The practical implementation of tasks aimed at creating new grape varieties through methods of artificial morphogenesis requires strict adherence to technological stages and high precision in performing manipulations with the plant's generative organs. The technique of the breeding process includes the isolation of inflorescences, castration of buds, harvesting and preservation of pollen fertility, and subsequent directed pollination.

Self-Pollination (Inbreeding / Incompressibility)

To ensure guaranteed self-pollination of the selected variety and reliable prevention of accidental penetration of foreign pollen carried by the wind onto the stigmas, it is necessary to cover the inflorescence with a special isolator (parchment bag) a few days before the onset of flowering. Since grape pollen possesses extremely small dimensions (about 6 microns) and high volatility, increased requirements are imposed on the quality of isolator manufacturing.

In modern breeding practice, glued parchment isolators of the "clapper" (hlopushka) type are used exclusively. Old types of isolators in the form of bags sewn on a sewing machine are unsuitable for precise scientific work, since foreign airborne pollen freely penetrates inside through microscopic holes from the sewing needle, which completely annuls the reliability of the experiment. The technology for manufacturing a proper clapper-type isolator consists of the following stages:

  1. Sheets of dense, high-quality parchment are cut to a standard size of 23 × 32 cm.
  2. On a special wooden template block (with a diameter of 6 cm and a length of 22 cm), a tube is formed from the sheet of parchment, the edges of which are glued with wood glue (the use of ordinary stationery glue is unacceptable, as it dissolves during field rains).
  3. Into one end of the resulting tube, prior to its removal from the block, a plug of sterile cotton wool is placed. The parchment edge is tightly pressed against the cotton plug and rigidly tied with thin twine, leaving two free ends for subsequent fixation, as shown in Fig. 55. After removing the template block, the isolator is ready for use.

The process of placing the isolator onto the inflorescence requires extreme accuracy. Opened flowers must be completely absent from the isolated inflorescence. If individual opened buds are found during inspection, they must be removed immediately using fine tweezers. As a preventive measure for disinfection, the inflorescence can be briefly dipped into a container with alcohol, after which it should be carefully blotted with sterile cotton wool. A even layer of cotton wool is wound onto the base of the inflorescence peduncle. Then, the parchment isolator is placed on top of it and tied tightly with twine directly at the location of the cotton pad. It is recommended to orient the isolator with its longitudinal glued seam facing downward. For protection against gusts of wind, the free upper end of the isolator is securely tied to the nearest stable shoot or leaf petiole. If the parchment proves to be excessively rigid, which prevents hermetic tying around the peduncle, the lower edge of the isolator should be slightly moistened with water beforehand. The formed clusters can remain in the isolators until the berries are completely ripe; however, it is physiologically more correct after the completion of flowering to replace the dense parchment bag with a light gauze one. If a breach in the parchment or a displacement of the cotton plug is discovered during routine field checks, this inflorescence is culled, and the results of the experiment are liquidated.

Hybridization (Crossbreeding)

The successful execution of controlled interspecific or intraspecific hybridization of grapes is based on the precise performance of three key technological stages: timely blocking of the possibility of self-pollination and accidental cross-pollination of the maternal flower, proper harvesting of viable paternal pollen, and precise artificial application of it to the stigma of the pistil.

To prepare the maternal component, an absolutely healthy vine typical of the variety is selected along with a normally developed fruitful shoot. If there are several inflorescences on the shoot, it is advisable to leave only one lower inflorescence, removing the others to optimize nutrition. If a variety with a functionally female flower type (possessing sterile pollen) is used as the maternal form, the procedure of removing anthers (castration) is excluded. To protect such an inflorescence from accidental cross-pollination by the wind, it is sufficient to isolate it with a parchment bag prior to flowering according to the method described above.

If the maternal variety is hermaphroditic (bisexual), performing castration is strictly mandatory. In most bisexual grape varieties, pollen maturation and fertilization of the stigma occur directly under the closed corolla-cap before it is shed (cleistogamy). To prevent this self-pollination, castration is carried out 6–7 days before the expected onset of natural flowering, when the anthers have not yet ruptured. The degree of maturity of the anthers is determined visually by the change in their color and by test-rubbing several of them between the fingers.

