Plant Propagation: Only the best of the best

Imagine you’re a novice botanist, but a friend of yours is older, wiser and has collected quite a nice display of exotic plants you admire. This wise friend has been growing for decades and seems to have collected his rare beauties from all corners of the earth. You know that unless you wander for years through the rainforest for seeds, or spend hundreds of dollars with mail order catalogues, you’ll never attain such a collection. So you ask your old friend the magical question: How might I start such a collection without traveling to the most remote stretches of the earth? The horticultural wizard begins to explain the answer to your enigmatic question. Selective propagation is the key. He cuts a branch off a miniature orange tree and hands it to you. “Good luck,” he chuckles.

Propagation is the practice by which any plant species can be replicated so that an exact genetic copy is produced or the best traits of two plants can be crossed. Plants reproduce by sexual and asexual methods, but keep in mind that plants are very diverse and no set of ‘rules’ is universal. With a little effort and understanding of plant physiology, any hobby gardener can fill their greenhouse with unique plants for little to no cost. Propagation includes such techniques like manual pollination, grafting, cloning, or tissue culture. Asexual propagation is most specific by allowing a highly successful plant to be genetically cloned. Anyone can propagate their favorite plant. Scientists use tissue culture to obtain genetically engineered strains just as your grandmother could split the root mass of a houseplant to share with a neighbor.

Sexual reproduction is the union of a male and female, typically encouraged by the wind, insect, or animal that transfers pollen to an egg inside the pistil. Once this union is complete, an embryo is formed and eventually develops into a seed. This transfer of genetic material leads to great variation, typically random, and results in a ‘wild type’ organism, a plant normally exhibited in nature. Wild type crossings are not concerned with elaborate colors or heavy yielding fruit, as they possess all dominant traits, good and bad, allowing them to survive in a wide range of conditions. Survival of the fittest ensures that very bad traits rarely reproduce successfully and die out. Manual pollination will combine the best traits of chosen plants.

Luckily, both pollen and egg are haploid (n), possessing half the genome of the whole organism. Upon pollination the seed becomes diploid (2n) and contains the complete genetic compliment, similar to animals. This is fortunate for two reasons. First, it enables researchers to engineer either pollen or egg and allows a direct correlation of gene sequence (genotype) to actual expression (phenotype). Second, manual pollination almost guarantees that observed dominant traits will be transmitted to the egg, whose dominant traits will also be incorporated. There are a few mechanisms which allow botanists to introduce new genes to produce specific strains, and they will be discussed later.

Asexual propagation is a magical phenomenon of all plants. Plant tissue is composed of three types of cells: parenchyma, collenchyma, and sclerenchyma. Collenchyma cells are for structural stability, are living at maturity, non-lignified and are commonly experienced as the ‘strings’ in a celery stalk. Sclerenchyma cells are also for structural rigidity, are dead at maturity, are lignified and compose sclereids (bark) and fibers (cotton). However, the majority of plant tissue is made of parenchyma cells that serve as specialized tissues of photosynthesis, respiration and protein sysnthesis. They compose roots, leaves, stems, shoots, fruits and seeds. Most importantly, they are the major component of meristems, zones of stem cells that give rise to all specialized parts of a plant. Parenchyma cells retain the ability to dedifferentiate and redifferentiate, meaning they can stop acting as a leaf and regenerate roots if the proper stimulus is provided. Multi-cell propagation is known as tissue culture and is the most complicated form of asexual propagation, although it theoretically allows any plant to be reproduced.

Meristematic zones experience indeterminant growth — that is, they will continue to grow, undifferentiated, as long as the plant is alive. This is why California Redwoods are enormous and thousands of years old. Plants will continue to grow unless limited by their environment or physical stress. These meristems contain a small number of totipotent cells capable of regenerating a whole plant, and also give rise to differentiated cells specialized to carry out a certain function. There are four meristematic zones: Apical, Axillary, Lateral, and Intercalary. Tissue for asexual reproduction must be taken from one of these zones, ideally the apical or axillary meristems.

