Seed Sowing Manual
|
OrchidSource Laboratory and Nursery orchidsource@gmail.com
|
|
| The purpose of this manual is to provide a guide to the first time tissue culture practitioner. However many of the suggestions may be of some special use for the old-timer as well. It should be pointed out that there are many ways to do the same thing. Each laboratory person may find an easier way to accomplish the same task. Until you have become acquainted with sterile procedures, I would suggest that you keep close to the procedures outlined in this manual. Lets assume that you have made your first cross and the seed is to ripen soon. Congratulations to you for you have embarked in the most interesting phase of growing orchids. The results of your hybridization will add greatly to your enjoyment of growing. In the next chapters, we will be discussing procedures in seed sowing as well as replating. We will also discuss the types of equipment which you may use, buy or make. |
Depending on the intent of the user, a glove box can be made very easily and inexpensively. My first glove box was made out of an old jewelry display case from a pharmacy. I used the three clear sides of the plastic case as the box and covered the ends with some greenhouse plastic. I cut holes into the ends and attached some plastic kitchen gloves with duct tape. Then I would use the kitchen table top or some other hard table top as a work surface. In such a device you can seed sow or replate about half a dozen flasks at a time! The cost of the box is less than ten dollars. You can make one out of plexiglass, or other materials which you may have some preferences towards, or make a more preferable laminar flow clean air station. This is a lot more expensive, but gives one lots of freedom of movement as well as efficiency. Either way you will still be able to get the job done.
Now that you have the equipment to do the seed sowing and replating, we should discuss procedures on cleaning the green pod. Oh by the way, I suggest attempting to sow green pods the first time around, as success rates are highest when you start with a sterile capsule. You may however over stay with a pod and have the thing open up quite unexpectedly and be faced with a lot of loose contaminated seed. But lets understand the principles in green pod sowing and later discuss the cleaning of contaminated seed.
First lets prepare our seed pods with some record keeping magic. It helps to devise some type of numbering system, so that you don't have to write out long names of crosses all the time. Of course, you must write the names somewhere. The system that I like most is to obtain a ledger book with the cross names and assigned numbering system. You may like to use some date code or other system of numbers and letters in combinations. But it is very important that you use at least four numbers or numbers and letters in combination to avoid duplication or confusion. In your ledger book write the name of the cross after the number assigned and record the date the cross was made and the date sown and leave room to record information about the results of the cross etc. This log book will become a valuable tool as a reference for future crosses and to evaluate the success of your stud plants as parents etc.
Next lets mark our pods with an indelible pen or laundry marker with a fine
tip with your assigned number. Of course this number will be placed on the
seed sowing flask or vessel as well. I find it quite easy to group no more
than a half dozen pods at a time to be sown in our glove box. 
Next we will need some tools. You will need some
disposable scalpels, a tooth brush, 2 pint sized Tupperware containers with
lid or its equivalent container, and a gallon of laundry bleach. If you are
working in a laminar hood, you might want to wear some kitchen gloves with
the cuffs rolled up to stop any bleach solutions from dripping down your arms
and burning your skin. Incidentally the scalpels are sold bin many hospital
supply stores. Get ones with plastic handles and fixed blades. The ones with
straight blades are preferable to ones with hooked blades for seed sowing.
Since the pods are marked with an indelible ink, we can soak them in a solution of laundry bleach (30 % in tap water) in the Tupperware container for 10 or 15 minutes. Now take the tooth brush and scrub the pod and let it soak for a while again. While all this is going on you should be soaking the scalpels with a 30% solution of bleach also. Now grab the stem end of the seed pod with your left hand and with your right hand pick up 1 of the scalpels and raise the blade up so that the bleach will flow down towards your gloved hand, draining back and away from the cutting blade for a moment. Now you can cut a water melon cut out of the seed plod exposing the seed. Now lets take 1 step backwards in the procedure. The seed sowing flasks which you will be placing the seed into in a moment or so should be covered with a foil cap at the time of sterilization (at least 4 layers thick). Just before attempting to open the pod you will remove the foil cap with the stopper in 1 piece and place them as a unit on a ledge, and out of the way. If you are using a glove box, you should spray the entire box with a fine mist of 30% bleach solution with a hand mister or old Windex sprayer. If you are in a laminar flow bench you don't have to do any of these things. Now back to the seed pod. With the scalpel blade scrape out a portion of the seed in the opened pod and drop it into the opened seed sowing flask. I don't trust myself and place another portion in a second seed flask, in case I might get something into the first flask and loose the cross etc., but suit yourself! Then re-stopper the flask and keep your fingers crossed for about 2 to 6 weeks. Hopefully we will be seeing some green in there.
