How To Germinate Cannabis Seeds With Hydrogen Peroxide

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THE BASICS Like almost everything else about growing excellent Cannabis, germinating your seeds successfully is pretty simple. We’ve used this very basic, inexpensive method for many years and have shared it among thousands of growers worldwide who have all had excellent results with no issues. First make a mix in the Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa Cannabis seed germination is an important process for growers and researchers alike. Many

Germination Tips

Like almost everything else about growing excellent Cannabis, germinating your seeds successfully is pretty simple. We’ve used this very basic, inexpensive method for many years and have shared it among thousands of growers worldwide who have all had excellent results with no issues.

First make a mix in the ratio of 1/3 3% hydrogen peroxide to 2/3 distilled water.

It’s important to use only distilled water – some of the common chemicals in tap water can stunt, warp or kill your plant even well past seedling stage when you’ve already spent weeks tending it.

Then soak your seeds in the distilled water/hydrogen peroxide mixture overnight. If you’re growing multiple varieties, be sure to soak them separately. (Don’t laugh. It’s happened.)

Next, pour some of the soak water onto really absorbent paper towels, then wring or squeeze them out lightly and lay them flat. We use 2-3 towels layered together to make a nice thick absorbent nest.

The best seeds often sink and compromised seeds sometimes float, but there are always exceptions to the rule.

Then sprinkle the soaked seeds, using a clean spoon or gloved fingers, onto the moist paper towels, not crowding the seeds.

Then fold the moistened towels over the seeds to make a flat little package.

If you’re sprouting more than 12 seeds at a time, make more than one package – don’t crowd them.

Then put the moist towel and seeds flat inside a closed, unzipped storage-size plastic baggie, laying it flat somewhere away from direct light at room temperature.

In two or three days the seeds will sprout – maybe not all at once, but that’s not a problem because within 24 hours of the first seed sprouting all the others will have sprouted too. Keep your inspection peeks short as not to dehydrate the paper towel.

When the seeds each have a 3/4” root and the halves of the shell are beginning to open noticeably, meaning the embryo leaves are swelling inside, they’re ready.

You then want to move each sprouted seed into a Jiffy Cube you’ve prepared by making a small hole using a pencil or chopstick. Again – be sure you used distilled water to hydrate your Jiffy Cubes and of course use it everywhere else during germination.

Replanted into Jiffys:

Using your fingers, pick up the seed very gently by its shell, and avoid touching the sprouting root as you transfer it into the jiffy cube.

Also remember what’s unfolding inside that little shell as you handle it, be super delicate and stay conscious of the life emerging inside.

Now place the sprouting seed root-down in the hole letting it settle in naturally with the top of the seed even with the top of the hole – never push it down.

If it doesn’t nest right in, lift it out and poke the hole a little deeper. The cells at the tip of that little root hold the most miraculous ecosystem of emerging life one can imagine and while they are incredibly tough in nature they are also vulnerable to our mis-handling.

Now all you have to do is let the new sprouts do their thing they do so well, with a little help from their friends. With a day or so they will raise up their first leaves. Keep them lightly misted if you’re in a dry environment but don’t over-do it.

In a couple of days when they’ve developed their first set of true leaves, not the embryo leaves, it’s time to put them into larger pots with living soil and a mild fertilizer and then let the plants fully establish themselves.

3 Days Later:

10 Days Later:

The same Skunk #1 x Haze seeds are available at:

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Is using distilled water really important for germination?

Using distilled water is probably the most important part of the germination process because well water, tap water, bottled water and even rainwater contain traces of contaminants that can inhibit germination and later vegetative growth and flowering.

High chlorine in tap water, herbicides in well water, phthalates in bottled water, as well as many other kinds of common water contaminants can kill or damage germinating seeds and will definitely affect your plant’s health, yield and flower quality.

Can I remove these contaminants with a garden hose filter?

