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GENETICS AND BREEDING OF STEVIA Introduction

Stevia (Stevia rebaudiana) is a medicinal plants belongs Asteraceae family and member of 154 in the genus stevia.

It originated in South America (native to brazil)(Alhady, 2011).

Commercially produced in Paraguay, Brazil, Japan, Korea, Thailand, China and South East Asia (Goyal et al, 2010). Distributed all most all over the world .

Branched bushy shrub and herbaceous perennial plant

Family:asteraceae Genus :stevia Species:stevia rebaudiana

It is diploid and has 11 chromosome pairs,

characteristic for most of the south american

members of the genus (akintunde and

mohamad, 2011).

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The leaves are intense green with lanceolate shape growing diametrically opposite on the stem.Reach up to 1m in height when cultivated.

is an obligate short-day plant with a critical day length of *13 h

(Mohamed et al. 2011; Ceunen et al. 2012 and

its flowering is induced when days become shorter. delayed flowering

with long days allows more time for glycoside accumulation. a long photoperiod stimulates leaf growth and steviol glycoside production.

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Its Pollination Behavior is self-

incompatible and probably insect

pollinated (Yadav,et al 2011

It has versatile medicinal uses without any side effects 100%

natural, 100% non-caloric, 100% sweet tasting plant that can be

safely used in herbal medicines; tonics for diabetic patients and

also in the daily usage products that focus the interest towards

Stevia in worldwide.

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stevioside is range between

100 to 300 times sweeter than

table sugar and the natural

form of stevia is approximately

20 to 30 times sweeter natural

sugar (Homayauni,2012).

INTRODUCTION CONT..

USES CONT..

Currently, stevia is used both directly and after processing as a sweetener for tea, chocolate, jam, cookies, ice cream, juice and other soft drinks and yoghurt as well for treating heartburn and other ailments.

Used around the world for its pleasant taste, as well as for its increasingly researched potential for inhibiting fat absorption and lowering blood pressure.

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Therefore, all over world there is increasing tendency towards consuming natural products and thus living a natural life.

It is attractive as a natural sweetener to diabetics and others on carbohydrate controlled diets.

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GENETICS AND BREEDING OF STEVIA

We need better Stevia plants now, which will produce more

leaves per plant, more glycosides in the leaves and should be

tough enough to fight all the diseases and environmental

stresses.

So, genetic improvement of the Stevia plants and development

of high yielding varieties is the topmost priority.

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GENETICS AND BREEDING OF STEVIA CONT..

Most of our aim to improve character are we are going

Higher leaf yield per unit area of farm

Longer and wider leaves

plants with higher number of shoots instead of long

slender plant with single shoot

Closely spaced stem internodes,

No apical dominance

Ability to grow vigorously even when they are planted at

a very close spacing between them

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GENETICS AND BREEDING OF STEVIA CONT..

Higher leaf to stem ratio 

Higher of specific glycosides And steviol glycoside content in

the leaves 

Rapid Growth Rate Better photosynthetic activity 

Resistance to pest and diseases 

Resistance to environmental stresses

  To develop the varieties with less sensitivity towards variation in

day length are less prone to flowering. This is a very important

desirable characteristic in stevia (Chatterjee, 2015).11

Less frequency of flowering

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Productivity up

Cost of production

down

1. Selection

Pure Line Selection

In nature, no two plants of same species are not necessarily

absolute replica of each other and is always there is some genetic

variation between all individuals.

These variations occur naturally due to small errors in copying all

the genetic information during reproduction.

That plant can be selected for further reproduction and cultivation.

This process is called pure line selection (Chatteere,2015). 

Pure line selection is only possible with plants which are

pollinated by themselves and each offspring is a replica of the

parent do not have any genetic variation.

Stevia is a cross pollinated plant – they generally cannot produce

seeds through self pollination and every stevia plant is different

from their parents and siblings (offspring having one a

brother/sister and both parents in common

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So, for stevia, Mass Selection method is adopted. Mass selection

could be called appropriately the granddaddy of all plant selection

methods, because farmers of many different cultures have used it

for centuries to improve many crops. Specifically, mass selection

is a breeding method where the decision to select a plant as a

parent of the next generation is based on the performance of that

plant

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In stevia, selection is an effective method of obtaining improved

varieties. For more than thirty years of breeding stevia, the steviol

glycoside levels in leaves has been raised from 2–10% to even

20% of dry mass (Yadav and Guleria, 2012)

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Selection of potential plants with desirable characteristics from a large

population is often a difficult task.

