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
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.
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
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.
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.
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.
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.
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.
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.
The authors declare that they have no competing interests.
Readers should cite both the Bio-protocol article and the original research article where this protocol was used.
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]
How To Germinate Old Cannabis Seeds
We can only expect to use the old seeds with the cost of seeds floating above potheads’ reach. However, improper or long-term storage of seeds can cause infertility – and they cannot germinate. However, how do we let those old cannabis seeds come to life again? This guide will help us convert the relics into a sea of green sections of robust cannabis fields!
Sorting Old Cannabis Seeds
The first step in any farming process is to make sure we have the right seeds, and the same goes for weeds. When harvesting autoflowering marijuana seeds, everything is preserved, and nothing is lost. This means that all types of seeds are ripe and immature. How do we distinguish good seeds from bad ones? You are considering the following.
Sorting Seeds By Color And Shape
Whether we obtained the seeds from a seed bank, a store, or a retailer, different cannabis seed strains have different colors. Mature cannabis seeds acquire a dark coat, while immature ones are brighter and usually white. The most visible colors are brown, tan, and sometimes black. In contrast, bright yellows and whites quickly identify immature seeds.
Cannabis seeds are known for their aesthetic properties and shape. Round and symmetrical seeds are best. Larger seeds have a reasonable rate of germination compared to small seeds, which tend to be immature.
Classification Of Seeds By Hardness
Although the seeds have taken some time inside storage containers, ripe weeds seeds’ hardness is not compromised. Hard and tough seeds with a smooth shell guarantee a reasonable degree of germination. Also, pay attention to wavy and cracked seeds. They will lose time and energy and will not germinate after planting.
Ways to Germinate Old Cannabis Seeds
Below we will go through 3 popular methods used to germinate old weeds seeds. Remember that we have to do the part by trying to keep the temperature between 26 ° C – 28 ° because that is where the clones seem to season-best. You must also ensure that the seed is kept in a dark area, as light can slow down the germination process.
Method 1 – Scarification
The first method is scarification. This includes causing injury to the seeds’ outer shell to allow water and air to enter, which is essential for germination. For manual scarification, we will need a container or box lined with sandpaper or any coarse material to scar the seeds’ outer surface. Put the seeds in a container or box and shake. After a while, we will find that the roots become dull, and we can see parts of the sources inside the container. Once we scare the outer shell of the cannabis seeds, we can germinate them as usual.
Method 2 – Carbonated water
The second method involves the use of carbonated water with a pH below 7. This slightly acidic solution absorbs the outer layer of the seeds. Put the seeds in a container full of carbonated water and wait for about two hours. The solution loosens the seed coat and allows it to absorb water, which helps germinate.
Method 3 – Mixture of hydrogen peroxide
Method 3 requires that we use a mixture of hydrogen peroxide to soften the seed’s outer skin. It would be best if we were careful when mixing the peroxide solution, as we can burn the seed, and it will never germinate.
To use a mixture of hydrogen peroxide for the germination process, use 1 to 2 drops of 99% hydrogen peroxide in a glass of water. After soaking for 24 hours, the outer shell is softened enough to germinate the seeds.
In addition to chemical and mechanical scarification, we can use other DIY methods to loosen seeds. For example, we can use a small knife to scratch or open the seed coat. We can explore different approaches as long as we do not damage the seed embryo.
- One part 3% hydrogen peroxide with six parts water.
- One part 4.5% hydrogen peroxide with nine parts water.
- One part 6% hydrogen peroxide with 12 parts water.
- One part 30% hydrogen peroxide with 60 parts water.
Always Use Clean, Fresh Water
Clean and clean water contains oxygen and hydrogen molecules. These are two life-supporting elements necessary for germination. Soaking the seeds for at least 12 hours allows water to enter living cells, a process known as osmosis. Now that the internal conditions promote germination, the semen embryo expands and breaks out of the protective sheath.
Old cannabis seeds sometimes pose a challenge for germination. You can sort ripe seeds, release the hard protective layer by various methods, try new chemical germination enhancers, or use biocatalysts. Similarly, soaking the clean, soft seeds in clean water will push the embryo out of its protective blanket. After trying some of the above processes to germinate the old cannabis seeds, we can be sure to grow healthy and living plants and expect good yields.
The Best Way Germinating Marijuana Seeds?
We asked 8 Experts + 143 MSB Growers!
Do you think there is 1 way to make your ladies sprout? Think again!
We asked 8 Experts & 143 MSB Growers:
What is your Preferred Germination Method?
We were blown away by the number of variations we received!
Perhaps your new best way of Germinating Marijuana Seeds is amongst them!
- Study Best Way Germinating Marijuana Seeds
- Dr. Dina, Queen of cannabis
- Ed rosenthal
- James Loud, Loud Genetics
- Jennifer Martin, Cultivation Sector Consulting
- Jesce Horton, LOWD
- Melanie Carruthers, 7acres
- Ryan Douglas, Ryan Douglas Cultivation
- Rudy Ellenbogen. Whole Grow
- Preferred Germination Method MSB Community
- Conclusion: What is the Best Way to Germinate Marijuana Seeds?
Study on Best Way Germinating Marijuana Seeds
Here at Marijuana Seed Breeders, we get many questions. From Beginner to Expert.
We love helping growers.
In fact, we try to address frequently asked questions into articles so that everyone, even non-MSB Growers can benefit.
