RU2080056C1 - Method for selection of hemp plants - Google Patents

Abstract

FIELD: agricultural technology. SUBSTANCE: according to method, hemp plants are grown and selected in conditions of natural climate alternated with growing in conditions of artificial climate which intensifies accumulation of cannaboids in plants. At growing in conditions of artificial climate, introduced in soil are mineral fertilizers N $$$ P $$$ K $$$, moisture content of soil is maintained at level of 55-65% as of its full moisture capacity, and air temperature - 42 C at photoperiod of 11.6-12.3 h. Growing in conditions of artificial climate is carried out in autumn-winter period, and taken for growing is only part of selected material required and sufficient for its evaluation. Selected in conditions of artificial climate only that selection material in which all descendants have stable number of ferrous hairs on bracts not exceeding 8 pieces on 1 sq.mm of their surface. EFFECT: improved commercial properties. 3 cl, 10 tbl

Description

 The invention relates to agriculture, in particular to the selection of hemp plants with improved economic properties.

 A known method of selection of hemp plants, including targeted hybridization and the formation of parent plants, their multiple assessment by a set of characters and properties in breeding nurseries and differentiation of the best of them to create new varieties. To obtain selection material with a low content of cannabinoids, promising individuals are selected according to the characteristic that takes into account the number of glandular hairs on the bracts, which is inversely proportional to the number of undesired cannabinoid compounds accumulated in plants / Virovets V.G. Gorshkova L.M. Senchenko G.I. Sazhko M. M. Guidelines for hemp breeding to reduce the content of cannabinoids. M. 1985, p. 14 /.

 The disadvantage of this known method is that it is long, requires 12-15 years of breeding, which leads to significant material and labor costs.

 The technical result achieved by the implementation of the present invention is a sharp reduction in the timing of selection, material and labor costs, obtaining for 1 2 seasons of breeding material with improved economically valuable characteristics.

This is achieved by the fact that in the method of selection of hemp plants, including growing plants in a natural climate, assessing the quality of the breeding material and its selection by the number of glandular hairs on the bracts, a distinctive feature is that growing and selection in the natural climate alternate with its growing and selection under such artificial climate conditions that intensify the accumulation of cannabinoids in plants. Such artificial conditions may include the following:
the introduction of mineral fertilizers N _190-210 P _90-110 K _230-250 into the soil;
maintaining soil moisture at 55 65% of its full moisture capacity,
maintaining the air temperature at 38 42 ^o C,
the use of a photoperiod of 11.5 to 12.5 hours

 It is advisable to grow plants in an artificial climate in the autumn-winter period and at the same time grow only part of the selected breeding material, necessary and sufficient for its assessment.

In an artificial climate, that material is selected whose entire offspring has a stable amount of glandular hairs on the bracts, not exceeding 8 pieces per 1 mm ^{2 of} their surface.

 It was found that when selecting hemp breeding material only on the basis of the number of glandular hairs on the bracts, by the method according to the invention, not only cannabinoids are reduced in the selected hemp plants, but also an unexpected improvement in many other economically valuable traits. This greatly simplifies the selection process and allows you to quickly get the necessary breeding material.

 Example 1. It is known that tetrahydrocannabinol / THC /, synthesized and accumulated in hemp plants, has a high psychotomimetic activity. In 1987 1988, special reconnaissance experiments were carried out using an SUVR-1 accelerated plant growing rack to study the effect of various levels of mineral nutrition on the content of tetrahydrocannabinol in dioecious hemp plants of the Glukhovskaya 10 variety. For this purpose, pre-calibrated and prepared metal vessels of the same volume / 18x22 cm /, stuffed thoroughly mixed with the necessary fertilizers soil. Sowing of seeds was carried out in wells of a depth of 2.0 cm in 20 pieces. on the vessel. Watering the plants was carried out with tap water, based on the optimal humidity established for the experiment, according to the mass of the vessels.

 The scheme and results of the experiment are given in table 1.

