Aqua Botanic - Tissue Culture Propagation of Aquatic Plants

The Spotlight Tissue Culture Propagation of Aquatic Plants: An Overview, Plus "How to do it in your kitchen"

by Carol M. Stiff [Best viewed in Netscape 4.0 and above]

Wetlands are critical to the health of our environment and much attention has been paid lately to their restoration. Finding sufficient numbers of aquatic plants to supply these restoration projects is not easy, and micropropagation has been suggested as a solution to increasing the numbers of these valuable plants (Burgess 1996). This paper will discuss some of the basics of plant tissue culture, how it has been applied to aquatic plants, including specific examples, a bibliography of publications and websites, and, finally, how you can conduct this with simple equipment and supplies in your kitchen.

Micropropagation involves the mass production of plants, from small parts (such as shoot tips, axillary buds, nodes, rhizomes) in sterile controlled environments. The main advantage of this technique, also referred to as in vitro propagation or plant tissue culture, is that a small piece of plant can produce hundreds of new plants which can be identical to the "mother" plant. Plant produced in tissue culture also tend to be disease free. While this does not eliminate disease once the plants are in the outdoor environment, it does give the plants a healthier start and a better chance of defense

While theoretically any plant part can produce new plants from single cells, the plant part, or "explant" of choice, is a terminal or axillary bud. These tend to produce clones of the mother plant and therefore should exhibit the desirable characteristics of the source plants. Other explants can be used, such as leaves, flowers, internode, embryos, etc, but there is a greater tendency of variation to occur.

The major steps in plant tissue culture involve:

Establishing an explant in a sterile culture environment free of contamination from bacteria and fungi
Inducing a proliferation of multiple shoots
Transferring these shoots to fresh media to induce further growth
Rooting in vitro or in soil
Acclimating the plantlets to the outside environment.

Explants are usually rinsed in dilute commercial bleach with a few drops of detergent to get rid of contaminants. The strength of the disinfecting solution and the duration of soaking the explant in the disinfectant will vary from species to species. Other disinfectants, such as calcium hypochlorite, alcohol, PPM, or even antibiotics, may be used in the procedure. Explants are rinsed in sterile water and cultured on a medium that will support the growth of the meristem or other plant part. See:

http://www.kitchenculturekit.com/africanviolet.htm

http://www.kitchenculturekit.com/avleafdevelop.htm

The type of medium used will also vary with the species that is being cultured.
Biocides, such as PPM, may also be included in the medium to minimize contamination.

Plant growth regulators may be included in the media to induce shoot growth or rooting. These should be used with caution as they can induce variation in the resulting plants.
Once a clean culture is established and multiple shoots have been produced, these can be aseptically separated and placed in fresh culture to continue growth or to induce root formation. A single plant part can produce hundreds to thousands of offspring in a single year.
Plantlets, whether rooted in tissue culture, or in soil, must then be acclimated to the outside world (lower humidity, higher temperatures, diseases and pests). Plantlets that have been transferred to soil are usually kept in a humid environment for a few days. Gradually the humidity is lowered until the plant can tolerate the "real world".

Tissue culture techniques can also be used on seeds that are hard store, or are difficult to germinate in nature. Eelgrass seeds can be surface sterilized and stored for several years without damage from fungi or bacteria. Cord grass has a germination inhibitor, abscisic acid, that needs to be leached out before germination occurs. Under certain tissue culture conditions, the inhibitor can be overcome and germination occurs immediately.

Michael Kane, Ph.D., at the University of Florida, the expert in aquatic plant tissue culture, has many papers on the various procedures used with different species. Some of the specifics are listed below.

Explants that he has used include: nodes, internodes, aerial plants, seeds, embryos. The explants are usually rinsed for ½ to 1 hour in running water to get rid of debris. This if often followed by a short dip/soak in ethanol and then a longer soak in dilute bleach solution. Basal Murashige and Skoog (MS) medium is commonly used with different cytokinins added to stimulate shoot production. The information in the tables below represent a small part of the entire published papers available. It is recommended that you consult the original paper before proceeding further. A bibliography of papers cited is at the end of the document.

Examples of Proven Methods of Aquatic Tissue Culture

Common name/species	Explant	Clean-up procedure	Media	Source
American lotus (Nelumbo lutea) See website below.	Excised embryos from immature flowers	Immature flower receptacles with embedded semi-mature fruit were collected. Fruit was removed from the receptacles and surface sterilized by immersion in 50% ethanol for 1 min followed by immersion in 50% commercial bleach and then 3 rinses in sterile water. Longitudinal incisions were made through fruit and seed coats. Seed coats were pryed apart with forceps. Cotyledons removed and the embryonic axis was cultured in liquid medium	Half strength MS medium with100 mg inositol, 0.4 mg thiamine, 100 mg/l GA3, and 30 g sucrose, liquid medium. Divided and subcultured after 28 days Suggested Kitchen Method: Make one quart of MS medium with vitamins and sucrose, dilute by 1/2; to each quart add 1 tablespoon sugar, 1 ml PPM, and 100 mg GA3	Kane, et al. 1988

Common name/species	Explant	Clean-up procedure	Media	Source
Cryptocoryne lucens aquatic plant	Aerial plants that were induced by rooting rhizome cuttings in soiless mix	Aerial plants were defoliated, divided into 1 cm nodal explants and rinsed for 1 hour in tap water. Explants surface sterilized in 50% ethanol for 1 min, agitated in 25% commercial bleach solution with few drops detergent for 12 minutes; rinsed 3 times in sterile distilled water.	Basal MS with 0.45 mg BAP and 0.1 mg NAA and 0.8% agar. Subculture single node and triple node explants at 4 week intervals to basal MS with 4.5 mg BAP and 0.1 mg NAA. Rooted without auxins in planting medium with high humidity. Flowering occurred 30 weeks post transplant	Kane, et al.1990

Common name/species	Explant	Clean-up procedure	Media	Source
Epiphytic orchid (Encyclia boothiana var. erythronioides)	Seed capsule contain- ing sterile seed	Intact seed capsules, 6-7 months of age are rinsed in cold water for 15 min and surface sterilized in 70% ethanol for 1 minutes and in 50% commercial bleach plus few drops detergent for 12 min. Capsules were rinsed 3 times in sterile water	Basal MS or Knudson C	Sten berg and Kane, 1998

Common name/species	Explant	Clean-up procedure	Media	Source
Parrotfeather (Myriophyllum aquaticum)	nodal seg- ments; inter- node segments	Aerial stem segments were surface sterilized in 20% commercial bleach with few drops detergent; rinsed three times in sterile water	Liquid half MS with 8 mg 2iP	Kane, et al., 1991


Shoots developing from parrotfeather internode sections cultured for 7 days on medium with 2iP.		Elongating shoot developing from parrotfeather shoot tip cultured on medium without hormones.

Common name/species	Explant	Clean-up procedure	Media	Source
Pickerelweed (Pontederia cordata)	shoot	Same as above		Kane and Philman, 1997

Common name/species	Explant	Clean-up procedure	Media	Source
Water lily (Nymphaea spp.)	Epiphyllous plantlets excised from donor leaves	Rinse explants in flowing tap water 30 min, remove trichomes, rinse in water again for 30 min, 90 sec in 50% ethanol, 5 min in deionized water, agitate in 25% commercial bleach solution with few drops detergent for 12 minutes; rinse 3 times in sterile distilled water.	Liquid basal MS with 2 mg 2iP and 0.5 mg IAA for 5 weeks; same medium solidified with agar for 4 weeks. Subculture to membrane rafts with same medium except 0.66 mg TDZ is substituted for 2iP. After 5 weeks, produced non-vitrified aerial leaves with epiphyllous plantlets Note: membrane rafts are expensive for the hobbyist; try using cotton balls in liquid medium (instead of agar)	Jenks, et al. 1990

Difficult to clean explants can also be treated with a biocide to get rid of contaminants. See the information below (from the Plant Cell Technology website):

Poster Presentation by Dr. Assaf Guri, Plant Cell Technology, Inc. the XXXVII Congresso Viterbo 28-30 Settembre 1998, organized by the Societa Italiana di Fisiologia Vegetale titled: Advances in Controlling Plant Tissue Culture Contamination Using the New Biocide PPM Disinfection of Aquatic Plants

Contamination in cultures of aquatic plants is a major problem. Using PPM in a decontamination pre-treatment helped to solve the problem. This work was done in cooperation with Dr. Nathalie Calero Belyjo of Canada.

Small aquatic Cryptocoryne lucens plantlets ( less than 1 cm. long and 0.2 cm. wide) that were contaminated with endophytic bacteria were used in this test
Plantlets were incubated in a disinfection medium consisting of 1/4 MS salts with 0 - 40 ml/L PPM.
Incubation times was 48 hours or 156 hours at room temperature.
Plantlets were then transferred to full strength MS medium with 3% sucrose, 5mg/L BAP , and 0.5 or 2ml/L PPM.
The plantlets were cultured in this medium for one month, and then transferred to similar medium without PPM
Each treatment contained 6 tubes with two plantlets per tube.
After 15 days, the turbidity of the culture medium (without PPM) was observed and visibly recorded as: 1 - Clear, 2 - Translucent, or 3 - Cloudy. The data is summarized in Table 2.
Plantlets incubated in 40ml/L PPM for 156 hrs., cultured for 30 days in media with 0.5 or 2 ml/L PPM, and then cultured in medium without PPM, were still clear and free of contaminants.

To see the complete abstract, go to:
www.kitchenculturekit.com/ppmnew.htm

Table 2. Disinfection experiments of aquatic plantlets of Cryptocoryne lucens consisted of incubating in various concentrations of PPM + 1/4 MS salts for 48 or 156 hours. Plantlets were subsequently cultured on multiplication medium containing 0.5 or 2 ml PPM for 4 weeks, and then transferred to PPM-free medium to test for growth of microorganisms. Relative growth was measured by visual assessment of the turbidity of the medium: 1 = clear; 2=translucent; 3=cloudy.

Concentration of PPM (ml/L) in disinfection medium	Time (hrs.) in the disinfection medium	Concentration of PPM (ml/L) in culture medium	Turbidity Values in the PPM-free medium after 4 weeks

0	156	0.5	3
0	156	2.0	3
7.5	48	0.5	3
7.5	48	2.0	3
7.5	156	0.5	3
7.5	156	2.0	3
15	48	0.5	3
15	48	2.0	3
15	156	0.5	3
15	156	2.0	2
20	48	0.5	3
20	48	2.0	3
20	156	0.5	2
20	156	2.0	2
40	48	0.5	3
40	48	2.0	2
40	156	0.5	1
40	156	2.0	1

Plant tissue culture in your kitchen?

How does all of this translate into something the hobbyist can use?

Plant tissue culture can be done in the kitchen, using a microwave oven to sterilize media, PPM, a biocide, to minimize contamination, and a simple box to limit dust falling into culture vessels (see: www.kitchenculturekit.com ).

Premixed plant media can be purchased, but with some experimentation, homemade media might suffice for your particular plant. See recipe below.

Some chemicals are difficult to find even at the garden stores, such as the cytokinins benzylaminopurine and isopentyladenine, but these can be purchased in small, inexpensive quantities. You may find that your particular plant does not require them!


Most supplies needed for home tissue culture can be found in your kitchen: bleach, detergent, vinegar, sugar, plate, knife,etc.		A simple plastic box will serve as a clean area. Pint jars can be used to hold alcohol, bleach, and sterile water for disinfecting plants.

What do I use for culture vessels instead of test tubes and flasks?

Baby food jars make great culture vessels. They can be sterilized in a pressure cooker with the original metal caps or in a microwave with polypropylene caps. Various food containers, such as Gladware, can also be used as culture vessels and processed in the microwave. If you are not sure how a particular plastic will hold up in the microwave, be sure to do a test run and have a paper plate under the item.

Where do I get distilled water? How do I sterilize it?

Tap water, or filtered water, such as Brita, can be used. Water can be microwaved for 10 minutes or processed in the pressure cooker for 20-30 minutes.

How do I make plant medium? How do I know which medium to use for my particular species?

Directions for making homemade medium can be found below. Determining which medium to use will require some research: surf the net, ask others on the "home tissue culture listserv", or go to the library.

How do I sterilize my forceps and knife? What do I use for a sterile cutting surface?

In the university labs, we prefer using ethanol for sterilizing instruments. It smells better than isopropanol and seems to be gentler on your hands and on the plants. In our home labs, isopropanol, purchased at WalMart or Kmart will do just fine. Forceps and knives should be dipped in the isopropanol, shaken to rid of excess liquid, and then used to cut the sterilized plant material. A small salad plant can be wiped off with isopropanol and will serve as a sterile cutting surface.

Where do I put my cultures once that are started?

Michael Kane's papers recommend maintaining the cultures at about 25 C under a 16 hour photoperiod provided by cool-white fluorescent tubes. Basically that translates to: room temperature, shop lights from Kmart or WalMart, and metal shelves from which to hang the light units. Lights should be about 9-12 inches from the baby food jars. Never put your cultures on the window sill or in direct sunlight. The temperature extremes will be too great.

Media Recipes (Kitchen Style)

Ingredients for Basal MS medium:

MS medium package

2 tablespoons table sugar

1 ml PPM

Agar

tap water or distilled water

vinegar and antacid tablet to adjust pH

Optional hormones

Ingredients for "Homemade" Medium"

1 teaspoon Peter's fertilizer (20-20-20)

2 tablespoon sugar

1 multivitamin pill (do not crush - allow to partially dissolve only and then remove after a few minutes)

1 ml PPM

Agar

tap water or distilled water

vinegar and antacid tablet to adjust pH

Optional hormones

Instructions for Media Preparation:

Fill quart jar with about 3 cups water

Add MS medium (or Peter's fertilizer) and 2 tablespoons table sugar and mix well

While wearing gloves and goggles, add pre-measured PPM and any necessary hormones to the quart jar; mix well. Bring volume to 1 quart with more water.

Test the pH of the solution by dipping the edge of a piece of pH paper into the solution. A pH of 5.5 to 6.0 is preferred. Compare the color of the wet pH paper to the pH color chart:

If the pH is too low ("acidic"), add a pinch of baking soda. Mix well and test the pH again.

If the pH is too high ("basic"), add a few milliliters of vinegar. Stir to mix and test the pH again.

Continue this process until the pH is between 5.5 and 6.0.

Add 3 tablespoons of liquid medium to each baby food jar using a plastic measuring tablespoon.

Add 1/16 teaspoon PhytoTechnology Lab's Agar, or one level "pink Baskin-Robbins" spoon to each baby food jar.

Place the polypropylene baby food jar caps on the jars and press to tighten. These can be processed in the microwave for about 3 minutes (see Stiff, 1998) or in a pressure cooker for 30 minutes. Metal baby food jar caps can be used in a pressure cooker.

Cleaning up Plant Material

While concentrations of bleach used and the duration of soaking time in the bleach will vary from plant to plant, the basic methodology is the same.

>Bleach solutions are made in the following way. Safety clothing and equipment are recommended (gloves, glasses, apron, old clothers, shoes, and a respirator if you have a chlorine allergy)

Note that all commercial bleach is not the same. The common commercial bleach in the States is 5.25% sodium hypochlorite. This concentration will vary in other countries so be aware of the concentration of the solution you are using. The following is based on used U.S. bleach:

10% bleach	=	1/4 cup bleach + 2 1/4 cup water + few drops detergent
25% bleach	=	1/4 cup bleach + 3/4 cup water + few drops detergent
50% bleach	=	1/2 cup bleach + 1/2 cup water + few drops detergent

The following website(s) show the basics of how to clean up plant material for tissue culture:

www.kitchenculturekit.com/africanviolet.htm

References of Interest

Ailstock, M.S., W.J. Fleming, and T.J. Cooke. 1991. The char- acterization of axenic culture systems suitable for plant propagation and experimental studies of the submerged aquatic angiosperm Potamogeton pectinatus (sago pondweed). Estuaries 14: 57-64.

Bird, K.T. 1993. Salinity effects on Ruppia maritima L. cultured in vitro. Botanica Marina 36:23-28.

Bird, K.T. and J.Jewett-Smith. 1994. Development of a medium and culture system for in vitro propagation of the sea grass Halophile engelmannii. Can. J. Bot. 72: 1503-1510.

