Biometric characteristics of fruits and leaves of Cornus officinalis Siebold et Zucc. genotypes in the M.M. Gryshko National Botanical Garden of the NAS of Ukraine

In the context of global climate change, the current strategy of agroeconomics focuses on the introduction of unique plant species and the selection of new commercially important cultivars adapted to the dramatic weather changes. Cornus officinalis (Cornaceae) has Chinese origin, its reintroduction at the M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine started in 1993. The objectives of this research were: to investigate the biometric parameters of fruits and leaves of C. officinalis genotypes, C. officinalis × C. mas hybrid ‘Etude’ and genotype from the grafting C. officinalis on C.  mas under cultivation in the M.M. Gryshko National Botanical Garden, National Academy of Sciences of Ukraine, and to determine the degree of adaptation of C. officinalis to the climatic conditions of Ukraine (in particular, Right-Bank Forest Steppe) for selection of promising genotypes for further breeding work. Material and methods. We used: a) 26-year-old maternal plant obtained from a two-year-old seedling in 1993 received from the nursery “Northwoods Wholesale Nursery” Mollala (Oregon, USA), where it was grown as an ornamental plant; b) seedlings of the maternal plant; c) cultivar Etude, which is an artificial hybrid from crossing C. officinalis × C. mas; and d) genotype obtained from grafting C. officinalis on C. mas. In our experiment, the maternal plant is indicated as G-01, while other plants – as G-02–G-08 genotypes. We determined the biometric parameters of the fruit (length, diameter, and weight), endocarp (length, diameter, and weight), pedicel (length and thickness), leaf blade (length, width, and the number of lateral veins) and petiole (length, width, and thickness). We examined the dynamics of fruit and endocarp formation during the season (genotypes G-01–G-03 and G-05) and compared the biometric characteristics of the fruit of genotypes G-01–G-05 from crops of two years, 2010 and 2018, which were most favorable in weather conditions. We have processed quantitative data in the PAST 2.10 software. The differences between the samples were estimated using the Tukey-Kramer test. The degree of variability was determined by the coefficient of variation. To assess the level of variability, we used the classification of Mamaev (1975). Results. We have found that the largest fruits in 2010 were observed in the genotype G-01 and the smallest – in the genotype G-03. The coefficient of variation of the linear parameters of the fruit and endocarp was in the range 5.7–10.1 %; the level of variability was very low or low. The variability of fruit weight and endocarp was high; the coefficient of variation was from 7.0 up to 28.3 %. The amplitude of the linear parameters of the leaf was wide (coefficient of variation was from 9.8 to 31.0 %). The cultivar Etude differed from other C. officinalis genotypes in size and weight of (M = 1800 mg, max = 2400 mg) and a much wider amplitude of variation in the length (17.9–22.6 vs. 14.3–18.2 mm) of the fruit. The largest leaves were in the genotypes G-08 and G-01, and the smallest – in the genotype G-02. The cultivar Etude did not differ so much by the mean leaf morphometric indices and number of veins, but it demonstrated one of the broadest leaf blades (51.5 mm). https://doi.org/10.46341/PI2020006 UDC 575.22:582.788.1:581.45+581.47(477:282.485)


Introduction
Modern climate change is related to rising temperatures, increasing carbon dioxide concentrations, the widespread melting of snow and ice, and raising the world's ocean level (Schneider, 2004;Shindell, 2007;Ceccarelli et al., 2010). Droughts have increased in Ukraine over the past 15 years, both in intensity and frequency, that affected yields. Further climate change may result in increased temperature extremes, especially in the southern part of Ukraine (Fileccia et al., 2014). One of the ways of adaptation to adverse climate changes is the minimization of the risk of crop losses by the introduction of new plant species and the selection of new cultivars resistant to sudden weather changes, heat shock and drought (Crossa et al., 2017;Taunk et al., 2017;Ortiz, 2018). In this aspect, Cornus L. species, the introduction, and acclimatization of which have already begun in the M.M. Gryshko National Botanical Garden of the NAS of Ukraine (NBG) are very promising (Klymenko et al., 2017a, b).
