This document provides suggestions for successful plant micropropagation based on a literature review. It discusses selecting appropriate plant species and explant locations, developing nutrient-rich media with the proper pH and additives to promote growth while preventing contamination, and maintaining sterile conditions throughout the process. Key factors include using leaf explants from orchids and other easily propagated plants, adding specific macronutrients, vitamins, auxins, and cytokinins to the media, adjusting the pH to 5-6, and thoroughly sterilizing explants and materials. Attention to all stages from initial plant and explant selection to media composition and sterile technique can optimize micropropagation outcomes.

1. Literature Review:
Suggestions for Successful
Plant Micropropagation
Bailey Banbury
September 6, 2015
Brigham Young University – Hawaii
55-220 Kulanui Street
Laie, Hawaii 96762

2. I. Table of Contents
I. Table of Contents………………………………………………………………………..1
II. Introduction…………………………………………………………………………….2
III. Plant Selection……………………………………………………………………..2 - 4
A. Species
B. Explant Location
IV. Media………………………………………………………………………....……4 - 9
A. Microbe Prevention
B. Nutrients
i. Macronutrients
ii. Micronutrients
iii. Vitamins
iv. Auxins
v. Cytokinins
C. Activated Charcoal
D. Gelling Agents
E. pH
V. Sterilization……………………………………………………………….………9 - 10
A. Environment
B. Materials
C. Explants
VI. Growth Period……………………………………………………………………….11
A. Temperature
B. Lighting
C. Contamination
V. Works Cited……………………………………………………………………...12 - 14
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3. II. Introduction
Nearly all plant cells retain the property of totipotentcy, which is the capability of
a single cell to develop by regeneration into an entire organism (Vasil and Hildebrandt
1965). Drawing parallels to the human stem cell, this property allows plant cells to be
useful in many different practices, ranging from agricultural mass quantity replication to
developments in medicine. One major utilization of totipotent plant cells is plant
micropropagation. Micropropagation is defined as “the aseptic culture of cells, pieces of
tissue, or organs,” (Polking and Stephens 1995). While different methods of propagation
do exist, this writing will focus on the technique of plant tissue culturing. Plant tissue
culturing involves the use of an explant to regenerate identical cells. However, plants
require extremely sensitive and variable conditions for ideal propagation. Below are
suggestions, based on extensive literature review, for conditions which might promote
optimum success of plant tissue culturing.
III. Plant Selection
A. SPECIES
The first component of successfully propagating a plant is proper plant selection.
Selecting a proper plant may seem difficult, as some plants are simply not suited for
propagation. In a book entitled Plant Cell and Tissue Culture, the authors state that, “Not
all plants lend themselves well to in vitro culture. It is a mystery why members of one
taxonomic family respond to in vitro culture more actively than those of another,” (Vasil
and Thorpe 2013). This implies that plants which are known to propagate effectively
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4. simply become established as such through documented successful experimentation. The
first plant to be successfully propagated on a commercial scale was the orchid,
specifically the Cymbidium genus (Arditti and Krikorian 1996). Orchids have continued
to be propagated on a mass scale with much success. While other plants can be
effectively propagated (Wimber 1963), the orchid was the first and continues to be one of
the most frequently propagated plants because of its visual appeal, relatively low cost,
and accessibility.
B. EXPLANT LOCATION
Plants can be propagated through many different techniques. Some of these
techniques include stem tip culture, seed germination, and tissue culture. Varied
techniques require various different explant origin locations. In other words, to perform a
seed germination, the initial plant cutting will come from a different part of the parent
plant than the explant of a tissue culture (Arditti and Krikorian 1996). The ideal
technique for propagation seems to be tissue culture, due to simple explant extraction, as
well as the
fundamental property that the explant is simply a portion of a leaf of a parent plant
(Churchill et al. 1973). In Churchill’s writings as well as a comprehensive reading
entitled Micropropagation of Orchids, it is further specified that for leaf tissue culture,
the portion of the leaf which the explant is trimmed from does matter. Arditti states that
success with these procedures is heavily dependent upon removal of explants prior to full
leaf tip differentiation and loss of callus formation ability (Arditti 2008). Forming callus
tissue is critical towards development, causing the leaf tip selection of explants to be an
important part of selection. Arditti also explains that a major advantage of leaf-tip
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5. cultures is that the actual removal of the explant does not endanger the parent plant,
which can happen with other propagative techniques. Aside from leaf tips, the node of
orchids have also been used for successful propagation (Deb and Pongener 2012).
