Leaf morphological and anatomical structure of pregenerative individuals of Ferula tadshikorum in ex situ conditions

of morphological and anatomical structure of the leaf of pregenerative individuals of Ferula tadshikorum in ex situ conditions. Acta


Introduction
The genus Ferula L. belongs to the tribe Scandiceae Spreng. (subtribe Ferulinae Engl.) The genus includes 180-185 species of perennial monocarpic and polycarpic herbaceous plants, usually with a tall and thick stem and a strong root system. Most of them are used in traditional eastern and folk medicine (Korovin, 1947;Panahi et al., 2018;Plunkett et al., 2018). Ferula species are a rich source of biologically active compounds, including coumarin derivatives, sesquiterpene compounds, esters or resinous substances, flavonoids, less often saponins (Barnaulov et al., 1974;Golovina et al., 1978;Rakhmankulov et al., 1981;Zhou et al., 2017;Mohammadhosseinia et al., 2019). The use of these biologically active components with antimicrobial and insecticidal effects can provide new strategies for the development of drugs and "green" pesticides, and protect endangered plant resources (Zhou et al., 2017). The literature contains ample information on the study of the pharmacological activity of isolated biologically active compounds from various organs of Ferula species. Many new secondary metabolites found in Ferula species belong to classes of natural products with interesting biological activities from antiproliferative to anti-inflammatory and neuroprotective (Mohammadhosseinia et al., 2019). The soluble lipid fraction from the roots of Ferula ferulaeoides (Steud.) Korovin revealed antiviral activity against the Hepatitis B virus (Zhai et al., 2011). Volatile components of the leaves of Ferula vesceritensis Coss. & Durieu ex Trab. showed antibacterial activity of essential oils against gram-positive and gram-negative bacteria (Zellagui et al., 2012). Compounds of other Ferula species (F. vesceritensis Coss. & Durieu ex Trab., F. assa-foetida (L.) Falc., F. gummosa Boiss., F. communis L., F. sinkiangensis K.M. Shen.) showed cytotoxic effects, and the main mechanisms of action are associated with slowing cell growth, making them an interesting choice as an adjuvant therapy for certain diseases (Iranshahi et al., 2018). Ferula tadshikorum Pimenov has been used as a medicinal species in folk medicine from ancient times in treating cough, meteorism, seizures, arteriosclerosis, cataracts, nervous and mental disorders. In addition, the plant has strong anti-helminthic properties, and it is used as a spice in cooking. Both underground (milky root juice solidified in the air) and the plant's aboveground parts are used as medicinal raw materials (Sadykov, 2003;Small, 2012;Sharopov, 2018). F. tadshikorum is distributed in the southwestern Pamiro-Alai, and is endemic to southern Uzbekistan and southern Tajikistan. F. tadshikorum is not included in the Umbelliferae of the multivolume monograph "Flora of Uzbekistan" (Korovin, 1959). For the first time in 2017, when conducting resource studies of medicinal plants in southern Uzbekistan, O. Khojimatov et al. (2018) collected the herbarium material Ferula tadshikorum, which was identified as a new species for the flora of Uzbekistan, and its species identity was also confirmed by the mono-graph of the Umbelliferae family of Central Asia, Prof. M.G. Pimenov. Unsustainable human economic activity often leads to a deterioration of the vegetation cover, and as a result leads to a reduction in the number of plant populations, and possibly complete extinction. During the last two decades, due to the strong exploitation of natural populations of the species for the sake of obtaining gum resin from the roots, the condition has worsened, and the species was included in the latest edition of the Red Book of the Republic of Uzbekistan with status 3 (endangered) (Makhmudov, 2019). To preserve natural populations and prevent a further decrease in the number of Ferula tadshikorum, it is necessary to study the biological features of this valuable medicinal plant, by