CHAPTER 1: Crop and Steviol Glycoside Improvement in Stevia by Breeding

Worldwide demand for both stevia leaves and purified steviol glycosides (SVglys) is steadily increasing,¹  and it is expected to increase further in the future, as metabolic disorders such as type-II diabetes and obesity are becoming ever more prevalent. Despite the stevia global market size being characterised by rapid progress, the agricultural production of this crop is still problematic and insufficient to meet the growing global demand. Stevia yields still remain low and unstable in many countries, where the crop is of recent domestication and/or performed by small- and medium-sized farmers, because of high input costs (in particular for planting and establishment), lack of suitable adapted and available varieties, limited expertise in the cultivation, poor disease control and lack of irrigation. To improve the competitiveness of stevia production it is important to produce improved, higher-performing and yielding crops, that are more resistant to drought, extreme weather conditions and a range of biotic stresses.

Therefore, genetic improvement with the development of varieties with higher leaf and SVgly yields and greater resistance to abiotic and biotic stresses, in comparison with the most and currently known cultivars, are important goals in stevia breeding.

The general objectives of stevia selection and breeding are: (1) to increase the leaf yield and its stability, (2) to ensure high quality value in terms of SVgly content and composition, and (3) to produce types with greater resistance and resilience against disturbance and stress, and targeted to specific growing conditions and farming needs.

The main desired traits, to reach through breeding program strategies in stevia, can be summarised as:

• high leaf yield per unit area

• high leaf-to-stem ratio

• rapid growth rate and regrowth capacity

• enhanced photosynthetic activity

• high SVgly content in the leaves

• high content of specific SVglys (e.g. rebaudioside A – Reb A)

• high adaptability to a wide range of pedoclimatic conditions

• resistance to environmental stresses, pest and diseases

• photoperiod insensitivity

• self-compatibility for viable seed production.

Different characteristics are associated with dry leaf yield such as leaf thickness, number of leaves per plant, leaf surface area, number of branches.^2–5  S. rebaudiana leaves are relatively small, simple, opposite, subsessile, with large variability both in shape and size, ranging from oblanceolate to lanceolate or ovate.⁶  It is possible to select high yielding varieties or hybrids characterised by longer and broader leaves, higher specific leaf area and thickness. The plants should have compact stature and green erect stems, upright and multiple branched. A reduced apical dominance with a higher number of branches, instead of a long slender plant with a single shoot, is preferred. Stem internodes should be closely spaced, to accommodate more leaves on the stems. Plants bearing upright leaves, instead of horizontal, have a better interception and utilisation of light, and are preferred in high plant density crop cultivation.

The stems of stevia plants do not contain appreciable quantities of SVglys, which are mainly accumulated in the leaves.^7,8  Hence, varieties that have a higher leaf-to-stem ratio are desirable because they yield higher quantities of SVglys per unit of harvested plant biomass.

Equally relevant, is a higher relative growth rate and a better capacity to regrow after each harvest, giving new leafy shoots rapidly, thus allowing multiple harvests per year. Other desirable agronomic characteristics include lodging resistance.

Dark green leaves, with a higher chlorophyll content and photosynthetic rate per unit leaf area, that show better efficiency in converting solar energy in leaf biomass and SVglys is another important characteristic⁹  that should be considered in breeding strategies.

Although originating from the Paraguayan highland of Amambay, between 22° and 25° S latitude and 55° to 56° W longitude,^10–15  stevia can be grown over a wide range of climatic conditions, from semi-humid, subtropical to temperate zones.¹⁶  Once established, it can tolerate mild frosts, but not long periods of hard frosts, which kill the roots of the plant. This lack of winter hardiness means that stevia can be grown as a perennial (three to five years) in temperate to warm climates but as an annual in colder regions.¹⁶ 

Despite its suitability for general cultivation over a wide range of environmental conditions, a wider adaptability with particular attention to cold and drought resistance, is still considered to be an important objective in stevia plant breeding, because it helps in stabilising the crop production over regions and seasons.

Stevia plants suffer from several abiotic factors such as drought, extreme temperatures like heat, cold and frost, wind, flooding, soil salinity, excessive light intensity and UV exposition. Stevia plants vary considerably in their tolerance to abiotic stresses. The development of drought-resistant cultivars of stevia through selection and breeding is of considerable economic value for increasing crop production in areas with low precipitation or without proper irrigation. The development of drought tolerance in plants is the result of the overall expression of many traits in a specific environment, including both drought escape (rapid development to complete a life cycle before drought) and drought avoidance (reducing water loss to prevent dehydration) strategies. Water stress reduces photosynthesis in the leaves of stevia due to stomata closure in the short-term, and due to photo-inhibition damage and to the inactivation of RuBisCO enzyme (ribulose-1,5-bisphosphate carboxylase/oxygenase) in the long-term.⁹  In this context, physiological and biochemical traits that contribute to enhance water use efficiency and photosynthesis at an early stage can be very useful. In semi-arid regions, resistance/tolerance to salinity is one of the major priorities to be addressed. Salinity reduces plant growth and yield, as well as SVgly content of the leaves. Since stevia shows variability in what concerns the sensitivity or tolerance to salt stress,^17–19  future research should focus on molecular, physiological and metabolic aspects of this stress tolerance to facilitate the development of cultivars with an inherent capacity to withstand salinity stress.

In temperate and cool regions, low temperature is the primary abiotic stress that limits crop productivity, since stevia is very sensitive to hard frosts especially in the first year of growth. The possibility to select superior overwintering varieties makes it possible to grow stevia as a perennial, also under mid- to high-latitude regions. This contributes to both enhance the productivity and to reduce cultivation costs for planting. Recent breeding programs have targeted the development of cultivars that can withstand multiple stresses by assembling series of genes from different parents into a single genotype by a phenomenon called gene pyramiding.

