The RTU Plant Biotech Project


Linkages to Websites on Plant Tissue Culture
June 4, 2010, 5:59 am
Filed under: Uncategorized

http://aggie-horticulture.tamu.edu/tisscult/microprop/microprop.html

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Training on Banana Tissue Culture
June 2, 2010, 6:53 am
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The PLANT BIOTECH LAB conducts  2-day Banana Tissue Culture Training for interested participants.   For information, kindly contact us at the Plant Biotechnology Laboratory, 3rd floor, Research Building, Rizal Technological University Compound, Boni Avenue, Mandaluyong City, Philippines.  Registration Fee is PhP3,000.00 per person inclusive of lab materials, handouts and kits.  Snacks & Meals are excluded.

For further details, please contact us at  : (+632) 534-8267 Local 135 or email at norby.bautista@gmail.com



Establishing an Orchid Laboratory for Conservation

Link to the RTU Plant Biotech Lab

Link to the Main RTU Website  http://www.rtu.edu.ph/

Link to the Philippine Orchid Society Website  http://philippineorchidsocietyph.org/

REQUIREMENTS IN ESTABLISHING A SIMPLE ORCHID LABORATORY FOR CONSERVATION PROJECT

 

Orchids are intriguing plants to grow because of their exotic beauty and mystery. A hobbyist usually starts with nature plants, and after learning the basics of their cultural requirements, will then proceed to grow established seedlings, then to seedling in community pots and maybe up to the ready-to-compot flasks. One of the hobbyist’s is to see this plants grow and multiply. He will then be tempted to grow to his own seedlings. For a hobbyist’s one of the great joys for life is to watch dust-like orchid seeds planted in mother flasks slowly turn green, enlarge, turn into Protocorms and grow into seedlings. Also, having a laboratory will mean that the number of plants you will have will be limited only by your growing space, since orchids produce thousands of seeds.

 

Establishing an orchid laboratory need not to be too expensive nowadays since there are already a lot of substitutes for expensive chemicals and equipment, also there are now a lot of gadgets, glass wares, culture vessels chemicals and equipment to choose form. However a hobbyist’s needs to train first in the basic of orchids micropropagation in order to foresee problems and to know how to trouble shoot them.

 

A trial laboratory could be remodeled inside your home, garage, warehouse, office or a portion of your green house. What is important is the cleanliness of the site, which should be away from source of chemicals (pesticides), dirt and pollution. A correct laboratory design and good practices by the staff usually minimized contamination, the number one problem of most tissue cultured laboratories. Through careful planning it could be made expensive, feasible, applicable and clean. Tissue culture laboratories usually consist of 3 parts, namely (1) a preparation area, (2) a transfer or inoculating room. The order is base on their increasing level of cleanliness with the inner most areawith less foot traffic. In extreme or temporary circumstances, these 3 areas could occupy the same room, but long term operating efficiency and need for cleanliness dictate that each be in a separate room.

 

A preparation room resembled a kitchen, with deep sink, running water, refrigerator, balance or scale, a working table, a pressure cooker or autoclave, gas tove (heavy duty) a lots of shelves and cabinet to store lab utensils, glass wares, bottles and chemical reagents, here are some division of this area:

 

a. General washing area- this is most accessible from the outside. This is where raw and non sterile plants part are cleaned and where lab utensils and glass ware are washed. Even if it is not sterile area. It has to be keep clean and orderly since it provide entry to other rooms. It usually a large an deep sink, a drying

and draining rack.

 

b. Media storage and preparation – This area is where reagent , glass wares, and other necessary things for preparing the media are kept. A working table which is resistant to chemical is located beside it. Other equipment includes a triple beam balance or top loading balance with accuracy up to 0.01 gram,

analytical balance*, storage tank for distilled water, blender/osterizer, distilling apparatus*, pH meter, stove/ hotplate stirrer*,refrigerator, and a micro filtration system*, In this area, stocks solution are made and medium are prepared.

 

c. Sterilization area – This where prepared media and lab instruments are sterilized. This is also where contamination cultures are decontaminated (the fungal or bacterial contaminant is killed) Equipment like the pressure cooker, gas

stove and LPG tank are located here. If possible a separate pressure cooker for sterilizing and another for decontamination is recommended. A lab cart or plastic baskets are useful in transforming culture bottles within the laboratory rooms.

