 gene, which is known to be involved in thethree step oxidation of kaurene to kaurenoic acid(Helliwell et al. 1999). The synthesis of kaurenoic acidis a key step in the synthesis of both steviol andgibberellic acid (Brandle et al. 2002).The ent-kaurene skeleton of chloroplast diterpene

glycosides, which are produced in large quantities inthe leaves of Stevia rebaudiana, is formed via the recentlydiscovered 2-C-methyl-D-erythritol 4-phosphate path-way. The enzymes catalyzing the first two steps ofthis pathway, 1-deoxy-D-xylulose-5-phosphate synthase(DXS) and 1-deoxy-D-xylulose-5-phosphate reductoi-somerase (DXR), were characterized. The cDNA-derived amino acid sequences for DXS andDXR contain716 and 474 residues, encoding polypeptides of about76.6 and 51 kDa, respectively. DXS and DXR fromStevia both contain an N-terminal plastid targetingsequence and show high homology to other known plantDXS and DXR enzymes (Totte et al. 2003).

Glycoside Content in Different Plant PartsPlant organs contain different amounts of the sweetglycosides, which decline in the following order: leaves,flowers, stem, seeds and roots. Roots are the onlyorgans that do not contain stevioside. The sweetness inthe leaves is two times higher than that in inflorescence(Dwivedi 1999). Sekaran et al. (2007) reported thatindividual tissues of stevia appear to differ significantly,


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with the stevioside content declining in a different order:leavesshootsrootsflowers. The fact that the high-est stevioside content is found in the leaves suggests thatthey serve as the main tissue for both synthesis andprimary accumulation of stevioside compounds. Thelargest amount of stevioside was found in the upperyoung, actively growing shoot sections, whereas thelowest senescent shoot sections exhibited the smallestamount of such compounds. During ontogeny, agradual increase in the stevioside concentration wasobserved in both mature leaves and stems, and thisprocess lasted to the budding phase at the onset offlowering (Bondarev et al. 2003).According to Bondarev et al. (2003), the lower,

mature leaves of stevia have fewer glands per leafsurface area than the upper, younger leaves, i.e., thereis a positive correlation between gland distributiondensity and steviol glycoside content. This argues infavor of possible increased accumulation, by 30 to 170%of glycosides, in upper young leaves as compared withlower senescent ones. Depending on the clone, theportion of the rebaudioside-A in the total glycosidescontent appeared to be increased as well. Duringontogeny, little variation in the glycoside content isalso found in roots. In these organs, from the vegetativephase to flowering, a gradual decrease in the glycosidecontent was observed. During the fruit developmentstage the levels of glycosides were found to revert to theinitial level. However, in roots the total glycosidescontent never exceed 0.1% (Bondarev et al. 2003).Kang and Lee (1981) demonstrated that the maximalcontent of stevioside in leaves is achieved during theformation of flower buds and it then gradually declines.All this information may indicate that the steviolglycosides are transported to generative organs. Similarresults were obtained for ecdisteroids in Rhaponticumcarthamoides, Ajuga reptans and Serratula coronata(Vereskovskii et al. 1983; Revina et al. 1986; Tomaset al. 1993; Anufrieva et al. 1998).At the whole-plant level, steviol glycosides tend to

accumulate in tissues as they age, so that older lowerleaves contain more sweetener than younger upperleaves. Since, chloroplasts are important in precursorsynthesis, those tissues devoid of chlorolphyll, such asroots and lower stems, contain no or trace amounts ofglycosides. Once flowering is initiated, glycoside con-centrations in the leaves start declining (Singh and Rao2005). Stems of stevia plants contain little or nosweeteners, although it is suggested that they maycontain some flavour enhancers, odourisers and otheragents of potential use for improving foodstuffs oralcoholic beverages (Singh and Rao 2005). As stemsmature and lose colour, any steviosides present dis-sipate. The structure, development and chemical contentof stevia roots have also received attention, oftenassociated with culturing procedures (Yamazaki andFlores 1989; Yamazaki et al. 1991; Zubenko et al. 1995).

Environmental EffectThe growth and flowering of stevia are affected byradiation, daylength, temperature, soil moisture, andwind (sh et al. 2006). Stevia is grown as a perennialcrop in subtropical regions, including parts of theUnited States, and as an annual crop in mid to highlatitude regions (Goettemoeller and Ching 1999). Theresults indicate that yield depends mainly on thegenetic characters of the plant, the phenotypic expres-sion of which is influenced by climatic and environ-mental factors (Metivier and Viana 1979; Ermakov andKotechetov 1996). Moreover, synthesis of terpenesis affected by climatic and environmental factors(Langston and Leopold 1954). Chen et al. (1978) studiedthe seasonal variation in stevioside content. Tateo et al.(1998) opined that environmental and agronomic factorshave more influence on stevioside production. The idealclimate for stevia is a semi-humid subtropical withtemperatures ranging from 6.0 to 438C with anaverage of 238C (Brandle and Rosa 1992). Researchconducted in Egypt revealed that climatic conditions,such as temperature, and length and intensity of photo-period, greatly affect stevia production and quality, asevident from the remarkable increase in yield during thesummer compared with winter (Allam et al. 2001).Long-day conditions, as compared with short days,

