Lecture 3 - Plant (Risha)

7/23/2019 Lecture 3 - Plant (Risha)

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Plant and Mammalian Cell

Technology

(BSB 3163)

Part 2: Plant Cell Technology

Topic 3: Applications to Plant Breeding ~

Somaclonal Variation

Sept - Dec 2011


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3.0 Somaclonal Variation

Somaclonal variation is a general phenomenon of all plant regeneration

systems that involve a callus phase

Somaclonal variation : soma refers to somatic tissue and clonal as

difference observed within the clones. Variations ~ phenotypic differences; choromosomal rearrangements,

biochemicalandmolecular changes.

Definition: Genetic or epigenetic changes induced during the callus

phase of i n v i t r o cultured plant cells -- sometimes visible as a changed

phenotype in regenerated plants.

In tissue cultures, such changes can be a problem as the main objective of

tissue culture is to raise genetically stable cultures.

However, investigations have shown that plant tissue culture undergo

frequent genetic changes and they are expressed in the form of variant traits

in regenerated plants

3.1 Introduction


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3.1 Introduction

There are two general types of somaclonal variation

(a) Genetic changes, heritable(alter the DNA)

Transmitted to the next generation Important for crop improvement

Analysis of R0, R1, R2 progenies leading to true breeding

variants

(b) Epigenetic ~stable, but non-heritablechanges (alter gene

expression) Temporary changes and ultimately reversible, e.g. changes in

gene expression such as hormone habituation of cell cultures

e.g. cold resistance in Nicotiana sylvestris.

Might persist through the life of the regenerated plant





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3.2 Type of somaclonal variation

(a) Genetic changes:

Point mutations (e.g. Adh mutants in wheat)

Cytoplasmic (maternal inheritance)

Gene amplification (e.g. gene copy number)

Activation of transposable element

Cytogenetics (changes to genome structure)

Aneuploidygain or loss of 1 or more chromosomes

Polyploidygain or loss of an entire genome

Translocationarms of chromosomes switched

Inversionpiece of chromosome inverted




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(b) Epigenetic:

Change in phenotype that isnt stable during sexual

propagation May or may not be stable during asexual propagation

Usually undesirable in a breeding program, not always

undesirable in propagation

Habituation (most studied epigenetic change)

Define as loss of exogenous requirement for a growth

factor (usually a PGR); e.g., auxin or cytokinin habituation

Detection:callus may lose requirement for a PGR in the

process of several transfers to fresh medium




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(b) Epigenetic:

Habituation (most studied epigenetic change)

CharacteristicsOften occur gradually

Are regularly reversible (especially in regenerated

plants)

Are not seed-transmitted




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3.3 Possible causes

Pre-existing cellular differences

If mother plant is originally a chimeras, i.e. composed of cells of

different genetic origin, different cell layers if the meristematic

tissue might have different genetic composition

Common from callus initiated from explants containing

differentiated and matured tissues with specialized functions

Polyploid cells give more variability than diploids

3.0 Somaclonal Variation3.0 Somaclonal Variation


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3.3 Possible causes

Tissue culture induced variation

The dedifferentiation and redifferentiation process:

Axillary shoot proliferation vs. organogenesis andembryogenesis

Hypothesis ofDAmato

Somaclonal variants are rare in micropropagated plants

(when multiplication is by axillary branching of shoot tips /

buds) More common during shoot organogenesis and somatic

embryogenesis (especially with callus phase)



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3.3 Possible causes


Theculture environment

Tissue culture is inherently stressful to cultured plant cellsTemperature

Length of culture: ploidy changes increase with increase

lengths of culture

Nutrient depletion favor the development of abnormal

cells (shortage of precursor necessary for rapid nucleicacid biosynthesis)

Composition of culture medium

Some growth regulators trigger polyploidy in vitro



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3.3 Possible causes



Environment stress is known to cause:DNA methylation: methylation of cytosine is known to

cause gene inactivation; this may occur during the

redifferentiation process

Most mutational events directly or indirectly related to

alternations in the state of DNA methylationA decrease in methylation correlates with increased gene

activity



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3.3 Possible causes



Environment stress is known to cause:

Gene amplification: can result in increase gene

expression

Transpositional changes

Inadequate control of the cell cycle (errors in microtubule

synthesis, spindle formation)Importance of PGRs

Scant evidence of direct mutagenic action

More evidence for transient modifications of

phenotype (e.g. dwarfing)



