Growers have discovered the secrets that the horticulture industry has known for decades, that plant tissue culture is the most efficient method of producing pest-free clones. Dedicated mother plants are eliminated and new plants are generated in jars or from their rooted plants thereof.
The secret to plant tissue culture is using sugar as an energy source. The tiny cuttings absorb sugar, nutrients and vitamins directly through their surface and grow in a self-contained environment while hormones in the media encourage branching and keep the new nodes tight. A proper vessel will keep the plants small and disease-free, saving time, light and money. Preservatives should be added to the medium to reduce the possibility of mold. The new branches and nodes can become the sources of thousands of cuttings.
The following items should be included in a tissue culture kit:
Using a sterile technique begins with the setup of a clean handling and plant growing room with smooth cleanable surfaces and HEPA air filtration, alcohol sprays and dips for hands and instruments that will touch plants, and surface decontamination of plants going into culture.
Start by setting up the clean area and hood where you will cut and handle plants. The hood protects the plants and tubes from mold spores that may be floating in the air. Set the storage container on its side in a clean, draft-free location. A clean kitchen, bathroom or grow tent are good locations. Light and grow the tubes in the same area as the clean hood to take advantage of the clean air.
Set up the HEPA filter between the door and the work area and start running. Assemble the grow lights and wipe down the room, rack, and hood with sanitizer. Sanitize the plate and set in center of the hood, toward the back at arm’s reach. The filter will capture items released into the air when cleaning.
Trimming the plant cuttings eliminates extra foliage and removes parts exposed to mold spores at the same time they are made into small cleanable pieces. The washed pieces are then trimmed to remove the bleached ends and placed in the tubes to grow.
Select clean, healthy vegetative branch tips. Use non-flowering lower growth from pruned veg plants to preserve flowering tops. The thin neglected branch tips thrive in the rich nutrient solution and triple in size or more. Trim into 3-inch pieces and remove leaves, leaving 1/2” of leaf stem above the node. Cut ends will be
trimmed once more after sanitizing to remove bleached cut tissue. Leave one inch or more below lowest node to be trimmed so that lowest node will be located above where the media surface meets the air. This node is often the most aggressive as it is near the sugar medium and the oxygen to metabolize the sugar.
Put the pieces into the washing jar. Run some tap water into the jar and shake gently to wash off any loose material. Add a drop of simple detergent and stir to dissolve oils and begin killing mold and bacteria. Drain the soapy water and add the sanitizer from the culture tubes and swirl every 10 minutes. Some bleach will get on your hands, cleaning them at the same time. The objective is to clean the plants and the vessel before going to the clean hood.
Dip tools in sanitizer and spill some into the plate to create a small puddle to trim cutting in. Take one cutting out of the jar with forceps and prepare to trim it again. Hold forceps as far back as you can and try to work with your hands as low as possible without reaching over the plate. You will often pass tools to your other hand across the front of the plate. Shake off residual sanitizer drops. Keep tools at least 3 or 4 inches inside of hood. Gloves are good if you have them.
With the node cutting in the center of the plate, pass the forceps to your other hand and take the scalpel in your working hand, holding as far back on the tool as you can. Trim away the bleached ends of the leaf stems, or petioles, about 1/4”. Do not damage the bud inside the node, the growing point that will develop into your new plant, unless it will fall beneath or just above the media level, in which case remove it as it will be deformed by the direct contact with the hormone.
Point the upper end of the node to the right side of the plate, put the scalpel back in the sanitizer, and pass the forceps back to your working hand. Open the tube and lid with one hand with the lid protecting the tube opening. This is a skill you will develop over time and is worth practicing dry before working with live plants. It is ok to pre-loosen the lid with two hands before working with live plants. Just do not take anything out of the hood when working with open tubes and do not lean in over them. Put tools back in sanitizer if you need your hands free.