increase internode length, leaf area, and dry weight, andreduce the interval between the appearances of succes-sive leaf pairs in S. rebaudiana. Total soluble leaf sugars,protein, and stevioside content are also augmented inboth absolute and relative terms and the biosynthesis ofsteviol, the aglucone present in stevioside, is increasedby 45%. The concentration of glycoside in the leaves ofstevia increases when the plants are grown under longdays. Since glycoside synthesis is reduced at or justbefore flowering, delaying flowering with long daysallows more time for glycoside accumulation (Metivierand Viana 1979, 2005; Singh and Rao 2005). Sekaranet al. (2007) have shown a rebaudioside A/steviosideratio of 1.65 in ex vitro green leaves and 0.91 in in vitroshoots. Only severe Ca deficiency caused reduction inthe glycoside concentration (DeLima et al. 1997). Thechemical content of the last five fully expanded leaf pairsshowed the plant nutritional status (Utumi et al. 1999).

CYTOLOGYThe genus stevia shows great variation in chromosomenumber. The chromosome number of Stevia rebaudiana(2n22), previously reported by Frederico et al. (1996)and Monteiro (1980, 1982), has been confirmed (Fig. 5)for various strains. However, strains with 2n33and 2n44 (representing triploid and tetraploid cyto-types) also occur, which show a high degree of malesterility owing to the chromosomal abnormalities duringgamete formation. Cytological studies on many speciesof genus Stevia, performed by Grashoff et al. (1972),concluded that in North America, all the shrubby specieshave a gametic chromosome number of n12, while,


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herbaceous species with flower heads in a lax paniculatecluster have n11. Further, although most reportsindicate that n11 (2n22), values of 2n24, 33, 34,44, 48, 66, 70 have also been observed (Darlington andWylie 1955; Bolkhoviskikh et al. 1969; Moore 1973,1974, 1977; Goldblatt 1981, 1984, 1985, 1988; Goldblattand Johnson 1990, 1991, 1996, 1998; Oliveira et al. 2004).Galiano (1987) considered Stevia as a multibasic genus,with x11, x12 and x17, while, Frederico et al.(1996) considered its basic chromosome number asx11. Accordingly, this variation reflects numerical(aneuploidy and polyploidy) and possibly structuralchanges, mainly pericentric inversions (Frederico et al.1996). Aneuploidy also occurs in Picris babylonica, aplant that belongs to the same family as Stevia (Malallahet al. 2001). All South American species of stevia studiedare diploid with the exception of hexaploid S. elatior(2n66) from Colombia (Jansen et al. 1984). There is apredominance of the basic chromosome number x11among stevia species from South America, with onlythree species (S. lucida from Colombia, one populationof S. jujuyensis from Argentina, and S. organensis fromBrazil) having x12 (Coleman 1968; Galiano 1987;Galiano and Hunziker 1987). These three species mayhave originated by ascending aneuploidy from specieswith x11. The main mechanism in the evolution of theSouth American species of stevia is probably chromo-some inversions, with a small amount of aneuploidy andpolyploidy.The chromosomal morphology of six Brazilian species

of Stevia, including S. rebaudiana, was studied byFrederico et al. (1996). They observed that karyotypeswere very similar in chromosome number (2n22) andsize (1.02.4 mm). On the other hand, the comparativeanalysis of arm ratios of each karyotype, revealed thatevery species has a difference in arm ratios for at leastone chromosome pair. Even in species with the samekaryotypic formula, the sm pairs were located in

different positions in the karyotypes. The distinctpattern strongly suggests the occurance of pericentricinversions as the rule in the divergence of Brazilianspecies of stevia. Most of the chromosomes weremetacentric, with a variable number of submedianones. Only S. ophryophylla and S. rebaudiana had apair with a subterminal centromere (Frederico et al.1996). These results of the chromosome size andcentromere position were confirmed by Oliveira et al.(2004). Chromosome lengths were similar in otherstrains with 2n22 or 2n44. The presence of anucleolus organizing region on the short arm of thethird major chromosome pair was also confirmedfor S. rebaudiana. In the Brazilian species of stevia,the main mechanism of chromosomal evolution isalso by pericentric inversion. Thus, Frederico et al.(1996) concluded that, in addition to the numericchanges, structural rearrangements have played animportant role in the chromosomal evolution of thetribe Eupatorieae.Based on pollen viability, normal meiosis can be

