AGRICULTURAL BIOLOGY, ISSN 2412-0324 (English ed. Online) 2018, V. 53, ¹ 3, pp. 624-633 (SEL’SKOKHOZYAISTVENNAYA BIOLOGIYA) ISSN 0131-6397 (Russian ed. Print) ISSN 2313-4836 (Russian ed. Online)

UDC 633.811:582.71:581.3 doi: 10.15389/agrobiology.2018.3.624eng doi: 10.15389/agrobiology.2018.3.624rus

SOME FEATURES OF EMBRYOLOGY OF Rosa spinosissima L., R. canina L. AND R. ½ damascena Mill. INTACT AND VIRUS-INFECTED

S.V. SHEVCHENKO, T.N. KUZMINA

Nikita Botanical Gardens — National Scientific Center RAS, Federal Agency for Scientific Organizations, 52, ul. Ni- kitskii spusk, pgt Nikita, Yalta, Republic of Crimea, 298648 Russia, e-mail [email protected] ( corres- ponding author), [email protected] ORCID: Shevchenko S.V. orcid.org/0000-0002-0534-5548 Kuzmina T.N. orcid.org/0000-0002-4218-6080 The authors declare no conflict of interests Acknowledgements: Supported financially by Russian Science Foundation (grant ¹ 14-50-00079) Received April 5, 2017

A b s t r a c t

Due to high decorative and economically valuable traits, have long been used in gar- den and park construction, and as medicinal and aromatic raw materials. Selection carried out with roses for many years, has resulted in more than 30 thousand cultivars. To create new forms and vari- eties, it is necessary to in-depth know species biology, including developments of generative ele- ments. This paper shows the study of male and female generative structure formation in two wild species of Rosa genus, Rosa spinosissima L. and R. canina L., naturally grown on the north-eastern slope of Mount Chatyr-Dag, and in two R. ½ damascena Mill. cultivars, which may be involved in breeding as parental plants. As a result, we have determined the main embryological types of genera- tive sphere formation in these species. It has been shown that the anthers are four-nested with two thecas, the wall of microsporangium is formed centripetal. A formed wall of microsporangium con- sists of epidermis, endothecium, 2-3 middle layers and tapetum, which is a derivative of a secondary parietal tissue. A tetrad of microspores is formed simultaneously. Differentiation mitosis in microspores leads to the formation of 2 celled pollen grains. Mature pollen grains are 2 celled with 3 pores. Micro- sporangium wall of the mature anther is represented by the epidermis covered with cuticle, fibrous endothecium and remnants of a middle layer. Morphogenicity and viability of mature pollen of these species and varieties have been established (abnormal pollen grains can be up to 30 %). Pollen sizes were measured. The diameter of pollen grains varies from 19 to 30 microns. The influence of viral infection on the formation of pollen grains has been shown, which causes an increase in the number of pollen abnormalities, changes the pollen grain size and ultimately leads to pollen grain degenera- tion. Defective pollen grains can be up to 50 %. Gynoecium of consists of many free carpels, each contains 1-2 ovules. Rosa ovule is anatropous, bitegmal, crassinucellate. Multicellular hypostase with thick-walled cells is formed in chalasa area. Micropyle is formed by an internal integument, which in its development is ahead of the outer one. Archesporium is polycelled and differentiated in the subepidermal layer of ovule. As a result of meiosis, linear tetrads of megaspores are mostly formed, of which chalasal or epichalasal megaspores were functionally active to derive embryo sacs of Polygonum-type. Mature embryo sacs are 8 nucleate and 7 celled. Egg apparatus consists of egg and 2 synergids, antipods are represented by three cells. Majority of embryo sacs gradually are obliterated, and only one of them matures. The studied species of genus Rosa are entomophilous plants pollinated by insects. It has been concluded that in selection, while choosing parental forms for hybridization, attention should be paid not only to the viability of their generative structures, but also to viral infection, since the presence of pathogens has led to a significant decrease in reproductive ability.

Keywords: Rosa sp., male and female generative structures, viral infection, reproductive ability.

Due to the variety of forms, abundant and long flowering, the rose has long been used in garden and park construction. The first information about the cultivation of roses was found for 4 thousand years BC; later, more than 30 thou- sand cultivars with decorative [1-3] and valuable medicinal characteristics [4-7] were created. Nowadays, the range of roses resistant to environmental stress fac- tors, diseases, and pests is expanding [8-10]. Cytological and genetic investigations 624

