Hello. Jeremiah generously gave me some Nepenthes Attenboroughii seeds to try my hand at in vitro culturing. I promised to share any progress, so here it is. I frequent this forum often, so it is only fair that perhaps someone can benefit. Let me preface this by saying that I have never performed plant tissue culture before. This is simply for fun. Also, I have only recently been growing Nepenthes, ~ 3 months. However, I do routinely culture a host of other organisms from all the kingdoms. Like a first time parent, I worried over every minute detail. Luckily, Bigbella has been a great help to alleviate some of these OCD tendencies. Per his advice, I tried to find something else to fulfill my neuroticism, which was not that difficult. As if on cue, germination did not occur until I was able to forget about them. Hahaaa. This posting is a little overdue but I wanted it to be complete, rather than rushed and necessitate editing.
Nepenthes Attenboroughii in vitro sowed on 11/25/12, germinated on 12/12/12
1/4MS + 0.5mg/L BAP + 0.2% gelrite
Nepenthes Attenboroughii sowed on 11/25/12, germinated on 12/12/12
(Mix was washed and sterilized. Ampac trichoderma flowerable 1tsp/gal seed soak and then sprayed over mix)
I am the kind of person who likes a detailed protocol if I am performing a new technique. This was a learning experience for me so I hope this information can benefit someone who is trying it for the first time. I had a lot of questions concerning the range of acceptable conditions. And most of these answers can be found on the various CP forums if one searches diligently enough. You do not need all the equipment I have listed. I simply used what I would normally use for my work. If you don't have something, be creative. Necessity is the mother of all inventions and I have MacGyvered many things in lab that had sufficed as an equal substitute. My protocol is, simply that. Everyone does what works for them and it need not be the same to achieve success. This is always true in a lab setting where protocols followed from one lab do not directly translate to another.
1/4X Murashige and Skoog ph 5.6 (homemade. Stored at 4C)
100X Vitamin Mix (individual reagents from Sigma. Stored in aliquots at -20C)
60% Sucrose (Sigma. Sterile filtered with Millipore 0.5L filtering device. Stored at RT)
Plant Preservative Mixture (Plant Cell Technology, Inc. Stored at 4C)
100mM Sterile Petri Plates (VWR)
1.5ml, 5ml, 15ml, 50ml Sterile centrifuge tubes (VWR)
MK-5 Vessels (Caisson Labs)
5ml, 10ml, 25ml, 50ml serological pipets (VWR)
p20, p200, p1000 Pipetman (Gilson Pipetman)
MBP Art Filter tips for pipetman
BAP (Sigma) Prepared as a 3mg/ml solution in 70% ethanol. Add 100% ethanol and then the necessary volume of sterile h20. Stored at -20C in aliquots.
Baxter sterile inoculating loops
Black Gold horticultural washed sand -> washed 3X in 10X volume of sterile h20.
Black Gold sphagnum peat moss
Ampac Trichoderma Atrioviride flowerable
Ampac Trichoderma Atroviride granular (Use considerably less than 0.5% (V/V) if using in peat mix. I don't know how much though as I switched to flowerable after hyphae explosion.)
In a glass wide mouth bottle add desired volume of H2O. On a stir plate with a stir bar on medium speed, add salts one by one and the PPM. Add 0.1M HCl to bring ph to 5.6. Take out the stir bar and add 0.2% gelzan. Place a piece of autoclave tape and unscrew cap a little (If you close completely you will get a little bomb or the glass vessel will crack). Autoclave for 20 minutes. Remove media from the autoclave and bring it to the laminar flow hood or where your clean work space is. Put on some gloves and spray your hands with 70% isopropanol/ethanol. Next generously spay down bottle with 70% isopropanol/ethanol. Place the bottle of media under the hood and take off the cap and just place it over the bottle. Let the media cool until it is warm to the touch. Thaw out the necessary aliquots of the 100X vitamin mix and PGRs. Add sucrose to a final concentration of 3%. Swirl to mix. Add PGRs and vitamins using pipets. The media should still be warm enough to not begin solidifying, but also not be steaming hot (This is to limit the amount of condensation inside the vessel). Use a serological pipet to distribute 50ml to each sterile MK-5 vessel.
*When I made the media the first time I saw that the ph would fluctuate if the stirring was set to a higher speed. For all subsequent media preparations, I would set the stir speed to where the ph meter would no longer drift. I tired this on 3 different ph meters in my lab and our neighboring lab.
Discard any media in the vessel and rinse with tap h2o. Soak in 10% bleack for at least 10 minutes. Rinse 10X volume with tap h20 or until you can not smell the bleach anymore. Rinse with DI/RO/milliQ h2o. Allow to dry. Autoclave for 20 minutes with indicator tape. Dry under flow hood if there is condensation in the vessel.
I use the same sterilization technique as Bigbella, a 8% PPM solution in 1/4 MS sterile salts, no PH/PGRS/sucrose. Place seeds on a piece of paper and funnel them into a sterile 5ml plastic tube. Add 2ml of 8% PPM solution and secure the cap. Place the tube laying down on an orbital shaker at 20rpm for 4 hours. Allow the tube to rock back and forth. The volume of 8% PPM used is dependent on the number of seeds being sterilized, which in turn will determine the tube size. You don't need an orbital shaker, you can let it sit and manually agitate it whenever it suits your fancy.
Spray the 5ml tube of seeds with 70% isopropanol/ethanol. Layout (1) 100mM sterile plate/ batch of seeds. Jiggle the tube of seeds to dislodge seeds from sticking to the sides. Pour the seeds and the sterilization solution onto the petri plate. Use a sterile inoculating loop to "fish" out the seeds by its TAIL.
*Attenboroughii seeds do not display the typical thin spindles so this was not as important. However, fishing out the seeds by the tail will allow the spindle to pierce through the water's surface tension (like diving vs. cannonballing).
Temperature and Light Conditions
Both in vivo and in vitro germination is performed in a VWR isotemp incubator set to 22.5C. In vitro vessel temperature range is 78.4F-80.2F. In vivo vessel temperature (32oz polysyrene food containers) is 81-85F. Light source is ~8'' to the top of media/soil. Germination light cycle is 16hr ON/ 8 hr OFF. Light is provided by: 13watt, 620 lumens 21’’ radionic hightech F13T5CW 4200K + 8watt, 320 lumens 13’’ F8T5CW 4200K + 10watt, 695 lumens eclipse compact flourescent light bulb 5100K. The total lumens is 1635 lumens enclosed in a 24’’X14’’ area enclose with 2mm mylar film.
Temperature is monitored with a alcohol thermometer in a MK-5 vessel and by a infrared temperature gun.
In summary, I believe in vitro is easier because of the lower maintanance involved. I have had several of my sowed seeds in vivo necessitate washing and resowing. This process takes me about 3-4 hours for a 100 seeds: pipet tip/steel poker to get off the the trichoderma, several washes in sterile h2o, and then replating onto new sterilized peat/sand + diluted tricho drench. The problem involved using trichoderma spray that had been sitting on the lab bench for a couple of days. The trichoderma spray was therefore more concentrated than I had desired. Now, I throw away the spay that I don't use (>90%) and remake fresh when I need it.
However, seed germination in both the in vitro and in vivo occurred at about the same time. I have 2 in vitro cultures: #1 sowed on 11/25/12 and germinated on 12/12/12, #2 sowed on 11/29/12 and germinated on 12/18/12. The seeds in #1 were originally sown in vivo but then suffered a trichoderma explosion and in my panicked state I decided to kill off all the extra organisms and throw it on jelly.
I told DonH I would do a round of Cindy (crossfit) for every new sprout.