Feminized Seeds: Production and STS Calculator

Feminized seeds grow into female plants. The pollen that sired them came from a female plant itself. A female does not form pollen on her own, though. She is usually sprayed with silver thiosulfate, STS for short. In a trial on four hemp cultivars, three applications made 95 to 100 % of the flowers at the shoot tips male. Shoot tips are the uppermost ends of the shoots.

Why do the offspring turn out female?

Because every pollen grain of a female plant passes on only an X chromosome. Chromosomes carry the genetic material in the cell nucleus. Female plants have two X, males one X and one Y. If such a pollen grain reaches a female flower, the result is again a seed with two X. In the trials, all tested offspring of such pollinations were female. In one trial this was confirmed on the seedlings by PCR. This genetic test detects a male-specific stretch of the genome.

The same thing happens unplanned when a female plant turns hermaphrodite. She forms anthers of her own and pollinates herself. Anthers are the organs in which pollen ripens. Seed from such flowers gave only genetically female plants in the trial. Seed from a cross with a male gave roughly half males in two lines. “Feminized” has nothing to do with “autoflowering”. Autoflowering means flowering by age rather than by day length.

Choose feminized seed if you want no males. Hermaphrodites remain possible.
With autoflower cultivars, check both traits separately. The two traits occur independently of each other.

What makes a female plant form pollen?

Usually an agent that blocks the action of ethylene. Ethylene is a gaseous signalling substance of the plant and promotes female flowers. Without its action, a genetic female also sets male flowers. STS forms from the silver salt silver nitrate and the salt sodium thiosulfate in a ratio of one to four. Almost all of the silver is then dissolved, each silver particle bound to two thiosulfate particles. In this form it moves readily in the vessels through which the plant distributes water. Plain silver nitrate hardly moves there. Where it acts has been studied in thale cress, the model plant of botany. There the plant recognises ethylene at a binding site, the receptor. Silver takes the place of copper in this receptor. Ethylene still binds, but the signal is not passed on. This has not been measured in cannabis.

The other agents worked less well or harmed the plant. In a comparison on one cultivar STS came first, ahead of AVG. Cobalt nitrate, a metal salt, and 1-MCP came well behind. AVG blocks the formation of ethylene. As a drop on the shoot tip it produced 132 male flowers in one trial, as a spray hardly any. At higher doses the tips turned yellow. 1-MCP is a gas that acts at the same receptor. In two trials it did not produce a single male flower. Colloidal silver consists of finely dispersed silver particles in water. At 30 ppm, parts per million, it worked only when sprayed daily until flowering. A single application had no effect. The growth hormone gibberellic acid, GA3 for short, is the oldest route. In 1972, 50 µg per plant given through a wick over ten days induced 33.8 male flowers on average. A week later the plant was already forming female flowers again. Sprayed at 0.01 %, GA3 had no effect at all in another trial. Short days means twelve hours of darkness or more, long days correspondingly shorter nights. Given at the switch to short days in a further trial, it made the inflorescences stretched and loose. THC and CBD, the two main active compounds, fell as well.

Choose STS as the agent.
Use colloidal silver only if you can spray daily until flowering.
Skip GA3 and 1-MCP. In the view of the authors of one study, GA3 is largely replaced by STS in practice.

How do you prepare and spray the STS solution?

You mix STS yourself from two salts sold by laboratory or chemical suppliers. The spray is a 3 mM solution, applied three times one week apart. Concentrations are given in millimoles per litre, mM for short. 0.1 M is 100 mM. One plant with three applications of 400 ml each needs 180 ml of stock solution, so two batches. One batch of 100 ml makes, by calculation, a little over 660 ml of spray. Following the recipe below, you buy 1.7 g of silver nitrate for this. Add 3.16 g of anhydrous sodium thiosulfate or 4.96 g of sodium thiosulfate pentahydrate. By calculation only 0.61 g of silver nitrate and 2.28 g or 3.57 g of sodium thiosulfate end up in the spray. The calculator below works out other amounts. The shopping list:

Silver nitrate (AgNO₃).
Sodium thiosulfate (Na₂S₂O₃), anhydrous or as the pentahydrate (Na₂S₂O₃ · 5 H₂O). The label says which form is in the container. The pentahydrate contains water of crystallisation and supplies less thiosulfate per gram.
Distilled water, as in the trials.
A wetting agent, so that the solution stays on the leaves. One trial used 0.1 % Tween 20.
A precision scale reading to 0.01 g, a measuring cylinder, two dark bottles and a spray bottle.

Solution A, silver nitrate 0.1 M: dissolve 1.7 g of silver nitrate in distilled water and make up to 100 ml.
Solution B, sodium thiosulfate 0.1 M: dissolve 3.16 g anhydrous or 4.96 g pentahydrate and make up to 200 ml. That covers two batches of stock solution.
Keep both solutions in the dark until use.
Stock solution, 20 mM STS: pour 20 ml of solution A slowly into 80 ml of solution B, in that order. This gives 100 ml. Make two batches for three applications of 400 ml. It keeps for up to a month in the refrigerator. Preparing it fresh is recommended.
Spray solution, 3 mM: make up 150 ml of stock solution to 1 l with distilled water and add the wetting agent.