Dry seed sowing is another story. Once a seed pod has opened and spilled its seed, or split open even slightly, you must assume that the seed is now contaminated with bacteria or spores of fungus. The biggest problem is fungus spores. Spores are coated with an impervious coat that protects them from chemicals and heat to a great extent, until the right environment is present for their bloom. In order to sterilize the seeds of orchids, you must expose the seeds to a sterilant capable of killing the live bacteria and bloomed fungus, but not kill off the seeds at the same time! There is a number of chemicals used for this purpose, and most of them are some type of chlorine salt with sodium or potassium. I have found excellent success with common household bleach. The trick is to expose the seed and villains to the solution long enough to kill the enemies, but not the seeds. I have found that a 5 percent V/V of household chlorox made with sterilized distilled water, when exposed to the contaminated seeds for a total time of 10 minutes and followed with a double rinse of sterile distilled water works in 70 or 80 percent of all contaminated seed sowing cases.
The exact procedure is simple. First label 1 or 2 mother flasks (seed sowing flasks) with the code name of the seed cross you wish to decontaminate. Next label a sterile vial or tissue culture tube which has been autoclaved and the mouth and cap, down about 2 or 3 inches onto the neck covered with a 4 layer thick foil cover. Into the sterile vial place a half portion of the collected seed and add a 5 % solution of chlorox solution and agitate intermittently. In the last 2 minutes let the seed settle or float to the top without agitation. Then decant off the excess solution about a half minute from the ten minute mark. At the last moment add sterile distilled water to the seeds in the vial and again agitate for 1 minute. Then again decant the solution and repeat the process again, but do not decant all of the solution. Open the seed sowing flask('s) and agitate the remaining seed and water to suspend the seed and pour the suspension into the seed sowing (mother) flasks and re-stopper them along with their foil caps. If all goes well you will be seeing germination in a few weeks. If you fail in your first attempt, you can retry the remaining seed you have saved from the pod. If that fails you will have to remake the cross next year, so record the results in your ledger book, to tell you the outcome of the attempt to make this cross etc. Keeping good records is very important in this business of hybridizing.
Once you have successfully make your seed sowing flasks, you must find a warm (70 to 75 degree f.), and well lighted place to grow them. I suggest artificial light, incandescent or fluorescent for a period of 8 hours, or daylight, if well filtered. Do not try to mimic the natural sun light period with fluorescent lighting in tissue culture, as this will only slow growth and cause problems with phenol production, a sign of stress, and toxicity to the developing protocorms.
After a few weeks, your seed should be germinating. But don't rush replating!
Your protocorms should be large enough to be handled with your replating tools
and allow for easy handling and separation. Do not overcrowd replate flasks!
Most plants including orchids do not do well if they are competing strongly
for the same space and food with many others. 
So when you begin transplanting into your replate flasks,
consider the number that will grow well in the space in their flask. If in
doubt underplant! Also consider the number of plants that you would like to
have when they grow up. Remember that when the plants are small they take
up little space, but grow up they will!
We will need some tools to do this job called replate forks. I like to make my own out of welders stainless steel rod cut to length and flattened into a knife or fork or hook end with a hammer. You might like to heat one end with a torch, or just pound the end cold. Also you will need a mister bottle with a solution of chlorox of about 30%V/V. You will also need a couple of rectangular Tupperware or glass dishes to soak your replate forks in prior to working your mother flasks , gloves, and a quart bottle full of sterile distilled water, capped with a four layer thick aluminum foil cover.
