Growing plants and animals by cloning

Introduction: (Initial Observation)

Cloning is the process of making a genetically identical organism through nonsexual means.

A cloned organism has the same physical and biological properties as its parent. For example, if a black sheep is resistant to certain disease. It’s clone will also be black and will be resistant to the same disease.

About the image: Rodent genome modification methods are being developed at Advanced Cell Technology by their inventors, Dr. Teruhiko Wakayama and Tony Perry. The mice on the image contain the gene for a green fluorescent protein, put there by a novel method called metaphase II transgenesis. This means that they glow green under ‘black light’ (long-wavelength UV) light. But producing green mice is just the first step using an easily identified marker gene; more importantly, the method can be used to introduce genes to study disease and produce more effective medicines. The cloning and transgenesis methods pioneered by Drs. Wakayama and Perry will help ACT to achieve this more quickly.

In this project you will study about different methods of cloning and experiment cloning a plant and an animal.

Information Gathering:

Find out about cells, nuclei, chromosomes and genes. Read books, magazines or ask professionals who might know in order to learn about creation of clones in nature. Study about different methods of cloning. Keep track of where you got your information from.

The following are samples of the information that you may gather:

Cloning is the process of making a genetically identical organism through nonsexual means. It has been used for many years to produce plants (even growing a plant from a cutting is a type of cloning). Animal cloning has been the subject of scientific experiments for years, but garnered little attention until the birth of the first cloned mammal in 1997, a sheep named Dolly. Since Dolly, several scientists have cloned other animals, including cows and mice. The recent success in cloning animals has sparked fierce debates among scientists, politicians and the general public about the use and morality of cloning plants, animals and possibly humans.

Nature has been cloning organisms for billions of years. For example, when a strawberry plant sends out a runner (a form of modified stem), a new plant grows where the runner takes root. That new plant is a clone. Similar cloning occurs in grass, potatoes and onions.

People have been cloning plants in one way or another for thousands of years. For example, when you take a leaf cutting from a plant and grow it into a new plant (vegetative propagation), you are cloning the original plant because the new plant has the same genetic makeup as the donor plant. Vegetative propagation works because the end of the cutting forms a mass of non-specialized cells called a callus. With luck, the callus will grow, divide and form various specialized cells (roots, stems), eventually forming a new plant.

More recently, scientists have been able to clone plants by taking pieces of specialized roots, breaking them up into root cells and growing the root cells in a nutrient-rich culture. In culture, the specialized cells become unspecialized (dedifferentiated) into calluses. The calluses can then be stimulated with the appropriate plant hormones to grow into new plants that are identical to the original plant from which the root pieces were taken.

This procedure, called tissue culture propagation, has been widely used by horticulturists to grow prized orchids and other rare flowers.

Plants are not the only organisms that can be cloned naturally. The unfertilized eggs of some animals (small invertebrates, worms, some species of fish, lizards and frogs) can develop into full-grown adults under certain environmental conditions — usually a chemical stimulus of some kind. This process is called parthenogenesis, and the offspring are clones of the females that laid the eggs.

Another example of natural cloning is identical twins. Although they are genetically different from their parents, identical twins are naturally occurring clones of each other.

Scientists have experimented with animal cloning, but have never been able to stimulate a specialized (differentiated) cell to produce a new organism directly. Instead, they rely on transplanting the genetic information from a specialized cell into an unfertilized egg cell whose genetic information has been destroyed or physically removed.