Crop plants are attacked by various diseases and insects, resulting in considerable yield losses. Genetic resistance is the cheapest and the best method for minimising such losses. Fortunately, there are few pests and diseases recorded in stevia, except for a limited number of fungal diseases. The fungal diseases Septoria leaf spot (Septoria steviae), Alternaria leaf spot (Alternaria alternata), stem rot (Sclerotium delphinii Welch.), root rot (Sclerotium rolfsii), powdery mildew (Erysiphe cichoracearum DC), damping-off (Rhizoctonia solani Kuehn.) and Sclerotinia sclerotiorum have been reported.^5,20–25  Septoria steviae and Sclerotinia sclerotiorum are the two most damaging and widespread fungi,^1,26  to be reported in stevia grown in Canada,²⁶  India,²⁷  and Italy.²⁸  There is a need to develop resistant or tolerant varieties through the use of resistant donor parents available in the gene pool.

The biological cycle of S. rebaudiana is photoperiod-dependent and latitude is the major factor affecting plant reproduction. Flowering in stevia has adverse effects on SVgly yield. In fact, the optimal time to harvest the leaves is at the onset of flowering, when the accumulation of SVglys reaches its peak.^16,29–31  When a stevia plant flowers, its SVgly content decreases. Stevia is a short-day plant with a critical day length for flowering of about 13 h.³²  Thus, the varieties with less sensitivity towards variation in day length are less prone to flowering, even when grown at medium–high latitudes. This is a very important desirable characteristic in stevia, therefore, selection may identify photo-insensitive varieties in the future.

Stevia is characterised by a complex mechanism of seed multiplication. The species is hermaphroditic, highly cross-pollinated, photoperiodically sensitive, and produces tiny flowers in small capitula with five white tubular flowers.^5,33  These flowers have sporophytic self-incompatibility.^33–35  Seed yield and poor germination ability is one of the major constraints caused by self-incompatibility.

S. rebaudiana produces two types of achenes, black and tan coloured.^35–38  The black type is produced by cross-pollination and is characterised by high-germination, while the tan achenes, which do not have the seed embryo, are originated from self-pollination.³⁵  Even if self-incompatibility is a genetic mechanism, which prevent and thus encourage allogamy, it can be problematic for breeding techniques to be employed for crop improvement. The possibility to select self-compatible lines enables more efficient breeding techniques, and to select cultivars to be easily reproduced by seed.

More than 100 compounds have been identified in S. rebaudiana, the best known of which are the SVglys, particularly stevioside (Stev) and Reb A, being the most abundant.³⁹  SVglys are tetracyclic ent-kaurene diterpenoids and share four steps of their biosynthesis with the gibberellin pathway.^40–42  The accumulation of SVglys occurs in active photosynthetic tissues (mainly in leaves). Therefore, quality is related to the improvement of SVgly content and of specific SVglys (namely Reb A, D and M) as a measure of taste. A higher content of antioxidant compounds is important, too.

The SVgly content in S. rebaudiana leaves varies according to genotype, phenological stage, and growth conditions.^5,26,43,44  The original wild varieties had a 2–3% total SVgly content of leaf dry mass.⁵  Breeding and selection have produced varieties with higher total Stev content up to 16–20% of leaf dry matter compared with the common lines of 8–10%.⁴⁵  The total SVgly content may be further improved up to 30%.⁵ 

Stevia accumulates more than 30 SVglys in varying concentrations. The best known include Stev, Reb A–G (Reb A–G), steviolmonoside, steviolbioside, rubusoside, dulcoside A (Dulc A).^46–48  The two major SVglys, Stev and Reb A, account for more than 90% of the total SVglys found in stevia leaves.^39,48,49  Stev is estimated to be 110–270 times sweeter than sucrose but has a well-known liquorice off-taste and lingering sweet aftertaste, which reduces its acceptability. Reb A is reported as being 140 to 400 times sweeter than sucrose and more pleasant-tasting than Stev.^50,51  The quality, as a measure of taste, is usually defined as the ratio of Reb A to Stev. If Reb A is present in equal quantities to Stev, it appears that the aftertaste is eliminated.⁵  Therefore, plants with a higher proportion of Reb A and less Stev are required to improve taste. Genotypes/lines descriptions indicate that sufficient genetic variability exists to make significant genetic gains in Reb A content and the RebA-to-Stev ratio.^43,52–55 

Morita and Yucheng⁵⁶  developed a variety of S. rebaudiana, by repetitive breeding and selection, which contained 2.56 times more Reb A than Stev. Nowadays, there are several varieties that can have Reb A content up to 80% of the total SVglys, with very little bitterness and aftertaste.^4,56  Other SVglys, besides Reb A, are of interest for their good taste. Among these, of particular interest are Reb D and Reb M (also known as Reb X), which have a more desirable taste profile than Reb A, even when present in tiny quantities (less than 0.5% by dry leaf weight).^57,58  It has been reported that Reb D, at the same concentration, has less astringency and bitterness taste perception attributes than Reb A, and behaves more like sucrose in terms of sweetness properties.⁵⁹  Therefore, there is great interest in increasing the Reb D concentration in stevia leaves, developing improved varieties. Stevia varieties containing high levels of Reb D have been recently developed, for example, the patented variety named “Stevia plants with an increased rebaudioside D content” (patent no. WO 2014146084 A1, Table 1.1).