inferred. Although pairing at diakinesis was normal,with the formation of bivalents in all diploids, in none ofthe S. rebaudiana strains studied was the pollen viable.High rates of tetrad normality (93%) were alsoobserved in the diploid strains. The lowest tetradnormality rates were those of the tetraploid (80%) andtriploid (64.5%) strains. Because of irregularities thatproduce unbalanced gametes during meiosis, polyploidswith an odd number of chromosome sets have a highlevel of sterility (Lawrence 1980). The low tetradnormality rates and the lack of viable pollen in thetriploid and tetraploid cytotypes may be related to thepairing of multivalents (tetravalents and trivalents) atdiakinesis. Other meiotic abnormalities, such as irregu-lar chromosomal disjunction in anaphase, could alsoexplain these results. However, as with the diploidcytotypes, the lack of viable pollen cannot necessarilybe linked with regular pairing at diakinesis (bivalents)and high rates of tetrad normality.Oliveira et al. (2004) studied pairing at diakinesis of

diploid, triploid and tetraploid cytotypes with 2n22,2n33 and 2n44, respectively. For diploid strains,pairing at diakinesis was n11II, which agrees with theliterature, While, pairing at diakinesis for the triploidand tetraploid strains was n11III and n11IV, respec-tively. This multivalent formation suggests that thesestrains may have an autopolyploid origin. Unlikethe above cytotypes, which occur spontaneouslyin nature, the Stevia cytotypes analyzed here wereobtained artificially by inducing polyploidy (Valois1992). Multivalents at diakinesis would be expected inautopolyploids, since the latter are derived from a singlegenome and result in three or four homologous sets ofchromosomes in triploid and tetraploids, respectively(Stace 1980).

Fig. 5. Chromosome count of diploid stevia plant.


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GENETIC INHERITANCEThe magnitude of heritable variation is of utmostimportance since it has relevance to selection response.Stevia produces steviol glycoside sweeteners in its leavesthat are up to 240 times sweeter than sugar. Under-standing the genetic basis of glycoside proportions willaid in their manipulation through plant breeding. Theexperiments conducted by Brandle (1999) were focusedon the genetic control of the proportions of two of theseglycosides, rebaudioside-A and rebaudioside-C. Thestudy was conducted using F2 population from crossesbetween two sets of parents with divergent glycosideprofiles. Segregation in the first set of F2s showed thatthe presence/absence of rebaudioside-A is controlled bya single dominant gene, but that the actual proportionsof rebaudioside-A may be controlled by multiple loci oralleles. In a second cross, proportions of rebaudioside-Aand rebaudioside-C were found to co-segregate andwere shown to be controlled by a single additive gene.This result suggests that both rebaudioside-A and -C aresynthesized by the same enzyme. The results were usedto propose a model for glycosylation of steviol glyco-sides (Brandle 1999). The presence of significant herit-ability (h2) for three economically important characters,i.e., leaf yield, leaf:stem ratio and stevioside (62.1, 78.8and 76.6, respectively) clearly suggested that geneticimprovement of stevia is possible (Brandle and Rosa1992). These high heritabilities enable selection andbreeding programs, aimed at higher yield, to achievesubstantial gains.

Linkage MapTo lay a foundation for molecular breeding efforts, thefirst genetic linkage map for S. rebaudiana was con-structed by Yao et al. (1999) based on RAPD markers.This information will be useful to those interested indeveloping marker-assisted selection procedures andquantitative trait analysis as well as provide a startingpoint for those interested in genome organization instevia. Despite the fact that the Compositae is one of thelargest and most diverse families of flowering plants,there has been little research involving molecularmarkers, largely because the family possesses very fewmajor crop species (Kesseli and Michelmore 1996). Thiswork represents the first detailed genetic study everconducted in this genus and one of only a few conductedamong the members of the Compositae. Yao et al.(1999) constructed the genetic linkage map forS. rebaudiana using segregation data from a pseudotest-cross F1 population. A total of 183 randomlyamplified polymorphic DNA (RAPD) markers wereanalysed and assembled into 21 linkage groups coveringa total distance of 1389 cM, with an average distancebetween markers of 7.6 cM. The 11 largest linkagegroups consisted of 419 loci, ranging in length from 56to 174 cM, and accounting for 75% of the total mapdistance. Of the primers that showed amplificationproducts, 35.5% detected polymorphic loci and 62.5%

of those marker loci segregated in a 1:1 ratio, indicatinga high level of genetic diversity in stevia. Most ofthe RAPD markers segregated in normal Mendelianfashion. Similar findings have been demonstratedin other cross-pollinated species (Grattapaglia andSederoff 1994). This may enable stevia breeders toconduct marker-assisted selection with genes that arefound closely linked to the markers in the map.In many linkage maps, loci are often found to be

concentrated in certain areas (hot spots) of a few linkagegroups (Paterson 1996; Keim et al. 1997). In compar-ison, the marker loci in the stevia linkage map weredistributed more evenly and may better represent of thewhole genome (Yao et al. 1999). Genetic diversitydetected with RAPDs varies with species and crosses(Beaumont et al. 1996). It appears that the limitedbreeding efforts undertaken to date have not signifi-cantly reduced levels of genetic diversity among thestevia breeding lines. This may be partly because steviahas not undergone a great deal of selection (Yao et al.1999).Construction of the stevia genetic linkage map has

laid a foundation on which to conduct marker-assistedselection in stevia. The next step is to associategenes involved with economically significant traits tothe RAPD markers and to convert those markers intoeasily scorable PCR-based markers such as SCARs.Mapping newly cloned cDNAs from stevia genesinvolved in glycoside synthesis, such as copalyl dipho-sphate synthase, can be addressed using cleaved ampli-fied polymorphic sequences markers and will lead to abetter understanding of the genomic organization ofsecondary metabolism (Richman et al. 1998; Koniecznyand Ausubel 1993). Further, marker development (e.g.,using AFLP or the unmapped RAPD markers segregat-ing 3:1) that will allow the resolution of the stevia mapinto 11 linkage groups is also an important goal for thefuture.

