Tipos de propagacion in vitro



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PROPAGACIÓN DE ORQUÍDEAS

 

TIPOS DE PROPAGACION IN VITRO

Las técnicas de propagación in vitro de orquídeas pueden ser de dos tipos:

1. PROPAGACION POR SEMILLAS

a. Por semilla Proveniente de Cápsula Dehiscente

Se utilizan semillas de cápsulas dehiscentes. Las semillas son esterilizadas con una solución de Hipoclorito de calcio o Hipoclorito de sodio (lejía comercial) en concentraciones que varían entre 0.5 a 10 %, por 10-30 minutos, tiempo que depende del tipo de semillas que se esta usando. Para favorecer la esterilización de las semillas es conveniente usar un agente que rompa la tensión superficial de la solución esterilizante (Tween-20), este es un agente que permite el mejor contacto del esterilizante con la semilla. Se utiliza Tween-20 a razón de dos gotas por cada 100 ml, pero este puede también ser reemplazado por detergente líquido casero. Las soluciones de Hipoclorito de calcio o de sodio son los esterilizantes más usados, aunque puede emplearse también peróxido de hidrógeno al 3% por 30 minutos (Show 1985).

Luego de la esterilización las semillas son enjuagadas tres veces en agua destilada estéril, para posteriormente ser sembrada en los medios de cultivo.

b. Por Semilla Proveniente De Cápsula No Dehiscente (Cápsula Verde)

En este caso se siembran semillas provenientes de cápsulas so dehiscentes. La cápsula es separada de la planta madre, se eliminan las partes que no serán necesarias o que puedan ser una fuente de contaminación (restos de pétalos y de columna), se enjuaga con agua y detergente y se sumerge por unos segundos en alcohol etílico al 75%. Luego se estiriliza sumergiéndola en una solución de Hipoclorito de calcio al 2 o 4 % por 20 minutos.

La siembra se realiza directamente sin necesidad de enjuague. Para abrir la cápsula se hacen dos cortes longitudinales a lo largo de la sutura de dehiscencia y dos cortes transversales, se retira el pedazo de cápsula seccionado y se procede a la siembra.

2. PROPAGACION VEGETATIVA O CLONAL

Se han desarrollado distintos métodos de propagación por meristemas dependiendo del tipo de explante brotes vegetativos en crecimiento, los cuales son esterilizados en Hipoclorito de calcio o de sodio al 4% por 20-30 minutos. Luego se enjuagan en agua destilada estéril y se procede al aislamiento y siembra de los meristemas o de las yemas.

Otro tipo de vegetación vegetativa se lleva a cabo utilizando las yemas de los brotes de vara floral, esta técnica ha sido aplicada con éxito en Cymbidium y Phalaenopsis (Intuwong et. la., 1972). Consiste en el corte de nudos que contienen yemas dormantes, su esterilización, enjuague y siembra en medios de cultivo con hormonas que inducen la formación de nuevas plántulas a partir de esas yemas.

Se ha usado también ápices foliares jóvenes de plántulas cultivada in vitro para la obtención de protocormos en Cattleya con éxito solo en el caso de utilizar como explante hojas muy jóvenes (Churchill. et. al., 1971). Los trabajos de propagación de especies como Cattleya y Plalaenopsis, abriendo la posibilidad de propagar especies hortícolas importantes sin afectar los órganos vegetativos de las mismas.



CONDICIONES DE CULTIVO

Existe una gran diversidad de condiciones y de medios usados en el cultivo in vitro de orquídeas. El estado dl medio de cultivo (líquido o sólido) depende de la especie con la que se este trabajando y debe ser determinado empíricamente. Generalmente la proliferaci_2n es mayor y más rápida en medio líquido y la diferenciación se ve favorecida en substartos o soportes sólidos.

Aún cuando es recomendable la agitación del medio líquido, se han obtenido éxitos con cultivos estacionarios (Kako, 1973; citado por Arditti, 1993) y con otros medios líquido agitados solo ocasionalmente (Arditt, 1993). La tasas de agitación rotatoria recomendads varían desde 0.25 r.p.m. (Jasper, 1996) hasta 200 r.p.m. (Scully, 1996). las condiciones apropiadas deben ser determinadas experimentalmente. la agitación del medio de cultivo favorece la proliferación de protocormos debido a la eliminación de la polaridad, retardo o prevención del desarrollo de raíces y brotes (Scully, 1967; Wimber, 1963, 1965), mejor aireación y dilución rápida de metabolitos tóxicos (Arditti, 1993).

