Plant Breeding Lecture 6-Starch-Sucrose-Fructans-Final PDF

25/03/2015
Breeding for carbohydrates
Luisa Trindade
Bioresource (PBR 31306)
Plant components
Primary
metabolites
Carbohydrates
Lipids
Membranes
Oils, waxes
Proteins and free aminoacids

Secondary metabolites
Easily degradable sugars: starch, sucrose, fructans

Insoluble sugars: plant cell walls: (hemi) cellulose, pectin
Vitamins
Flavonoids / terpenoids
Water and minerals
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Program for today
Easily fermentable sugars:

Starch: potato
Sucrose: sugarbeet and sugarcane
Fructans: grasses
Plant cell walls

Maize as a model
Starch
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Starch crops
The main sources of starch are Europe are:

Cereal crops
Rice
Maize
Wheat
Potato
The Netherlands is the largest

producer world wide
Potato is the major starch crop in The
Netherlands
...in other part of the world other crops are used: e.g
cassava, taro...
Breeding targets in starch crops
Starch yield
Starch composition
Starch granule size
Starch properties
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Breeding for starch yield
What are the key factors behind starch yield?

1. Starch synthases expression play a (small) role depending on
the crop and growth conditions
Overexpression of glycogen
synthases in wheat lead to
increase in starch content:
in line 72 only at high
temperature
In line 79 at both high
and low temperature
In this approach the biosynthesis of starch is stimulated/induced
Reference: Burrell. J Exp.Bot. 54(382): 451-456

2. Very recently reported: starch content is inversely correlated
with sugar content in potato:
High sugar content ->Low starch content
in the tuber
The sugars are probably originary from

starch.
This approach focuses on reduction of

starch degradation
What are the factors that influence starch

degradation?
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In approach 1 - target
genes in red
Synthases
In approach 2 target
genes in blue
Phosphorylation
Amylases
Debranching
Reference: Schreiber et al. 2014. 4 (10): 1797-1811
Breeding for starch content in potato
What are potato characteristics:

Potato is a cross pollinator
Tetraploid (most varieties)
Highly heterozygous
Clonally propagated crop
Tools available
Genome sequence
Genetic maps of different diploid
populations and markers for
different traits
Starch content is correlated with

underwater weight
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Traditionally breeding for starch content targeted:

High yield of tubers
High underwater weight
Breeding methods
1. Crosses between diverse genotypes
2. Selection of genotypes with good performance (for both
yield and resistance to Phytophthora)
3. Different rounds of selection and generation of elite lines

4. Clonal propagation of elite genotypes
Current/modern breeding for starch content targeted:

High yield of tubers
High underwater weight
Breeding methods: Marker assisted selection

1. Identification of genetic markers (QTLs) for the above traits
2. Identification of strong alleles

3. Introgression of the right alleles in elite lines
Future breeding: make use of diploid lines

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Starch quality
Starch quality
Starch needs to be derivatized before it can be used for

different application
modified starch
pregelatinized
acid-thinned
oxidized
hydroxyalkyl ethers
esterified
monophosphates
treatment
(example)
heat/moisture
acid
hypochlorite
advantage
food use
non-food
use
cold-water soluble
pie fillings, instant products,

coatings
gums, jellies
oil-drilling,
mining
low hot paste

viscosity, high gel
viscosity
increased clarity,
reduced set-back
gravy, sauces
thickeners,
jellies
propylene
oxide
acetic
anhydride
increased clarity and

stability
salad dressings,
pie fillings
reduced set-back,
increased clarity,
forms films/fibres
instant foods,
frozen foods
phosphoric
acid
increased stability
to freeze/thaw
cycles
increased stability
to heat, pH, freeze
/thaw cycles
increased cold-water
solubility/dispersability, increased
binding to negatively
charged materials
frozen foods,
infant formulae
cross-linked
phosphorous
oxychloride
cationic starches
tertiary or
quaternary
amines
wide range of
canned and
frozen foods
textiles,
laundry
starch
paper, textiles, spray
starch,
adhesives
paper and
textile sizes
textiles,
paper,
packaging,
film
paper, textiles, metal
refining
paper, metal
sequestrants
paper, mining
Derivatization implies:
Use of chemicals
Energy
Reduces the quality of
starch
Improvement of starch in
plant has many
advantages:
Environmental impact
Improve quality of
starch
More economical
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Starch granule composition
Amylose: 20-30%
Mw. 5105 to 106
Amylopectin: 70-80%
Mw. 107 to 108
Proteins
Lipids
Phosphate
Contaminating
substances
Possibly starch modifications
Amylose and amylopectin ratio

Size of the granules
Length of the amylopectin chains
Phosphate content
Protein and lipids content
Expression of heterologous proteins:
New polymers
New linkages
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Potato amylose free starch
What happens when you remove amylose?