The genus Cornus is relatively uniform in flower structure and leaf morphology, but very heterogeneous in the structure of inflorescences and morphology of bracts and fruits (Murrell, 1993;Zhang et al., 2013;Murrell & Poindexter, 2016). This fact results in the delimitation of four to ten sections and subgenera, which sometimes are considered as separate genera (Wangerin, 1910;Poyarkova, 1951;Takhtajan, 1987).
Cornus s. str. comprises four species: C. chinensis Wangerin (native to Central China), C. sessilis Torr. ex Dur. (naturally growing in the western part of North America (California and Oregon), C. officinalis Siebold et Zucc. (aboriginal to the north of Central and South-Eastern China, where occurs in forested areas at 400 to 1500 (2100) m altitude; also cultivated in Japan and Korea) and C. mas L. (the natural range includes Southern and partly Central Europe to the Balkans, Asia Minor, and the Caucasus). In Ukraine, C. mas grows naturally Biometrics of Cornus officinalis genotypes in the M.M. Gryshko National Botanical Garden in the Crimea, as well as on the narrow Transnistria strip from the western border of Ivano-Frankivsk to the northern part of the Odesa region. In addition to the Crimean range, C. mas is distributed in mountain forests and hillsides in Transcarpathia. One such locality is in the vicinity of Botar village and covers the area of about 30 hectares. It is one of the largest natural populations of C. mas not only in Ukraine but also in Europe (Fodor, 1974). In some areas of Cherkasy and Kirovograd regions, there are isolated natural locations, and relict 'cornelian cherry hills' have been preserved along the Dnipro River (Kleopov, 1990).
Cornus chinensis and C. sessilis are difficult for cultivation and are almost not applied in selection work (Weaver, 1976). However, C. chinensis is used in Chinese folk medicine in the same way as C. officinalis (Xiang & Boufford, 2005). C. mas is one of the most studied species of the genus and, at the same time, one of the most valuable fruiting representatives of the Cornaceae family (Klymenko et al., 2017a(Klymenko et al., , b, 2019. Its cultivated habitat covers almost all Europe, partly North America and other regions. C. mas is widely cultivated in private farms in Austria, Italy, France, Poland, Czech Republic, Slovakia, and especially in Bulgaria. It has been used for hundreds of years as a medicinal plant in Bulgaria, and many varieties have already been created there (Klimenko, 2004;Klymenko et al., 2017a, b).
In Ukraine, cornelian cherry trees are common in the private and farm gardens of Zakarpattia, Ternopil, Vinnytsia, Cherkasy, Zaporizhia, Khmelnytskyi, Poltava, and Kyiv Oblasts and Crimea. The northern border of the successful fruiting cornelian cherry passes through Chernihiv -Hlukhiv. In Ukraine, the cultivars of C. mas are widely introduced in horticulture (Klimenko, 2004). In particular, the most significant gene pool of cultivars and forms of this species (30 cultivars and about 100 forms) was collected at the NBG. Hence, the most cornelian cherry assortment in Ukraine consists of cultivars that originated from NBG breeding.
Asian-originated species C. officinalis is not widespread outside the natural range. In Korea, and especially in Japan (locally called sandzaki), medicinal cornel is cultivated for edible fruits and as a medicinal plant (Cao et al., 2016;Li et al., 2012). Since the 1870s, it has also been cultivated, mostly in botanical gardens, in Europe (Czerwińska & Melzig, 2018) and North America (Ferguson, 1966;Xiang & Boufford, 2005). It is interesting that in the UK, C. officinalis is more frequently cultivated as a garden plant than C. mas (Klymenko et al., 2017a).