Las intensidades de luz usadas tanto para cultivo de semillas como para cultivo de meristemas o yemas van desde la oscuridad hasta los 300 lux, generalmente obtenidos con fluorescentes y algunas veces complementados con lamparas incandescentes. Los fotoperiodos más adecuados para la germinación de semillas y cultivo de tejido de plántulas son también adedecuados para el cultivo de meristemas (Arditti, 1993).

Tanto para el cultivo de meristemas como para el cultivo de semillas el rango de pH más adecuado para el cultivo in vitro de orquídeas es de 4.8 a 5.5. Cuando el pH del medio de cultivo es muy ácido antes del autoclavado, puede ocurrir la hidrólisis de algunos componentes del medio, lo que puede resultar en la formación de soluciones semisólidas y la producción de algunos compuestos tóxicos para el explante (Arditti, 1993).

Las temperaturas apropiadas para el cultivo varían desde 24 a 26°C. Aunque temperaturas superiores también pueden ser toleradas por los cultivos, algunas especies pueden tener una menor tolerancia o requerimientos específicos.

 

SUSTANCIAS ORGANICAS USADAS EN LA GERMINACION ASIMBIOTICA DE SEMILLAS DE ORQUIDEAS

Un gran número de aditivos se usan para favorecer la germinación asimbiótica de semillas de orquídeas y el subsecuente desarrollo y diferenciación de los protocormos (Arditti, 1967). Los más frecuentemente usados son el endospermo líquido de coco, el jugo de tomate y el fruto del plátano. Steward and Simmonds (1954) reportaron que las capas formativas del fruto del plátano poseen sustancias qu estimulan la división de la células de zanahoria; con solo esta evidencia disponible, Khalifah llegó a la conclusión de que estas sustancias responsables de la división celular eran las citokininas.

Van y Steward (1975) encontarron que el fruto del plátano contiene pequeñas cantidades de compuestos inductores de la división celular [zeatina, zeatin-ribosido y 6-( 2-isopentilamino) purina] pero que comparados con el endosperma líquido del coco su actividad era extremadamente baja: 20 ml. de este aditivo daban un arespuesta similar a 500 g de plátano. Sin embargo, existen evidencia de que el efecto del plátano sobre el desarrollo de las orquídeas puede deberse a la presencia de otros compuestos distintos a las citokininas, pues se ha reportado la presencia de compuestos afines a giberelinas y auxinas en el mismo (Khalifah, 1966).

Valmayor (1972) reportó que el endosperma líquido del coco solamente permite una pobre diferenciación en protocormos de Dendrobium, vanda y Cattleya, mientras que su combinación con extractos de plátano resulta en un buen desarrollo de raíces y tallos.

  

HOME ORCHID FLASKING

Orchid flasking is the process of growing orchids from seed. Although the following techniques are not common lab procedures, they have worked fine for me, and should for you too. If I discover a better way to perform a certain technique, I will place the updated procedure in the appropriate category.

There is quite a bit of controversy concerning flasking by amateur growers. The ones opposed to it say that the amateur hasn't a full understanding on hybridizing, thus making inferior crosses. These crosses would then lower the overall quality of orchids in general. The world would be overrun with poor quality plants of questionable parentage.

The other side suggests that many of the very popular crosses were made by such growers, and sometimes purely by accident. Conservationists also argue that the ones that flask species, are actually helping to save certain orchids that are in danger of loosing their natural habitat, or that already have.

Which ever side is correct, flasking is an invaluable learning process in the culture of orchids.

Flasking Case: A flasking case can be as simple, or elaborate as you wish. One can be constructed out of an ordinary box, or plexiglass. The later would be more durable if you plan to do alot of flasking. Even a fish tank, laid on it's side with some plastic draped over the front will work. There are ready made glove boxes that can be bought from many firms that are nice, but sometimes expensive.

What I use, is a fish tank. I have it laid on it's side, then taken a piece of plexiglass cut to fit the opening, that have arm holes cut in it. I then secure long rubber gloves with duct tape (that stuff is wonderful) to the holes. I also fashioned longer sleeves, so I could reach all the way to the back of the tank. When flasking, the plexiglass top is taped fully to the tank, making an airtight seal. This setup cost me about $50, and has worked very well.