In January 2016, Reb M was positively approved by EFSA's Panel on Food Additives and Nutrient Sources and has been included among the 10 SVglys approved for use in Europe. After this approval, high-purity Reb M is marketed for food and beverage applications.⁵⁸ 

Beyond SVglys, stevia leaves contain other important constituents, such as minerals, vitamins, phenolic compounds (mainly hydroxycinnamic acids and flavonoids), alkaloids, water soluble chlorophylls and xanthophylls, with potential beneficial effects on human health.^48,60–64  Flavonoids (flavonols and flavones) are particularly important for their high antioxidant capacity,^48,65  as well as phenolic acids, like chlorogenic acid.^48,66  Even if variation in this desirable character is often produced by differences in environmental and agronomic conditions, genetic variation also plays an important role in affecting the total amount and composition of flavonoids.

Caligari⁶⁷  stated “the breeder generally uses the natural variation that already exists within the species and he needs to measure any character in order to observe variation in its expression”. Often, this variation reflects not just variation produced by differences in the environment in which the plant grows, but also genetic variation, which is heritable. Sources of variation, in fact, include environmental variation, genetic variation and interaction of genetic and environmental variation. Generally, plant breeders must distinguish among these sources of variation for the character of interest in order to effectively select and transmit the desired character to subsequent generations. Consequently, in order to effectively manipulate quantitative and qualitative traits of interest in stevia, the breeders need to understand the nature and the extent of their genetic and environmental control. What the breeder really observes is the phenotype of the plant, but he needs to select the genotype.⁶⁷  The success of a breeding program in stevia depends on the selection of potential plants with desirable characteristics from the populations, and, therefore, on the ability to predict the genotypic value of the selected plants, starting from the phenotypic value or from some other selectable criterion.⁶⁸  The phenotype of a plant is modelled as a function of its genotype as modified by the environment.

Some characters are more responsible or sensitive to growing conditions than others. It is known that in stevia, SVglys accumulation and composition, widely vary according to phenological stage and growth conditions (photoperiod, irradiance, temperature, and available nutrients),^{8,9,16,26,65,69–77}  as well as according to agronomic practices adopted during cultivation (such as fertilisation, water availability and salinity, plant density, harvest time and frequency).^{9,18,19,31,44,78–84} 

The degree of sensitivity and the potential plant response to the environment is determined by the genetic composition of the individual plant or population of plants. So, genetic variability is essential in order to make progress in stevia cultivar improvement.

Today, there are about 90 varieties of S. rebaudiana developed throughout the world.^16,40,85  The most known and studied stevia varieties are Criolla and Morita II; the first seems to be the original stevia variety native to Paraguay, while the second variety has been selected for its high Reb A content. Other known and cultivated varieties are Eirete, a hybrid developed in Paraguay for intensive cultivation; Morita III, obtained from Morita II and characterised by low water requirements; and Katupyry, a recently selected variety in Paraguay for growing in arid soils, characterised by high sweetening power. Also, several new stevia varieties with improved desirable traits have been released, such as AKH L1; AKH L4; SW 201; SW 107, and others (see Table 1.1).

The genetic divergence among stevia genotypes plays an important role in the selection of parents having wider variability for different traits. Consequently, the study of phenotypic and genotypic diversity plays a key role in order to develop a plant breeding program for stevia.⁶  In stevia, considerable genetic and phenotypic variability has been observed, with particular regard to plant size, flowering period, leaf yield and SVgly content and composition.^5,20,86–89 

In Figure 1.1, an example of phenotypic variability between two stevia genotypes, belonging to the germplasm collection of the Department of Agriculture, Food and Environment of the University of Pisa, is reported. This natural variability could be partially due to the largely self-incompatible nature of the flowers, as reported by Handro et al.⁹⁰  and Midmore and Rank.⁹¹ 

As the varietal diversity in stevia is reportedly quite high, researchers have attempted to enhance their knowledge about this variability and to develop a breeding strategy, studying the genotypic and phenotypic variability of several genotypes under different environmental and agro-climatic conditions.

Since a better understanding of the genotypic control of SVgly composition could be of interest for plant breeding, initial investigations on the genotypic variability for SVgly composition within a population have established three clusters: (1) plants containing mainly glucose(glc)-type glycosides (Stev and Reb A); (2) plants containing mainly rhamnose(rhm)-type glycosides (Reb C, Dulc A) and (3) plants containing nearly equivalent quantities of glc- and rhm-type glycosides.⁹²  As a result of differential glycosylation, each SVgly has distinctive organoleptic and biological properties. It is known that a sugar unit or a carboxyl group in C19 position and a sugar or a hydroxyl group in C13 position, are essential for sweetness.⁹³  However, rhamnosylation decreases the organoleptic properties, and the resulting sweetness and the taste quality of the rhamnosylated SVglys (such as Dulc A and Reb C) is inferior to their glycosylated counterparts.⁹⁴ 

