Stevia Rebaudiana: A Review On The Improvement of Stevia ( (Bertoni) )

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 (2n 22) of genus Stevia
Cav., which consists of approximately 230 species of herbaceous, shrub and sub-shrub plants. Leaves of stevia produce
Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by 190.104.112.42 on 04/15/20
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
For personal use only.
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’amélioration génétique du stévia [Stevia rebaudiana (Bertoni)],
tour d’horizon. Can. J. Plant Sci. 91: 127. Stevia rebaudiana (Bertoni) est une herbacée vivace (2n22) du genre Stevia
Cav., lequel compte environ 230 espèces végétales de type herbacé, arbustif ou sous-arbustif. Les feuilles du stévia
produisent des glycosides diterpène (stévioside et rébaudiosides), non nutritifs et non toxiques, mais au très fort pouvoir
sucrant, susceptibles de remplacer le sucrose et d’autres édulcorants artificiels, car ils sont 300 fois plus doux que le sucrose.
Outre cette propriété, le stévia possède des vertus médicinales. Le stévia n’est pas une plante autogame et la pollinisation
s’effectue grâce à des insectes. De tous les glycosides que produisent les feuilles du stévia, le rébaudioside-A présente un
intérêt particulier, car son profil de sapidité est le plus désirable, le stévioside laissant un arrière-goût amer. La création de
nouvelles variétés de S. rebaudiana plus riches en rébaudioside-A et contenant moins de stévioside est le but principal que
recherchent les améliorateurs, qui souhaitent accroı̂tre la production de ces édulcorants naturels et en favoriser l’utilisation.
Un seul gène additif, co-ségrégant, commande la proportion de rébaudioside-A et C produite, ce qui laisse supposer
l’intervention du même enzyme dans la synthèse. Le stévioside et le rébaudioside-A sont négativement corrélés, alors qu’il
existe une corrélation positive entre le rébaudioside-A et le rébiaudioside-C. La meilleure façon de renforcer les caractères
recherchés dans une espèce à pollinisation aussi croisée que le stévia consiste à recourir aux méthodes classiques
d’amélioration génétique comme la sélection et l’hybridation des génotypes intéressants. On a déjà homologué plusieurs
cultivars créés de cette manière, et produisant une quantité supérieure de tel ou tel glycoside, notamment RSIT 94-1306,
Abbreviations: BAP, benzylaminopurine; CDP, ( )-copalyl

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

2 CANADIAN JOURNAL OF PLANT SCIENCE
RSIT 94-75 et RSIT 95-166-1. On peut aussi recourir à des variétés composées ou synthétiques pour saisir une partie de
l’hétérosis existante, en raison du fort degré naturel de croisement extérieur et de l’absence d’un système efficace régulant la
pollinisation. Des variétés synthétiques et composées comme AC Black Bird et PTA-444 ont d’ailleurs déjà été réalisées. La
polyploı̈die entraı̂ne une meilleure capacité d’adaptation des plantes, tout en engendrant de plus gros organes et cellules.
Les tétraploı̈des se caractérisent par des feuilles plus grandes et épaisses, dont la biomasse et le rendement pourraient
dépasser ceux des souches diploı̈des. L’amélioration par mutation pourrait aussi donner lieu à une amélioration des
caractères recherchés, au sein d’une population peu variable. Le recours à certaines méthodes biotechnologiques comme la
culture tissulaire pour la multiplication massive des meilleurs génotypes, la culture d’anthères pour le développement
d’homozygotes purs à double haploı̈die et l’usage de marqueurs moléculaires pour identifier l’emplacement des locus
associés à la synthèse du rébaudioside-A pourrait offrir d’autres possibilités aux améliorateurs. Enfin, comprendre le
mécanisme et les voies de la biosynthèse des glycosides du stéviol permettrait d’en bonifier le profil par une régulation en
amont ou en aval des gènes.
Mots clés: Stevia, glycosides diterpēne, rēbaudioside A, sēlection

Stevia rebaudiana (Bertoni) is a herbaceous perennial Brazil, Malaysia and Paraguay. Currently, Stevia
plant of the Asteraceae family, native to Paraguay is consumed in Japan, Brazil, Korea, Israel, the
(South America). Stevioside, the major sweetener pre- United States of America, Argentina, China, Canada,
sent in leaf and stem tissues of stevia, was first seriously Paraguay and Indonesia (Crammer and Ikan 1986;
considered as a sugar substitute in the early 1970s by a Singh and Rao 2005) and to date there have been no
Japanese consortium formed for the purpose of com- reports of adverse effects from its use (Kinghorn and
mercializing stevioside and stevia extracts (Kinghorn Soejarto 1985; Brandle and Rosa 1992).
and Soejarto 1985). Diterpene glycosides produced by In the past, the main commercial constraint for
stevia leaves are many times sweeter than sucrose. They the stevia industry was the ban on its use in food
can be utilized as a substitute to sucrose (Robinson products as a food additive in the United States of
1930; Soejarto et al. 1982, 1983; Lyakhoukin et al. 1993; America, although its use as a dietary supplement was
Matsui 1996; Megeji et al. 2005; Sekaran et al. 2007); approved by the Food and Drug Administration in
they are natural sources of non-caloric sweetener and 1995 (Bespalhok-Filho and Hattori 1997). In India, the
alternatives to the synthetic sweetening agents that are plant was introduced at the University of Agricultural
now available to the diet conscious consumers. Randi Sciences, Bangalore, during the late 1990s, and studies
(1980) reviewed the potential uses of Stevia rebaudiana, on its adaptability were initiated. Research focused
which produces stevioside, a non-caloric sweetener that on cultivation rather than crop improvement. Later,
does not metabolize in the human body. The sweet the Institute of Himalayan Bioresource Technology
compounds pass through the digestive process without (CSIR), Palampur, introduced two accessions for do-
chemically breaking down, making stevia safe for those mestication and cultivation in Himachal Pradesh. As
who need to control their blood sugar level (Strauss well as cultivation, research has now been aimed at crop
1995). This is more important, especially in the context improvement through conventional breeding and bio-
of the current social movement towards more natural technological approaches. Products like stevia sweetener
foods (Brandle and Rosa 1992; Kamalakannan et al. will increasingly be in demand due to consumer interest
2007). With the increased incidence of diabetes in India in natural products. Such demand will need to be
and abroad, and growing concern over the safety of supported by varieties of stevia improved for agrono-
some chemical sweeteners, there is a need for a natural mical traits as well as for higher quantities and quality of
non-caloric sweetener with acceptable taste and health iterpene glycosides, such as rebaudioside-A, which does
properties. In addition to its non-caloric sweetening not have an bitter aftertaste. The purpose of this review
properties, it has many therapeutic values: it is as is to summarize the existing literature for the improve-
antihyperglycaemic, anticancerous (Jeppensen et al. ment of stevia through conventional plant breeding and
2002, 2003), and antihypersensitive (Chan et al. 1998; selection and modern biotechnological approaches to
Jeppensen et al. 2002), it has contraceptive properties provide a baseline for further improvement.
(Melis 1999), and prevents dental caries (Fujita and
Edahira 1979). It can also inhibit bacterial and fungal ORIGIN AND ANTIQUITY
growth (Rojas and Miranda 2002). Commercial exploi- The genus Stevia Cav. consists of approximately
tation of stevia increased when Japanese researchers 150200 species of herbaceous, shrub and sub-shrub
developed a series of processes for the extraction and plants (Gentry 1996) and is one of the most distinctive
refinement of sweeteners from its leaves. Research has genera within the tribe Eupatorieae, mainly because
also made possible simpler, water-only extraction pro- of the morphological uniformity of its flowers and
cesses (similar to sugar processing) in place of the older capitula, which consist of five tubular flowers and
solvent extraction technology. The main producers of five involucral bracts (King and Robinson 1987); it
stevia are Japan, China, Taiwan, Thailand, Korea, is distributed from the southwestern United States
YADAV ET AL. * A REVIEW ON IMPROVEMENT OF STEVIA 3
southward through Mexico and Central America. It also a mixture of plants of both types. The cultivated
occurs from non-Amazonian South America, southward plants are reported to be more vigorous (Shock 1982;
to Central Argentina (King and Robinson 1987). In Frederico et al. 1996).
Brazil, 36 species have been found, distributed mainly in
southern and central regions (Frederico et al. 1996). Kingdom Plantae
Stevia rebaudiana originated in the highland regions of Subkingdom Tracheobionta
northeastern Paraguay (on the Brazilian border), be- Superdivision Spermatophyta
tween latitudes 238 and 248S, where the unique sweet- Division Magnoliophyta
ening power of its leaves and its medicinal properties Class Magnoliopsida
have been known by the local Guarani Indians many Subclass Asteridae
hundreds of years (Chan et al. 1998; Melis 1999; Group Monochlamydae
Jeppensen et al. 2002, 2003; Srimaroeng et al. 2005). Order Asterales
The Guarani Indians called the plant ‘‘kaa he-he’’, Family Asteraceae
which translates as ‘‘sweet herb’’, and used it as (Compositae