Flasking Tools: These are simple tools that are used in the process of replating, and removing the seedlings from the flask. Thin stainless steel rod, bent into a small hoop, and pounded flat will aid you in the tasks of replating, and deflasking. They can also be used if you need to go into the flask to remove small bits of contaminates. More on that later. Make at least 3-4 of each, so you can be sterilizing one set while using the other.

Besides the replating tools, you will need a small spray bottle for spraying the inside of the box with bleach solution. Two shallow containers for sterilizing, and rinsing tools, a vile for sterilizing and rinsing seed, a pipett, a scalpel, tweezers, toothbrush, and a beaker graduated in milliliters for measuring solutions.

Flasks: There are a number of containers that can be used for sowing, and re-plating. For sowing, I use baby food jars with either rubber stoppers, or Magenta B-Caps...non vented for sowing seed. This is because to germinate, seeds don't need a gaseous exchange. The media keeps longer, and doesn't dry out. Therefore, you can make a number of mother flasks ahead of time that will be ready when you need to sow seed. I used to use the lids that came with the jars, but found that after 1-2 uses the lids did not work any more, so I went to the types listed above.

For re-plating, again any vessel can be used. Glass is preferable, as it can be sterilized again, and again. Select a container that is big enough for the growth of seedlings. While the sowing flasks don't have to be vented, the re-plate flasks should be. After the protocorms begin to grow, they need a gaseous exchange for photosynthesis to take place effectively. There are many ways to do this. A drilled rubber stopper with cotton stuffed in the hole is the most common. Or, just a simple hole punched in the lid with cotton stuffed in it. The rubber stopper is a better way to go, because mold spores and bacteria have further to travel before entering the flask. There are even specialized tops that can be purchased that have a membrane to let gases pass through.

Pollination: The pollination of orchids is fairly easy once you know what you are looking for. I won't go into crosses here, just the basics of pollination. Hybridizing will be left up to your imagination.

To pollinate a plant, all you need is a pencil, toothpick, or a piece of bamboo, and of course...the flower! Take the "tool" and hook it under the anther cap. Pull it towards you. The cap will fall away, and the pollen will be stuck to the tool. Do the same process on another flower. Take the pollen from the second flower, and press it onto the stigmatic surface of the first flower. Pull away, and the pollen should be stuck in place. If not try again utill it sticks. There! You have a pollinated flower! Now all you have to do is wait for the capsule to form.

Pod Times: The time that it takes for seed to ripen in the capsule varies from genera to genera. I have included a table for the various orchid species.

Medium: The medium is what's used to sow the seeds on. It has a number of ingredients to promote growth, and agar to make it jell, so the seeds stay on top of the medium.

There are a number of companies that have ready mixed mediums. All you do is mix in distilled water, and follow the instructions.

Seed Sterilization: Once the pod has split or opened, the seed must be assumed to be contaminated. Before it can be sown, it has to be sterilized. To do this, take the seed and place it in a vile, or test tube inside the flasking case. To this add a 5% bleach solution with a drop of dish soap as a wetting agent and agitate continuously for 10 min. Just before the end of this time, decant off the bleach solution and add some sterilized distilled water. Agitate for about 1 min, and then decant once more. Repeat this step one more time. If you don't use a pipett, pour off all but a small amount of water, so you can pour the remaining water and seed into the flask. If a pipett is used, then just leave the water in the vile, and let the seed settle to the bottom. I prefer to use a pipett, as it offers more control.

Seed Sowing: There are two procedures for seed sowing. One is the green-pod method. This is the easiest, as you don't have to sterilize he seed. The second is the dry-pod method, done with the above procedure.

First you must prepare the case for your work. Place all the tools, flasks, and other equipment you will be using during the flasking process inside the case. Spray a fine mist of 10% bleach solution all over the inside of the case, including the flask tops. Let set for 5 min for it to take effect. Using the pipett, suck up a small amount of seed and some water. Crack open a flask, insert the pipet into the flask without touching the sides. Eject the seed, and water into the flask. Re-stop the flask, and swirl the mixture to spread the seed around. Do the remaining flasks.