Burgess, Carla B. 1995. Wetland plants from test tubes. North Carolina Sea Grant, Publication #U.C.-SG-95-08. 36 pages. [North Carolina Sea Grant, Box 8605, NC State University, Raleigh, NC 27695-8605]

Cook, DA, DM Decker, and JL Gallagher. 1989. Regeneration of Kosteletzya virginica L. Press. (Seahorse Mallow) from callus cultures. Plant Cell, Tissue and Organ Culture 17: 111-119.

Jenks, M., M. Kane, F. Marasca, D. McConnell, and T. Sheeran. 1990. In vitro establishment and epiphyllum plantlets regeneration of Nymphaea 'Daubeniana'. HortScience 25(12): 1664.

Kane, M.E. and E.F. Gilman. 1991. In vitro propagation and bioassay systems for evaluating growth regulator effects on Myriophyllum species. J. Aquatic Plant Manage. 29-32.

Kane, M.E., E.F. Gilman, and M.A. Jenks. 1991. Regenerative capacity of Myriophyllum aquaticum tissues cultured in vitro.

Kane, M.E., E.F. Gilman, M.A. Jenks, and T. Sheeran. 1990. Micropropagation of the aquatic plant Cryptocoryne lucens. HortScience 25(6): 687-689.

Kane, M.E. and N.L. Philman. 1997. In vitro propagation and selection of superior wetland plants for habitat restoration. Combined Proc. Inter. Plant Propagator's Society, Vol. 47: 556-560.

Kane, M.E., N.L. Philman, C.A. Bartuska and D.B. McConnell. 1993. Growth regulator effects on in vitro shoot regeneration of Crassula hemsii. J. Aquatic Plant Manage. 31: 59-64.

Kane, M.E., T.J. Sheeran, F.H. Ferwerda. 1988. In vitro growth of American lotus embryos. HortScience 23(3):611-613.

Nash, Helen and Steve Stroupe. 1998. Aquatic Plants and Their Cultivation: A complete guide for water gardeners. Sterling Publishing Co., Inc., New York. 224 p. Great photos

Stenberg, M.L. and M.E. Kane. 1998. In vitro seed germination and greenhouse cultivation of Encyclia boothiana var erthyronioides, an endangered Florida orchid. Lindleyana 13 (2): 101-112.

Stiff, Carol M. 1998. Plant tissue culture for the classroom and home. Kitchen Culture Kits, Inc. Version 981201. 72 pages.

Straub, P.F., DM Decker, and JL Gallagher. 1988. Tissue culture and long term regeneration of Phragmites austalis (Cav.) Trin ex Steud. Plant Cell, Tissue and Organ Culture 15: 73-78.

Straub, P.F, DM Decker, and JL Gallagher. 1989. Tissue culture and regeneration of Distichlis spicata (Gramineae). Am. J. Bot. 76: 1448-1451.

Straub, P.F, DM Decker, and JL Gallagher. 1992. Characterization of tissue culture initiation and plant regeneration in Sporobolus virginicus. Am. J. Bot. 79: 1119-1125.

Websites of Interest American lotus
http://aquat1.ifas.ufl.edu/nelupic.html

Aquatic Botany
http://www.aquabotanic.com/

Center for Aquatic and Invasive Plants (includes great photographs):
http://aquat1.ifas.ufl.edu/

The Crypts Pages
http://users.bart.nl/~crypts/index.html

Michael Kane, Ph.D., Professor, University of Florida
http://hort.ifas.ufl.edu/people/tkane.htm

North Carolina State University Aquatic Botany Laboratory:
http://www.pfiesteria.org/

Orchid Seed Culture by Chu and Mudge (An excellent laboratory report with wonderful photos)
http://www.cals.cornell.edu/dept/flori/hort400/orchid/orchid1.html

Parrotfeather
http://aquat1.ifas.ufl.edu/myaqpic.html

Pickerelweed
http://aquat1.ifas.ufl.edu/pocopic.html

Plant Preservative Mixture
http://www.kitchenculturekit.com/ppm.htm

Water Lily
http://aquat1.ifas.ufl.edu/nyodpic.html

Please contact Carol M. Stiff if you need a publication translated into "kitchen langauge" or need further information. Visit the Kitchen Culture Kits, Inc. website for other resources.