RU2485755C1 - Method of growing planting material - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: invention relates to plant production, forestry, aesthetic forestry and agriculture, namely to nursery. The method of growing planting material includes harvesting the shoots, their sterilisation, induction of development of basic auxiliary branch on primary explants, rooting of isolated shoots and their multiplication in vitro. Transfer of the shoots in non-sterile conditions and planting into the soil. Micro cutting grafting is carried out on a nutrient medium WPM - Woody Plant Medium with a reduced content of BAM - benzylaminopurine 0.2 mg/l, and adding PS - gibberellic acid, 0.2 mg/l. The planting of test tube microplants in non-sterile conditions is carried out without their prior adaptation to the conditions of the climatic chamber.

EFFECT: use of this method enables to produce high-quality two-year planting material in conditions of the open ground.

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Description

The invention relates to crop production, forestry, forest park and agriculture, namely to nursery.

The invention solves the problem of producing high-quality planting material with high rates of growth and increase the survival rate of seedlings.

A known method of propagation of planting material of woody plants, including the cultivation of shoots in vitro on an artificial nutrient medium, testing by enzyme-linked immunosorbent assay, plant grafting (RF patent No. 2118486, IPC A01G 01/00, publ. 09/10/1998). However, this method is expensive.

An in vitro plant propagation method was selected as a prototype, including planting the lateral shoots of plants in vessels filled with agarized medium without a bottom for initial rooting, placing the rooted shoots in a container filled with perlite soaked in liquid sterile medium (RF patent No. 2277773, IPC А01Н 04 / 00, publ. 06/20/2006). However, this method is very laborious.

The method is intended for the mass replication of economically valuable varieties of woody plants that are difficult to cut in the traditional way, and the cultivation of high-quality standard planting material to create plantation stands, shelterbelts, forest lands.

To this end, plant regeneration is carried out on the basis of proliferation of axillary meristems (direct distillation of axillary shoots), their rooting and multiplication of the resulting microplants, i.e. using a breeding model that excludes the callus formation stage. In this case, nodal segments of actively growing summer non-lignified (rather than winter lignified) shoots are used as explants, the use of hormonal nutrient media is limited to only one period of cultivation (one month) of the primary explants of adult trees, and at the stage of rooting of microprobes, their growing and animation, one is used the same hormone-free environments.

The proposed method of growing planting material is as follows.

Stage 1 - Harvesting shoots, isolation of explants and obtaining aseptic viable crops.

Harvesting of shoots: annual growing non-lignified (green) poplar shoots of 40 ... 50 cm in size with axillary vegetative buds are harvested in the beginning - mid-June from the middle part of the crown of adult selected trees. Shoots with well-defined axillary buds and internodes are selected. Cut shoots are placed in jars with tap water in a refrigerator (temperature 10 ... 12 ° C), where they are kept for 3 ... 5 days for their cold pretreatment before isolation of the explants. Every three days, sections are updated and water is changed.

Sterilization of shoots. First, preliminary preparation of the shoots is carried out. To do this, shoot segments of 10 cm in size are thoroughly washed with warm water and laundry soap, and then subjected to phased sterilization:

1) Surface sterilization with commercial 2% Domestos solution (heated to 30 ... 40 ° C) for 15 minutes, followed by washing in running tap water (at least 20 minutes). It is carried out in non-sterile conditions.

2) Basic sterilization of the shoots under aseptic conditions (in the conditions of a laminar box) with a 0.025% solution of merthiolate in combination with the Belizna liquid disinfectant solution (7% solution) for 15 minutes, followed by 5-fold washing with sterile distilled water. For better wetting of the shoots during the main sterilization, it is recommended to add Tween 20 (0.08 ... 0.12%) to the mortar solution.

Stage 2 - Induction of the development of the main axillary shoot on primary explants.

Sterilized shoots are cut under aseptic conditions into segments of 1.5-2 cm in size with one axillary kidney (culture explants). Explants under aseptic conditions (in a laminar box) are placed vertically or slightly inclined one at a time in culture vessels (glass biological tubes) with WPM culture medium (medium No. 1) with the addition of 6-BAP (6-benzylaminopurine) 0.5 mg / l, HA (gibberellic acid) 0.2 mg / l.