Brandle and Rosa⁴³  showed that economically important breeding traits of stevia are characterised with high variability within populations and high heritability. These traits include leaf yield (h² = 62.1), leaf-to-stem ratio (h² = 78.8) and content of SVglys (h² = 76.6). Due to high heritability they are susceptible to modification by selection.⁴³  Brandle⁴⁹  studied the genetic control of the proportions of Reb A and Reb C, suggesting that these two compounds, equally glycosylated, differing only in the nature of one sugar unit, are synthesised by the same enzyme. These findings have been recently confirmed by Barbet-Massin et al.⁵³  in a study aimed to evaluate the genotypic variability for SVgly content and composition in several stevia genotypes. In this two-year experiment, a set of 96 stevia genotypes was randomly chosen in a population of Criolla, reproduced by stem cuttings and then transplanted in open field conditions, at the INP-EI Purpan Nursery Station, Toulouse, France. Five of these 96 genotypes were also transplanted at the INP-EI Purpan Experimental Station, Seysses, France. The genotypes showed a high variability in SVgly content and composition, with higher Reb A than Stev content in some genotypes, whereas other genotypes had a very high proportion of minor SVglys. In addition, while SVgly content varied among the different environments here tested, SVgly composition remained stable, indicating a higher genotypic determinism for this trait than for total content. Lankes et al.⁹⁵  studied the performance of six stevia genotypes in temperate European climatic conditions, in comparison with Gawi, a genotype selected by the University of Bonn. The six genotypes were provided by the European Stevia Association (EUSTAS). The results showed that Gawi was well adapted to the environmental conditions of European temperate zones, but genotype C was able to outperform Gawi in terms of leaf yield and Reb A content. Anami et al.⁹⁶  evaluated 15 stevia clones (selected for their high concentration of total SVglys and considerable Reb A to Stev ratio) and characterised genetic divergence in order to select genitors for hybridisation in breeding programs. Clones were evaluated in field experiments in the North region of Paraná, adopting a randomised complete blocks scheme with three repetitions. These authors observed an ample genetic variability among the 15 stevia clones for plant height, fresh and dry matter production, number of branches per plant, SVgly concentration and Reb A/Stev ratio, and they concluded that the Reb A/Stev ratio and total SVgly concentration contributed the most for genetic divergence (79% and 12%, respectively). Four of the 15 clones showed high mean genetic divergence in relation to the whole genotypic pool studied. Therefore, the authors stated that hybridisations among these four clones could provide a generation of segregated populations with high genetic potential for superior individuals' selection.⁹⁶ 

For selection and breeding purposes in stevia, information about the interrelationships among characters are of crucial importance for the following reasons: (1) to have a good choice of characters for selection of desirable genotypes under planned breeding program for higher yield, yield components and qualitative traits; (2) to have information about the knowledge of nature and magnitude of variation existing in available breeding materials and (3) to know the association among characters. In stevia, high estimates of heritability, genotypic and phenotypic variability were recorded for several important characters. Several studies have been carried out in stevia in order to find character associations potentially useful in assisting selection programs and to demonstrate the dependence of yield on various growth parameters, as well as Stev content.^{2–4,43,97–99} 

First reports⁴³  demonstrated the presence of significant heritable variation for some important characters and underlined the possibility of the genetic improvement for this species. In particular, Brandle and Rosa,⁴³  studying a landrace stevia cultivar (imported from China and grown in Delhi Research Station, Ontario, Canada), found that there was high heritability for three economically important characters: leaf yield, leaf–stem ratio, and Stev concentration. Furthermore, the authors observed that Stev concentrations were uncorrelated to yield or leaf–stem ratio, indicating that concurrent improvement of agronomic and chemical characteristics was possible. Several authors^2–4  observed that, in stevia, plant leaf yield was proportional to branch number, leaf number and (not always) plant height. Tateo et al.¹⁰⁰  found that total Stev content was positively correlated with the leaf–stem ratio, while Shyu et al.⁹⁹  concluded that leaf thickness and Reb A/Stev ratio were positively correlated. High Reb A content was found to be also linked to large leaf area, high net photosynthetic rate, high chlorophyll and protein content.¹⁰¹  Nakamura and Tamura⁹²  found positive correlations between Dulc A and Stev, as well as between Reb A and C; on the contrary, negative correlations have been observed between Stev and Reb A and between Dulc A and Reb C.

The genus stevia shows great variation in chromosome number.^5,35  Although most reports indicate that n = 11 (2n = 22),^{86,89,102,103}  values of 2n = 24, 33, 34, 44, 48, 66, 70 have also been observed.^104–116  Among these, strains with 2n = 33 and 2n = 44 (representing triploid and tetraploid cytotypes) showed a high degree of male sterility owing to the chromosomal abnormalities during gamete formation.⁴  The first and extensive cytological study on many species of the genus stevia (belonging to the following Series: Corymbosae 16 species counted out of 38, Fruticosae 9/25; Podocephalae 5/16) was performed by Grashoff et al.¹¹⁷  These authors found that, in North America, the shrubby species presented a gametic chromosome number of n = 12; the herbaceous species with flower heads in a lax paniculate cluster had a number of n = 11 (with no aneuploidy), while species in compact corymbose cluster had 2n = 34 univalents with aneuploidy.¹¹⁸  This latter group of species can be considered a triploid derivative of n = 11,¹¹⁹  due to the widespread occurrence of apomixis among the North American species. For South American species, Grashoff et al.¹¹⁷  established that the species were diploid with the exception of hexaploid S. elatior (2n = 66) from Colombia.¹²⁰  The studies carried out on this topic indicate a clear predominance of the basic chromosome number x = 11 among stevia species from South America, with only three species (S. lucida from Colombia, one population of S. jujuyensis from Argentina, and S. organensis from Brazil) showing x = 12.^5,121–123  As reported by Frederico et al.,¹⁰²  these three species may have originated by ascending aneuploidy from species with x = 11. This implies that the main mechanism in the evolution of the South American species of stevia is probably chromosome inversions, with a small amount of aneuploidy and polyploidy.^5,102  Regarding to chromosomal morphology, Frederico et al.¹⁰²  studied the karyotypes of six stevia species from southern Brazil, utilising root tip metaphase. These authors found that all the species analysed were diploid, with a chromosome number of 2n = 22, with the basic chromosome number of x = 11. The chromosomes of the karyotypes were also similar in size (1.0–2.4 µm) and most of them were metacentric, with a variable number of submedian ones. Only S. ophryophylla and S. rebaudiana had a pair with a subterminal centromere.¹⁰²  Frederico et al.¹⁰²  suggested the occurrence of pericentric inversion as the rule in the divergence of Brazilian species of stevia. More recently, de Oliveira et al.⁸⁹  determined the chromosome number, the meiotic behaviour and morphological features, in diploid and polyploid cytotypes of S. rebaudiana. In this study, tetrad analysis and pollen viability as well as stomata size, pollen diameter, and some vegetative (leaf size and plant height) and reproductive (inflorescences per plant) features, were also assessed. These authors⁸⁹  confirmed for S. rebaudiana the chromosome number of 2n = 22 (n = 11), previously reported by Frederico et al.¹⁰²  and Monteiro,^86,103  with the exception of two strains, characterised 2n = 33 and 2n = 44, and representing triploid and tetraploid cytotypes, respectively. de Oliveira et al.⁸⁹  observed for S. rebaudiana the presence of a nucleolus organising region on the short arm of the third major chromosome pair, confirming previous reports.¹⁰² 

