SU1704715A1 - Method for buckwheat propagation in vitro - Google Patents

Abstract

The invention relates to agriculture and can be used in the selection of plants for the mass reproduction of valuable specimens, in studies on the physiology and genetics of plants. The aim of the invention is to preserve the high regeneration capacity of callus tissue during long-term subculturing. In the method of propagation of buckwheat in vitro, including isolating explants, cultivating them on a nutrient medium to obtain primary callus tissue and subsequent transplanting it into a nutrient medium to induce organogenesis and to obtain regenerated plants. As explants, immature embryos with a size of 0.5-3.0 mm are isolated, which are cultivated to obtain primary callus tissue for 40-60 days, while cultivation of explants and subculture of callus tissue is carried out on a nutrient medium of Hamburg B 5. containing mg / l: nicotinic acid 0.1-0.5 and additionally bacto-agar 7000-8000: casein hydrolyzate 1500-2000; IAA 0.1-0.5; NUS 0.3-0.5, kinetin 0.2-0.4. At the same time, for subculturing, primary callus tissue is selected, represented by dense globular formations of white, light yellow colors or soft lumps of gray or light brown color, including brilliant dense structures of white or yellow color. The invention allows preserving the high regenerative capacity of callus cultures for 18 months. At the same time, the yield of regenerants per 1 g of callus tissue on cultural buckwheat is 3.5 pcs, and Tatar buckwheat is 23.0 pcs. 3 tab., 1 Il. with vj oh vj ate

Description

The invention relates to agriculture and can be used in the selection of plants for the mass reproduction of valuable specimens, in studies on the physiology and genetics of plants.

The purpose of the invention is to preserve the high regenerative ability of callus tissue during long-term subculture.

The goal is achieved by those. that in the method of propagation of buckwheat in vitro, including isolating explants, cultivating them in a nutrient medium to obtain primary callus tissue and subsequent transplanting it to a nutrient medium for inducing organogenesis and obtaining regenerated plants, unripe embryos are extracted as explants

0.5-3.0 mm in size, which are cultivated to obtain primary callus tissue for 40-60 days, while cultivation of explants and subculturation of callus tissue is carried out on Hamburg nutrient medium B 5, mg / l: NaH2P04150 KMOz 2500 (MNFRSM 134 MgS04 7H20 250 CaCla 2H20 150 NZVOZ 3.0 MnS04 H20 10.0 CoCla 6H20 0.025 Sysode H20 0.025 ZnSCU 7H20 2.0 Na2Mo4 2H20 0.25 KJ 0.75 FeS04 7H20 28.0 Thiamine-HCI 10.0 Pyridoxine-HCI 1.0 Nicotine 4Q20 Pyridoxine-HCI 1.0 Nicotine, Nicotine 4H20 28.0 Thiamine-HCI 10.0 Pyridoxine-HCI 1.0 2,4-D 2.0 Saharaoa 20,000 for subculturing, select the primary callus tissue, represented by dense globular formations of white th, light yellow or soft lumps of gray or light brown, including dense structure lustrous white or yellow.

The method is carried out as follows.

Fruits of buckwheat after 4-13 days after pollination are treated with 70% ethyl alcohol for 0.5-3 minutes (depending on the age of the embryo) and then 3-10 minutes, in a 0.1% solution of diacide, followed by a triple washing with sterile distilled water. A germ 0.5–3 mm long is isolated under aseptic conditions by prepair needles and placed on a callusogenic medium. Incubate for 40-80 days in the dark at 25 ± 2 ° С in Petri dishes. During this time, the primary callus is formed on the explants and various morphological types of callus are possible, since before 40 days certain callus morphotypes have not yet been detected, and after 10 days the tissue necrosis begins and the output of calluses with high morphogenic capacity decreases (Table 1) .

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Studies show that in the culture of buckwheat callus tissue, there is a close relationship between the source of the explant (embryo of a certain length), the morphology of callus cultures and the ability of these cultures to regenerate.

Cultural buckwheat calluses (Fig. 1), according to their morphological and histological characteristics, can be conventionally divided into 4 main types (A - heterogeneous, B - globulary, C - loose. D - densely globular). As can be seen from FIG. 1, heterogeneous and dense-globular cultures have the highest kidney-forming activity. However, morphological instability is characteristic of heterogeneous cultured buckwheat calluses: with an increase in the cultivation time, the culture decays into new morphological types and, as a result, it goes into the discharge of globular or loose cultures. In addition, organogenesis in heterogeneous calluses of cultivated buckwheat

0 is often accompanied by the appearance of morphogenesis anomalies (the formation of anthocyanin-containing leaf-like outgrowths, the appearance of threadlike light green shoots resembling moss).

5 Unlike heterogeneous callusoo, the absolute majority of dense-globular calli (8 out of 9) retains a typical morphology during the year and, when converted to an organogenic environment, forms

0 kidneys. Other types of calli - globular, decomposing during cultivation into separate, unrelated globules, form roots on an organogenic medium and very rarely buds: loose cultures or

5 are not capable of organogenesis at all, or they form separate roots.

Due to the fact that heterogeneous calli are not morphologically stable, globular and friable, although

0 retain morphological features for a long time of cultivation, but are not capable of budding, it is advisable to use calluses of dense-globular type for regeneration of plants of cultivated buckwheat.

