Cannabis Seed Development

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How long does it take to germinate cannabis seeds? When to transplant seedlings? When to start vegging and flowering? Grow your knowledge about the lifecycle of the cannabis plant. Marijuana Cultivation/Producing Seeds Producing Seeds [ edit | edit source ] Sooner or later every grower is going to want to produce marijuana seeds. Developing a new stable strain is beyond The use of cannabis plants as a source of therapeutic compounds is gaining great importance since restrictions on its growth and use are gradually reduced throughout the world. Intensification of medical cannabis production stimulated breeding activities aimed at developing new, improved cultivars with precisely defined and stable cannabinoid profiles. Breeding medical cannabis rely significantly on manipulation of sex expression, since only genetically female plants are cultivated. Sex is determined by a pair of sex chromosomes (female XX and male XY), but the presence of Y chromosome is not the sole determining factor for the absence of male flowers on genetically female plants. The effects of several exogenous substances, such as silver thiosulfate, gibberellic acid and colloidal silver, on medical cannabis sex expression were therefore analysed in this study. Substances in various concentrations were tested within 20 different treatments on two high CBD breeding lines. Our results showed that spraying whole plants with STS once is more efficient than application of STS on shoot tips, while spraying plants with 0.01 % gibberellic acid and intensive cutting is ineffective in stimulating the production of male flowers. Additionally, spraying whole plants with colloidal silver was also shown to be effective in induction of male flowers on genetically female plants, since it produced up to 379 male flowers per plant. The viability and fertility of the induced male flowers were confirmed by FDA staining of pollen grains, by in vitro and in vivo germination tests of pollen, by counting the number of seeds developed after hybridisation, and by evaluating germination rates of developed seeds. Finally, the established protocol was implemented for crossing selected genetically female plants. The cannabinoid profile of the progeny was compared to the profile of the parental population and an improvement in biochemical profile of the breeding line was confirmed. The progeny had a higher and more uniform tCBD to tTHC ratio (up to 29.6; average 21.33±0.39) compared to the original population (up to 18.8; average 7.83±1.03). This is the first comprehensive scientific report on induction of fertile male flowers on genetically female plants of medical cannabis (Cannabis sativa L.).

Cannabis growth stages breakdown

Growing cannabis can seem easier when the process is broken down into the 4 main cannabis growth stages. These are the cannabis seed germination, seedling, veg and bloom. At each stage the requirements for nutrients, light and water will vary. The experienced cannabis grower will know how to give the right environmental conditions at each of the stages of cannabis growth. However cannabis plants will always teach you something new to improve the way you grow. Dive into this guide to learn all you should know about the life cycle of cannabis!

Summary:
How long is a cannabis full grow cycle on average?
Cannabis germination stage (2-10 days)
Cannabis seedling stage (2-3 weeks)
Cannabis vegetative stage (3-15 weeks)
Cannabis flowering stage (7-14 weeks)
Other important cannabis life cycle considerations

How long is a cannabis full grow cycle on average?

There are 4 main stages of the cannabis life cycle as it transitions from seed to harvest. Germination is often defined as the time taken from planting the cannabis seed to the point where it has produced it’s first cotyledon leaf pair. These are the first ‘baby’ (non-serrated) leaf set which is formed as the seed germinates.

Cannabis growth stages Average duration
Germination stage 2-10 days
Seedling stage 1-3 weeks
Vegetative stage 1-15 weeks
Flowering stage 7-14 weeks*

The cannabis plant life cycle for a fast growing autoflower seed variety such as as Auto Blueberry or Auto Blackberry Kush could be as little as 9 weeks from seed to harvest.

Or it could be a 6 months cannabis life cycle for an outdoor seed variety. The indoor cannabis grower has full control over their plants and the environment. This allows indoor feminised seed growers to dictate the length of vegetative growth, which in turn will affect final plant size, yield and overall life cycle.

The length of the cannabis full grow cycle will depend on your choice of cannabis seeds (autoflower seeds vs feminised seeds) and whether you grow them indoors or outdoors.

Related:
Autoflower seed vs feminised seed outdoor cannabis growing

Cannabis germination stage (2-10 days)

Cannabis seeds are typically small, hard and dry. The colours vary from light to dark brown. The first cannabis plant stages take place after the seed has germinated. During seed germination the shell of the seed is initially softened by the moist germination conditions. It’s important to provide moist, but never soaked, conditions for cannabis seed germination in order to achieve maximum germination rates from your precious seeds.

Cannabis seeds should be germinated in dark conditions and don’t need any nutrients initially. Water is sufficient for the first few days. The tap root will emerge from the cannabis seed and grow downwards. The first set of cotyledon leaves will emerge and the cannabis grow cycle has begun! Note that these leaves don’t have the ‘normal’ serrated edges which you will see on all subsequent leaves. As all this is happening the cannabis root system starts to form.

It can take around 2-10 days for seed germination to occur. Occasionally, cannabis seeds can take up to 2 weeks to germinate. Eventually you will see the first set of ‘true’ cannabis leaves with serrated edges appear. For many growers, this represents the end of the cannabis germination stage and the start of the seedling stage.

Related:
Dark vs white cannabis seed germination test

How long does it take to germinate cannabis seeds?

It can vary from one cannabis seed to another. Usually you can expect seeds to germinate somewhere around 2-10 days after you begin the germination process. Occasionally you can get cannabis seeds to germinate in just one day. Sometimes it can take around 2 weeks. But usually you can expect to wait around 2-10 days for your cannabis seeds to germinate.

Can you speed up the germination process?

Not really. You need to provide good cannabis seed germination conditions and then wait for nature to do her work. If you have bought good quality cannabis seeds from a proven supplier than you can expect cannabis seed germination rates of 90%+.

Many growers have accidentally killed their plants during germination by trying to speed things up by a day or two. It definitely isn’t recommended to e.g. sand-paper your seeds to reduce the shell thickness in an attempt to speed up germination. Nor is it recommended to try to force the shell off the plant during germination. Instead, just be patient and allow the cannabis genetics to do their work.

When to transplant cannabis seedlings?

If you have germinated your cannabis seeds with the moist cotton pad method then you will simply place the germinated seedlings in your grow medium (e.g. soil or coco fibre) or your grow system (e.g. DWC or NFT hydroponic system).

Many growers that use e.g. autoflowering cannabis seeds will simply put their seedling into the final grow container. This avoids the need to repeatedly transplant the seedling to progressively larger containers. In the case of an autoflower strain with a limited lifetime, this process allows the auto to focus all the available time on growth. No plant time is spent adapting during repeated transplants, allowing your auto to reach maximum potential.

Those that grow photoperiod feminised seeds indoors can choose when the blooming process starts simply by reducing daylight hours. That gives them more time to spend progressively potting up and transplanting into gradually larger containers if they wish.

Related:
Cotton pads germination video tutorial

Cannabis seedling stage (2-3 weeks)

For the next 2-3 weeks after germination, the cannabis seedling will grow. The cannabis root system is essential for healthy growth and development. Experienced growers aim to deliver fully optimised grow conditions in order to maximise root growth. Give the roots waterlogged cold soil and they won’t grow well, this may result in a permanently stunted plant.

Above ground, the cannabis seedling will continue to grow. With each new set of leaves you may notice progressively more ‘blades’ or fingers on the leaves. Initially you may see 3 fingers, then 5 or 7 etc. During the life cycle of cannabis, the seedling needs less water and nutrition than it does in subsequent veg growth and flowering stages. This is one of the most delicate cannabis growing stages. The seedling needs little water and minimal nutrients.

If growing in light mix soil there you may not need to consider any grow nutrients until a week after the first set of serrated leaves emerge. Generally, a light mix soil has enough nutrition for the first couple of weeks after cannabis seed germination. After that many growers use root stimulator and grow nutrients.

If you are growing in hydroponics or coco fibre you may already be carefully using very light nutrients on young seedlings. You may prefer to use specialist low strength seedling nutrients at this stage. The goal is to keep the cannabis seedling in the nutrient sweet spot without over feeding or underfeeding. If seedlings are given excessive nutrients it can ‘burn’ the plant, permanently limiting future growth.

Related:
Everything you need to know about cannabis roots

How long does the cannabis seedling stage last?

