Feminized Seeds in Cannabis – Genetics, Production and Limits

Feminized seeds are seeds that grow into female plants because the pollen that sired them already came from a female plant. A female cannabis plant does not form pollen on its own. It is made to set male flowers with a spray solution, and the most common agent for this is silver thiosulfate, STS for short.

How well that can work is shown by a trial on four hemp cultivars: after three applications one week apart, 95 to 100 % of the flowers on the inflorescences at the shoot tips were male. Anyone who puts this pollen on female flowers harvests feminized seed. Whether there is enough usable pollen depends on the cultivar, though, and timing matters as well. The sections below go through agents, preparation, timing and choice of plant; a calculator takes care of weights and dates.

Why the offspring turn out female

Female cannabis plants carry two X chromosomes, males one X and one Y. Chromosomes carry the genetic material in the cell nucleus, and in cannabis this pair decides the sex. When a genetically female plant forms pollen, every pollen grain can pass on only an X. If it reaches a female flower, the result is again a seed with two X. In the trials this article draws on, all tested offspring of such pollinations were female; in one of them this was confirmed on the seedlings by PCR, a genetic test that detects a male-specific stretch of the genome.

The same thing happens unplanned when a stressed female plant turns hermaphrodite, forms anthers of its own, the organs in which pollen ripens, and pollinates itself. Seed from such flowers gave only genetically female plants in that trial, while seed from a cross with a male gave roughly half male plants in two lines.

“Feminized” concerns only the sex of the offspring. It has nothing to do with “autoflowering”, flowering by age rather than by day length; the two traits occur independently of each other.

What makes a female plant form pollen

Behind the sex of the flowers lies ethylene, a gaseous signalling substance of the plant. It promotes female flowers. If its action is blocked, a genetically female plant also sets male flowers, and most agents rely on this.

Silver thiosulfate forms when silver nitrate, a silver salt, is mixed with sodium thiosulfate in a ratio of one to four. Almost all of the silver is then held in a dissolved complex in which one silver particle is bound to two thiosulfate particles. In this form the silver moves readily in the vascular system, the vessels through which the plant distributes water and nutrients; plain silver nitrate hardly moves there. Where it acts has been studied in thale cress, the model plant of botany. The receptor, the binding site at which the plant recognises ethylene, needs a copper particle there, and silver can take its place. Ethylene still binds, but the signal is not passed on. Whether cannabis works the same way has not been measured.

The other agents worked less well in the trials or harmed the plant. In a comparison on one cultivar STS came out best, ahead of AVG, a substance that blocks the formation of ethylene, and well ahead of cobalt nitrate and 1-MCP, a gas that acts at the same receptor. Colloidal silver, finely dispersed silver particles in water, produced male flowers at 30 ppm (parts per million) only when sprayed daily until flowering; a single application had no effect. AVG worked as a drop on the shoot tip, where in one trial it produced 132 male flowers in total, but hardly as a spray, and at higher doses the tips turned yellow. 1-MCP did not produce a single male flower in two trials.

Gibberellic acid, GA3 for short, a growth hormone, is the oldest route. In a trial from 1972, 50 µg per plant, spread over ten days and given through a wick at the shoot tip, induced an average of 33.8 male flowers per plant; a week later the plants were already forming female flowers again. Sprayed at 0.01 % it had no effect at all in another trial. Where it works it comes at a price: in a further study that applied it at the switch to short days, the inflorescences grew stretched and loose, and their THC and CBD content fell. The authors of that study therefore regard the route as largely replaced by STS in practice.

Preparing and spraying the STS solution

STS is made from two stock solutions that are later only diluted. The concentration is given in millimoles per litre, mM for short, an amount by number of particles: 1 mM of silver is, by calculation, 108 mg of silver in one litre, roughly what four to five grains of rice weigh. For a 0.1 molar silver nitrate solution, that is 100 mM, 1.7 g are dissolved in 100 ml of water. The same strength of sodium thiosulfate takes 1.58 g of the anhydrous salt or, by calculation, 2.48 g of the pentahydrate, a form with bound water of crystallisation that contains less thiosulfate per gram. Keep both solutions in the dark until use.

Then pour 20 ml of the silver solution slowly into 80 ml of the thiosulfate solution, in that order. This gives 100 ml of stock solution at 20 mM. It keeps for up to a month in the refrigerator, but preparing it fresh is recommended. For one litre of spray solution at 3 mM, 150 ml of this stock is made up to one litre with water; the spray solution is thus 15 % stock.

At 3 mM, applied three times one week apart, 95 to 100 % of the flowers at the shoot tips were male in the trial mentioned above. At a tenth of that, 0.3 mM, the share was only 42 to 91 %. In another trial a single application of 1.5 mM was enough for three cultivars if it came early; even there three applications worked better than one. More brings nothing: 20 mM caused yellow spots and leaf loss. Too little does not work at all. A treatment with 100 µg of STS per plant in total did not produce a single male flower in one trial; by calculation that is less than a thousandth of the silver in a single application of 400 ml at 3 mM.