Plant breeders employ a variety of techniques to improve genetic composition of the crops and different breeding approaches are required for self-pollinating, cross-pollinating and clonally-propagated plants. In cross-pollinated species, like stevia, the selection of new cultivars involves the change of the gene frequency of desirable alleles within a population of mixed genotypes, trying to maintain a high degree of heterozygosity.⁶⁷ 

At the beginning, the development of new varieties of stevia with a higher content of Reb A and a reduced content of Stev was the primary aim of plant breeders.^{43,49,52–54,95,96,124–126}  At the same time, also the morphological characteristics, such as plant height, branches and leaf number, leaf area index have been taken into account, together with the photoperiodic needs of stevia for its vegetative development, in order to ensure optimal production of SVglys in the different latitudes. Other characteristics such as tolerance or resistance to disease and pests, or to drought or high soil pH values should be further considered.⁵ 

There are several methods for genetic improvements and the most breeding programs, for stevia, are based on conventional plant breeding approaches such as selection and intercrossing among various desirable genotypes, along with chemical profiling for improving quality traits. Since all traits of a plant are controlled by genes located on chromosomes, conventional plant breeding can be considered as the manipulation of the combination of chromosomes, through pure line selection, hybridisation and polyploidy (increased number of chromosome sets).

Mass selection is a very simple breeding scheme, that allows to create a new population by cross-pollinating two different existing open-pollinating populations.¹²⁷  A representative set of individuals from each population will be taken to be crossed. The seed that results from such a set of crosses is grown under field conditions over a number of seasons.¹²⁸ 

As reported by Yadav et al.,⁵  three decades of breeding and selection programs in stevia have allowed to increase SVgly concentration in the leaves by as much as 20%.⁴⁵  At the beginning, the countries that have been researching more in stevia variety improvement were Japan, China, Korea, and Taiwan, reporting success in their breeding/selection programs with the releases of new varieties with enhanced SVgly content and higher leaf yields.^5,129  Some examples of these varieties/cultivar selections and releases are:

• Sunweon 2, characterised by high leaf yield and Stev content¹³⁰ 

• Sunweon 11, with a high percentage of Reb A⁵² 

• K1, K2 and K3, characterised by high crop yield and favourable RebA–Stev ratio¹³¹ 

• Zongping, with increased Reb A and Stev content in the leaves¹³¹ 

• SM4, with high yield and RebA–Stev ratio.¹⁰¹ 

Some of these selections, although very high yielding, are self-incompatible and can only be reproduced vegetatively.⁵²  This limits their commercial use, although they may be useful for breeding new hybrids.

Patent applications for superior stevia plant types, in particular with improved sweetening power and taste, have been filed and registered (especially in USA and Japan) as results of selection, intercrossing and hybridisation.⁵  In the US and the EU, the number of patent applications regarding new stevia varieties and SVglys, strongly increased after the JECFA evaluation on the safety of SVglys in 2008. Over 1000 patent applications concerning stevia had been filed by the end of 2014. Based on Espacenet data, eight companies (PureCircle®, Pepsi Co, Coca-Cola®, DSM, Evolva, McNeil Nutritionals LLC, Suntory Holdings and Cargill®) have made the most patent applications focused on SVgly production methods.¹³²  With their patents, these companies are able to control the whole market for SVglys.¹³² 

Examples of patent applications (from 1998) for several new stevia varieties have been reported and are shown in Table 1.1.

The well-known stevia varieties obtained through selection and intercrossing are RSIT 94-1306,¹³³  RSIT 94-751,¹³⁴  and RSIT 95-166-13 ²⁶  (Table 1.1). The first two were obtained from seeds of a “landrace” variety, collected from China and are characterised by improved content of SVglys. RSIT 95-166-13 was developed as a unique combination of characteristics, which distinguished it from its parents and all other stevia varieties due to its high and unique Reb C–Stev ratio. Due to stevia self-incompatibility, these varieties cannot be reproduced using a seed-based production system, and they have to be vegetatively propagated by shoot-tip and stem cuttings.⁵ 

The selection was based on phenotypic traits, which depend very much on the breeding conditions (i.e. environmental conditions, such as soil and weather) and on the plant age.¹³⁵  It is estimated that, in the case of young seedlings, only 20–30% of the variability is genetically determined.^5,135  Therefore, selection should be made with mature plants, which means a longer time before obtaining the results.¹³⁵ 