 

The second is the transfer room. It is a clean room which contains the transfer chamber. It is in the are where the technician do the inoculation of seeds into mother flasks and sub culturing of Protocorms into reflasking medium. The transfer chamber could be in the form of a laminar flow hood or a still – air inoculating box. A large glass aquarium titled on its side and the opening covered

with clear plastic sheet with two holes for hand is just find. If the area could always be kept clean, a transfer chamber could also be made from white cotton cloth (from flour bag) dipped in 20 % Clorox suspended above a small in working table in one corner of the room. The surfaces have to be cleaned with Clorox solution and the air current minimized. The transfer is room is ideally air conditioned. It is fumigated every 6 months. It is useful that the UV lamp is installed with the fluorescent light in the chamber. It is open 30 minutes before work to contact sterilized culture bottles and the working surface. Before working inside, Clorox and ethyl solution is wipe on the surface. An alcohol lamp, a beaker of ethyl or denatured alcohol, forcep and scapel are place inside the chamber.

 

It is also helpful to install the sliding door with glass windows inside the transfer and culture room to separate them from the preparation room. Sliding doors are recommended compare the ordinary wooden doors since it will not cause air draft when open or closed.  This would minimized the entry of contaminants.

 

The third room is to culture room. It is also a clean room, similar to the transfer room, with lots of shelves, air conditioned, and well lighted (ideally 16 hours light and 8 hours dark photoperiod with the aid of the timer switch or manually switched on and off) the long hours in light will prevent dormancy in orchids being cultured.

 

Culture bottles with plant are placed in these shelves to grow. The temperature is lowered to about 24-29 degrees C. in order to minimized contamination. It is the least accessible room for the out side environment. An observation area (with microscope) with in the culture room could be an accessory. This helpful in observing the viability of seeds through a microscope. The culture room should be protected from windows since outdoor light and temperature reduce the ability to control the growing environment.

 

Potting Shed/Acclimatization area located outside the laboratory or inside the green house is a necessity in order to help adjust plant from living in the laboratory to living in the natural environment. Plant inside the bottle (in vitro) are usually use to high  humidity, a sterile environment and low light intensity. They are like human babies that need to be gradually adapted in order to survive in the outside world. Thus the culture bottle or flask with plant would have to stay in the said area for one month before they are transferred out of though bottle . the Potting shed usually has 50 – 80% shade and protected from rain. It also has work benches, plant trays, and a cabinet for plant nursery tools, rooting hormones, fungicides and insecticides.

 

Precautions & Practices in the Laboratory:

 

1. To prevent fire, circuit breakers (which controls the flow of electricity going into fluorescent lamps) are installed for the culture room and a fire extinguisher is handy.

2. Only personnel working in these areas should be allowed to enter except occasional visitors who are previously tipped on how to conduct themselves in these areas.

3. Avoid entertaining friends and relatives in the work areas.

4. Avoid eating, drinking, smoking and applying cosmetic in work areas. Food scraps attract rodents, cockroaches, ants and insects which spread contamination.

5. Mop transfer and culture room floors with 10% Clorox /Lysol. As much as possible, do not sweep or vacuum clean since it will cause dust to be suspended in the air . at the preparation area, it is all right to sweep dust.

6. Monitor contamination regularly fumigate if necessary. Removed contaminated culture and decontaminate them in pressure cooker before cleaning the bottles.

7. Wash hand with soap and water and rinse with 70% rubbing alcohol before inoculating and handling cultures. Wash hands after handling chemicals.

8. Disinfect laminar flow hood before using. Turn the UV lamp 30 minute before using and wipe surface with 10% Clorox or 70% ethyl alcohol. Be careful not to work in laminar flow hood if the UV lamp is switched on.

9. Sterile all instruments used for handling tissues inside inoculating chamber: forceps, scalpel, scissors wrapped in aluminum foil or paper and sterilized with media 15 psi at 121 degree’s Celsius.

10. Flame instrument like scalpel, forceps, scooped and scraper before use. Dip in 70% alcohol and flame in alcohol lamp. They can be reused by reflaming.

11. Keep 95% alcohol away from open flame.

12. Practice extreme care with chemicals.

13. Before opening the pressure cooker, make sure the pressure cooker has dropped to 0 and the temperature below 100 degrees Celsius.