Spray until the solution drips off the leaves. For plants 1 to 1.5 m tall that came to about 400 ml per plant and application. Spraying the whole plant worked better than treating only the shoot tips. Anyone who wants to convert a single branch should know that the agent moves: treating only the tips already caused leaf damage across the whole plant. Whether the untreated branches then also set male flowers has not been measured in any trial.

STS solution calculator

The calculator shows how much silver nitrate and sodium thiosulfate a spray solution of the chosen concentration needs, diluted from the stock solution or weighed in directly, and sets out the application dates and the approximate timing of pollen and seed.

Inputs

In the trials 1.5 to 3 mM worked; 0.3 mM worked only in part, 20 mM damaged the leaves.
Spray until it drips off; plants 1 to 1.5 m tall took about 400 ml.
Preparation
Sodium thiosulfate as

Result

    Dates

      Handle and store silver nitrate and sodium thiosulfate according to the safety data sheet of the product.

      Values calculated from the molar masses: silver nitrate 169.87 g/mol, sodium thiosulfate 158.11 g/mol, as pentahydrate 248.18 g/mol. Stock solution after the Sigma-Aldrich protocol: 20 ml of 0.1 M silver nitrate in 80 ml of 0.1 M sodium thiosulfate give 100 ml at 20 mM.

      Timing, indoors and outdoors

      What counts is the plant’s stage of development. Indoors, flowering begins on the day the timer is switched to short days, to uninterrupted darkness of twelve hours or more. Treated early in short days, the conversion was reliable. A single application in mid-flowering was not enough in one trial, and even three applications at that point produced a few male flowers in only 26 to 57 % of the plants. A review even recommends spraying while still in the vegetative phase and keeping the plants under long days for one more week before short days begin.

      Outdoors there is no such switching day. After the summer solstice the nights grow a little longer every day, and each cultivar switches at a different point, as the article on flowering induction describes. The change stretches over days. In a growth-chamber trial an inflorescence began in more than half of the plants only after three short days in a row, and back under long days it broke up again. The start can be seen at the shoot tip: three or more pairs of stigmas, the white hairs of the female flower, stand close together there; this is how the chamber trial defined a visible inflorescence. Single flowers in the leaf axils do not count, as an older plant forms them under long days too.

      The first application therefore belongs at the start of this change or shortly before, with the other two following one week apart. No field trial has matched the applications to this gradual transition; the rule is derived from the indoor trials. For cultivars that switch outdoors shortly after the summer solstice, the applications fall in July. Since about four weeks pass from the first spray to pollen and another three to four from pollination to ripe seed, the seed of such a cultivar ripens, by calculation, between late August and mid-September.

      Donor plant, pollen and pollination

      The plant that is to supply the pollen must be reliably female. A review advises letting it flower beforehand and checking at every node that only female flowers appear, or confirming its sex by a genetic test. Beyond that, its health counts, along with the traits it is meant to pass on.

      How much pollen a plant delivers after treatment depends strongly on the cultivar. Of five treated hemp cultivars, one delivered pollen in amounts similar to a true male, three fell in between and one formed none. The usable share is poorer as well: 37 to 83 % of the grains from the treated plants were misshapen, none from the males. In a germination test on culture medium 0 to 13 % of the grains sprouted, against 10 and 19 % for the two males. Anyone treating a cultivar for the first time should therefore make a trial run.

      Male flowers appeared about three weeks after the first application, pollen after about four. Under short days the converted flowers opened after about 25 days, those of true males after 18. Many converted flowers do not open at all or release their pollen only reluctantly. Pollen is collected from the open flowers, in one of the trials by tapping them gently over a sheet of paper.

      If the pollen goes onto the same plant or onto a cutting of it, a genetically identical copy, that is selfing, and the seeds form the S1 generation. If it goes onto another female plant, the result is a cross whose offspring are female as well. From self-pollinated hermaphrodite flowers, ripe seed was present three to four weeks after pollination, 22 to 48 per inflorescence, and it germinated at 90 to 95 %. Seed from the pollen of STS-treated plants germinated at 64.3 % in another trial, a little under two in three.

      Feminized does not automatically mean stable

      Feminized seed says nothing about the tendency to turn hermaphrodite. Whether a plant forms anthers under stress is, as far as is known today, partly heritable. What has been measured is what continued selfing can do: in a line whose generations were grown side by side, the share of abnormal flowers rose from 0.4 % in the starting generation to 13.7 % in the sixth selfed generation, more than thirtyfold. Anyone who works over several generations only with pollen from treated plants is therefore also selecting for the ability to convert.

      For a single S1 generation there is no measurement of this. One trial found no measurable loss of genetic diversity after one round of selfing. The quality of the seed thus depends on the Breeding Stability of the parents, which can be checked beforehand with a stress test.

      Sources