Many growers consider the first 2-3 weeks after germination to be the cannabis seedling stage. These are the first couple of weeks where the seedling is most vulnerable. The seedling may only be a few inches/cm tall with a couple of sets of true (non-cotyledon) leaves.

Lighting levels don’t need to be particularly intense for cannabis seedlings, for the technically minded PPFD levels of 200-400 should be adequate. Many use T5 fluorescent tubes for cannabis seedling lighting. The delicate young seedling leaf tissue can be damaged by the intense light levels which you will need in later cannabis flowering stages.

If you do see your cannabis seedlings stretching a little too much it can help to reduce the distance between the plants and the light. With higher light intensities, the stretching should reduce. If your seedling suffer elongated stems you can gently prop up your seedling with some small wooden supports, such as toothpicks (or similar)

What does a healthy cannabis seedling look like?

You can expect a short, squat plant. The cotyledon leaves will be small in comparison to the emerging ‘true’ leaves and you will notice new leaf sets emerging from the central growing point of your plant (the ‘apex’). The colour should be a vibrant green. Any signs of yellowing is a signal that something is wrong. If your seedling has brown leaf tips it’s a sign that you have overfed your seedlings and ‘burned’ the plant. This is never a good sign and can temporarily or permanently restrict future growth.

If you have a healthy cannabis seedling it will have all the basics in place for future growth. The roots should have the space and nutrients/minerals required to grow a larger frame. The leaves will be ready to grow and absorb more light which will power future photosynthesis. Your plant is set for vegetative growth and will be ready for more light, nutrients and water.

Autoflower seed growers may already have their plant in the final grow container at this stage.

Related:
Top 10 germination and seedling mistakes

Cannabis vegetative stage (3-15 weeks)

Vegetative growth is the indoor cannabis growing stage where roots, branches and leaves grow but no buds are formed. Indoor growers often use 18-24 hours of daily light whether they are using autoflower seeds or photoperiod feminised cannabis seeds.

During vegetative growth the cannabis plants gradually grow in both height and width. Nitrogen rich nutrients are particularly useful in the vegetative growth stage. The first sets of cannabis leaves grow gradually larger and new leaf sets are formed. As the plant grows it’s requirements for nutrients, water and light will all increase. Light levels can be increased from around 200 PPFD to nearer 400-600 PPFD – your light manufacturer should be able to detail the PPFD levels at various hanging heights.

How long should a cannabis plant stay in veg?

Those growing photoperiod feminised cannabis seeds can select the length of the vegetative growth stage. Often it’s around 4-6 weeks for many growers. But some growers, e.g. SCROG growers (Screen Of Green method), prefer very long periods of ‘veg’ growth, in extreme cases up to 15 weeks or so. However, SOG growers (Sea Of Green method), may not give their plants any veg growth and instead put them straight into bloom conditions.

Those growing autoflower seeds will not be able to dictate the length of the vegetative growth phase. Instead the autoflower seed genetics will determine the point at which it automatically transitions from veg to bloom. It does this without any change or alteration to the light cycle. Autoflowering cannabis seeds grow from seed to harvest under the same light cycle, typically 20 hours of daily light. Photoperiod feminised cannabis strains only commence bloom when indoor light hours are reduced to 12 per day.

Related:
SOG vs SCROG cannabis growing

How does a healthy cannabis plant in veg look like?

Much depends on the length of time the plant has been in the vegetative growth stage for. A feminised strain with 15 weeks of veg growth could have filled a very large SCROG screen. Whereas an autoflower plant in veg may be perhaps 10-20cm tall, perhaps 3 weeks old and ready to start stretching once bloom begins. Much depends on your cannabis genetics and grow style. But you can expect to see a medium-sized plant with healthy green foliage, but no buds.

Why does my cannabis plant want to flower in the vegetative stage?

The cannabis flowering stage follows veg growth. Cannabis plants are genetically geared towards bloom. It’s the only chance for cannabis to produce seeds and produce the next generation of plants. You may see pre-flowers at the nodes between the stem and branches. Autoflower genetics don’t hold back. As soon as they are ready, autos start to transition from veg to bloom. During this process the auto exhibits features of both veg and bloom.

Cannabis flowering stage (7-14 weeks)

During the cannabis flowering stages, the female plant produces buds and resin. The flowering stage follows the vegetative growth stage. When growing autoflowering cannabis seeds, the transition from the vegetative stage to the flowering stage happens automatically (hence the name, autoflowering).

When growing photoperiod feminised cannabis seeds, bloom begins indoors when the daily light hours are decreased to 12. Outdoors, feminised strains sense the shortened daylight hours as autumn/fall approaches and bloom begins. However if you are growing at equatorial regions the plants can sense the short days immediately.

During the cannabis flowering stage, the plant will require gradually increased levels of nutrients and water. Phosphorus will be required in greater quantities as the plant biochemistry changes. During the cannabis flowering stage the plant biomass can increase dramatically.

Intense light levels can be used in bloom, often with PPFD levels of 600-900. More light can deliver heavier yields. Some professional cannabis growers used PPFD levels of around/over 1000 and may also supplement with Carbon Dioxide to further boost yields.

The length of the flowering stage depends on the genetics. 7 weeks of bloom is required by fast flowering indica strains such as Bubba Island Kush seeds. But a slow blooming Haze may require upwards of 14 weeks in bloom.

The following cannabis flowering stages are shown in week by week pictures, below.

How to tell if a cannabis plant is ready to bloom?

Knowing when your plant is ready to be flipped from veg growth to bloom is one of the most important decisions you will make. But first you may wish to consider a few points related to the timing of the cannabis flowering stage.

• The height of your grow room may be a limiting factor. If you have restricted vertical growing space you may prefer to have minimal veg time.

• Are you growing indica or sativa cannabis seeds? Sativa strains may stretch dramatically during bloom. Take this into account when deciding if your cannabis plant is ready to bloom.

• Growing clones or seeds? Clones don’t always have well established root systems and can take a while to create one before being flipped into bloom.

• Outdoor plants can also be forced into early bloom if you have a greenhouse equipped with blackout blinds. Otherwise, they will choose their own moment to bloom as daylight hours shorten.

• Which growing method are you using? If using the SCROG method you may wish to wait and give the plant a long veg stage. If using the SOG method you may want to offer minimal veg time or even none at all and grow from seed to harvest under 12/12 light

• Which region/climate are you growing in? If you’re growing in tropical equatorial regions you may want the most sativa dominant strains with the most stretch. That’s because the plants go into flowering more-or-less immediately. In more temperate climates, the plants commence bloom as daylight hours shorten. For Northern Hemisphere growers (Europe, USA etc) this often happens around August.

Related:
Power Plant grown from seed to harvest under 12/12 light

How long does it take for cannabis to start blooming after switching the light cycle?

Once you switch the light cycle to 12/12 (12 hours of daily light) the plant undergoes plant hormone changes as it senses the shorter days.

The plant hormones cause the plant to prepare for bloom. Over the following week or so you will see the changes on the cannabis plant as she gets ready to stretch and produce flowers. You may see female pre-flowers producing a couple of pistils (hairs) at the node between the stem and a branch.

However you can expect to wait 1-2 weeks before you start to see flowers starting to appear. With certain sativa and hybrid strains it can take 3-6 weeks before any significant flower formation.

How does a healthy cannabis plant in flowering look like?

Initially you may notice areas of light green foliage at the eventual points where buds will eventually form. You may also notice the plants stretching, with increased internodal distance.

Growers monitor their cannabis flowering stages week by week. Some like to consider the cannabis flowering stage as 3 separate mini-phases; early bloom, mid bloom and late bloom. As your cannabis plant flowers the weight of buds and resin should increase as harvest point approaches.

During the cannabis flowering stage you will notice that your plant appetite for nutrients reaches maximum as it produces bigger buds and more cannabinoid-containing resin. The ratio of required nutrients will change too. Less Nitrogen (N) is required and increasing amounts of Phosphorus (P) and Potassium (K) are needed to support heavy harvests of compact flowers.

During the first few weeks of bloom your plant will stretch. A sativa may stretch to 2-3 times the height it was at the end of veg. You will see increasing amounts of pistils being produced and the buds start to form and eventually fatten up. After around 5 weeks of bloom the cannabis plants have generally stopped stretching and the buds start to get larger.