Castration is performed using fine anatomical tweezers, as illustrated in Fig. 56. Depending on the variety, the cap, along with the anthers firmly attached to it, is removed from each bud in either one or two motions. Full castration of every single bud on the inflorescence is not required, as a significant portion of them will not functionally develop anyway. It is sufficient to carefully castrate 100 to 150 of the central buds, while completely removing all remaining lateral and apical buds with the tweezers. The operation is conducted in the early morning hours in the complete absence of wind. In the presence of strong wind, castration may be performed exclusively inside special portable structures that protect the operational field from incoming foreign airborne pollen. Immediately after the completion of castration, a parchment isolator is placed over the inflorescence.

Harvesting and Preservation of Pollen

If the designated paternal variety blossoms simultaneously with the castrated maternal variety, preliminary harvesting and drying of pollen are not required. In this case, it is sufficient to isolate several inflorescences on the paternal vine well in advance, and at the moment of crossbreeding, cut them and perform pollination directly with the fresh paternal inflorescence.

In case of non-coincidence of flowering periods, regulatory techniques are applied:

  • The paternal variety flowers later than the maternal variety: to accelerate its vegetation in the spring, a glass box made of greenhouse frames is mounted over the vine or shoot. To delay the flowering of the maternal variety, the shoots are covered with straw and ice in the spring, or previously rooted layers grown in boxes from the preceding year are utilized, maintaining them in a cold cellar.
  • The paternal variety flowers earlier than the maternal variety: advance collection and conservation of the paternal pollen are carried out.

For pollen harvesting, the inflorescences of the paternal variety are isolated with parchment prior to flowering. At the peak of flowering, they are cut along with the bags and brought into the laboratory. Using tweezers, each bud is carefully opened and the anthers are stripped onto sheets of clean paper. The opened buds and isolated anthers are dried on the paper in a dry room for 3–4 days, after which the released dry pollen is thoroughly sifted through a fine silk sieve. The finished fertile pollen is transferred into clean glass test tubes, which are then stoppered with hygroscopic cotton wool and stored in a cool, dry place until the maternal variety reaches readiness.

Artificial Pollination

Artificial pollination of the castrated maternal inflorescence is performed approximately 5 days after castration. An indicator of the pistils' readiness for fertilization is the onset of the secretion of small drops of transparent fluid on their stigmas. To achieve maximum crossbreeding efficiency, it is recommended to repeat the pollination procedure after 2–3 days.

To perform the manipulation, the parchment isolator placed on the maternal inflorescence after castration is carefully opened. Opening can be done either from the top or from the bottom. High efficiency is also demonstrated by a method in which a small technological window is carefully cut in the side wall of the parchment bag, through which the pollen is introduced via a long brush, after which the hole is hermetically sealed with a fresh piece of parchment. The dry harvested pollen is applied to the stigmas using a fine watercolor brush, a sterile cotton swab, or directly with the inflorescence of the paternal variety. Immediately upon completion of artificial pollination, a label indicating the unique experiment number and the exact formula of the crossbreeding is placed inside the isolator. Concurrently, all data (date, time, names of parental varieties) are entered into the breeding journal under the corresponding ordinal number. Control verification of the hybridization results is carried out twice: first, immediately after the end of flowering (at the moment of replacing the dense parchment bag with a gauze one), and second, during the harvest of the ripe crop.

Preparation of Seeds for Sowing

Formed clusters of hybrid berries or berries obtained as a result of inbreeding can remain in isolators or gauze bags until late winter, suspended from the ceiling in a cool room. In their free time, breeders perform the manual extraction and cleaning of seeds from the berry pulp for each breeding combination separately. The separated seeds are thoroughly washed in clean water, slightly dried at room temperature, and poured into paper packets, on which the unique experiment number, the exact name of the crossbreeding combination, and the total number of seeds are written. In these packets, the seeds are preserved until spring sowing in a cool, not very dry place.