IV. Media
The required chemical needs within a medium drastically vary plant to plant.
Bhojwani and Ranzdan made not that there is not a single medium which can be
suggested as being entirely satisfactory for all types of plant tissues and organs (2013).
However, it should be noted that this medium is critical toward success in plant culture,
which is largely determined by the quality of nutrient media (Vasil and Thorpe 2013). By
effectively researching and manipulating the media for plant tissue culture, explants can
better thrive after transfer.
A. MICROBE PREVENTION
Perhaps one of the most common and frustrating complications of plant
micropropagation is contamination through fungal, bacterial, or other microbial means. It
may be implied that there are stock plants which are pathogen free, however Lineberger
states these plants can only be speculated to be free of pathogens, as little research
documenting viral, bacterial, or fungal diseases transmitted through propagation is
available (Lineberger). While means are also taken through sterilization, this section is
about effective measures to prevent microbes, solely by manipulation of the media rather
than environment. There is no clear ruling towards the use of antibiotics within media in
micropropagation, mainly because the sensitivities and succeptabilities of each plant can
drastically vary (Vasil and Thorpe 2013). In one study performed on Cattleya and
Stanhopea orchids, researchers sought to determine the effects of phytotoxicity of 25
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6. various fungicides, bactericides, and compounds on the orchid media (Thurston et al.
1979). These substances were combined in 9 different ways, and then employed on the
orchid seedlings. While each of the instances did prevent contamination, multiple
seedlings had impaired development. This is a glimpse into the difficulty that antibiotics
pose, which is their toxicity to both intended microbes and the unintended explant. Plant
Tissue Culture: Techniques and Experiments states that in general, the addition of
antibiotics has not been very useful, because they can be toxic to the explant (Smith
2012). On the other hand, some propagative techniques have benefitted from the use of
chemical compounds to prevent microbes. Vasil and Thorpe state that generally,
Carbendazim and Fenbendazole can both be used safely at a 30 mg/L dosage, and that 20
mg/L of Imazalil can typically be effective in most plants (2013). Perhaps the best
method for preventing bacteria and fungus solely within the media of various specific
plant species can be found in the work of Lifert and Waites from 1990. This reading
provides extensive reviews of various plants along with their physical and chemical
properties, and effective microbe prevention. Another component towards bacterial
growth in explants stems not from surface microorganisms, but those within the actual
culture. In a published work entitled Plant Cell Culture Protocols, the authors highlight
this issue stating that when selecting plants for tissue culture, the main question is not
whether they are infected on the surface with microorganisms that can be eliminated by
surface sterilization of the explant; but whether there are inter or intra-cellular endophytes
that may enter the cultures to cause contamination (Loyola-Vargas and Vazquez-Flota
2006). Endophytic contamination presents an incredible problem to agricultural
micropropagative techniques, which are aimed at mass cloning of edible plants. However,
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7. such complications are much more evident and crucial in mass traditional agricultural
practices than laboratory propagation. When performing tissue culturing of orchids,
which use leaf explants, the reading goes on to state that leaf and petiole explants can be
used from plants whose tissues are considered to give rise to genetically stable shoots.
The criterion of visible expression of contamination is generally used as an indicator of
non-aseptic status. To summarize, some literature emphasizes the toxicity to explants
caused by antibiotics and antifungals typically outweigh their beneficial role. However,
this can not be used as a blanket statement, as each plant’s chemical properties vary
drastically.
