school seminar

8/8/2019 School Seminar

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Controlled Elimination ofChromosomes in Callus

Cultures of Potato Hybrids(Solanum acaule × S. phureja )

Massih Forootan

University of Reading



Contents

Potato: Important features related to breeding projects

Major restrictions in potato breeding

Conventional breeding methods

Chromosome instability and application in potato breeding

Experimental manipulation of chromosome instability



POTATO (Solanum tuberosum L.)

Solanaceae (Nightshades)

Fourth major crop (After maize, rice, wheat)

Rich in vitamin C, protein, and calcium

High yields



Tetraploid (2n=4X=48)

Clonal reproduction

Heterozygote

Susceptible to diseases

SO:

Disease resistance is an important objective for breeding

POTATO (Solanum tuberosum L.)

Features of important cultivars



Potato Br eeding

High yield

Susceptible

High yield

Resistant

Low yield

Resistant

Low yield

Susceptible

We typically select elite clones from a very large population

×P arent 2

Low yield

Resistant

P arent 1

High yield

Susceptible

Problem 1:

Heter ozygote par ents will r esult in unwanted segr egation



Potato Br eeding

Problem 2:

Most disease r esistance genetic r esour ces ar e f ound in diploid species

bulbocastanum canasense demissum polyadenium vernei

Late blight

Potato Virus X

Potato Leaf Roll Virus



Potato Br eeding

Problem 2:

Most disease r esistance genetic r esour ces ar e f ound in diploid species

Reduced quality

Reduced fertility

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Gametes

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Diploid

Cultivar

Hybrid

Tetraploid-Diploid cross: Imbalance between parental ploidy levels

Needs prolonged backcrossing with

tetraploid potatoes



Potato Br eeding

Equalising parental polidy level: Exploiting dihaploid inducer s

Certain Solanum phureja clones identified as dihaploid inducers

Why this happens?

egg cells are parthenogenically developed

dihaploid inducer chromosomes are eliminated

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pollen

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Dihaploid inducer

Cultivar

Dihaploid

egg



Potato Br eeding

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Dihaploidinducer

Cultivar

×Dihaploid

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Diploid

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×

Hybrid A

Hybrid B(produced like A)

Equalising parental polidy level: Exploiting dihaploid inducer s

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llllllllllllHybrid ABdoubled

Colchicine

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Hybrid ABCD

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llllllllllllllllllllllllHybrid CDdoubled

Same procedure

for two other traits C and D


Hybrid AB



Potato Br eeding

Pr otoplast Fusion: A different approach to bypass ploidy barrier







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protoplastisolation

protoplastisolation

fusion

regeneration

Hybrids will contain ALL genetic

material from BOTH parents

But still needs extensive

backcrossing



Potato Br eeding

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Hybrid AB

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Dihaploidinducer

Cultivar

×Dihaploid

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Diploid

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×

Hybrid A

Hybrid B(produced like A)

Pr otoplast Fusion: A different approach to bypass ploidy barrier

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llllllllllllHybrid ABCD

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Hybrid CDfusion



Potato Br eeding

A quick r eview

Most cultivars are tetraploid and susceptible to diseases

Many disease resistant species are diploid

A cross between tetraploid cultivar and diploid can introducedesired traits into the cultivar

The imbalanced tetraploid-diploid cross may result in loss of

quality and fertility

Using dihaploid inducers and/or somatic hybridisation

may help to overcome ploidy level problems



Potato Br eeding

But...

Many unwanted traits will also be introduced into new cultivar

Extensive backcrossing needed to remove unwanted genes from

wild relative

Cultivar quality will be reduced



An alter native:

Chromosome loss has been observed in rapid dividing tissues

Callus

3-15 days after protoplast culture in potato somatic

hybrids (Ramulu, ¶84)

Embr yo

1-3 days after embryo formation in barley hybrids

(Bennett, ¶76)

Root tips

during root growth in aneuploid potato (Wilkinson µ92)

Chromosomes are eliminated in early stage of cell development

Can we exploit natur al instability of chr omosomes in hybr ids?



Chr omosome Elimination



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callus



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protoplast

callus


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ll

Exploiting chromosome instability in breeding program

protoplast

fusion

selection of cloneslacking unwanted

chromosomesidentify aneuploid clonewith resistance-carryingalien chromosome only

Success of this

appr oach r elies on an under standing

of f actor s causing

chr omosome

instability



Factor s inf luencing elimination:

Nucleolar organiser region (NOR)

May result in nondisjunction when active (Pijnacker, ¶87)

Growth regulatorse.g. 2,4-D may cause chromosome breakage (Ganesh, ¶77)

Ploidy Level


Intracellular

Environmental

Irradiation

X-ray can increase rate of elimination (Oberwalder, ¶97)



Cytological causes: (Focus of this study)

Spindle faults:

Different chromosome size (chromosomes fail to attach spindles)

Cell cycle faults:

Spatial arrangement (chromosomes fail to go to spindle poles)

Asynchrony in cell cycle of the two parents

Nuclear fragmentation




Aim of this study

Studying the nature of chromosome instability in callus culture using molecular markers

Investigating possibility of manipulating chromosome elimination

Str ategy

a. Produce callus-derived hybrid regenerants

b. Studying genome stability

Identifying suitable markers

Screening the population for possible genome instability

Confirmation through cytological observations



×

S. acaule

(4x)

S. phureja

(2x)

Hybrid

(3x)

Plant Mater ial



Molecular Tool

Microsatellites (S

impleS

equence Repeats)

Suitable microsatellites for this study:

Chromosome specific (presence/absence of microsat is an inde

xfor presence/absence of chromosome)

Descriminate different alleles (i.e. chromosomes of the same linkage group)

Different

product size

in PCR



ª ª



% of chromosome loss under different temperature regimes

0%

5%

10%

15%

20%

25%

30%

22°C 24°C 26°C

% of chromosome loss in different temperature regimes



% of chr omosome loss over par ental genome

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

acaule phur eja

% of chr omosome loss or iginated f r om par ental mater ial



% of c r omosome loss under diff erent temperature re imes,

over parental genot pe and location gr oup

0%

5%

10%

15%

20%

25%

30%

35%

40%

I II V VI VII IX X XI

Lo a on roup

22°C 24°C 26°C

a au e

phurea

L nkage group

% of chromosome loss at d fferent temper ature reg mes w th d fferent linkage groups



ª



In pr ogr ess...

Confirmation through cytological tests (chromosome counting & flowcyto)

Analysing the results from colchicine level test

Investigating possible correlation between stability of two chromosomes



Summar y

Chromosome loss can occur during callus formation

It can be exploited to produce genotypes with less undesirable traits

Cell cycle faults and spindle faults are two possible causes for

chromosome instabilty

Callus cultures were treated by different temperature regimes and

colchicine levels to monitor the chromosome stability

Higher temperature (26°C) showed more chromosome loss than low

temperature (22°C), however control (24°C) showed irregular

behaviour.

No preferrence toward either of parental chromosomes were observed.

Frequency of microsat slippage and chimera were low.



THANK YOU!

Dr. Andr ew Wetten

Dr. Mike Wilkinson

Dr. Joel Allainguillaume

Mrs Moy Robson

Dr. George Gibbings

My Family

PSL staff

Colleagues

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