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Biotechnology in ornamental plant
breeding and production
István Dániel Mosonyi
Using biotechnology in plant production – in which cases?
Aims Methods
Production of propagation material -micropropagation-cell and tissue culture
-artificial seeds, encapsulation
Virus elimination -meristem culture
-micrografting
Gene pool conservation -cryopreservation
-minimal growth technique
Breeding of new cultivars -in vitro mutagenesis
-induction of polyploidity
-protoplastfusion
-genetic transformation
Production of secondary metabolites
(not by ornamentals)
-cell and tissue culture
-genetic transformation
The significance of micropropagation
Israel: carnation,
chrysanthemum, gladiolus
Arab countries: date palm,
banana, potato, strawberry, pistachio, rose
India: mainly woody
plants, ornamentals, banana
China: forestry plants
Ornamentals, fruits
Every year 700 millions of plants are produced in vitro (data from 2003).
Colombia, Ecuador,
Brazil: ornamentals
0
20
40
60
80
100
120
2005 2006 2007 2008 2009 2010
Nu
mb
er
of
pla
nts
(mil
lio
np
cs)
Phalaenopsis
Anthurium
Kalanchoe
Rosa
Hyacinthus
Chrysanthemum
Dracaena
Ficus
Spathiphyllum
Cyclamen
Begonia
Poinsettia
Amount of ornamental pot plants in Dutch Flower Auction
ADVANTAGES DISADVANTAGES
Requires little space Big loss if contamination occurs
Certifiable pathogen-free plants Somaclonal variability cause problems
Recontamination is unlikely Loss during aclimatization
Homogenous quality High demand cost
Independence from season/climate Requires laboratory background
Continuous production Requires qualified labour force
Especially good for recalcitrant species
Mass propagation of new genotypes
Can be robotized and automated
Fast method
Cultures can be easily transported
Methods of micropropagation
Methods of micropropagation
1. Organogenesis
(meristem-, node-, shoot tip-, adventitious shoot culture)
Explant: meristem or bud (leaf primordia containing bud)
Development: - direct morphogenesis
(shoot developing directly from explant)
- indirect morphogenesis
(callus formation first followed by organ formation from callus)
- Many new plants in a short time period
- Meristem culture is suitable for virus elimination
- Genetic stability is high
- Can be done on solid media
Methods of micropropagation
2. Somatic embryogenesis
Explant: somatic cell
Development: - direct embryogenesis
(embryo develops directly from the somatic cell)
- indirect embryogenesis
(non embryogenic cell duplication before embryo structure forms)
- Germination of embryo requires exactly defined environmental conditions
- Liquid medium needed for development of embryo
- Ideal for bioreactor systems
- Suitable for cryopreservation (gene bank storage)
- Suitable for artificial (synthetic) seeds, genetic transformation
- Establishment and maintenance (germination) of somatic embryos are difficult
- High risk of genetic instability
Micropropagation methods of selected ornamentals
Organogenesis Somatic embryogenesis
Alocasia spp.
Anthurium spp.
Begonia spp.
Chrysanthemum spp.
Dracaena spp.
Dianthus caryophyllus
Ficus spp.
Gerbera jamesonii
Hortensia spp.
Pelargonium spp.
Rhododendron spp.
Rosa hybrida
Saintpaulia ionantha
Spathiphyllum spp.
Begonia gracilis
Chrysanthemum grandiflorum
Cyclamen persicum
Euphorbia pulcherrima
Rosa hybrida
Rosa rugosa
Saintpaulia ionantha
Bioreactor (Fermentor) technology
Bioreactor definition:is a manufactured or engineered device or system that
supports a biologically active environment. Primarily it is a
vessel in which chemical or biological process is carried out
which involves organisms or biochemically active substances.
In plant biotechnology it is a device meant to grow cells or
tissues.