Main stem and roots enlarge by primary growth from apical meristems located near their tips, mainly by cell elongation. These cells are self-perpetuating and not only produce tissue that form the body of the plant, but also continuously replicate themselves. Self regenerating cells are termed initials, and are comparable to animal stem cells. As apical meristems divide they leave behind a radial pattern of cells which also continue to divide as they are pushed out of the primary meristem. A cell’s position in this radial pattern is what ultimately decides its fate of specialization, not its ancestry. Transitional meristems are subsequently formed and give rise to further specialized tissue like vascular, epidermal, and ground tissue. Vegetative meristems preserve a fresh and pure bank of DNA for further cell replications, however, floral meristems become dedicated tissue once flowering has been initiated and lose their totipotent ability.

Axillary meristems are secondary meristems similar in structure and function to apical meristems, although often suppressed in plants showing apical dominance. They are formed in the junction of leaves to the stem and generate new shoots. Lateral meristems are also secondary and form as a cylinder of meristematic cells. They are responsible for increasing the girth of stems and roots. They produce additional xylem and phloem, and cork cambium, a protective layer called the periderm that replaces the epidermis. Intercalary meristems are not present in all plants but are responsible for the regeneration of grass after it has been mowed.

Asexual reproduction by taking cuttings of fresh growing tips are the primary method of cloning. Wounding of plant tissues initiate cell divisions at the wound site and typically generate callus tissue of new, unorganized, relatively undifferentiated cells. Since the majority of the plant remains intact, providing the proper environmental stimulus will queue these cells to specialize and generate new roots. Tissue culture experiments began in the 1930s, when Philip White demonstrated that tomato roots can be grown indefinitely in a simple nutrient medium of sucrose, mineral salts, and a few vitamins. However, these roots would not generate a whole plant.

In 1948, Caplin and Steward revived White’s studies. They used the original nutrient medium, though now supplemented with the hormone auxin and 20% coconut milk. This media combination produced amazing results by regenerating a whole plant from solely root tissue. Many years later in 1955, Skoog and Miller identified a small molecule, kinetin, derived from heat denatured DNA that would stimulate tobacco parenchyma cells to proliferate in culture. A new class of hormone was discovered, cytokinins, that induce dramatic cell growth and differentiation when combined with auxin in appropriate ratios. High levels of auxin to cytokinin stimulate the formation of roots, whereas high levels of cytokinin to auxin lead to the formation of shoots. Equal levels continue the growth of undifferentiated callus tissue.

The dawning of modern tissue culture was born. Now researchers could produce a whole plant from only a few genetically engineered cells, or indefinitely study engineered cells by continuous culture and experimentation. In 1981, Matzke and Chilton discovered a new method of transfecting (the incorporation of new genes into a plant’s genome) plant cells. They observed how a naturally occurring bacterium, Agrobacterium tumefaciens, infects a wounded plant and induces a tumor disorder known as crown gall. The bacterium possesses a large genetic ring called a plasmid that is not necessary for sustaining its life. Instead it infects its host with this plasmid, inducing the crown gall tumor, stimulating the production of auxin and cytokinin in the infected tissue resulting in uncontrolled tumor growth. Interestingly, crown gall tissue can be grown on a nutrient agar plate without supplemental hormones. Because plant cells have rigid cell walls, the bacterium can be killed leaving plant cells unharmed with the bacterium’s plasmid still intact in the host plant genome. In essence, the bacterium serves as a vector for transmitting its plasmid and thus gave researchers a new tool for introducing engineered genes into a plant genome, termed the Ti plasmid (tumor-inducing plasmid).

Since the Ti plasmid is not required for survival of the bacteria, the tumor causing portion of the plasmid can be removed and replaced by any desirable sequence of appropriate length. This is done by removing the gene sequence responsible for stimulating auxin and cytokinin production. Additionally, an antibiotic resistant gene is also added for selection purposes. When this bacterium infects a wounded plant, it incorporates the new plasmid into the host genome. The infected cells are then transplanted onto a nutrient agar plate containing auxin, cytokinin and an antibiotic to select against and kill any plant cells that have not been successfully transfected. New engineered cells can then be transplanted one more time where they are stimulated to regenerate a new whole plant. This procedure must be done in a sterile environment because air borne bacteria replicate faster than plant cells and will out-compete and kill plant tissue.