Yes most of them you can. There are a variety of relatively inexpensive filters that screw onto the end of the garden hose that you use to fill containers and water your plants. Some have replaceable screens, while others use granular activated charcoal.

There are also screw-on systems with carbon block filters that run $20-$30 and are very effective at reducing chlorine and chloramine, and at removing pesticides, heavy metals, and herbicides.

What’s some of the science behind using Hydrogen Peroxide for germination?

Here are two core articles from the PubMed database:

“Different Modes of Hydrogen Peroxide Action During Seed Germination”

Development and Standardization of Rapid and Efficient Seed Germination Protocol for Cannabis sativa

Cannabis seed germination is an important process for growers and researchers alike. Many biotechnological applications require a reliable sterile method for seed germination. This protocol outlines a seed germination procedure for Cannabis sativa using a hydrogen peroxide (H2O2) solution as liquid germination media. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in an H2O2 solution of different concentrations; 1% H2O2 solution showed the fastest and the most efficient germination. This protocol also exhibited high germination efficiency for very old cannabis seeds with lower viability. Overall, this protocol demonstrates superior germination compared to water control and reduces the risk of contamination, making it suitable for tissue culture and other sensitive applications.

Keywords: Cannabis sativa, Rapid germination, Hydrogen peroxide, Seed sterilization, Seedling development

Background

Cannabis sativa, otherwise known as marijuana or hemp, is an annual primarily dioecious flowering plant in which male/female sex is determined by heteromorphic chromosomes (X and Y) ( Gaudet et al., 2020 ). Cannabis is grown for a variety of agricultural uses; nearly all parts of cannabis plant are used, seeds for food, stem for fiber, and flowers/leaves for medicine. Flowers produce a mix of cannabinoids and aromatic compounds valued for their therapeutic and recreational effects ( Chandra et al., 2017 ). Cannabis plants are propagated either clonally through cuttings or via seed germination. Seed germination is very important for researchers, breeders, and growers alike, especially since seeds from elite cultivars can be very expensive and valuable. Additionally, older seeds may have a reduced germination rate while bacterial and fungal contamination can compromise germination, especially when seeds are germinated for tissue culture propagation. To address these issues, we have developed a rapid, sterile, and efficient seed germination protocol using a 1% hydrogen peroxide (H2O2) solution. In this protocol, all three steps including seed sterilization, germination, and seedlings development were carried out in a 1% H2O2 solution. This presents a significant advantage over other sterilants, such as mercuric chloride or bleach, which require additional washing of seeds and a separate germination step on MS solid medium. Our protocol resulted in faster germination and increased seed germination percentage as compared to water control, with no bacterial or fungal contamination, making it suitable for tissue culture and other sensitive applications. In comparison to previous germination methods which take between 4-7 days for radicle appearance and 5-15 days for seedling development ( Wielgus et al., 2008 and references therein), our germination method resulted in radicle appearance in 1 day and allowed us to obtain cannabis seedlings in a very short period (3-7 days) with minimal efforts. This protocol is also very efficient for germination of very old cannabis seeds with lower viability.

Materials and Reagents

All seeds were harvested in our laboratory. Blueberry seeds were not older than 6 months, when employed in the experiments. Finola and X59 seeds were more than 5 years old.

See also  Cannabis Plant Seed Pods

Equipment

Growth chamber (Sanyo MLR-350, catalog number: 859-600-06): 24 °C, 18 h light/6 h dark cycle, light intensity 200 μmol·m -2 ·sec -1

Procedure

Seed germination assay

Soak seeds overnight in various concentrations of hydrogen peroxide solution (liquid germination media or germination solutions) as well as in sterile water control (H2O, 1% H2O2, 3% H2O2, 5% H2O2, or 10% H2O2) in 15 or 50 ml screw-cap (Falcon tube). Falcon tubes with submerged seeds in various germination solutions were kept in the dark at room temperature.

Next day, record the percentage of germinated seeds in germination solution (appearance of radicle is considered as germination event) and add fresh respective germination solution after removal of old solution simply by pouring out.