In Tatar buckwheat, there are two types of callus cultures that differ in morphology: heterogeneous calli and heterogeneous calli with brilliant white or

0 yellow. The first type of callus is the root-forming one, while the second, when transferred to the organogenic environment, actively forms the kidneys. Morphological characteristics and ability to regenerate in heterogeneous cultures

5 with dense structures maintained for 18 months. and more.

Thus, in order to preserve the high regenerative capacity of calli during their long cultivation, it is necessary to select calluses of a certain morphotype — tightly globular (Table 2, 1 morphotype) or heterogeneous with brilliant dense structures of white or yellow color (morphotype 2).

As can be seen from the table. 2 (variant 8), the yield of such calluses correlates with the embryo's age; they occur only on embryos 0.5–3 mm in length in the case of cultural buckwheat and on embryos 0.5–2 mm in length in the case of Tatar buckwheat.

Due to the fact that the described types of calli retain a stable morphology and high regenerative capacity for at least 18 hours (Table 3), only such callus morphotypes are selected for subsequent subculturing.

Cultures are transplanted every 25–40 days, Hamburg B 5 medium, optionally containing, mg / L: nicotinic acid, 0.1–0.5; bacto agar 7000-8000; and casein hydrolyzate 1500-2000; IUKO, 1-0.5; NUKO.3-0,5; kinetin 0.2-0.4.

As can be seen from the table. 4, this medium is the most optimal. A decrease in the concentration of the listed components (variant of medium 1) reduces the growth rate of callus cultures and contributes to the formation of globular-type calli. Concentrations of components that are super-optimal (option 4) promote the formation of roots and an increase in the yield of loose calli. Globular and loose cultures (Fig. 1) are not capable of bud formation.

Using the original Hamburg B 5 medium to maintain buckwheat calluses with high morphogenetic potential is unacceptable. On this medium, the appearance of various types of calli can be induced, incl. and densely globular, however, the further growth of calluses of this morphotype cannot be maintained: after 1–2 x passages on the initial medium of Hamburg, B 5 calluses become dark brown. Morforlogically callus is represented by numerous globular formations. The histological analysis of such calluses revealed their specific organization: in the center of a separate globule there is a meristematic zone, surrounded by a lining of large parenchymal cells.

This type of callus on the Hamburg B 5 medium (without modifications) could be maintained for at least a year, however it differs by slow growth and forms only roots on the regeneration medium.

When callus is grown on the initial medium of Hamburg B 5, certain types of tissues are selected, it is not possible to support the growth of calluses with high morphogenetic potentials (capable of forming buds),

Kidney formation is obtained on a Hamburg B 5 nutrient medium containing 1-2.5 mg / l of BAP and 0.1-0.2 mg / l of IAA as phytohormones. 1.5–3 weeks after transplantation of callus cultures to the organogenic medium, a mass occurrence of stem buds is noted. In order to obtain the required volume of homogeneous material, the kidney is propagated by the induction of adventitious shoots using a regeneration medium or a medium with a low content of BAP or zeatin (1-0.5 mg / l). The use of the latter medium in the passage, prior to transplanting the regenerant into rooting, promotes the elongation of the stem, which facilitates the isolation of a single shoot from the clone of the kidneys and its rooting on agar. 5-6 days after planting on the root formation medium, the first roots appear in the plants, after 2-2.5 weeks, the regenerants have 4-5 leaves and are ready for transplantation into the bud. Rooted regenerant plants, 5-6 cm in height, are planted in paper cups under a glass cap; for 1-1.5 weeks, the plants are hardened, gradually increasing the duration of their stay in the open air, after it is transferred to a greenhouse or growing house and grown hunt until full maturity of seeds. Upon receipt and cultivation of calluses Fagopyrum esculentum and Fagopyrum tatarlcum by the described method, the high regeneration ability of callus cultures is maintained for at least 18 months.

The method was tested on two types of buckwheat: cultural (variety Kazansk coarse grain) and Tatar (K-17). The use of the proposed method allows the accelerated reproduction of unique genotypes of buckwheat, as well as increasing the yield of plants - buckwheat regenerants with a wide range of variability due to maintaining a high regenerative ability of calli during their long-term cultivation.

Claims (1)

Invention Formula The method of reproduction of buckwheat in vitro, including isolating explants, cultivating them on a nutrient medium to obtain primary callus tissue, subculturing primary callus tissue and subsequent transplanting it to a nutrient medium for the induction of organogenesis and the production of regenerated plants, which is, In order to preserve the high regenerative capacity of callus tissue during long-term subculturing, immature embryos with a size of 0.5–3.0 mm are cultured as explants, which are cultivated to the ground tim primary callus tissue during 40-60 days, the explants culturing and subculturing the callus tissue is carried out in a nutrient medium Gamborg B 5, which contained 0.1-0.5 mg / l nicotinic sour 1500 2000 mg / l of casein hydrolyzate, 0.1-0.5 mg / l IAA, 0.3-0.5 mg / l NAA, 0.2-0.4 mg / l kinetin and 7000-8000 mg / l bacto-agar, and for subculturing primary callus tissue is selected, represented by dense globular formations of white, light yellow color or soft lumps of gray or light brown color, including shiny dense structures of white or yellow color . Table 1 The effect of the duration of cultivation of explants on the output of D-calli Dependence of the output of calluses of a certain type on the length of the embryo table 2  - the variable components are indicated, the Hamburg B 5 nutrient medium is taken as the basis (clause 5). Table 3 Table 4 Nollo nashlu V 5В С 12 Continued cultivation