Stratification of seeds in sand, as practiced by pomologists, is not strictly mandatory for grapes. Before spring sowing, grape seeds must be subjected to prolonged smoking in water for 6–10 days, with the water being changed daily. Optimally, the water temperature should be maintained in the range of 24–30°R (degrees Réaumur). Exceptionally high field germination results are achieved if preliminary germination of seeds is carried out in moist sand inside a greenhouse at a temperature of 24–30°R about two weeks before the expected sowing. Coarse clean sand is mixed with seeds in a volume ratio of approximately 2:1. Sowing is performed immediately as soon as the seed coat cracks and the tip of the root breaks through. With ordinary dry sowing, seeds emerge in 3–4 weeks, whereas seeds that have undergone preliminary germination yield uniform shoots within a few days.

Growing Seedlings

Sowing of prepared seeds can be carried out in a greenhouse, in hotbeds, or directly into open ground. For the accelerated forcing of hybrid seedlings until the phase of first fruiting (which is especially critical for central and northern viticulture regions), growing in the protected ground of greenhouses shows the best results. Thus, in greenhouses near London, gardeners (as indicated by Pynaert) managed to bring hybrid grape seedlings to fruiting in a record short time — 18 months. However, it must be taken into account that a new variety formed in the artificial conditions of a greenhouse will be adapted to the greenhouse environment, and its behavior in open ground remains unpredictable.

The great breeder I. V. Michurin quite rightly pointed out that the directed cultivation and education of hybrid seedlings must take place under the natural climatic conditions for which the new variety is being bred. For this reason, in the vast majority of cases, seedlings must be grown under open ground conditions — either by direct sowing of seeds in a permanent place or via the pricking out of seedlings grown in greenhouses or hotbeds. Mass experience has shown that in large-scale sowings (especially under southern conditions with dry winds and frequent droughts), the best results are obtained by direct sowing into the ground without pricking out, as transplanting young plants under these conditions leads to their mass mortality (loss). In the central and northern zones, on the contrary, to accelerate vegetation, it is advisable to sow germinated seeds in greenhouses and warm hotbeds in early spring, and then carefully prick out the seedlings to a permanent place in the field.

In mass sowings, the work is carried out in autumn or in spring with the onset of stable warm weather. Seeds are embedded to a depth of 2–3 cm. Sowing is performed in regular rows with row spacings of 1 m; in the row, seeds are placed at a distance of 2–3 cm from each other. The technology of caring for seedlings in the first 1–2 years should ensure maximum vegetative growth, but without creating artificial conditions unusual for the given region. Transplanting seedlings during this period is not recommended; in case of extreme necessity, it is performed with special care, preserving a large root ball of earth. In arid locations, moderate watering and the application of nitrogen fertilizers in the first year of vegetation are mandatory, followed by phosphorus fertilizers in subsequent years. Excessive abuse of fertilizers, as I. V. Michurin warned, is unacceptable, as it renders the plant delicate.

Pruning of young seedlings in the first years of vegetation is subjected to a strict scheme:

  • First year: pruning is conducted in such a way that only 1 main shoot grows in the first year.
  • Second year: they proceed to the development and formation of the trunk, for which 2 to 4 strong shoots are left to grow.
  • Third year: long pruning of the formed canes is carried out. On the breeding nursery, the rules of agricultural technology usually applied in the specific viticulture region are strictly observed.

The entry of seedlings into the fruiting phase depends on their individual hereditary properties and the conditions of vine education. Seedlings usually begin to flower in the 3rd year, with the bulk of the hybrid population flowering in the 5th–7th year of vegetation. Currently, all breeding stations working with grapes are conducting research to develop a method for accelerating the passage of developmental stages in young seedlings.

Organization of Selection

The specific location, exact timing of seed sowing, and the general layout of the breeding nursery depend directly on the adopted system of selection organization. Taking into account the set task and the material and technical base, breeding selection of grapes is divided into three main types: 1) selection under natural conditions, 2) selection under natural conditions with the application of provocation, 3) selection under laboratory conditions.