Many commercial crops including cereal grains, tobacco, corn and rice have been engineered to have advantageous traits like disease and herbicide resistance, salt and drought tolerance, and increased nutritional value. Transformed plants can also be produced by a method called vacuum infiltration. In this procedure, seeds or whole plants are submerged in a liquid containing engineered Agrobacterium, and all air is removed from intercellular spaces, effectively allowing the bacterium to penetrate tissues to rapidly generate new transformants.

Methodology

Sexual Propagation:

Plant propagation by sexual methods is commonly the easiest and cheapest way to generate a large number of plants. Commercial agriculture could not survive without the production of fertile seeds, although specialized farmers do sow individual plants obtained through asexual practices. The most beneficial sexual propagation methods are manual pollination of strong specimens to obtain genetically rich seeds or obtaining genetically pure seeds from a reliable seed bank.

When sorting through a seed bank catalogue, select a variety that best suits your growing conditions. The United States has many temperate zones and growing seasons vary greatly. Specialized seeds may be available with shorter maturation times for northern climates; others may have been bread with disease resistance or enhanced salt tolerance. Make sure the seeds are fresh, as old seeds have lower germination rates and may exhibit stunted growth. Commonly, horticulturist organizations have seed sharing networks that take great pride in preserving heirloom varieties, manually pollinating the best plants for many generations.

To preserve the genetics from a private collection, first consider the type of pollination system the plant uses. Species like peas and snapdragons have perfect flowers and may be inbred. These are termed self-compatible in that they will readily pollinate themselves within the flower or flowers of the same plant. Monoecious plants like corn have imperfect flowers and must be cross pollinated by different flowers of the same plant or neighboring plants. Dioecious plants like mulberry, cottonwood, and willow also have imperfect flowers and must be cross-pollinated by other plants. It should be noted that cross-pollination induces a condition known as hybrid vigor because no one likes to be inbred, and crossing two strong parents often results in a superior generation.

When the method of pollination has been determined, wait for the pollen sacs to mature enough to easily release pollen when shaken. This window of opportunity is often small and attention should be dedicated to timing. Male plants are usually taller to aide in ‘raining’ pollen and these organs typically mature before female ones, making them more noticeable. If one father is chosen to be the donor, gently tie a bag around the pollen stalk and shake the pollen into the bag, being careful not to allow undesirable contamination. A paint brush may be used to gently transfer pollen to the chosen female pistil.

Seeds are produced in different locations depending on plant species. In fruit, the seeds should be removed, washed, and stored in a cool, dry location. Some seeds are small and hard to find. These flowers should be cut and kept in a bag in a warm, dry place until the flower completely dries and allows the seeds to be shaken out. Store seeds in a refrigerator to prevent germination which requires warmth and moisture. Chilling seeds before germination breaks dormancy and is a method called stratification, as it mimics a winter freeze and spring thaw, aiding the seed coat to harden in cold and then crack with moisture expansion. Scarification of seeds, by rubbing them with sandpaper also aids in germination by damaging the seed shell. In nature, this process is performed while passing through an animal’s digestive tract.

Seedlings need be treated with a gentle touch. Very weak nutrient solution should be used if even at all. Low light conditions should be kept as photosynthetic rates are not very high and cellular damage can occur under high light intensity. Watering seedling is a fine balance as young roots can not handle a high water load nor can they tolerate drought very long. Before transplanting outside, seedlings should be acclimated by slowly introducing them to an outdoor environment of low night temperatures. A seedling box can be built to serve as a mini greenhouse to trap moisture and warmth during this process. A late spring frost will most definitely kill young seedlings and attention should again be dedicated to timing.

Asexual Propagation:

Cuttings

Softwood stems respond best to this method, although semi-woody stems are also suitable. Woody stems have lignified bark on their surface and are composed of waterproof, dead cells, although new shoots of woody plants have softer periderms and may sprout roots. Water and hormones need to be able to penetrate the stem periderm to stimulate cortex parenchyma cells. Roots can only penetrate softer tissue and will not sprout through lignified bark.

Select a newly growing shoot approximately 3-5” long with enough leaves to keep it alive. Flowering shoots will not exhibit vegetative meristem growth and are already dedicated to maturation. Leaves serve as water storage for a shoot without roots and also are the site of energy and cytokinin production. High humidity should be maintained to retard dehydration (using a hood is a good idea). Cut the shoot just below a node because they are actually axial meristems and have the magic potential for root differentiation. Use a clean, sharp scissor or knife and make a diagonal cut to provide maximum surface area. Dip the roots in a hormone solution of auxin and/or cytokinin. An antifungal agent could help but is not necessary. Rooting should take place in two to four weeks depending on species.