Keep seeds soaked in the same solution for 3 more days in the dark at room temperature and record the percentage of germinated seeds every day.

Thereafter, germinated seeds/seedlings were transferred with or without seed coats from H2O2 solution to MS medium plates to observe the growth of H2O2 solution-germinated seeds/seedlings on MS medium. To transfer, first germinated seeds/seedlings were poured together with H2O2 solution from the Falcon tube to the empty petri plate. Then seedlings were transferred to sterile paper by using forceps to remove excess H2O2 solution. Finally, the germinated seeds/seedlings were transferred to MS media plate by using forceps. The whole transfer process has been carried out in the laminar flow hood.

Parafilm sealed MS medium plates with germinated seeds/seedlings are then transferred to the growth chamber (24 °C, 18 h light/6 h dark cycle and light intensity 200 μmol·m -2 ·sec -1 ) for 3 days to observe the growth and survival of H2O2 solution germinated seeds/seedlings on MS medium.

The H2O2 solution-germinated seeds/seedlings growth was also observed in soil. Pro-Mix HP Mycorrhizae Growing Medium used for soil experiment. The cannabis seeds were soaked in the H2O2 solution (germination solutions) for four days and thereafter, germinated seeds/seedlings were transferred from H2O2 solution to soil pot (Pro-Mix HP Mycorrhizae Growing Medium) to observe the growth and survival of H2O2 solution germinated seeds/seedlings on soil. The soil pots were transferred to the growth chamber (24 °C, 18 h light/6 h dark cycle and light intensity 200 μmol·m -2 ·sec -1 ). The photographs were taken on day 12.

Data analysis

Mean seed germination percentage under various concentrations of H2O2 solution as well as water control were calculated in an excel sheet. Data were shown as mean ± SE.

Results

In this study, we have described a rapid and efficient seed germination protocol for Cannabis sativa. The brief description of this protocol has been reported in Sorokin et al. (2020) . In the current study, we have standardized the optimum concentration of hydrogen peroxide (H2O2) solution media for efficient sterilization and rapid germination. We have tested various concentrations of H2O2 solution as well as sterile water control (H2O, 1% H2O2, 3% H2O2, 5% H2O2, or 10% H2O2) for sterilization and germination efficiency. All three steps of germination (seed sterilization, germination, and seedlings development) were carried out in various concentrations of H2O2 solution and seeds were kept in liquid media for four days. Hydrogen peroxide presents several significant advantages over mercuric chloride or bleach sterilants, which require additional seed washing, and separate germination/seedling development step in Murashige and Skoog (MS) agar medium ( Sorokin et al., 2020 ). The 1% H2O2 solution showed rapid and higher germination than higher H2O2 concentrations solution and water control at day 1 ( Figure 1 ). On day 1, 1% H2O2 solution exhibited 82.5% germination as compared to 22.5% germination for 3% H2O2 group, 17.5% germination for 5% H2O2 group and 47.5% germination in water control group ( Figure 1B ). Interestingly, 10% H2O2 did not show any germination on day 1 due to its toxic effect ( Figure 1 ). In 1% H2O2 solution, radicle appearance (germination) occurred within 24 h and seedling development (two fully developed cotyledons and two immature true leaves stage) occurred in 72-96 h ( Figure 1A ). In comparison to previous germination methods which take between 4-7 days for radicle appearance and 5-15 days for seedling development ( Wielgus et al., 2008 and references therein), our germination method resulted in radicle appearance in 1 day and allowed us to obtain cannabis seedlings in a very short period (3-7 days) with minimal efforts ( Figures 1 -2). Considering the possible toxic effect of H2O2 (since germinated seeds/seedlings stayed continuously in H2O2 solution for 4 days), we have checked further survival of germinated seeds/seedlings on MS media and soil ( Figures 2 -3). On MS media, 1% H2O2 solution seedlings survived better than other treatments ( Figure 2 ). The water germinated seeds exhibited contamination and did not survive on MS media ( Figure 2 ). Similarly, due to the toxic effect of higher concentration of H2O2, the 10% H2O2 germinated seeds did not survive on MS media ( Figure 2 ). The 1% H2O2 solution seedlings also survived well on soil ( Figure 3 ). Apart from this, we have also tested our method for more than 5-years old cannabis seeds with lower viability, which demonstrated that 1% H2O2 solution medium exhibited a very high germination percentage (~50%) as compared to water control (~10%) ( Figure 4 ). In conclusion, we have developed a rapid and efficient method for C. sativa seed germination under sterile conditions for tissue culture and other sensitive applications.