Character AssociationsInformation and understanding of the interrelationshipsamong characters are important to aid selection and setlimits of each economic character that a breeder canchoose without adversely affecting another importantcharacter. Several authors have studied the dependenceof yield on various growth parameters as well asstevioside content (Buana and Goenadi 1985; Shu andWang 1988; Buana 1989; Nishiyama et al. 1991; Brandleand Rosa, 1992; Shyu 1994; Chalapathi et al. 1998,1999b; Truong et al. 1999; Utumi et al. 1999). Someinteresting correlations have been found which can assistselection programmes (Table 1). Plant height and leafnumber at the second and fourth week after plantingwere positively correlated with biomass production ina greenhouse experiment conducted by Buana andGoenadi (1985). In another study, Buana (1989)reported that plant height had no significant correlationwith production, leaf number, or branch number in the


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first 4 wk. A positive correlation between total solublecarbohydrates and stevioside content was established byNishiyama et al. (1991). Stevioside content was uncor-related with yield or leaf:stem ratio (Brandle and Rosa1992). Further, dry leaf yield was correlated with leafsize and thickness and content of rebaudioside-A washighly correlated with leaf thickness (Shyu 1994).The dry yield of stevia was positively correlated with

plant height, number of branches, leaves per plant anddry matter accumulation. About 96.88% of the totalvariation in dry leaf yield was explained by a linearfunction of these four characters (Chalapathi et al.1998). Shu and Wang (1988) showed that leaf dry weightper plant had the greatest influence on yield. Steviosidecontent is influenced by both leaf surface and number ofroots; however, the leaf surface has more influence onstevioside content than the number of roots (Truonget al. 1999). The chemical content of the last fullyexpanded leaf pair was well correlated with plantnutrient status (Utumi et al. 1999).Furthermore, stevioside concentrations were uncorrelated

with yield or leaf:stem ratio indicating that concurrentimprovement of agronomic and chemical characteristicsis possible (Brandle and Rosa 1992). The observedcorrelation suggests that the carbohydrate reserve inthe leaves of stevia is found mainly in the form ofditerpenic glycosides of the stevioside type. The para-meters obtained from this correlation allowed theestablishment of a simple method to determine thestevioside content in dry stevia leaves (Nishiyama

et al. 1991). Plant leaf yield is proportional to branchnumber, leaf number and (not always) plant height(Buana and Goenadi 1985; Shu and Wang 1988; Buana1989). Total stevioside content is positively correlatedwith leaf:stem ratio (Tateo et al. 1998). Leaf thickness ispositively correlated with rebaudioside-A:stevioside ra-tio (Shyu 1994). The total stevioside content of leaves atthe seedling stage and when mature is not correlated,making plant selection at the seedling stage ineffective.High rebaudioside-A content is also linked to large leafarea, high net photosynthetic rate, high chlorophylland protein content (Weng et al. 1996). Nakamura andTamura (1985) reported that the levels of dulcoside-A and stevioside and rebaudioside-A, and -C arepositively correlated with each other, while steviosideand rebaudioside-A, and dulcoside and rebaudioside-Care negatively correlated with each other.

PHENOTYPIC VARIABILITYIn the wild populations of Stevia rebaudiana, there isgreat variation in phenotype and leaf analysis. Thecollections made as part of the various breeding andselection research programs have invariably included arange of genotypes and selections of plants with distinctlevels of steviosides in their leaves. Shock (1982) planted200 lines for survival testing and screened 17 linesfor productivity. The stevioside content of leaves canvary substantially (416%) between individual plants,even after a selection program has been continued forsome time (Bian 1981; Nakamura and Tamura 1985).