The History of Cloning

1938	Cloning is Envisioned	Hans Spemann proposed a “fantastical experiment” in which one removes the nucleus from a cell of a late-stage embryo, juvenile or adult, and transplants it into an egg.
1952	1st Cloning Experiment with Frogs	Robert Briggs and T.J.King add the nucleus from an advanced frog embryo cell to a frog egg. No development.
1970	Another Frog Experiment, Better Results	John Gurdon tried the same procedure. The eggs developed into tadpoles but died after they were ready to begin feeding. He later showed that transplanted nuclei reverted to an embryonic state. This was a milestone because, even though the frogs never reached adulthood, he replaced the nucleus of a frog egg with that of another cell from a different frog. At this stage, scientists could transfer nuclei from adult cells to egg cells, but the frogs only developed to tadpoles and always died.
1981	“Cloning of Mice”	Karl Illmensee and Peter Hoppe reported that they had produced normal mice from mouse embryo cells. After a lengthy inquiry, it was discovered that they had faked the results.
1982	Research Stalls	James McGrath and Davor Solter reported that they could not repeat the mouse-cloning experiment and conclude that once mouse embryos have reached the two-cell stage, they cannot be used for cloning. Others confirm their results.
1984	1st Embryo Cloning with Sheep	Steen Willadsen reports that he cloned a live lamb from immature sheep embryo cells. Others later replicate his experiment using a variety of animals, including cattle, pigs, goats, rabbits, and rhesus monkeys.
1994	1st Cloning of More Advanced Embryo Cells	Neal First clones calves from embryos that have grown to at least 120 cells.
1996	Groundwork Laid for Cloning of Adult Sheep	Ian Wilmut repeats Dr. First’s experiment with sheep, but puts embryo cells into a resting state before transferring their nuclei to sheep eggs. The eggs develop into normal embryos and then into lambs.
1997	Adult Sheep Cloned	Dr. Wilmut reports that he had cloned a 6-year-old adult sheep from an udder cell. Dolly, the cloned sheep, was the only one to survive from 277 eggs that had been fused with adult cells.
1997	Bull Calf Cloned	ABS Global, Inc. produce “Gene”, a 6-month-old bull calf, from its proprietary cloning technology. They also announce the formation of Infigen, Inc., to commercialize applications of cloning technologies in the cattle breeding, pharmaceutical, nutraceuticaal and xenotransplantation fields.

Information Source: New York Times, 3 March 1997, pp. A20-22.

The Three Types of Cloning:

Embryo cloning: This is a medical technique which produces monozygotic (identical) twins or triplets. It duplicates the process that nature uses to produce twins or triplets. One or more cells are removed from a fertilized embryo and encouraged to develop into one or more duplicate embryos. Twins or triplets are thus formed, with identical DNA. This has been done for many years on various species of animals; only very limited experimentation has been done on humans.

Adult DNA cloning

(a.k.a. reproductive cloning)

This technique which is intended to produce a duplicate of an existing animal. It has been used to clone a sheep and other mammals. The DNA from an ovum is removed and replaced with the DNA from a cell removed from an adult animal. Then, the fertilized ovum, now called a pre-embryo, is implanted in a womb and allowed to develop into a new animal. As of 2002-JAN, It had not been tried on humans. It is specifically forbidden by law in many countries. There are rumors that Dr. Severino Aninori has successfully initiated a pregnancy through reproductive cloning. It has the potential of producing a twin of an existing person. Based on previous animal studies, it also has the potential of producing severe genetic defects. For the latter reason alone, many medical ethicists consider it to be a profoundly immoral procedure when done on humans.

Therapeutic cloning (a.k.a. biomedical cloning): This is a procedure whose initial stages are identical to adult DNA cloning. However, the stem cells are removed from the pre-embryo with the intent of producing tissue or a whole organ for transplant back into the person who supplied the DNA. The pre-embryo dies in the process. The goal of therapeutic cloning is to produce a healthy copy of a sick person’s tissue or organ for transplant. This technique would be vastly superior to relying on organ transplants from other people. The supply would be unlimited, so there would be no waiting lists. The tissue or organ would have the sick person’s original DNA; the patient would not have to take immunosuppressant drugs for the rest of their life, as is now required after transplants. There would not be any danger of organ rejection.

CLONING PLANTS

ASEXUAL PROPAGATION OF PLANTS IS A FORM OF CLONING

Asexual propagation does not involve exchange of genetic material, so it almost always produces plants that are identical to a single parent. Delicious apple and Bartlett pear are two examples of species that have been asexually propagated for decades. Asexual propagation methods include cuttings, layering, division, grafting, budding and tissue culture.