If our objective is selecting plants with higher steviol glycoside content or

higher rebaudioside

A content, we need to collect leaves from all plants, process them for analysis

and assay the steviol glycoside content in the leaves with high performance

liquid chromatography.

Moreover, for doing this analysis, the plants are to be grown to full maturity.

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Short-cut methods for selection of better plants from a large population

Likewise, for getting an accurate assessment of biomass production

potential, the plants are to be harvested at maturity and the leaf yield

is to be measured.

Thus, we need to wait for 3 – 4 months after transplantation, and

then we can collect the data.

 

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Selection cont..

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There are certain other easily identifiable characteristics

of Stevia plants, which are correlated with steviol glycoside

profile and ultimate biomass yield.

These characteristics can be spotted through visual

inspection quite early in the growth stage.

Through this process we can identify potential plants

quickly and select them as candidate for further study.

Selection cont..

This phenomenon of association of one character with another is

termed “Character Association”which is a very important

genetic tool.

There is a hardcore technical term for it – “Phenotypic Marker

Based Selection”.  “Phenotypic” characteristics of a plant is its

outward appearance or measurable attributes like plant height, leaf

yield, glycoside content – which is associated are associated with

some “Genotypes”, i.e. presence or absence of specific genes.20

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Selection cont..Selection cont..Selection cont..

Often two different mass selected lines can have two different

desirable characteristics.

These two desired characteristics can be combined together by

cross-breeding those two plants to develop an offspring which

exhibits both the characteristics simultaneously. This process is

called Hybridization.

Plant hybrids are created by cross breeding of plants of different species or genus.

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www.ju.edu.et2. Hybridization/crossing

Intentional, controlled production of hybrids allows for

introducing new genes into the gene pool, what contributes to

enriching the genetic diversity.

This phenomenon is positive for homogenous populations of

crops, where it can enhance heterosis.

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HYBRIDIZATION CONT… Hybridization/crossing cont…

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HYBRIDIZATION CONT…

Short cut method to hybridize stevia.

2. Hybridization/crossing

3.POLYPLOIDISATION

Polyploidisation is successfully used for improving yields of

many crops and plants are often characterized with higher

adaptability to environmental conditions and larger organs and

cells.

Triploidity in stevia is linked with higher content of a

rebaudioside and in larger leaves (Shuichi et al, 2001). Where

stevia tetraploids have bigger and thicker leaves, what can

potentially lead to increase in biomass yields.

All the poliploids also had nonfunctional pollen (Oliveira, et al,

2004).

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Triploid plants (3n) were produced by mating tetraployed plants

(4n) with normal diploid plants (2n).

The triploid plants showed higher Reb A content. The tetraploid

plants had larger leaves (Sanyo K, 1990; Shuichi et al, 2001)

The plants produced as a result of cross breeding of specimen

with different chromosome numbers are most often either

completely or partially sterile.

Stevia triploids are obtained by placing seeds in colchicine

solution or by cross breeding a tetraploid female plant with a

male diploid.

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Anther is the part of the flower which produces pollen. The

number of chromosomes in pollens of a plant is half of that in

normal (somatic) cells. Thus, in plant physiological terms, pollens

are “haploid”. Since pollens are produced in anther through a

specific type of cell division (meiosis), it is a rich source of

haploid cells (Chatterjee, 2015).

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This method is of great importance in stevia, since development

of a pure line in stevia is very problematic due to its “self-

incompatibility”.

Pollen from a stevia plant cannot be used to fertilize its own

ovule.

Thus, a pure-line cannot be started from a single plant. Anther

culture provides a method for starting a pure-line from a single

stevia plant (Garnighian,2012). 28

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Random mutagenesis In this method, chemical agents or radiation is used to randomly

alter the DNA in seeds of plants to produce “mutants”.