Table 1. List of some important correlations

Characters Correlation Characters References

Plant height, Leaf number ve Biomass production Buana and Goenadi (1985)Plant height Uncorrelated Production, leaf number,

branch numberBuana (1989)

Stevioside content ve Total soluble carbohydrates Nishiyama et al. (1991)Uncorrelated Yield, leaf:stem ratio Brandle and Rosa (1992)

Dry leaf yield ve Leaf size and thickness Shyu (1994)Rebaudioside-A ve Leaf thickness Shyu (1994)Dry yield ve Plant height, number of branches,

leaves per plant and dry matterChalapathi et al. (1998)

Leaf dry weight per plant ve Yield. Shu and Wang (1988)Stevioside content ve Leaf surface, number of roots Truong et al. (1999)Chemical content of last fullyexpanded leaf pair

ve Plant nutrient status Utumi et al. (1999)

Stevioside Uncorrelated Yield, leaf:stem ratio Brandle and Rosa (1992)Plant leaf yield ve Branch number, leaf number Buana (1989); Buana and Goenadi

(1985); Shu and Wang (1988)ve (not always) Plant height

Total stevioside content ve Leaf/stem ratio Tateo et al. (1998)Leaf thickness ve Rebaudioside-A/Stevioside ratio Shyu (1994)Stevioside content atseedling stage

Uncorrelated Stevioside content at maturity Weng et al. (1996)

Rebaudioside-A content ve Leaf area, net photosynthetic rate,chlorophyll and protein content

Weng et al. (1996)

Dulcoside-A ve Stevioside Nakamura and Tamura (1985)Rebaudioside-A ve Rebaudioside-C Nakamura and Tamura (1985)Stevioside ve Rebaudioside-A Nakamura and Tamura (1985)Dulcoside ve Rebaudioside-C Nakamura and Tamura (1985)


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This natural variability could be partially due to thelargely out-crossing nature of the species (Handro et al.1993).Monteiro (1980) studied the phenotypic differences

present in the population and was unable to separatethem into a valid taxonomic variety. There are alsoreports of irregular quantitative and qualitative produc-tion of the sweetening molecules from cultivatedS. rebaudiana. The phenotypic variability within thepopulation is linked to the open pollination behavior ofthe species (Tateo et al. 1998). Nurhaimi and Toruan(1995) showed somaclonal variations in DNA finger-prints between six groups of plantlets. It has beenreported from China, that, in a sample of plants fromone clone, stevioside varied from 1.48 to 6.98% andrebaudioside-A from 4.5 to 12.1%, with total glycosidevarying from 10.26 to 19.57% (Huang et al. 1995). Inanother report from China, total sweet glycoside con-centration in some lines has been reported to be as highas 20.5%, and in separate cultivar rebaudioside-A:stevioside ratios of 9:1 have been disclosed (Morita1987; Shizhen 1995). Such variability in the raw materialwould permit the use of conventional extraction meth-ods to produce a stevia sweetener with more than 85%rebaudioside-A, without the need to recrystalize indivi-dual glycosides. Much of the morphological variabilityhas been observed in the population of S. rebaudianaunder study at the Institute of Himalayan BioresourceTechnology, Palampur. It also exhibits considerablevariability for stevioside content, which may vary from2 to 10% (Megeji et al. 2005). The genetic improvementof stevia is only possible through the characterization ofthe available variability at the morphological, chemicaland biochemical, cytogenetic and molecular levels, inorder to utilize the information to develop an ideal planttype. Stevia grown at the Delhi Research Station(Ontario, Canada) had 1.22 times as much leaf dryweight as stem dry weight, this same ratio was about0.67 in California (Brandle and Rosa 1992).

DISEASE RESISTANCEStevia is known to be free from attacks by insects, whichmay be due to its inherent sweetness acting as a repellent.Therefore, insecticides are not required at an essentialbasis as in other crops, which helps in producing organicStevia. The fungal diseases Septoria leaf spot (Septoriasteviae), Alterneria leaf spot (Alternaria alternata), stemrot (Sclerotium dephinii Welch.), root rot (Sclerotiumrolfsii), powdery mildew (Erysiphe cichoracearum DC),damping-off (Rhizoctonia solani Kuehn.) and Sclerotiniasclerotoirum have been reported (Ishiba et al. 1982;Lovering and Reeleeder 1996; Chang et al. 1997; Thomas2000; Megeji et al. 2005; Kamalakannan et al. 2007)(Table 2). There is a need to develop and identifyresistant sources to develop varieties resistant to ortolerant of these diseases.

BREEDING OBJECTIVESBreeding programmes for stevia should be aimed atimproving total glycoside content and rebaudioside-A:stevioside ratio with higher leaf yield. So far, plantbreeding efforts with stevia have been largely focusedon improving leaf yield and rebaudioside-A concentra-tion in the leaves. High leaf:stem ratios are desirablein cultivated stevia because of the low steviosideconcentrations (B5 mg g1) in stem tissue. Cultivardescriptions indicate that sufficient genetic vari-ability exists to make significant genetic gains in leafyield, rebaudioside-A content and the rebaudioside-A:stevioside ratio (Lee et al. 1982; Morita 1987; Brandleand Rosa 1992; Shizhen 1995) (Table 3).The native rebaudioside-A:stevioside ratio in Stevia

rebaudiana leaves is usually about 0.5 or less. Thepredominance of stevioside gives a characteristic bitteraftertaste to the crude extract. Conversely, the mostvaluable extracts are those that have rebaudioside-A asthe major component, because of its organoleptic andphysicochemical features, i.e., it has the best profilerelative to all other glycosides, and is more soluble inwater (Ahmed and Dobberstein 1982; Crammer andIkan 1986; Huang et al. 1995), allowing a greatervariety of formulations. Therefore, the developmentand phytochemical characterization of new varieties ofS. rebaudiana with higher levels of rebaudioside-A is aprimary aim of plant breeders concerned with theimprovement and utilisation of this source of naturalsweeteners (Yao et al. 1999; Dacome et al. 2005; Sekaranet al. 2007). With the high level of natural variability dueto constant out-crossing, breeders are able to improve thelevel of sweeteners in the leaves and alter the rebaudio-side-A:stevioside ratio (Shu 1989; Huang et al. 1995).