CUTTINGS

Cuttings involve removing a piece from the parent plant and that piece then regrows the lost parts or tissues. Both woody and herbaceous plants are asexually propagated by cuttings of stems, leaves and roots. New plants can be grown from parts of plants because each living plant cell contains the ability to duplicate all plant parts and functions. Mature cells can change into MERISTEMATIC (mare-ah-ste-MAT-ick) cells that are found at rapid growth sites like buds.

There are many types of cuttings. Often, a plant can be propagated by more than one method of cutting. Some plants will reproduce readily from cuttings and others take a considerable amount of time and care.

STOCK PLANTS are the parent plants used in asexual propagation. Stock plants must be in excellent health and should possess characteristics desirable for production of new plants. Herbaceous cuttings are those taken from nonwoody plants, such as perennials and houseplants.

Softwood cuttings are pieces of new growth taken from woody stock plants. These cuttings must be taken before the new growth starts to harden. Hardwood cuttings are taken from tissue which has become woody. Other forms of cuttings are leaf cuttings and root cuttings.

The gardener must try to duplicate the conditions needed for a plant to root from a cutting. High humidity, indirect light and soil temperatures of 70 to 80 degrees F are best for most cuttings. These conditions may be created by keeping cuttings enclosed under glass or in plastic bags in dappled shade. Cuttings must be shielded from direct sunlight, especially if they are under glass or plastic.

STEM CUTTINGS

When a cutting is made, injured xylem and phloem cells plug the tubes so that precious fluids are not lost. Usually a CALLUS forms at the cut. Cells near the callus area reorganize to form adventitious roots.

Stem cuttings are the most commonly used method to produce houseplants. Select vigorous, new growth with no flower buds. Stem sections should be free of diseases and insects. Each cutting should be 2 to 4 inches long and have 2 or 3 leaves attached.

Make a cut 1/4 inch below a leaf node and pull off the leaves that are at the nodes that will be below the surface of the rooting medium. ROOTING HORMONE helps to stimulate rooting, but is optional. Pour a small amount of the rooting hormone into a clean container to prevent contamination of all of your rooting hormone. Dip the base of the stem, including the node area, into the rooting powder. The stem should be dry when dipped.

Commercial rooting products often include a fungicide. This is a good idea given the damp conditions required for rooting success. Tap off excess powder, since too much hormone can inhibit rooting.

Poke a hole in the medium before inserting the cutting to avoid loss of the rooting hormone. Insert treated cutting in a moist rooting medium. A suitable rooting medium is half perlite and half sphagnum peat moss. Any disinfested container with drainage is acceptable for use.

Cover container and cutting with a plastic bag tent to maintain high humidity. Place unit in a warm area with indirect light. Check the rooting medium every few days to make sure it remains moist. Rooting can take from a few days up to several months.

After a few weeks, test for rooting by gently tugging at the cutting. If there is resistance, rooting has started and the plastic cover may be removed. More detailed instructions on stem cuttings are provided in Fact Sheet 1226, Reference Prop.1.

LEAF CUTTINGS

In this method, a leaf blade or leaf with petiole is used to propagate new plants. The same steps are followed as for stem cuttings. Choose a healthy leaf from a vigorously growing plant. Cut it close to the stem with a sharp, disinfested razor or knife. Trim off 1/4 of the leaf and dip into rooting hormone, if desired. Insert the leaf into rooting medium so that 1/3 of the leaf is below the surface.

One or many new small plants form at the base of the leaf. African violet leaves will produce many new plants. Begonia leaves can be divided into segments for propagation; however each leaf piece must contain a major vein. With leaf cuttings, the original leaf is not a part of the new plant and is usually discarded.

Many succulent plants, such as sedum, jade, and peperomia, can be propagated by leaf cutting. Some plants such as Kalanchoe pinnata or K. bryophyllum, piggy-back or air plants, form their own new plantlets in this fashion.

ROOT CUTTINGS

Cultivation of root cuttings probably started after gardeners observed new plants growing from pieces of root accidentally left behind in the soil. Take cuttings from newer root growth. Make cuttings 1 to 4 inches long from roots that are 1/4 to 1/2 inch in diameter. Be sure that the roots collected are from the chosen plant and not neighboring plants.