As harvest approaches, the pistils start appearing as orange rather than white.

How to tell when your cannabis buds are ready for harvest?

Your cannabis seed supplier should give an indication of the approximate length of the flowering stage. This is a guideline rather than a fixed rule and it may indicate the typical earliest harvest point rather than the recommended harvest time.

Environmental conditions and the specific phenotype will determine the actual harvest date. In addition, you may have a preference for early, mid or late harvested buds.

There are various cannabis bud growth stages. They start small and gradually pack on weight and resin as they grow. Some growers like the slightly heavy effects (and more generous yields) offered by allowing the buds a week or two extra in bloom.

There are different cannabis trichomes stages to consider. Immature buds tend to have clear trichomes. As the buds approach harvest the trichomes become cloudy and eventually start to produce amber (or even red) colourations. Many harvest their buds as the trichomes are transitioning from clear to cloudy.

Related:
Understanding cannabis trichomes

What to do in case of early or late flowering?

If your plant flowers early you can look forward to an earlier harvest. A late flowering plant will generally have enjoyed more time for veg growth, so you may be able to look forward to a heavier harvest.

When growing autoflower seeds you may find some plants will be ready to harvest a week or two before the slower phenotypes. Remember each plant is different. Try to time the harvest so that you have buds of the perfect maturity level for your personal tastes. Some growers love the lively energetic buzz from an early harvested plant. Other growers will always wait an extra couple of weeks to ensure that their plants have a high proportion of amber trichomes which can produce heavier effects.

See also  Cannabis Seeds Uk

What really matters to the home grower is that they:

• are growing the best cannabis seeds for them personal needs and,

• they select the optimised harvest date which provides maximum enjoyment and satisfaction for their recreational or medical needs.

Other important cannabis life cycle considerations

There are different cannabis growth stages as well as different cannabis flowering stages. The experienced grower understands the various environmental, nutrient and lighting requirements at the various stages of cannabis growth.

What stage of growth does cannabis produce trichomes?

This can depend on the specific cannabis seeds being grown. Trichomes can be seen even on young plants though they can be microscopically small. As the plant matures the amount of trichomes increases dramatically. When growing photoperiod feminised cannabis seeds you may see the first trichomes around 3 weeks into bloom. Over the following month trichome production is heavy and gives the plant a frosty appearance, as if sprinkled with sugar.

When growing autoflower seeds, trichome production tends to start around 4 weeks after germination. In the following weeks, trichome production steps up a gear as the buds gain weight. Aroma also increases as more and more trichomes are produced.

What stage of growth does cannabis stop growing?

During flowering, most photoperiod cannabis plants stop stretching after around 4-5 weeks. After that point most of the growth happens on the buds. For autoflower plants, stretch tends to stop around 6-7 weeks after germination. At that point the bulk of the plants energy is focussed on bud growth and resin production.

But it’s worth adding that these figures are only approximate. Much depends on the specific cannabis genetics that you are growing, your environmental conditions and the grow method.

How long should the harvested buds be left to dry?

Harvested buds are typically left for 7-14 days to dry before being transferred to the curing jars. When the branches ‘snap’ (rather than bend) it’s an indication that the plant is dry enough for curing to begin.

How long should the harvested buds be cured?

Many would say that a month or two is a realistic minimum to allow the taste and aromas to fully develop. Keeping your cured buds in jars even longer isn’t an issue. Many people feel that a 6-month cure with your jars in cool/dark conditions is a great way to maximise taste and aroma.

How to keep a consistent cannabis growth timeline?

When growing photoperiod feminised cannabis seeds the growth timeline is up to you. You can offer minimal veg growth for a SOG grow or several months for a SCROG grow.

Autoflower seeds, on the other hand, have a cannabis growth timeline of their own. They, not you, decide when to start blooming. Good quality auto seed suppliers should be able to give you a good idea whether they are likely to have a growth lifecycle as fast as 9 weeks e.g. Auto Blueberry seeds or a slow growth lifecycle of 12-15 weeks e.g. Auto Ultimate seeds. Note that the autos which take longer to grow may well deliver very heavy yields.

Outdoor cannabis growers should note that the different regions you live in can also determine how many daylight hours per day you will have. That will have a huge influence on your outdoor cannabis lifecycle (or growth stages). Equatorial cannabis growers have 12/12 light (or thereabouts) almost all year round. This means you won’t have any veg time at all if you grow outdoors.

Understanding the cannabis growth stages is key

With an increased understanding of the different cannabis growth stages you will find your control and enjoyment of cannabis cultivation will increase. As well as optimising your grow environment and improving your understanding of the cannabis grow cycle be sure to select the best cannabis seeds for your personal grow situation. The choice of cannabis seeds may seem large and possibly confusing.

If so please check out the Dutch Passion Seed Finder which asks a few simple questions before recommending the seeds which best fit your needs.

Marijuana Cultivation/Producing Seeds

Producing Seeds [ edit | edit source ]

Sooner or later every grower is going to want to produce marijuana seeds. Developing a new stable strain is beyond the scope of this discussion and requires the ability to grow hundreds or even thousands of breeding plants. However, just about any grower can manage to preserve some genetics by growing f2 seeds where they have crossed a male and female of the same strain, or can produce a simple cross which would be referred to as strain1xstrain2 for instance white widow crossed with ak-47 would be referred to as a WW x AK-47. You can produce some excellent seed and excellent marijuana this way.

To Feminise or not to Feminise [ edit | edit source ]

There are numerous myths surrounding feminized seeds. Feminizing seeds are a bit more work than simply crossing two plants naturally. However it will save you a lot of time in the end. If you make fem seeds properly then there is no increased chance of hermaphrodites and all seeds will be female. This means no wasted time and effort growing males and it means that all your viable seeds produce useful plants. Since roughly half of normal seeds are male this effectively doubles the number of seeds you have.

Feminized seeds are bred to contain no male chromosomes, which will be able to produce the crop of resinous buds sought by most growers. For gardeners who require a quick and easy cultivation process, feminized seeds are the ideal choice. Some medicinal cannabis users may be deterred from growing their own supply because of the perceived difficulty of growing or of identifying the different genders and removing males early in the blooming period. Feminized seed-strains offer a simple solution to these issues, as there is no need to spend time in the first weeks of flowering checking for male plants.

Other times you will have no choice but to produce feminized seed because it will be a female plants genetics that you want to preserve and you won’t have any males. Perhaps you received these genetics via clone or didn’t keep males.

The new thing on the market for commercial Cannabis cultivation are auto-flowering feminized strains. By crossing of the cannabis ruderalis with Sativa and Indica strains many cultivators have created interesting hybrids which boast benefits from both sides of these families.

The first ‘auto-flowering cannabis seed’ which came on the market a few years ago was the Lowryder #1. This was a hybrid between a Cannabis ruderalis, a William’s Wonder and a Northern Lights #2. This strain was marketed by ‘The Joint Doctor’ and was honestly speaking not very impressive. The genetics of the ruderalis was still highly present which caused for a very low yield and little psychoactive effect. Not very attractive.

Auto-flowering cannabis and the easily distributed seed have opened a whole new market in the world of the online grow-shop, making it easy for home growers with shortage of space to grow rewarding cannabis plants in many different varieties. To grow plants indoors, a growing medium (e.g. soil or growing Potting soil, irrigation (water), fertilizer (nutrients), light and atmosphere need to be supplied to the plant.

Auto-flowers have been rising in popularity fast and there are now auto flower growers communities. These Web properties allow users to get information on how to grow these non photo-sensitive plants and what are the best practices when producing and germinating auto-flower seeds.

Selecting Suitable Parents [ edit | edit source ]

There are a number of important characteristics when selecting parents. First are you making fem seeds? If you are then both parents will be female. This makes things easier. If not then the best you can do is select a male with characteristics in common with the females you hope to achieve from the seed.

Obviously potency, yield, and psychoactive effects are critical to the selection process. But some other important traits are size, odor, taste, resistance to mold and contaminants, early finishing and consistency.