sweetener for their green herbal tea ‘‘mate’’, and as a formerly)
flavour enhancer (Soejarto et al. 1983). In the native Subfamily Asteroideae
state it grows on the edges of marshes or in grassland Tribe Eupatorieae
communities on soils with shallow water tables (Shock Genus Stevia
1982). It is indigenous to the Rio Monday Valley of the Species rebaudiana
Amambay moutain region at altitudes between 200 and
500 m. The climate can be considered as semi-humid
subtropical, with temperatures ranging from 6 to Some other related species of Stevia rebaudiana are
438C, with an average of 238C, and rainfall ranging Stevia eupatoria, Stevia lemmonii Lemmon’s stevia,
from 1500 to 1800 mm per annum. In 1943, the first Stevia micrantha annual stevia, Stevia ovata var.
seeds were exported to the United Kingdom where it texana roundleaf candyleaf, Stevia plummerae
could not be brought under cultivation. In 1968 it was Plummer’s stevia, Stevia plummerae var. alba, Stevia
exported to Japan, and from there awareness of and rhombifolia Kunth, Stevia salicifolia willow-leaf
cultivation of the plant spread throughout the world dtevia, Stevia serrata sawtooth stevia, Stevia viscida
(Lewis 1992). By now, the crop has been introduced to viscid stevia, Stevia commixta, Stevia satureiaefilia,
many countries, including Brazil, Korea, Mexico, the Stevia leptophylla, Stevia myriadenia, Stevia ophryphylla,
United States of America, Indonesia, Tanzania, Canada Stevia selloi, Stevia nepetifolia, Stevia oligophylla, Stevia
and India (Lee et al. 1979; Donalisio et al. 1982; Shock origanoides and Stevia triflora.
1982; Goenadi 1983; Saxena and Ming 1988; Brandle
and Rosa 1992; Fors 1995). BOTANICAL DESCRIPTION
CLASSIFICATION OF STEVIA Floral Biology
Stevia rebaudiana is one of the 950 genera of the Flower Structure
Asteraceae family (Soejarto et al. 1983; Lester 1999). A The inflorescence is loosely paniculate with the heads
systematic study of the North and Central American appearing opposite the bracts in irregular sympodial
species of Stevia was done by Grashoff (1972). Stevia cymes. They are arranged in indeterminate heads. The
consists of a group of annual and perennial herbs, flowers are small (1517 mm) and white (Marsolais et al.
subshurbs and shrubs that occur in mountain regions, 1998; Dwivedi 1999) with pale purple throat corollas.
open forests, borders of rivers and dry valleys (Robinson The tiny white florets are perfect (hermaphrodite)
1930). Its first botanical description is attributed to having both male and female organs, borne in small
M. S. Bertoni. The plant was first called Eupatorium corymbs of two to six florets (Goettemoeller and Ching
rebaudianum Bert. in honour of Rebaudi, the first 1999). The plant can initiate flowering after a minimum
chemist to study the chemical characteristics of the of four true leaves have formed. The plant takes more
substances extracted. Its name was later changed to the than a month to pass through the various developmental
current one, and it was recommended not only for food flower stages (Fig. 1) and produce all its flowers
production, but also for the medicinal effects that were (Taiariol 2004; sh et al. 2006).
attributed to it (Bertoni 1905). Although there are about
230 species in the genus, only S. rebaudiana gave the
sweetest essence (Soejarto et al. 1983), while other Anther, Pollen and Stigma
species contain other biochemicals of interest (Frederico Anthers are small, five in number. The pollen can
et al. 1996). It is a perennial herb with an extensive be highly allergic. Using the acetocarmine technique,
root system and brittle stems, producing small, ellipti- Monteiro (1980) observed that in some diploid indivi-
cal leaves. Under some environmental conditions duals of S. rebaudiana the pollen viability was 65%,
and management situations it behaves as an annual or which differs from the results of Oliveira et al. (2004)
Fig. 1. Different stages of flower opening.
with no viable pollen grains. Stigma is bi-lobed/ Zaidan et al. (1980) identified three photoperiod classes
bifurcated from the middle and style is surrounded by based on the daylength. According to Valio and Rocha
anthers (Fig. 2). (1977), a minimum of two inductive short-day cycles are
necessary for flowering induction. It can be induced to
Reproduction and Pollination Behaviour flower from the four-leaf-pairs stage onwards. Flower-
Stevia is self-incompatible (Miyagawa et al. 1986; ing is more precocious in the 8-h photoperiod, but
Chalapathi et al. 1997) and probably insect polli- plants remained vegetative under an 8-h photoperiod
nated (Oddone 1997). It has been reported that the with interrupted night (Valio and Rocha 1977).
amount of selfing ranged from 0 to 0.5%, while out-
crossing ranged from 0.7 to 68.7%, indicating that the Seed
self-incompatibility system is operating (Katayama et al. Seeds are contained in slender achenes about 3 mm in
1976; Maiti and Purohit 2008). Since stevia is self- length. Each achene has about 20 persistent pappus
incompatible, seed collected from an individual plant bristles (Goettemoeller and Ching 1999). Seeds have a
would represent a half-sib family. very small endosperm and are dispersed in the wind via
Grashoff (1974) and Monteiro (1980) reported hairy pappus. Shock (1982), Duke (1993), Carneiro et al.
agamospermy in certain genotypes of S. rebaudiana. (1997) and Lester (1999) reported a poor and highly
Sexual and apomictic plants of S. rebaudiana produce variable percentage of viable seeds. Fertile seeds are
normal and malformed pollen, respectively. According usually dark coloured, whereas infertile seeds are usually
to Monteiro (1980), the presence of apomixis in pale or clear (Fig. 3a, b) (Felippe 1978; Monteiro 1980;
S. rebaudiana shown by embryological studies may be Oddone 1997, 1999; Goettemoeller and Ching 1999).
related to specific physiological and/or ecological fac- Seeds are very small (1000 seeds weigh 0.31.0 g) and as
tors. Analysis of sporogenesis allows the detection of a result seedlings are slow to develop, reaching a size
irregularities that can lead to the formation of inviable suitable for transplanting to the field at 4560 d
gametes. (Colombus 1997; Brandle et al. 1998a). Goettemoeller
and Ching (1999) revealed that some active manipula-
Photoperiod and Flowering Time tion of the blossoms is necessary to achieve pollination.
Stevia is a short-day plant that flowers from January to Seed yields of up to 8.1 kg ha1 have been recorded, but
March in the southern hemisphere and from September it is common to achieve less than 50% germination.
to December in the northern hemisphere. Flowering Seed production in 1 ha would be enough for leaf
under short-day conditions should occur 54104 d production in 200 ha (Lester 1999). Agamospermy, i.e.,
following transplanting, depending on the daylength the asexual formation of seeds (Lumaret 1988), could
sensitivity of the cultivar. The variability for photoper- explain the reproductive capacity of this species, even if
iod sensitivity is large, ranging from 8 h to 14 h (Valio one takes into account the total lack of viable pollen
and Rocha 1977; Zaidan et al. 1980; Chalapathi 1997). observed.
a b c d
Fig. 2. (a) Stevia anthers, (b) germinated pollen with pollen tube, (c) stigma and (d) stigma coming out of anthers.
determinants for the rooting percentage and growth.

Cuttings with four pairs of leaves rooted poorly,
especially in February. Cuttings with two pairs of leaves
rooted best in February and those with three pairs of
leaves in April (Zubenko et al. 1991a; Ramesh et al.
2006). Cuttings taken in late winter rooted better than
those taken at other times (Carvalho and Zaidan 1995).
a b The sprouting percentage and shoot growth is also
affected by the length of the cuttings. The sprouting
percentage and shoot growth of sprouted cuttings were
Fig. 3. Stevia seeds (a) infertile seeds are usually pale or clear significantly higher with 15-cm-long cuttings compared
and (b) fertile seeds are dark coloured. with 7.5-cm-long cuttings (Chalapathi et al. 2001).
The superior performance of the 15-cm-long cuttings

Leaf may be because they have double the quantity of food
Stevia has an alternate leaf arrangement and herbacious reserves compared to 7.5-cm-long cuttings. Stolz (1968)
growth habit (Singh and Rao 2005). Leaves are small, indicated the necessity of higher quantities of starch,
sessile, lanceolate to oblanceolate, oblong, serate above carbohydrates, sugar and phenolic compounds for root
the middle and somewhat folded upwards. Trichomes initiation.
on the leaf surface are of two distinct sizes, large Rooting of cuttings can sometimes be stimulated by
(45 mm) and small (2.5 mm) (Shaffert and Chebotar the use of growth regulators (Zubenko et al. 1991b;
1994). 9 stevia, the leaf area index (LAI) 80 d after Carvalho and Zaidan 1995; Kornienko and Parfenov
sowing was 4.83 (Fronza and Folegatti 2003). Stevia 1996; Kornilova and Kalashnikova 1996; Bondarev
leaves vary widely in quality due to many environmental et al. 1998). Some growth regulators can sometimes
factors, including soil conditions, irrigation methods, influence (increase) the concentration of steviosides in
sunlight, air purity, farming practices, sanitation, pro- the leaves (Chen and Li 1993; Acuna et al. 1997).
cessing and storage. The leaf has a pleasantly sweet, Sprouting, however, was not influenced by NAA or
refereshing taste that can linger in the mouth for hours. IBA treatment at 500 ppm. IBA or NAA at a higher
The material contains the sweet components, sur- concentration of 1000 ppm, however, caused complete
rounded by the bitter components in the veins (Maiti inhibition of rooting and sprouting of cuttings, which
and Purohit 2008). may be due to the injury caused to the callus tissue. Shin
and Lee (1979) also reported that higher concentrations
PROPAGATION of IBA inhibited the rooting and sprouting of chry-
a) Vegetative Propagation santhemum cuttings due to the injury caused to the
tissues. Zubenko et al. (1991b) found better rooting
Shoot Cuttings and sprouting of stevia cuttings by prolonged dipping
Propagation of stevia is usually done by stem cuttings, of cuttings in 50 ppm IBA solution. Pre-treatment
which root easily, but require high labour inputs. Some of cuttings with paclobutrazol at 50 or 100 ppm was
plant varieties/selections produce virtually no viable found to be an effective growth regulator treatment
seed and vegetative propagation is the only way of for the induction of roots and sprouts from stem
multiplication. Shock (1982) opined that stem cuttings cuttings. Shoot vigour index is an important indication
were the prime means for the propagation of stevia. The of vigorous growth of sprouted cuttings. Paclobutrazol-
direct planting of stem cuttings in the field was found to induced sprouts were found to be more vigorous than
have a limited success (Chalapathi et al. 1999a). In IBA- or NAA-treated cuttings (Chalapathi et al. 2001).
general, a vigorous branch is cut at the base with a sharp
blade and planting in the field, keeping two to three
nodes above the soil. The cut portion of the branch is Micro-propagation
dipped in neem oil or any other fungicide (Maiti and Many different parts of the plant viz., leaves, auxiliary
Purohit 2008). Cuttings of new stems and shoots can be shoots, root-neck sprouts, shoot primordia, internodal
propagated successfully (Lee et al. 1979; Shock 1982; explants etc., can be used successfully for tissue culture
Gvasaliya et al. 1990; Nishiyama et al. 1991). Gvasaliya propagation (Handro et al. 1977; Yang and Chang 1979;
et al. (1990) reported that nearly 98100% rooting can Miyagawa et al. 1986; Ferreira and Handro 1987a;
be obtained, when the current year’s cuttings are taken Bespalhok-Filho et al. 1992, 1993; Swanson et al.
from the leaf axils. Further, the location on the plant 1992; Akita et al. 1994; Kornilova and Kalashnikova
from which cuttings are taken can also affect growth 1996; Constantinovici and Cachita 1997). In vitro
and rooting. Cuttings from the top part of the main multiplication has frequently been used to multiply
shoot with four internodes generally gave the best individually selected or bred clones and successful
results (Tirtoboma 1988). However, the pair of leaves procedures have been documented (Handro et al. 1977;
in the cuttings as well as the season also acts as Ferreira and Handro 1988; Kornilova and Kalashnikova
Shade can reduce total growth, delay flowering time and

reduce the rate of flowering (Slamet and Tahardi 1988).
Direct seeding to the field is not practiced, but may be a
requirement for large-scale commercial production. Lee
et al. (1979) found that plants from seeds were less
productive in the first year than those from cuttings.
SEED VIABILITY AND GERMINATION