 

Some Tips:

1. Separate ballast of fluorescent light into upper part of shelves or outside lab. Ballast usually emit heat.

2. Make sure your lab is comfortable to work. A tissue culture laboratory is usually similar to that of a hospital or operating room environment. To prevent if from being boring, cold and morbid (from the smell of Clorox or Lysol ), it is helpful to play music through a cassette recorder.

3. If the area outside is muddy, a foot dip is copper sulfate solution adjacent to the lab door entry is recommended. A cemented pathway to the lab is also preferred.

4. Different clean slipper are used in the 3 areas of the lab. This rule has to be strictly followed to prevent entry of contamination.

5. Laboratory shelves and walls are wiped Clorox solution once a week or month.

Contamination rates usually climb up during the rainy season. As much as possible do most of your inoculation and sub culturing during summer or take extra precaution on sterilizing during the rainy season. Also test and check for air back drafts in the laminar flow hood or inoculating chamber by placing 6 opened sterilized culture bottles (without plant yet) for 1 minute and placed back the covers. If these bottles become contaminated after 3-7 days, then, there might be air back drafts in your transfer chamber.

 

Substitutes

One of the great things in orchid embryo culture is that some techniques or materials could be substitute or improved. In this way, the cost of operation is lowered. Some of the equipment and gadgets that can be substitute are the following:

1. Laminar Flow Hood – can be substituted by a wooden transfer box with glass viewing window or large glass aquarium titled on its side, with a plastic cover with the holes for hands. However, a laminar flow is still very reliable.

2. Pressure cooker- can be substituted by using a large kaldero. This process is called Tantalization, wherein the culture medium is sterilized by steaming for 1 hours, then letting it cool down but the cover is not opened. the next day, it is repeated until the third day. It is not as reliable as the pressure cooker though.

3. Petri dish – can be substitute by ½ sheet used bond paper (PLDT Directory book will do) sterilized inside polypropylene plastic bags.

4. Culture vessels. – (defend on size of plant) Gilbey’s ketsup, mayonnaise, Gerber, or dextrose bottles.

5. Bottles Caps/Rubber stoppers- cotton plugs, plastic caps, metal caps, or polypropylene plastic sheets with rubber bands.

6. Chemicals for medium — Foliar Fertilizers, sugar, ferrous sulfate tablets, young coconut water, banana homogenate, tomato puree, gelatin, baby’s vitamins and distilled water.

7. Distilled water — Wilkins or Rain water (clean)

8. Culture shelves — book shelves

9. Fluorescent lights in culture room — make the roof into clear plastic roofs instead of GI (similar to greenhouse in construction) in order for sunlight to enter and the shelves made of durable metal mesh, with wooden frame. In this way, sunlight will pass through all the layers of the shelves.

 

References:

Kyte, Lydiane. 1987. Plants from Test Tubes: An Introduction to Micropropagation. rev. ed. Portland, Oregon: Timber Press, pp. 20-35.

 

Hicks, Aaron. March 22, 1999. Personal communication with, through email (ahicks@nmt.edu) Subject: Re: Thank you for your reply.

 

Bergman, Fred. March 22, 1999. Personal communication with, cited by Aaron Hicks. Subject: From seed sowing: the easy way (1996) In Orchid Review 104: 23-25 URL: http://vergers.com/culture/flaskii.htm

 

Hicks, Aaron. March 22, 1999. Personal communication with, cited a compiled compiled communication with Bob@argon.berkeley.edu dated July 12-29, 1993

 

Del Rosario, Aurora G. 1994. Personal communication with. Department of Horticulture, CA-UPLB, College, Laguna

 

* * *

* Optional materials, can be substituted.

 

Author can be contacted at : E mail:  norby_b@rocketmail.com

 



The Conservation of Philippine Orchid Species Project
February 6, 2008, 2:12 pm
Filed under: Uncategorized | Tags: , ,

The Conservation of Philippine Orchid Species Project of Rizal Technolocial University started in 1995 with the help of the Philippine Orchid Society. Its main objective is to create a genuine orchid conservation program for Philippine Orchid species with coordination with the Philippine Orchid Society, other garden NGO’s (Philippine Horticultural Society, Ferns & Nature Society of the Phililippines), government institutions, agricultural State Universities and Colleges and the then created Orchid Conservation Network of the Philippines. 

Click Here to View Orchid Researches at RTU