As can be seen, an increase in doses and ratios of N RK is accompanied by a significant increase in the content of THC in plants. The optimal dose should be recognized as N _190-210 P _90-110 K _230-250 as contributing to the maximum accumulation of THC in hemp plants.

 Example 2. We studied the effect of soil moisture on the formation of THC in plants / table 2/.

From the data table. Figure 2 shows that under the artificial climate created with the help of the SUVR-1 installation, in all variants of the experiment, a decrease in soil moisture leads to an adequate increase in the THC content in plants. Given that at a soil moisture content of 35-45% of the total moisture capacity, a significant part of the plants die, we consider the option with a moisture content of 55 65% to be the most practical
Example 3. We studied the influence of the temperature factor on the formation of THC in plants / tab. 3/.

An analysis of the data in Table 3 shows that with increasing air temperature in the room, the synthesis of THC in plants increases. The most optimal is the air temperature equal to 35 42 ^o C.

 Example 4. We studied the effect of daylight hours on the content of THC in plants / table 4/.

 It was found that there is no significant dependence between the lighting regime and the process of THC formation in plants. However, it is important for practice that with a 12-hour photothermal regime compared with a 17-hour full biological ripeness of plants occurs on the 97th day, or 21 days earlier.

Thus, in order to enhance the process of THC synthesis in plants at the SUVR-1 installation, the following photothermal regimen is most optimal: the ratio of mineral fertilizers N _190-210 P _90-110 K _230-250 mg per 1 kg of dry soil; soil moisture 55-65% of the total moisture capacity, indoor air temperature 38-42 ^o C, photoperiod 11.5-12.5 hours of illumination of plants.

 Example 5. The crop of each elite plant was divided into two parts "halves". One of them was used for growing plants in the autumn-winter period in an artificial climate at the SUVR-1 installation, and the other was kept in reserve. Families reproduced under laboratory conditions were compared with each other by the content of tetrahydrocannabinol, while the worst were rejected, and families with a small amount of THC were left for further selection. Due to the fact that in the conditions of artificial climate it is possible to pollinate the best families with undesirable forms, the seeds of reserve “halves” were used for sowing next year. In a breeding nursery that was closed in the second year under natural conditions, the material was double-evaluated for the presence of cannabinoids: the first time before the flowering phase to regulate the hybridization process between plants of greatest interest for selection, and the second time during seed maturation for the final differentiation of phenotypically best forms with a minimum amount of THC. The results obtained were compared with the known method / table 5/.

 From the data table. Figure 5 shows that in allogamous populations, the method of reserves / "halves" /, combined with the installation of an artificial climate, leads to a significant shift in the sign of "THC content" in the right direction. Moreover, each cycle of continuous selection gives a more significant change in the trait than each cycle of family-group selection. It has been shown that almost two cannons of continuous selection are sufficient for hemp to isolate phenotypes with a homozygous state of recessive alleles responsible for the synthesis of THC from the population and to create relatively constant biotypes based on them without active cannabinoid compounds. The known method is time-consuming and takes up to 12-15 years to breed varieties with minimum indicators of the content of cannabinoid compounds.

 Example 6. A new method is as follows.

First, in the nursery of the source material in the field, in the phase of maturation of 75% of the seeds in the inflorescences, phenotypically the best elite plants are selected by visual inspection. In addition, each selected plant is labeled with its serial number and gender. In laboratory conditions, each elite plant is individually evaluated according to the degree of pubescence of the bracts with glandular hairs of the capitate-stem type on long legs with a head from yellow-orange to brown. To this end, two well-formed and developed bracts along with seeds are selected from the inflorescence of the plant with tweezers. Subsequently, using a stereoscopic microscope of the MBS-2 system at a magnification of 4x7 in reflected light, the number of glandular hairs on the dorsal side of the bract is counted on an area of 1 mm ^{2 of the} surface. The required working area on the surface of the bract is determined using an ocular mesh placed in one of the microscope’s ocular tubes, counting is done in 16 squares of the ocular mesh, which in the field of view of the microscope at this magnification limit the area of 1 mm ² on the surface of the bract. The counting data is recorded in the journal under the number corresponding to the number of the elite plant.