Two types of seeds are found in stevia, black and tan-
coloured, with black seeds being heavier than tan seeds.
Goettemoeller and Ching (1999) investigated the low
seed germination of stevia and tested the viability of

seeds based on tetrazolium chloride, finding that the
Fig. 4. Micro-propagation of stevia from nodal explants. viability of black seeds was much higher than that of
tan-coloured seeds, i.e., 76.7 vs. 8.3%, respectively.
1996; Carneiro et al. 1997; Bondarev et al. 1998; Cross-pollination and self-pollination was accomplished
Nepovim 1998). Explants from leaf, nodal (Fig. 4) and by transferring pollen with a bumble bee (Bombus
inter-nodal segments were cultured on MS medium impatiens) thorax on the end of a toothpick. The
containing 2,4-D at 2.0, 3.0, 4.0 and 5.0 mg L 1 for influence of pollination treatments as well as the effect
callus induction. Inter-nodal segments initiated callus of light and darkness during germination were also
formation earlier than node and leaf. The greatest evaluated. Light increases the germination of black seed
amount of callus was produced in MS medium with 3.0 but not of tan seed. This suggests that tan seeds
mg L 1 2,4-D, whereas, MS medium with 5.0 mg L 1 represent inviable seed that are produced without
2,4-D gave the poorest callus (Salim-Uddin et al. 2006). fertilization.
The plant growth and stevioside content in the leaves of
The germination study of seeds obtained from five

the plants grown from stem cuttings or tissue culture pollination treatments: cross-pollination by bumble bees
were more uniform than the plant grown from seeds. The (78.3%); cross-pollination by hand (92.0%); cross-
number of roots, shoot biomass and stevioside content pollination by wind (68.3%); self-pollination by hand
were greater in the vegetatively grown plants (Truong (93.3%) and control (36.3%) suggested that incompat-
and Valicek 1999). ibility is not a factor in these clones. However, all
pollination treatments increase seed germination of
Seed Propagation black seed over the control, suggesting that some active
Reproduction in the wild is mainly by seed, manipulation of the blossoms is necessary to achieve
but germination and establishment from seed are pollination (Goettemoeller and Ching 1999).
often poor and sometimes unsuccessful (Shaffert and Germination rates of stevia seeds vary greatly. It can
Chebotar 1994). Seeds germinate within 710 d after take 46 d to reach two-thirds of the final germination
sowing. Propagation through seeds is not a common of 6290% at 258C (Shock 1982; Carneiro and Guedes
method of propagation owing to the problem of low 1992; Chen and Shu 1995; Takahashi et al. 1996).
seed production and poor germination capacity. Using Germination requires at least 208C and often more
seed to establish crops of stevia is more successful than 258C; light generally increases germination. In-
in tropical climates, where there is no climatic restriction creased temperature (408C on moist paper) for less than
on the length of the growing season. In northern
24 h can accelerate germination, but reduce total
climates the shorter growing season necessitates seedling
germination (Tanaka 1985). In Japan, seed remained
establishment in a glasshouse/greenhouse prior to the
growing season. Stevia flowers need to be fertilized viable for up to 3 yr when stored under low humidity
by pollen from another plant to produce viable seed. and in darkness (Kawatani et al. 1977). This contrasts
A high density of bees (three to four hives per hectare) is with claims of optimum storage at 08C still producing a
recommended for good seed production (Oddone 1999). 50% loss of viability after 3 yr (Brandle et al. 1998a). In
Harvesting of immature seed may also contribute to one experiment, seeds collected in the field and green-
poor germination (Colombus 1997). house were compared for germination. The greenhouse-
Seed production and fertility studies suggest that collected seeds had a 90% germination rate, whereas
high germination rates are possible from selected lines field collected seeds gave only 34% germination
(Carneiro and Guedes 1992). Timing of flowering, seed (Carneiro 1996). Total quantities of viable seed pro-
harvest and pollination methods play an important role duced in 1 yr are uncertain. Seed yield up to 8 kg ha1
in seed production (Melis and Sainati 1991; Strauss with 50% germination would be sufficient for 200 ha of
1995). Rain at flowering can also reduce seed setting. crop (Lester 1999).
GLYCOSIDES 1985). It is also non-calorific, non-fermentable and does

Eight diterpene glycosides with sweetening proper- not darken upon cooking (Crammer and Ikan 1986).
ties have been identified in leaf tissues of stevia. These There are reports of stevioside content (total glyco-
are synthesized, at least in the initial stages, using the sides) ranging between 4 and 20% on a dry weight
same pathway as gibberellic acid, an important plant basis, depending on the cultivar and growing condi-
hormone (Singh and Rao 2005). The four major sweet- tions (Kennely 2002; Starrat et al. 2002). The sweetening
eners are stevioside, rebaudioside-A, rebaudioside-C and potency (sucrose1) of stevioside, rebaudioside-A,
dulcoside-A. According to Kinghorn (1987) the sweet- rebaudioside-B, rebaudioside-C, rebaudioside-D,
ness of these compounds relative to sucrose are 210, 242, rebaudioside-E, dulcoside-A and steviolbioside are
30 and 30 times, respectively. The two main glycosides 250300, 350450, 300350, 50120, 200300, 520300,
are stevioside, traditionally 510% of the dry weight of 50120 and 100125, respectively (Crammer and Ikan
the leaves, and rebaudioside-A (Reb-A), 24%; these 1986). The essential oil composition of the aerial parts of
five different Stevia rebaudiana genotypes cultivated in
are the sweetest compounds. There are also other re-

lated compounds including minor glycosides, such as on the Tuscan coast (Italy) was examined by CC
rebaudioside-B, rebaudioside-C (12%), rebaudioside- and GC/MS. Forty different components were identified
D, rebaudioside-E, rebaudioside-F, dulcoside-A, and the main constituents in all studying samples
dulcoside-C and steviolbioside, as well as flavonoid were spathulenol (13.440.9%), caryophyllene oxide
glycosides, coumarins, cinnamic acids, phenylpropa- (1.318.7%), beta-caryophyllene (2.116.0%) and beta-
noids and some essential oils (Erik et al. 1956; Erich pinene (5.521.5%) (Cioni et al. 2006). In general,
et al. 1961; Harry et al. 1956; Hiroshi et al. 1976; Masur Paraguan leaves contain the highest concentration
et al. 1977; Yohei and Masataka 1978; Rajbhandari and (913%) of the sweet steviosides/rebaudiosides mole-
Roberts 1983; Makapugay et al. 1984; Crammer and cules, Chinese stevia contains only 56%, and Indian
Ikan 1986; Kinghorn 1987; Tsanava et al. 1989; Shaffert stevia is midway between these. Under Indian condi-
and Chebotar 1994; Putieva and Saatov 1997; Dzyuba tions stevioside concentration was about 9.08% of the
1998; Dacome et al. 2005; Sekaran et al. 2007). dry weight of leaves (Ashwini 1996; Chalapathi 1996).
Among the components of stevia, one, called The genotypes under study at the Institute of Himalayan
Bioresource Technology show considerable morpholo-

rebaudioside-A, is of particular interest because it has
gical variation. Stevioside content is higher in Accession
the most desirable flavour profile (DuBois 2000). Stevio-
1 compared with Accession 2 (Megeji et al. 2005).
side traditionally makes up the majority of the sweetener
(6070% of the total glycosides content) and is assessed
Biosynthesis of Steviol Glycosides
as being 110270 times sweeter than sugar. It is also
The steviol glycoside and gibberellin pathways diverge
responsible for the bitter aftertaste, sometimes reported
at kaurene. In stevia, kaurene is converted to steviol, the
as a ‘‘licorice’’ taste. As well as sweetness, stevioside may ‘‘backbone’’ of the sweet glycosides, then glucosylated
have a lingering effect or certain degree of pungency, or rhaminosylated to form the principle sweeteners. The
which is not appreciated by the majority of people, and precursor compounds are synthesized in the chloroplast,
which reduces its acceptability. Rebaudioside-A is and from there are transported to the endoplasmic
usually present as 3040% of total sweetener and has reticulum, Golgi apparatus and then vacuolated. The
the sweetest taste, assessed as 180400 times sweeter purpose of these compounds in the stevia plant is not yet
than sugar with no bitter aftertaste (licorice taste or clear, but their high concentration in the leaf and
lingering effect). the conservation of the pathway within the species
The ratio of rebaudioside-A to stevioside is the indicate that, at some point in evoluntionary time, their
accepted measure of sweetness quality; the more presence conferred significant advantage upon those
rebaudioside-A the better. If rebaudioside-A is present individuals that possessed them. Some researchers feel
in equal quantities to stevioside, it appears that the that they act to repel certain insects and others speculate
aftertaste is eliminated. The minor glycosides are that it is an elaborate means of controlling levels of
considered to be less sweet, 3080 times sweeter than gibberellic acid (Smith and Van-Stadin 1992).
sugar (Crammer and Ikan 1986; Brandle 1999; Oddone Expressed sequence tags (EST) are a powerful tool
1999). The sweetening effect of these compounds is that has emerged from genomics research. Expressed
purely taste; they are undigested and no part of the sequence tag collections can reveal gene expression
chemical is absorbed by the body. They are therefore of patterns, gene regulation and sequence diversity. Now,
no nutritional value (Hutapea 1997). The yield of that enriched libraries and efficient high-throughput
sweetening compounds in leaf tissue can vary according sequencing is widely available, ESTs have also become
to method of propagation (Tamura et al. 1984a), an effective means of gene discovery in focused meta-
daylength (Metivier and Viana 1979) and agronomic bolic situations (Sterky et al. 1998; Ohlrogge and
practices (Shock 1982). Unlike many low-calorie sweet- Benning 2000). This concept was first applied to the
eners, stevioside is stable at high temperatures (1008C) isolation of oleate hydroxylase from castor (Van de
and over a range of pH values (Kinghorn and Soejarto Loo et al. 1995). Since then, ESTs have been used to find
new genes from 1-deoxy-D-xylulose 5-phosphate (DXP) glucosylated, which forms stevioside (Shibata et al. 1991,
pathway of different crops. It is now clear that 1995). The pathway terminates with the glucosylation of
transcriptome analysis can be used to identify highly stevioside, which forms rebaudioside A. Rhamnosylated
expressed genes that are involved in many different glycosides can also be formed by the addition of a UDP
metabolic events (Brandle et al. 2002). Stevia rebaudiana rhamnose moiety to steviolmonoside (Richman et al.
leaves can accumulate high concentrations (up to 30%) 1999). The enzymes involved in steviol glucosylation
of seven different glycosides derived from the tetracyclic have been partially characterized and there is an under-
diterpene steviol (Brandle et al. 1998a). Their intense standing of the inheritance of certain glycoside patterns;
sweetness and close structural relationship to gibberellic however, corresponding genes have not been isolated
acid, coupled with the highly active nature of the (Shibata et al. 1991; Richman et al. 1999). In an effort to
pathway have fostered interest in steviol glycoside create a resource for gene discovery and to understand
biosynthesis and metabolism (Richman et al. 1999; the synthesis of steviol glycosides, Brandle et al. (2002)
Totté et al. 2000). In S. rebaudiana, these compounds sequenced 5548 random cDNAs from a S. rebaudiana
are synthesized exclusively in the mesophyll cells of leaf library. With electronic probes, database searches
leaves and are undetectable in the roots. Although the and differential representation, candidate genes for 70%
adaptive value of steviol glycosides is not known, it is of the steps in the steviol glycoside biosynthetic pathway
clear that S. rebaudiana has committed a very large have been searched.
portion of total metabolism to their synthesis, making The average GC content of the ESTs was 42.5%,
S. rebaudiana a good candidate for an EST-based gene similar to what has been found with Arabidopsis ESTs
discovery effort. Despite this amazing metabolic cap- (Asamizu et al. 2000). Of the 278 ESTs classified into the
ability, only a few genes involved in the biosynthesis of ‘‘secondary metabolism’’ category, 62 were candidates
steviol glycosides have been isolated and characterized for diterpene glycoside synthesis. No members of the
(Richman et al. 1999; Brandle et al. 2002). mevalonic acid pathway were identified among the
Recent experiments have shown that the early steps in S. rebaudiana leaf ESTs. This supports the work of
steviol biosynthesis involve the plastid localized DXP Totte et al. (2000), and further proves that the DXP
pathway and not the mevalonate pathway (Totté et al. pathway is used to synthesize IDP for conversion to
2000). Therefore, the first step in the steviol glycoside steviol. Candidates were identified for 70% of the
biosynthetic pathway is the formation of DXP from steps in the pathway from pyruvate and glyceraldehydes
pyruvate and glyceraldehyde 3-phosphate by thiamine 3-phosphate to rebaudioside A. The most highly repre-
phosphate-dependent DXP synthase (Lange et al. 1998; sented pathway EST was an orthologue of the Arabi-
Eisenreich et al. 2001). It has also been found that dopsis GA3 gene, which is known to be involved in the
dimethylallyl diphosphate (DMADP) is not necessarily three step oxidation of kaurene to kaurenoic acid
the committed precursor of isopentenyl diphosphate (Helliwell et al. 1999). The synthesis of kaurenoic acid
(IDP) and that IDP and DMADP may arise from is a key step in the synthesis of both steviol and
separate syntheses (Arigoni et al. 1999; Rodriguez- gibberellic acid (Brandle et al. 2002).
Concepción et al. 2000; Brandle et al. 2002). The ent-kaurene skeleton of chloroplast diterpene
Isopentenyl diphosphate and DMADP are con- glycosides, which are produced in large quantities in
verted to geranylgeranyl diphosphate (GGDP) by the leaves of Stevia rebaudiana, is formed via the recently
GGDP synthase via three successive condensation reac- discovered 2-C-methyl-D-erythritol 4-phosphate path-
tions (McGarvey and Croteau 1995). Like all diterpenes, way. The enzymes catalyzing the first two steps of
steviol is synthesized from GGDP, first by protonation- this pathway, 1-deoxy-D-xylulose-5-phosphate synthase
initiated cyclization to ()-copalyl diphosphate (CDP) (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoi-
by CDP synthase (Richman et al. 1999; Hedden and somerase (DXR), were characterized. The cDNA-
Phillips 2000). Next, ()-kaurene is produced from derived amino acid sequences for DXS and DXR contain
CDP by an ionization-dependent cyclization catalyzed 716 and 474 residues, encoding polypeptides of about
by ()-kaurene synthase (Richman et al. 1999). 76.6 and 51 kDa, respectively. DXS and DXR from
()-Kaurene is then oxidized at the C-19 position to Stevia both contain an N-terminal plastid targeting
()-kaurenoic acid, by a novel P450 mono-oxygenase sequence and show high homology to other known plant
(Helliwell et al. 1999). Steviol is produced by the DXS and DXR enzymes (Totté et al. 2003).
hydroxylation of ()-kaurenoic acid at the C-13 posi-
tion, but the gene for this FAD-dependent mono- Glycoside Content in Different Plant Parts
xygenase has not yet been isolated (Kim et al. 1996). Plant organs contain different amounts of the sweet
The two oxygenated functional groups of steviol, the glycosides, which decline in the following order: leaves,
C-19 carboxylate and the C-13 alcohol, provide attach- flowers, stem, seeds and roots. Roots are the only
ment points for the sugar side chains that determine organs that do not contain stevioside. The sweetness in
the identity of the different glycosides. The C-13 alcohol the leaves is two times higher than that in inflorescence
is successively glucosylated, first yielding steviolmono- (Dwivedi 1999). Sekaran et al. (2007) reported that
side then steviol-bioside, next the C-19 carboxylate is individual tissues of stevia appear to differ significantly,
with the stevioside content declining in a different order: Environmental Effect