propagating seed material in ex situ conditions and conducting further monitoring to determine the viability of sprouted individuals. An important mechanism for the conservation of plant species is primarily the need to organize protected natural areas in natural ecosystems or create the artificial reserves, among which botanical gardens have a leading role. Botanical gardens need to pay special attention to creating plant expositions including Red-listed species and conduct educational work with the public. For the first time, the Tashkent Botanical Garden at the Institute of Botany of the Academy of Sciences of the Republic of Uzbekistan, established experimental sites to study the adaptability of F. tadshikorum to ex situ conditions. According to our data, in ex situ conditions, the length of the juvenile phase of F. tadshikorum development is much reduced. According to literary data, in natural conditions the maximum age of the species can reach 6-7 years (Rakhmonov, 2017), and in introduced conditions, some individuals in the first year of life go into the immature phase of development (Khamraeva et al., 2019). Shortening the pre-generative period of promising medicinal plants during cultivation is considered an important biological feature from an economic perspective, for the rapid production of medicinal raw materials from the underground or aboveground part. To identify the biological stability of the species in new environmental conditions, it is necessary to study the rates of ontogenesis, phenology, growth processes from which assimilating organs predefine the normal vital state of the plant. The main function of leaves is the production of organic substances, which eventually accumulate in underground organs, and a study of the structure of this organ will determine the range of its ecological adaptability to completely different environmental conditions. The present study aimed to conduct a comparative study to detect the diagnostic signs in the morphological and anatomical structure of leaves of the individuals in the pre-generative age state F. tadshikorum in ex situ conditions.

Material and methods
F. tadshikorum grows in the middle mountain zone in the territories of the Kashkadarya and Surkhandarya regions of Uzbekistan on loess and finely ground soils and limestone, along dry river valleys and on river terraces, at altitudes of 1400-1800 m above sea level. The places of collection of seeds for subsequent cultivation under introduced conditions and a detailed description of the studied age states of Ferula tadshikorum were given by us in a previously published work (Khamraeva et al., 2019). The material for studying the morpho-anatomical structure of leaves of different ages was collected from experimental sites of the Tashkent Botanical Garden in 2019-2020 and preserved in 70% ethyl alcohol. The transverse sections of the leaf plate and petiole from the middle part of the leaves were stained with methylene blue and enclosed in glycerol gelatin according to the conventional method (Barykina, Chubatova, 2005). A Canon A 2300 was used to photograph the crosssections. Statistical processing of quantitative data was carried out by G.N. Zaitsev (Zaitsev, 1990) using MS Excel. For each anatomical parameter of the leaf blade and petiole, the arithmetic mean was calculated from 30 values.
The soil and climatic conditions of the Botanical Garden are described in detail in I.V. Belolipov (1989). The garden is located in the northeastern part of Tashkent at an altitude of 480 m above sea level. The soil is an ancient rusty typical gray soil. The climate of Tashkent is sharply continental, characterized by dryness, significant daily temperature fluctuations, hot summers, dry warm autumn and moderately cold winters. The absolute minimum temperature is... -25.8°С, the absolute maximum is... + 44.6°С. According to long-term data, the main rainfall is 380-440 mm, which falls during the autumn-winter-spring period. At the beginning of the vegetation period, watering and weeding were carried out twice in the experimental areas.

