genetics and breeding of stevia short communication write up

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2. 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 .

3.  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.

4.  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

5.  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..

7. 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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8.  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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9. 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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10. 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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11. 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).
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Less frequency of flowering

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Productivity up
Cost of
production
down

13. 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).

14.  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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15.  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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16.  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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17.  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

18.  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..

20.  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.
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Selection cont..Selection cont..Selection cont..

21.  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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2. Hybridization/crossing

22.  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 CONT…Hybridization/crossing cont…

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HYBRIDIZATION CONT…HYBRIDIZATION CONT…

2. Hybridization/crossing

24. 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)

26.  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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27.  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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28.  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).
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29. 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
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30.  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.
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31.  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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32.  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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33.  (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
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35.  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 varieties
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36.  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

37.  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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38.  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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39.  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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40.  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

41.  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
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PROFUSE THANKS FOR YOUR
ATTENTION!!!