B. NUTRIENTS:
i. Macronutrients
Macronutrients required within the media include C, H, O, N, P, S, Ca, K, and
Mg. Of those, Carbon is the most crucial. It’s important to note that unlike typical plants,
explant cells in culture are not typically photosynthetic (Smith 2012). The ideal
supplemental Carbon source for media is sucrose at 3% (w/v) ratio (Bhojwani and
Razdan 1996, Deb and Pongener 2012, Churchill et al. 1973), although glucose and
fructose are both also frequently used at the same concentration (Bhojwani and Razdan
1996, 21).
ii. Micronutrients
Micronutrients for media use include Fe, Mn, Cu, Zn, B, and Mo (Bhojwani and
Razdan 1996). These vary by plant, however, and play a less crucial role than
macronutrients.
iii. Vitamins
7

8. Vitamins are often added to media for plant tissue cultures. One method suggests
storing them in the freezer before use, and includes Nicotinic acid 100 mg/mL, Thiamine
HCl 1000 mg/mL, and Myo-Insitol 10,000 mg/mL (Masawa 1994). Bhojwani and
Razdan also noted that some plants require filtering the vitamins used in a microfilter, as
they may be too delicate or heat labile to autoclave. Again, these may vary by different
plant type (Davidson College 2012).
iv. Auxins
Auxins are plant growth hormones, which have been determined to play a large
role in plant growth and development. Auxins promote cell division, and can be synthetic
or naturally occurring. Interestingly, extracts from coconuts have been identified to
contain auxins, which have been proven to promote growth (Dix and Staden 1982,
Mauney et al. 1952). Mauney et al. explain the benefit of using coconut water, as it can
effortlessly be incorporated in orchid media with no loss of activity as a result of
autoclaving (1952). Autoclaving is usually done at 121 degrees Celsius for 15 minutes.
Leva and Rinaldi state that the common auxins used in plant tissue culture media include
indole-3-acetic acid (AA), indole-3-butricacide (IBA), 2,4-dichlorophenoxy-acetic acid
(2,4-D) and naphthalene-acetic acid (NAA) (Leva, Rinaldi ). Lea and Rinaldi state 2,4-D
promoted growth and activity 12 times higher than IAA. It should be noted that when
using IAA, solutions should be prepared fresh at time of media preparation rather than
storing for long periods of time, because it loses its growth properties. Another auxin
commonly incorporated to plant growth media include BAP (Bhojwani and Razdan 1996,
Deb and Pongener 2012, Churchill et al. 1973). Churchill suggests a concentration of
0.5mg/L of BAP and 1 mg/L of 2,4-D. 2,4-D appears to be most effective.
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9. v. Cytokinins
Bhojwani and Razdan also explain the importance of cytokinins, stating that in
tissue culture media, cytokinins are incorporated mainly for cell division and
differentiation of adventitious shoots from callus and organs (1996). When cytokines and
auxins are both present, their concentration dictates the plant tissue development. A low
concentration of auxins in the presence of a high concentration of cytokinins will produce
shoot development, whereas a high concentration of auxins in the presence of a low
concentration of cytokinins will produce root development. An equal balance of cytokine
and auxin concentration induces callus development. A study was completed on
Dendrobiums, in which it was identified that after 60 days, explants which contained no
cytokinins became necrotic, while those with cytokinins induced embryo formation from
leaf explants (Chung et al. 2005).
C. ACTIVATED CHARCOAL:
Adding activated charcoal to plant tissue culture media is a technique that was
implemented by Peter Werkmeister in 1970. The charcoal adsorbs toxic substances which
form in plant media, after release from the explant (Chugh et al. 2009). Chugh et al.
explain that this is necessary, because when explants are isolated from mature plants,
they release phenolics. When oxidized, phenolics become toxic to the tissue culture cells.
Activated charcoal is also sometimes added to micropropagation media because while
inhibiting growth in soybeans (Leva and Renaldi 2012), charcoal promotes plant tissue
growth when used as a medium additive (Loyola-Vargas and Vazquez-Flota 2006).
D. GELLING AGENTS:
Overall, between a 0.6 to 1.6% agar (depending on the plant) is the most
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10. commonly used gelling agent and corresponding concentration to make suitable media on
Petri dishes for explants. Other agents included 0.1 – 0.3% Gelrite (Vasil and Thorpe
2013), and a reasoning that at higher concentrations, the medium becomes physically
hard, and does not allow the diffusion of nutrients into the tissues, (Bhojwani and Razdan
1996).