Aims/possibilities:
-Mass production of cells – yeast, bacteria, plant cells
-Production of cell components – enzymes, nucleic acids, polysaccharides
-Production of metabolites – ethanol, lactic acid, vinegar, antibiotics
-Simple substrate conversion – glucose -> fructose
-Multi substrate conversion – biological cleaning of wastewater
Comparison of bacterial and plant cell cultures
Characteristics Bacterial cellculture
Plant cell culture
Size of cells 0,5 – 5 µm Larger with 1 order of magnitude
Change of cell shape Negligible Considerable
Status of cells Individiual Forming aggregates
Duplication time of cells 1-2 hours 2-3 days
Aeration requirements High Low
Mechanical sensitivity of cells
Low High
The requirements of plant cell cultures and choosing a
suitable bioreactor:
- Proper / sufficient aeration
- Liquid stirring with low mechanical tension (cell sensitivity!)
- Proper possibilities for material inlet and outlet (cell aggregates!)
Suitable bioreactors for plant cell cultures:
- Stirred tank reactor with mechanical mixing
- Bubble column reactor with pneumatical mixing
- Air-lift reactor (pneumatical mixing)
From 1980 the already established bioreactor systems were modified to be
suitable for plant cell cultures.
Stirred-tank reactor with mechanical mixing
Advantages Disadvantages
Commonly used High shear stress around
the impeller
Useful for high viscously
cell culture
High capital and
operational cost
High aeration Heat generation due to
mechanical mixing
Good fluid mixing High energy cost due to
mechanic agitation
Ease of compliance with
cGMP requirements
High contamination risk
with mechanical seal
Bubble column reactors with pneumatical mixing
Advantages Disadvantages
Suitable for plant and
animal cells
Poor oxygen mass transfer
ability
Easy to construct and
scale up
Poor fluid mixing in highly
viscous cultures compared
with stirred tank reactors
Low operational cost Serious foaming under
high aeration conditions
Low contamination risk
Low shear stress
No heat generation
Air-lift reactors with pneumatical mixing (very similar to bubble column)
Advantages Disadvantages
Multiple choice of internal draft
tubes
Same as by bubble columns
Better aeration than bubble column
Circulating flow pattern
Comparison of individual vessel and bioreactor systems
Characteristics Individual
vessels
Bioreactors
Manipulation of cultures Manual Automatized
Nutrients available Limited Can be refilled
Space for grow Small Large
Alteration of air ingredients Difficult Easy
Specific space requirements
(for 1 plantlet)
High Low
Specific costs Higher Lower
Suspension culture of Oncidium ‘Sugar Sweet’ PLBs (protocorm like bodies)
Production of Spathiphyllum cannifolium in modified air-lift reactors
1. Immersion without net
2. Immersion with net
3. Partial immersion
Production of oriental hybrid Lilium ‘Casablanca’ in bubble bioreactor
Synthetic seed technology
synthetic (artificial) seed
somatic embryo or rarely shoot tip
artificial endosperm
Shell: Sodium alginate, dipped in CaCl2 solution + nutrients, hormons,
biocids (fungicid, bactericid)
Synthetic seed preparation
A. The explants are sucked with a pipette together with sodium-alginate
B. A drop of Na-alginate containing an explant is going to be released in thecomplexing solution
C. Synseeds during the hardeningD. Recover and washing the synseeds
from the complexing solutionE. Sequoia sempervirens (27 days)F. Tilia cordata (21 days)G. Photinia fraserii (20 days)
H. Nerium oleander (21 days)
Application area of synthetic seeds
-Fast propagation of horticultural cultivars (substitution of F1 hybrids!)