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3.4 Mutagens

Physical Mutagens (irradiation)

Neutrons, Alpha rays

Densely ionizing (Cannon balls), mostly chromosomeaberrations

Gamma, Beta, X-rays

Sparsely ionizing (Bullets), chromosome aberrations &point mutations

UV radiation

Non-ionizing, cause point mutations (if any), lowpenetrating

Chemical Mutagens (carcinogens)

Many different chemicals

Most are highly toxic, usually result in point mutations



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3.5 Traditional mutation breeding procedures

Treat seed with mutagen (irradiation or chemical)

Target: 50% kill

Grow-out 1st generation (R1) plants Evaluation for dominant mutations possible, but most are

recessive

Grow-out R2 plants

Evaluate for recessive mutations

Expect segregation

Progeny test to select true mutants

Prove mutation is stable and heritable



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3.6 Detection and isolation of somaclonal variants

Two basis:

Visual analysis of cultures:morphologically distinct cells such

as non-green cells or cells that accumulate anthocyanins andother plant pigments are detected visually.

Experimental analysis of cultures: isolate herbicide and

antibiotic resistance variants, plant cells grown on media

containing wild type cells of culture. Surviving cells are

subcultured and retested for growth on a herbicide or

antibiotic supplemented medium. This will eliminate remaining

wild type cells that may have inadvertently survived on the

first round of selection.



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3.6 Detection and isolation of somaclonal variants

Examples of experimental analysis of cultures:

Screening

Observation of large number of cells or plants for the detection of variants

Normally for mutants for yield or yield traits (production for biochemicals) 1st generation plant (R1) are scored for the identification of variant plants

2nd generation plant (R2) are evaluated for confirmation

Cell selection

Selection pressure is applied which permits the preferential

survival/growth of variant cells Selection methods:

Direct selection, e.g. resistant to herbicides

Rescue method, e.g. low temperature resistant cells

Stepwise selection, e.g. salt resistant cells

Double selection, e.g. antibiotic resistance with chlorophyll developed



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In direct selection, the cells resistant to the selection pressure

survive and divide to form colonies; the wild type cells are killed by

the selection agent. This is the most common selection method. It is

used for the isolation of cells resistant to toxins (produced by

pathogens), herbicides, elevated salt concentration, antibiotics,

amino acid analogues etc.

In the rescue method, the wild type cells are killedby the selection

agent, while the variant cells remain alivebut, usually, do not divide

due to the unfavourable environment. The selection agent is then

removed to recover the variant cells. This approach has been used

to recover low temperature and aluminium resistant variant cells.


Selection methods:



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The selection pressure, e.g., salt concentration, may be gradually increased

from a relatively low level to the cytotoxic level. The resistant clones isolated at

each stage are subjected to the higher selection pressure. Such a selection

approach is called stepwise selection. It may often favour gene amplification(which is unstable) or mutations in the organelle DNA.

In some cases, it may be feasible to select for survival and/or growth on one

hand and some other feature reflecting resistance to the selection pressure on

the other; this is called double selection. An example of double selection is

provided by the selection for resistance to the antibiotic streptomycin, which

inhibits chlorophyll development in cultured cells. The selection was based on

cell survival and colony formation in the presence of streptomycin (one feature)

as well as for the development of green colour in these colonies (second

feature; only green colonies were selected).


Selection methods:



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3.7 Applications

Herbicide Tolerance

Tolerance: able to grow in the presence of the herbicideeitherthe target enzyme or altered form of enzyme

Most successful application of somaclonal variation have

been herbicide tolerance Glyphosate tolerantpetunia, carrot, tobacco and tomato

[elevated EPSP (enolpyruvyl shikimate phosphatesynthase)]

Imazaquin (Sceptor) tolerantmaize

Disease Resistant variants

Plant cell cultures are exposed to lethal concentrations oftoxins involved in disease development (Add toxin orculture filtrate to growth media)

Fiji disease resistant sugarcane, exposed plant to culturefiltrate ofHelminthosporium sp.

Late blight resistant potato, exposed plant to culture

filtrate ofPhytophthora infestans



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3.7 Applications

Stress resistant variants

Add or subject cultures to selection agent

Salt tolerant alfalfa and citrus fruits to be cultivated at saline soil

Aluminium resistant, e.g. N. plimbaginifolia

Low temperature resistant e.g. chilies High yield variants

Alfalfa variety Sigma,tobacco, corn, tomato etc

Variants for efficient nutrient utilization

Tomatoes with increase rate of phosphate uptake so can grow in phosphatedeficient condition

Specific product accumulators

Screen for specific product produced

Lysine in cereals

Variants for morphology

Potato of different tuber shape

Rice for plant height

Geranium for better flower

http://www.hort.purdue.edu/ext/senior/vegetabl/images/large/potatowhite.jpghttp://www.hort.purdue.edu/ext/senior/vegetabl/images/large/potatowhite.jpghttp://www.hort.purdue.edu/ext/senior/vegetabl/images/large/potatored.jpghttp://www.hort.purdue.edu/ext/senior/vegetabl/images/large/potatored.jpg


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3.8 Advantages & Disadvantages

Advantages:

Frequency of useful somaclonal variations is high

New mutations may be isolated which were not available in the

germplasm or through mutagenesis

Time frame is shorter as compared to conventional mutation breeding

Free from undesirable features e.g. sterility

Effective selection at cell level

Relatively small effort, time, cost and space requirements

Only approach for the isolation of biochemical mutants, especiallyauxotrophic (plant that is unable to synthesize a particular organic

compound required for its growth) mutants in plants

Not subject to regulatory requirements (or consumer attitudes) of

genetically engineered plants



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3.8 Advantages & Disadvantages

Disadvantages:

Many mutations are non-heritable

Requires dominant mutation (or double recessive mutation); mostmutations are recessive

Can avoid this constraint by not applying selection pressure in culture,

but you loose the advantage of high through-put screening have to

grow out all regenerated plants, produce seed, and evaluate the R2

Alternative: perform on haploid cell lines

Many selected plants show undesirable features e.g. reduced

fertility, growth and overall performance



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Students Activities 3

Case Study: Application of somaclonal variation and in v i t r o

selection to rice improvement (By: J. Bouharmont, A.

Dekeyser, V. Van Sint Jan and Y. S. Dogbe In RICE

GENETICS II Proceedings of the Second International Rice

Genetics Symposium).

Task:

Read the abstract provided and based on the knowledge orapplications that you have learnt on somaclonal variation

topic, answer the questions given.

Students Activities 3

(Directed-self Learning)


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Abstract:Somaclonal variation was observed in the greenhouse and in the field

on plants regenerated from calli and on their progeny. Many plantlets were

recovered from cells cultivated on medium with 1.5% NaCl. Several selected

plants showed even higher salt tolerance. A few cell lines survived at a

sublethal NaCl concentration (1.75%).However, the progeny of one plant

from such selection did not express improved salt tolerance. Adding Al2(SO4)3

to the culture medium reduced cell proliferation. Calli were also affected by

other medium modifications required for Al solubilization. Some plants

regenerated from calli selected on a modified medium, with or without Al,

expressed a degree of Al tolerance. Selection for cold tolerance was

attempted by long-term culturing of calli at sublethal temperatures (11-13 C).

Some plants regenerated from cell selection and tested under hydroponlc

conditions showed improved cold tolerance. Progeny of plants regenerated

for the different stresses are being field-tested in Africa. Although incomplete,

these experiments confirm the potential of somaclonal variation in rice

improvement and the applicability of in vitro selection for stress tolerance.

Case Study: Application of somaclonal variation and in v it ro

selection to rice improvement


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Questions

How many type of selection pressures were implemented to the rice

cultivars?

Based on the abstract, if you are given rice cultivars A and B; design

an experiment on how are you going to implement the selection

pressures mention above in the format of a flow chart.

Tips: In the lecture note, the sources of genetic variability can be due

to pre-existing cellular differences and / or tissue culture inducedvariation. Which of the above was used in the experiment?

Which method of cell selection did the researchers use?


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Traditional Mutation Breeding

100 paddy

Seeds

(F0)

Gamma irradiation

(200Gy)

Grow under water stress

No seeds survived

(Drought resistant gene

is influenced by

dominant allele)

Grow under normal water condition 50 seeds survived / growth

Adult plants (F1)

Obtained seeds

Grow under water stress

Some can grow

Adult plants (F2)

Some die

Continue growing the plants (water stress) until F3 and F4 generations

Test for stability

Lecture 3 - Plant (Risha)

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