have shown the possibility to obtain polyploid forms [11-13]; the in vitro culture method has been widely used for the cultivation and reproduction of valuable gen- otypes of roses in recent decades [14-17]. It is important to mention that different species of the Rosa genus [18-20] can be used as medicinal plants. The Rosa genus has a wide geographic range, is widely studied and actively used in hybridization [21-24]. The genus includes 10 sections, including Pimpinel- lifoliae and Caninae, the representatives of which, along with others, serve as the original forms of garden varieties [6, 25-28]. The active development of industry and agriculture, the increase in air and soil pollution often lead to abnormal development of the generative sphere of plants; therefore, when conducting breeding, careful selection of parental forms is necessary. In this paper, the authors have presented the results of the investigation of the processes of male and female gametophyte formation in two prospective initial forms used in roses selection, Rosa spinosissima L. and R. canina L., and two high-yielding varieties of essential-oil-bearing rose (R.½ damascena Mill.), Dzhalita and Festivalnaya. In addition, the quality of pollen among visually healthy and virus-infected plants was assessed. The aim was to identify the features of the development and construc- tion of generative structures for the representatives of the three species from the Rosa genus in normal and viral infection. Techniques. Collection of samples from Rosa spinosissima and R. canina for making permanent preparations and observation of blooming were carried out in Crimea in 2016 under natural vegetation of the studied species in different populations on the North-Eastern slope of the Chatyr-Dag mountain at the alti- tude of 750-868 m above the sea level. The biomaterial of Dzhalita and Festival- naya cultivars (R. ½ damascena) was taken in the collection of Nikitsky botanical garden. To make permanent cytoembryological preparations, buds at different stages of development were fixed in the Carnoy mixture containing 96% aqueous solution of ethyl alcohol, chloroform, and glacial acetic acid in the 6:3:1 ratio. The material was dehydrated with butyl alcohol and xylene and then enclosed in paraffin [34, 35]. Paraffin serial sections with the thickness of 10-12 microns were prepared with a rotary microtome MRTU (Russia). The preparations were stained with methyl green-pironin with additional staining with Alcian blue [38] and viewed (light microscopes Jenaval and AxioScope A. 1, Carl Zeiss Micros- copy GmbH, Germany). PowerShot A550 digital camera (Canon, Inc., Malay- sia) and AxioCamERc5s image analysis system (Carl Zeiss Microscopy GmbH, Germany) were used for documentation. Temporary preparations of average pollen samples colored by 1% aceto- orcein were used for cyto-morphological characterization of pollen grains of R. canina, R. spinosissima, and R. ½ damascena. Morphologically normal and defective grains were counted. The first group includes the grains with homoge- neous coloring, a clearly expressed core of the vegetative cell, and the generative cell. Pollen grains with visual signs of cytoplasm and nuclei destruction of vege- tative and generative cells were considered defective. Their percent was determined as a ratio of the number of pollen grains with abnomalities to the total number of the recorded pollen grains for every species or variety. The di- ameter of pollen grains was measured on permanent preparations of average pol- len samples [37] (AxioVision Rel.4.8 software, Carl Zeiss Microscopy GmbH, Germany). Statistically significant differences in the proportion of defective pollen grains from anthers of R. ½ damascena with visual signs of infection and asymptomatic ones were determined by φ-angle transformation of Fischer (Fφ) 625 if p < 0.05 [36]. Data processing, with descriptive statistics and comparison of samples on the diameter of plants pollen grains with and without viral infection, was carried out with Statistica 6.0 (StatSoft Inc., USA), using Student's t- criterion. The arithmetic mean values (M) and standard errors of means (±SEM) are presented. Results. Rosa spinosissima L. (syn. Rosa pimpinellifolia L.) is a shrub up to 2 m with prickly shoots. Buzzes are of different sizes, thin, straight, usually ex- tended at the base. Leaves are with 5-11 folioles and naked stipules. are single, on long pedicels, sepals are simple, up to 15 mm long, remaining with fruits. Corollas are white, up to 5 cm in diameter, petals are sinuate, hypanthia are globular, baculums form a large head of the stigma. Fruits are up to 15 mm long, spherical or flattened-spherical, ripened, of dark color (up to black). De- spite the rather extensive geographic range, the population size of R. spinosissima is reduced, so it was listed in the Red Books of some regions, such as Kursk Re- gion and the Republic of Khakassia [29, 30]. R. spinosissima is the ancestor of many varieties, in particular, the extensive group of Scott's roses, which are known since 1600 and, according to modern ideas, belong to the Hybrid Spi- nosissima class [31]. In the past, many authors examined R. spinosissima and R. pimpinellifolia as different species, but molecular studies of samples from natu- ral populations of Great Britain have shown that they are of one species [10]. Rose varieties selected on the basis of R. spinosissima are resistant to diseases and frost [32]. Rosa canina L. is a deciduous shrub, reaching a height of 2.0-2.5 m. Shoots are thick, arched curved. Buzzes are rare, sickle-shaped, with a very short base. Flowers are single or 3-5 flowers collected in the apical cymose inflo- rescence. The corolla is white or bright pink, with a diameter of 5-8 cm; sepals are broad-lanceolate, with abundant pinnate appendages, bend back after - ing and fall off long before fruits ripening [33]. The fruits are smooth, glossy, orange-red with many pilose seeds. Rosa ½ damascena is a hybrid form of R. gallica and R. moschata. DNA analysis also showed the presence of the third species, R. fedtschenkoana, in the genealogy of this hybrid. The Dzhalita variety is a shrub reaching a height of 2 m; flowers are pink with a lilac hue, 6-7 cm in diameter with soft orange-yellow petals, have a delicate aroma. The Festivalnaya variety is a shrub up to 170 cm of height, with pink or pale pink flowers, 5-6 cm in diameter, with a strong aroma. All the studied wild species and varieties are entomophilous plants, pol- len of which is carried by insects. As a result of effective processes of pollination and fertilization, aggregate accessory fruits formed by stone fruits enclosed in fleshy hypanthiums, bright color of which attracts birds, develop. The rose blooms in May-June in the Crimea. Flowers are actinomorphic, ambisexual, 5- membered, R. spinosissima and Dzhalita and Festivalnaya have fragrant flowers, R. canina flowers are odorless. The androecium is represented by a great number of stamens, located in circles. First, the outer stamens were opened, then the inner ones. Anthers are 4-locular, 2-sided. The placentoid juts out inside the microsporangium. The microsporangium wall is formed centripetal. First, the archesporial cell differentiated in the subepidermal layer, which formed the pri- mary parietal and sporogenous cells by fission. The latter forms multi-layer spo- rogenous tissue. The parietal cell forms secondary parietal cells by division, one of which produces tapetum, and the other, continuing to divide, gives endothe- cium and middle layers. The formed microsporangium wall consists of the epidermis, endothecium, 2-3 middle layers, and tapetum (Fig. 1, B). After all divisions in the sporogenous tissue, the cells form a separate structure, surround- ed by deposited callose, and microsporocytes appear in which meiosis and simul- taneous formation of the microspore tetrad occur. The arrangement of micro- 626