In China, C. officinalis has been grown and widely used in traditional medicine for thousands of years (Czerwińska & Melzig, 2018). And now, it is cultivated there as a nationally protected medicinal plant thanks to its medical and commercial importance (Li et al., 2012). In Japan, plantings of seedlings of wild and cultivated genotypes are used to harvest medicinal raw materials. The cultivation of plants beyond their natural growth is of great importance. It is based on the individual variability of these plants. Therefore, valuable forms were selected from nature and transferred to the culture. The diversity of the chosen forms is explained by the fact that the plantations in amateur gardening are mostly represented by seedlingderived material, which is heterozygous. Taking into account that cornelian cherries are cross-pollinated plants, the seeds of the original forms usually do not preserve in such collections.
Pharmacological studies showed that vegetative raw materials of C. officinalis have neuroprotective, anti-diabetic, antiinflammatory, antioxidant, and cardiovascular effects. The biochemical composition and medicinal properties of C. officinalis fruits have been extensively studied, especially in Japan, Korea (Krośniak et al., 2010;Kryvoruchko & Kryvoruchko 2018) and China (Cao et al., 2016;Ji et al., 2019). There was almost no breeding work for this species. Only in China explorations on the phenotypic and genetic diversity of local cultivars and wild genotypes are conducting (Li et al., 2012). Before the 1990s, the collection, evaluation, conservation, and use of C. officinalis germplasm had a high scientific priority. The Laboratory of Development of Endangered Chinese Raw Narcotic Resources (North-Western China) has now collected 73 samples of nine germplasm types. Morphology and phytochemistry confirmed the differences among these germplasm types (Li et al., 2012), and, hence, are promising tools for their investigation.
For Ukraine, C. officinalis is a new and promising food and medicinal plant. It is cultivated only in the botanical gardens, in particular in the O.V. Fomin Botanical Garden of Taras Shevchenko National University of Kyiv. The introduction of C. officinalis in the NBG is closely related to the establishment of the collection of Cornaceae at the Dendrology Department, considering the possibility of its comprehensive application (i.e., as fruiting, medicinal, ornamental and timber plants). C. officinalis was a part of the first batch of Cornus species, the mass planting of which began in early 1949. Unfortunately, in subsequent years, this collection, including C. officinalis, died due to an incorrectly selected planting place and lack of irrigation (at the early stages it is essential), which caused insufficient winter hardiness and, finally, death of plants. Later, in 1993, C. officinalis was successfully reintroduced at the NBG. Since then, comprehensive and intensive introductory, biological and ecological studies of this species are carrying out at the Department of Acclimatization of Fruit Plants.
The morpho-biological features of C. officinalis and C. mas are similar, with differences in leaves and fruits. The fruits are similar in shape and color. However, fruits of C. officinalis are smaller but remain on the plant for a long time after full ripening. The leaves of C. officinalis are glossy, dark green, rounded, or rounded ovate with elongatedpointed and often twisted apex, wedge-shaped base, and tufts of brown hairs in the corners of the largest lateral veins. In C. mas, leaves are bluish-green, oval, or elliptical with a gradually pointed apex and a rounded or wedge-shaped base and tufts of colorless hairs in the corners of the largest lateral veins.
The NBG is currently breeding this species to use the results of spontaneous intraspecific variation in the selection of the most promising forms. The work is focused on obtaining the interspecific hybrids of C. officinalis × C. mas that produce large long-maturing fruits.
In particular, at the NBG, C. officinalis plants were artificially pollinated with C. mas pollen (a mixture of pollen of 'Lukyanovsky' and 'Olena' cultivars) and thus obtained a cultivar Etude.
For the mass vegetative propagation of cultivars of cornelian cherry, different species of the Cornaceae family were tested as rootstocks. The best compatibility for vegetative propagation has been found in C. mas and C. officinalis. Ideal growth of grafted components of both C. mas on C. officinalis and C. officinalis on C. mas (85-90 %) was observed, which is explained by their close phylogenetic affinity. Other Cornaceae species (C. kousa Bürger ex Miq, C. florida L., C. nuttallii Audubon, Swida spp.) were found incompatible with C. mas and C. officinalis.