Interesting breeding target!!
Bakery creams ( 20% expensive alginates)

Fruit filling (shiny texture)
Powdered soups, sauces & processed

meat products (salt stability)
Noodles
(hydration at lower
temperatures)
Snacks ( crispness)
Breeding for amylose-free potato
How do you identify amylose

free lines?
Based on phenotype
Wild type
Amylose-free
Iodine staining
Based on genotype
Search for lines where
GBSS has been mutated
Induce mutations in GBSS

(Tilling)
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Breeding for amylose-free potato
Breeding strategy: mutation breeding (non-GM)

As potato is a highly heterozygous and tetraploid crop it is
difficult to combine all the desired traits in one genotype
One tool is to do the first breeding steps in diploid (or even

monoploids) to generate a line homozygous for non-functional
GBSS
Introduce the non-functional GBSS in an elite line (this can

take many years!)
A GM approach is much faster and effective in this case
Amflora
Attempts to breed for high-amylose

starch in potato
Advantages of high amylose

starches:
Form strong gels

Applications in jelly-gum
candies and coating of
deep-fried foods
No 100% amylose potato

could be achieved
Probably lethal
Large impact on tuber yield

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25/03/2015
Starch phosphate content
Where is P in starch?
Amylopectin: C3 and C6 positions
What is the effect of P on starch prop.?

Higher phosphate content
High swelling power
Increased solubility
Resistance starch effective in lowering glycemic index
Low phosphate content

Higher digestibility
Low viscosity
Breeding for phosphate content
The genes involved in phosphorylation of starch are

known: GWD (glucan-water dikinase), PWD
(phophoglucan water dikinase)
Phosphatases are known: sex4 (starch excess)

Phosphate content is not only important for starch
properties but for survival of plant:
Paradox:
P is needed for starch for degradation in the plant
P is reducing digestibility of starch in humans
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25/03/2015
Breeding for phosphate content
Breeding approach: Reverse genetics (candidate gene)

Breeding strategy:
1. Alleles of GWD, PWD and SEX4 have been identified in a wide
collection of potato genotypes
2. These same genotypes have been characterized for P content

3. Alleles correlating with high or low P content have been
identified
4. Epistatic effects of starch phosphorylation are being analysed

(specially effect of reduced P on yield)
5. Positive and strong alleles will be introgressed in potato elite

lines
Tailoring of potato starch (GM)

CBM25
Streptococcus downei
Mutansucrase gtfi
L. mesenteroides
Dextransucrase
dsrs
A. thaliana
Water dikinase
E. coli
Branching
enzyme (GlgB)
L. mesenteroides
Alternansucrase asr
Human - Laforin
Bacterial Glucansucrases
Expression of different heterologous proteins lead to changes in

starch granule morphology and properties of starch
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Sugarbeet
Crop characteristics:
Cross pollinator
Most commonly diploid and tetraploid
Originally self-incompatible
Root has high sucrose (sugar) content
Sugar content in sugar beet
History:
Modern sugarbeets are originated from selections made in the
middle of the 18th century from fodderbeets grown in then
German Silesia
Breeding of sugarbeet was intensified during the blockade of

shipments of cane sugar to Europe by the British during the
Napoleonic wars
The sugar extraction process developed in the 19th century has

created a market for sugarbeet and thus breeding of this crop
enforced
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25/03/2015
Breeding sugarbeet
Breeding of the first varieties of sugar beet ware done by

mass selection
These varieties were followed by better ones produced
by anisoploid (triploid) synthetic varieties resulting from

crossing diploid and tetraploid lines
Diploid male sterile x tetraploid pollinators
Current sugarbeet varieties are diploid hybrids

1. Making diploid inbred parents containing the required traits
(sugar content, resistance to pathogens, and sugar quality)
2. making hybrid seeds

Synthetic: refers to varieties that result from crossing of multiple (>2) parents
Breeding sugarbeet
Breeding targets:
Total root yield
Sucrose yield
More recently breeding
for sugar quality has
become a target.
Current yield in The Netherlands:

Average root yield ~ 60 tons/ha
Average sugar yield ~ 10 tons/ha (16%)
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25/03/2015
Sugarcane
Perennial grass
Genetics: 2n=80 chromosomes
Breeding is a great challenge!!
Comprises 6 species but Saccharum

officinarum is the major contributor of
high sucrose in modern varieties
Contains a lot of duplications and

unpaired chromosomes
Breeding targets:
Sugar yield!
Ratooning performance, resistance to
pathogens, low fibre, cane yield.
Further reading: Jackson. 2005. 92: 277290
Breeding sugarcane
Breeding of sugarcane happen in three phases:

1. Crossing and selecting among S. officinarum clones: these
varieties had high sugar content and low fibre content, but
they were susceptible to diseases and lacked vigour
2. Development of interspecific hybrids between S. officinarum

and other species (mostly S. spontaneum): these were
vigorous and with good ratooning performance but they had
lower sucrose levels. By backcrossing the hybrids with S.
officinarum rubust high-sugar-high-yielding varieties were
generated: wonder cane (ancestry of all modern varieties)
3. Exploitation of interspecific hybrids from phase 2 by recurrent

selection
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Breeding sugarcane
Most modern varieties are made from a small number

of original progenitors
Narrow genetic basis of sugarcane breeding programs
Limited possibilities for introgression or improvement

of new traits
Breeding sugarcane
Sugar yield/ha has increased

But...that was not to increase
in sugar content but due to
improvements in total
biomass yield
Future:
Increase genetic basis in
breeding programs
Developing genetic markers

for different positive and
negative traits
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25/03/2015
Fructans in plants
Fructans are the major form of storage carbohydrates in grasses:
Are important to sustain regrowth of the plant after defoliation

Nutritive value of feed
When extracted can be used as sweeteners as humans are not
able to degrade fructans
Breeding fructans in wheat
Genetic markers (QTLs) for fructan yield in wheat grain have been
(recently) identified
Breeding strategy: Marker

Assisted Selection (MAS)
Identify the genes underneath

these QTLs would create tools
for breeding in other crops
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25/03/2015
Summary
Starch, sucrose and fructans are the main storage forms of easily
degradable sugars and they are specific to each crop.
Each crops has different characteristics and different

genetic/breeding tools are available this determines breeding
strategy
Breeding for sugar/starch content is often indirect. The real target is

crop yield
Starch biosynthetic pathway is well characterized and starch quality

has been mainly tackled using candidate gene approaches both GM
and non-GM.
Breeding of polyploids (potato and sugarcane) is much more difficult

than diploids species
One (future) line of potato breeding is through diploids

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25/03/2015

Breeding for carbohydrates

Bioresource (PBR 31306)

Proteins and free aminoacids

Easily degradable sugars: starch, sucrose, fructans

Water and minerals

Bioresource (PBR 31306)

Program for today

Easily fermentable sugars:

Plant cell walls

Bioresource (PBR 31306)

Bioresource (PBR 31306)

The main sources of starch are Europe are:

The Netherlands is the largest

Breeding targets in starch crops

Bioresource (PBR 31306)

Breeding for starch yield

What are the key factors behind starch yield?

Reference: Burrell. J Exp.Bot. 54(382): 451-456

Breeding for starch yield

The sugars are probably originary from

This approach focuses on reduction of

What are the factors that influence starch

Breeding for starch yield

Bioresource (PBR 31306)

Reference: Schreiber et al. 2014. 4 (10): 1797-1811

Breeding for starch content in potato

What are potato characteristics:

Starch content is correlated with

Breeding for starch content in potato

Traditionally breeding for starch content targeted:

3. Different rounds of selection and generation of elite lines

Bioresource (PBR 31306)

Breeding for starch content in potato

Current/modern breeding for starch content targeted:

Breeding methods: Marker assisted selection

2. Identification of strong alleles

Future breeding: make use of diploid lines

Bioresource (PBR 31306)

Starch needs to be derivatized before it can be used for

pie fillings, instant products,

low hot paste

increased clarity and

Bioresource (PBR 31306)

Starch granule composition

Possibly starch modifications

Amylose and amylopectin ratio

Potato amylose free starch

What happens when you remove amylose?

Bakery creams ( 20% expensive alginates)

Powdered soups, sauces & processed

Bioresource (PBR 31306)

Breeding for amylose-free potato

How do you identify amylose

Induce mutations in GBSS

Breeding for amylose-free potato

Breeding strategy: mutation breeding (non-GM)

One tool is to do the first breeding steps in diploid (or even

Introduce the non-functional GBSS in an elite line (this can

A GM approach is much faster and effective in this case

Attempts to breed for high-amylose

Advantages of high amylose