Cuttings should be taken during the dormant season when roots have large carbohydrate supplies. However, they also may be taken throughout the growing season. Cut straight through the end of the root closest to the stem. Cut the other end on a slant. This allows you to remember which end is the top (the straight cut) and which is the bottom (the diagonal cut).

Store cuttings from dormant roots for 3 weeks in moist rooting medium at 40 degrees F. Remove from storage and plant upright in the growing medium. Keep moist and warm, in a bright location until growth and weather permit acclimatizing to the outdoors.

If root cuttings are taken during active growth, skip the storage period and place cuttings directly in the rooting medium. For smaller plants, take 1- to 2-inch sections. Place cuttings horizontally a half inch below the surface of the rooting medium. These cuttings should be handled indoors or in a HOTBED. The fine roots of many perennials are used for propagation. Root cuttings of some variegated plants will lose their variegation.

MICROPROPAGATION OR TISSUE CULTURE

Each plant cell has the potential to grow into a new plant exactly like the parent. This fact coupled with technical advances, specialized equipment and sterile laboratory conditions has produced modern tissue culture.

In tissue culture, individual or small groups of plant cells are manipulated so they each produce a new plant. A tiny piece of bud, leaf or stem can produce incredible numbers of new plants in a small space in a short time.

The advantages of tissue culture, in addition to speed and efficiency of propagation, include production of disease-free plants. New plants can be made available to the public more quickly because of tissue culture.

However, there are some problems with spontaneous mutations which naturally occur. In tissue culture, the incidence of these mutations is greatly increased.

Conditions for tissue culture are very exacting. Absolutely sterile conditions must be maintained. Temperature, light, humidity and atmosphere are strictly controlled with electronic sensors and computerized controls.

Question/ Purpose:

What do you want to find out? Write a statement that describes what you want to do. Use your observations and questions to write the statement.

The purpose of this project is to understand and display different methods of cloning.

You may choose to change this project from a display project to an experimental project. Some of the questions that I propose for your experimental project are:

1. What is the best temperature for cloning flatworms by cutting?
2. What invertebrates can be cloned by cutting? (Test at least 3 different invertebrates).
3. What plants can be cloned and propagated by stem cutting?

If you have access to high quality microscopes and laboratory equipment, you may choose to do more complex (College level) experiments. The following are some sample questions:

1. What are the best conditions for cell starvation? (As you know, cell starvation substitutes removal of nucleus from an egg cell. In this method nucleus shrinks and disappears by starvation.)
2. What is the best voltage for cell fusion?

Identify Variables:

When you think you know what variables may be involved, think about ways to change one at a time. If you change more than one at a time, you will not know what variable is causing your observation. Sometimes variables are linked and work together to cause something. At first, try to choose variables that you think act independently of each other.

A display project does not need variables! If you do this project as an experimental project, you will then need to define variables. For example imagine your question is: What is the best temperature for cloning flatworms by cutting?

In this case you define variables as follows:

Independent variable is temperature. Dependent variable is the rate of success in regeneration of new flatworms from flatworm pieces. Controlled variables are the type of flatworm, the size and orientation of pieces and experiment procedures.

Hypothesis:

Based on your gathered information, make an educated guess about what types of things affect the system you are working with. Identifying variables is necessary before you can make a hypothesis.

A display project does not need a hypothesis, however if you are doing this as an experimental project, the following is a sample hypothesis:

The rate of regeneration of flatworm from pieces will be maximized at warm temperatures (85ºF). My hypothesis is based on my gathered information and an online document suggesting to lower the temperature in an aquarium to prevent regeneration of flatworms.

Experiment Design:

Design an experiment to test each hypothesis. Make a step-by-step list of what you will do to answer each question. This list is called an experimental procedure. For an experiment to give answers you can trust, it must have a “control.” A control is an additional experimental trial or run. It is a separate experiment, done exactly like the others. The only difference is that no experimental variables are changed. A control is a neutral “reference point” for comparison that allows you to see what changing a variable does by comparing it to not changing anything. Dependable controls are sometimes very hard to develop. They can be the hardest part of a project. Without a control you cannot be sure that changing the variable causes your observations. A series of experiments that includes a control is called a “controlled experiment.”