BREEDING METHODS

Germplasm Introduction, Collectionand ConservationGermplasm is a very important material for the im-provement of crops. Introduction of germplasm fromone area to another continues to be an important activityfor breeding, particularly in developing countries. It isgenerally used as source of superior genes and increasinggenetic diversity in the germplasm for breeding pro-grammes. Introductions can be used directly as commer-cial cultivars. Adapting exotic germplasm is, however, along-term programme. Intermating should be carried outfor several generations and selection pressure appliedgradually for desirable gene combinations.Institutions around the world that have undertaken

research and/or appraisal studies on stevia have col-lected seed and plant material from Paraguay in its wild,natural environment (Grashoff 1972; Bian 1981; Shock1982; Soejarto et al. 1983; Suhendi 1989; Tateo et al.1998). The rationale behind seed collection is to con-serve genes and not genotypes, since, in stevia, due toheterozygosity, no genotype is true breeding. Two


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genotypes of S. rebaudiana, Accessions I and II, whichare morphologically diverse with respect to their growthhabit and sweetness are being maintained and multipliedat the Institute of Himalayan Bioresource Techno-logy. Further selections for desirable plant types arebeing performed in segregating progenies of individualselections.

SelectionThe success of stevia breeding depends on the choice ofparents, making crosses, raising adequate populationand further selections. In the wild, the total glycoside

concentration in stevia leaves typically varies from 2 to10% on a dry weight basis. Nearly three decades ofbreeding and selection have increased glycoside concen-tration in stevia leaves by as much as 20% (Huang et al.1995). However, this improvement was based on phe-notypic selection for total glycoside concentration instevia leaves, which is heavily influenced by environ-mental conditions, such as soil and weather. Moreimportantly, it requires selection relatively late in thegrowing season. Selection at the early seedling stage isleast effective, because seedlings are so influenced by theenvironment that only 2030% of the variability isgenetic, and measurements are based on expensive andtedious high performance liquid chromatography(HPLC) procedures (Brandle and Rosa 1992). As aresult, selection for plants producing high amounts ofglycoside is expensive, time consuming, and relativelyinefficient (Yao et al. 1999).Countries that have been researching stevia for some

time, especially Japan, China, Korea, Taiwan andRussia, have all reported success in their breeding/selection programmes and have released new varietieswith improved glycoside content and higher yields(Table 4). Most breeding programs are based on crossbreeding and selection. Vegetative propagation andcloning have frequently been used to multiply individu-ally selected plants. Some of these selections, althoughvery high yielding, are self-incompatible and can only bereproduced vegetatively (Lee et al. 1982). This limitstheir commercial use, although they may be useful forbreeding new hybrids.Attempts made elsewhere in the world have resulted in

patents for the superior plant types (Table 5). A cultivarwith a rebaudioside-A:stevioside ratio of 0.96:1, com-pared with 0.36:1 in the starting material, was developedwith total glycosides of 22.4% (Lee et al. 1982). Otherplants have been developed that exhibited rebaudioside-A:stevioside ratios as high as 9.1:1, but total steviolglycosides were 10.1% (Morita 1987). Again, due to selfincompatibility, the cultivar could not be reproducedusing a seed-based production system. The costs asso-ciated with clonal propagation limit the general applic-ability for large-scale production of stevia plants.

Table 2. Various diseases reported in stevia

Disease Causal organism Symptoms

Septoria leaf spot Septoria steviae Depressed, angular, shiny olive grey foliar lesions are formed that rapidly coalesced and oftensurrounded by a chlorotic halo. Leaves quickly become necrotic and often drop off the plant.

Alterneria leaf spot Alternaria alternate Initially appears as small circular spots, light brown in colour. Later, many became irregular anddark brown to grey, while others remain circular with concentric rings or zones.

Root rot disease Sclerotium rolfsii Yellowing and drooping of leaves, with wilting of plants and white cottony mycelial growth at thecollar region takes place. The mycelial growth spread to the stem and roots, with associated tissuerotting. Brown sclerotia appear on the diseased areas.

Stem rot Sclerotinia sclerotiorum Brown lesions on the stem, near the soil line are formed, followed by wilting and eventually by thecomplete collapse of affected individuals.

