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 Why Are Your Cannabis Seeds Not Germinating? Many first-time growers and inexperienced planters often experience this problem when trying to cultivate cannabis seed. They end up languishing and White cannabis seeds vs dark cannabis seeds. Should cannabis seed germination and growth be affected by seed colour or seed appearance?
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]
Why Are Your Cannabis Seeds Not Germinating?
Many first-time growers and inexperienced planters often experience this problem when trying to cultivate cannabis seed. They end up languishing and wondering why their effort is not yielding since a cannabis seed has a pretty high germination rate (99%). However, many things might not promote the germination process.
Generally, cannabis seeds are powerful and grow pretty fast. If the seeds do not germinate, there is likely a problem with the germination method. This article will explore many things that can go wrong when trying to cultivate cannabis seeds.
The chances of having a viable seed increase significantly if the genetics is good. This is pretty important as it can avoid wasting energy and effort trying to germinate a bad seed. When you buy good quality seeds from trusted banks, you can breathe a sigh of relief, knowing that your actions will not be wasted.
As a result, it is not a good idea to grow seeds that you stumbled on inside a cannabis bag you got. Such seeds could be infertile and may not germinate. If it eventually germinates, you might not like cannabis. Make sure to buy the right seed from reputable stores and increase the chances of germination.
Direct Soil Germination
This is one of the top reasons your cannabis seed is not germinating. If you do not water the substrate before sowing the seeds and water afterward, there is a high probability that the seeds will not grow. This is not surprising as the seeds could be buried too deep, and adding water after sowing worsens this.
You can obtain good results by germinating the seeds in jiffy pellets, peat plugs, or using kitchen paper and later transplant them to a pot or the soil when the small seedlings come out. Make sure to care for your tender plant at this stage. Limit irrigation, support the right temperature and use Northern lights when growing indoors.
Wrong Temperature and Humidity
The ideal temperature for germinating cannabis seeds is high humidity and high-temperature levels. It is essential for people in tropical countries to provide external support of heat to get the temperature within reasonable and acceptable ranges. This is where heated greenhouses come in. Not only do they provide the right temperature, but they can get the humidity level to the ideal range essential for germinations. A cannabis seed needs 70% relative humidity and an average temperature of about 270C for germination.
If the temperature and relative humidity ranges are lower than the ideal value specified, the growth rate will be slow and unsuccessful. Excessive range, on the other hand, can lead to fungal problems.
Seeds Planted too Deep.
The amount of energy available to a germinating cannabis seed is limited. As a result, if planted too deep, it might not have the required energy to make its way up. A seed planted underground will have no light and is not producing food yet since no photosynthesis. Planting seed too deep creates extra stress for the seedling as migration to the surface becomes an issue.
As a result, you should plant your cannabis seed around 2 to 3 cm down or half inches deep. With this, they have enough room to grow taproots. This might be one of the reasons your cannabis seed didn’t grow.
Using Old or Unsterilized Pots and Soils
A fungus is one of the greatest enemies that might affect the germination of cannabis seed. If you reuse soil and it is not sterilized, there is a high chance of mold and other unhealthy organisms like insects and bacteria. As a result, the seeds may not sprout at all. Even if they grow, these organisms might kill them after a couple of days. For instance, a sprouting seedling might suddenly bend and change color to brown in a process known as damping off. This is common when you supply the seed with too much water when there is poor drainage or the aeration is poor.
Due to this, consider planting seedlings in a sterilized pot as the tendency of containing harmful organisms is low. Also, make sure the containers are clean as it can reduce the tendency of fungus.
Planting lots of Seed in one Pot
Planting a lot of seed in a single container might seem like a good idea due to the ability to save cost. This is like shooting yourself in the leg, as it might lead to a wasted effort. Even if the seeds germinate, they will compete for limited space, nutrients, and water. A cannabis plant needs enough space for its root to travel down without restriction to prevent severe intermodal distance.
Limited space translates to severe competition not only for nutrients but light as well. This is not a good idea if you want to get the best from your cannabis seed. If the seed eventually germinates, they will be weak, which will affect the plants and overall output.
Cannabis Seed left underwater for a long time.
Some growers prefer to soak their cannabis seeds before planting them. This is an essential step to break the dormancy and soften the shell to prepare the seed for planting. With a softshell, the taproot will easily break the body and push its way out of the shell. Typically, the seed will be put inside a glass of water and left until it sinks before the grower plants it. However, this is not as simple as it sounds.
A cannabis seed needs to be continually breathing, and a fully submerged seed will not get the necessary air supply. This might kill the seed if left for long inside the water. Once the seed sinks, take them inside the paper towel as soon as possible.
There are many intricacies involved in germinating a cannabis seed. Getting it wrong with any of the systems might not make the cannabis seed grow. You can arm yourself with these essential tips to ensure that your cannabis plants germinate. Use these tips as a guide when trying to germinate your cannabis seed. Also, if your seed does not germinate, this guide can help you troubleshoot.
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Dark vs white cannabis seed germination test
One common question from growers is whether white cannabis seeds or dark cannabis seeds have different germination rates. When you compare cannabis seeds you may have noticed that the seeds are rarely identical. Instead you may see a range of different sizes and colours. The differences in size, colour and appearance often produce debate among growers. Why do the cannabis seeds look different and do these differences result in slightly different growth characteristics in the plants which grow from them?