spores in the tetrad, like in species of Malus genus [35], is tetrahedral and isobi- lateral (see Fig. 1, C, D).

A B

C D

E Fig. 1. Fragments of the microsporangium of Ro- sa spinosissima L. at different stages of develop- ment: A — division of archesporial cells, B — the formed microsporangium wall, C, D — mi- crospore tetrads, E — differentiating mitosis (the preparations are colored with methyl green- pironin followed by Alcian blue, a microscope AxioScope A.1, Carl Zeiss Microscopy GmbH, Germany).

The tapetum is single-layer; its cells are large, binuclear. The first signs of degradation of the tapetum

cells are observed during meiosis and formation of microspores tetrad, and their full lysis occurs during formation of pollen grains. The cells of the middle layer, adjacent to tapetum, degenerate, the cells of the second layer become flat (see Fig. 1, E). Outer stamens outgrew inner ones. When the outer anthers con- tained 2-cell pollen grains, tetrad decay was observed in the inner ones. At the stage of differentiating mitosis, the microsporangium wall consists of epidermis covered with cuticle, endothecium with fibrous thickenings on the walls of cells, and flattened cells of the middle layer (see Fig. 1, E). The microsporangium wall of the mature anther contains epidermis cov- ered with cuticle, fibrous endothecium and the remnants of the middle layer (Fig. 2, A, B). Mature pollen grains are 2-cell; most of them were morphologi- cally normal, but some were defective. Among R. canina the number of defective pollen grains could be up to 30%, among R. spinosissima approximately 15% (Table 1). Male germ cells formed in pollen tubes when they germinated through 627 the tissues of the baculum.

A B

C D

E F

G Fig. 2. Fragments of the microsporangium and megasporangium of Rosa canina: A, B — fragments of the microsporangium in mature anthers with pollen (a micro- scope AxioScope A.1, Carl Zeiss Mi- croscopy Gmb", Germany), C — arche- sporial cells in ovules, D — division of archesporial cells, E, F — meiosis in megasporocytes, G — functioning epi- chalasal megaspore (a microscope Je- naval, Carl Zeiss Microscopy GmbH, Germany). Preparation staining with methyl green-pironin followed by Alci- an blue, ½400 magnfication.

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1. Pollen formation in different cultivars of roses with a viral infection and without its symptoms (the Republic of Crimea, 2016) Total number Morphologically nor- Defective PG Species, cultivar F of PG, pcs mal PG, pcs Total, pcs % φ Rosa canina 3195 2230 1065 31.4 R. spinosissima 2390 1050 340 14.3 R. ½ damascenа: Dzhalita no symptoms 1030 730 300 29.0 13.64 infection 3260 1880 1380 42.3* Festivalnaya no symptoms 1240 870 370 29.8 204.39 infection 2400 1100 1300 54.1* N o t e. PG — pollen grains, F — the value of the Fisher criterion obtained with the use of the method  [36]. Gaps mean that this indicator was not determined. * Differences between the number of morphologically normal pollen grains when infected with viruses and without symptoms of infection are statistically significant at p < 0.05.

Rose gynoecium consisted of many free carpels, containing 1-2 ovules per each. The pistil is straight; the baculum is with a cephalated stigma. The ovule is quite large, anatropous, two-sided, crassinucellate. The multicellular hypostasis with thick-walled cells formed in the chalazal zone. The micropyle was formed by the internal tunicle, which in its development was ahead of the external. Rosa sp. has multicellular archesporium as well as many other species of [40]. As a result of the archesporium division, parietal and sporogenic cells are formed (see Fig. 2, C, D), the latter is transformed into a megasporo- cyte, and the nucellus develops from the parietal cell. As a result of meiosis (see Fig. 2, E, F), ordered tetrads of megaspores are formed. Chalazal or epichalasal megaspores usually were the functioning ones (see Fig. 2, F), from which 7-cell 8-nuclear polygonum-type embryo sac developed. Several embryo sacs could be in the ovule, but only one developed to the mature condition.

A B

Fig. 3. The leaves of essential-oil-bearing rose (Rosa damascena) of the Dzhalita (A) and Festivalnaya (B) varieties with symptoms of viral infection (discoloration and wrinkles) (the Republic of Crimea, Nikitsky botanical garden, 2016).

Biogenic and abiogenic stress factors, including virus damage, affect the physiological and biochemical parameters of plants, which violate the metabolic balance and affect the anatomical and morphological characteristics. Changes in leaf blade traits, such as spotting, wrinkling, and dwarfism, are used for visual assessment of plant infection and its viability in general [41-44]. The state of the male gametophyte, characterized by the morphological maturity of pollen grains, 629 is one of the most accessible and effective signs, giving an idea of the degree of stress effects of various factors on the plant and its viability [43, 44]. Previously, the diagnosis of viral pathogens in the plant material of essential-oil-bearing rose (R. ½ damascena) revealed that most of the selected material (up to 70%) was affected by viral pathogens of different origin [45]. The external manifestation of virus infection is most noticeable on the leaves and flowers with wrinkles, inter- veinal chlorosis, and spotting of different nature (Fig. 3), which was the result of changes in the state of their internal structures. 2. Morphometric parameters of pollen grains of Rosa canina and R. ½ damascena with a viral infection and without its symptoms (the Republic of Crimea, 2016) Species, cultivar Plant condition M±SEM, μm min-max, μm σ Сv, % t R. canina No symptoms 30.88±0.18 24.18-38.45 2.41 7.80 26.140 Infection 22.91±0.25 19.16-39.92 2.37 10.34* R. ½ damascena: Dzhalita No symptoms 24.69±0.11 20.18-30.62 1.70 6.89 6.430 Infection 25.76±0.13 21.21-35.12 2.01 7.08* Festivalnaya No symptoms 24.56±0.11 20.40-31.65 1.76 7.17 0.096 Infection 24.55±0.10 19.91-30.25 1.62 6.60 N o t e. M —the arithmetic mean, SEM — standard error of the mean, min-max — minimum and maximum values of the selection, σ — the mean square deviation, Cv — coefficient of variation, t — Student criterion at n = 130. * Differences in the diameter of the pollen grains of the average samples when infected with viruses and without symptoms of infection are statistically significant at p < 0.05.

Morphometric analysis of the male generative structures of the virus- infected and visually clean plant of essential-oil-bearing rose varieties, and R. canina showed that infection leads to the increase in the number of abnormal pollen grains and change in their sizes (see Tables 1, 2). Among plants without symptoms of infection, more morphologically normal pollen grains were formed, and among plants with signs of infection, the proportion of abnormal and defec- tive pollen grains was increased, which may indicate potential violations in the genesis of particular elements of the flower. Thus, according to the main embryological features, Rosa spinosissima and R. canina, as well as the studied varieties of R. ½ damascena, are similar to other members of Rosaceae family and especially of Rosoideae subfamily. They are characterized by the centripetal development of the microsporangium wall, the simultaneous type of formation of microspore tetrad, 2-cell pollen grains, multicellular archesporium, and the formation of several embryo sacs in the ov- ule, most of which obliterate at different stages of development. The viability of the generative structures of the species presented in this paper can provide a normal reproductive process, which is very important for their use in selection in order to obtain stress-resistant varieties. During selection of the initial forms for hybridization, it is necessary to pay attention not only to the viability of the gen- erative sphere of plants but also to their infection because the presence of patho- gens in the plant leads to the decrease in its reproductive ability.

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