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!