At the NBG, C. officinalis plants and cultivar Etude bloom profusely and produce fruits. They are undemanding to cultivation conditions, grow successfully on various soils (from acidic to alkaline), and are characterized by high winter resistance. These plants are also highly decorative (Klymenko et al., 2017a).
The introduction experiment on the acclimatization of C. officinalis in Ukraine continues. Present work aimed: a) to investigate the biometric parameters of fruits and leaves of C. officinalis genotypes, C. officinalis × C. mas (cv. 'Etude') and genotype from the grafting C. officinalis on C. mas under cultivation in NBG; and b) to determine the degree of adaptation of C. officinalis to the local climatic conditions (in particular, Right-Bank Forest Steppe of Ukraine) and selection of promising genotypes for future breeding work.

Biological material
For the study we used: a) 26-year-old maternal plant obtained from a two-year-old seedling received in 1993 from the nursery "Northwoods Wholesale Nursery" Mollala (Oregon, USA), where it was grown as an ornamental plant; b) seedlings of the mother plant; c) cultivar Etude, which is an artificial hybrid from crossing C. officinalis × C. mas; and d) genotype obtained from grafting C. officinalis on C. mas. In our experiment, the maternal plant is indicated as G-01, while other plants as G-02-G-08 genotypes respectively (Table 1).

Biometric analysis
The following quantitative traits were used to test selected genotypes: length, diameter, and weight of the fruit and endocarp; length and Biometrics of Cornus officinalis genotypes in the M.M. Gryshko National Botanical Garden width of pedicel; length, width, and a number of lateral veins of the leaf blade; length, width, and thickness of the petiole. We examined the dynamics of fruit and endocarp formation during the season (for genotypes G-01-G-03 and G-05) and compared the biometric characteristics of the fruit of genotypes G-01-G-05 from crops of two years, 2010 and 2018, which were the most favorable in weather-climatic conditions for fruiting plants. The morphometric parameters of the leaf were examined in the same genotypes with the exception of G-04.

Statistical analysis
We have processed quantitative data using the free data processing software PAST 2. 10 (Hammer et al. 2001). We used the main descriptors to characterize all the samples: the arithmetic mean and its error (M ± m), the minimum and maximum values (minmax), and the coefficient of variation (CV, %). The differences between the samples were estimated using the Tukey-Kramer test. The degree of variability was determined by the coefficient of variation. To assess the level of variability, we used the classification of Mamaev (1975).

Results and discussion
Fruit biometric parameters Genotypes of C. officinalis (Table 2; Fig. 1). In 2010, the average length of fruits varied from 13.8 to 16.5 mm, and diameter from 7.7 to 10.5 mm. The average length of endocarp was 12.2-13.5 mm, and average diameter -4.6-5.4 mm. The average length of pedicels was 7.7-10.2 mm, and average width -0.2-0.6 mm. During October 2010, in G-01-G-03 genotypes, the mean value of fruit size and endocarp increased by increasing the number of large fruits. At the end of the autumn season of 2010 (03.11.2010), the largest fruits were in the maternal plant (genotype G-01), and significantly smallest -in the genotype G-03. Under favorable weather conditions, fruits of G-01, G-02, G-03, and G-04 genotypes were even larger, and the amplitude of traits shifted toward higher values (      dimensions of the fruit and endocarp were stable. Their coefficient of variation (CV) in most genotypes did not exceed 7 %, which corresponds to a very low level of variability. Rarely CV varied from 7.1 % to 10.1 %. The average values of fruit weight and endocarp of the C. officinalis genotypes were 600-1000 and 200-300 mg, respectively. The amplitude of variation in fruit weight in 2010 was 400-1800 mg, and in 2018 it shifted toward larger values, but the magnitude of variation was smaller, 1000-1700 mg. Endocarp weight varied within 100-400 mg. The lowest weight, 200 mg, was fixed for the genotype G-02. In 2010 (03.11.10), CV for the fruit weight was 11.8-20.1 % (mean variability), and in 2018 (01.11.18), it decreased significantly to 8.4-9.4 % (low variability). The smallest variation was observed in fruit weight, while endocarp weight was characterized by a high level of variability with a CV reaching 7.0-27.9 %.
