Literature Review:Suggestions for Successful Plant Micropropagation

Bailey Banbury

September 6, 2015

Brigham Young University – Hawaii55-220 Kulanui StreetLaie, Hawaii 96762

I. Table of Contents

I. Table of Contents………………………………………………………………………..1

II. Introduction…………………………………………………………………………….2

III. Plant Selection……………………………………………………………………..2 - 4A. SpeciesB. Explant Location

IV. Media………………………………………………………………………....……4 - 9A. Microbe PreventionB. Nutrients

i. Macronutrientsii. Micronutrientsiii. Vitaminsiv. Auxinsv. Cytokinins

C. Activated CharcoalD. Gelling AgentsE. pH

V. Sterilization……………………………………………………………….………9 - 10A. EnvironmentB. MaterialsC. Explants

VI. Growth Period……………………………………………………………………….11A. TemperatureB. LightingC. Contamination

V. Works Cited……………………………………………………………………...12 - 14

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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

simply become established as such through documented successful experimentation. The

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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

cultures is that the actual removal of the explant does not endanger the parent plant,

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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

various fungicides, bactericides, and compounds on the orchid media (Thurston et al.

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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,

such complications are much more evident and crucial in mass traditional agricultural

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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

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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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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:

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Overall, between a 0.6 to 1.6% agar (depending on the plant) is the most

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,

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Cassells 1997, Thurston et al. 1973, and Fogh 2012). Doing so kills almost all

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

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plants. Churchill et al. 1973 suggest CaOCl as such an agent. All protocols end in 3 to 5

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

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spread and cause complete contamination, inhibiting growth and survival. Check for

contamination daily, which will usually present itself as an obvious physical change.

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.

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Deb, C.R. and A. Pongener. 2012. Development of a Cost Effective In Vitro

Regenerative Protocol of Cymbidium aloifolium (L.) Using Nodal Segments As

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.

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Thurston, K., Spencer, S., and J. Arditti. 1979. Phytotoxicity of Fungicides and

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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