-Propagation of sterile / seedless cultivars
-Gene bank storage of a given genotype
Some tested (still in research phase but working) synthetic seeds in ornamentals:
-Syringa vulgaris axillary buds
-Lilium longiflorum bulblets
-Cyclamen persicum somatic embryos
-Pelargonium hortorum somatic embryos
-Camellia japonica somatic embryos
-Sequoia sempervirens somatic embryos
-Betula pendula nodal segments
In many species the aim of sterile culture is mainly the virus elimination:
-carnation (CarMV, CERV, CRSV, CVMV, CNFV, CIRSV, CLV, CCMV…)
-chrysanth (CBV, TAV, CMV, TSWV, CSNV, CVCV, CSVd, CCMVd)
-hortensia (HRSV, TRSV, TNV, TSWV, CMV, HLV, AMV, HMV)
- Heat treatment:60 days 37 °C, 16 h light, 50-60% RHphysiological background: at high temperature the intensity of
gene silencing is increasing
- Meristem preparation: the youngest parts does not carry viruses
under microscope (10-16x magnification)with frequently changed knives
Hortensia can not tolerate the temperature above 30 °C in vitro:treatment with ribavirin (inhibits the replication of potex viruses)
Virus elimination
• Biotest – sensitivity is excellent, but requires lot of space and time
• Immunological tests: ELISA – enzyme linked immunosorbent assay:
tobamoviruses are easily detectable, other virus groups are onthe edge of detectability with this method– false-negative results!
viroids can not be detected by ELISA(chrysanths have stunt viroid, chlorotic mottle viroid)
• Detection of nucleic acid: PCR – polymerase chain reactionvery accurate, disadvantage: requires accredited labour force(because of radioactive isotopes)
suitable for mass testing (without radioactive labeling)
Characteristics of in vitro plants in virus diagnostics:
- Few inhibitor compound – eases diagnostics
- Lower virus concentraion – more difficult to detect
Viral diagnosis
Transfer shoot tips on stocks – in vitro or in vivo
In vitro application:
- rejuvenating woody plants
- elimination of viruses in infected plants
- examination of graft incompatibility
- conducting in vitro resistance screening
Process:
- rootstock: seedlings, rooted cuttings, both of them raised in vitro
- cut off the tip of rootstock
- putting the scion to the rootstock in a reverse T-shaped cut- or simply fit them together followed by gluing:
with 1% agar-solution
Micrografting
6 week old apple micrograft
A – perfect fusion B – tissue of the stock declines
C – unwanted shoot on stock D – 28 days after grafting
Micrografting Opuntia ficus-indica to another O. sp.
Cryopreservation for gene pool conservation
Cryopreservation: storage in very low temperature (-196 °C = -321 °F)
Aim: timing the cultures or storing them as a gene pool
Main problem to be avoid: formation of ice crystals during freezing
Solution: appropriate freezing technique, use of cryoprotectants
Cryoprotectants: glycerine, dimethylsulfoxide (DMSO), ethylene-glycol („antifreeze”)
protects the membranes from damage
Process of freezing:
• Immersion of plant material in cryoprotectant solution (cryoprotectant could be
also incorporated in growing medium)
• Optionally : dehydration (no water should be left which would form ice crystals)
• Slow cooling (0,2 °C/min) in cryoprotectant solution until temp. reaches ~ -40 °C
• Immersion in liquid nitrogen (-196 °C)
Plant breeders can work with genotype already established.
Traits can be combined with crossing two parent plant.
BUTIf a particular crossing is not possible, or breeders want a new trait, mutation can be a good source for enhancing the variability.
Frequency of natural occuring mutation is very low
One should use mutation induction techniques (= mutagenesis)under in vitro environment:
1.Chemical – with mutagen compounds
2.Physical – with ionizing radiation
In vitro mutagenesis
1. Chemical mutagenesis: the most effective mutagens
ethyl methanesulfonate –
methyl methanesulfonate – alkylizing the DNA bases
N-ethyl-N-nitrosourea
formaldehyde
2. Physical mutagenesis: ionizing radiation 20-60 Gy gamma / (X)- rays (1 Gy = 1 J/kg energy, absorbed dose)(6 Gy is already deadly!)