Morphogenic potencies (the formation of well-developed axillary shoots) are more fully manifested when explants are cultured on WPM nutrient medium supplemented with a 0.5 mg / L BAP and 0.2 mg / L HA (Table 1). Adding HA to the nutrient medium allows not only increasing the number of morphogenic crops (for example, 1.7 times for poplar Khopersky 1 (77.4% versus 44.3% without HA) and 1.9 times for poplar Priyarsky), but also promotes elongation (growing) of induced shoots. In this case, shoots are formed within a month from the start of cultivation, characterized by good growth in height (4 ... 8 cm) with normally developed leaf blades, and the absence of signs of somaclonal variation. The low content of BAP in the medium (0.2 mg / l) at the initial stage of cultivation is not enough for the effective induction and development of primary shoots. A higher content of BAP in the medium (1.0 mg / l) inhibits the development of explants and the growth of shoots in height.

To increase the number of induced shoots, it is recommended that primary explants be recultivated with a cut axillary shoot (removal of apical dominance) on WPM medium with a reduced BAP content of 0.2 mg / L and the addition of HA 0.2 mg / L. In this case, during the month of cultivation, intensive multiple formation of additional shoots occurs, characterized by good growth (3 ... 5 cm) and normal development. The number of shoots per 1 explant is 9.6 with a variation from 6 to 14 shoots.

Table 1 Morphogenetic activity of primary explants of summer cuttings of gray poplar depending on the hormonal composition of the nutrient medium WPM No. p / p BAP mg / L HA mg / L % of explants that formed the main shoot Hopersky 1 Priyarsky one 0.2 - 8.2 ± 0.5 5.1 ± 0.3 2 0.2 0.2 17.5 ± 1.6 10.8 ± 1.5 3 0.5 - 44.3 ± 4.4 28.0 ± 3.3 four 0.5 0.2 77.4 ± 3.9 * 54.4 ± 3.6 * 5 1.0 - 21.8 ± 1.5 12.3 ± 0.7 6 1.0 0.2 44.3 ± 4.1 31.2 ± 2.7

Stage 3 - Rooting of isolated shoots and their multiplication (clonal micropropagation) in vitro.

The formed axillary or induced additional shoots (reaching a height of 1.5 ... 3 cm) are isolated and transferred to a medium for spontaneous rooting of WPM or 1/2 WPM (with a reduced content of macrosalts) without hormones. Larger shoots before planting are cut into microcrops with one axillary kidney size of 1.5 ... 3 cm.

For microclonal propagation, it is recommended to use all micro-stems of the regenerant plant, which can increase work productivity. This is due to the absence of significant differences between cuttings of different tiers of one plant (upper, middle, lower part of the shoot) in terms of the effectiveness of their rooting in vitro. All single-node segments are well rooted and have normal growth.

The multiplication of microplants (multiple microcrossing, carried out to increase the number of clones) is carried out once a month or once every 2 months on nutrient media WPM or 1/2 WPM without hormones. To do this, microcranes 1 ... 1.5 cm long, containing one axillary kidney, are placed in 20 pieces in one plastic container.

The conversion of microplants to non-sterile conditions must be carried out without preliminary adaptation in the conditions of the climatic chamber, which allows to increase the safety of regenerants by 20%.

Direct planting of plants from a test tube culture in the soil involves the absence of an intermediate stage of growing in the laboratory. Microplants were transferred directly from the culture vessels to the non-sterile substrate of the greenhouse in the spring and summer (May-July). Plants adapted in open culture vessels are removed and dive into straight grooves made in well-loosened and leveled soil.

Shed with a weak solution of potassium permanganate (KMnO ₄ ), sprinkled with soil, compacted. Placing plants - from 100 to 200 pieces per 1 m in straight rows. The row spacing is not less than 10 cm. This placement promotes high-quality and timely care (loosening, weeding) during the first growing season, which, in turn, increases the survival rate of test plants in the soil.

To obtain standard planting material (two-year-old seedlings), plants wintered in a greenhouse in early spring are transplanted to an open area. At this stage, special attention should be paid to planting dates, on which not only their survival, but also growth processes substantially depend.

During the growing season, caring for the plants is necessary (weeding, loosening, fertilizing with mineral fertilizers, for example, Kemira universal), and in hot and dry summers, daily watering.

The proposed method contributes to the maximum degree of preservation of the economic and genetic value of the original specimens and allows to obtain high-quality homogeneous (standard) two-year-old planting material in open ground conditions.

Claims (1)

A method of growing planting material, including harvesting shoots, their sterilization, induction of the development of the main axillary shoot on primary explants, rooting of isolated shoots, their mobilization in vitro and translation into non-sterile conditions, characterized in that microcrossing is carried out on WPM - Woody Plant nutrient medium Medium, with a reduced content of BAM - benzylaminopurin 0.2 mg / l and the addition of HA - gibberellic acid 0.2 mg / l, and the planting of test tube microplants in non-sterile conditions is carried out without them ritual adaptation to the conditions of the climate chamber.