On May 31, 1996, you wrote:
>Finally, I have been reading up on tissue culture techniques for
>various species, the slide show provided by Rick Walker being
>invaluable, and have tried some on Nepenthes leaf cuttings. The
>whole procedure being undertaken in aseptic conditions in a laminar
>flow cabinet, but I got some wicked fungal cultures!! Notably a vivid
>orange one and a particularly rampant grey beastie! This happened in
>all ten cultures, surely not by chance. What I would like to know is,
>if they are systemic fungi how can I get rid of them before culturing?
Culture contamination due to systemic fungal sources is rare and is more
likely due to the insufficient disinfestation of the epidermis. Did the
fungal hyphae arise from the cut surface of the explants, especially from
the cut vienal tissue? This symptom may indicate a systemic infection
source. Or did the hyphae grow from the surface of the expalnts in
general? This would most probably indicate an epidermal source. Hyphal
growth can ususally be observed in Stage I cultures three to five days
following explant disinfestation. But that is of course what we don't want
to see! I am assuming the most probable scenario of explant epidermis
fungal contamination here in this discussion.
>The explants were sterilised by washing in distilled water, dipping in
>70% ethanol(2-3secs), 15mins in dilute Sodium hypochlorite solution
>then rinsing with distilled water again. Any thoughts?
Your disinfesting protocol is standard and should yield at least a low
percentage of "clean" cultures providing all of the myriad of other
interacting factors are controlled. Just how dilute was your "dilute
sodium hypochlorite solution"? A low success rate can be attributable to
the duration of the disinfecting treatment being too short relative to the
disinfectant concentration. Also, I am assuming that your rinsing with
sterile distilled water.
An often neglected stock plant pretreatment (Stage 0), is to grow the stock
plant indoors for at least two weeks in a low humidity environment,
prferably air conditioned, while providing all of the other necessary
growing conditions optimally, before harvesting your explants. This will
understandably stress a Nepenthes plant, but it is a proven technique to
significantly reduce the micro flora and fauna of the stock plant
epidermis.
Another method to try is using other disinfectants, either singly, or in
combination, at various durations and concentrations to find the best
treatment to achieve asepsis under your conditions. Other disinfectants
might be calcium hypochlorite, mercuric chloride (handle this with extreme
care!), or the relatively unknown chlorous acid or chlorine dioxide that is
a ligand solution of sodium chlorite and lactic acid, known in the trade as
Alcide LD. I have been having good success with this disinfectant since
1981, and even better success since modifying the dilutant as 35%
isopropanol rather than just water per the Anthurium explant disinfestation
research of Dr. Michael Tanabe of the University of Hawaii, Hilo. Drop me
a line if you would like more information.
I am curious if you there is a published or unpublished protocol that you
are using for regenerating adventitious plants from Nepenthes leaf
expalnts? I would be happy to know of this, if you care to share. Any and
all comments, objections, and /or observations are welcome to my basic
generalities of Stage I disinfestation.
Regards, Scott Hyndman
Apopka, Florida
marva@nebula.ispace.com