Table 3. Characters for improvement/utilization through breeding

Sr. no. Characters Reference

Steviol glycoside characters1 Total glycosides content Sys et al. (1998)2 Rebaudioside-A content Marsolais et al. (1998)3 Rebaudioside-A: stevioside

ratioBrandle (2001), Morita andYucheng (1998)

Yield contributing characters4 Leaf:stem ratio Brandle and Rosa (1992)5 Leaf size/area index6 Leaf thickness7 Number of leaves/plant or

branch8 Number of branches/plant9 Internode length10 Stalk weight and thickness11 Leaf angle12 Photoperiod sensitivity/short

day plantLester (1999), Valio andRocha (1977)

13 Plant vigour/growth rate Borie (2000), Andolfiet al. (2002)

14 Seed germination percent Barathi (2003), Carneiroet al. (1997), Duke (1993),Shock (1982)

15 Seed viability for shortduration

Marcavillaca (1985)

16 Self-incompatibility Chalapathi et al. (1997)17 Lodging susceptibility18 Drought tolerance Jia (1984)19 Sensitive to water logging20 Asynchronous seed maturity

and seed dispersal21 Disease resistance22 Poor tolerant to high soil pH Shock (1982)


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Development of Stevia Plant RSIT 94-1306and RSIT 94-751Sys et al. (1998) and Marsolais et al. (1998) set out todevelop stevia plants with high concentrations ofindividual steviol glycosides that could be extractedand recombined in ratios suitable for specific productuses. Landrace stevia has a combination of steviolglycosides that is not optimal for all product applica-tions. In 1989, seed of a landrace variety from China wasintroduced at Agriculture and Agri-Food Canada, DelhiResearch Station, Ontario, Canada. Superior plantswere selected from this population and designated SR1to SR15 (breeding procedure shown in Fig. 6). Theseplants were inter-crossed and half-sib seed was collectedfrom each parent plant.The half-sib families were evaluated in a genetic

heritability trial (Brandle and Rosa 1992). RSIT94-1306 was selected from the SR2 half-sib populationand RSIT 94-751 was selected from the SR13 half-sibpopulation on the basis of agronomic traits and steviolglycoside profile, based on replicated trials. RSIT

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

Development of RSIT 95-166-13Brandle et al. (1998b) developed RSIT 95-166-13 as aunique combination of characteristics, which distin-guished it from its parents and all other stevia varietiesfor a high rebaudioside-C:stevioside ratio. Differentseed germplasm accessions of stevia from China weregrown for evaluation at the Delhi Research Station,Agriculture and Agri-Food Canada. Plant samplesRSIT 94-1838, RSIT 94-1833, and RSIT 94-1560 wereretained as a result of the higher than average concen-trations of rebaudioside-C in their leaves on a dryweight basis. A plant (RSIT 94-1829) selected from thevariety Brazil Zairai was also retained, because its leaveshad higher than average concentrations of rebaudioside-C. Brazil Zairai is an open-pollinated landrace variety ofstevia obtained from the Japanese National GermplasmDepository. These four clones were used as parents andintercrossed. Half-sib seeds collected from clone RSIT

Table 4. Some varieties/cultivar selections and releases

Year Country Reference Variety Features

1979 Korea Lee et al. (1978) Suweon 2 High yield and steviosides1982 Korea Lee et al. (1982) Suweon 11 Thick leaves, high Reb%1989 China Shu (1989) Yunri, Yunbing1995 China Shu (1995) Zongping Highest Rebaudioside and stevioside1994 Taiwan Shyu (1994) K1, K2, K3. High yield, better Rebaudioside:stevioside ratio1994 Indonesia Suhendi (1989) BPP721996 China Weng et al. (1996) SM4 High yield and Rebaudioside:stevioside ratio1996 Russia Kornienko and Parfenov (1996) Ramonskaya Slastena2000 India IHBT (CSIR) annual report Madhuguna High yield and total glycoside content2000 India IHBT (CSIR) annual report Madhuguni High yield and total glycoside content

Table 5. List of patents

Title Country Inventer Year Patent no.

Stevia rebaudiana with altered steviol glycoside composition USA Brandle, J. 2001 6,255,557Stevia rebaudiana with altered steviol glycoside composition USA Brandle, J. 1999 PCT-WO99/49724Variety of Stevia rebaudiana Bertoni USA Morita, T. and Yucheng, B. 1998 6031157Stevia plant named RSIT 94-751 USA Marsolais, A. A.; Brandle, J.

and Sys, E. A.1998 PP10,564

Stevia plant named RSIT 94-1306 USA Sys, E. A.; Marsolais, A. A.and Brandle, J.

1998 PP10,562

Stevia plant named RSIT 95-166-13 USA Brandle, J.; Sys, E. A. andMarsolais, A. A.

1998 PP10,563

Extraction of sweet compounds from Stevia rebaudiana Bertoni USA 1999 5,972,120Method of cultivating hybrid new variety via systematicallybreeding stevia rebaudiana clone parental plant.

Wang, Q 2006 CN1985575

Japan Morita 1984a, b JP-59034848;JP-59034826

Japan Morita 1985 JP-60160823Japan Morita 1986 JP-61202667Japan Nakazato, T 1987 JP-62096025Japan Nakazato, T 1988 JP-63173531Europe Stevia co. Inc. 1985 EPA0154235

New triploid of Stevia Rebaudiana Bertoni- containssweet diterpenoid.