E. pH
At pH values outside the accepted range, explants do not take up their nutrients,
and will die (Churchill et al. 1973, Dodds and Roberts 1985, Vasil and Thorpe 2013).
Similar to other factors, the appropriate media pH varies by plant species. However, the
acceptable range for most plants falls between 5.0 and 6.0 (Polking and Stephens 1995,
Bhojwani and Razdan 1996, Vasil and Thorpe 2013, Smith 2012, Thurston et al. 1973,
Deb and Pongener 2012, Churchill et al. 1973, Dodds and Roberts 1985).
V. Sterilization
A. ENVIRONMENT:
In addition to the most sterile media possible, a sterile environment is also
required to prevent contamination. The most ideal setting for a sterile environment is a
laminar flow hood (Polking and Stephens 1995, Vasil and Thorpe 2013, Fogh 2012).
However, these can be costly, and as Loyola-Vargas et al. previously mentioned, proper
aspectic technique truly can prevent contamination, and has been proven as such. Perhaps
the most common technique to do such in terms of the environment includes using an
absolute cleansing of the surface with 70% ethanol (Vasil and Thorpe 2013, Smith 2012,
Cassells 1997, Thurston et al. 1973, and Fogh 2012). Doing so kills almost all
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11. microorganisms that may be detrimental to the plant.
B. MATERIALS:
Instruments are most commonly autoclaved at 120-122 degrees Celsius for 15
minutes. However, Vasil and Thorpe also present the suggestion of immersing
instruments in absolute ethanol, and then flaming them for sufficient sterility.
C. EXPLANTS:
Sterilizing explants is one of the most important yet diverse components of plant
micropropagation. Similar to other elements of micropropagation, this component of
protocol largely depends on the species of plant at hand. A rinse of the explants is used
after they are cut. The elements of this rinse vary drastically. However, one common
theme between many protocols seems to be the importance of agitation (Polking and
Stephens 1995, Smith 2012, Deb and Pongener 2012, Churchill et al. 1973, Arditti 2008,
and Dodds and Roberts 1985). This literature states agitation enhances the contact of the
liquid rinsing agents. Specifically practical towards species of orchids, after being rinsed
with sterile water to get rid of soil and obvious surface contaminants and 70% ethanol,
explants can be washed in a detergent of concentration 0.1% v/v, such as basic
dishwashing detergent, Tween-20, or Extran laboratory detergent for about 2 minutes
(Arditti 2008, Chugh et al 2009, Polking and Stephens 1995, Churchill et al. 1973,
Masawa 1994). Following this, a 2 minute sterile water rinse is used. Smith and Masawa
both state an additional disinfecting agent can be useful, such as 5.25% NaOCl. Smith
also suggests 3-10% H2O2 or PPM, but also warns that this can be toxic to plant tissue.
Polking and Stephens also cite the use of bleach, while others warn of it’s toxicity to
plants. Churchill et al. 1973 suggest CaOCl as such an agent. All protocols end in 3 to 5
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12. rinses of sterile water.
VI. Growth Period
After plating explants on Petri dishes of media, they are wrapped in Parafilm and
stored, while monitored for growth. During this period, specific conditions are set for
optimum growth and contamination prevention.
A. TEMPERATURE:
The ideal temperature is somewhere within the range of 24 and 26 degrees
Celsius (Polking and Stephens 1995, Chugh et al. 2009, Vasil and Thorpe 2013, Deb and
Pongener 2012, Churchill et al. 1973,
B. LIGHTING:
Lighting is another important component towards explant survival. In general,
plants are required to receive a photoperiod of approximately 12 – 18 hours per day
(Vasil and Thorpe, Polking and Stephens 1995, Chugh et al. 2009, Deb and Pongener
2012, Churchill et al. 1973) depending on different species. One other interesting
notation on lighting is put forth by Masawa, who states that “present technology dictates
the use of stainless steel tanks for growth of plant cells on an industrial scale, thus in
general, eliminating the use of light,” (1994). It will be interesting to see how light usage
transforms in the future of micropropagation techniques.
