How To Germinate Cannabis Seeds With Hydrogen Peroxide

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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 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 Seeds are expensive, having them all germinate is achievable with these easy germination techniques. You'll be growing in no time!

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.

Conclusion

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.

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.

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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.

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]

How to Germinate Cannabis Seeds

So you’ve gotten hold of some cannabis seeds and want to make sure they all pop? Seeds are expensive and having them all germinate is achievable, provided they were bred and stored correctly. There are various methods of germination that different cultivators will swear by, from pre soak solutions to the type of medium used.

General Requirements for Germination

Consider a seed falling off a tree in winter. By the time spring comes around the seed may be slightly covered in a light soil, temperatures have risen and rains are starting to fall. These are the basics conditions for a seed to germinate; warmth, water, air and some sort of darkness or cover.

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The Seed Soak Germination

This is a part of germination where the seed is soaked for a period of time. Soaking can be in anything from distilled or RO water, to some form of special solution. Solutions can vary from beneficial microbial inoculation or a solution of hydrogen peroxide. The time a seed is soaked can vary from a few minutes to hours or even a day. The soft seed is then carefully transplanted to the growing medium of choice to sprout and grow. It should be noted that seeds can drown and we wouldn’t advise submerging seeds for more than 24 hours.

Hydrogen Peroxide Solution

Hydrogen peroxide is used to sterilize the outer shell of the seed and increase oxygen levels around it. Oxygen is important for gemination and is not uncommon for cultivators to rinse seeds in a weak solution for up to 30 minutes. Add 3ml of 50% hydrogen peroxide to 250ml of water for this method of soaking. Be sure to rinse the seeds well with clean water after soaking. Microbial life of all kinds won’t survive this method, whether it be unwanted fungi spores and bacteria or beneficial microbes.

Beneficial Microbe Solution

An alternative solution is dechlorinated water and beneficial microbes. The seed is then placed in the solution for anything from 30 minutes to a couple of hours. Beneficial organisms fight any “bad guys” around and coat the seed with “good guys.” The microbes will colonize the seedling’s root zone creating a symbiotic relationship between them. Rinsing with clean water afterwards is not necessary.

Paper Towel “Soak”

Many did it at school with paper towels or cotton wool. Seeds are placed in-between soft, water retaining materials in a warm place until a tap root is seen. Paper towels and cotton wool can dry out quickly so check on the moisture content regularly.

Once the tap root reveals itself, gently transfer it to a growing medium or starter plug. Place the sprouted seed tap root down and cover lightly. When transplanting seeds, minimize exposing the root to the elements especially sunlight. Sterilize all tools used and be sure not to break the tap root during transplant, as that can happen too.

Starter Plug Germination

Starter plugs range from rock wool to peat pellets and natural sponges of different kinds. What’s important to note is not to mix your mediums. For example, don’t germinate seeds in rock wool when planning a coco peat finish. A peat pellet or natural sponge would be better suited. For a sterile or inoculated plug to germinate in, add either peroxide or microbes to the water used to soak or expand with.

Rock Wool

An old favourite able to hold both air and water well. Produced by the heating of basalt rock and calcium and spun into a sheet. Rock wool naturally has a high pH of 8 and should be soaked in water with a pH of 5.5 – 6 for 30 minutes. Shake excess water off and place the seeds in the designated hole. Pinch the hole closed and place the rock wool in a warm area. Keep the rock wool plug moist but not overly soaked.

Peat Pellet

Popular in recent years the coco peat pellet is expanded with water and surrounded by a soft material with an opening at the top. It’s pH is naturally around 5.5 but any water or solution used to expand the pellet should also be 5.5. Place the seed gently 1cm to 5mm inside the pellet and cover lightly. Make sure the pellet stays moist but not soaked, allowing for water to drain off.

Natural Sponges

A sponge shaped like rock wool but made from organic materials like coco peat. When produced, they are pH ready at around 5.5 and in some cases when sold in packs, are already sufficiently soaked too. Like above, place the seed gently inside and place in a warm area making sure they stay moist.

Direct Sowing Germination

You may want to sow your seed directly in the ground in the garden. For that, choose the sunniest spot with a bit of space around as cannabis can get big outdoors. You can soak your seed like above or just get it in there by placing it 5mm – 1cm deep in loosened soil. Keep the area moist and protected, even covered with something clear if preferred.

Cannabis plants become sexually mature and ready to flower from around 6-8 weeks strain depending. This allows for seeds to be planted from spring though to mid-late summer, the longer you wait though, the smaller the yield. Plant your seed in mid spring for vigorous growth during the summer months and a big harvest come autumn.

Conclusion

These are a few of the basic methods used to germinate cannabis seeds although the options are endless. Each method will vary in the time it takes for the seed to break ground, anything from a day or two, to two weeks outdoors with direct sowing. Starting off in the same fashion as vegetable seeds in seed trays are also options for germinating cannabis seeds.

Here are a few more tips for germinating cannabis seed:

If using a propagation dome or some sort of closed environment to increase humidity, the air should be changed daily.

The use of heat mats can greatly increase germination rates, the right temperature can make all the difference.

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