Application results:
- Rose – EMS
-> color variants
- Chrysanthemum – irradiation
-> cold tolerant mutants
-> 7-10 days earlier flowering
-> flower color and form variants
- Euphorbia pulcherrima – irradiation
-> better tolerance against low temperature
In vitro mutagenesis
Optimara EverFloris™ - space violets
NASA research program – examination the effect of cosmic rays and weightlessness
Optimara EverFloris™ - space violets
NASA - Long Duration Exposure Facility
Optimara EverFloris™ - space violets
- 25 000 pcs of Saintpaulia seeds were sent into the space
- they were on Earth orbit nearly 6 years (between 1984-1990)
- 80% of them kept their ability to germinate
- 4 new traits were selected from the mutant seedlings
Wavy / scalloped
leavesRippled / overcurling
leaves
Multi-branched
flowersUp to 50% larger
EverFloris™ cultivars: http://www.optimara.com/everfloris.html
Optimara EverFloris™
Optimara EverFloris™
Optimara EverFloris™
Optimara EverFloris™
Optimara EverFloris™
Multiplying the chromosome sets in a plant cell:
- creation of new morphological treats
- overcome crossing barriers: making dihaploid plants (to have homozygotic genotype)
Execution: use of anti-mitotic agents (colchicine, oryzaline, trifluraline) –inhibit the separation of chromosomes in mitosis
Example: diploid – tetraploid Hemerocallis cvs.- more robust habit- larger flower- more pigment – more intense color
Induction of polyploidism
Protoplast: a cell without cell wall (removing the cell wall artificially )
Making:
- Selecting the cells from in vitro culture (sterile environment)
- Digesting the cell wall with the help of enzymes
- Osmotic protection of protoplasts with so called plasmolitics:(in the solution of carbohydrates which cannot be metabolised: mannitol, xylitol, sorbitol)
- Their mechanical vulnerability is high – careful manipulation
- Centrifugation –because of low density they flow in the supernatant
- Growing protoplasts on medium
Fusioning: glue two protoplast together, they can be whatever cells from distantly related species as well but the results can not be guaranteed (electrofusion/PEG method)heterokaryon – is a cell that contains multiple, genetically different nuclei
Asymmetric development – the genome of one parent is coming over
Not aimed intervention – the result is random
Protoplast fusion (Somatic fusion)
• Transfer the cold tolerance from
Lavatera thuringiaca to
Hibiscus rosa-sinensis
• Making interspecific hybrid
between oriental hybrid lily
‘Acapulco and Shirotae’ and
L. formolongi ‘Hikucho’
Image: Cyclamen alpinum and
Cyclamen mirabile hybrid
regeneration from fused
protoplasts
Protoplast fusion – research and applications
Introducing foreign DNA in a host cell
Requires in vitro environment:
PLANT – CELL – PLANT system
Aimed intervention: introducing a DNA of a specific feature into a host cell
Methods:
Genetic transformation
Indirect gene transfer Direct gene transfer
Agrobacterium spp.
viral vectors
to protoplast to intact cell
Chemical treatment
(PEG, CaCl2 )
Electroporation
Microinjection
Ultrasonic treatment
Macroinjection
Microneedle technique
Biolistic particle delivery
Genetic transformation – application examples
Plant Trans gene Feature
Rosa hybrid Ace-AMP1 Resistance to powdery
mildew
Rosa DFR (from Iris, Viola) Blue color
Petunia rol C More compact habit,
better branching
Petunia CHS A New pattern on flower
petals
Pelargonium Ace-AMP1 Resistance to powdery
mildew
Rhododendron Ace-AMP1 Resistance to powdery
mildew
L-phenylalanine trans-cinnamic
acid
p-coumaric
acid
p-coumaryl coA
tetrahydroxy-chalcone
naringenin
dihydrokaempfer
ol
dihydroquercetin
eriodictyol
kaempferolquercetin
dihydromyricetin
leucopelargonidinleucocyanidin leucodelphinidin
pelargonidincyanidin delphinidin
PAL C4H 4-CL
CHS
CHIF3’H
F3H
F3’H
F3H
FLS
DFR
ANS
FLS
DFR DFR
ANS ANS
F3’H
flavanones
dihydroflavonols
flavonols
anthocyanins
Biosynthesis of flavonoids (the phenylpropanoid pathway)
F3’5’H
FNSFNS
chalcones … …
……
Thank you for your attention!