Morphological structure of the leaf
We have previously studied the initial stages of the ontogenesis of Ferula tadshikorum in the introduction and given a detailed description of the morphological structure of the aboveground and underground parts (Khamraeva et al., 2019). According to this data, the leaf of juvenile plants of the first year of life is simple, diamond-shaped, the plate is elongated, finely serrated along the edge, 4-5 cm long and 2-2.5 cm wide, the petiole 3.0-4.0 cm long. As it grows 1-3 (5) rosette leaves form on the rosette shoot (Fig. 1a). Rosette leaves are of various lengths, from 14 to 24 cm, with the leaf plate 9-12 cm long, 4-5 cm wide, and petioles 5-12 cm long. Immature individuals of the first year of life have 4-6 simple leaves, with one triple leaf 17-25 (28) cm long, lobes obovate or wide-oval, petioles 8-16 cm long (Fig.  1b). Immature plants of the second year of life develop 1-2 scaly leaves, 7-11 cm long, which quickly wilt after the appearance of rosette leaves. Rosette leaves range from 3 to 6, of which 1-2 are simple, and 2-3 triple-dissected. In some plants more complex 5-6-lobed leaves develop (Fig. 1c, d). Triple-dissected leaves show obovate or wide oval lobes, leaf plates 15-26 cm long, petioles 12-16 cm long, while 5-6-lobed leaves have elongated oval, ovoid or broad ovate segments, leaf plates are wide-triangular in outline, up to 27 cm long, the segments 10-16 cm long, 4-6 cm wide, the primary segments with short petioles, the others sessile, with petioles up to 14 cm long.

Anatomical structure of the leaves of the juvenile plant of the first year
On the cross-section, the sheet along the entire plate is unevenly thickened, platelike, bare ( Fig. 2 a, e). Collenchyma in the central vein area is located in the ribs, on the abaxial side of 9-15 rows, adaxial -5-6 rows, and in the lateral part -above and under the bundles, only on the abaxial side above large bundles are more multirow. The central vein area on the abaxial part shows three prominent widely spaced protrusions, and the adaxial part is closely spaced, three more or less prominent protrusions and is more thickened due to multilayer aquiferous parenchyma. In the area of large lateral veins, the leaf is biconvex (Fig. 2 d). The mesophyll consists of isolateral palisade. Under the epidermis on the abaxial side is a hypoderm single layer formed by rounded-oval cells. The palisade parenchyma on the abaxial side is 2-layered, with tabloid cells. On the adaxial side it is also 2-layered, the cells are more or less elongated (Table 1). The spongy parenchyma is 2-3-layered, with horizontally elongated homogeneous cells (Table 1).
The aquiferous parenchyma is only found in the central vein area, multilayered, consisting of up to 10 layers of various sizes thin-walled rounded-oval cells. The abaxial and adaxial epidermis are single-layered, their external walls are thickened, and both epidermises are covered with a toothed cuticle (Table 1). The central vein is represented by two vascular bundles, on the abaxial side an extensive bundle, and the adaxial part a horizontally oriented more minor (Fig. 2 b, c). Two lateral vascular bundles are located close by one above the other, and one vascular bundle is located in the side parts of the leaf in the central plane. A large main vascular bundle is represented by ten vessels, of which four are large. The vascular bundles are collateral. One secretory duct is found throughout the leaf above and below the vascular bundles, in the adaxial part with 6-7, in the area of the central vein with 10 epithelial cells, and in the abaxial part with 7-8, in the area of the central vein with 11 epithelial cells (Table 1). A large main bundle on the xylem side is accompanied by two small secretory ducts with six epithelial cells. At the edge of the leaf, the vascular bundle is surrounded by lining cells and there is one secretory duct on the abaxial part with 7-8 epithelial cells (Fig. 2 e).