In the trial mentioned above, 3 mM in three applications made 95 to 100 % of the tip flowers male. A tenth of that, 0.3 mM, gave only 42 to 91 %. In another trial a single early application of 1.5 mM was enough for three cultivars. Three applications worked better there too. On three high-THC cultivars, 3 mM in three applications made 80.5 to 94.7 % of the flowers change sex. The first application came at the switch to short days. More brings nothing: 20 mM caused yellow spots and leaf loss. Too little does not work at all. A total of 100 µg of STS per plant 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 application.
Spray right after the lights go off, so the leaves do not burn. Outdoors this corresponds to dusk, which has not been tested in the field.
Spray the whole plant. That worked better than treating only the shoot tips.
Even partial treatment acts on the whole plant. Treating only the shoot tips already caused leaf damage across the whole plant.
For self-pollination you can treat a single branch. In a breeding programme only one branch was sprayed, three times two days apart at 6 mM. Whether the other branches then flower male has not been measured.

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

Concentration (mM) In the trials 1.5 to 3 mM worked; 0.3 mM worked only in part, 20 mM damaged the leaves.

Spray solution per plant and application (ml) Spray until it drips off; plants 1 to 1.5 m tall took about 400 ml.

Plants

Applications

Interval between applications (days)

First application on

Preparationfrom stock solution weigh in directly

Sodium thiosulfate asanhydrous salt pentahydrate

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.

When do you spray, indoors and outdoors?

Early in flowering. Indoors it begins on the day the timer switches to short days. Short days means twelve hours or more of uninterrupted darkness. Treated early in short days, sex reversal was reliable. In mid-flowering a single application was not enough in one trial. Even three applications then gave a few male flowers in only 26 to 57 % of the plants. In a review, spraying is recommended already in the vegetative phase. The plants then stay under long days for one more week.

Outdoors there is no switching day. After the summer solstice the nights grow a little longer every day. Flowering induction is the plant’s transition into flower. When it sets in depends on the cultivar. The transition 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. Back under long days it broke up again. The start shows at the shoot tip: three or more pairs of stigmas stand close together there. Stigmas are the white hairs of the female flower. Single flowers in the leaf axils do not count. An older plant forms them under long days too.

Indoors, give the first application on the day of the switch to short days, the other two one week apart.
Or, following the review, spray in the vegetative phase and then give one more week of long days.
Outdoors, place the first application at the start of the transition or shortly before. No field trial exists, the rule comes from indoor trials.
For cultivars that switch shortly after the summer solstice, plan the applications for July.
Allow about four weeks from the first spray to pollen, then another three to four to ripe seed. Seed ripening was measured on self-pollinated hermaphrodite flowers.
Outdoors the seed of such cultivars then ripens between the second half of August and mid-September by calculation.

Which plant makes a good donor?

A reliably female plant that shows no male flowers untreated. STS works however prone a plant is. So the treatment does not show whether the donor tends to form male flowers on her own. That tendency is inherited. Daughters of females with male flowers formed them more often. In two trials it was 34 % instead of 23 % and 13.7 % instead of 4.9 % of females.

A feminized line is not automatically stable either. “Feminized” concerns only the sex of the offspring, not the tendency to turn hermaphrodite. In a breeding programme, 16 accessions were selfed over up to six generations. From the fourth generation on, abnormal flowers appeared in all lines. These included flowers with organs of both sexes or flowers in the wrong place. Fewer seeds formed as well. In one line with seven generations side by side, the share of abnormal flowers rose from 0.4 % to 13.7 %. That is more than thirtyfold. This line was monoecious and carried male and female flowers on one plant. In such lines treatment was not always needed for selfing. In lines from female plants, the treated plants often delivered very little pollen in later generations. Eight of 22 such attempts failed because of it. It is suspected that the ability to reverse sex is selected along the way and spontaneous male flowers become more frequent. This was not measured; the suspicion comes from the authors of the breeding programme. For a single S1 generation there is no measurement of the tendency to turn hermaphrodite. After one round of selfing, no measurable loss of genetic diversity turned up in one trial.

Let the donor flower untreated beforehand and check every node. Alternatively, confirm her sex by a genetic test.
Drop a plant with even one male flower as a donor. After that, health counts, along with the traits she is meant to pass on.
Do not self a line again and again over many generations.
From the fourth selfed generation on, look specifically for abnormal flowers.
Check the breeding stability of a donor beforehand, as described in the article on stress testing.

How do you collect and transfer the pollen?

You collect it from the open male flowers, about four weeks after the first application. How much pollen a treated plant delivers depends strongly on the cultivar. Of five cultivars, one delivered about as much as a true male. Three fell in between, and one formed none. Of the pollen from treated plants, 37 to 83 % of the grains were misshapen, none from the males. In a germination test on culture medium, 0 to 13 % of the grains sprouted. For two males it was 10 and 19 %. Male flowers appeared about three weeks after the first application, pollen after about four. The male flowers of treated plants opened after about 25 days, those of true males after 18. Many do not open at all or release their pollen only reluctantly.

With a new cultivar, make a trial run first.
Collect pollen from open flowers, for example by tapping them gently over a sheet of paper.
Put the pollen onto the same plant or a cutting of it, a genetically identical copy. That is selfing, and the seeds form the S1 generation.
For a cross, put the pollen onto another female plant. Its offspring are female as well.
Plan for germination: seed from STS pollen germinated at 64.3 % in one trial, a little under two in three.
Expect ripe seed from about three to four weeks after pollination. This was measured on self-pollinated hermaphrodite flowers with 22 to 48 seeds per inflorescence and 90 to 95 % germination.