Experiment 1: Cloning Flatworms (Planarian)

Flatworms can be found in many different sizes and shapes. They can be as small as a few millimeters up to 60-70 millimeters. If you choose to do this experiment, you need to find and catch your own flatworms. I don’t know of any place who may sell these animals.

If you live in an area with no access to shallow lakes or streams, you may try a similar experiment on other invertebrates such as jellyfish and sponges that can be purchased from some pet shops and aquarium stores.

Most flatworms are plain; however, some flatworms have fantastic colors.

Most flatworms are plain; however, some flatworms have fantastic colors.

To catch flatworms you need to know where they live and how they look.

Flatworms like to lurk in dark places in waterways.

As their name suggests, they are wormlike and flat without segments on their body. They have soft skin with hair, generally down the side. The smaller species of Flatworm wave their hair to propel themselves! The larger species move across the bottom of a waterway in a gliding fashion, helped by muscular waves that ripple down their body, but cannot swim.

Flatworms are found in streams and shallow parts of lakes. They live in dark places on the surface of rocks and plants.

What they eat:

Flatworms are mostly carnivorous and prey on invertebrates small enough to be captured. They also scavenge on the dead bodies of animals that sink to the bottom. Flatworms tend to live where there are lots of dead plant and animal remains to feed on. This means that sites of organic pollution are good for Flatworms.

How to catch it?

They creep and crawl on the coral reef and under rocks. If you want to catch a flatworm to look at, you have to pick up a rock and drop water on the worm. It will slowly get washed off. If you try to pull it off the rock, it will rip. A flatworm is as thick as folder paper. Some of them move fast and some of them move very slow. Flatworms do not move by themselves they have these little little hairs called cilia that help it to slide along with on this mucus from the worm. People are dangerous to them because sometimes they rip them off and they can be torn into pieces easily. Some are very clear, they camouflage themselves really well. Many people think that Flatworms are Nudibranchs. A Nudibranch is a Seaslug and a Flatworm comes from the family group called the Platyhelminthes. They are in a different worm group because they are not Annelids which means that they are not segmented.

It is possible to trap the flatworms by placing bin-bags of compost around and collecting any worms which congregate underneath.

When your flatworm is ready for experiment, perform the experiment. Note that the experiment of Planarian Regeneration has been performed by Harriet Randolph, professor of Biology at Bryn Mawr College.

Safety Notes: Some species of flatworm and some species of jellyfish are poisonous. Do not touch them while doing such experiments. Wear latex gloves if necessary.

Procedure:

Use a sterile surgery blade or utility knife to cut (transversely) a flatworm into three pieces. (If your knife is not sterile, insert it it ethyl alcohol for a few minutes or hold it in flame a few seconds).

Place all three pieces back in water and make daily observations for up to 15 days. Record your observations to show the progress of regeneration.

To make sure that your cloning experiment will be successful, you may better do this experiment simultaneously on more than one flatworm. Just make sure that you keep the pieces of each flatworm in a separate container. When pieces of each flatworm regenerate and become a new flatworm, they will all be clones of the original flatworm and they will all be genetically identical.

Varieties and extended results:

Below, several of the figures from Randolph’s manuscript are presented to illustrate the variety of ways in which she cut planarians (Planaria maculata) to challenge their regeneration abilities.

Animals were cut transversely (Fig. 1) and longitudinally (Fig. 2) and in most cases, each of the obtained pieces was capable of regenerating the missing halves.

In Fig. 4, the animals were cut both transversely and longitudinally. Randolph repeated this experiment several times and reported that ” in all cases each piece lived. Regeneration of all the missing parts took place…”

In Fig. 5, the animals were cut transversely into eight different fragments and ” all the missing parts were regenerated. The new individuals lived and seemed entirely normal…”

The experiment shown in Fig. 6 was designed to test the size limitations of planarian regeneration. As Randolph reported ” a piece only large enough to be seen with the naked eye will develop into a perfect whole”.

