Chapter 6

SHOOT CULTURE PROCEDURES

MICROPROPAGATION
Concepts
1. Introduction
2. Shoot apical meristem
3. In vitro culture of shoot meristems
4. Meristem and meristem tip culture
5. Shoot and node culture
6. Micropropagation
7. Micropropagation stage
Stage 0: Donor plant selection and preparation
Stage I: Establishment of aseptic cultures
Stage II: Proliferation of axillary shoots
Stage III: Pre-transplant (rooting)
Stage IV: Transfer to natural environment
Concepts

Shoot meristem cells retain the embryo capacity for unlimited division

Isolated smaller meristem explants require more complex culture media for survival

Meristem and meristem tip culture are methods for disease eradication

Shoot culture provides a means to multiply periclinal chimeras

Cytokinin disrupt apical dominance and enhance axillary shoot production

Increased auxin concentration increases rooting percentage and root number, but
decreases root elongation

Negative carryover effects of auxins used for Stage III rooting may effect ex vitro
survival and growth of plantlets
 1. INTRODUCTION

Micropropagation if defined as true-to-type propagation of selected genotype using in

vitro.

Four basic methods are used to propagate plants in vitro depending on the species and
cultural conditions that can be achieved by the following:
(1) Enhanced axillary shoot proliferation (shoot culture)
(2) Node culture
(3) De novo formation of adventitious shoots through shoot organogenesis
(4) Nonzygotic embryogenesis

Currently, the most frequently used micropropagation method for production utilizes
enhanced axillary shoot proliferation from cultured meristems

This method provides genetic stability and is easily attained for many species

Consequently, the shoot culture method has played an important role in development of
a worldwide industry that produces more than 350 million plants annually

Besides propagation, shoot meristems are cultured in vitro for 2 other purposes:
(1) The production of pathogen-eradicated plants
(2) The preservation of pathogen-eradicated germplasm
 2. SHOOT APICAL MERISTEMS

Shoot growth in mature plants is restricted to specialized regions that exhibit little differentiation,
and in which the cells retain the embryogenic capacity for unlimited division

These regions, called apical meristems, are located in the apices of the main and lateral buds of
the plants

Cells derived from these apical meristems subsequently undergo differentiation to form the main
tissues of the plant body

Due to their highly organized structure, apical meristems tend to be genetically stable

There exist significantly differences in the shape and size of shoot apices among different
taxonomy plant groups (Fahn, 1974)

A typical dicotyledonous shoot apical meristem consists of a layered dome of actively dividing
cells located at the extreme tip of a shoot, and measures about 0.1-0.2mm in diameter and 0.2-
0.3mm in length

The apical meristem has no connection to the vascular system of the stem

Below the apical meristem, localized areas of cell division and elongation represent sites of a
newly developing leaf primodia (fig11.1)

Lateral buds, each containing an apical meristem, develop within the axils of the subtending leaves.
Lateral buds is usually inhibited by apical dominance of the terminal shoot tip

Organized shoot growth from the apical meristem of plants is potentially unlimited and is said to be
indeterminate; and, however, shoot apical meristems may form determinate organs such as flowers
 Fig 6.1: Diagrammatic representation of a dicotyledonous shoot tip.
The shoot tip comprise the apical meristem, subtending leaf primordial, and lateral buds
 Structure of apical meristem

Dicotyledon:
- Having 2 layers: L1, L2 (tunica)
- Having 1 layer: L3 (corpus)
Monocotyledon:
- Having 2-3 layers
L1: leaf epidermis, stem epidermis, flower epidermis
L2: palissade parenchyma and margin of the leaf gamets, petals, stems
L3:spongy parenchyma of the leaf, veins, roots, central parts of the stem
 3. IN VITRO CULTURE OF SHOOT MERISTEMS

In 1920s, unsuccess to culture isolated shoot meristems in aseptic conditions

By mid-1940s, sustained growth and maintenance of organization of cultured shoot

meristems through repeated subculture was achieved for several species

Ball (1946) provided the first detailed procedure for the isolation and production of plants from
cultured shoot meristem tips and the successful transfer of rooted plalets into soil

These studies demonstrated the feasibility of regeneration of shoots from cultured shoot
tips, the procedure typically yielded unbranched shoots

Several important findings facilitated application of in vitro culture techniques for large-scale
clonal propagation from meristem
(1) Culture meristems for virus-eradiated plants for free-virus plant (Morel & Martin, 1952; Styer
& Chin, 1984)
(2) Culture shoot tip for rapid production of orchid (Morel, 1960, 1964) through the cultured
meristems become disorganized and form spheroid protocorm-like bodies (PLB) that are
actually nonzygotic embryos

The final discovery was the elucidation of the role of cytokinin in the inhibition of apical
dominance (Wickson & Thimann, 1958) and application to enhance of axillary shoot
production in vitro

Application of this method was expedited by development of improved culture media that
supported the propagation of a wide diversity of plant species (Murashige & Skoog, 1962; Lloyd
& McCown, 1980)
Apical meristem culture
Iris meristem culture
Regeneration of cucumber meristem
 4. MERISTEM AND MERISTEM TIP CULTURE

Meristem and meristem tip culture are used to generate pathogen-eradiated shoots
that subsequently serve as propagules for in vitro propagation

4.1. Meristem culture

Culture of the apical meristematic dome alone (fig11.2) from either terminal or lateral
buds, for the purpose of pathogen elimination, is termed meristem culture

In reality, true meristem culture is rarely used because isolated meristems of many
species exhibit low survival rates and increased chance of genetic variability
following callus formation and indirect shoot organogenesis

Pathogen elimination can often be accomplished by culture of relatively larger (0.2-

0.5mm) meristem tip explants excised from plants that have undergo thermo- or
chemotherapy

4.2. Meristem tip culture

The meristem tip is comprised of the meristem + 1/2 subtending leaf primodia
(fig11.2). This procedure termed meristem tip culture.
Fig 6.2: Micropropagation stages for production by shoot culture
Cultivation of potato meristem
Potato: Internode - Lateral bud - Meristem

Chemotherapy
Contamination
Heat therapy
 5. SHOOT AND NODE CULTURE
Although not the most sufficient procedure, propagation from axillary shoots has proven to be
a reliable method for the micropropagation of a large number of species (Kurtz etal, 1991)

Depending on the species, 2 methods, shoot and node culture, are used. Both methods rely on
the stimulation of axillary shoot growth from lateral buds following disruption of apical
dominance of the shoot apex

5.1. Shoot culture

Shoot culture (shoot tip culture) referes to the in vitro propagation by repeated enhanced
formation of axillary shoots from shoot tip or lateral buds cultured on medium supplemented
with PGRs, usually a cytokinin (George, 1993)

The axillary shoots produced are either subdivided into shoot tip and nodal segments that
serve as secondary explants for further proliferation or are treated as microcuttings for
rooting

In some species, modified storage organs such as miniaturized tubers or corms (fig11.3)
develop under inductive culture conditions from axillary shoots or rhizomes and may serve as
the propagule for either direct planting or long-term storage

When either verified pathogen-free stock plants are used or when pathogen elimination is not
a concern, relatively large (1-20mm) shoot tip or lateral bud primary explants (fig11.2) can be
used for cultures

Adventages of using larger shoot tips include greater survival, more rapid growth response,
and the presence of more axillary buds
 Fig 6.3: In some species, such as Sagittaria latifolia L.,
in vitro multiplication may occur through production of shoots (left) and corm (right),
depending on culture conditions. Bar=10mm
5.2. Node culture

Node culture, a simplified form of shoot culture, is another method for production from pre-
existing meristems

Numerous plants such as potato do not respond well to cytokinin stimulate of axillary shoot
proliferation observed in the micropropagation of many crops

Axillary shoot growth is promoted by the culture of either intact shoots (from meristem tip
cultures) positioned horizontally on the medium (in vitro layering) or single or multiple node
segments

Typically, single elongated unbranched shoots, comprised of multiple nodes, are rapidly
produced

These shoots (microcuttings) are either rooted or acclimatized to ex vitro conditions or

repeatedly subdivided into nodal cuttings to initiate additional cultures

Although node culture is the simplest method, it is associated with the least genetic
variation
Node culture
 Trouble shooting
Somaclonal variation

Benefits:
plant improvement

Disadvantage:
require clonal uniformity

Reduce:
subculture
explant status
Less using 2.4D
 6. MICROPROPAGATION
6.1. Why we micropropagation ?

Rapid clonal in vitro propagation of plants from cells, tissues or organs cultured
aseptically on defined media contained in culture vessels maintained under
controlled conditions of light and temperature
• Micropropagation is used commercially to asexually propagate plants,
millions of new plants can be derived from a single plant
• This rapid multiplication allows breeders and growers to introduce new
cultivars much earlier than they could by using conventional propagation
techniques, such as cuttings.
• Micropropagation also can be used to establish and maintain virus-free plant
stock.
• Micropropagation differs from all other conventional propagation methods
in that aseptic conditions are essential to achieve success
 6.2. Micropropagation Applications

* Rapid increase of stock of new varieties

* Elimination of diseases
* Cloning of plant types not easily propagated by conventional
methods (few offshoots/ sprouts/ seeds; date palms, ferns,
nandinas)
* Propagules have enhanced growth features (multibranched
character; Ficus, Syngonium)
6.3. Clone and Cloning

Genetically identical assemblage of individuals propagated entirely by

vegetative means from a single plant.
6.4. Conventional Propagation
* Cuttings
* Budding, grafting
* Layering
6.5. Conventional Propagation Advantages

* Equipment costs minimal

* Little experience or technical expertise needed
* Inexpensive
* Specialized techniques for growth control
(e.g. grafting onto dwarfing rootstocks)
6.6. Micropropagation Advantages

* From one to many propagules rapidly

* Multiplication in controlled lab conditions
* Continuous propagation year round
* Potential for disease-free propagules
* Inexpensive per plant once established
* Precise crop production scheduling
* Reduce stock plant space
* Long-term germplasm storage
* Production of difficult-to-propagate species
6.7. Micropropagation Disadvantages

* Specialized equipment/facilities required

* More technical expertise required
* Protocols not optimized for all species
* Plants produced may not fit industry standards
* Relatively expensive to set up?
6.8. Explant
Cell, tissue or organ of a plant that is used to
start in vitro cultures
Many different explants can be used for
micropropagation, but axillary buds and
meristems are most commonly used

6.9. Choice of explant

Desirable properties of an explant
Easily sterilizable
Juvenile
Responsive to culture
Shoot tips
Axillary buds
Seeds
Hypocotyl (from germinated seed)
Leaves
 6.10. Methods of micropropagation
Samples = meristem, shoot-tip, interrnode

1. Axillary branching >95% of all micropropagation

( Internode-cuttings ) Genetically stable
Simple and straightforward
SAMPLES

2. Adventitious shoot formation Efficient but prone to genetic

( Multiple-shoots ) instability
 Axillary shoot proliferation
Growth of axillary buds stimulated by cytokinin treatment;
shoots arise mostly from pre-existing meristems
 7. MICROPROPAGATION STAGES

Murashige (1974) originally described 3 basic Stages (I-III) for successful

micropropagation. Deberg & Maene (1981) to includes Stage 0 because there are
contamination problems often associated with inoculation of primary explants

It is now agreed that there are 5 Stages (0-IV) critical to successful micropropagation

These Stages not only describe the procedure steps in the micropropagation
process, but also represent points at which the cultural environnment is altered
(Miller & Murashige, 1976)

This system has been adopted by most commercial and research laboratories
as it simplifies production scheduling, accounting, and cost analysis (Kurtz etal,
1991) (fig11.2)
Stage 0: Donor plant selection and preparation
Stage I: Establishment of aseptic cultures
Stage II: Proliferation of axillary shoots
Stage III: Pre-transplant (rooting)
Stage IV: Transfer to natural environment
 Stage 0: Donor plant selection and preparation
Explant quality and subsequent responsiveness in vitro is significantly influenced by the
phytosanitary and physical conditions of the donor plant (Deberg & Maene, 1981; Read,
1988)

Phytosanitary conditions
(1) Selection and maintenance of the stock plants in cleans, controlled conditions for
reducing the propability of disease
(2) Maintenance of pathogen-tested stock plant under conditions of relative humidity, drip
irrigation and antibiotic spray

Physiological status
(1) Trimming to stimulate lateral shoot growth
(2) Pretreatment sprays containing cytokinin or gibberellic acid
(3) Use of forcing solutions containing 2% sucrose and 200 mg/l 8-hydroxyquinoline
citrate for induction of bud break and delivery of growth regulators to target explant
tissues (Read, 1988)
 Stage 0 : Donor plants

Bud decapitalization

Based irrigation

Soaking in GA3 solution

Yellow leaves
Stimulation by kinetin
 In vivo / In vitro - Branching

In vivo

In vitro
 In vivo / In vitro - Adventitous

Adventitious
In vivo

Adventitous
In vitro
 In vivo / In vitro – Crown gall

Crown gall in vitro:

Crown gall in vivo callus production
 In vivo / In vitro – Callus

Unorganised growth of cell Unorganised growth of cell

(callus) in vivo (callus) in vitro
Contamination
Heat therapy
 Stage I: Establishment of aseptic cultures
Prepare explants  sterilization  culture in vitro  growth room (fig11.2)

Preparation of meristem explants

(1) Explantation time (juvenile and young physiological status)
(2) Position of the explant on the stem
(3) Explant size
(4) Polyphenol oxidation

Sterilization
(1) Explants were washed under with soap tap water  washed by tap water
(2) Prepare explants: 10-20mm in hood  sterilization
(3) Sterilization by alcohol (70%)  washed by distilled water in 3 times
(4) Sterilization by Ca/Na-hypoclhorite or the other agents (HgCl2, antibiotics)  washed by
distilled water in 3 times
(5) Prepare explants for in vitro culture  shoot tip (0.5-5mm) node (0.5-15mm)

Cultured medium
(1) Choose the MS (1962) first
(2) Using cytokinin (BA, 2iP)
(3) Using combination of cytokinin and auxin (IAA, IBA, NAA)
(4) Antioxidants: activated charcoal, PVP, ascorbic acid and citric acid (150mg/l)

Growth room
(1) Temperature 25+2oC, humidity 65-70%, light intensity: 33μM/m2/s
(2) Lighting: 8-12hrs
 A piece of plant tissue (called an explant) is:-
-cut from the plant,
-disinfested (removal of surface contaminants), and
-placed on a medium.

A medium typically contains mineral salts,

sucrose, and a solidifying agent such as agar.
The objective of this stage is to achieve an
aseptic culture. An aseptic culture is one
without contaminating bacteria or fungi.
Fig 6.4: Micropropagation stages for production by shoot culture
 PROPAGULES IN MICROPROPAGATION

Single-node cutting method

- low cytokinin
* Meristem - BA
* Shoot tip
* Internode

Multiple-shoots method
- Cytokinin / Auxin
 MICROPROPAGATION
Single node cutting

Peper

Potato

Grape
 MICROPROPAGATION
Microsorium pteopus Multiple-Shoots

Aloe

Alternathera
 MICROPROPAGATION
Multiple-Shoots
Agave

Zantedeschia

Larix
 MICROPROPAGATION
Multiple-Shoots

Sugarbeet

Cataceae

Tulip
 BROWNING - Remedies

Browning

Prevention
of browning

Removing phenolic compounds

Modifying redox potential
Inactivating phenolase enzymes
Reducing phenolase activity and substract available
 BROWNING – Remedies
Removing phenolic compounds

PVP (polyvynyl pyrrolydon)

Charcoal
Contamination – Cure by ELISA
 Stage II: Proliferation of axillary shoots

Characteristic of shoot system

After Stage 0, the shoot were regenerated in vitro after 30-60-day that appeared as (fig11.4):
(1) A single unbranched shoot system (potato, sweet potato, teak…)  use nodes
(2) A multiple-shoots system (banana, sugarcane, pineapple…)  use shoots

Cultured medium
(1) MS (1962) for agricultural plants and WPM (1980) for forest trees
(2) Single node cuttings: non-PGRs
(3) Multiple-shoots: a combination of cytokinin (0.1-3mg/l) and auxin (0.1-0.5mg/l) (fig11.5/6)
(4) Antioxidants: activated charcoal, PVP, ascorbic acid, citric acid
(5) Organic matter: coconut water, banana, potato…

Subculture (proliferation)
(1) Single node cuttings: using 1-2 node to produce unbranched shoots
(2) Multiple-shoots: divided to 1-3 cluster and cultured on proliferrated medium to produce
multiple-shoots
(3) Subculture interval: 21-45-days

Growth room
(1) Temperature 25+2oC, humidity 65-70%, light intensity: 33μM/m2/s
(2) Lighting: 8-12hrs
A growing explant can be induced to produce vegetative shoots by
including a cytokinin in the medium. A cytokinin is a plant growth
regulator that promotes shoot formation from growing plant cells
Fig 6.4: (a) Stage II shoot multiplication is achieved by repeated formation of axillary
shoot clusters from leaf explants containing lateral buds (Aronia arbutifolia L.).
Depending on the species, individual microcuttings or shoot clusters may be rooted and acclimatized ex
vitro. Bar=10mm. (b) For maximum survival. Stage III rooting may be required prior to acclimatization to
ex vitro conditions. (c) Rooted and acclimatized Stage IV plants
Stage II : Micropropagation – Cutting technique
Stage II : Micropropagation – Multiple-shoots technique
Stage II : Micropropagation - Double layer technique

Knop (macro=1/2)
Charcoal
Sugar
Auxin
Reduce production cost – Robotization (1)
Reduce production cost – Robotization (2)
 Reduce production cost – Bioreactor (3)

Nalgene

RITA (Cirad)

Using bioreactor for micropropagation

Life reactor (Osmotek)
 Stage III: Pre-transplant (rooting)

Process
(1) Elongation of shoots prior to rooting
(2) Rooting of individual shoots or multiple-shoots
(3) Fulfilling dormancy requirements of storage organs by cold treatment
(4) Pre-hardening cultures to increase survival

Regeneration
(1) Single node cuttings: regeneration to a single unbranched shoot
(2) Multiple-shoots: separation into each shoot (including leaves)

Cultured medium
(1) MS (1962) for agricultural plants and WPM (1980) for forest trees
(2) Single node cuttings: non-PGRs or auxin in low concentrations (0.1-0.5mg/l)
(3) Multiple-shoots: auxin (0.1-0.5mg/l) (fig11.7/8)
(4) Antioxidants
(5) Organic matter: coconut water, banana, potato…

Growth room
(1) Temperature 25+2oC, humidity 65-70%, light intensity: 33μM/m2/s
(2) Lighting: 8-12hrs

Natural conditions
Indirect sunlight (33-80μM/m2/s)
Growing shoots can be induced to produce adventitious roots by including
an auxin in the medium. Auxins are plant growth regulators that promote
root formation. For easily rooted plants, an auxin is usually not necessary
and many commercial labs will skip this step.
 Stage III: Rooting
Olive

Amarylis

Prunus
 Stage III : Rooting

Iris

Heliathus annus

Varietgated hosta
Stage IV: Transfer to natural environment for acclimatization

Hardening under indirectly:

natural light = (33-66μM/m2/s),
temperature = 28+2oC

Acclimatization in nursery:
light intensity = 3000lux,
humidity = 100%,
shaded cover = 15-day,
temperature = 28+2oC
A growing, rooted shoot can be removed from tissue culture and
placed in soil. When this is done, the humidity must be gradually
reduced over time because tissue-cultured plants are extremely
susceptible to wilting
Ultimate success of shoot culture depends on
ability to acclimatize vigorously growing
quality plants from in vitro to ex vitro conditions
Stage IV: Acclimatization – Mist house
Stage IV : Acclimatization - tunels
Stage IV : Acclimatization – humiditiy devices
Stage IV : Acclimatization – multi-layer cells
Stage IV : Acclimatization - substract
Stage IV : Acclimatization - transplanting
 Micropropagation of Rose
Stage 0 : Preparation of explant and meristem tip culture

ROSE

Cut young buds

Rose plant irrigated in base

Explant sterilization

Release the leaves

 Stage I : Meristem tip and internode culture

ROSE

Preparing the nodes

Cut the shoot tip

Culture ininvitro Finish sterilization

 Stage II : Induction of multiple shoots

ROSE

In vitro multiple-shoots Release the base

Divide to small cluster

Release the weaken shoots
 Stage III : Micropropagation
via multiple-shoot culture tecnique

ROSE

Propagule

Culture into bottle

Explants for proliferation

Stage IV : Acclimatization

ROSE
Micropropagation of Banana
Micropropagation of Banana
Micropropagation of Banana
 Micropropagation of Banana

1mos

11mos
10mos
2mos

4mos

9mos
 Micropropagation of Banana

11mos
Micropropagation of Banana
Micropropagation of Banana
Micropropagation of Banana
National potato seed system
 Agricultural characteristics

1. Important food plant

2. Infected by pathogen (fungi and virus)
3. Seed storage through 9 months under tropical climate
4. Fast degeneration by pathogens and environments leading low
yield and quality
5. Dalat city with mild climate favored for seed production around
the year
6. Application of plant biotechnology for set up national potato seed
production system
 National potato seed system

1. Meristem tip culture + chomotherapy

2. In vitro micropropagation
3. G0 seed production
4. G1 seed production
5. G2 seed production
6. Commercial production
Scheme of potato micropropagation
Meristem tip culture
Conservation - Exchange
Micropropagation via cutting technique
Micropropagation
 Micropropagation
via liquid culture technique
Certified seed production
 Micropropagation on sand tray

Step - 1

Step - 2
Micropropagation on soil row

Step - 3
 Potato plantlets

Step - 4
G0 seed production - minituberlets

Step - 5
G1 seed production - tuberlets
G2 seed production - tubers
Potato commercial production