Anatomical structure of the leaves of the immature plant of the first year
On the cross-section, the sheet along the entire plate is unevenly thickened, plateshaped, abaxially pubescent with simple hairs (Fig. 3 a, b). Collenchyma in the central vein area is located in the ribs, on the abaxial side 7-18 rows, adaxial 7-10 rows, and in the lateral part -above and under the bundles, more multi-row only on the abaxial side above large bundles (Fig. 3 b, c). In the central vein area on the abaxial part there are three widely spaced, strongly prominent and two more or less prominent protrusions, and on the adaxial part with three slightly spaced more or less prominent protrusions. In the central part, the leaf is even thicker due to the multilayering of the aquifer parenchyma than in juvenile plants, and is biconvex in the area of the lateral veins. The mesophyll has the isolateral palisades. Under the epidermis on the abaxial side is a single-layer hypoderm formed by rounded oval cells. The palisade parenchyma on the abaxial side consists of 2-layered tabloid cells. On the adaxial side, it is also 2-layered, consisting of elongated cells (Table 1). The spongy parenchyma is 2-3-layered, with horizontally elongated small cells (Fig. 3 d, e; Table 1). The aquifer parenchyma is found only in the central vein area, multilayered, consisting of thin-walled round-oval cells of various sizes in up to 15 layers. The abaxial and adaxial epidermis are single-layered, their external walls thickened, and both epidermises are covered with a toothed cuticle ( Table 1). The vascular bundles are of the collateral type. The central vein is represented by two vascular bundles, on the abaxial side by a large bundle, and the adaxial part is also a large double, from oppositely oriented bundles (Fig. 3 b, c). The double bundle consists of 13-15 vessels, of which seven are large; the second bundle of 8-10 vessels, of which five are large. Near the phloem there are two small secretory ducts with 5-6 epithelial cells. On two sides of the central vein one above the other there are two side bundles; on the adaxial side the bundle is inverted. One vascular bundle is arranged in the central plane of the side parts of the leaf. Large secretory ducts with up to 12 epithelial cells, and smaller ones with up to 6 epithelial cells are located above and below the lateral vascular bundles. In the central part of the leaf on the abaxial side, the secretory ducts have up to 11-13 epithelial cells, with adaxial up to 9-10 epithelial cells (Table 1). At the edge of the leaf, secretory ducts in the abaxial part with 8, and with adaxial -6 epithelial cells (Fig. 3 e).

Anatomical structure of the leaf of an immature plant of the second year
On the cross-section, the sheet is plate-shaped, rounded-triangular in the central vein area, pubescent on the abaxial side with simple hairs (Fig. 4 a). Collenchyma in the area of central vein is located in the ribs on the abaxial side, consisting of 9-20 rows, adaxial -7-18 rows, and in the lateral part above and under the bundles, on the abaxial side above the large bundles they are more multi-rowed (Fig. 4 b). In the central vein area, the leaf is thicker due to aquifer parenchyma than in immature individuals of the first year of vegetation, which is biconvex in the area of the lateral   veins. The mesophyll is isolateral palisade. Under the epidermis in the abaxial and adaxial parts there is a single-layer hypoderm with rounded-oval cells. The palisade parenchyma in the abaxial part is 2-layered, with shortened cells; in the adaxial part it also consists of 2 layers of elongated cells (Table 1). The sponge parenchyma is 4-5 layered, with horizontally elongated small cells (Table 1). The aquifer parenchyma is found only in the central vein area, with thin-walled rounded oval cells of various sizes in up to 22 layers (Fig. 4 c, d). The abaxial and adaxial epidermis are singlelayered, their external walls thickened, and both epidermises are covered with a toothed cuticle (Table 1). Vascular bundles of collateral type. The central vein is multi-bundles, represented by several large and small peripheral and one central vascular bundle. Peripheral bundles are located opposite the collenchyma strands and carry one secretory duct on the phloem side. On the phloem side, bundles are deep fibers, in large multilayer bundles (Fig. 4 b, d). There is one vascular bundle in the side parts of the leaf in the central plane (Fig. 4 e). Secretory ducts are located above and below the lateral vascular bundles, from the abaxial to 10 epithelial cells, from adaxial to 9 (Table 1). Vascular bundles in the central part of the leaf on the abaxial side have up to 10-11 epithelial cells, on adaxial up to 8-9 epithelial cells. The edge of the leaf on the abaxial side is one secretory duct with seven epithelial cells (Fig. 4 e).