Japan Sanyo Kokusaku Pulp Co. 1990 JP-2242622


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

Population Improvement

Recurrent SelectionRecurrent Selection is useful for improving quantita-tively inherited characters in cross-pollinated species. InS. rebaudiana, where self-incompatibility ensures a highdegree of heterogeneity, recurrent selection is the mosteffective method of increasing foliage yield as well astotal glycoside content. In population improvement,plant breeders aim to increase the frequency of desirablealleles through the selection of superior recombinants.Recurrent selection typically starts with harvestingindividual open-pollinated plants from the source po-pulation. A portion of the seed of these plants is saved asa reserve. The agronomic characters of progeny rows are

evaluated visually, and superior rows are identified. Theselected rows are harvested separately. Equal quantitiesof reserve seed from selected single plants, based onprogeny performance, is composited. The first recurrentselection cycle starts when the new composite is grownin an isolated plot where random mating among theplants is allowed. A bulk seed sample is harvested fromthe remaining plants of each composite for use inreplicated yield trials to determine the response toselection in each recurrent selection cycle for thecharacteristics under improvement. Recurrent selectionis continued until a reasonable response to selection isachieved. Each cycle of recurrent selection produces anew population that may be a potential cultivar. Theimproved population with increased frequency of desir-able alleles can be used as a cultivar per se or as a sourceto identify superior individual genotypes.

Development of Synthetics and CompositesThere is a need to develop a stevia cultivar that isenriched in rebaudioside-A and has a high steviolglycoside content that can be produced using a relativelylow-cost method based on transplants produced fromseed. Therefore, a synthetic cultivar produced by inter-crossing clones or sibbed lines obtained from a breedingpopulation during cycles of recurrent selection is re-quired. In order to develop a synthetic variety more thanone line is required and the lines or clones are typicallytested for combining ability, preserved for future synth-esis of the synthetic cultivar, as well as combined byrandom crossing. Such synthetic cultivars are intendedfor use in crop production systems. Because of the highdegree of natural out-crossing and the absence of anefficient system of pollination control, composites andsynthetics are used to capture part of the availableheterosis. This is the most practical and effectivebreeding method, and there is a large effort aimedat establishing stevia as a crop in Japan as well as anumber of other countries based on developing syntheticcultivar.

Synthetic Cultivar AC Black BirdBrandle (2001) developed the synthetic cultivar ACBlack Bird (breeding procedure shown in Fig. 7),which is characterized by exhibiting a high level oftotal glycosides (at least 14%), and a high ratio ofrebaudioside-A to stevioside (at least 9.1:1). In order tocreate parents for the synthetic cultivar, crosses weremade among a number of single plants and a largenumber of progeny were planted out to the field,and selections were made among those progeny.Leaves sampled from those selected plants were ana-lyzed for glycoside concentration as well as composition,and selections that were high in glycosides, withrebaudioside-A to stevioside ratios of at least about9.3:1 (denoted as A to D), were inter-crossed in thegreenhouse and seeds were collected from the maternalparents. At the same time, cuttings were taken from

In summer 1994

In 1989, seeds of landrace var. from china

1000 plants grown

SR1 - SR15

Intercrossed & half-sibseeds collected

15 Plants selected

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

In summer 1990

In summer 1991

These half-sib families wereevaluated in genetic heritability trial

SR 2 SR 13

RSIT 94-1306 RSIT 94-751

Stevioside (%) = 17.25Reb-A = 0.0Total glycosides = 18.37

Stevioside (%) = 4.88Reb-A = 11.82Total glycosides = 18.08

Cuttings evaluatedCuttings evaluated

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


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the plants so that they could be used to duplicatethe synthetic as required. Seed from the maternalparents was retained as half-sib families and a portionbulked to create a synthetic cultivar AC Blackbird. Thehalf-sib families, the bulked sample and the parentalclones were evaluated in a replicated field trial. Theplants so obtained are characterized as exhibiting highlevels of total steviol glycosides and being enriched inrebaudioside-A. It is preferred that the cultivar seed beproduced from at least two intermating genotypes.

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

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

IIIIIIIIIIIIIIIIIIIIIIIIIII1...12

OxO OxO OxO OxOOxO OxO OxO OxO

Selection among these families

Large no. of progeny planted in field

Crossing among no.of selected plants

Seeds collected

20 plants from each family selected for HPLC analysis

60 plants in each family

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

Cuttings to duplicate the synthetic as required

AC Black bird(Synthetic cultivar)

Stevioside (%) = 1.27Reb-A = 12.49Total = 15.06Reb-A/Stev = 9.96

Parental clones, Selections, half-sib families of selections and synthetic cultivar evaluated in replicated field trial

A B C D

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

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

Sexual inter-crossing

IIII

Fig. 7. Breedin

a review on the improvement of stevia

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