With the cutting on the plate directly in line with the forceps, not crossways like chopsticks, lift the node and place directly down the center of the tube. Replace forceps in sanitizer and close lid onto tube, if you are using screw tops turn back about ¼ turn from tightened. There will be room in the threads for the plant to exchange air at the same time reducing the hazard of contamination.
Continue until each tube contains one cutting. Wipe off plate and drain sanitizer puddle after every few cuttings, when the debris can get in the way. Optionally, wrap the neck of the tubes with tape, where the lid meets the tube, with the plastic wrap as an extra layer of protection. It may be necessary to spool out some of the wrap before using it on the jar.
Grow the cultures under a small fluorescent fixture like a T5 or LED in a clean area at about four to six watts per square foot. Maintain temperature in the mid-70’s and around 50% humidity. Evaluate the tubes for contamination that may appear as cloudiness, growth in the liquid surface, or odd coloration. Remove spoiled tubes and clean elsewhere. Plants should take a few weeks to grow to three or more times their original size and develop leaves in vitro (in tubes).
Tube clones are ready to root when leaves have appeared inside the tube and the stem in the solution has gotten wider. Inspect and clean all apparently clean cuttings by emptying into a colander in the sink and rinsing gently to remove any sugar solution. Remove yellow leaves and use a sharp blade to make a 45° cut across bottom of cutting. The widened stem of the tube cuttings should appear rough as a result of the natural rooting hormone in the cutting conflicting with the branching hormone of the growth solution. We have also rinsed tube clones in a solution of 50ppm chlorine where there was risk of contamination.
Root your tube-grown clones in a high humidity chamber with thermostat-controlled bottom heat and gentle air circulation. Start by using the rooting plugs and system you have used for regular rooting with an extra emphasis on humidity as the tube-grown tissues are thin and have not developed their waxy cuticle and stomatal flexing. Maintain a high humidity inside and outside of a perforated dome and an 80-degree bottom heat. Roots may appear as early as 9 days but are more common after two weeks.
One good technique is to keep plug trays in mesh bottom 10 by 20 prop trays and to preload the plug holes with 1) a drop of diversified root-inhabiting organism to penetrate the cube and colonize roots when they develop, and 2) a drop of root hormone gel. The strategy is to add the hormone second as a drop where it will coat the inside of the hole and gravity will keep it in contact with the rough stem and exposed cambium cut at the bottom. Humidity will be maintained and do not bottom water the tc clones until the roots have penetrated the cubes or you are certain they have all developed.
After roots have developed, you may plant as regular rooted cuttings, or as we prefer, plant up into three-inch pots and allow the first few dominant shoots to develop. I’ve had success taking shoots at six to eight inches, leaving a single node behind, as new cuttings by planting directly into fresh rooting plugs. The rooted clones from tissue culture are different from regular clones primarily because of the density of shoots at the top of the tiny cutting. I like to give the secondary branches a chance to add to the efficient candelabra shape at the same time as getting extra apical clones with all of the advantages of the tissue culture that it was just derived from. Usually one to three such clones can be taken from each rooted tube plants in each 18-site propagation tray.
Eight to eleven weeks after cuttings were placed into tubes, the pruned and trained veg plants are ready. Select the top ten or fifteen percent of rooted clones to be the clone donors of the next generation and give the rest to the cultivators to flower. Keep them in the clean environment and take tall dominant cuttings as they develop and smaller apical cuttings to introduce into fresh tissue culture tubes. Second generation cuttings have already been evaluated for disease and are much more successful than the first generation. Such small plants as the rooted cuttings, clone donors and veg plants are best maintained in rack systems for immediate distribution at the cultivator’s convenience. Growth can be slowed or increased with temperature and light levels.
Plants grown in media tubes without contamination are most likely free of disease organisms and possess all the advantages of tissue culture. The plants have directly absorbed the balanced nutrients and hormones creating sturdy bushy starts and activating genes that may have been dormant. Stage 1 Introduction clones may be removed from the tubes, rinsed and planted in plug trays in a high humidity environment, creating your new stock. Take clippings from these veg plants to create new tissue culture starts.