Later T. H. Morgan would show that a fragment 1/279th the size of the original organism is competent to regenerate a whole animal, thus confirming and extending Randolph’s results.

Planaria culture can be purchased from Home Training Tools website or from http://wardsci.com/default.asp.

Experiment 2: Cloning Plants by stem cutting

Introduction:

There are several advantages to propagating plants by cloning (root cutting, stem cutting, layering, …):

1. The new plant will be identical to the parent plant. For example, if the parent plant has multi-color foliage, the new plant grown from the cutting will have the same foliage. If the parent plant is female, the new plant will also be female. Propagating a plant by cloning will allow you to keep the special characteristics of that plant. Plants grown from seed will often be different from the parent plant and from each other.

2. Propagating a new plant via cloning avoids the difficulties of propagating by seed. For example, by using cuttings you could propagate a young tree that has not yet flowered (and thus has not yet produced seed), a male tree, or a sterile plant such as a navel orange. Additionally, some seeds are difficult to germinate, taking two to three years for the seedling to appear.

3. A new plant grown by from a cutting, layering or other methods of cloning will frequently mature faster and flower sooner than a plant grown from a seed.

Cuttings can be made from any part of the plant. Most frequently, however, either a stem or leaf is used. A stem cutting includes a piece of stem plus any attached leaves or buds. Thus, the stem cutting only needs to form new roots to be a complete, independent plant. A leaf cutting uses just the leaf, so both new roots and new stems must be formed to create a new plant.

Procedure:

Select a healthy garden flowers or houseplant such as Pothos for this experiment.

1. Cut off a piece of stem, 3-8 inches long. There should be at least three sets of leaves on the cutting.

2. Trim the cutting in the following way:

a. Make the bottom cut just below a node (a node is where the leaf and/or the bud joins the stem).

b. Remove 1/2 to 2/3 of the leaves, starting from the bottom of the cutting. Cut large leaves in half.

c. Remove all flowers, flower buds, and fruits if any.

3. (optional) Dip the lower inch of the cutting in rooting hormone.

4. In a pot of damp, but drained, rooting mix, make a hole for the cutting using a pencil. Put the cutting in the hole and firm the rooting mix around it. If any leaves are touching the surface of the mix, trim them back. Several cuttings can be placed in the same pot as long as their leaves do not touch.

5. Enclose the pot in a plastic bag, making sure the bag does not touch the leaves.

(Insert straws or wooden sticks around the edge of the pot to hold the bag away from the cutting. Place the pot in a bright area, but out of direct sunlight, so the leaves will receive the light they need but the plant will not get overly hot. The plastic bag insures that humidity around the leaves remains high, which slows the rate of water loss.)

6. Place the pot in a warm, bright spot but out of direct sunlight. Every few days, check the rooting mix to make sure it is damp, and water as necessary. Discard any water that collects in the bottom of the bag.

7. After two or three weeks, check to see if roots have formed by working your hand under the cutting and gently lifting (Figure 3). If no roots have formed, or if they are very small, firm the cutting back into the mix, rebag, and check for roots again in one to two weeks.

8. Once roots have formed, slowly decrease the humidity around the plant by untying the plastic bag and then opening it a little more each day. When it is growing well without a plastic bag, pot in a good quality potting mix and move to its permanent location.

Checklist for a successful experiment:

Start with cuttings that contain as much water as possible. Water the plant well the day before and take the cutting before the heat of the day reduces water content.

Once the cutting is harvested, excessive water loss must be prevented. To minimize water loss:

1. Process the cutting immediately. If this is not possible, stand the cut end in water or place the cutting in a plastic bag with a damp paper towel and store out of direct sun. If the plant is frost-tolerant, store the bagged cutting in the refrigerator.

2. Remove some of the leaves. Most of the water will be lost through the leaves, so by decreasing the leaf surface you also decrease the amount of water loss. A general rule of thumb is to remove 1/2 to 2/3 of the leaves. Cut remaining leaves in half if they are large.

