ANATOMICAL AND HISTOCHEMICAL FEATURES OF THE SHOOTS OF PROSPECTIVE VARIETIES OF ROSA DAMASCENA MILL

Abstract

Over the last few decades, biotechnological research of polyphenols synthesis in plant explants on the stage of introduction to the in vitro culture greatly grew in its significance. Rosa damascena Mill. refers to the plants that contain a large number of secondary metabolites in their composition. There is a whole variety of bioactive phenolic compounds that are synthesized in the leaves of plants of Rosaсеае Juss. Family: flavonoids aglycones - quercetin and kaempferol and their glycosides, catechol, pyrogallol, eugenol, Proanthocyanidins, major of which is epicatechin. In young stems can be found ferulic and gallic acids, epicatechines, galotanin and dimers of proanthocyanidins. In the tissues of lignified stems accumulate flavan-3-ol (-) - epicatechin monomers, dimers and polymers proanthocyanidins. Intensive synthesis of secondary metabolites accompanies the traumatic stress and ensures the protection of damaged surfaces from the potentially dangerous pathogens in case of their excessive accumulation slows down the regeneration of tissues and creates the conditions for irreversible destructive processes and premature cell death, which lead to the loss of valuable plant material.

Aim of the research: to study the anatomical features of the shoots of prospective varieties of Rosa damascena Mill. Lan`, Lada and Raduga of Ukrainian selection, and to establish methods of successful adaptation of explants to the in vitro conditions.

Methods and materials

For the research were used shoots of Rosa damascena Mill. varieties Lan`, Lada and Raduga, 20-30 cm long with axillary buds, that were taken from 2-year donor plants and shoots of regenerant plants from in vitro culture. Anatomical- histochemical analysis of stems was performed on cross sections of second internodes of annual shoots using Nikon Eclipse E-200 microscope. The structure of plant tissues was studied on 20-30 microns thick native specimens. To identify the catechins in tissues were used 1% vanillin reagent and 5% hydrochloric acid. Phenolic compounds were determined using 5% aqueous solution of ferric chloride. Localization of basic proteins was determined by Bonkheh using Bromothymol blue. M-lignin deposition was determined by permanganate reactions.

Results and discussion

The results of histochemical analysis revealed that spare polysaccharides intensively accumulate in the tissues of stems of Lan` variety. In regenerant plants starch was detected only in the cells of the primary cortex. Topologically, the primary cortex consists of exocortex, that has several layers of cells, medicortex, represented by large parenchyma cells, and endocortex, that is made of several layers of cells. Exocortex lies under the epidermis of the stem in the form of multilayer, usually continuous circle, whose cells eventually differentiate into plate type collenchyma. But in the in vitro conditions the given process slows down considerably due to the specific mixotrophic nutrition and hormonal status of plants by which the formation of secondary cell walls is inhibited. Consequently, the extraaxillare fiber bundles are insufficiently developed. Secondary cell walls of sclerenchyma are slightly stiff, though with minor deposits of M lignin. Medicortex is represented by large chlorophyll cells of 30-50 microns in diameter. In in vitro conditions, outside layer of cortical cells of plants contains the largest deposits of starch. Localization of proteins is spatially associated with the concentration of main mass of catechins and other polyphenols, including oxybenzoic and oxycinnamic acids, tannins. According to the results of histochemical analysis, the deposition of catechins most actively occurs in medicortex of primary cortex, medullary ray cells and phloem. However, epicatechines are usually concentrated in areas of primary phloem. In the xylem catechins are found only in parenchyma of medullary rays.

It should be mentioned that the activation of polyphenol synthesis occurs in response to traumatic tissue damage of primary explants when the plants are introduced into the in vitro culture. Exactly this stage is the most critical in obtaining of viable explants.

Oxidation and polymerization of phenolic products leads to inactivation of enzymes, blocking the xylem transport systems that provide power to cambium cells and apical meristems.

Conclusions

It was shown that condensed tannins and catechins rapidly accumulate in the primary cortex, cells of medullary rays and phloem parenchyma of annual shoots of Rosa damascena Mill. varieties Lan`, Lada and Raduga. During traumatic stress in the living tissues the processes of oxidation and polymerization of polyphenols are activated, that lead to blocking of lateral nutrients transportation, affecting the viability of primary tissue explants. Localization of proteins is spatially associated with the concentration of main mass of catechins and other polyphenols.

During cultivation of regenerant plants under in vitro conditions, stem tissues of Rosa damascena Mill.A acquire the juvenile properties. Identified anatomical and histochemical features of shoots are associated with the specific character of mixotrophic nutrition and hormonal status of plants by which the formation of secondary cell walls is inhibited.