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Germination of 6-month-old seeds of Blueberry variety in various concentrations of hydrogen peroxide solution and water control.

A. Representative photographs of germinated seeds/seedlings in the H2O2 solution of various concentrations or water control on day 1 to day 4. B. Comparison of germination percentage between the various concentrations of H2O2 solution or water control. Data are shown as mean ± SE (n = 4). In each replicate, 30 seeds were used.

Representative photographs of growth and survival of H2O2 solutions germinated seeds/seedlings of Blueberry variety on MS media.

The Blueberry variety seeds were soaked in the H2O2 solution (germination solutions) for four days and thereafter, germinated seeds/seedlings were transferred from H2O2 solution to MS medium plates to observe the growth and survival of H2O2 solution germinated seeds/seedlings on MS medium. The photographs were taken at day 0 (just after transfer to MS medium plates), day 1 (after 24 h of the transfer to MS medium plates), and day 3 (after 72 h of the transfer to MS medium plates) on MS media.

Representative photograph of Blueberry variety young plantlet growing in soil (Pro-Mix HP Mycorrhizae Growing Medium).

The Blueberry variety seeds were soaked in the H2O2 solution (germination solutions) for four days and thereafter, germinated seeds/seedlings were transferred from H2O2 solution to soil pot (Pro-Mix HP Mycorrhizae Growing Medium) to observe the growth and survival of H2O2 solution germinated seeds/seedlings on soil. The photographs were taken on day 12.

Germination of 5-years old seeds of Finola and X59 varieties in 1% hydrogen peroxide solution and water control.

Comparison of germination percentage between 1% H2O2 solution media and water control. Data are shown as mean ± SE (n = 5). In each replicate, around 30 seeds were used.

Recipes

4.43 g Murashige & Skoog Basal Medium with Vitamins

Adjust pH to 5.7 with KOH and sterilize by autoclaving at 121 °C for 40 min. 25 ml of MS media on each Petri plate.

Acknowledgments

This protocol is derived from Sorokin et al. (2020). We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and MITACS for funding our work.

Competing interests

The authors declare that they have no competing interests.

Citation

Readers should cite both the Bio-protocol article and the original research article where this protocol was used.

References

1. Chandra S., Lata H. and ElSohly M. A.(2017). Cannabis sativa L.-botany and biotechnology. Chandra, S., Lata, H. and ElSohly, M. A.(Eds.). Springer International Publishing: Cham, Switzerland. ISBN: 9783319545639. [Google Scholar]

2. Gaudet D., Yadav N. S., Sorokin A., Bilichak A. and Kovalchuk I.(2020). Development and optimization of a germination assay and long-term storage for Cannabis sativa pollen . Plants 9 : 665. [PMC free article] [PubMed] [Google Scholar]

3. Sorokin A., Yadav N. S., Gaudet D. and Kovalchuk I.(2020). Transient expression of the β-glucuronidase gene in Cannabis sativa varieties . Plant Signal Behav 15 ( 8 ): 1780037. [PMC free article] [PubMed] [Google Scholar]

4. Wielgus K., Luwanska A., Lassocinski W. and Kaczmarek Z.(2008). Estimation of Cannabis sativa L. tissue culture conditions essential for callus induction and plant regeneration . J Nat Fibers 5 : 199-207. [Google Scholar]

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