In order to improve quantitatively inherited characteristics in stevia—a cross-pollinated species characterised by high differentiation of a given trait within the population—recurrent phenotypic selection tends to be more effective than mass selection.^54,127,135 

This technique systematically increases the frequency of favourable alleles and maintains the genetic variation within a population to permit continual progress from selection. Most methods of recurrent selection include three phases conducted in a repetitive manner: development of progenies, evaluation of progenies in replicated trials and recombination of the superior progenies based on the evaluation trials.¹³⁶ 

A synthetic cultivar refers to a cultivar produced by intercrossing clones or sibbed lines obtained from a breeding population during several cycles of recurrent selection. Caligari⁶⁷  stated that “a synthetic cultivar basically gives rise to the same end result as an open-pollinated cultivar, the main difference being that a synthetic cultivar is continually reproduced from specific parents, whereas if it is left to open-pollinate to produce over generations, it will change its genetic make-up as a population”. A synthetic cultivar is propagated for only a limited number of generations and then must be reconstituted from the parental stock.

In the case of stevia, the development of synthetic varieties appears to be the most suitable method for the development of a variety enriched in Reb A and having a high SVgly content.⁵  A synthetic variety, ‘AC Black Bird’, has been developed by Brandle.¹³⁷  This cultivar is characterised by a total SVgly content ranging from 14.0–18.8% with a high Reb A–Stev ratio of at least 9.1 : 1 and up to 11.0 : 1.¹³⁷  In order to achieve the parents for the synthetic cultivar, crosses were made among a high number of progeny plants, grown in the field, and the selection was made among these plants. Seeds were collected and a large number of progeny (12 families and 60 plants for each family) were obtained and planted in the field. Twenty plants from each family were selected by HPLC analysis for their SVgly content. Then, four of these plants were further selected on the basis of their SVgly content and composition (low Stev, high Reb A, and high Reb A-to-Stev ratio). These four selected plants were enclosed and cross pollinated by bees, and, at the end of the reproductive phase, their seeds were mixed together, and the new progeny obtained.

Another synthetic stevia cultivar was also developed by Morita and Yucheng,⁵⁶  the ATCC Accession No. PTA-444. These breeders, through repetitive crossing and selection, obtained a variety with at least 2.56 times more Reb A than Stev and capable of being cultivated by seed propagation, differently to “AC Black Bird” variety, that is self-sterile and can be only bred vegetatively.¹³⁵ 

Manipulation of ploidy is considered as a valuable tool in breeding programs of many plants,¹³⁸  in order to increase the organ size, and the adaptability of individuals and to improve the agronomic yield.^139,140  Polyploidy often generates variants that may possess useful characteristic by doubling the gene products.¹³⁸ 

First studies showed that triploid stevia plants (3n)—produced by mating tetraploid plants (4n) with normal diploid plants (2n)—were characterised by higher Reb A content and larger leaves than normal diploid plants.^141,142  However, Shaopan et al.,¹⁴³  Yadav et al.¹⁴⁰  and de Oliveira et al.⁸⁹  revealed that tetraploids in stevia determine larger leaf size, thickness and chlorophyll content with increased biomass in comparison with diploid strains. These findings underline that tetraploid stevia genotypes can represent important germplasm for further improvement in terms of biomass yield and SVgly content. Rameshsing et al.¹³⁹  developed different stevia mutants using colchicine at different concentrations and confirmed that colchicine represents an effective polyploidising agent in creating new stevia variants with improved biomass and SVglys. Hedge et al.¹⁴⁴  recorded higher numbers of secondary branches, greater leaf thickness and area, delayed flowering and higher SVgly content in polyploid stevia plants in comparison with normal diploids. In addition, these authors found that yield performances of all induced polyploids were better compare to diploids and, among the polyploids, mixoploids showed the best yielding ability. de Oliveira et al.⁸⁹  studied the pollen viability of triploid and tetraploid strains and observed that all polyploids had non-functional pollen. In particular, these authors observed that the higher the ploidy number, the greater the size of the pollen and the stomata and the lower their number per unit area. In addition, they found that the triploid strain produced the shortest plants and the lowest number of inflorescences, whereas the tetraploid strain had the largest leaves.⁸⁹ 

Anther culture, usually carried out at the beginning of breeding program, leads to haploid plants from which doubled haploids/homozygous plants can be developed by colchicine treatment in a short time.⁵  Through anther culture, by developing haploid progenies, dominant and recessive characters can be separated and varieties with novel characters can be developed. This method is relevant for stevia, since development of a pure line in stevia is very problematic due to its self-incompatibility. Through anther culture, pure-line from a single stevia plant can be obtained. Flachsland et al.¹⁴⁵  successfully carried out stevia plant regeneration from anthers, even if cytological studies of root tips from regenerated plants have been revealed a normal diploid number of chromosomes (2n = 22). Garnighian¹⁴⁶  developed a patent for the stevia variety “T60”, starting from anther culture of the Paraguayan variety “Criolla”, to produce a haploid line with enhanced SVgly content. This haploid line had undergone a colchicine treatment in order to obtain diploid fertile plants.