Anatomical structure of the petiole of a juvenile plant of the first year
On the cross-section, the petiole is almost rounded, weakly ribbed, nominally on the adaxial side with two tubercles, strengthened by a collinear up to 10-12 rows (Fig. 5 a, c). Collenchyma strains from 7 to 12-13 rows, which alternate with 2 place three-layered chlorenchyma (Fig. 5 b). The epidermis is single-layered, covered with a cuticle, and the outer and inner walls are thickened. Under the epidermis lies a single layer of sub-epidermis whose cells contain chlorophyll grains. Around the periphery there are nine collateral-type vascular bundles, of which four are small, one is large and four are mediums. In the central part there is one vascular bundle of medium size. A large peripheral bundle carries ten large vessels and in the xylem part two secretory ducts with 6-7 epithelial cells ( Table 2). The peripheral vascular bundles from the phloem part have one secretory duct with 6-10 epithelial cells, which corresponds to the size of the bundles. The main part of the petiole is represented by thin-walled rounded-oval parenchymal cells. The part phloem of the central bundle has one secretory duct with six epithelial cells.

Anatomical structure of the petiole of an immature plant of first year
On the cross-section, the petiole is rounded ovate, ribbed, nominally on the adaxial part with a small recess, collenchyma in two tubercles up to 15-18 rows (Fig. 5 d,  f). The collenchyma in ribs has 8 to 20-22 rows, with large size above large bundles, smaller ones above small ones. Collenchyma strains alternate with chlorenchyma, which is a 3-4 layer. The epidermis is single-layered, covered with a cuticle, the outer and inner walls are thickened, and under it is a single-layer sub-epidermis, the cells of which contain chloroplasts. Peripheral tufts are 17, of which 11 are large, 6 smalls, all of the collateral type. There are two vascular bundles in the central part (Fig. 5 e). Above the phloem are bast fibers, above them secretory ducts with ten epithelial cells (Table 2). There are 1 or 2 secretory ducts with 6-8 epithelial cells in some large peripheral bundles of the xylem part. Above each peripheral bundle there is also one secretory duct with 10-11 epithelial cells, in small bundles up to 8. The main part of the petiole is represented by thin-walled rounded oval parenchymal cells.

Anatomical structure of the petiole of an immature plant of the second year
On the cross-section there is an ellipsoidal petiole. The epidermis is single-layered, the surface is covered with simple hairs, and the outer and inner walls are thickened. Under it is one layer of sub-epidermis with numerous chlorophyll grains. Collenchyma strains correspond to the sizes of peripheral bundles, which alternate with 4-5 layered chlorenchyma (Fig. 5 g, i). Vascular bundles are of the collateral type. Peripheral vascular bundles are represented by alternating large, medium and small numerous bundles. There are five parallel vascular bundles of various sizes in the petiole's central part, and perpendicular to the middle bundle 2 small side bundles (Fig. 5 h). Sometimes one of the central bundles is obliquely oriented concerning the rest of the central bundles. The deep fibers form caps above the phloem of the bundles; the central bundles are less multilayered. Above the peripheral bundles is one secretory duct with 10-12 epithelial cells. Numerous secretory ducts with 6-8 epithelial cells are located in the parenchymal part of the petiole. In large peripheral bundles on the xylem side there are two secretory ducts. Growing conditions or diagnostic, morphological and anatomical features of vegetative and generative organs of some species of the genus Ferula have been studied (Akalın et al., 2020;Safina et al., 2014;Wang et al., 2016, Khamraeva et al., 2018Sagyndukova, Imanbaeva, 2020). As a result of studying the anatomical structure of the above ground vegetative organs of the three ceno-populations of the rare and endemic species Ferula iliensis Krasn. ex Korovin found that in the stems developed mechanical and assimilation tissues located in alternating areas along the periphery directly under the epidermis, differing in volume among individuals from the different ceno-populations associated with different environmental con- ditions (Akhmetova, 2013). When studying the morphological features of leaves, inflorescences, fruits, and the anatomical structure of mericarp in Ferula caspica M. Bieb. and Ferula szowitsiana DC., diagnostic species-specific features of these structures were identified (Tuncay et al., 2019). For Ferula foetida, the diagnostic features of the leaf are the shape and structure of the epidermis cells, the