 The selection of plants for selection to reduce or increase the content of cannabinoids is carried out, respectively, according to a small or a large number of glandular hairs per unit surface area of the bracts, according to our proposed scale for assessing plants according to the content of THC / table.6/.

 At the next stage, the seeds of plants selected to continue breeding are divided into two parts: one half of them at the rate of 10 pcs. per vessel is used to test offspring in an artificial climatic environment at the SUVR-1 installation, and the other for field assessment.

At the SUVR-1 installation, plants are grown in metal vessels of the same volume / 18 • 22 cm /, pre-scoured and packed with soil carefully mixed with mineral fertilizers / N ₂₀₀ P ₁₀₀ K ₂₄₀ mg per 1 kg of dry soil /. Sowing of seeds is carried out in holes with a depth of 2.0 cm. Watering of vessels is carried out systematically at the same hours with tap water according to their weight until the optimum humidity is established 60.0% of the total moisture capacity. From the moment the seedlings start, the air temperature is maintained at +40 ^o C, and the photoperiod is set to 12 hours.

 In the ripening phase, 75% of the seeds in the inflorescences are counted according to the method described above for the number of glandular hairs on the bracts of all plants grown in vessels on the SUVR-1 installation.

 Table 7 shows the form of an approximate entry in the log of the results of counting glandular hairs on the bracts of plants of the inter-varietal hemp hybrid / Chuvash local x Single-house Bernburg / x YSO-31.

 From table 7 it is seen that in the first elite plant under the conditions of the optimal phytothermal regime in the offspring, the sign “THC content” practically does not change. This serves as evidence that in this case, indeed, the recessive allele that controls the development of the trait is in a homozygous state. Therefore, this elite plant is the most valuable source material for breeding.

 In the second elite plant in the offspring, a high heterogeneity and variability of the trait is noticeable: such material, of course, is subject to culling and exclusion from the selection process to reduce the content of cannabinoids.

 Based on similar analyzes of the results of evaluations, which, if necessary, can be repeated, the seeds of the remaining reserve halves of the most valuable families are ultimately used for sowing under natural conditions.

 Example 7. In 1987, hemp began breeding to create varieties without active cannabinoid compounds. For this purpose, in the field conditions in the hybrid / Chuvash local x Single-house Bernburg) x YSO-31 population, in the phase of ripening of 75% of seeds in inflorescences, 300 phenotypically best elite plants were selected by visual inspection, which were labeled with the serial number and gender.

The content of tetrahydrocannabinol (THC) in plants was judged by the degree of pubescence of their bracts with glandular head-stem type glandular hairs on long legs with a head from yellow-orange to brown according to our proposed scale / see table 6/. For this, from the inflorescence of each plant using tweezers, two developed bracts along with seeds were selected. Then, using a stereoscopic microscope MBS-2 / magnification 4x7 /, the number of glandular hairs was counted on the dorsal side of the bract on an area of 1 mm ^{2 of the} surface. The indicated working area was established using an ocular mesh placed in one of the eyepiece tubes of the microscope. The calculation was made in 16 squares of the ocular network, which in the field of view of the microscope at this magnification limit on the surface of the bract an area equal to 1 mm ² . The counting data was recorded in a journal under the number corresponding to the number of the elite plant.

The results of the evaluation of elite material selected in the field are presented in table. eight
Due to the fact that the program of our work was aimed at reducing the content of cannabinoids, from the original elite plants, only those with no more than 8 pieces on their bracts were used to continue the selection process. glandular hairs on 1 mm ² surface. There were 115 such plants, all other biotypes with a large number of glandular hairs on the bracts were rejected, and they did not participate in further breeding work to reduce the content of cannabinoids.