Collecting and Storing Pollen [ edit | edit source ]

In order to collect pollen you simply put down newspaper around the base of the plant. The pollen will fall from the plant onto the newspaper. You can then put this newspaper into a plastic bag and store it in the refrigerator or freeze it. Pollen will keep for a few months in the refrigerator and can be used on the next crop. Filtering the pollen through a silkscreen, drying, and freezing can extend viability for decades. At least one reader indicates success using pollen treated in this manner and stored at -30 c for 17 years. The chance of viability does decrease with time, even in the freezer, so the more fresh the pollen the better. Wrapping the pollen in a layer of aluminum foil and then a layer of plastic should help to protect it from freezer burn. Additionally, oxygen evacuation such as with a heavy gas like nitrogen or vacuum sealing should provide additional assurance of preservation.

Pollinating a Plant [ edit | edit source ]

To pollinate a plant you can brush the pollen on a flower with a cotton swab or you can take the plastic bag, then wrap the flower inside it and shake, trapping the pollen inside for easier transportation. In this way you can selectively pollinate plants and even individual buds and branches.

Male Isolation [ edit | edit source ]

A male plant or a plant with male flowers will pollinate your entire crop rendering it seedy. You probably don’t want THAT many seeds so how can you avoid it? Moving the male to another room might work but if that other room shares an air path via ducting or air conditioning then pollen may still find its way. One technique is to construct a male isolation chamber.

A male isolation chamber is simply a transparent container such as a large plastic storage tub turned on its side (available at your local megamart). Get a good sized PC fan that can be powered with pretty much any 12v wall adapter, by splicing together the + (yellow or red on fan, usually dotted on power adapter) and the – wires (black on fan, usually dotted power adapter) just twist with the like wire on the other device and then seal up the connection with electric tape. Then take a filtrate filter and cut out squares that fit the back of the pc fan so that the fan pulls (rather than pushes) air through the filter. Tape several layers of filter to the back of the pc fan so all the air goes through the filter. Now cut a large hole in the top of the plastic container and mount the pc fan over top of it so it pulls air out the box. You can use silicon sealant, latex, whatever you’ve got that gives a good tight seal.

This can be used as is, or you can cut a small intake in the bottom to improve airflow. Pollen won’t be able to escape the intake as long as the fan is moving but you might put filter paper over the intake to protect against fan failures. You can also use grommets to seal holes and run tubing into the chamber in order to water hydroponically from a reservoir outside the chamber. Otherwise you will need to remove the whole chamber to a safe location in order to water the plant or maintain a reservoir kept inside the chamber.

Making Feminized Seed [ edit | edit source ]

To make feminized seed you must induce male flowers in a female plant. There is all sorts of information on the Internet about doing this with light stress (light interruptions during flowering) and other forms of stress. The best of the stress techniques is to simply keep the plant in the flowering stage well past ripeness and it will produce a flower (with seed).

Stress techniques will work but whatever genetic weakness caused the plants to produce a male flower under stress will be carried on to the seeds. This means the resulting seeds have a known tendency to produce hermaphrodites. Fortunately, environmental stress is not the only way to produce male flowers in a female plant.

The ideal way to produce feminized seed through hormonal alteration of the plant. By adding or inhibiting plant hormones you can cause the plant to produce male flowers. Because you did not select a plant that produces male flowers under stress there is no genetic predisposition to hermaphroditism in the seed vs plants bred between a male and female parent. There are actually a few ways to do this, the easiest I will list here.

Colloidal Silver (CS) [ edit | edit source ]

This is the least expensive and most privacy conscious way to produce fem seed. CS has gotten a bad name because there is so much bad information spread around about its production and concentrations. It doesn’t help that there are those who believe in drinking low concentration colloidal silver for good health and there is information mixed in about how to produce that low concentration food grade product. Follow the information here and you will consistently produce effective CS and know how to apply it to get consistent results.

Simply construct a generator using a 9-12 v power supply (DC output, if it says AC then its no good) that can deliver at least 250ma (most wall wart type power supplies work, batteries are not recommended since their output varies over time). The supply will have a positive and negative lead, attach silver to each lead (contrary to Internet rumors, you aren’t drinking this so cheap 925(92.5%) Stirling silver is more than pure enough. You can expose the leads by clipping off the round plug at the end and splitting the wires, one will be positive and the other negative just like any old battery. Submerge both leads about 2-3 inches apart in a glass of distilled water (roughly 8 oz). Let this run for 8-24 hrs (until the liquid reads 12-15 ppm) and when you return the liquid will be a purple or silver hue and there may be some precipitate on the bottom.

This liquid is called colloidal silver. It is nothing more or less than fine particles of silver suspended in water so it is a completely natural solution. It is safe to handle without any special precautions. [ citation needed ] The silver inhibits female flowering hormones in cannabis and so the result is that male flowering hormone dominates and male flowers are produced.

To use the silver, spray on a plant or branch three days prior to switching the lights to 12/12 and continue spraying every three days until you see the first male flowers. Repeated applications after the first flowers appear may result in more male flowers and therefore more pollen. As the plant matures it will produce pollen that can be collected and used to pollinate any female flower (including flowers on the same plant).

Silver Thiosulfate (STS) [ edit | edit source ]

Silver Thiosulfate is a substance that has similar principle, application and results of CS, but is more difficult to make. STS is more difficult to acquire, but it can still be obtained directly from a chemical supply company. STS is not an expensive or controlled substance.

Gibberellic Acid (GA3) [ edit | edit source ]

This is probably the most popular way to produce feminized seed, but at the same time the least effective. GA3 is a plant hormone that also causes the plant to stretch uncontrollably. It can be purchased readily in powdered form, a quick search reveals numerous sources on e-bay for as little as $15. Simply add to water to reach 100ppm concentration and spray the plant daily for 10 days during flowering and male flowers will be produced.

Production of Feminized Seeds of High CBD Cannabis sativa L. by Manipulation of Sex Expression and Its Application to Breeding

The use of the cannabis plant as a source of therapeutic compounds is gaining great importance since restrictions on its growth and use are gradually reduced throughout the world. Intensification of medical (drug type) cannabis production stimulated breeding activities aimed at developing new, improved cultivars with precisely defined, and stable cannabinoid profiles. The effects of several exogenous substances, known to be involved in sex expressions, such as silver thiosulfate (STS), gibberellic acid (GA), and colloidal silver, were analyzed in this study. Various concentrations were tested within 23 different treatments on two high cannabidiol (CBD) breeding populations. Our results showed that spraying whole plants with STS once is more efficient than the application of STS on shoot tips while spraying plants with 0.01% GA and intensive cutting is ineffective in stimulating the production of male flowers. Additionally, spraying whole plants with colloidal silver was also shown to be effective in the induction of male flowers on female plants, since it produced up to 379 male flowers per plant. The viability and fertility of the induced male flowers were confirmed by fluorescein diacetate (FDA) staining of pollen grains, in vitro and in vivo germination tests of pollen, counting the number of seeds developed after hybridization, and evaluating germination rates of developed seeds. Finally, one established protocol was implemented for crossing selected female plants. The cannabinoid profile of the progeny was compared with the profile of the parental population and an improvement in the biochemical profile of the breeding population was confirmed. The progeny had a higher and more uniform total CBD (tCBD) to total tetrahydrocannabinol (tTHC) ratio (up to 29.6; average 21.33 ± 0.39) compared with the original population (up to 18.8; average 7.83 ± 1.03). This is the first comprehensive report on the induction of fertile male flowers on female plants from dioecious medical cannabis (Cannabis sativa L.).

Introduction

Cannabis (Cannabis sativa L.) naturally shows sexual dimorphism with a small proportion of monoecism. In the past, it was mostly cultivated for fiber and grain, but nowadays, the plant is gaining importance in the medicinal industry due to its production of unique cannabinoids (Andre et al., 2016). They are produced in the trichomes on flower bracts of female inflorescences (Small, 2015; Andre et al., 2016). Most pharmaceutically important cannabinoids are cannabidiol (CBD) and the psychoactive tetrahydrocannabinol (THC) (Δ-9-THC) (Freeman et al., 2019). The relative content (in % of dry weight) of the latter divides cannabis genotypes into two groups: (i) industrial cannabis, commonly known as hemp or fiber-type hemp (defined as containing < 0.2% THC by dry weight in Europe) and commonly grown as a field crop and (ii) medical cannabis, marijuana or drug type cannabis (with >0.2% THC) (The European Commission, 2014), cultivated under strict legal restrictions.

