Stevia Global Business Scenario

Genetic Improvement of Stevia

Why we need genetic improvement in Stevia ?

Now, the profitability of the Stevia sector is mostly governed by the economic efficiency of its farming. To remain competitive in the Stevia market, we need to derive the maximum yield from our farming against reasonable crop management expenses. We now need more and more quantity of leaves from unit area of our Stevia farms and we need maximum amount of sweet glycosides in the leaves. Moreover, to reduce our input costs in farming, we need robust plants with resistance to diseases and environmental stresses.

 

It’s a real ropewalk now !

 

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.

Yeah……That’s pretty obvious, but what’s the scope of your “demystification” here?

At first glance, the matter of genetic improvement of Stevia may seem very high-tech and beyond comprehension of any grassroots level farmer. But, at least a basic idea about the developments achieved in this sector is required, to achieve sustainable economic rewards from Stevia farming.

At first, the farmer should understand what the basic requirements for producing salable leaves are and what yield he should target for the economic feasibility of the farming venture.

 

These information may enable the farmers to –

Specifically ask for varieties with particular genetic characteristics at the time of buying planting materials from vendors.

Secure official guarantee for the performance of the plants from the vendors with provision for recovery of damages from the vendors in case of underperformance of the variety in field.

Get acquainted with basic techniques and terminologies of plant genetic improvement process, so that they are not bamboozled by technical jargons hurled against them.

Make crucial technical contribution in the farm level genetic improvement program.

What the farmers need ?

We need Stevia varieties with the following characteristics –


Higher leaf yield per unit area of farm. 

 

That means –

  1. Longer and wider leaves

  2. Bushy plants with higher number of shoots instead of long slender plant with single shoot

  3. Closely spaced stem internodes, to accommodate more leaves on the stems

  4. No apical dominance – that means the bud at the top of the leading shoot of the plant should not be very dominant to make the plant tall and slender without many branches.

  5. Ability to grow vigorously even when they are planted at a very close spacing between them.

 

Higher leaf : stem ratio – The stems of stevia plants do not contain appreciable quantities of steviol glycosides. Hence, the varieties which has higher leaf : stem ratio ultimately yield higher quantities of glycoside per unit harvested plant biomass. 


Higher steviol glycoside content in the leaves - The original wild varieties often had 2 – 3 % total steviol glycoside in the leaves (% of dry matter basis), but, now, the leaf steviol glycoside content may reach up to 18 – 20% in some varieties.


Higher content of specific glycosides - As we all know, Reb A has been the most sought after glycoside. So, we need plant varieties which yield more Reb A and less of other glycosides. In the wild varieties, the Reb A content was sometimes as low as 10% of the total glycosides. Nowadays, there are several varieties which can have Reb A content up to 80% of the total glycosides.


Rapid Growth Rate – which represents the speed with which the plant can regenerate leafy shots after they are harvested. Varieties with higher growth rate can yield more number of harvests per year.


Better photosynthetic activity – which is the ability for efficiently converting solar energy into leaf biomass and sweet glycosides


Resistance to pest and diseases – so that we need to spend less money on pesticides and the crop is not affected by any pest and diseases.


Resistance to environmental stresses – so that the plant can tolerate the following conditions without significant adverse effect on the yield


a.    Drought
b.    Waterlogging
c.    Extremes of temperature
d.    Salinity, acidity and alkalinity of soil


Less frequency of flowering – Flowering in stevia always has adverse effect on steviol glycoside yield. Whenever stevia plant flowers, its steviol glycoside content goes down. So, frequent flowering is not desirable in Stevia. Flowering is generally triggered by specific day length (photoperiod). Thus, the varieties with less sensitivity towards variation in day length are less prone to flowering. This is a very important desirable characteristic in stevia.  

How the Genetic Improvement is done ?

There are several methods for genetic improvements. The most commonly adopted processes are described below.

Classical Plant breeding:

It involves identifying and selecting desirable characteristic in different individual plants of same species and combining these into one individual plant.

 

Since 1900, Mendel's laws of genetics provided the scientific basis for plant breeding. All the inherent characteristics of a plant are controlled by genes. Genes are located in coiled thread like structures in the nucleus of the cells – which are called chromosomes. Conventional plant breeding can be considered as the manipulation of the combination of chromosomes. In general, there are three main procedures to manipulate plant chromosome combination.

Selection

In nature, no two plants of same species are not necessarily absolute replica of each other. There is always some genetic variation between all individuals. These variations occur naturally due to small errors in copying all the genetic information during reproduction. This phenomena is called “natural mutation”. So, we can look for individual plants in wild or in farms which has the characteristics desired by us. That plant can be selected for further reproduction and cultivation. This process is called Pure Line Selection. Pure line selection is only possible with plants which are pollinated by themselves and each offspring is a replica of the parent. Thus, siblings reproduced by a specific parent do not have any genetic variation. 