One of these questions is about germination: what is the best way for germinating marijuana seeds?
Interesting one, because perhaps is our best way, not your best way. Therefore we created the article How to germinate weed seeds.
In the article we give steps for 5 frequently used cannabis germination methods:
- Glass of water
- Wet towel
- Directly in soil
- Stone wool blocks
- Using a starter kit
We also give pros and cons to every method. So this should give you enough information to determine what is the best fit for your next growth.
Which Germination Method is the most used?
Every spouted seed counts. To more plants the better, right?
That’s why we are interested to understand how marijuana growers around the world germinate their cannabis seeds.
When we ask people this question we get a lot of different answers. Which germination method is the most used one?
We had an idea, but we didn’t know for sure.
It was time for a survey amongst our beloved MSB Community. We also reached out to experts in the field to learn more about their preferred method.
We were blown away by the number of enthusiastic replies! Both from our MSB community as well as the experts.
Yes, sure we received replies, just confirming one of the 5 methods.
But the larger group of people actually took the time to reveal their ‘secret’ to cannabis seed germination!
But first: let look at the statistics from our own research.
Results Preferred Marijuana Seed Germination Study
Without further ado, the most favorite germination method amongst 143 growers is:
#1. Wet Towel (37.3%).
#2. Other. 21.1% of the growers follow another germination routine.
#3. Directly in Soil. A surprisingly high percentage (19%) put their precious seeds directly in soil!
#4. Starter kits. 10% use starter kits (like Spongepot).
#5. Glass of water is the best way for 8.5% of the growers.
#6. Stonewool (3.5%).
Here is the breakdown in a chart:
Preferred Germination Method Experts
Ok, enough of the statistic, it’s germination time.
Let’s start with Expert advice!
First off: even though they were busy, we received kind feedback from the Experts. This shows again how approachable and awesome the people in our growing community are,
We received feedback from these experts from the field (sorted in alphabetical order):
#1. Dr. Dina, The Queen of Cannabis, Co-Owner of AHHS WeHo
#2. Ed Rosenthal, The Guru of Ganja
#3. James Loud, Founder of Loud Genetics Loud Seeds
#4. Jennifer Martin, Founder Cultivation Sector Consulting LLC
#5. Jesce Horton, Founder and CEO LOWD
#6. Melanie Carruthers, Director of Propagation 7ACRES
#7. Ryan Douglas, Cannabis Growth Consultant Ryan Douglas Cultivation
#8. Rudy Ellenbogen, Founder and CEO Whole Grow
Ready? Let’s dive right in!
Dr. Dina, The Queen of Cannabis, Co-Owner of AHHS WeHo
When someone gets a nickname from Snoop Dogg, and when that person is referred to as The Queen of Cannabis, the Mona Lisa of Mary Jane as well as The Queen of Weed AND the main character of the 8 season series Weeds is based on their person, then you know that you are dealing with a true expert.
I prefer to germinate My seeds in root gel (clonex) and stick them in cubes. Paper towels work well too, but I think the root gel gives the seeds the extra push to become a strong plant.
– Dr. Dina, The Queen of Cannabis
Ed Rosenthal, Author, Educator, Social Activist, And Legalization Pioneer
I use starter trays and plugs from iHort – EXcel T-50 and 32 Star Excel trays to start seeds. They come pre-moistened, with a little hole to drop a seed.
I use the 50 per tray when I will be transplanting within two weeks and the 32 per tray for growing up to 3 weeks.
When I’m using fresh seed, I just drop them into the hole in the cube.
With stale seeds, first I soak them in a 0.5% hydrogen peroxide (H2O2) solution for 3-4 hours. The recipe is 1 part drugstore 3% H2O2 to 5 parts water. This sterilizes their surfaces. Then they soak in mycos and liquid kelp solution for about 6 hours. Then they are placed in the cubes.
Upon first signs of germination, I water them with mycos and kelp to promote root growth. The next day they are watered with a weak combo of veg and flowering formula about 400 PPM.
– Ed Rosenthal, The Guru of Ganja
James Loud, Founder of Loud Genetics
Well it’s a loaded question because it depends on scale.
I sterilize in an h2o2 Soak then germinate in a jiffy #7 typically. However, large scale I prefer a needle/vacuum seeder to place the seeds evenly in the substrate.
Well and with autoflower I believe direct sow in optimal conditions can yield better results.
– James Loud, Loud Seeds
Jennifer Martin, Owner Cultivation Sector Consulting LLC
I use coco plugs. I gave up the wet paper towel method years ago because managing the moisture was too cumbersome.
I buy coco plugs that come in the 50-cell sheet, make a light nutrient mix, put all of the plugs in a pitcher of the nutrient mix, squeeze them to fully saturate them with the mix, put them back in the cell sheet, push a seed into each hole, and put a dome on the tray under the light.
Then I just keep the plugs moist. The seeds start to pop in about 3 days. Some take up to a week. I leave them in there until they overgrow the tray, then transplant into 3″ pots.
Jesce Horton, Founder and CEO, LOWD
I’m a water glass guy. I like to know before planting which seeds are more vigorous and then assess based on that benchmark throughout the growth process.
I carefully pull the seeds with a spoon and drop them into a coco mixture a day or two after they pop.
– Jesce Horton, LOWD
Melanie Carruthers, Director of Propagation, 7ACRES
Our processes at 7ACRES for seed germination use a combination of a glass of water and wet towel.