C. CONTAMINATION:
If contamination occurs, it is important to either replate the explant on a new
media plate, or attempt to cut the affected area off, as the microorganisms can quickly
spread and cause complete contamination, inhibiting growth and survival. Check for
contamination daily, which will usually present itself as an obvious physical change.
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13. V. Works Cited
Arditti, J. 2008. Micropropagation of Orchids. 2nd
Edition. Massachusets, Blackwell
Publishing. 1523 pp.
Arditti, J. and A. Krikorian. 1996. Orchid Micropropagation: The Path From
Laboratory to Commercialization and an Account of Several Unappreciated
Investigators. Botanical Journal of the Linnean Society 122: 183-241.
Bhojwani, S.S. and M.K. Razdan. 1996. Plant Tissue Culture: Theory and Practice.
Delhi: University of Delhi. 511 pp.
Cassells, A.C. 1997. Pathogen and Microbial Contamination Management in
Micropropagation. Volume 12. Dordrecht: Springer Netherlands. 300 pp.
Chugh, S., Guha, S., and U. Rao. 2009. Micropropagation of Orchids: A Review on the
Potential of Different Explants
Chung, H., Chen, J. and W.C. Chang. 2005. Cytokinins Induce Direct Somatic
Embryogenesis of Dendrobium Chiengmai Pinka dn Subsequent Plant
Regeneration. In Vitro Cellular Development Biology - Plant 41(6): 765-769.
Churchill, M.E., Ball, E. and J. Arditti. 1973. Tissue Culture of Orchids, Methods for
Leaf Tips. New Phytologist. 72(1): 161-166.
Chugh S., Satyakam G., and U. Rao. 2009. Micropropagation of Orchids: A Review on
the Potential of Different Explants. Scientia 122(4):507-520.
Davidson College. Plant Tissue Culture. 2002.
Deb, C.R. and A. Pongener. 2012. Development of a Cost Effective In Vitro
Regenerative Protocol of Cymbidium aloifolium (L.) Using Nodal Segments As
13

14. An Explants Source. International Journal of Chemical and Biochemical Sciences
1:77-84.
Dix, L. and V. Staden. 1982. Auxin and Gibberlilins-Like Substances in Coconut Milk
and Malt Extract. Plant Cell Tissue and Organ Culture 1(1): 239-245.
Dodds, J. and L. Roberts. 1985. Experiments in Plant Tissue Culture. 2nd
Edition. New
York, Cambridge University Press. 232pp.
Fogh, J. 2012. Contamination in Tissue Culture. New York: Academic Press. 300 pp.
Leva, A. and M. Rinaldi. 2012. Recent Advances in Plant In Vitro Culture. Croatia,
InTech, 320pp.
Lineberger, R. D. The Many Dimensions of Plant Tissue Culture Research. Texas A&M
University.
Loyola-Vargas V.M. and F. Vazquez-Flota. 2006. Plant Cell Culture Protocols. 2nd
Edition. New York, Humana Press. 393 pp.
Mauney, J., Hillman, W.S., Miller, C., and M.F. Skoog. 1952. Bioassay, Purification, and
Properties of a Growth Factor From Coconut. Physiologia Plantarum 5(4):79-85.
Misawa, M. 1994. Plant Tissue Culture: An Alternative For Production of Useful
Metabolite. Food and Agriculture Organization Agricultural Services Bulletin
108.
Polking, G. and L. Stephens. 1995. Plant Micropropagation Using African Violet Leaves.
Smith, R. 2012. Plant Tissue Culture: Techniques and Experiments. 3rd
Edition.
London: Academic Press. 208 pp.
Thurston, K., Spencer, S., and J. Arditti. 1979. Phytotoxicity of Fungicides and
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15. Bactericides in Orchid Culture Media. American Journal of Botany 66(7): 825-
835.
Vasil, I.K. and T. Thorpe. 2013. Plant Cell and Tissue Culture. 594 pp.
Wimber, D. E. 1963. Clonal Multiplication of Cymbidiums Through Tissue Culture of the
Shoot Meristem. American Orchid Society Bulletin 32:105-107.
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