Preventing Disease

Take cuttings only from healthy plants. To prevent the spread of disease, use clean tools and pots (clean with 10% bleach, rinse, and let dry thoroughly). Use fresh soilless potting mix since garden soil can harbor plant diseases.

Encouraging Root Formation

Just like leaves, the roots of plants need air to live. Rooting mix that is continuously waterlogged is devoid of air and cuttings will rot rather than form roots. A mixture of 50% vermiculite/50% perlite holds sufficient air and water to support good root growth, but any well-drained soilless potting mix is acceptable. If your cuttings frequently rot before they root, you know the mix is staying too wet. Add vermiculite or perlite to increase its air- holding capacity.

Cuttings use energy to form new roots. If the cutting has leaves, most of the energy comes from photosynthesis. Expose these cuttings to bright light, but not direct sunlight, during the rooting period. If you use hardwood cuttings that have no leaves, the energy will come from reserves stored in the woody stem. For best results, select shoots that are robust for the species. Since you want all the energy to go into the new roots, make sure you cut off any flowers or fruits that would compete for energy.

Auxin, a naturally occurring plant hormone, stimulates root formation. Several synthetic forms of auxin are sold as “rooting hormone.” Though some plants will root readily without treatment, application of rooting hormone to the base of the cutting will often improve your chance for success. Two synthetic auxins, IBA (indolebutyric acid) and NAA (naphthaleneacetic acid) are most frequently used. They are available in several concentrations and in both liquid and powder form. 1,000 ppm (0.1%) is used most often for herbaceous and softwood cuttings; 3,000 ppm (0.3%) and 8,000 ppm (0.8%) are used for semi-hardwood and hardwood cuttings. Liquid formulations can be used at low or high concentration for softwood or hardwood cuttings, respectively. To determine the appropriate concentration for your cutting, follow the instructions on the product label and the general guidelines just given, or consult the references listed at the end of this publication.

To use rooting hormone, place the amount needed in a separate container. Any material that remains after treating the cuttings should be discarded, not returned to the original container. These precautions will prevent contamination of the entire bottle of rooting hormone.

Cuttings will root more quickly and reliably in warm rooting mix. Keep your cuttings between 65°F and 75°F, avoiding excessive heat. If your area is too cold, consider a heating mat or cable especially designed for this purpose.

Experiment 3: Cloning plant by Root Cuttings

Introduction: Root cuttings probably started after gardeners observed

new plants growing from pieces of root accidentally left behind in the soil. Cuttings taken from roots may be used to clone and propagate some plants. Cuttings are usually taken when the plant is dormant and the roots contain the most stored energy. Each root produces two to three new stems and each stem then produces its own roots. The original root cutting disintegrates. Best results from root cuttings are likely if cuttings are taken
in late winter or early spring. Geraniums, Blackberries, Raspberries, phlox, baby’s breath, oriental poppy and many other plants can be propagated by root cuttings; however, in this experiment we clone and propagate potato by root cutting.

Procedure:

Cut a healthy fresh potato into about one ounce pieces with an eye and/or bud on each piece. Plant potato pieces three to four inches deep in the soil. Water as needed to keep the soil moist (not wet). In a good season, they should sprout and grow roots in a matter of days.

Encouraging Root Formation

Just like leaves, the roots of plants need air to live. Rooting mix that is continuously waterlogged is devoid of air and cuttings will rot rather than form roots. A mixture of 50% vermiculite/50% perlite holds sufficient air and water to support good root growth, but any well-drained soilless potting mix is acceptable. If your cuttings frequently rot before they root, you know the mix is staying too wet. Add vermiculite or perlite to increase its air- holding capacity.

Cuttings use energy to form new roots. If the cutting has leaves, most of the energy comes from photosynthesis. Expose these cuttings to bright light, but not direct sunlight, during the rooting period.