The “mutants” have slightly different genetic make-up than the

original plant and thus may express some desirable traits in

some cases. The chemical agents used for this “mutagenesis” are

often ethyl methanesulfonate (EMS) or nitroso guanidine

(NTG). Hard x-ray, gamma-ray and neutron beams 29

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Development in use of molecular markers and identification of

markers linked with specific traits creates new possibilities and ways

of plant breeding.

It allows for discovering desired traits earlier on in the plant

development by their link to easily detectable molecular marker.

This eliminates the need to produce mature plants and shortens

considerably the time needed for evaluation of the traits of a given

specimen. 30

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Moreover, it is possible to run the selection process on smaller populations. Genetic maps for stevia were created with the use of RAPD (Randomly Amplified Polymorphic DNA) technique in 1999(Sun, 2001).

Constructing genetic maps will enable to the molecular selection techniques in breeding of stevia based on genetic markers and will lay the grounds for studies on the stevia genome organization and metabolism

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This technique is clean and safe. The only disadvantage of this process is that serious damage can be happened to the cellular tissue. In this method, also known as the “Micro-Projectile Bombardment” or “Biolistic (Chaattree, 2015).

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(Mubarak,et al,2015)study Improving sweet leaf (Stevia

rebaudiana var. Bertoni) resistance to bialaphos herbicide via bar

gene transfer indicated the feasibility of transformation method

for S. Rebaudiana indicated this efficient transformation system

was used to transfer the bar gene, as a selectable marker, to

improve S. Rebaudiana resistance to herbicides. Resistance to

bialaphos herbicide was also clearly evidenced in transformed

plants in vitro 33

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Theoretically, in Stevia, if the genes responsible for production of ent

Kaurenoic acid is enhanced or “over expressed”, that may result into

high concentration of that ent Kaurenoic acid in the plant cells.

Then, if the genes responsible for production of Gibberellin is

“silenced” or blocked, it may result into higher availability of ent

Kaurenoic acid for conversion into steviol glycosides.

Thus, silencing of Gibberellin biosynthesis pathway may be a very

potential genetic engineering objective for development of improved

Stevia varieties35

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There are about 90 varieties of stevia rebaudiana

developed all around the world. Basically all these

varieties have been developed for different climate

requirements, many times these varieties perform

strangely in different climate conditions (Aleksandra et

al,2015)

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GENETICS AND BREEDING OF STEVIA

Stevia breeding lines RSIT 94-1306 and RSIT 751 with improved content of glycosides were obtained with controlled cross breeding and selection method described by Brandle and Rosa (1992) (Sun,2001).

Sys et al. (1998) and Marsolais et al. (1998) developed stevia plants with high concentrations of individual steviol glycosides that could be extracted and recombined in ratios suitable for specific product uses. They started with seeds of a “landrace” variety (variety grown in a particular location and adopted to the local environmental conditions) collected from China. Their methodology mostly involved mass selection. 37

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J. Brandle developed this caltivar in 2001. This caltivar is characterized by high level of total glycosides (at least 14%), and a high ratio of rebaudioside-A to stevioside (at least 9.1:1)

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Triployed plants (3n) were produced by mating tetraployed plants (4n) with normal diploid plants (2n). The triployed plants showed higher Reb A content. The tetraployed plants had larger leaves (Sanyo K, 1990; Shuichi et al, 2001).

Polyploid plants recorded higher numbers of secondary branches, more leaf thickness and area, delayed flowering and higher steviol glycoside content in leaf (Hegde et al, 2015)

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Khan et al (2016) has reported success in generating stevia mutants by EMS and Gamma-ray and they observed that some the mutant showed higher Reb-A and Stevioside content (200% increase). The mutant strains showed higher expression of some important enzymes (UDP Glucosyl transferase family) involved in the steviol glycoside biosynthesis pathway

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Achievements

Nader R. Abdelsalam et al.2016 study genetic improvement through selection of different stevia rebaudiana genotypes on nineteen stevia accessions were collected from healthy plants of nature population and selected randomly.

Reported high significant variations between all tested genotypes and there is high genetic diversity between these genotypes and this might be, due to the open pollination for these genotypes for several years and theses genotypes could used in the future in breeding program 41

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PROFUSE THANKS FOR YOUR

ATTENTION!!!