Sex of C. sativa L. (2n = 20) is genetically determined by one pair of sex chromosomes X and Y, where male gender of dioecious plants is determined by heterogametic XY chromosomes, while dioecious female and monoecious or hermaphrodite plants exhibit homogametic chromosomes XX (Moliterni et al., 2004; van Bakel et al., 2011; Divashuk et al., 2014; Faux et al., 2014). The ratio of female to male flowers in a single monoecious cannabis plant is highly variable and ranges from predominantly male flowers to predominantly female flowers (Faux et al., 2014). Moreover, dioecious cannabis plants can produce flowers of the opposite sex as determined by their sex chromosomes (Moliterni et al., 2004). Due to instability of the sexual phenotypes across generations of XX plants, and the quantitative nature of sex expression, it was hypothesized that sex expression is a polygenic trait (Faux et al., 2013, 2014; Faux and Bertin, 2014). A first association mapping study of sex determination was performed in 2016 (Faux et al., 2016) on three biparental hemp populations (two dioecious and one monoecious) using 71 amplified fragment length polymorphism (AFLP) markers. It identified five quantitative trait loci (QTLs) associated with sex expressions that were putatively located on sex chromosomes. Recently, Petit and colleagues (Petit et al., 2020) published the results of a GWAS (Genome-Wide Association Study) analysis for characterization of the genetic architecture underpinning sex determination in hemp. They used a set of 600 K single-nucleotide polymorphism (SNP) markers on a panel of 123 hemp accessions (monoecious and dioecious), tested in three contrasting environments across Europe with contrasting photoperiod regimes. They identified two QTLs for sex determination across locations that contained transcription factors and genes involved in regulating the balance of phytohormones, especially auxins and gibberellic acid (GA). Two auxin response factor genes (arf2 and arf5), bZIP transcription factor 16-like, and gene gibberellic acid insensitive (GAI) that codes for the DELLA RGL1-like (repressor of giberellic acid-like) protein were identified in QTLSex_det1 for sex determination. These genes are involved in the balance of the phytohormones auxins and gibberellic acid (GA), which are known to play an active role in the sex expression (male or female) in many crops, such as hemp or spinach. The lack of a complete genome sequence did not allow to map of the QTLSex_det1 in any specific chromosome (Petit et al., 2020).

The findings confirmed previous reports that several factors, like sex-determining genes, sex chromosomes, epigenetic control by DNA methylation, and microRNAs, and physiological regulation with phytohormones influence sex expression of predetermined cannabis plants (Galoch, 1978; Dellaporta and Calderon-Urrea, 1993; Hall et al., 2012; Punja and Holmes, 2020). Several studies with hormonal manipulation confirmed gender reversal in C. sativa L. and proved bipotency of sexually predetermined dioecious cannabis plants. It has been shown that gibberellins induce maleness in plants, while ethylene, cytokinins, and auxins stimulate the formation of female flowers on genetically male plants (Ainsworth, 2000). Galoch (1978) showed that indole-3-acetic acid (IAA), kinetin (up to 100 μg/plant), and ethylene-releasing compound ethrel (up to 500 μg/plant) enhanced the feminization of male plants. Abscisic acid (ABA) was completely ineffective in sexing both male and female hemp when used alone. GA3 (up to 100 μg/plant) promoted masculinization of female plants while having no effect on sex change in male plants. Similarly, Ram and Jaiswal (1972) earlier found that male plants showed no change in sex expression when treated with gibberellins (up to 100 μg/plant), but female plants developed male flowers with normal stamens and viable pollen grains. Besides, environmental factors such as temperature, photoperiod, light conditions, nutrient deficiency, and mechanical stresses (e.g., damages) can influence sex expression and induce monoecism (Ram and Sett, 1979). As reviewed in Truta et al. (2007) and Petit et al. (2020), the ratio of different phytohormones plays a crucial role in the sex expression of hemp. External treatment of GA to spinach, for example, affects the expression of the GAI gene, which is a transcription factor of the DELLA family. It is highly expressed in female inflorescences and acts as a repressor of the expression of B-class homeotic genes, which are masculinizing factors. B-class genes stimulate male organ formation and simultaneously suppress the development of female organs in the flowers (Petit et al., 2020).

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Cannabis sex determination could be modified by applying exogenous growth regulators or chemicals, which can influence the ratio of endogenous hormones and hence the incidence of sex organs (Truta et al., 2007). Silver compounds such as silver nitrate (AgNO3) or silver thiosulfate (Ag2S2O3; STS) have been found to have masculine effects in many plant species, e.g., in Coccinia grandis (Devani et al., 2017), Cucumis sativus (Den Nijs and Visser, 1980), Silene latifolia (Law et al., 2002), Cucumis melo (Owens et al., 1980), and also Cannabis sativa. Ram and Sett (1982) applied 50, 100, and 150 μg of silver nitrate and 25, 50, and 100 μg of STS to shoot tips of female cannabis plants. Both silver compounds successfully evoked the formation of male flowers, but STS was more effective than AgNO3. 100 μg of STS caused the highest number of fully altered male flowers, which was significantly higher than the number of reduced male, intersexual, and female flowers. On the other hand, the treatment of shoot tip with 100 μg AgNO3 resulted in more than half the lower number of male flowers, with the highest amount of AgNO3 (150 μg) being ineffective in altering sex expression. Furthermore, pollen from all induced male flowers was viable in vitro and also successfully induced seed set. Lubell and Brand (2018) published the results of using 3 and 0.3 mM STS to induce male flowers in genetically female hemp plants of four strains. They sprayed three times at 7-day intervals and counted flowers (male and female) on terminal buds, not whole plants. They determined the percentage of male flowers to all flowers and the masculinization rate. The authors confirmed the successful induction of male flowers in hemp strains. Regarding the percentage of inflorescences with male flowers, their best two hemp strains yielded up to ≈15% no male inflorescences, regardless of the STS concentration used. In the books by Green (2005) and Rosenthal (2010), the authors suggest a method for making 0.3 mM STS and spraying the entire female plant until the solution drips from the plant. There is no quantitative evidence of the success of the method used. More recently, two other studies have also successfully used STS to induce male flowers: DiMatteo et al. (2020) sprayed 3 mM of STS until runoff three times at 7-day intervals after exposing the plants to short-day conditions for 12 h. Adal et al. (2021) applied 20 ml of STS (2.5 μg/ml) to whole plants on the first and third day after the start of 12-h lighting and fertilization on a foliar basis. However, these studies aimed to investigate some other aspects of male sex induction in cannabis rather than the establishment of the sex induction protocol, so no detailed data on the success of the sex reversal were presented. As far as we know, no scientific study has used colloidal silver for sex reversal in cannabis and not in other plants species. However, this method is very well known in the cannabis industry and a lot of information is available on the internet. Several other chemicals have shown alteration of cannabis sex expression, e.g., female plants treated with 75 μg of aminoethoxyvinylglycine formed only male and no intersexual flowers (Ram and Sett, 1981). Foliar spraying of male cannabis plants with 960 ppm 2-chloroethanephosphonic acid caused the highest formation of the fertile female flower (Ram and Jaiswal, 1970). A total of 100 μg/plant of cobalt chloride applied to the shoot tip triggers male sex expression in the female plants of cannabis (Ram and Sett, 1979). The mode of action of these chemicals in plants is not yet entirely deciphered. Truta et al. (2007) hypothesized that these external factors probably indirectly affect the level of endogenous auxins, which have a regulatory role on factors controlling sexual organs differentiation. The authors concluded that sex determination genes balance endogenous hormonal levels via signal transduction mechanism and thus enable sex reversion in sexually bipotent floral primordia. A comprehensive study of gene expression during flower development in cannabis was recently published by Adal et al. (2021), who discovered approximately 200 genes that were potentially involved in the production of male flowers in female plants. Although the exact role of all these genes was not examined further, the study opened many possibilities for further studies of the genetic background of sex expression in cannabis.

Manipulation of sex expression is of paramount importance in breeding medical cannabis, since only genetically and phenotypically female plants are used in commercial cultivation. It enables self-pollination and crossing of female plants for obtaining pure lines and feminized seeds, respectively (Ram and Sett, 1982). Upon germination, the latter produce entirely female progeny that is used for the production of female flowers. Most cannabis sex manipulation studies are performed on fiber-type hemp (Ram and Sett, 1982; Lubell and Brand, 2018; DiMatteo et al., 2020), and knowledge about the efficiency of various exogenous factors and application methods for inducing sex conversion in medical cannabis is needed.

The aim of our investigation was to test different sex manipulation methods (chemical, hormonal, and physiological) for induction of male flowers on female plants of medical cannabis and to evaluate their efficiency based on the number of male inflorescences and male flowers, by evaluation of pollen viability, germination potential in vitro and in vivo, and seed set. In addition, the selected treatment was implemented in a breeding program for crossing a population of female plants of a high CBD breeding population of medical cannabis to verify the usefulness of such treatments in the high-valued medical cannabis industry.

Materials and Methods

Plant Material and Growing Conditions

The experiment was carried out using plants of two breeding populations of medical cannabis, namely MX-CBD-11 and MX-CBD-707, owned by MGC Pharmaceuticals Ltd. and described in Mestinšek Mubi et al. (2020). They were grown as part of a joint research project between the Biotechnical Faculty of the University of Ljubljana and MGC Pharmaceuticals under license from the Slovenian Ministry of Health.

The mother plants (48 different genotypes of MX-CBD-707 and 31 different genotypes of MX-CBD-11) were grown from feminized seeds. Rooted cuttings were made from lateral shoots of mother plants. All plants were grown in 3.5 L pots (substrate Kekkila, Finland) in a step-in growth chamber under 24–26°C and 16/8 light/dark regime. The light was ensured by using 600-W high-pressure sodium (HPS) lamps (Phantom HPS 600W; Hydrofarm, Petaluma, CA, United States). At the vegetative stage, plants were fertilized with a mixture of vegetative fertilizer (NPK 4-1-2) and CalMag (N-Ca-Mg 2-5-2.5) + microelements in 1:1 proportion. After 31 (Experiment 1) or 60 (Experiment 2) days of vegetative growth, the plants were fertilized with a mixture of flowering fertilizer (NPK 1-3-5) and CalMag (N-Ca-Mg 2-5-2.5) + microelements in 1:1 proportion and subjected to a 12/12 photoperiod.

Design and Performance of the Experiments

In the first experiment, eight different treatments were applied using two different growth regulators in different concentrations and modes of application, along with one physiological treatment (cut) and control (no application) (Table 1). The concentrations (amounts) of STS and GA were chosen based on literature data (Ram and Sett, 1982; Green, 2005; Rosenthal, 2010) and the variant “cut” was based on the recommendations from the growers. The experiment was designed as a randomized complete block design with two factors: seven male induction treatments and control on two breeding populations, with six replicates (potted plants) for each combination of factors.

Table 1. Treatments of the first experiment of male flowers induction on female plants of Cannabis sativa.

A total of 20 mM STS was prepared by mixing 0.1 M AgNO3 and 0.1 M Na2S2O3 in a molar ratio of 1:4. 0.7 mM STS was prepared by 30x dilution of 20 mM STS with water. GA3 (Duchefa) was dissolved in double-distilled water and applied in 0.01% concentration. Spraying with STS or GA3 (treatments 1, 2, and 6) was performed once at the beginning of the experiment until runoff. For treatments 3, 4, and 5, 10 μl of STS stock solutions (1, 2, and 3 μg/μl, respectively) were applied for five consecutive days on the apical shoot tip until final amounts of STS were reached (50, 100, and 150 μg of STS, respectively) (Figure 1A). For treatment 7, plants were cut down to the height of the first two nodes. Plants from the eighth treatment represented a control group and no treatment was performed. When the treatments were applied and the experiment began, the plants (age of 31 days) were put on under a 12/12 light/dark regime to induce flowering.

Figure 1. The induction of male flowers on female plants of medical cannabis. (A) Application of silver thiosulfate (STS) on shoot tip. (B) Yellow spots on the leaves 1 week after spraying with 20 mM STS. (C) Male inflorescence at full flowering. (D) In vivo germination of pollen. (E) In vitro germination of pollen. (F) The occurrence of male flowers on female plants of breeding population MX-CBD-707 after spraying with 30 ppm colloidal silver every day. (G) Viable pollen stained with fluorescein diacetate (FDA). (H) Developing seeds after pollination of a control plant.

In the second experiment (Table 2), 45 plants of breeding population MX-CBD-707 were treated for male sex induction. After 60 days on vegetative growth, they were first exposed to three different lighting regimes (henceforth referred to as “pretreatments;” 15 plants per pretreatment), which was followed by four different treatments: spraying whole plants with STS (Green, 2005), spraying whole plants with colloidal silver once, or every day until anthesis (recommendation of grower), and control (non-treated plants).

Table 2. Combinations of pretreatments, treatments, and growing photoperiods used in the second experiment of male flowers induction.

After the application of silver solutions, the plants from almost all combinations of pretreatment and treatment were exposed to a 12/12 light/dark regime to induce flowering. After application of 0.3 mM STS on whole plants, a stress-inducing photoperiod with 96 h of light and 72 h of the dark was tested and compared with the results of the same STS treatment followed by a normal 12-h photoperiod.

Measurements of Response Variables

In the first experiment, five different response variables were analyzed, namely:

(2) A number of nodes per plant, both expressed as a ratio between the final state (measurement/count at the end of the experiment) and the initial state (counted at the start of the experiment prior to (pre)treatments). In this way, not only the final morphology of the plants was taken into account, but also the initial state of the plants.

(3) Number of all inflorescences per plant.

(4) The number of inflorescences with one male flower or more per plant.

(5) The number of male flowers per plant. Variables were counted 31 days after the beginning of the experiment.

In the second experiment, the number of male flowers on breeding population MX-CBD-707 was counted 37 days after the beginning of the experiment.

Viability and Germination of Pollen

Several tests were performed to verify the viability of pollen developed in induced male flowers. Pollen was stained with FDA at a final concentration of 1 μg/ml and analyzed under an epi-fluorescent microscope (Nikon Eclipse 80i) with filter sets for the detection of green fluorescence. The germination of pollen was first tested in vitro on solidified germination medium composed of 170 g/l sucrose, 0.1 g/l H3BO3, 0.432 g/l Ca(NO3)2 ∗ 4H2O, and pH 7.0. The Petri dishes were incubated in the dark at room temperature for 24 h and the results were detected under the microscope. Furthermore, in vivo germination of pollen was tested by pollinating female flowers of control, not treated, and plants. The stigmas of pollinated female flowers were collected after 24 h, stained with 1% aniline blue in 0.1 N Na3PO4, as described by Murovec and Bohanec (2013), and analyzed under the epi-fluorescent microscope with filter sets for the detection of blue fluorescence. Finally, pollen from some of the treatments was used for pollination of female control plants, and the number of developing seeds was counted 2 weeks after pollination.

Statistical Analysis

Both sex induction experiments were performed once and analyzed as a two-factorial experiment, where, the main effect of factors and their interaction was statistically quantified using ANOVA. Before analysis, each response variable was tested for assumptions about normal distribution and homogeneity of the treatment variances by Levene’s test. In the case of non-homogeneity of variances, data were transformed to sqrt(y). Significant differences in mean values indicated by ANOVA were evaluated using Tukey’s test (α = 0.05). All statistical analyses were performed using the agricolae package in the statistical software program R version 3.2.5 (R Core Team, 2019). Data are presented as untransformed means ± SE. Graphs were drawn in the Microsoft Excel program.

Implementation of Sex Manipulation for Breeding Medical Cannabis

In order to verify the usefulness of our approach for breeding medical cannabis, we first analyzed the cannabinoid content in inflorescences of 48 mother plants of breeding population MX-CBD-707. The high performance liquid chromatography (HPLC) analysis was performed as described by Gul et al. (2015), with modifications described in Laznik et al. (2020).

Based on the results, 23 mother plants with high total CBD (tCBD) and low total THC (tTHC) content with a ratio of tCBD:tTHC > 13 were selected for further breeding. From each selected mother plant, two clones were produced and cultured under vegetative conditions in separate chambers. One clone per mother plant was exposed to flowering conditions of light and fertilization and was sprayed with 30 ppm colloidal silver every day until the appearance of the male flower. The other clone was exposed to a flowering regime without any treatment in order to stimulate female flowering. The masculinized and non-treated plants were joined in the same flowering room upon the occurrence of male flowers on treated plants and left to cross-pollinate due to forced ventilation in the flowering chamber.

Mature seeds were collected, soaked in water for 12 h in the dark at room temperature, and then sown in polystyrene plates with 84 holes in the substrate Kekkila (Finland). The polystyrene plates were incubated at 25°C with a photoperiod of 16/8 days/nights and 60% humidity. The emerged seedlings were clonally propagated and the clones of 74 genetically different seedlings were analyzed for their cannabinoid content in inflorescences as described above. Plants from this breeding experiment were grown in the vegetative and flowering stages like the other plants in this study (described in section “Plant Material and Growing Conditions”).

Results

Experiment 1

Silver Thiosulfate Negatively Effects the Growth of Plants and Morphology

On the plants from treatment 1 (sprayed with 20 mM STS), yellow spots on the leaves were observed 1 week after application, and then the spots started to dry (Figure 1B). The plants began to lose leaves after 3 weeks of flowering. The plants from treatment 2 (sprayed with 0.7 mM STS) had fewer yellowish spots and dry leaves. Their growth and development were not as inhibited as those of plants from treatment 1.

Treatments 3, 4, and 5 (application of 50, 100, and 150 μg STS on shoot tip, respectively) also caused some physiological responses. Three weeks after application, the young leaves, which were not fully developed at the time of treatment, began to show injuries and deformations. The leaves began to dry throughout the plant, not only at the shoot tip, where STS was applied. The intensity of these injuries coincided with the amount of STS applied at the shoot tip. The higher the amount of STS applied to the shoot tip, the more severe effect it had to plant morphology and fitness. Plants from treatment 6 (sprayed with 0.01% GA) began to grow in length and intensive elongation of internodes was observed.

Male inflorescences began to appear 3 weeks after treating female plants. They were first observed in treatment 1 (20 mM STS, sprayed), followed by the appearance of male flowers on plants from treatments 2 (0.7 mM STS, sprayed), 5, 4, and 3 (application of 150, 100, and 50 μg STS on shoot tip, respectively) at intervals of 3 days as the treatments are listed. Male flowers began to open 4 weeks after treating the plants and pollen began to spread (Figure 1C). On the plants from treatments 6 (GA3), 7 (cut), and the control, only a few male flowers were observed. Plants from all treatments developed female flowers as well. No hermaphrodite flowers (i.e., pistillate flowers containing also anthers) were observed.

Different Treatments Induced the Formation of Male Flowers on Female Plants

The breeding population and the treatment had a statistically significant influence on the ratio of plants height, the ratio of the number of nodes, number of all inflorescences, and number of male inflorescences with only one exception (Table 3). No interaction between breeding population and treatment was found for mentioned variables.

Table 3. Influence of breeding population and treatment on the ratio of plants height, the ratio of the number of nodes, number of all inflorescences, and number of male inflorescences.

The Number of Male Flowers Was Influenced by the Interaction Effect Between Both Factors

Statistically significant interaction (p = 0.0393) was found between main factors for the number of male flowers per plant (Figure 2). The highest number of male flowers (for both breeding populations) was observed after treatments 1 and 2 (sprayed with 20 and 0.7 mM STS), followed by treatments 3, 4, and 5 (application 50, 100, and 150 μg STS on shoot tip, respectively). The last three treatments (6 – GA3, 7 – cut, and control) produced a significantly lower number of male flowers. In all the eight tested treatments, the breeding population MX-CBD-11 developed a higher number of male flowers compared with MX-CBD-707 (Figure 2).

Figure 2. The number of induced male flowers per plant is indicated by the interaction effect between breeding population and treatment. Mean values followed by different letters are significantly different at the 5% level of probability (Tukey). Horizontal bars represent SE (± SE).

Experiment 2

Colloidal Silver Induced Formation of Fertile Male Flowers on Female Plants

In the second experiment, the effect of different pretreatments (168 h light, 168 h dark, and alternation of 18/6 light/dark) before application of 0.3 mM STS or 30 ppm colloidal silver was studied. After spraying the whole plants, they were exposed to a constant 12/12 light/dark photoperiod or to a stress-inducing photoperiod (one treatment). Pretreatment, as well as treatment, had a statistically significant influence on the number of male flowers, but their interaction was not observed (Table 4). The highest average number of male flowers per plant (339) was achieved after pretreating plants at the usual light regime for vegetative growth (18 h of light and 6 h of darkness), while incubation in darkness for 168 h caused the lowest appearance of male flowers. Among the tested treatments, 0.3 mM STS caused the highest formation of male flowers, followed by the same treatment and exposing plants to stress-inducing light regimes. In contrast to spraying of 0.3 mM STS, which induces male flowering after only one application, the colloidal silver had to be sprayed every day until the formation of male flowers and yielded on an average 293 male flowers per plant. Spraying plants with colloidal silver only once produced a negligible number of male flowers, the results being practically equal to the results of control plants, which were not sprayed with any silver solutions (Tables 4, 5). The appearance of induced male flowers and the viability of pollen are shown in Figures 1F,G.

Table 4. Influence of pretreatment and treatment on a number of male flowers per plant of MX-CBD-707.

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Table 5. Number of male flowers per plant and number of germinated pollen cells in vitro after the induction of male flowering on female plants of MX-CBD-707.

Pollen Successfully Germinated in vitro and in vivo

The in vitro germination test showed that the induced male flowers produced viable pollen that is able to germinate in vitro on solidified germination medium (Figure 1E and Table 5).

The germination ability of pollen was confirmed also with in vivo pollination of female flowers. After 24 h, the germinating pollen tubes were clearly visible on stigmas stained with aniline blue (Figure 1D).

In order to verify the ability of pollen to fertilize female flowers and produce feminized seeds, the pollen was collected from treated plants of breeding population MX-CBD-707 and used for pollination of different shoots of one control (non-treated) plant. Two weeks after pollination, the number of developing feminized seeds was counted, which is presented in Table 6 and Figure 1H.

Table 6. Number of developing seeds 2 weeks after pollination with pollen from plants MX-CBD-707 induced with different light (pre)treatments and silver applications.

Breeding MX-CBD-707

Analysis of cannabinoid profile revealed that 48 plants of MX-CBD-707 contained between 3.47 and 11.70% and 0.41 to 9.91% of tCBD and tTHC, respectively. The ratios between tCBD and tTHC thus varied between 0.9 and 18.8, which represents an almost 21 fold difference. In order to stabilize CBD extraction from female flowers, we selected 23 plants with tCBD to tTHC ratios above 13.

These mother plants were cloned, induced to produce male flowers by spraying them with 30 ppm colloidal silver every day, and left to cross-pollinate in a contained flowering chamber. The colloidal silver treatment was chosen based on our results obtained in the above described experiments, which demonstrated the best performance in terms of the number of in vitro germinated pollen grains and of the number of developing seeds (Tables 5, 6). Seeds were left on plants until maturity when they were sown, and a 64.3% germination rate was recorded. The cannabinoid analysis of 74 seedlings showed that their flowers contained from 1.77 to 24.34% and 0.09 to 0.85% of tCBD and tTHC, respectively. The ratio between tCBD and tTHC varied between 13.26 and 29.58 (Figure 3).

Figure 3. Distribution of the ratios between tCBD and tTHC in 48 mother plants of breeding population MX-CBD-707 (left-blue) and 74 of their progeny (right-green).

Discussion

Alteration of the reproduction system in cannabis, in the form of the appearance of male flowers on female plants, is a useful phenomenon in cannabis breeding. It enables self-pollination and/or crossing plants that are genetically female. Moreover, it leads to offspring seeds that are entirely feminized. As such, they are highly valuable in medical cannabis production, which relies exclusively on phenotypically female plants (Soler et al., 2017).

Ethylene is a known gaseous plant hormone, which is involved in sex expression in plants. It promotes femaleness and inhibitors of ethylene biosynthesis or ethylene response suppress the development of female reproductive organs, thus promoting masculinity (Kumar et al., 2009). The mode of action was partly elucidated recently in cucumber and melon, where, Tao et al. (2018) demonstrated that ethylene signaling is directly involved in interaction among sex determination-related genes by controlling ethylene-responsive transcription factors CsERF110 and CmERF110 (Tao et al., 2018). Silver ions from STS and colloidal silver act as ethylene antagonists, thus blocking its function, and in this way probably enable male sex induction (Ram and Sett, 1982). Ram and Sett (1982) showed for the first time that STS is capable of male sex induction on wild accession of C. sativa L. They also discovered that STS was more efficient compared to AgNO3, probably due to the faster transport of STS through plants. Recently, Adal et al. (2021) used STS for the chemical induction of male flowers on female plants. They identified over 10,500 differentially expressed genes, of which, around 200 are potentially responsible for male flower development on female plants. Their study confirmed that sex determination in cannabis flowers is controlled primarily at the genetic level. However, the expressed genes appeared to be involved in several pathways, such as phytohormone signaling, floral development, metabolism of lipids, sugar, and others, implying that the process of sex expression in cannabis plants occurs at multiple levels.

In our experiment, 23 different treatments (chemical using STS or colloidal silver; hormonal using GA; and physiological by intensive cutting) were used for induction of male flowers on female plants, in order to evaluate their influence on sex expression in medical cannabis. Plant height and number of nodes, which assessed the influence of treatments on plant growth, showed no negative influence as a result of our treatments. Although Ram and Sett (1982) reported that after application of STS on shoot tips the treated plants became black, the young leaves became decolorized, wilted, and deformed, etc., less pronounced effects of STS on plant growth and morphology were observed in our study. Even the highest amount of STS added on the shoot tip in our research (150 μg) had no negative influence on plant growth (Table 3), while application of 100 μg on the shoot tip in the study of Ram and Sett (1982) caused a total collapse of the shoot tip, decreased leaf area, and reduced plant growth in height after treatment. The number of male flowers per plant in our study was similar to the number they counted for the same treatment (application of 100 μg; up to 110 male flowers), but our research also showed that applying STS to whole plants is more efficient than the application of STS to the shoot tips.

The interaction effect between genotype and treatment on a number of male flowers in this study proved that genotype affects the success of male flower induction. Overall, we observed a higher number of male flowers developed per plant compared with the study of Ram and Sett (1982), who treated fiber-type hemp plants; while in our experiment, plants of medical cannabis were used. Besides, we applied STS by spraying whole plants, where Ram and Sett (1982) add STS to shoot tip only and this could also be the reason for the obtained variation. Differences in maleness induction were also observed by comparing our results with Lubell and Brand (2018), who used STS for the induction of male flowers on female hemp plants. Although the number of male flowers in their study was not exactly counted, they determined up to ≈85% of inflorescences with male flowers, where in our study, only approximately 55% of inflorescences contained male flowers. Lubell and Brand (2018) found that one (out of four tested) hemp strain was more prone to sex conversion and exhibited a higher level of masculinization. The phenomenon of genotype dependency on sex induction was also observed by Moliterni et al. (2004), who noticed that European hemp varieties exhibit different stages of resistance to sex reversion treatments.

The assumption about genotype specificity for sex reversion was confirmed by the results of our first experiment, in which breeding population MX-CBD-11 outperformed MX-CBD-707 in terms of male flower production after all seven different treatments. Since the treatments were identical for both breeding populations and performed simultaneously under the same flowering conditions, the results clearly demonstrate genotype dependency of physio-morphologic response to silver compounds.

Comparison of pollen from naturally male hemp plants and masculinized female ones showed that the latter produce a significantly higher number of irregular or misshapen pollen grains that are inefficient in dispersal from anthers and had a lower germination rate (DiMatteo et al., 2020). In our study, a comparison between pollen from masculinized female plants and male plants was not possible, because the medical breeding populations contained exclusively female plants. We, therefore, decided to test pollen viability and its germinability in vitro and in vivo and, finally, evaluated seed set after pollination of female plants with pollen from masculinized plants. Our results (Figure 1 and Tables 5, 6) confirmed the viability and functionality of the pollen developed on masculinized female plants. It demonstrated that colloidal silver is also very efficient for the induction of male flowers (as reported in Table 5), and this is the first report about the induction of male flowers on female plants of cannabis with colloidal silver.

On the other hand, spraying with hormone GA3 had no significant effects on male flower induction in our medical cannabis plants, as was also shown by Sarath and Ram (1978). Although, Chailakhyan (1979) demonstrated that GA3 has a strong effect on the appearance of male flowers, they applied the hormone through roots of very young cannabis plants, which is impractical for breeding purposes. In addition to chemical and hormonal triggers, physiological stress caused by mechanical damage was also expected to increase the likelihood of sex reversion (Clarke, 1999). However, intensive cut, which was one of our treatments, did not produce positive results in terms of induction of male flowers.

Some male flowers appeared on the control plants, where no staminate flowers were expected. It is possible that unwanted drift of STS from treated plants to the control plants occurred in the growing room since strong ventilation was used during growth. On the other hand, it has been recently shown by Punja and Holmes (2020) that male flowers can form spontaneously on up to 10% of female plants, therefore, the possibility of unwanted spontaneous sex conversion cannot be excluded.

Finally, the protocol using colloidal silver was successfully used for breeding female plants of medical cannabis breeding population MX-CBD-707. Entirely, the female progeny was obtained after crossing the parental population of females induced to form male flowers, thus confirming the theory of producing exclusively feminized seeds when crossing only XX plants (Green, 2005). Furthermore, a significant improvement of tCBD/tTHC ratio was observed (with a maximal tCBD content of 29.58% measured in a plant with a tTHC as low as 0.82%) after crossing only 23 selected parental plants. It shows that crossing plants selected based on their chemotype profile improves the genetic constitution of the breeding population and consequently enables the development of new varieties with improved cannabinoid profiles.

Conclusion

To the best of our knowledge, this is the first comprehensive scientific report on the induction of fertile male flowers on female plants of medical cannabis (Cannabis sativa L.). Previous reports on gender manipulation in cannabis were performed on fiber-hemp genotypes (Ram and Sett, 1982; Lubell and Brand, 2018; DiMatteo et al., 2020), while methods for medical cannabis have been shared among growers for years, but the information was not obtained in a manner based on scientific methodologies.

Data Availability Statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Author Contributions

MF and JM conceived and designed the study and wrote the manuscript. MS performed the experiments. MF, MS, and JM analyzed the data. All authors read and approved the manuscript.

Funding

This work was carried out in the framework of the scientific-research project “Breeding medical cannabis (Cannabis sativa L.)” in collaboration between the Biotechnical Faculty of the University of Ljubljana, Slovenia, and MGC Pharmaceuticals Ltd. This study received funding from MGC Pharmaceuticals Ltd. The funder was not involved in the study design, collection, analysis, and interpretation of data, the writing of this article, or the decision to submit it for publication. The research was also supported by research programme P4-0077 and infrastructural centre IC RRC-AG (IO-0022-0481-001) of the Slovenian Research Agency.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank Sinja Svetik, Špela Mestinšek Mubi, Liza Zavrl (from the Biotechnical Faculty), and Irena Pribošič (from MGC Pharmaceuticals Ltd.) for their technical assistance during the experiments.

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Keywords : Cannabis sativa L., sex manipulation, silver thiosulfate, cannabidiol, high CBD medical cannabis, feminized seed, cannabinoids

Citation: Flajšman M, Slapnik M and Murovec J (2021) Production of Feminized Seeds of High CBD Cannabis sativa L. by Manipulation of Sex Expression and Its Application to Breeding. Front. Plant Sci. 12:718092. doi: 10.3389/fpls.2021.718092

Received: 31 May 2021; Accepted: 30 September 2021;
Published: 01 November 2021.

Zamir Punja, Simon Fraser University, Canada
Ayelign M. Adal, University of British Columbia Okanagan, Canada
Jordi Petit Pedró, Polytechnic University of Valencia, Spain

Copyright © 2021 Flajšman, Slapnik and Murovec. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

This article is part of the Research Topic

Behind the Smoke and Mirrors: Reflections on Improving Cannabis Production and Investigating Medical Potential

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