The cultivar Etude, within average values of the size and weight of its fruits, was almost indistinguishable from maternal plant and seedlings of C. officinalis (Table 2). However, the fruits of 'Etude' in 2018 were much larger (length reached 19.9 mm, and diameter -11.1 mm) than those of the maternal plant and seedlings. The cultivar Etude significantly differed by increased variation in fetal length (17.9-22.6 vs. 14.3-18.2 mm), but was almost stable by fruit diameter ( Table 2) Biometrics of Cornus officinalis genotypes in the M.M. Gryshko National Botanical Garden pedicels in the cultivar Etude varied from 12.4 to 14.0 mm, while in genotypes of C. officinalis -from 9.0 to 11.6 mm. The pedicels of 'Etude' were slightly thicker.

Biometric parameters of leaves
Genotypes of C. officinalis (Table 3, Fig. 1). The largest leaf blades had the genotypes G-08 and G-01, and the smallest -the genotype G-02. Genotype G-02 had the smallest span of variation in linear dimensions of leaf length and width, 25.3 and 19.7 mm, respectively. Corresponding leaf indices of other C. officinalis genotypes varied within 38.6-49.9 and 26.1-43.1 mm, respectively. The width of the leaf varied more than the length (ranges were 7.2-13.5, and 9.8-18.9 mm, respectively). The mean values of the number of veins were 12.9-14.0 pieces per one leaf, and the min-max values in the studied genotypes were 10-18 pieces. The longest petioles had the leaves of genotype G-08, and the shortest -the leaves of genotype G-02. The level of variation in length was low or intermediate.
The coefficient of variation was in the range of 10.3-18.1%. Comparing to the length, the width and thickness of the petiole were more variable, and CV reached 26.7 and 31.0 %, respectively. Cultivar Etude (Table 3, Fig. 1). The mean, minimum and maximum values and CV of leaf blade size and petiole did not differ significantly from respective values of C. officinalis genotypes. However, the variation of the width was much larger (with registered 51.5 mm), whereas G-01 has only 35.4 mm, and G-07 -only 43.1 mm. The number of veins on the leaf blade of 'Etude' also did not go beyond the amplitude of variation designated for other genotypes. Leaves of C. officinalis and cultivar Etude were found relatively more variable in size, with a low or intermediate level of variation. The leaves of genotype G-08 had the highest length and width and low CV. The genotype G-05 was characterized by medium-sized leaf blades and petioles with low or medium variability, compared to other C. officinalis genotypes.
As a result of the study, the biometric parameters of the fruits, endocarp, and pedicel were established in four genotypes of C. officinalis and the cultivar Etude, and leaf blade and petiole -in six genotypes of C. officinalis and the cultivar Etude (Tables  2 & 3).
It is known that for the selection of promising cultivars of fruiting plants, a complex of indicators is applying. In particular, fruit parameters play a crucial role in the selection process. However, leaves, which in the process of photosynthesis, ensure the existence of the plant and accumulation of nutrients in the fruits, are also important. Quantitative traits are especially convenient to control the condition of plants at all stages of introduction and breeding -from initial testing to variety testing, naturalization, and 'complete' acclimatization.
