I am working on endophytes in the Cannabis family and have run into a problem when surface sterilizing hemp seeds. They will not germinate with various protocols i've used on other plants. I have tried 40% commercial bleach solution for 10-30 minutes with and without 1-3 Minutes of 70% ETOH before or after the bleach solution, 5-6 rinses with sterile water afterwards. The seeds have high germination rates when simply strewn on media or peat lite without any surface sterilization. I had thought maybe there was a microbe being removed that was essential for germination and after inoculating with my isolated bacteria I still did not have germination. I'm wondering if it is the concentration of bleach, the time, or the RPM on the shaker.
I have experimented with H2O2 from some papers for sprouting hemp, but they did not actually get the seeds clean with 1% as reported in the paper. I have tried concentrations from 1-9% H2O2 and while I do see germination initiate, it stops abruptly when radicle emerges about 1mm.
Dear April, thank you for asking this interesting technical question. As an inorganic chemist I'm far from being a proven expert in this particular field of research. However, it often pays off searching the "Publications" section of RG directly for helpful references. For example, the following relevant articles have been posted as public full texts on RG and can be freely downloaded as pdf files:
A Quick Viability Test Protocol for Hemp (CannabisSativaL.) Seeds
Article A Quick Viability Test Protocol for Hemp (CannabisSativaL.) Seeds
and
Establishing surface sterilization protocol for nodal culture of Solanecio biafrae
Article Establishing surface sterilization protocol for nodal cultur...
Also please have a look at the following Open Access article:
In Vitro Germination and Tissue Culture Protocol Establishment For Canabbis sativa L.
https://bt.iamr.ac.in/wp-content/uploads/2019/11/In-Vitro-Germination-and-Tissue-Culture-Protocol-Establishment-For-Canabbis-sativa-L.pdf
(see attached pdf file)
Good luck with your research work!
For the surface sterilization of seeds we used 1.4% (w/v) of NaOCl for 5 minutes and extensively washed in sterile water and placed on sterile filters… have a look at Journal of Biological Chemistry 272: pp 1534- 1540, 1997.
You can try successively lower concentrations of bleach. I think it needs to be much lower than 40%, and also make sure you have viable seeds using the flotation test (viable seeds sink)
Exploring the bioactive compounds from endophytic bacteria of a medicinal plant: Ephedra foliata (Ephedrales: Ephedraceae)
So far I didn't sterilized hemp seeds but can recommend surface sterilization with NaDDC in concentrations of 200-1000ppm for 2-6 hours depending on species followed by washing with sterile water, worked well for a lot of genus e.g Thelymitra, Nepenthes, Drosera and some others
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