leaves shoots roots flowers. The fact that the high- The growth and flowering of stevia are affected by
est stevioside content is found in the leaves suggests that radiation, daylength, temperature, soil moisture, and
they serve as the main tissue for both synthesis and wind (sh et al. 2006). Stevia is grown as a perennial
primary accumulation of stevioside compounds. The crop in subtropical regions, including parts of the
largest amount of stevioside was found in the upper United States, and as an annual crop in mid to high
young, actively growing shoot sections, whereas the latitude regions (Goettemoeller and Ching 1999). The
lowest senescent shoot sections exhibited the smallest results indicate that yield depends mainly on the
amount of such compounds. During ontogeny, a genetic characters of the plant, the phenotypic expres-
gradual increase in the stevioside concentration was sion of which is influenced by climatic and environ-
observed in both mature leaves and stems, and this mental factors (Metivier and Viana 1979; Ermakov and
process lasted to the budding phase at the onset of Kotechetov 1996). Moreover, synthesis of terpenes
is affected by climatic and environmental factors
flowering (Bondarev et al. 2003).

According to Bondarev et al. (2003), the lower, (Langston and Leopold 1954). Chen et al. (1978) studied
mature leaves of stevia have fewer glands per leaf the seasonal variation in stevioside content. Tateo et al.
surface area than the upper, younger leaves, i.e., there (1998) opined that environmental and agronomic factors
is a positive correlation between gland distribution have more influence on stevioside production. The ideal
density and steviol glycoside content. This argues in climate for stevia is a semi-humid subtropical with
favor of possible increased accumulation, by 30 to 170% temperatures ranging from 6.0 to 438C with an
of glycosides, in upper young leaves as compared with average of 238C (Brandle and Rosa 1992). Research
lower senescent ones. Depending on the clone, the conducted in Egypt revealed that climatic conditions,
portion of the rebaudioside-A in the total glycosides such as temperature, and length and intensity of photo-
content appeared to be increased as well. During period, greatly affect stevia production and quality, as
ontogeny, little variation in the glycoside content is evident from the remarkable increase in yield during the
also found in roots. In these organs, from the vegetative summer compared with winter (Allam et al. 2001).
phase to flowering, a gradual decrease in the glycoside Long-day conditions, as compared with short days,
increase internode length, leaf area, and dry weight, and

content was observed. During the fruit development
reduce the interval between the appearances of succes-
stage the levels of glycosides were found to revert to the
sive leaf pairs in S. rebaudiana. Total soluble leaf sugars,
initial level. However, in roots the total glycosides
protein, and stevioside content are also augmented in
content never exceed 0.1% (Bondarev et al. 2003).
both absolute and relative terms and the biosynthesis of
Kang and Lee (1981) demonstrated that the maximal
steviol, the aglucone present in stevioside, is increased
content of stevioside in leaves is achieved during the by 45%. The concentration of glycoside in the leaves of
formation of flower buds and it then gradually declines. stevia increases when the plants are grown under long
All this information may indicate that the steviol days. Since glycoside synthesis is reduced at or just
glycosides are transported to generative organs. Similar before flowering, delaying flowering with long days
results were obtained for ecdisteroids in Rhaponticum allows more time for glycoside accumulation (Metivier
carthamoides, Ajuga reptans and Serratula coronata and Viana 1979, 2005; Singh and Rao 2005). Sekaran
(Vereskovskii et al. 1983; Revina et al. 1986; Tomas et al. (2007) have shown a rebaudioside A/stevioside
et al. 1993; Anufrieva et al. 1998). ratio of 1.65 in ex vitro green leaves and 0.91 in in vitro
At the whole-plant level, steviol glycosides tend to shoots. Only severe Ca deficiency caused reduction in
accumulate in tissues as they age, so that older lower the glycoside concentration (DeLima et al. 1997). The
leaves contain more sweetener than younger upper chemical content of the last five fully expanded leaf pairs
leaves. Since, chloroplasts are important in precursor showed the plant nutritional status (Utumi et al. 1999).
synthesis, those tissues devoid of chlorolphyll, such as
roots and lower stems, contain no or trace amounts of CYTOLOGY
glycosides. Once flowering is initiated, glycoside con- The genus stevia shows great variation in chromosome
centrations in the leaves start declining (Singh and Rao number. The chromosome number of Stevia rebaudiana
2005). Stems of stevia plants contain little or no (2n 22), previously reported by Frederico et al. (1996)
sweeteners, although it is suggested that they may and Monteiro (1980, 1982), has been confirmed (Fig. 5)
contain some flavour enhancers, odourisers and other for various strains. However, strains with 2n 33
agents of potential use for improving foodstuffs or and 2n 44 (representing triploid and tetraploid cyto-
alcoholic beverages (Singh and Rao 2005). As stems types) also occur, which show a high degree of male
mature and lose colour, any steviosides present dis- sterility owing to the chromosomal abnormalities during
sipate. The structure, development and chemical content gamete formation. Cytological studies on many species
of stevia roots have also received attention, often of genus Stevia, performed by Grashoff et al. (1972),
associated with culturing procedures (Yamazaki and concluded that in North America, all the shrubby species
Flores 1989; Yamazaki et al. 1991; Zubenko et al. 1995). have a gametic chromosome number of n 12, while,
different positions in the karyotypes. The distinct

pattern strongly suggests the occurance of pericentric
inversions as the rule in the divergence of Brazilian
species of stevia. Most of the chromosomes were
metacentric, with a variable number of submedian
ones. Only S. ophryophylla and S. rebaudiana had a
pair with a subterminal centromere (Frederico et al.
1996). These results of the chromosome size and
centromere position were confirmed by Oliveira et al.
(2004). Chromosome lengths were similar in other
strains with 2n 22 or 2n44. The presence of a
nucleolus organizing region on the short arm of the
third major chromosome pair was also confirmed

for S. rebaudiana. In the Brazilian species of stevia,
the main mechanism of chromosomal evolution is
also by pericentric inversion. Thus, Frederico et al.
Fig. 5. Chromosome count of diploid stevia plant. (1996) concluded that, in addition to the numeric
changes, structural rearrangements have played an
important role in the chromosomal evolution of the
herbaceous species with flower heads in a lax paniculate tribe Eupatorieae.
cluster have n11. Further, although most reports Based on pollen viability, normal meiosis can be
indicate that n 11 (2n 22), values of 2n 24, 33, 34, inferred. Although pairing at diakinesis was normal,
44, 48, 66, 70 have also been observed (Darlington and with the formation of bivalents in all diploids, in none of
Wylie 1955; Bolkhoviskikh et al. 1969; Moore 1973, the S. rebaudiana strains studied was the pollen viable.
1974, 1977; Goldblatt 1981, 1984, 1985, 1988; Goldblatt High rates of tetrad normality (93%) were also
and Johnson 1990, 1991, 1996, 1998; Oliveira et al. 2004).
observed in the diploid strains. The lowest tetrad

Galiano (1987) considered Stevia as a multibasic genus,
normality rates were those of the tetraploid (80%) and
with x11, x 12 and x 17, while, Frederico et al.
triploid (64.5%) strains. Because of irregularities that
(1996) considered its basic chromosome number as
produce unbalanced gametes during meiosis, polyploids
x 11. Accordingly, this variation reflects numerical
with an odd number of chromosome sets have a high
(aneuploidy and polyploidy) and possibly structural
level of sterility (Lawrence 1980). The low tetrad
changes, mainly pericentric inversions (Frederico et al.
1996). Aneuploidy also occurs in Picris babylonica, a normality rates and the lack of viable pollen in the
plant that belongs to the same family as Stevia (Malallah triploid and tetraploid cytotypes may be related to the
et al. 2001). All South American species of stevia studied pairing of multivalents (tetravalents and trivalents) at
are diploid with the exception of hexaploid S. elatior diakinesis. Other meiotic abnormalities, such as irregu-
(2n66) from Colombia (Jansen et al. 1984). There is a lar chromosomal disjunction in anaphase, could also
predominance of the basic chromosome number x 11 explain these results. However, as with the diploid
among stevia species from South America, with only cytotypes, the lack of viable pollen cannot necessarily
three species (S. lucida from Colombia, one population be linked with regular pairing at diakinesis (bivalents)
of S. jujuyensis from Argentina, and S. organensis from and high rates of tetrad normality.
Brazil) having x 12 (Coleman 1968; Galiano 1987; Oliveira et al. (2004) studied pairing at diakinesis of
Galiano and Hunziker 1987). These three species may diploid, triploid and tetraploid cytotypes with 2n22,
have originated by ascending aneuploidy from species 2n 33 and 2n 44, respectively. For diploid strains,
with x 11. The main mechanism in the evolution of the pairing at diakinesis was n11II, which agrees with the
South American species of stevia is probably chromo- literature, While, pairing at diakinesis for the triploid
some inversions, with a small amount of aneuploidy and and tetraploid strains was n 11III and n 11IV, respec-
polyploidy. tively. This multivalent formation suggests that these
The chromosomal morphology of six Brazilian species strains may have an autopolyploid origin. Unlike
of Stevia, including S. rebaudiana, was studied by the above cytotypes, which occur spontaneously
Frederico et al. (1996). They observed that karyotypes in nature, the Stevia cytotypes analyzed here were
were very similar in chromosome number (2n 22) and obtained artificially by inducing polyploidy (Valois
size (1.02.4 mm). On the other hand, the comparative 1992). Multivalents at diakinesis would be expected in
analysis of arm ratios of each karyotype, revealed that autopolyploids, since the latter are derived from a single
every species has a difference in arm ratios for at least genome and result in three or four homologous sets of
one chromosome pair. Even in species with the same chromosomes in triploid and tetraploids, respectively
karyotypic formula, the sm pairs were located in (Stace 1980).
GENETIC INHERITANCE of those marker loci segregated in a 1:1 ratio, indicating

The magnitude of heritable variation is of utmost a high level of genetic diversity in stevia. Most of
importance since it has relevance to selection response. the RAPD markers segregated in normal Mendelian
Stevia produces steviol glycoside sweeteners in its leaves fashion. Similar findings have been demonstrated
that are up to 240 times sweeter than sugar. Under- in other cross-pollinated species (Grattapaglia and
standing the genetic basis of glycoside proportions will Sederoff 1994). This may enable stevia breeders to
aid in their manipulation through plant breeding. The conduct marker-assisted selection with genes that are
experiments conducted by Brandle (1999) were focused found closely linked to the markers in the map.
on the genetic control of the proportions of two of these In many linkage maps, loci are often found to be
glycosides, rebaudioside-A and rebaudioside-C. The concentrated in certain areas (hot spots) of a few linkage
study was conducted using F2 population from crosses groups (Paterson 1996; Keim et al. 1997). In compar-
between two sets of parents with divergent glycoside ison, the marker loci in the stevia linkage map were
profiles. Segregation in the first set of F2s showed that distributed more evenly and may better represent of the
the presence/absence of rebaudioside-A is controlled by whole genome (Yao et al. 1999). Genetic diversity
a single dominant gene, but that the actual proportions detected with RAPDs varies with species and crosses
of rebaudioside-A may be controlled by multiple loci or (Beaumont et al. 1996). It appears that the limited
alleles. In a second cross, proportions of rebaudioside-A breeding efforts undertaken to date have not signifi-
and rebaudioside-C were found to co-segregate and cantly reduced levels of genetic diversity among the
were shown to be controlled by a single additive gene. stevia breeding lines. This may be partly because stevia
This result suggests that both rebaudioside-A and -C are has not undergone a great deal of selection (Yao et al.
synthesized by the same enzyme. The results were used 1999).
to propose a model for glycosylation of steviol glyco- Construction of the stevia genetic linkage map has
sides (Brandle 1999). The presence of significant herit- laid a foundation on which to conduct marker-assisted
ability (h2) for three economically important characters, selection in stevia. The next step is to associate
i.e., leaf yield, leaf:stem ratio and stevioside (62.1, 78.8 genes involved with economically significant traits to
and 76.6, respectively) clearly suggested that genetic the RAPD markers and to convert those markers into
improvement of stevia is possible (Brandle and Rosa easily scorable PCR-based markers such as SCARs.
1992). These high heritabilities enable selection and Mapping newly cloned cDNAs from stevia genes
breeding programs, aimed at higher yield, to achieve involved in glycoside synthesis, such as copalyl dipho-
substantial gains. sphate synthase, can be addressed using cleaved ampli-
fied polymorphic sequences markers and will lead to a
Linkage Map better understanding of the genomic organization of
To lay a foundation for molecular breeding efforts, the secondary metabolism (Richman et al. 1998; Konieczny
first genetic linkage map for S. rebaudiana was con- and Ausubel 1993). Further, marker development (e.g.,
structed by Yao et al. (1999) based on RAPD markers. using AFLP or the unmapped RAPD markers segregat-
This information will be useful to those interested in ing 3:1) that will allow the resolution of the stevia map
developing marker-assisted selection procedures and into 11 linkage groups is also an important goal for the
quantitative trait analysis as well as provide a starting future.
point for those interested in genome organization in
stevia. Despite the fact that the Compositae is one of the Character Associations
largest and most diverse families of flowering plants, Information and understanding of the interrelationships
there has been little research involving molecular among characters are important to aid selection and set
markers, largely because the family possesses very few limits of each economic character that a breeder can
major crop species (Kesseli and Michelmore 1996). This choose without adversely affecting another important
work represents the first detailed genetic study ever character. Several authors have studied the dependence
conducted in this genus and one of only a few conducted of yield on various growth parameters as well as
among the members of the Compositae. Yao et al. stevioside content (Buana and Goenadi 1985; Shu and
(1999) constructed the genetic linkage map for Wang 1988; Buana 1989; Nishiyama et al. 1991; Brandle
S. rebaudiana using segregation data from a pseudo and Rosa, 1992; Shyu 1994; Chalapathi et al. 1998,
test-cross F1 population. A total of 183 randomly 1999b; Truong et al. 1999; Utumi et al. 1999). Some
amplified polymorphic DNA (RAPD) markers were interesting correlations have been found which can assist
analysed and assembled into 21 linkage groups covering selection programmes (Table 1). Plant height and leaf
a total distance of 1389 cM, with an average distance number at the second and fourth week after planting
between markers of 7.6 cM. The 11 largest linkage were positively correlated with biomass production in
groups consisted of 419 loci, ranging in length from 56 a greenhouse experiment conducted by Buana and
to 174 cM, and accounting for 75% of the total map Goenadi (1985). In another study, Buana (1989)
distance. Of the primers that showed amplification reported that plant height had no significant correlation
products, 35.5% detected polymorphic loci and 62.5% with production, leaf number, or branch number in the
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, Buana (1989)
branch number
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, Chalapathi et al. (1998)
leaves per plant and dry matter
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 fully ve Plant nutrient status Utumi et al. (1999)
expanded leaf pair
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 at Uncorrelated Stevioside content at maturity Weng et al. (1996)
seedling stage
Rebaudioside-A content ve Leaf area, net photosynthetic rate, Weng et al. (1996)
chlorophyll and protein content
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)
first 4 wk. A positive correlation between total soluble et al. 1991). Plant leaf yield is proportional to branch
carbohydrates and stevioside content was established by number, leaf number and (not always) plant height
Nishiyama et al. (1991). Stevioside content was uncor- (Buana and Goenadi 1985; Shu and Wang 1988; Buana
related with yield or leaf:stem ratio (Brandle and Rosa 1989). Total stevioside content is positively correlated
1992). Further, dry leaf yield was correlated with leaf with leaf:stem ratio (Tateo et al. 1998). Leaf thickness is
size and thickness and content of rebaudioside-A was positively correlated with rebaudioside-A:stevioside ra-
highly correlated with leaf thickness (Shyu 1994). tio (Shyu 1994). The total stevioside content of leaves at
The dry yield of stevia was positively correlated with the seedling stage and when mature is not correlated,
plant height, number of branches, leaves per plant and making plant selection at the seedling stage ineffective.
dry matter accumulation. About 96.88% of the total High rebaudioside-A content is also linked to large leaf
variation in dry leaf yield was explained by a linear area, high net photosynthetic rate, high chlorophyll
function of these four characters (Chalapathi et al. and protein content (Weng et al. 1996). Nakamura and
1998). Shu and Wang (1988) showed that leaf dry weight Tamura (1985) reported that the levels of dulcoside-
per plant had the greatest influence on yield. Stevioside A and stevioside and rebaudioside-A, and -C are
content is influenced by both leaf surface and number of positively correlated with each other, while stevioside
roots; however, the leaf surface has more influence on and rebaudioside-A, and dulcoside and rebaudioside-C
stevioside content than the number of roots (Truong are negatively correlated with each other.
et al. 1999). The chemical content of the last fully
expanded leaf pair was well correlated with plant PHENOTYPIC VARIABILITY
nutrient status (Utumi et al. 1999). In the wild populations of Stevia rebaudiana, there is
Furthermore, stevioside concentrations were uncorrelated great variation in phenotype and leaf analysis. The
with yield or leaf:stem ratio indicating that concurrent collections made as part of the various breeding and
improvement of agronomic and chemical characteristics selection research programs have invariably included a
is possible (Brandle and Rosa 1992). The observed range of genotypes and selections of plants with distinct
correlation suggests that the carbohydrate reserve in levels of steviosides in their leaves. Shock (1982) planted
the leaves of stevia is found mainly in the form of 200 lines for survival testing and screened 17 lines
diterpenic glycosides of the stevioside type. The para- for productivity. The stevioside content of leaves can
meters obtained from this correlation allowed the vary substantially (416%) between individual plants,
establishment of a simple method to determine the even after a selection program has been continued for
stevioside content in dry stevia leaves (Nishiyama some time (Bian 1981; Nakamura and Tamura 1985).
This natural variability could be partially due to the BREEDING OBJECTIVES

largely out-crossing nature of the species (Handro et al. Breeding programmes for stevia should be aimed at
1993). improving total glycoside content and rebaudioside-
Monteiro (1980) studied the phenotypic differences A:stevioside ratio with higher leaf yield. So far, plant
present in the population and was unable to separate breeding efforts with stevia have been largely focused
them into a valid taxonomic variety. There are also on improving leaf yield and rebaudioside-A concentra-
reports of irregular quantitative and qualitative production in the leaves. High leaf:stem ratios are desirable
tion of the sweetening molecules from cultivated in cultivated stevia because of the low stevioside
S. rebaudiana. The phenotypic variability within the concentrations (B5 mg g1) in stem tissue. Cultivar
population is linked to the open pollination behavior of descriptions indicate that sufficient genetic vari-
the species (Tateo et al. 1998). Nurhaimi and Toruan ability exists to make significant genetic gains in leaf
(1995) showed somaclonal variations in DNA finger- yield, rebaudioside-A content and the rebaudioside-
A:stevioside ratio (Lee et al. 1982; Morita 1987; Brandle
prints between six groups of plantlets. It has been

reported from China, that, in a sample of plants from and Rosa 1992; Shizhen 1995) (Table 3).
one clone, stevioside varied from 1.48 to 6.98% and The native rebaudioside-A:stevioside ratio in Stevia
rebaudioside-A from 4.5 to 12.1%, with total glycoside rebaudiana leaves is usually about 0.5 or less. The
varying from 10.26 to 19.57% (Huang et al. 1995). In predominance of stevioside gives a characteristic bitter
another report from China, total sweet glycoside con- aftertaste to the crude extract. Conversely, the most
centration in some lines has been reported to be as high valuable extracts are those that have rebaudioside-A as
as 20.5%, and in separate cultivar rebaudioside-A: the major component, because of its organoleptic and
stevioside ratios of 9:1 have been disclosed (Morita physicochemical features, i.e., it has the best profile
1987; Shizhen 1995). Such variability in the raw material relative to all other glycosides, and is more soluble in
would permit the use of conventional extraction meth- water (Ahmed and Dobberstein 1982; Crammer and
ods to produce a stevia sweetener with more than 85% Ikan 1986; Huang et al. 1995), allowing a greater
rebaudioside-A, without the need to recrystalize indivi- variety of formulations. Therefore, the development
and phytochemical characterization of new varieties of
dual glycosides. Much of the morphological variability
S. rebaudiana with higher levels of rebaudioside-A is a

has been observed in the population of S. rebaudiana
primary aim of plant breeders concerned with the
under study at the Institute of Himalayan Bioresource
improvement and utilisation of this source of natural
Technology, Palampur. It also exhibits considerable
sweeteners (Yao et al. 1999; Dacome et al. 2005; Sekaran
variability for stevioside content, which may vary from et al. 2007). With the high level of natural variability due
2 to 10% (Megeji et al. 2005). The genetic improvement to constant out-crossing, breeders are able to improve the
of stevia is only possible through the characterization of level of sweeteners in the leaves and alter the rebaudio-
the available variability at the morphological, chemical side-A:stevioside ratio (Shu 1989; Huang et al. 1995).
and biochemical, cytogenetic and molecular levels, in
order to utilize the information to develop an ideal plant
type. Stevia grown at the Delhi Research Station BREEDING METHODS
(Ontario, Canada) had 1.22 times as much leaf dry Germplasm Introduction, Collection
weight as stem dry weight, this same ratio was about and Conservation
0.67 in California (Brandle and Rosa 1992). Germplasm is a very important material for the im-
provement of crops. Introduction of germplasm from
one area to another continues to be an important activity
DISEASE RESISTANCE for breeding, particularly in developing countries. It is
Stevia is known to be free from attacks by insects, which generally used as source of superior genes and increasing
may be due to its inherent sweetness acting as a repellent. genetic diversity in the germplasm for breeding pro-
Therefore, insecticides are not required at an essential grammes. Introductions can be used directly as commer-
basis as in other crops, which helps in producing organic cial cultivars. Adapting exotic germplasm is, however, a
Stevia. The fungal diseases Septoria leaf spot (Septoria long-term programme. Intermating should be carried out
steviae), Alterneria leaf spot (Alternaria alternata), stem for several generations and selection pressure applied
rot (Sclerotium dephinii Welch.), root rot (Sclerotium gradually for desirable gene combinations.
rolfsii), powdery mildew (Erysiphe cichoracearum DC), Institutions around the world that have undertaken
damping-off (Rhizoctonia solani Kuehn.) and Sclerotinia research and/or appraisal studies on stevia have col-
sclerotoirum have been reported (Ishiba et al. 1982; lected seed and plant material from Paraguay in its wild,
Lovering and Reeleeder 1996; Chang et al. 1997; Thomas natural environment (Grashoff 1972; Bian 1981; Shock
2000; Megeji et al. 2005; Kamalakannan et al. 2007) 1982; Soejarto et al. 1983; Suhendi 1989; Tateo et al.
(Table 2). There is a need to develop and identify 1998). The rationale behind seed collection is to con-
resistant sources to develop varieties resistant to or serve genes and not genotypes, since, in stevia, due to
tolerant of these diseases. heterozygosity, no genotype is true breeding. Two
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 often
surrounded 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 and
dark 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 the
collar region takes place. The mycelial growth spread to the stem and roots, with associated tissue
rotting. Brown sclerotia appear on the diseased areas.
Stem rot Sclerotinia sclerotiorum Brown lesions on the stem, near the soil line are formed, followed by wilting and eventually by the
complete collapse of affected individuals.
genotypes of S. rebaudiana, Accessions I and II, which concentration in stevia leaves typically varies from 2 to
are morphologically diverse with respect to their growth 10% on a dry weight basis. Nearly three decades of
habit and sweetness are being maintained and multiplied breeding and selection have increased glycoside concen-
at the Institute of Himalayan Bioresource Techno- tration in stevia leaves by as much as 20% (Huang et al.
logy. Further selections for desirable plant types are 1995). However, this improvement was based on phe-
being performed in segregating progenies of individual notypic selection for total glycoside concentration in
selections. stevia leaves, which is heavily influenced by environ-
mental conditions, such as soil and weather. More
importantly, it requires selection relatively late in the
Selection
The success of stevia breeding depends on the choice of growing season. Selection at the early seedling stage is
parents, making crosses, raising adequate population least effective, because seedlings are so influenced by the
and further selections. In the wild, the total glycoside environment that only 2030% of the variability is
genetic, and measurements are based on expensive and

tedious high performance liquid chromatography
Table 3. Characters for improvement/utilization through breeding (HPLC) procedures (Brandle and Rosa 1992). As a
Sr. no. Characters Reference result, selection for plants producing high amounts of
glycoside is expensive, time consuming, and relatively
Steviol glycoside characters inefficient (Yao et al. 1999).
1 Total glycosides content Sys et al. (1998)
2 Rebaudioside-A content Marsolais et al. (1998) Countries that have been researching stevia for some
3 Rebaudioside-A: stevioside Brandle (2001), Morita and time, especially Japan, China, Korea, Taiwan and
ratio Yucheng (1998) Russia, have all reported success in their breeding/
Yield contributing characters selection programmes and have released new varieties
4 Leaf:stem ratio Brandle and Rosa (1992) with improved glycoside content and higher yields
5 Leaf size/area index
6 Leaf thickness (Table 4). Most breeding programs are based on cross
7 Number of leaves/plant or breeding and selection. Vegetative propagation and
branch cloning have frequently been used to multiply individu-
8 Number of branches/plant
9 Internode length
ally selected plants. Some of these selections, although
10 Stalk weight and thickness very high yielding, are self-incompatible and can only be
11 Leaf angle reproduced vegetatively (Lee et al. 1982). This limits
12 Photoperiod sensitivity/short Lester (1999), Valio and their commercial use, although they may be useful for
day plant Rocha (1977)
13 Plant vigour/growth rate Borie (2000), Andolfi
breeding new hybrids.
et al. (2002) Attempts made elsewhere in the world have resulted in
14 Seed germination percent Barathi (2003), Carneiro patents for the superior plant types (Table 5). A cultivar
et al. (1997), Duke (1993), with a rebaudioside-A:stevioside ratio of 0.96:1, com-
Shock (1982)
15 Seed viability for short Marcavillaca (1985)
pared with 0.36:1 in the starting material, was developed
duration with total glycosides of 22.4% (Lee et al. 1982). Other
16 Self-incompatibility Chalapathi et al. (1997) plants have been developed that exhibited rebaudioside-
17 Lodging susceptibility A:stevioside ratios as high as 9.1:1, but total steviol
18 Drought tolerance Jia (1984)
19 Sensitive to water logging glycosides were 10.1% (Morita 1987). Again, due to self
20 Asynchronous seed maturity incompatibility, the cultivar could not be reproduced
and seed dispersal using a seed-based production system. The costs asso-
21 Disease resistance ciated with clonal propagation limit the general applic-
22 Poor tolerant to high soil pH Shock (1982)
ability for large-scale production of stevia plants.
Table 4. Some varieties/cultivar selections and releases
Year Country Reference Variety Features
1979 Korea Lee et al. (1978) Suweon 2 High yield and steviosides
1982 Korea Lee et al. (1982) Suweon 11 Thick leaves, high Reb%
1989 China Shu (1989) Yunri, Yunbing
1995 China Shu (1995) Zongping Highest Rebaudioside and stevioside
1994 Taiwan Shyu (1994) K1, K2, K3. High yield, better Rebaudioside:stevioside ratio
1994 Indonesia Suhendi (1989) BPP72
1996 China Weng et al. (1996) SM4 High yield and Rebaudioside:stevioside ratio
1996 Russia Kornienko and Parfenov (1996) Ramonskaya Slastena
2000 India IHBT (CSIR) annual report Madhuguna High yield and total glycoside content
2000 India IHBT (CSIR) annual report Madhuguni High yield and total glycoside content
Development of Stevia Plant RSIT 94-1306 94-1306 and RSIT 94-751 were vegetatively propagated
and RSIT 94-751 by shoot-tip and stem cuttings.
Sys et al. (1998) and Marsolais et al. (1998) set out to
develop stevia plants with high concentrations of
Development of RSIT 95-166-13
individual steviol glycosides that could be extracted
Brandle et al. (1998b) developed RSIT 95-166-13 as a
and recombined in ratios suitable for specific product
unique combination of characteristics, which distin-
uses. Landrace stevia has a combination of steviol guished it from its parents and all other stevia varieties
glycosides that is not optimal for all product applica- for a high rebaudioside-C:stevioside ratio. Different
tions. In 1989, seed of a landrace variety from China was seed germplasm accessions of stevia from China were
introduced at Agriculture and Agri-Food Canada, Delhi grown for evaluation at the Delhi Research Station,
Research Station, Ontario, Canada. Superior plants Agriculture and Agri-Food Canada. Plant samples
were selected from this population and designated SR1 RSIT 94-1838, RSIT 94-1833, and RSIT 94-1560 were
to SR15 (breeding procedure shown in Fig. 6). These retained as a result of the higher than average concen-
plants were inter-crossed and half-sib seed was collected trations of rebaudioside-C in their leaves on a dry
from each parent plant. weight basis. A plant (RSIT 94-1829) selected from the
The half-sib families were evaluated in a genetic variety Brazil Zairai was also retained, because its leaves
heritability trial (Brandle and Rosa 1992). RSIT had higher than average concentrations of rebaudioside-
94-1306 was selected from the SR2 half-sib population C. Brazil Zairai is an open-pollinated landrace variety of
and RSIT 94-751 was selected from the SR13 half-sib stevia obtained from the Japanese National Germplasm
population on the basis of agronomic traits and steviol Depository. These four clones were used as parents and
glycoside profile, based on replicated trials. RSIT intercrossed. Half-sib seeds collected from clone RSIT
Table 5. List of patents
Title Country Inventer Year Patent no.
Stevia rebaudiana with altered steviol glycoside composition USA Brandle, J. 2001 6,255,557
Stevia rebaudiana with altered steviol glycoside composition USA Brandle, J. 1999 PCT-WO99/49724
Variety of Stevia rebaudiana Bertoni USA Morita, T. and Yucheng, B. 1998 6031157
Stevia plant named ‘RSIT 94-751 USA Marsolais, A. A.; Brandle, J. 1998 PP10,564
and Sys, E. A.
Stevia plant named ‘RSIT 94-1306 USA Sys, E. A.; Marsolais, A. A. 1998 PP10,562
and Brandle, J.
Stevia plant named ‘RSIT 95-166-13 USA Brandle, J.; Sys, E. A. and 1998 PP10,563
Marsolais, A. A.
Extraction of sweet compounds from Stevia rebaudiana Bertoni USA 1999 5,972,120
Method of cultivating hybrid new variety via systematically Wang, Q 2006 CN1985575
breeding stevia rebaudiana clone parental plant.
Japan Morita 1984a, b JP-59034848;
JP-59034826
Japan Morita 1985 JP-60160823
Japan Morita 1986 JP-61202667
Japan Nakazato, T 1987 JP-62096025
Japan Nakazato, T 1988 JP-63173531
Europe Stevia co. Inc. 1985 EPA0154235
New triploid of Stevia Rebaudiana Bertoni- contains Japan Sanyo Kokusaku Pulp Co. 1990 JP-2242622
sweet diterpenoid.
In 1989, seeds of landrace var. from china evaluated visually, and superior rows are identified. The
selected rows are harvested separately. Equal quantities
In summer 1990 1000 plants grown
of reserve seed from selected single plants, based on
15 Plants selected progeny performance, is composited. The first recurrent
selection cycle starts when the new composite is grown
SR1 - SR15 in an isolated plot where random mating among the
Crown dug-out & grown in green
plants is allowed. A bulk seed sample is harvested from
house in winter 1990-91 the remaining plants of each composite for use in
replicated yield trials to determine the response to
In summer 1991 Intercrossed & half-sib selection in each recurrent selection cycle for the
seeds collected characteristics under improvement. Recurrent selection
is continued until a reasonable response to selection is
achieved. Each cycle of recurrent selection produces a
new population that may be a potential cultivar. The

These half-sib families were
evaluated in genetic heritability trial
improved population with increased frequency of desir-
able alleles can be used as a cultivar per se or as a source
In summer 1994
to identify superior individual genotypes.
Development of Synthetics and Composites

SR 2 SR 13 There is a need to develop a stevia cultivar that is
enriched in rebaudioside-A and has a high steviol
glycoside content that can be produced using a relatively
low-cost method based on transplants produced from
seed. Therefore, a synthetic cultivar produced by inter-
RSIT 94-1306 RSIT 94-751 crossing clones or sibbed lines obtained from a breeding
Cuttings evaluated Cuttings evaluated
population during cycles of recurrent selection is re-
quired. In order to develop a synthetic variety more than

one line is required and the lines or clones are typically
Stevioside (%) = 17.25 Stevioside (%) = 4.88
tested for combining ability, preserved for future synth-
Reb-A = 0.0 Reb-A = 11.82 esis of the synthetic cultivar, as well as combined by
Total glycosides = 18.37 Total glycosides = 18.08 random crossing. Such synthetic cultivars are intended
for use in crop production systems. Because of the high
Fig. 6. Breeding procedure followed by Sys et al. (1998) and degree of natural out-crossing and the absence of an
Marsolais et al. (1998). efficient system of pollination control, composites and
synthetics are used to capture part of the available
94-1560 were designated half-sib population RSIT 95- heterosis. This is the most practical and effective
166. Plants from half-sib population RSIT 95-166 were breeding method, and there is a large effort aimed
evaluated in the field and one plant, RSIT 95-166-13, at establishing stevia as a crop in Japan as well as a
was selected on the basis of its novel steviol glycoside number of other countries based on developing synthetic
traits high in rebaudioside-C content. RSIT 95-16613 cultivar.
was vegetatively propagated at the Delhi Research
Station by shoot-tip and stem cuttings. Synthetic Cultivar AC Black Bird
Brandle (2001) developed the synthetic cultivar AC
Black Bird (breeding procedure shown in Fig. 7),
Population Improvement
which is characterized by exhibiting a high level of
Recurrent Selection total glycosides (at least 14%), and a high ratio of
Recurrent Selection is useful for improving quantita- rebaudioside-A to stevioside (at least 9.1:1). In order to
tively inherited characters in cross-pollinated species. In create parents for the synthetic cultivar, crosses were
S. rebaudiana, where self-incompatibility ensures a high made among a number of single plants and a large
degree of heterogeneity, recurrent selection is the most number of progeny were planted out to the field,
effective method of increasing foliage yield as well as and selections were made among those progeny.
total glycoside content. In population improvement, Leaves sampled from those selected plants were ana-
plant breeders aim to increase the frequency of desirable lyzed for glycoside concentration as well as composition,
alleles through the selection of superior recombinants. and selections that were high in glycosides, with
Recurrent selection typically starts with harvesting rebaudioside-A to stevioside ratios of at least about
individual open-pollinated plants from the source po- 9.3:1 (denoted as A to D), were inter-crossed in the
pulation. A portion of the seed of these plants is saved as greenhouse and seeds were collected from the maternal
a reserve. The agronomic characters of progeny rows are parents. At the same time, cuttings were taken from
In order to create parents for the synthetic cultivar, crosses were made among
a no. of single plants and a large no. of progeny planted out in field and
selections were made among those progeny
OxO OxO OxO OxO

Crossing among no.
OxO OxO OxO OxO of selected plants
Seeds collected
Large no. of progeny planted in field
IIIIIIIIIIIIIIIIIIIIIIIIIII 60 plants in each family

1……………………………………………...12
Selection among these families
20 plants from each family

selected for HPLC analysis
Selected high in glycosides

and reb-A/stevioside = 9.3:1
4 plants selected
(9.3:1) (A,B,C,D)
Isolated from field in green-house, trimmed
IIII inside 1.3x1m insect case screened with

10mm mesh. A hive of bumble bees was
used for inter-crossing
ABCD
Sexual inter-crossing
Cuttings to duplicate the
synthetic as required
AC Black bird
(Synthetic cultivar)
Parental clones, Selections, half-sib families of selections

and synthetic cultivar evaluated in replicated field trial
Stevioside (%) = 1.27

Reb-A = 12.49
Total = 15.06
Reb-A/Stev = 9.96
Fig. 7. Breeding procedure followed by Brandle (2001) for development of synthetic cultivar AC Black Bird.
the plants so that they could be used to duplicate Variety of Stevia ATCC Accession No. PTA-444
the synthetic as required. Seed from the maternal A variety of S. rebaudiana developed by Morita and
parents was retained as half-sib families and a portion Yucheng (1998), which contained 2.56 times or more
bulked to create a synthetic cultivar AC Blackbird. The rebaudioside-A than stevioside and is capable of being
half-sib families, the bulked sample and the parental cultivated by seed propagation, is produced from SF-6
clones were evaluated in a replicated field trial. The seed, having ATCC Accession No. PTA-444, or progeny
plants so obtained are characterized as exhibiting high thereof. Morita and Yucheng (1998) carried out stevia
levels of total steviol glycosides and being enriched in breeding by repetitive crossing and selection to obtain a
rebaudioside-A. It is preferred that the cultivar seed be variety with a high content ratio of rebaudioside-A to
produced from at least two intermating genotypes. stevioside. Hills (SF3 grade) containing 2.56 times or
more rebaudioside-A than stevioside were selected from

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

stable component proportion of rebaudioside-A.
Mutation Breeding
Mutagenesis is a potential tool to broaden variability
and to isolate desirable economic traits in a shorter
period compared with conventional breeding proce-
dures. The variability found in nature is due to the
accumulation of natural mutations that have occurred Fig. 8. Comparison for leaf size of (a) polyploid and (b) diploid
during the evolution of the plant. With the discovery of plant.
mutagenic agents, both physical and chemical, plant
breeders have the ability to induce variability and use it colchicine (an antimitotic agent) at concentration ran-
in their breeding programmes. Several mutagenic ging from 0.001% to 0.5% for 18 h. Polyploidization
agents, such as X-rays, g-rays, fast neutrons, thermal was confirmed in only two treatments, which corre-
neutrons and chemicals such as EMS, DES, MNUA, sponded to the lowest colchicine concentrations.
ENUA, MNU, ENU, can be used to produce useful Specifically, triploid plants of S. rebaudiana can
mutations. Irradiation with Cobalt-60 gamma rays has be produced by mating tetraploid female and diploid
been used to induce variation in breeding lines (Toruan- male parents. Triploid plants of S. rebaudiana contain a
Mathius et al. 1995). Characters of interest can only be large amount of rebaudioside-A, a very sweet diterpe-
improved through mutation breeding if the population noid (Sanyo 1990; Shuichi et al. 2001). The glycoside
shows less variability for the character concerned. quality of stevia is improved by using the polyploids.
Because leaves are the economically important part of Triploid and tetraploid plants had a lower tetrad
this crop, mutation breeding can play a very important normality rate than the diploids. All of the strains had
role in the improvement of stevia. Gamma irradiation of inviable pollen (Oliveira et al. 2004). Thus, the higher
the seeds of stevia does not affect germination, but at
the ploidy number, the greater the size of the pollen and
higher doses suppresses root development.
the stomata, and the lower their number per unit area.
The triploid strain produced the shortest plants and the
Polyploid Breeding
The induction of polyploidy to improve agronomic lowest number of inflorescences, whereas the tetraploid
yields is a process commonly used in plants of economic strain had the largest leaves. Analysis of variance
interest (Allard 1960) and has been applied to other revealed highly significant differences among the strains,
species, such as Nicandra physaloides (Gupta and Roy with a positive correlation between the level of ploidy
1986), coffee (Cruz et al. 1993), Clitoria ternatea and all of the morphological features examined (Oliveira
(Gandhi and Patil 1997) and orange (Romero-Aranda et al. 2004).
et al. 1997). Better adaptability of individuals and Breeding of triploid plants of stevia was conducted by
increased organ (Fig. 8) and cell sizes are usually Shuichi et al. (2001), and 42 triploid plants, grouped into
associated with polyploidy (Guerra 1988). Crossbreed- eight cultivars, were obtained. The chromosome number
ing between individuals with different numbers of of these plants, obtained by counting the chromosome
chromosomes usually leads to sterile progeny. Accord- number of root tip cells and by flow cytometry analyses,
ing to Allard (1960), in various agricultural plants with showed that the plants were triploid (2n33). In these
diploid and tetraploid individuals as parents, the triploid plants, the leaves, flowers and guard cells were
progeny are partially or completely sterile triploids larger than that in the plants classified as diploid. The
that do not bear seeds. leaf shape of the triploid plants was categorized as
Valois (1992) developed polyploid strains by soaking needle-like or wide needle-like. In thin-layer chromato-
the seeds of S. rebaudiana in nine treatments of graphy and HPLC analyses, seven kinds of triploid
plants, T1 to T7, obtained from crossing tetraploid growth, metabolism, etc., of dicotyledonous plants
SMX1W with a high rebaudioside-A content with seven (Yuang-ling and King-In 1978; Cheng et al. 1980;
kinds of diploid cultivars with a high rebaudioside-A Facciotti et al. 1985; Barwale et al. 1986; Christou
content, showed high rebaudioside-A contents with a et al. 1987; Fametaer et al. 1990; Dhir et al. 1991a, b;
ratio (rebaudioside-A:stevioside) of more than 0.63. On Ahsan et al. 2000).
the other hand, T-8, obtained from crossing tetraploid Plant regeneration from in vitro culture can be
KSW with a high stevioside content with diploid SMX6 achieved by embryogenesis or organogenesis. In stevia,
with a high RA content, showed high STV contents regeneration has been achieved by organogenesis from
(rebaudioside-A ratio 0.23). The seasonal changes in different explants, such as leaves (Yang and Chang
the total content of the sweeteners and the RA ratio of 1979; Ferreira and Handro 1987a, b), axillary shoots
the triploid plants showed that total content increased (Bespalhok-Filho et al. 1992), stem tips (Tamura et al.
until August, whereas the RA ratio remained constant 1984b), suspension cultures (Ferreira and Handro 1988)
(Shuichi et al. 2001). and anthers (Flachsland et al. 1996). Somatic embry-
ogenesis has previously been reported from leaves

Heterosis Breeding (Wada et al. 1981; Bespalhok-Filho et al. 1993) and
Hybrids offer an opportunity to mobilize greater genetic stems (Miyagawa et al. 1984; Bespalhok-Filho and
variation and heterotic response. Success in developing Hattori 1997). Somatic embryogenesis can also be
hybrids in any crop depends on the availability of obtained from floret explants of S. rebaudiana cultured
heterotic response for economic yield and economic on MS medium supplemented with 2,4-D (9.05 and
feasibility in terms of cost involved in seed production. 18.10 mM) and kinetin (0 to 9.29 mM). On 9.05 mM
Sun (2001) suggested a method for breeding hybridized 2,4-D supplemented medium maximum embryogenic
seed of S. rebaudiana that involves hybridizing between callus formation occurred in medium without kinetin.
female plants produced from cuttings and male plants On 18.10 mM 2,4-D supplemented medium the best
produced from seed and collecting seeds from plants treatment was 2.32 mM kinetin. Embryogenic callus
produced from cuttings. Its advantages are low cost, started at the base of the corolla and ovary (Bespalhok-
a high rebaudioside-A content and high output of Filho and Hattori 1997). The embryogenic callus is
stevioside. usually characterized by a light green or light yellow

Wang (2006) claims a method of cultivating a colour, compact structure and the presence of globular
new hybrid variety by systematically breeding a S. somatic embryos on its surface. The embryogenic callus
rebaudiana clone parental plant, which systematically formed first on the base of the ovary and/or corolla, and
breeds good single plants, selects and matches good then proliferated throughout the whole explant. Non-
combinations, propagates clone parental plants, hybri- embryogenic callus may also be present, characterized
dizes the producing seeds, and mixes the collected seeds. by a white colour and a hyperhydric appearance
Asexual propagation of the parental plant is adopted (Bespalhok-Filho and Hattori 1997). A synthetic auxin
to fix its good properties and make the parental plant such as 2,4-D or picloram is usually used for the
clone to process group hybridization, which has simple induction of somatic embryogenesis (Merkle et al.
programme and more rapid yielding efficiency than 1990). Auxin is needed to cause de-differentiation
traditional breeding method; the new variety of S. and to elicit totipotency (Terzi and Loschiavo 1990;
rebaudiana bred according to this method has strong Bespalhok-Filho and Hattori 1997).
resistance, high leaf output, and a high content of total
glycoside in the leaves. Anther Culture
Anther culture is usually used to obtain haploid plants
from which doubled haploids/homozygous plants can be
BIOTECHNOLOGICAL APPROACHES developed through colchicine treatment in a short time
Tissue Culture and also in crops where self-incompatibility is the
Seeds of stevia show a very low germination percentage limiting factor for the development of homozygous
(Felippe and Lucas 1971; Felippe et al. 1971; Monteiro plants or inbred lines. In this method, immature anthers
1980; Toffler and Orio 1981) and vegetative propagation from a differentiating population are grown or cultured
through cuttings is limited by the small number of on substrate. Anther culture is usually carried out at the
individuals (Sakaguchi and Kan 1982). Tissue culture is beginning of a breeding programme. Once the popula-
the only rapid process for the mass propagation of stevia, tions of plants homozygous for a particular trait are
and there have been a few reports of in vitro growth of available, new varieties are developed by hybridization
stevia (Miyagawa et al. 1986) and in vitro micropropa- and genetic studies can be carried out efficiently.
gation from shoot tips and leaves (Akita et al. 1994; Flachsland et al. (1996) regenerated plants from
Kornilova and Kalashnikova 1996; Constantinovici and anthers of S. rebaudiana cultured in vitro under
Cachita 1997; Sivaram and Mukundan 2003; Salim- defined conditions. Anthers (containing uninucleate
Uddin et al. 2006; Ahmed et al. 2007). Cell and tissue microspores) were induced to form callus when asepti-
culture techniques have been widely used to study the cally cultured on Murashige and Skoog liquid medium
supplemented with 0.1 to 1 mg L 1 BAP. Regeneration crop for the production of nonnutritive, nontoxic,
of shoots was readily achieved by transferring pieces of high-potency sweeteners. It has many other cura-
callus to fresh solid medium with the same composition. tive properties, such as the inhibition of bacterial
Shoots were induced to form roots upon transfer to and fungal growth, and it is an anti-cancerous, anti-
medium with 0.1 mg L1 NAA. Plantlets were success- hyperglycaemic, anti-hypersensitive agent, it prevents
fully potted, but cytological studies of root tips from dental caries and has contraceptive properties, as
regenerated plants revealed a normal diploid number of reported in the literature. In the recent past, research
chromosomes (2n22). The study implies that somatic has been conduced around the world on various aspects
cells of the anther wall respond to the high BAP of crop improvement, the development of new varieties,
concentration in the medium. propagation, seed production, cultivation, disease resis-
tance, improvement of glycosides quality and quantity.
Marker Assisted Selection The major problem of large-scale cultivation is
The development of molecular marker technology and the lack of quality planting material. Stevia is a self-
consequent identification of marker loci linked to incompatible plant, and seed-grown plants vary in their
important agronomic traits have created exciting new growth, quality and quantity of diterpene glycosides and
opportunities for plant breeders. Marker-assisted selec- desirable ratio of rebaudioside-A and stevioside, which
tion provides the potential for improving selection restrict its cultivation from seed. Generally, plants with
efficiency by allowing for earlier selection and reduced desirable characteristics are propagated by stem cuttings
plant population size (Staub et al. 1996). In the past and tissue culture practices, which limits the large-scale
decade the creation of genetic maps has been the production of planting material. The emphasis in future
foundation of this new plant breeding tool. One of the research should be on the development of new seed
priorities of plant genome mapping is the identification varieties with wider adaptability to different climatic
of genes associated with economically important traits conditions, better germination and viable seed produc-
and the use of this information for further improvement tion, better leaf:stem ratio and with a high content of
of crops. The value of molecular markers for the rebaudioside-A compared with other glycosides for
development of linkage maps and their use in the successful cropping and higher diterpene glycoside
analysis of economically important traits has been production.

amply demonstrated in both, field crops (Paterson
et al. 1991; Yu et al. 1991; Mackill et al. 1993; Paran Acuna, I., Nepovim, A. and Valicek, P. 1997. Micropropaga-
and Michelmore 1993) and forest trees (Groover et al. tion of plants of Stevia rebaudiana in vitrio and content of
1994; Bradshaw and Stettler 1995). Molecular linkage stevioside in leaves after application of growth regulators
maps have been constructed for most major crop plants under field conditions. Agric. Trop. Subtrop. 30: 5361.
(Staub et al. 1996; Paterson 1996); these maps provide a Ahmed, M. B., Salahin, M., Karim, R., Razvy, M. A., Hannan,
more direct method for the selection of desirable M. M., Sultana, R., Hossain, M. and Islam, R. 2007. An
qualitative and quantitative traits through their linkage efficient method for in vitro clonal propagation of a newly
to easily detectable genetic markers (Edwards et al. introduced sweetener plant (Stevia rebaudiana Bertoni) in
1987; Paran and Michelmore 1993; Mackill et al. 1993; Bangladesh. Am.-Euras. J. Sci. Res. 2: 121125.
Ahmed, M. S. and Dobberstein, R. H. 1982. Stevia rebaudiana
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types with a high content of rebaudioside-A with respect scale bioreactor. Plant Cell Rep. Jpn. 13: 180183.
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Stevia Rebaudiana: A Review On The Improvement of Stevia ( (Bertoni) )

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Stevia Rebaudiana: A Review On The Improvement of Stevia ( (Bertoni) )

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A review on the improvement of stevia

[Stevia rebaudiana (Bertoni)]

Abbreviations: BAP, benzylaminopurine; CDP, ( )-copalyl

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

Mots clés: Stevia, glycosides diterpēne, rēbaudioside A, sēlection

which translates as ‘‘sweet herb’’, and used it as (Compositae

Fig. 1. Different stages of ﬂower opening.

determinants for the rooting percentage and growth.

The superior performance of the 15-cm-long cuttings

Shade can reduce total growth, delay flowering time and

SEED VIABILITY AND GERMINATION

seed germination of stevia and tested the viability of

The germination study of seeds obtained from five

GLYCOSIDES 1985). It is also non-calorific, non-fermentable and does

are the sweetest compounds. There are also other re-

Bioresource Technology show considerable morpholo-

with the stevioside content declining in a different order: Environmental Effect

flowering (Bondarev et al. 2003).

increase internode length, leaf area, and dry weight, and

different positions in the karyotypes. The distinct

third major chromosome pair was also confirmed

observed in the diploid strains. The lowest tetrad

GENETIC INHERITANCE of those marker loci segregated in a 1:1 ratio, indicating

Table 1. List of some important correlations

Characters Correlation Characters References

This natural variability could be partially due to the BREEDING OBJECTIVES

prints between six groups of plantlets. It has been

S. rebaudiana with higher levels of rebaudioside-A is a

Table 2. Various diseases reported in stevia

Disease Causal organism Symptoms

genetic, and measurements are based on expensive and

Table 4. Some varieties/cultivar selections and releases

Year Country Reference Variety Features

Table 5. List of patents

Title Country Inventer Year Patent no.

new population that may be a potential cultivar. The

Development of Synthetics and Composites

quired. In order to develop a synthetic variety more than

OxO OxO OxO OxO

Large no. of progeny planted in field

IIIIIIIIIIIIIIIIIIIIIIIIIII 60 plants in each family

20 plants from each family

Selected high in glycosides

Isolated from field in green-house, trimmed

IIII inside 1.3x1m insect case screened with

Parental clones, Selections, half-sib families of selections

Stevioside (%) = 1.27

more rebaudioside-A than stevioside were selected from

or No. 109 (SF6-III) did not give hills containing a

ogenesis has previously been reported from leaves

stevioside. usually characterized by a light green or light yellow

analysis of economically important traits has been production.

39623966. Helment, V. 1961. Direct correlation of the diterpene alkaloids

for Stevia rebaudiana. Genome 42: 657661.

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