Green-pod sowing involves taking a seed capsule that hasn't opened up yet for the sowing process. Put the pod in the glove box, and soak it in the 5% bleach solution for about 5-10 min. Scrub the outside with a toothbrush during this time. Rinse in sterilized distilled water. Slice open the pod, and scrape the seed into the flask. Or you can scrape the seed into a vile of sterilized distilled water, and use a pipett to put the seeds into the flask. This will eliminate much of the fibrous material that's in the pod getting into the flask.

Flask Care: After the flasks have been sown, place them under fluorescent lights for 8 hours per day. Do not try to simulate daylight hours. This creates problems with the growing seed. Seeds should start to green in about 4-6 weeks. Some seed may take longer.

If mold, or other contaminates start to grow, it's best to just throw the contents out. But if the seeds are of particular value, you can go back in and remove the contaminates. Remove the spot, plus 1-2mm surrounding it. Place a drop or two of 5% bleach in the hole that's left. Keep in mind, that every time you go back into the flask, you increase the chance of contamination.

Replating: After the protocorms have reached a workable size, it's time to replate them onto the final medium.

Place inside the case both the mother flasks, and the replate flasks, replating tools, a container of 10% bleach solution, and one of sterilized distilled water, a spray bottle of 10% bleach solution. Place the re-plating tools in the bleach solution, and let soak for 5-10 minutes, then rince them in the distilled water. Remove the stopper from the mother flask, and the flask to receive the replate. Scoop out a small amount of protocorms with one of the clean tools, and place them in the replate flask. Don't spread them too heavy, as the young plants do not do well competing for the same space. Sterilize the tools often to prevent contaminating the replate flasks. I use a new tool for each flask I re-plate. After you are done, replace the stoppers tightly. Place them back under the light, and watch them grow up!

Deflasking: When the plants have grown to 5-6cm, and have 3 or more roots, they can be set out. Once your plants are ready to be removed from the flasks, open them and let them become acclimatized for 24 hours before removing them. The easiest way to remove them may be to break the flasks. This will reduce the chance of damage by being pulled through the neck of the flask. Wrap the flask in a towel, and gently break it open. Gently pull the plants apart and place in a bowl of luke warm water to wash off any bits of glass, and medium. Place the seedlings in a mixture of sphagnum and perlite. Spray with a fungicide/bactericide. The seedlings do better if grouped together, so put 5-10 per 3" pot. It's best to not fertilize them right away, but give them a solution of Superthrive for the first week, or two.

Seedling Care: The young plants will take from 3-5 years before their first flowering. To get them to that point, requires special care. To start out, put them in 70% shade for 2-3 weeks, then move to 50% shade for the same time. At this point, they may still need a little more shade than a mature plant, and need to watered more often, as they don't tolerate drought as well as mature plants. You'll just have to experiment with them. I have several Cattleya seedlings that I put in very bright light, and they're doing fine. Fertilize every two weeks with a balanced fertilizer, and repeat spray with a fungicide/bactericide every 3-4 weeks.



COLCHICINE AND ORCHIDS.

This material may be copied for personal use, or for use by societies or research groups, providing that it is accompanied by a notice acknowledging authorship.

Cedric Maunder

Firstly, let it be clear that colchicine is a dangerous substance which needs careful use and disposal.

N.B. In the UK, Sigma/Aldrich (and other suppliers too) will not supply colchicine to individuals who cannot provide evidence of qualifications and/or experience fitting them to handle dangerous substances.

None the less, for those so qualified, it is possible to use colchicine safely and successfully to promote polyploidy in orchid seedlings. Dr Donald Wimber has been a recognised authority on this business for many years. He was a co-author of a paper in the Lindleyana in 1988 which gave details of a controlled trial of polyploidy induction in paphiopedilum seedlings. Colchicine was used in three different concentrations for 3 to 25 days on protoplasts at three different stages of growth.

From this the authors found that :-

1, Protocorms should not be too advanced in growth when treated - up to the stage when they are green and just starting to produce the first leaves.

2, About 7 days in contact with the colchicine should produce good conversion rates, longer treatment caused many seedlings to die.

3, Filter-sterilised colchicine was effective at a concentration of 0.005%.

4, Heat-sterilised colchicine was effective at a concentrations of 0.05% and at 0.025%.

The paper gives a very thorough treatment with the results in great detail, but the above is a very rough abstract of the 'nitty-gritty' as it affects small scale use by the 'serious hobbyist'!

Colchicine is reported to be destroyed by heat, so sterilisation by cold filtering permits use of less colchicine per flask. However, suitable filtering equipment is not cheap - ( one has to pay for more than is likely to be used within a reasonable space of time) - so for small-time use at home it seems best to use a 0.025% or 0.05% strength colchicine solution, adding this to flasks before they are sterilised.

In fact, I have been informed by those who have considerable experience of using colchicine that it is quite heat-stable when used with agar and water only. (For the paper cited above, the colchicine was added to normal orchid medium and it may be that the salts in that medium were more responsible for instability of the colchicine than the exposure to heat. - Personal communication from Robert M. Hamilton.)

Sigma sell colchicine in lots of 100 milligrams (or larger). It arrives in a small plastic bottle inside a bag inside a large plastic bottle - clearly marked as containing toxic material. Inside the small bottle is what looks like, and is, a very small amount of fine white powder.

To avoid having to handle this powder it is best to make up a solution containing all 100 milligrams at one time. To produce a 0.025% solution use 400 ml. of water, or 200ml for 0.05%. The article I mentioned above describes using a normal nutrient medium, but for one week it seems adequate to use a plain agar solution to which the colchicine has been added. This should allow successful use of 0.025% concentrations. Dissolve 2.8 gm. of Sigma agar in 300 ml. of water, add the colchicine ( uncap the colchicine and pour a little of the remaining 100ml. of water into the colchicine bottle. Replace the cap and shake and swirl to ensure all the powder dissolves in the water. Pour into the agar solution and mix in. Add more water to the bottle and empty into the agar. The bottle and cap can then be rinsed again and disposed of safely (only minuscule amounts of colchicine can remain by then).

Finally, add the remaining water so that all of the 400 ml. is used in the mix.

This mix should be a fairly solid one. This ensures that it is not too difficult to remove the seedlings from the colchicine mix when the time comes.

It would be advisable to wear gloves and a mask when handling either the colchicine or medium containing it so as to avoid hazard if an untimely cough, sneeze or spill should occur.

As the medium is only to be used for a week only a small amount is needed for each bottle - I usually make this mix produce ten or eleven bottles. The bottles can be capped and heat sterilised by your usual method. I use baby food bottles with foil for the seals - the bottles do not need to be vented as the seedlings are only in them for a week and there is no banana or other ethylene producing ingredient in there!

After re-plating the protocorms, the bottles can be incubated for a week in your normal incubation set-up. After about a week (5 to 10 days) the protocorms are re-planted again into your favourite medium and treated from then on as usual. Be sure to label the bottles after re-plating as having been colchicine treated so as to avoid confusion later between treated and untreated seedlings.

Agar/colchicine bottles not used immediately can be stored for several weeks after capping and sterilising.

After the seedlings have been re-planted out of the colchicine mix, the used medium must be disposed of. It still contains colchicine. It should be disposed of safely - check your local regulations and comply with them - it isn't nice to damage the local fishes etc. In any case, I like to melt the medium in hot water and dilute it considerably before disposal.

So far I have treated a number of species and hybrids using this method. Most have had a reasonable survival rate - some plants appear more sensitive than others. As to whether I have many tetraploid seedlings; time will tell. None are flowering yet (or for some time to come) and none are yet large enough to have root tips chopped off for chromosome counting - the only way to be sure what the ploidy is. Compared with untreated members of the same batch of seedlings growth of these colchicine treated seedlings is slow at first but picks up later. The general experience is that colchicine treatment will convert around 25 per cent of the seedlings to the tetraploid state.

Bob Hamilton has been the main source for most of the information I have on this subject, including the above. Many thanks to him for this. See his many postings on this and other matters. ( Any errors in the above are my own - the information was all OK before I started messing with it!!! )

FLASKING FOR THE AMATEUR

John T Woolf

Florafest Orchids

PO Box 7011

Toowoomba Qld

Australia 4352

Some basics about in-vitro culture for the Amateur.

POLINATION OF PLANT :-

One takes pollen from the POLLEN PARENT and places it onto the stigmatic surface of the POD PARENT.

Pollen is found in a variety of places on the different Orchid Genera and can be dry (as Cymbidium) or wet (as Paphiopedilum).

Pollination may take a few days or a few weeks depending on genera, Fertilisation occurs when the pollen has grown down into the Ovary (seed capsule) this usually takes approximately two thirds of the period to maturity. After this GREEN POD culture can be started, Once the seed capsule has split one has to use the DRY SEED technique. It is far easier to flask Greenpods, you simply trim off any damaged areas and old flower parts, sterilise in 2% sodium (1/2 strength Snowwhite or similar) take into your clean air cabinet (or Home made box), cut seedpod open and decant seed into sterile flasks. For dry seed one has to sterilise individual seed then wash in sterile water before placing into sterile flasks, this can result in over sterilisation and you can kill the seed or under sterilisation and you can lose the seed to Fungi / Bacteria.

MERICLONING OR MERISTEMMING :-

This is the taking of a piece of tissue from a plant and reproducing many exactly (in theory) the same. The Explant as it is known will be washed in alcohol, sterile water, sodium, sterile water trimmed washed again and this treatment continued until you have a sterile minute piece of tissue to place in the sterile flask. Once it begins to grow it is then placed onto a multiplication media and continually cut up until you have produced the required number of plants.

THE FLASK :-

This can be any glass, plastic or similar jar that has a lid with a good seal and can be autoclaved in a pressure cooker. The lid has a hole drilled with a rubber tube packed with non-absorbent cotton wool inserted as a breather (or you can use a Teflon patch). The jar etc. MUST be clean, soak in sodium for 30 minutes and then rinse any dirt and sodium off as the sodium will kill plants if left in flask.

THE MEDIA :-

This can be purchased from firms such as Orchid Media Lab who specialise in orchid flasking media (and others see Orchids Australia) or make your own.

Some ingredients that we use are available only on permit from A Q I S .

A simple media.....

(1) 2 1/2 grams of G F 9 fertiliser - available from Groforce

(2) 5 beads of Calcium Nitrate - available from Groforce

(3) 6-7 grams of Agar Agar powder -available from health food shops

(4) 20 grams of sugar, (less for some genera) fructose or glucose

(5) 1/4 teaspoon activated charcoal (optional but required for some genera)

available from Selbys.

(6) 1 drop of Best Grow - available from Bacto ( we have tried Nufarm Growth

Formula with equal success)

(7) Peptone (optional) you will need a permit for this)

(8) 100 grams of ripe Cavendish bananas (put through a blender ). Other fruit used

for specific purposes are coconut milk, potato, tomato, pumpkin etc.

Make up to 1 LITRE with Demineralised water (available from supermarkets)

STERILISING THE MEDIA :-

This is then placed on a stove and bought to near boil, stirring continuously,decant into flasks and seal with prepared lid. This is then placed into pressure cooker for 15 minutes at 15 p s i to sterilise. Leave to cool, remove and tighten loose lids and place in a clean dry area until used. ( A new foam esky or icepak box is good)

THE CABINET :-

This can be a CLEAN AIR UNIT costing from $2.500.00 to $10.000.00 or a home made unit costing $50.00 depending on how serious you get or how many flasks you are going to produce.

Here are two examples.

(1) a wooden, metal or perspex box with a sliding glass top and two armholes.

(2) a foam icepak box with the top cut out and glass inserted with two armholes.

METHODS OF FLASKING :-

There are two different methods used,

(1) Using sodium hypochlorite

(2) using a Bunsen Burner

The inside of the unit should be washed with Alcohol or Sodium before starting.

Equipment needed includes,

1a. A plastic food container to hold sodium

1b. Or a Bunsen Burner

2. A petri dish or a saucer

3. A scalpel or knife (Stainless Steel)

4. A pair of tweezers (Stainless Steel)

5. A pair of rubber gloves and some Talcum Powder

6. A charcoal impregnated face mask

7. A plastic backed apron or some old clothes

8. Some cloth that will stand sodium (Kimtex Wipes if available)

9. You will find other tools as you become more adapt.

STORAGE OF FINISHED FLASKS :-

Once finished replating (placing seedlings on agar in flasks) place flasks in a dry area where they will get reasonable light from all sides (if possible or turn occasionally) or place under artificial lights.



REFERENCES

León Martinez, Marco. CONSERVACION DE ESOECIES PERUANAS DE ORQUIDEAS UTILIZANDO TECNICAS DE CULTIVOS DE TEJIDOS IN VITRO. Tesis para optar el Tiltulo de Biólogo. La Molina 1995.

Colchicine & Ordichs

http://www.ask/cochicine

Home Ordich Flasking



http://www.ask/homeordichflasking.com


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