Auxin, a naturally occurring plant hormone, stimulates root formation. Several synthetic forms of auxin are sold as “rooting hormone.” Though some plants will root readily without treatment, application of rooting hormone to the base of the cutting will often improve your chance for success. Two synthetic auxins, IBA (indolebutyric acid) and NAA (naphthaleneacetic acid) are most frequently used. They are available in several concentrations and in both liquid and powder form. 1,000 ppm (0.1%) is used most often for herbaceous and softwood cuttings; 3,000 ppm (0.3%) and 8,000 ppm (0.8%) are used for semi-hardwood and hardwood cuttings. Liquid formulations can be used at low or high concentration for softwood or hardwood cuttings, respectively. To determine the appropriate concentration for your cutting, follow the instructions on the product label and the general guidelines just given, or consult the references listed at the end of this publication.

To use rooting hormone, place the amount needed in a separate container. Any material that remains after treating the cuttings should be discarded, not returned to the original container. These precautions will prevent contamination of the entire bottle of rooting hormone.

Cuttings will root more quickly and reliably in warm rooting mix. Keep your cuttings between 65°F and 75°F, avoiding excessive heat. If your area is too cold, consider a heating mat or cable especially designed for this purpose.

Experiment 4: Make a display to show cloning animals by DNA cloning (Nucleus transfer)

Many diagrams for animal cloning can be found on the Internet. Use them to get an idea and then make your own diagram or display. The following are some of the samples:

http://www.txtwriter.com/Backgrounders/Cloning/Cloningpage1.html

http://static.howstuffworks.com/gif/cloning-frog.gif

http://static.howstuffworks.com/gif/cloning-sheep.gif

Materials and Equipment:

List of material depends on the experiments that you choose to do. Such a list can be extracted from the experiment section.

Planaria culture can be purchased from Home Training Tools website or from http://wardsci.com/default.asp.

Calculations:

No calculation is required for this project as a display project.

Summary of Results:

Summarize what happened. This can be in the form of a table of processed numerical data, or graphs. It could also be a written statement of what occurred during experiments.

It is from calculations using recorded data that tables and graphs are made. Studying tables and graphs, we can see trends that tell us how different variables cause our observations. Based on these trends, we can draw conclusions about the system under study. These conclusions help us confirm or deny our original hypothesis. Often, mathematical equations can be made from graphs. These equations allow us to predict how a change will affect the system without the need to do additional experiments. Advanced levels of experimental science rely heavily on graphical and mathematical analysis of data. At this level, science becomes even more interesting and powerful.

Conclusion:

Using the trends in your experimental data and your experimental observations, try to answer your original questions. Is your hypothesis correct? Now is the time to pull together what happened, and assess the experiments you did.

Related Questions & Answers:

What you have learned may allow you to answer other questions. Many questions are related. Several new questions may have occurred to you while doing experiments. You may now be able to understand or verify things that you discovered when gathering information for the project. Questions lead to more questions, which lead to additional hypothesis that need to be tested.

Possible Errors:

If you did not observe anything different than what happened with your control, the variable you changed may not affect the system you are investigating. If you did not observe a consistent, reproducible trend in your series of experimental runs there may be experimental errors affecting your results. The first thing to check is how you are making your measurements. Is the measurement method questionable or unreliable? Maybe you are reading a scale incorrectly, or maybe the measuring instrument is working erratically.

If you determine that experimental errors are influencing your results, carefully rethink the design of your experiments. Review each step of the procedure to find sources of potential errors. If possible, have a scientist review the procedure with you. Sometimes the designer of an experiment can miss the obvious.

References:

List of References

List of plants and their propagation method

http://www.txtwriter.com/Backgrounders/Cloning/Cloningpage1.html

http://home.hawaii.rr.com/johns/index.html

http://www.ntu.edu.au/faculties/science/sbes/sbi106/SBI106Inverts1/sld001.htm

http://cloning.tripod.com/intro.htm

http://www.religioustolerance.org/cloning.htm

http://www.ornl.gov/TechResources/Human_Genome/elsi/cloning.html#whatis

http://science.howstuffworks.com/cloning1.htm

http://planaria.neuro.utah.edu/sanchezsite/Randolph.htm

Don’t kill your plants