One tip that I like to use when the mother flask is really packed with protocorms that are competing heavily and are unlikely to grow evenly is to make a spread flask. A spread flask is an intermediate replate flask which contains a larger number of protocorms than the mother flask, but isn't a replate. This spread flask allows the protocorms to grow up a bit before the final replate is done. This also allows a lab person to size the plants a bit and makes a better and more evenly growing replate. Also when a spread flask is done, the time it takes to grow them to a size needed to replate is shortened and may save several weeks in growing time.
Begin by selecting your mother flask and gathering the number of replates you will want to transfer to, along with the tools into your glove box or clean air machine. Place your replate forks in one of the Tupperware vats or their equivalent in a solution of 30 percent chlorox and water. Let your instruments soak for 30 minutes while you are labeling your replate bottles. Also fill a spray bottle with a solution of 30 percent chlorox solution. I recommend that you use a numbering system of some type. You should use an indelible laundry felt pen and write on a plastic label or a laboratory type tape that will not peal. Attach the label in a secure way such as a wire or water resistant tape. Now we are about ready to open the mother flask. If you are working in a glove box, you will first need to clean the work area and remove any airborne spores or bacteria. So with gloves on and all the flasks in box ready to work on (labeled etc.), spray entire box areas with a fine mist of chlorox from the hand sprayer. Also spray the tops of the bottles that you will be opening, the sterile distilled water that will be used to rinse the forks off before use and lastly your gloved hands, which I am sure are in need of cleaning. Now that you have sprayed, lets let the mist settle and allow the chlorox to kill any little critters that are on any of the things mentioned.
Now open the mother flask or spread and the desired number of replates that are ready to be filled with protocorms. Place the foil covered stoppers in integral units on a ledge away from where the actual replate work is to be done and in a dry place towards the back of the work chamber. It is now important to begin thinking about where your hands are or were while you are doing the transplant work. This is part of the technique that you must develop in order to minimize the contaminations. As mentioned earlier, I like to use 2 vats to sterilize my replate forks prior to replating. In the first vat all the forks start and after they are used, they are moved to the second vat. When they are all used, I start all over with a 20 minute break to let the last fork soak in the number 1 vat. Next I remove a fork from the number 1 vat into my working hand and open a bottle of sterile distilled water and wash the fork by erecting the fork tip vertically to allow the water to drain towards the hands. I then insert the fork into the mother flask or spread and start working the protocorms apart and carrying them to the replate flask. I like to work the protocorms against the side of the flask, rather than on the media itself, which is soft and unable to support much pressure. It is good to count the number of protocorms removed and planted because it is easy to over plant and cause the transplant to fail to grow properly due to crowding. I usually plant between 25 to 35 plants into a 500 ml replate flask, depending on the surface area in the vessel you happen to be using. When you are done with planting 2 to 4 flasks, it is a good idea to exchange forks before going into the mother flask again. Let me back up a little though; try to limit the mother flask to a minimum of entries, so use 1 fork to move protocorms from the mother flask to the replates and 1 or more forks to do the actual replating etc. If there is an accidental touching of the fork onto something unclean, or mental lapse, you should exchange forks.
Now that the actual replating is done, you can enjoy watching the plants grow up. You should grow the replates and mother flasks in a warm (70-75 degree F.) dry place with 8 hours of artificial light at around 100 foot candles, or by natural light of around 300 foot candles measured at noon on a clear day. Do not try to mimic the duration of time with artificial light, as there are some important differences. I will not go into this in this article, but trust me for the moment.
I hope that you will find my comments and suggestions helpful and you will not have many difficulties in attempting your first seed sowing and replating work. Keep in mind that practice will perfect your techniques. Don't be afraid to try some variations on the techniques I have suggested. You may find a better way to do some of the things that I have described. Good luck!