At the NBG, investigations of biometric characteristics of fruits and leaves of nontraditional species of fruiting, medicinal, ornamental, and other commercially important plants are conducting. These investigations are mostly aimed at the evaluation of prospective ways of cultivation of the new plant varieties in conditions of temperate continental climate of the Right-Bank Forest Steppe (Klymenko et al., 2017a, b, c;Rakhmetov, 2018). In particular, statistical indicators of variability have been an integral part of bioecological research of C. mas and its numerous cultivars and forms, including those created at the NBG (Klimenko, 2004;Klymenko et al., 2019). Morphometric characteristics of the fruit were examined in different genotypes of Ziziphus jujuba Mill. The obtained biometric data showed that the fruits of the studied C. officinalis genotypes and the cultivar Etude have no notable variability. In most cases, the coefficient of variation corresponded to a very low or low level of variation (Mamaev, 1975). The most variable sign was endocarp weight (Table 3) Table 3. Biometric parameters of the leaves of investigated Cornus officinalis genotypes and cultivar Etude. There is still very little information on the biometric indices of C. officinalis fruits and leaves. This is probably because the introduction and breeding of this species are not widespread so much. The data about the size and weight of the fruit of nine types of germplasm, selected from 73 naturallyoccurring growth specimens in North-

Biometrics of Cornus officinalis genotypes in the M.M. Gryshko National Botanical Garden
Western China (collection of the Laboratory of Development of Endangered Chinese Raw Narcotic Resources) were reported (Li et al., 2012). The comparison shows that the limits of variation of the mean fruit size in Chinese and Kyiv genotypes are quite similar. The smallest size of the fruit is practically the same, and the maximum is larger in Chinese genotypes. In particular, the maximum length of the mature fruit in the G-04 was 16.7 mm, and in the two Chinese genotypes -19.1 mm. The same tendency has been observed for fruit weight. According to the Flora of China (Xiang & Boufford, 2005), the fruits of C. officinalis wild individuals may reach 12-18 mm long and 5-6 mm in diameter. The length of the fruits of investigated genotypes is within the reported range of variability for the wild species. Still, in most cases, it is closer to the upper limit of variation. However, the diameter of the investigated fruits is almost twice higher in comparison with wild species. Consequently, the fruits of the introduced plants, both Chinese and Kyiv, vary more in diameter than in length. The fruits of the cultivar Etude, which we examined, exceeded those of all nine Chinese germplasm types by the size (e.g., length 19.9 mm vs. 19.1 mm, respectively) and weight (1.8 mg vs. 1.6 mg, respectively). This indicates the prospect of further breeding work with 'Etude' in order to produce largefruited forms with long-fading after ripening fruits.
The variability in leaf quantitative traits is resulted from both their plasticity and genotype variation, and thus reflects the relationship between plant and climate (Royer et al., 2008). Our current research has confirmed this statement. We found that the quantitative parameters of leaves of the studied C. officinalis genotypes demonstrate higher variation comparing to the fruits. The biometric indices of the leaves, as well as the fruits, in the studied genotypes were closer (or beyond) to their variation in the wild species reported by Xiang & Boufford (2005). This fact, again, indicates that the genotypes of C. officinalis introduced into the NBG were sufficiently adapted to the temperate continental climate of the Right-Bank Forest Steppe of Ukraine. The leaves of cultivar Etude were almost indistinguishable from the leaves of C. officinalis genotypes. There was also no significant change in the number of largest lateral veins of the leaf because the wild individuals of C. officinalis and C. mas are quite similar in this respect. The leaves of C. mas are characterized by 6-10 (12) lateral veins (Poyarkova, 1951;Murrell & Poindexter, 2016), and the leavevs of C. officinalis -by 12-14 veins (Xiang & Boufford, 2005). Interesting was the nature of inheritance of other morphological features of the leaf blade by cultivar Etude. The shape of the leaf, especially its apex (gradually narrowed) and color (colorless, not brown) of trichomes in the cultivar Etude, are similar to C. mas. However, the number of lateral veins in 'Etude' is closer to the maternal species, C. officinalis. Thus, the cultivar Etude is characterized by the combined inheritance of leaf features.
Hybridization between Cornus taxa is still a poorly investigated phenomenon. