BIOT3113

Biotechnology 1
Tissue Culture laboratory Manual
2012
BIOT3113 BIOTECHNOLOGY I

PLANT TISSUE CULTURE

Lecturer, Resource Person: Dr. Sylvia Mitchel

INTRODUCTION

Plant tissue culture comprises a set of in vitro techniques, methods and strategies that are

part of the group of technologies called plant biotechnology.

Plant tissue culture can be used to propagate clones of a plant. There are various reasons

this may be done, among them: to create exact copies of plants that produce particularly

good flowers or fruits, or otherwise desirable traits for some purpose; to quickly produce

mature plants; to produce multiples of plants in the absence of seeds or necessary

pollinators to produce seeds. Plant tissue culture can also be used to regenerate whole

plants from plant cells that have been genetically modified.

Tissue culture technique provide a means for:

 Rejuvenation of the plants (provide new plants from mature adult tissue)

 Phytosanitary improvement - plant tissue culture can be used to obtain, maintain,

and mass propagate specific pathogen-free plants. The concept behind indexing
plants free of pests is closely linked to the concept of using tissue culture as a
selection system. Plant tissues known to be free of the pathogen under
consideration (viral, bacterial, or fungal) are physically selected as the explant for
tissue culture.

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  Rapid clonal propagation – mass production of plant with desirable traits. Also
called micropropagation.
 Somatic embryogenesis – another in vitro propagation method used mainly for
tree crops to obtain somatic embryos that germinate with a tap root.

 In vitro germplasm conservation.

 Selection of plants with enhanced stress or pest resistance: The search for these
superior individuals can be tremendously accelerated using in vitro systems. Such
systems can attempt to exploit the natural variability known to occur in plants or
variability can be induced by chemical or physical agents known to cause
mutations.

 Somatic Hybridization: Tissue culture techniques such as protoplast culture allow

for manipulations such as the fusion of plant cells from species that may be
incompatible as sexual crosses

REQUIREMENTS FOR TISSUE CULTURE:

• Growth chamber
• Planting material
• Media components
• Glassware -eg petri dishes, beakers, test tubes, jars
• Instruments -eg. Forceps, hot plates
• Lamina flow
• Distiller
• Autoclave

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MICROPROPAGATION
Micropropagation is the most common application of tissue culture. It is the art and
science of plant multiplication in vitro. The process includes a number of steps:

 stock plant care (Stage 0)

 explant selection, sterilization and initiation into culture (Stage I)
 establishment and multiplication of in vitro plantlets through a series of
subculturing (Stage II)
 rooting of in vitro plantlets (Stage III)
 acclimatization/hardening (Stage IV)

Micropropagation allows for the production of large numbers of plants from small pieces
(explant) of the stock plant in a relatively short period of time. Depending on the species
in question, the original explant may be a shoot tip, leaf, lateral bud, stem or root. It is
important that the explant has a preformed meristem so as to ensure genetic integrity. In
most cases, the original plant is not destroyed in the process. Once the explant is placed
in culture, proliferation of apical and lateral buds results in tremendous increases in the
number of shoots available for rooting. A single explant can be multiplied into several
thousand plants in less than one year. Once established, actively dividing cultures are a
continuous source of microcuttings that can result in plant production under greenhouse
conditions without seasonal interruption.

SAFETY PRECAUTIONS

Initiation and subculture of plant cultures must be performed in a laminar flow hood or a
‘clean’ area. Before beginning your work, turn on the blower for 20 minutes; wipe down
all surfaces with 70% ethanol (EOH), and ethanol-wash your hands. When working under
sterile conditions, avoid wearing jewelry (i.e. rings). Contaminants are carried by
particles, such as dead skin or fallen hair. You should try not to lean into the laminar
hood. Use only sterile utensils and culture vessels and something to cut on (filter paper,
petri dish) in the flow. Do not pass your hand over open petri-dishes. All culture vessels,

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test tubes, sterilized explants etc. should be opened only in the laminar flow hood.
Utensils should be sprayed with EOH prior to use, but should be flame sterilized when
used (metal or glass utensil only). To do so, dip the utensil in 75% EOH and pass it
through the flame. Be careful not to get EOH near your hands. Allow a few seconds for
the utensil to cool and test if it is cool enough for use by touching a section with agar. Hot
utensils may damage tissue. Also, be careful not to place a hot utensil in the EOH
solution as it may catch fire.

The following safety precautions should be observed:

• wear a protective lab coat at all times

• wash hands before and after handling cultures and before leaving the lab
• decontaminate work surfaces with disinfectant (before and after)
• use pipette aids to prevent ingestion and keep aerosols down to a minimum
• no eating, drinking, or smoking
• bottle and flask mouths should also be flame sterilized before and after pouring media.
Flame the mouth of the vessel for a few seconds by passing it over a flame several times.
This will prevent contaminants from the edges from being introduced into the medium.
• in the event of a fire in the hood, turn off the gas supply, if the fire is contained in a small
vessel, suffocate the fire by covering with a lid or a larger vessel; if your clothes catches
fire, drop to the floor and roll to smother the fire
• autoclave all contaminated materials

You are expected to write up your experiments (i.e. any changes to the lab manual as
well as your observations and results) for each report. Report should be handed at the
end of exercise 2 and 4.

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Some parameters to consider when doing micropropagation:
Selecting plant sources: some species or even clones are easier to grow in culture than
others. Some respond reluctantly to culture, some do not respond at all, and many plants
have never been tried.

Choosing a growth medium: price, convenience, type of plant and purpose of the
micropropagation all enter into the decision of media choice.

The process of plant micropropagation involves a complex sequence of growth stages:

initiation, establishment in culture, rooting and acclimatization. Differences in the
requirements of different species and different cultivars of the same species make the use
of a general protocol for micropropagation impossible.

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EXPERIMENT 1

INITIATION OF YAM VINES INTO CULTURE

Preparation of initiation media
Table 1. Composition and preparation of one litre of Murashige and Skoog (1962)
medium

Constituents in mg for 1 Concentration of stock Volume of stock per liter

Constituents liter of medium solution g/L of medium (ml)

Macronutrients
NH4NO3 1650 165 10
KNO3 1900 190 10
CaCl2 anhydrous 440 44 10
MgSO4 370 37 10
K2HPO4 170 17 10

Micronutrients
KI 0.83 0.83 1
H3BO3 6.20 6.20
MnSO4.H2O 16.9 16.9
ZnSO4.7H2O 8.60 8.60
Na2MoO4.5H2O 0.25 0.25
CuSO4.5H2O 0.025 0.025
CoCl2.6H2O 0.025 0.025

Iron source
Na2EDTA.2H2O 37.3 37.3 1
FeSO4..7H2O 27.8 27.8
Vitamins
Myo-inositol 100 100
Nicotinic acid 0.5 0.5 1
Pyridoxine-HCL 0.5 0.5
Thiamine-HCL 0.1 0.1
Glycine 2 2

Carbon source Add as solid: 30 g/l

Sucrose
Make up all stocks with analytical grade chemicals and distilled water. Store macro- and micronutrients,
iron and hormone stocks in the fridge (4 oC) and vitamins in 1 ml aliquots in the freezer (-20 oC).

Note that in the preparation of initiation media 0.5 mg/L of the cytokinin
benzylaminopurine is added before adjusting pH to 5.6.

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Table 2 Commonly used plant growth regulators

Class Name Abbr Mol. Preparation of Comments

Wt. stock solution
Auxins p-Chlorophenoxyacetic acid PCPA 186.6 Dissolve in IAA is
2,4-Dichlorophenoxyacetic 2,4-D 221.0 NaOH except 2,4- photosensitive and
acid D which is readily oxidized by
dissolved in explants
Indole-3-acetic acid IAA 175.2 ethanol
3-Indolebutyric acid IBA 203.2
Napthoxyacetic acid NAA 186.2
Cytokinins 6-Benzylaminopurine BAP 225.2 Dissolve in
N-Isopenylaminopurine 2iP 203.3 NaOH and make
up to volume with
6-Furfurylaminopurine K 215.5 distilled water
(kinetin)
Zeatin Z 219.2
Thidiazuron TDZ NaOH
Gibberelins Gibberellic acid GA3 346.4 Freely soluble in Gibberellic acid is
water thermolabile

Preparation of 1 litre of initiation media

• Measure about 500 ml of distilled water in a 2 litre measuring cylinder and place it on a
stirring block.
• Measure and add all macro- and micronutrients along with vitamin mix from stock
solutions.
• Add ferrous salt and inositol
• Add 30g sucrose and allow to dissolve
• Adjust pH 5.6
• Make final volume to 1 Litre
• Add 2 g of phytagel
• Cover the cylinder and dissolve the phytagel by heating in an autoclave for 10 minutes.
• Remove the media from autoclave and pour about 12 ml into each test tube(clean)
• Cap the tubes and autoclave immediately for 15 minutes at 121 °C and 200 psi.
• Remove autoclaved media and place directly into a sterile lamina flow to cool and
solidify.

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Initiation of nodal segments

Equipment:
Lamina flow
Forceps
Scalpels and blades
Bunsen burners

Glassware:
Petri dishes
Beakers

The processes of plant micropropagation involve a complex sequence of growth stages:

initiation, establishment in culture, rooting and acclimatization.

Today you will select, surface-sterilize and initiate nodes of Dioscorea sp. (yam) into
culture.

Selection and surface-sterilization of explant

Select vines that are young, sturdy and actively growing.
• Remove all leaves from vines and cut vines such that each cutting contains at least
one node. Discard tip and three youngest nodes.
• Surface-sterilize the nodes by swirling in a beaker of 70% ethanol for 4 minutes.
• Transfer nodes to 20% bleach solution and leave for twenty minutes with
continuous shaking.
• Pour off bleach solution and rinse three times with sterile distilled water.

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DEMONSTRATION OF ASEPTIC TECHNIQUE
The importance of maintaining a sterile environment during in vitro culture of
explants cannot be over-emphasized. Contamination in tissue culture can originate from
two sources, either from micro-organisms on the surface (exogenous) or inside the tissue
(endogenous, systemic) of explants, or through faulty procedures in the laboratory.
• The lamina flow cabinet provides an environment for aseptic transfer in plant
tissue work. The air is sterilized by filtration.

o Wash hands with soap followed by spraying with 75% ethanol or put on gloves.
o Work in the back 2/3rds of the cabinet; avoid sweeping arm movements over the
work area. Keep your head out of the cabinet. Do not remove hands and put them
back in without spraying them again with alcohol. Do not talk or sneeze into the
flow.
o Set up the materials needed on the sides of the cabinet (vessels on one side,
sterilized explants on the other). No material should be between your work area
and the back of the flow.
o Cover culture containers promptly after inoculation with explant.
o Do not wave hands over open petri dishes.

Initiation of explants into culture:

Working under the lamina flow and applying suitable aseptic techniques:
1. Cut away damaged ends ie. bleach-damaged ends of vine.
2. Place nodes on pre-prepared initiation media (already sterilized).
3. Cover the culture tubes
4. Seal culture tubes containing the explants using parafilm.
5. Label and store culture tubes on clean shelves in the growth room at controlled
temperature and lighting (i.e. 20-25 ºC &16 hrs of light and 8 hrs of darkness) .

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EXPERIMENT 2

TRANSFER OF PLANTLETS TO ESTABLISHMENT MEDIA

Assessment of tissue culture

The explants have now been on initiation media for two weeks, inspect and describe your
observations (make note of any growth of the new plantlets). The growth of bacterial or
fungal colonies indicates contamination.

Establishment
New shoots and roots start to develop from the explants initiated in the previous exercise.
New shoots become established in culture when they are excised from and are able to
grow independently of explants. After four weeks established shoots are transferred onto
multiplication media.
Working under the lamina flow and using aseptic techniques:
• Place the explants in a petri dish
• Cut new shoots from the explants
• Cut shoots into nodal segments and place the nodes onto media (about 4 nodes per
culture jar). This will encourage the growth of more shoots. This media contains
basal salts and 0.1mg/L BAP.
• Seal test tubes containing the nodes using parafilm.
• Label and store culture tubes on clean shelves in the growth room at controlled
temperature and lighting (i.e. 20-25 ºC and 16 hrs of light)

Questions

1. What are the possible reasons for the observed level of contamination?

2. Tissue culture has been exploited in the biotechnological improvement of a

number of crops. Write short notes on how this technique has been applied any
two crops.

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EXPERIMENT 3

TRANSFER OF PLANTLETS TO MULTIPLICATION MEDIA

Multiplication
In this stage there is rapid proliferation of shoots resulting in large number of shoots
being produced from a single shoot. Shoots that have been established in culture are
transferred onto multiplication media. This media again is similar to the
establishment used for yams. It should be noted that different plant species may
require an alteration in media composition for optimal growth.

Multiplication media: Murashige and Skoog salts and vitamins containing 30g/L
sucrose, supplemented with 0.1mg/L BAP and 2g/L phytagel adjusted to pH 5.6.

Working under the lamina flow and using aseptic techniques,

• Remove the established plantlets from culture vessel and place on a sterile Petri
dish
• Cut the shoots such that each new cutting has at least one node
• Transfer nodes to fresh multiplication media (about 4 nodes per culture jar)
• Seal test tubes containing the nodes using parafilm.
• Label and store test tubes on clean shelves in the growth room at controlled
temperature and lighting.

These shoots are sub-cultured every four weeks until the desired number of plantlets
is achieved. It has been shown that the low concentrations of growth regulators used
in the in vitro media for yam, produces a reasonable multiplication rate.

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EXPERIMENT 4

ROOTING AND ACCLIMATIZATION

Rooting
This is the stage where actual plantlets are produced. 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. However for easily rooted plants like
yam, an auxin is usually not necessary.
In this exercise plantlets that have been growing in culture on rooting media (in the case
of yams, rooting can be obtained by subculturing onto a medium devoid of hormones).
After rooting, plantlets are ready for acclimatization and field transfer.
• Working under the lamina flow and using aseptic techniques, transfer shoots that
had been growing on multiplication media for four weeks onto rooting media
(same formulation as multiplication media but free of BAP).
• Seal test tubes containing the shoots using parafilm.
• Label and store test tubes on clean shelves in the growth room at control
temperature and lighting.

Growth on rooting media for six weeks allows for adequate root development before
acclimatization. Plantlets are now ready to be transferred to shaded house.

Acclimatization
A growing, rooted shoot (plantlet) can be removed from tissue culture and placed in soil.
When this is done, the humidity must be gradually reduced over time until the plantlets
have adjusted to their new environment, since tissue cultured plants are extremely
susceptible to wilting.

This process of adjustment is called acclimatization and involves the growth of new
leaves and roots that will allow the plantlets to survive under external conditions. This

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period of acclimatization allows the plants to develop the necessary physiological and
anatomical structure necessary for survival in the external environment. This takes 6-8
weeks.

Procedures:
• Take the culture tubes containing the plantlets with well developed root system to
the raised beds which have an overhead sprinkler system.
• Working with one culture tube (or baby food jar) at a time, remove plantlets and
shake gently to remove culture growth medium.
• Gently rinse the roots with distilled water and plant in the soil.
• Press the soil around the root and water them in.
• Cover the plantlets with a clear plastic cup to maintain an area off high humidity
around the plantlets. Make sure to use the soil around the edge of the cup to keep
it down.
• After two weeks, remove the soil from around the cup, and completely remove
the following week. (Method as described in Mitchell 1995)
• The hardened plants can now be lifted out with the soil and placed in potting bags
for transport to the field. They will be ready for the field 6-8 weeks after transfer
from in vitro culture. It has been shown that after four weeks the plantlets have
developed the necessary anatomical and physiological structures that will allow
them to survive in the open field (Wheatley, 2000).

Questions
1. Acclimatization has been one of the major obstacles to the use of tissue
culture in crop improvement. Explain why this process is necessary for the ex
vivo survival of tissue culture derived plants.

2. A number of attempts have been made at optimizing the hardening/

acclimatization stage. Give two examples of attempts to improve
acclimatization and explain the principle behind these methods.

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References

1. Asemota, H.N., Iyare, O.A., Wheatley, A.O.., Ahmad, M.H. (1997)

Acclimatization of in vitro grown yam (Dioscorea spp.) plantlets and some
enzyme changes. Trop. Agri. 74:243-247.

2. Lineberger, R. The Many Dimensions of Plant Tissue Culture Research.

http://aggie-horticulture.tamu.edu/tisscult/pltissue/pltissue.html

3. Mantell, S.H. and Hugo, S.A. (1989) Effects of photoperiod, mineral strength,
inorganic ammonium, sucrose and cytokinin on root, shoot and microtuber
development in shoot cultures of Dioscorea alata L. and D. bulbifera L. yams.

4. Mitchell S.A. and Ahmad M.H. (1999) Morphological changes of Dioscorea

trifida cv Short Neck Yampie and D. cayenensis cv Round Leaf Yellow Yam
linked to the number and size of harvested tubers. J. Hort Science 74 (4): 531-
539.

5. Mitchell S.A., Asemota H.N. and Ahmad M.H. (1995a) Effects of explant source,
culture medium strength, and growth regulators on the in vitro propagation of
three Jamaican yams (Dioscorea cayenensis, D. trifida and D. rotundata). J. Sci.
Food Agric. 67: 173-180.

6. Mitchell S.A., Asemota H.N. and Ahmad M.H. (1995b) Factors affecting the in
vitro establishment of Jamaican yams (Dioscorea spp.) from nodal pieces. J. Sci.
Food Agric. 67: 541-550.

7. Mitchell S.A., Asemota H.N., Curtis B. and Ahmad M.H. (1995) Optimizing the
transfer of tissue cultured yam (Dioscorea spp.) plantlets to the terrestrial
environment. In: Proceedings of the Second Conference of the Faculty of Natural
Sciences, UWI, Mona, March 7-9, pg 19.

8. Mitchell, S.A. (2000) Development of a micropropagation system for Jamaican

yams (Dioscorea spp.): Initiation to Tuber production. Ph. D Thesis. University
of the West Indies, Mona. Jamaica.

9. Wheatley, A.O. (2000) Molecular assessments of some biochemical,

physiological and environmental factors affecting the lab to field transfer of in
vitro derived yam (Dioscorea spp.) plantlets and storage of the resultant tubers.
Ph. D Thesis. University of the West Indies, Mona. Jamaica.

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