In some plants, leaves may be propagated in the same way, but is more difficult. Include the leaf petiole and midrib as it harbors meristematic tissue. Roots will sprout from the base and new shoots will soon follow. The parent leaf may then be cut back as it is no longer required. Root mass division of a shrubby plant may involve splitting it along its axis and growing each as individual plants. A good rule of thumb is to divide fall flowering perennials in spring and spring/summer flowering perennials in fall. A technique known as layering can be used on some plants where a branch is bent to the ground and partially covered with dirt, leaving the tip exposed. Hold the shoot down with a rock and hopefully new roots will grow from the middle of the stem. This technique can be mimicked by wrapping moist sphagnum moss around a vertical stem.

Grafting

Grafting is most commonly used with hard wood, fruit bearing trees. This method includes joining a selected branch or bud section, called a scion, to the trunk of an existing root stock or vigorously growing tree. Orchard farmers use this technique when they find a tree of unique qualities because growing from seed rarely produces quality fruit trees. By cutting young branches ¼ to 1” thick, the scion is joined to a matching chunk removed from the trunk of a vigorous root stock. It is necessary that the underlying cambium layers are the same so that the union will merge and grow as a new branch of a different cultivar. This scion will retain the genetic composition of the original, hearty donor.

Overall, grafting involves the union of a new branch or bud to an existing tree. There are many ways to achieve a multi strain tree. Farmers will even fuse as many as three different strains of apple onto the same root stock. In general, four conditions must be met: the scion and rootstock must be compatible, each must be at the proper physiological stage, the cambial layers of the scion and stock must meet, and the graft union must be kept moist until the wound has healed. Commonly, the graft union is covered with wax, rubber tape, or a tar like substance to prevent desiccation. Mechanical support should be added when necessary.

Tissue Culture

Tissue culture requires specially prepared equipment and is usually left to professionals, researchers, or a wise and dedicated hobbyist. This method uses a few parenchyma cells to generate a whole plant and is therefore an easy method to generate many plants from a single source, when carefully performed. Tissue culture requires that all tools, media, and tissue be sterilized. Wrap tools and jars in tin foil to preserve sterility and do not tighten jar lids because they might break when pressure is built upon heating. A home gardener can perform this task by placing all supplies in a pressure cooker for 30 minutes or an oven at 400º for three hours. A sterile environment can be obtained by constructing a temporary hood, disinfected by alcohol, and work performed over a flame. The flame provides an upward draft of air and thus carries away contaminants from your sterile media. Additionally, tools may be flamed to ensure sterility. Good hygiene and clean hands are also very important.

Select an actively growing part of the plant as previously discussed. Plant tissue should be disinfected in one part bleach to eight parts water. Soak for 10 minutes and sterilely remove the outer tissue. Carefully cut the fresh tissue into ¼” cubes and transfer to a sterilized mason jar with culture media. Don’t forget the coconut milk and auxin or the tissue will not root! Warmth and gentle light will encourage growth. Contamination is easily noticeable and will kill plant tissue. Discard these cultures to prevent further contamination.

Proliferating the best nature has to offer is a good idea indeed. Why wait to see if unknown seeds produce beautiful flowers or large, tasty fruit? Take charge of your plants and only grow the best of what you can find. Borrow a branch from a friend, take a new cutting from a nature walk, purchase the best seeds yet developed, combine the best pollen with the brightest flowers and collect your own prize seeds, split a shrub and plant it everywhere, graft a new fruit bud to an existing tree, or get really adventurous and culture tissue from an exotic orchid. As one can see, the possibilities are endless and rather cost effective!

References

Moore, Clark, and Vodopich. 1998. Botany. 2nd Ed. McGraw-Hill, New York, NY.

Taiz, L., Zeiger, E. 1998. Plant Physiology. 2nd Ed. Sinauer Associates, Sunderland, MA.

For technique methodology visit: Arizona Master Gardner Manual:

http://ag.arizona.edu/pubs/garden/mg/