Tissue culture attempt number 2!
Well, now that some of my plants have started growing again and some of them actually put up flower stalks, I decided to go ahead and make my second attempt at tissue culture with new explants. I made my second attempt on February 11th, 2009. Last time I did tissue culture, back in November 2008, I created the media, gathered the explants and did all of the sterilization in the same day. It was way too much work. So this time I decided to make the media one day and do the explant gathering and sterilizing the next day. It worked out really well! I made the media on Monday, February 9th and then gathered the explants, did the sterilization, and put them in vitro on Tuesday, February 10th. Breaking it up into two days made things much easier. This time I actually made 3 liters of media and each batch was slightly different. All of them had 1/3 Murashige & Skoog (MS) because I used one 1L packet and split it evenly between 3 1L containers. Also, each liter had 20g of table sugar. Other than those similarities, all of the other ingredients were different. Below is a summary of them. I used one drop of food coloring this time in two of the 3 liters to be able to tell them apart: Blue formula--------------------- 1/3 MS 2 Tablespoons sugar 1 ml BAP pH = 5.6 Yellow formula--------------------- 1/3 MS 2 Tablespoons sugar 1 ml PPM 5 ml BAP 0.25 ml IBA pH = 5.6 Clear formula--------------------- 1/3 MS 2 Tablespoons sugar 1 ml PPM pH = 5.6 I put 50ml of each formula into baby food jars until there was no media left to distribute. Then I added one level measuring spoon "pinch" of agar to the jar, capped it, and then put it in the pressure cooker. After bringing the pressure cooker up to full pressure, which is 15PSI, I leave it going for 30 minutes and then turn off the heat. I do 30 minutes because I live in Boulder, Colorado where the elevation is 5300 feet above sea level. At sea level, I would do 25 minutes. After letting the pressure cooker come back to near room temperature, I remove the jars one at a time and swirl them sufficiently to make sure the agar is evenly distributed throughout the media. Then I put them in the fridge to cool and solidify. This time the media turned out darn near perfect! I was very happy with myself on that point. In addition to the media, there were several other things that I ran through the pressure cooker in order to sterilize. These items include: Pint size jar half full with H2O2 3 pint sized jars of 10% bleach 3 pint sized jars of sterilized water 5 tall baby food jars with sterilized water forceps, scalpel, and wire for sowing seeds All of these items were left in the pressure cooker overnight and carried into the sterile working area the next morning just before I started sterilizing the explants. The forceps, scalpel and wire were placed in 91% alcohol to keep them sterile. In order to gather the explants, I decided to repot my oldest Venus Flytraps that were growing the most this early in the year. Most of them were already putting out significant growth and some of them had quite large flower stalks. I gathered a couple dozen or so of the newest, largest leaves from the plants and all of the flower stalks that had grown, which was about 6 flower stalks. When gathering them, I gently pulled the leaves and flower stalks all the way down to the bottom of the rhizome and then off. One of the flower stalks was already nearly 6 inches tall! It is important to note that these plants had been indoors since October with the exception of a few days that were very mild and I put them outside. Gathering tissue that grew indoors gives you a significant sterile advantage to tissue growing outdoors. Outdoor tissue is much dirtier and therefore harder to sterilize. Also, all of the tissue was at most 4 weeks old, another advantage when it comes to easier sterilization. This time I decided to leave the explants much larger than I did in my first tissue culture attempt. The first time, I cut the explants into 1.5cm to 2cm, or 1/2" to 3/4", pieces. This time, I left all of the leaves the same size they were except I removed all of the traps. The flower stalks I cut into 3cm to 4cm, or 1" to 1.5", pieces. Here are the sterilization procedures that I used:For leaves==========1) Wash thoroughly in soapy water with my hands and made sure free of all dirt, etc.2) Rinse under running water (about 30 minutes) with a mason jar with cheese cloth on top placed under sink.3) Transfer to bowl and carry to work area.4) 5 minutes in H2O25) 10 minutes in 10% bleach (6 minutes for small pieces)6) 5 minutes rinse in sterile water For flower stalks=================Identical to above except 6 minutes in 10% bleach instead of 10 minutes. If the pieces of tissue were small, I would soak them for less than 10 minutes. This sterilization technique seemed to work well. After 4 days in vitro, the explants are still very green and healthy looking. In fact, it appears that the flower stalks are actually bending toward the light! Today is Saturday and I put the explants in vitro on Tuesday. I can't see any contamination yet, but I can see some phenolic oxidation on a few of the explants, but it's not terribly severe like it was the first time I tried tissue culture. I am VERY optimistic this time about getting an explant to take! So here are some photos that I took today (Saturday) after 4 days in vitro: You can see some cloudiness around the base of the leaf in photo 2. That is phenolic oxidation, or sometimes called phenolic bleeding. It is also apparent in the last photo directly above. However, it's not that bad in either case and those are the worst two cases I have of it this time. I think this bodes well! I'm planning on leaving the cultures in the dark until Tuesday. That will be one week after I put them in vitro. Then I'll move them to my terrarium which is on a 16 hour photoperiod. I sure hope that I get ONE of these explants to take! I'll post again soon with any further updates. UPDATES:February 20th Update