Marker-assisted selection (MAS) is a procedure that has been developed to avoid problems associated with phenotypic selection, replacing the selection of the phenotype by selection of genes, both directly and indirectly.¹⁴⁷  In fact, molecular markers are not influenced by the environment and are detected at any stage of plant development.¹⁴⁸  DNA markers are used to assess genetic diversity at various levels of taxon-species, inter and intra population and progeny. One of the most efficient molecular methods, in terms of ability to produce abundant polymorphic markers within a short time and at low cost, is the random amplified polymorphic DNA (RAPD) technique. Recently, RAPD has been used for estimation of genetic diversity in various plant species, collected from different geographical regions.¹⁴⁹  The first genetic linkage map for S. rebaudiana was constructed by Yao et al.¹⁵⁰  based on RAPD markers using segregation data from a pseudo test-cross F1 population. This information will be useful to those interested in developing marker-assisted selection procedures and quantitative trait analysis, as well as provide a starting point for those interested in genome organisation in stevia. Recently, Chester et al.¹⁵¹  evaluated the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India, using RAPD markers and high performance thin layer chromatography (HPTLC) analysis. Similarly, Thiyagarajan and Venkatachalam,¹⁴⁹  while investigating the genomic DNA polymorphism and phytochemical variation in stevia by RAPD-PCR and HPLC analysis, concluded that there was a strong correlation between the phytochemical variables and the DNA polymorphism data. The correlation between RAPD markers and chemotype highlights the potentiality of RAPD analysis as a reliable method for identification and authentication of high yield accessions of SVgly production.

With the aim to develop a breeding strategy to get cultivars adapted to organic production in South West of France, Hastoy et al.¹²⁵  evaluated the genetic variability using SSR (Simple Sequence Repeat) molecular markers in five genotypes from different origins, cultivated under greenhouse conditions, as well as well as in 33 additional genotypes whose leaves were harvested in the field. Molecular markers have been developed in order to classify the genetic diversity of the available stevia germplasm, selecting the SSR markers on the basis of the available sequences present in database. This preliminary study revealed a very great phenotypic and genetic variability in the tested genotypes. The authors concluded that the different genotypes could be classified through their genetic distance and the five SSR markers used in this experiment were sufficient to distinguish all the genotypes and allow partial classification of the genotypes into five different clusters.

New biotechnological tools, such as random mutagenesis, site direct mutagenesis, transgenic plants, Agrobacterium mediated gene transfer and silencing, can provide useful opportunities for improvement of desired traits in stevia. These techniques can bring innovations beyond the usual approaches, with consequent technical and scientific progress in plant breeding.

Induced mutation has been considered an important phenomenon which, when combined with in vitro studies, could act as a very handy tool for creating novel genetic variations with desirable traits.¹⁵²  In order to obtain this variance towards desirable genetic traits by means of DNA alteration, it is possible to use chemical (ethyl methanesulfonate, EMS; N-methyl-N′-nitro-N-nitrosoguanidine, NTG) or physical (gamma radiation, hard X-ray, neutron beam) agents. In any case, random mutagenesis by chemical or physical agents is a blind technique, and outcomes are often unpredictable. In fact, the induced mutation can deactivate or silence genes with some specific biological activity. The seeds of stevia exposed to gamma irradiation do not show changes in germination percentage, even if at very high doses root development can be suppressed.⁵  Toruan-Mathius et al.¹⁵³  have used Cobalt-60 γ-rays irradiation to induce variation in stevia breeding lines. The authors produced plants with the highest growth rate, normal morphology and highest Stev content by using gamma radiation at the dose of 1500 rad. Khan et al.¹⁵⁴  exposed stevia leaf explants to different doses of EMS and γ-ray. The results showed lower chlorophyll content, transpiration rate, CO₂ exchange rate and stomatal conductance in traded plants than in control (plants without any chemical or physical treatment). However, at harvesting time, phytochemical analysis showed that plants treated with γ-rays had double Reb A content with respect to the control plants, and the EMS-treated plants showed a 1.5- to 2.0-fold increase of both Stev and Reb A, in comparison with control plants. Khan et al.¹⁵⁴  found that SVgly enhancement was related to an increasing UGT74G1 (corresponding to Stev biosynthesis) and UGT76G1 (corresponding to Reb A biosynthesis) gene expression, in both EMS- and γ-ray-treated plants.

Site-directed mutagenesis is a molecular biological method and can be applied to any given plant for creating a specific mutation in a known sequence. This method makes largely use of the Expressed Sequence Tags (ESTs), which are contributing to the gene discovery in plant secondary metabolism, revealing gene expression patterns, gene regulation and sequence diversity.¹⁵⁵  Some studies were conducted about stevia ESTs, in order to collect a resource for gene discovery, understand and increase SVgly biosynthesis.^155–157  In particular, Brandle et al.¹⁵⁵  sequenced 5548 ESTs from a S. rebaudiana leaf cDNA library. In this study, ESTs involved in diterpene synthesis were identified underlining that the use of ESTs greatly facilitated the identification of candidate genes and increased the understanding of diterpene metabolism. Brandle et al.¹⁵⁵  grouped ESTs into functional categories and found that significant portions of the ESTs were specific for standard leaf metabolic pathways; energy and primary metabolism represented 17.6% and 13.1% of total transcripts, respectively. Regarding ‘secondary metabolism’, 278 ESTs were classified into this category and, among these ESTs, 62 were candidates for diterpene glycoside synthesis. Diterpene metabolism in stevia represented 1.1% of total transcripts. This study identified candidate genes for 70% of the known steps in the SVgly pathway, with kaurene oxidase the eighth most abundant EST in the collection.

Stevia is a cross-pollinated species that requires the presence of insect pollinators to produce viable seeds.^38,158  In order to obtain homogeneous population by selected plants, in-vitro propagation appears to be the best approach, even if it is not economically sustainable for large-scale production, because of its high costs and high labour inputs. Therefore, the possibility to have alternative methods for rapid production of homogeneous stevia populations, characterised by high and uniform yield of SVglys, is strongly needed. Therefore, transgenic engineering may represent an interesting resource. Despite the variety of genetic engineering techniques, few studies have been carried out on stevia. Mubarak et al.¹⁵⁹  produced transgenic stevia plants using biolistic gene gun protocol; carrying bar gene, as a selectable marker for herbicide bialaphos resistance¹⁶⁰  and GUS gene (Escherichia coli beta-glucuronidase gene), as a reporter marker for analysis of gene expression in transformed plants.¹⁶¹  The bar gene was originally cloned from Streptomyces hygroscopicus, an organism that produces the tripeptide bialaphos as a secondary metabolite. In the study of Mubarak et al.,¹⁵⁹  histochemical staining technique and PCR analysis were used to detect the presence of the GUS and bar genes in the putatively transformed tissues. The expression of GUS DNA, introduced into the plant cells, was monitored by using x-gluc (5-bromo-4-chloro-3-indolyl glucuronide) as a substrate for the GUS enzyme. The results demonstrated that the transformed S. rebaudiana explants were more resistant than non-transformed one, with 66.7% of transformed stevia plants able to survive at 3 mg L⁻¹ of bialaphos.

The ability of Agrobacterium tumefaciens to transfer DNA to plant cells has been utilised in plant genetic engineering. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell.¹⁶²  A. tumefaciens is capable to transfer a particular DNA segment (T-DNA) of the tumour-inducing (Ti) plasmid into the nucleus of infected cells, where it is subsequently stable integrated into the host genome and transcribed causing the crown gall disease.^163,164 

Khan et al.¹⁶⁵  reported Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation in S. rebaudiana. The development of somaclones via indirect regeneration was also evaluated to obtain varieties with improved traits, e.g. disease resistance, improved tolerance to climatic condition, and efficient yield of desirable phytomolecules.¹⁶⁵  In this experiment, transgenic plants were compared with four different somaclonal plants and mother plants through in vitro leaf culture. These authors expected that the random insertion of T-DNA into plant genome could affect the total SVgly content with the consequent possibility to develop an improved variety of stevia plants. The obtained results showed that inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 43% were monomorphic and 57% were polymorphic. Transgenic plant was found to be closer to mother plant, while, on the basis of steviol, Stev, and Reb A profile, somaclone S2 was found to be the best (with maximum Stev content) and maximum variability in ISSR profiling.

Gene silencing has always been a recommended tool to understand the functionality of genes.^166,167  RNA interference (RNAi) being the most commonly employed approach that involves preparation of inverted repeat construct of target gene and generation of transgenics.¹⁶⁷  Agrobacterium tumefaciens is being used as an efficient molecular vehicle to transform plants with silencing/overexpression constructs for transgenic development.¹⁶⁸  Guleria and Yadav¹⁶⁶  adopted a gene silencing approach in order to understand the genetic regulation of SVgly biosynthetic pathway in stevia plant, and its molecular mechanism and association with gibberellins. RNA interference (RNAi) was applied through AMTS. It is known that SVglys share common steps of their biosynthesis with the gibberellin pathway.^41,146  However, the genetic basis of SVgly biosynthesis and its relation with gibberellins is not well understood.¹⁶⁶  In the SVgly pathway, steps catalysed by enzymes encoded by SrKA13H, SrUGT85C2, SrUGT74G1 and SrUGT76G1 genes have been considered important for SVgly biosynthesis.¹⁶⁹  On the basis of these considerations, Guleria and Yadav¹⁶⁶  silenced SrKA13H and three SrUGTs (SrUGT85C2, SrUGT74G1 and SrUGT76G1) genes encoding ent-kaurenoic acid-13 hydroxylase and three UDP glycosyltransferases of SVgly biosynthesis pathway. The downregulation of the above-mentioned genes was found to negate the SVgly accumulation level. However, the total SVgly reduction was not more than 60% on AMTS of any of the four genes, suggesting the existence of alternate SVgly biosynthesis route. In particular, SrKA13H and SrUGT85C2 were identified as regulatory genes influencing carbon flux between SVgly and gibberellin biosynthesis. In fact, silencing of these two genes was found to block the metabolite flow of SVglys.

The worldwide demand for high potency and natural non-calorific sweeteners is expected to increase in the near future, which would necessitate new stevia cultivars with high quality yields of sweet compounds.

Great progress has been made so far in stevia breeding strategies. The data presented in this chapter clearly indicates the growing interest of scientists, researchers and plant breeders in S. rebaudiana genetic improvement through conventional and innovative biotechnological approaches. Stevia plant breeding will continue to be highly dependent on classical techniques but the adoption of new biotechnological approaches that will be used in parallel with the more classical ones will contribute to increase the efficiency and effectiveness of breeding efforts in this species.

However, despite the progress that has been made in stevia breeding strategies, some critical questions should be taken into consideration in the future research. First of all, a number of concerns have arisen due to the application of plant transformation technology with particular regards to new transgenic crops. Therefore, researchers and plant breeders should take into consideration these concerns and the regulations applied to plants obtained using recombinant DNA.

Another urgent question regards patents of new stevia varieties. Stevia varieties together with SVglys are the subject of intense patent activity. The International Union for the Protection of New Varieties of Plants (UPOV) database showed that there are some 40 applications worldwide for Plant Breeders Rights (31) or Plant Patents (9) concerning stevia.¹⁷⁰  Regarding patent applications for new stevia varieties, there is the issue with intellectual property of the indigenous Guaraní people in Paraguay and Brazil. In fact, since global demand for natural and sugar-free products is rapidly expanding, stevia plants are being grown and processed commercially in many countries outside Paraguay, especially in China. However, the Guaraní people's right to benefit from its use, as established under the Convention on Biological Diversity's Nagoya Protocol, is being ignored and is a clear case of biopiracy.¹³²