 

Stevia is a cross pollinated plant – they generally cannot produce seeds through self pollination. So, every stevia plant is different from their parents and siblings. 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. 

 

Let me explain this with an example. Let us consider that our objective is to develop a variety with higher leaves production. The strategy may be as follows - 

Hybridization

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

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

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. This process is tedious, costly and time consuming. Moreover, for doing this analysis, the plants are to be grown to full maturity.

 

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.

 

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.

 

This phenomenon of association of one character with another is termed “Character Association”. It 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.

 

A partial list of different Character Association in Stevia is given below.    

Some Success Stories

Development of Stevia Plant RSIT 94-1306 and RSIT 94-751

 

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. Basics of their work is described below :

Marsolais,A. A.,Brandle,J. and Sys,E. A. 1998. Stevia plant named ‘RSIT 94-751’ United States Patent PP10564

Sys,E. A.,Marsolais,A. A. and Brandle,J. 1998. Stevia plant named ‘RSIT 94-1306’ United States Patent PP10562

Development of synthetic cultivar AC Black Bird

 

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). The selection process is described below.  

Brandle,J. 2001. Stevia rebaudiana with altered steviol glycoside composition. U.S. Patent no. US 6255557 B1.

Changing the number of chromosomes in plant cells
Polyploidy Induction

A plant cell typically has two copies of a set of chromosomes. Mathematically speaking, a plant cell should have 2n number of chromosomes when n is the number of chromosome in one set. In an offspring born through sexual reproduction, n number of chromosomes comes from each parent and the resultant number of chromosomes becomes 2n. Plants having 2n number of chromosomes are called “Diploid”. During production of male and female germ cells, a special type of cell division takes place, which halves the number of chromosomes in those cells. Thus, a plant having 2n number of chromosomes in its cells produces pollen cell and female germ cells with n number of chromosomes in each of them. The germ cells having n number of chromosomes are termed “Haploid”.

 

It is possible to produce progeny plants with 4n or more number of chromosomes through a chemical treatment of seeds or growing tissues (meristem) of plants. A chemical – colchicine, is typically used to induct polyploidy in plants. The plants having 4n number of chromosomes are called “Tetraploid”. Plants having more sets of chromosomes than that of normal plants are called “Polyploid”.

 

In stevia, induction of polyploidy is often an important tool in its genetic improvement. In some cases, the polyploid plants have higher growth vigour, larger and thicker leaves and greater biomass production potential. Sometimes, polyploidy in plants have negative outcomes also.  Polyploid plants with odd number of sets of chromosomes (n, 3n, 5n etc.) are generally sterile and can only be multiplied vegetatively (through cuttings and tissue culture).

 

In case of Stevia n = 11. Thus number of chromosomes in normal diploid variety is 2x11=22

A brief account of work done on polyploidy induction in Stevia

 

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)

 

Tetraploids in stevia had significantly increased leaf size, thickness and chlorophyll content and reduced internode length (Yadav et al, 2013)

 

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)

 

References : 

 

Sanyo Kokusaku. 1990. New triploid of Stevia Rebaudiana Bertoni contains sweet diterpenoid. Patent Number(s): JP2242622-A; JP2748141-B2.

 

Shuichi,H.,Tsuneo,Y. and Satoshi,F. 2001. Breeding of triploid plants of stevia (Stevia rebaudiana Bertoni) with high rebaudioside A content. Jpn. J. Trop. Agric. 45: 281289.

 

Yadav, AK; Singh, S; Yadav, SC; Dhyani, D; Bhardwaj, G; Sharma, A, Singh, B. 2013. Induction and morpho-chemical characterization of Stevia rebaudiana colchiploids: Indian Journal of Agricultural Sciences 83 (2): 159–65,

 

Hegde SN, Rameshsing CN, Vasundhara M. 2015. Characterization of Stevia rebaudiana Bertoni polyploids for growth and quality; International Journal of Phytomedicines and Related Industries, Volume 7, Issue  3 : 188-195

Anther Culture

Stevia Anthers

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.

 

Anthers with immature pollens can be made to grow in tissue culture medium. Often a mass of undifferentiated plant tissue, commonly termed as callus can be grown in this way. The cells of this tissue are haploid, i.e. they have n number of chromosomes when the cells of the original plants contain 2n number of chromosomes. Whole plants can be grown from the callus by specific hormone treatment. The regenerated plants are also haploid.  

 

As we know now, each cell in all plants contains at least two copies of the same chromosome. Both the copies of the same chromosome contain same genes, but, one copy may contain a stronger (dominant) gene and another copy may contain a weaker gene (recessive) for certain characteristics. In this case, the characteristics coded by the dominant gene are expressed in the plants. Let us consider an example. Let us consider for a certain plant species, shorter height and smaller leaves are the dominant character and taller height and broader leaves are recessive character. So, in a homologous pair, if one of the chromosomes harbors genes for shorter height and smaller leaves and the other has genes for taller height and broader leaves, the outward appearance of the plant will always be with shorter height and smaller leaves. This dominant character will never allow the recessive but desirable character to express.

                                                                                              

Now, if we can separate those two chromosome pairs in a haploid progeny, the progeny having the chromosome with the desirable recessive characters will be expressed since those characters won’t get suppressed by the dominant characters in the other member of the pair.

 

Thus, through anther culture, by developing haploid progenies, dominant and recessive characters can be separated and varieties with novel characters can be developed.

 

Though the haploid plants can be grown up to maturity in laboratory in tissue culture conditions, they are not always robust for growing in fields and they are sterile. These haploid plants can be converted into diploid again by colchicine treatment. The diploid plants thus produced (autodiploid) can reproduce sexually and are robust enough for pot and field culture.

 

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.

 

Flachsland et al. (1996) tried to regenerate whole plants from anthers of Stevia, but ultimately ended up with diploid plants. Which indicated the plants was originated from diploid somatic cells of the anthers.

 

I could find another reference of haploid stevia plant regeneration in an US patent. In that patent it was claimed that a haploid line is generated by culturing anther of Paraguayan “Criola” variety. It was also reported that diploid plants were generated from the haploid line with colchicine treatment and the plant showed enhanced steviol glycoside content. The claim for real haploid generation in that study could not be confirmed in absence of flow-cytometric data for counting the number of chromosomes in the progenies.

 

References:

 

Flachsland,E.,Mroginski,L. and Davina,J. 1996. Regeneration of plants from anthers of Stevia rebaudiana Bertoni (Compositae) cultivated in vitro. Biocell 20: 87_90.

 

Garnighian, GV, 2012 Stevia plant named ‘T60’ US patent PP22593 P3

New Biotechnological Tools

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 are often used as physical method for DNA alteration.

 

Radiation or chemical damage of the plant genes often deactivates or silences genes of some specific biological activity. It may also remove natural silencing of some dormant genes. Thus, plants with new character develop.

 

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.

 

Random mutagenesis is a blind technique and outcomes are most of the time unpredictable. In the experiment described above, though the mutants showed higher steviol glycoside profile but their photosynthetic efficiency was compromised. However we can interesting genetic variation in the mutant strains and the mutant strains can be used for breeding with other stable cultivars. 

 

References:

Khan, SA;  Rahman, L; Verma,R; Shankar, K (2016) Physical and chemical mutagenesis in Stevia rebaudiana: variant generation with higher UGT expression and glycoside profiles but with low photosynthetic capabilities.: Acta Physiologiae Plantaram, 38:4

Site Directed Mutagenesis

 

There are some biotechnological methods which can edit specific single bits of genetic information in the whole plant genome. With this technology, it is possible to silence or express specific genetic character in the plant. Sometimes a specific physiological function of a plant species is suppressed by a gene. If the gene is inactivated, the suppressed physiological function may get expressed. Sometime this technology is used to inactivate the genes responsible for unwanted physiological functions.

 

Here are several methods for site directed mutagenesis. Some of them are described below

 

Chemical agents: Some chemical agents alter specific bits of the genetic code. The chemical used for this process are often aminopurine, nitrosoguanidine and bisulfate. They replace single genetic code bits (neucleotide pairs) into nonsense bits within a specific gene and thus inactivate the gene by “corrupting” the code.

 

Specific Nuclease enzymes: These enzymes break down specific regions of the DNA. When the DNA repairs itself within the cell, some portion of DNA may get lost or some non-coding DNA fragment may get inserted into the broken site. This also corrupts the gene coded by the DNA sequence.

 

Short Fragments of DNA (oligoneucleotides): If some specifically engineered DNA fragment is introduced in a replicating plant cell, it may get entangled with plant cell genomic DNA during DNA copying and copying errors may result. Thus, it is also an effective tool for corrupting and silencing specific genetic code.

Transgenic Plants:

 

There are some biotechnological tools, by which, scientists can artificially insert some genes into a plant. The resultant plant is called a “Genetically Modified (GM)” or “Transgenic” plant. The inserted gene may come from another unrelated plant or from completely different organism like bacteria or animals.


Classical plant breeding is a method of combining desirable genes from different plants within the same species or closely related species. But, through creation of transgenic plants, desirable genes from widely different organisms can be combined together in a plant.     


There are two plausible methods for inserting foreign genes into a plant:

Gene gun method:


In this method, also known as the “Micro-Projectile Bombardment” or “Biolistic” method is most commonly used in many cereal species. In this method, DNA fragment, coding the desirable gene, is bound to the tiny particles of Gold or Tungsten, which is subsequently shot into plant tissue or single plant cells, under high pressure using a sophisticated equipment. The accelerated particles penetrate both into the cell wall and membranes. 


The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a Petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into a plant chromosome.


This technique is clean and safe. The only disadvantage of this process is that serious damage can be happened to the cellular tissue.

Agrobacterium Mediated Gene Transfer

 

Agrobacterium tumefaciens is a soil bacterium which infects a variety of plants and produces crown gall disease. This bacterium is used for genetic transformation of different agricultural crops. Agrobacteria carry some unique genes, which they insert into plant cell genome when they infect a plant. That unique genes get incorporated into the plant genome and instruct the plant cell to produce foods for the bacteria and coax them for grow in an uncontrolled fashion like cancer. Thus, the bacteria hijack the metabolic activities of the plant cell. The gene cluster of Agrobacteria, which is transferred to the plants, is called T- DNA. It is a cluster of 15 genes.

 

In Agrobacteria, the T DNA is carried in short circular pieces of DNA which are dispersed in the cell and it is not a part of their principal genome, i.e. the chromosomal DNA. These short circular pieces of microbial DNA are called “plasmids”. The specific plasmid, which carries the T-DNA is called Ti  plasmid. In that plasmid, the T DNA sequence is flanked by two boundaries (a unique sequence of DNA). All the genes within this boundary are transferred to the plant. There are some other genes in that Ti  plasmid, which helps in the bacterial infection process. Those auxiliary genes are called Vir-Genes or virulence genes.

 

Agrobacteria is used to transfer desirable genes to the plant and they are used as a carrier or “vector” of extraneous genes. Two transfer desirable genes to the plants the following method is adopted.

 

  1. The disease causing Ti  plasmids are taken out from the bacteria.

  2. The plasmids are engineered to remove the disease causing genes keeping their boundaries intact. Thus the Ti plasmid is “disarmed”.

  3. Desirable genes (specific DNA sequence) are inserted into the region within the boundaries.

  4. The engineered Ti  plasmids are inserted into the bacterial cells.

  5. Then the target plant is infected by the bacteria carrying the engineered Ti  plasmid.

  6. The bacteria insert the genes within the boundary to the plant genome.

 

In this process, desirable genes from unrelated organisms can be transferred to the plant cell. This is a simplified description of the process without going into the technical details.

Agrobacterium Mediated Transient Gene Silencing:

 

Gibberellic acid is a naturally occurring plant hormone. It helps in plant growth and development, flowering, seed germination and various other physiological functions. All the growing organs of a plant are sites of Gibberellin biosynthesis. In Stevia, steviol glycoside production also shares a part of the biosynthetic pathway of Gibberellin. The common part of the biosynthetic pathway produces the common precursor, ent Kaurenoic acid. Then the pathway bifurcates into two separate independent pathways.  Gibberellin is produced at the end of one process and steviol glycoside is produced at the end of the other process.

 

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.

 

Craig Mello and Andrew Fire were awarded Nobel Prize in 2006 for discovering a unique biological process in which small fragments of nucleic acid, Ribo-Nucleic Acid (RNA) suppresses gene expression. The small RNA molecules interfere with the Gene decoding process. A RNA molecule with a specific nucleic acid sequence blocks only a unique gene without disturbing others. This process is called RNA interference and has become later an important biotechnological tool for genetic manipulation. 

 

Guleria and Yadav tried Gene silencing in Gibberellic acid synthesis pathway through RNA interferences. They inserted two genes into Stevia leaf cells which encoded the production of interfering RNA for two genes of ent Kaurenoic acid to Steviol glycoside synthesis pathway. The used "agroinfiltration" technique to infect leaf cells with agrobacteria. The technique involded injecting genetically engineered bacterial suspension into leaf cells under high pressure. The experiment was successful in partially blocking the steviol biosynthesis pathway. Thus, the potentiality of RNA interference in manipulating the pathway was established.    

Reference

 
Guleria P1, Yadav SK. (2013) Agrobacterium mediated transient gene silencing (AMTS) in Stevia rebaudiana: insights into steviol glycoside biosynthesis pathway: PLoS One. 2013 Sep 4;8(9):e74731. doi: 10.1371/journal.pone.0074731. eCollection 2013.

This page has been prepared according to the write up kindly provided by Dr. S. N. Chatterjee. Dr. Chatterjee is an eminent Geneticist and Molecular Biologist and has numerous original research publications to his credit. He was retired as Joint Director of Seribiotech Research Laboratory, Central Silk Board (Ministry of Textiles, Govt. of India).