 The selected prospective breeding numbers / 115 plants / were then divided into two equal parts for each class: 58 of them were used to organize the breeding process in a new way, and 57 in a known way.

To reproduce 58 breeding families in the autumn-winter period, metal vessels were used in the SUVR-1 installation. In this case, the number of the vessel corresponded to the number of the breeding family. Metal vessels, identical in volume / 18x22 cm / and pre-calibrated, were stuffed with carefully mixed soil with mineral fertilizers / N ₂₀₀ P ₁₀₀ K ₂₄₀ mg per 1 kg of dry soil /. 10 pieces were sown in each vessel. seeds into wells 2.0 cm deep. Watering the vessels was carried out at the same time with tap water according to their weight until a moisture content of 60.0% of the full moisture capacity was established. From the moment seedlings started in the room with the SUVR-1 installation, the air temperature was maintained at +40 ^o C, and the photoperiod was set for 12 hours.

 The selection numbers were estimated by the number of ferruginous hairs on the bracts, which were grown on the SUVR-1 plant under the above phytothermal regime, which enhances the isolation of tetrahydrocannabinol (THC) by plants, in the phase of complete seed ripening. The most valuable were considered those breeding families in whose populations the trait “THC content” did not change upwards. The seeds of the remaining reserve halves of such families in the spring were used for sowing in vivo. In this case, in a breeding nursery, the material was double-evaluated for the presence of cannabinoids: the first time before the flowering phase to regulate the hybridization process and the second time during the seed maturation period for the final differentiation of phenotypically better forms with a minimum amount of THC.

 The experiments showed that for cannabis, two cycles of continuous selection are enough to isolate from the population phenotypes with the homozygous state of the recessive alleles responsible for the synthesis of THC, and create relatively constant biotypes based on them without active cannabinoid compounds.

 The results of breeding work are presented in table 9.

 The data table. 9 show that the proposed method for two cycles of continuous selection allows you to create a population consisting of 93.6% of plants without active cannabinoid compounds. In addition, the new method doubles the selection process, increases the accuracy, objectivity and efficiency of selection.

 Based on the proposed method, a new breeding variety of hemp of the Central Russian type Chuvash monoecious has been created, which has been in competitive variety testing since 1990 and which has high rates for many economically valuable characteristics. / Table 10 /.

 From the table. Figure 10 shows that the new variety Chuvash monoecious significantly exceeds the zoned variety YSO-14 in seed yield / by 188.6% / and fiber yield / by 2.2% /. In addition, it is characterized by a short growing season / early ripening varieties YSO-14 for 11 days / and the absence of active cannabinoid compounds.

 Thus, the proposed method improves the evaluation and selection of breeding material by growing them in the autumn-winter period at the SUVR-1 unit under the phytothermal regime that is most optimal for the synthesis of THC in plants, which ultimately significantly accelerates the pace of selection work.

Claims (4)

 1. The method of selection of hemp plants, including growing plants in a natural climate, assessing the quality of breeding material and its selection by the number of ferruginous hairs on the bracts, characterized in that growing and selection in a natural climate alternate with its growing and selection in an artificial climate intensifying the accumulation of cannabinoids in plants. 2. The method according to claim 1, characterized in that when growing in an artificial climate, mineral fertilizers N 190-210 ^P 90-110 ^K 230-250 are applied to the soil, the soil humidity is maintained at 55–65% of its full moisture capacity, and air temperature 38 42 ^o With a photoperiod of 11.6 12.3 hours  3. The method according to claim 1, characterized in that the cultivation in an artificial climate is carried out in the autumn-winter period, and only part of the selected breeding material is grown, which is necessary and sufficient for its evaluation. 4. The method according to claim 1, characterized in that under the conditions of an artificial climate that selection material is selected, all of whose offspring has a stable amount of glandular hairs on the bracts, not exceeding 8 pieces per 1 mm ^{2 of} their surface.

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