Dark vs white cannabis seeds germination
In the video below you can see a germination comparison. On the left hand side, you can see 9 white Think Different autoflowering seeds. On the right hand side there are 9 dark coloured Think Different seeds.
The autoflower seeds all came from the same batch, meaning that they were produced from the same parent genetics under the same conditions. Seeds were chosen simply on the basis of their appearance. 9 white cannabis seeds and 9 dark coloured Think Different seeds were selected. They were planted in moist (but never soaked) soil and a time lapse video was made.
Note that the white cannabis seeds on the left hand side were the first seeds to germinate, with 8 out of 9 seeds sprouting pretty quickly. In the end all 9 seeds germinated on both sides, which was a 100% score. In terms of white seeds vs dark seeds (cannabis), there was no difference in the germination rates.
Does the colour of your cannabis seeds really matter?
The time lapse video shows that the colour of your cannabis seeds doesn’t have any impact on their germination rates. The colour of the shell casing is simply not an indication of the type of genetics contained inside. Nor is the colour or appearance of the exterior of the cannabis seed a good indicator of any other property, such as the future plant sex, size or potency. Only the genetic material inside the cannabis seed really determines the types of result that you can expect.
Black cannabis seeds germination test results
The dark cannabis seeds (on the right hand side in the video) showed the same germination rate as the white cannabis seeds (left hand side). However, the white cannabis seeds appeared to germinate a little bit faster than the dark cannabis seeds.
White cannabis seeds germination test results
Can white cannabis seeds germinate? One curious result from the white vs dark seeds cannabis video was that the white cannabis seeds appeared to germinate slightly faster than the dark cannabis seeds. This is interesting, since the seeds all contain the same Think Different autoflowering genetics from a recent cannabis seed batch.
One suggestion is that the white cannabis seeds may have a slightly softer shell, allowing the white cannabis seeds a slightly faster germination. The experiment was repeated twice more. On each occasion, the seed germination rates were the same, but the white seeds tended to germinate faster.
|The best ways to germinate cannabis seeds|
Why are some cannabis seeds white and some black?
Even cannabis seeds produced from the same branch of a plant can have quite different appearances. Size, as well as shape and colour can vary. Some cannabis strains can produce seeds that have certain appearance characteristics. White Widow seeds, for example, can often be very small and pale. Frisian Dew seeds can have a slightly grey colour.
There have been many theories about how the size, shape and colour of cannabis seeds can affect the plants which they eventually produce. In reality, it’s the cannabis genetics inside the seed case which really determines the results that you can expect. The shell of the seed and the markings on it are purely cosmetic and don’t give any clues to the results you can expect.
In the years before Dutch Passion invented feminised cannabis seeds, people would often try to predict which seeds would produce female plants. Of course, this was impossible. But it didn’t stop people going to great lengths, even measuring the weight of individual seeds in an attempt to derive some knowledge from the seeds physical appearance/size.
|What does the appearance of cannabis seeds indicate?|
Is the colour of seeds a cosmetic difference only?
Some dark cannabis seeds have black lines on them (tiger prints), giving them a distinctive appearance. Other seeds have no significant patterns or markings on them at all. Dark black cannabis seeds can be occasionally seen.
You may also see light vs dark cannabis seeds produced from the same plant. But it’s important to note that cosmetic differences between the seed casing is not a factor which defines how the cannabis seeds grow or germinate.
The genetic material in the soft plant tissue inside the seeds is what really determines the type, size, sex and potency of the cannabis you will eventually grow. These genetics, along with the environment you provide, will control the terpenes, cannabinoids, yield and potency of your eventual cannabis harvest.
Why are some cannabis seeds white and some black? It’s just the way that nature has evolved. Seeds from all kinds of plants and trees show similar variations in their appearance.
What should you look out for in a cannabis seed?
Rather than looking at the appearance of a cannabis seed, you should focus more on the quality of breeding which has gone into the seed. Some growers think that only the largest, tiger-striped cannabis seeds will go on to produce the best plants. Yet some strains will never produce seeds like that.
Time spent researching your cannabis seeds and cannabis seed supplier is time wisely spent. Look for a seed supplier with a good track record in cannabis breeding. Preferably look for a cannabis seed supplier that has been around for a long time with a few cannabis cups and plenty of online reference grows for you to check out before you spend your cash.
Knowing whether you prefer e.g. a Kush, a THC rich vs CBD rich strain, a fruity strain or a Skunk strain is a good place to start. If the array of cannabis seed choices can seem overwhelming, the following article may help you select the best strain for you.
|How to choose the best cannabis strains for you|
Once you have the best cannabis seeds for you, it’s worth ensuring you get maximum germination rates with a good germination method. The preferred Dutch Passion way to germinate cannabis seeds is with moist (but never soaked) cotton pads. If you spot any issue during the growth of your cannabis seeds, we highly recommend to refer to our illustrated guide featuring the key nutrient deficiencies and excesses symptoms and how to cure them.
White cannabis seeds vs dark cannabis seeds
We hope the germination video is a useful illustration of the fact that there is little useful information that can be conveyed by the colour or darkness of a cannabis seed.
Each cannabis strain can produce subtle differences in the size and appearance of their cannabis seeds. You can even expect to see differences between cannabis seeds produced from the same plant. Don’t worry about trying to read too much into cannabis seed appearance. Instead, do your research carefully and select the best cannabis seeds for your particular needs and your specific grow situation. Enjoy growing and good luck!