presence of simple one-and multicellular trichomes, the placement of vascular bundles and the structure of secretory ducts in the leaf blade and petiole, which differ slightly in the leaves of different age individuals (Imanbaeva et al., 2015). According to S. Rakhimov and Kh. Rakhmonov (2015), in juvenile plants under natural conditions of Southern Tajikistan, in the second and third years, two scale-shaped and one diamond-shaped rosette leaves are formed annually on the rosette shoot. From the fourth to the eighth-tenth year of life in immature plants, 3-4 scaly, and 3-6 assimilating rosette leaves are formed on each annual rosette shoot, the edges of which are wavy, sometimes cut, leaves are simple, pinnately-dissected. However, as our study in ex situ conditions showed, the absolute majority of juvenile individuals (95-97%) of F. tadshikorum go through the immature phase of development in the second year of vegetation, when the individuals have 1-2 scaly and 3-6 rosette leaves, of which 1-2 are simple and 2-3 triple-dissected, or some still pinnately-dissected with 5-6-lobes. Perhaps, in ex situ conditions with good moisture availability, less insolation and the absence of sharp daily fluctuations in air and soil temperature typical for mountain conditions, there is a significant reduction in the initial stages of development of the virginial period associated with new soil climatic conditions of growth. S. Rakhimov, Kh. Rakhmonov (2015) noted in natural individuals of Ferula tadshikorum different leaves according to the morphological structure. Studies on the morphogenesis and anatomical structure of the leaves in the closely related species Ferula foetida in Kyzylkum showed in juvenile and immature plants an isolateral palisade type of mesophyll, an increase in leaf pubescence on the adaxial side and an increase in the number of layers of aquifer and palisade parenchyma in the second year of vegetation (Butnik et al., 2009). According to our results, Ferula tadshikorum also has such signs in the structure of the leaf blade, as an isolateral palisade type of mesophyll in juvenile and immature individuals, the absence of pubescence in juvenile plants and its amplification on the adaxial side in immature individuals, and characteristic features, such as the multiplicity of the central vein, the multilayering of the aquifer parenchyma and the presence of bast fibers above the phloem of the bundles of the central vein in immature plants of the second year, the structure and location of secretory ducts. The petiole of the leaf in juvenile individuals of the first year is smaller, with a straightforward blade, and in immature plants of the second year of life it has a large, dissected leaf blade, and is characterized by the robust development of collenchyme weights and vascular bundles. Xeromorphic features in the anatomical structure of the petiole are manifested in the development of wide and narrow collenchyma strains in the ribs but differing in different age individuals in the number of rows, the thickening of the outer walls of the epidermal cells in juvenile individuals, which are most susceptible to stress-  ful environmental conditions, as well as in the development of additional central vascular bundles. The volume and number of central bundles increase with age. It should be especially noted that deep fibers are formed over the phloem of peripheral bundles and the phloem of central bundles in immature plants of the second year. Diagnostic features of the petiole are the shape on the cross-section of different-age individuals, which in juvenile plants are almost rounded, with two tubercles conventionally on the adaxial part, in immature plants of the first year are round ovate, with two tubercles, and in immature plants of the second year ellipsoidal. According to Kh. Rakhmonov (2017), the rosettes of shoots in species of the genus Ferula were formed quite early, when they developed in open habitats. This feature turned out to be adaptive in the xerophilic line of evolution of the genus Ferula. In this regard, the internal structure of the sheet is being rebuilt by the transition to an isolateral palisade structure, compaction of spongy tissue or reduction in the central vein area due to high air temperature differences during the day intense insolation in mountain conditions.
The results of our study showed that when plants transition from one age state to another, morphological changes in their structure, including the internal structure of the assimilated organ occur. For example, in the juvenile and immature state, the main elements of the leaf 's cover, mechanical and vascular tissues are laid, which are more powerfully developed in immature plants.