Selection under Natural Conditions. It is methodologically the most correct, since new varieties are formed and evaluated in the very same natural environment in which they will be cultivated in production. Selection under natural conditions is conducted according to indicators of stable yield, fruit quality, and early maturity. Following the 2nd year of vegetation, severe culling of seedlings is performed according to such breeding markers as the timing of the maturation of annual vine wood and the time of spring bud burst. In the very first year, all segregating defective recessives must be destroyed based on general habitus: dwarfs, albinos, variegated, and monstrous forms. If the breeding program is aimed at obtaining exclusively black varieties, then already in the first autumn, the required forms are selected by the characteristic red coloration of the leaves, completely culling seedlings with yellowing foliage (a marker for white berries). With the onset of the flowering phase, sterile male forms of vines are completely removed from the nursery. After the seedlings enter stable fruiting, their strict evaluation by fruit quality begins, which is initially conducted by tasting methods. In this way, table, raisin, and wine varieties are isolated. A complete technological evaluation becomes possible only after the selected seedlings have been vegetatively propagated.

Selection under Natural Conditions with the Application of Provocation. To accelerate the breeding process and eliminate dependence on accidental weather factors of a specific year, provocational methods are applied. The purpose of this method is to artificially and annually create conditions under which seedlings can reveal their hidden properties without waiting for the onset of a critical year in nature. For this purpose, seedlings are intentionally planted in harsh conditions of artificial stress:

  • In selection for drought resistance — the nursery is established on extremely arid plots without irrigation.
  • In selection for cold hardiness — the vines are intentionally left uncovered for the winter (without winter protection).
  • In selection for phylloxera and mildew resistance — the roots of the plants are artificially infected with phylloxera, and the leaves and infлоrescences are mass-infected with fungal diseases.

Selection for Winter Hardiness. Since the winter hardiness of grapes is largely determined by the maturation of the wood, selection should primarily be conducted according to this trait. I. V. Michurin pointed out that varieties with early wood maturation are more winter-hardy; furthermore, their fruits will also mature early, which is especially important for northern regions. Under northern conditions, with a long day, grapes produce long shoots that mature poorly. However, such a reaction to a long day is not the same for all varieties. Northern varieties (Riesling, Silvaner) are less sensitive in this respect than southern American species. On a southern short day, shoots grow more weakly and mature better. For the selection under southern conditions of varieties that give good wood maturation on a northern long day, seedlings can be tested under a day elongated by means of electric light. Field trials, as indicated, are performed by leaving the seedling nursery uncovered for the winter. In order not to destroy all the material, in the presence of other breeding tasks, it is necessary to allocate only a part of the seedlings for this experiment. Evaluation of winter hardiness is usually performed in the spring. Damage to buds and roots should be recorded separately. Seedlings suffering severely from winter frosts should be culled.

Selection for Mildew Resistance. This should be performed under field conditions with artificial infection of seedlings if mildew is weakly developed under natural conditions. Since the resistance of small seedlings is significantly less than that of adults, infection should be done after the seedlings have put out the 4th–5th leaf. Evaluation should be conducted according to the character of the damage. Resistant forms have only black dots or angular spots on the leaves. Unresistant ones have diffuse oily spots. Infection is performed by spraying seedlings with water (rainwater) containing mildew spores washed from infected leaves (Zotov, 1935).

Selection for Root Phylloxera Resistance. This is conducted by infecting the plot with phylloxera. Infection is performed in the middle of summer or toward the autumn of the first year, as small seedlings are more sensitive than adults. A leaf with phylloxera galls is placed under each seedling, and this is sufficient for infection. Evaluation of resistance is conducted after 1–2 years by excavating roots and determining the character of the damage.

Selection under Laboratory Conditions. It does not always yield a correct evaluation, as the testing conditions are too different from those available in the field. However, in some cases, the laboratory method can significantly accelerate work at early stages. For example, using a refrigerator, it is possible to determine the cold hardiness of seedlings' roots and shoots with high accuracy. It must be taken into account that during the winter, due to hardening, cold hardiness changes; the resistance of roots, old wood, and buds in winter, and of burst buds in spring, are different in different varieties and within the same variety. Also, under the controlled microclimate of a greenhouse, it is possible to create conditions favorable for the development of fungal diseases and artificially infect seedlings. Depending on the task facing the breeder and the available equipment, it is necessary to combine these methods, applying them in one sequence or another.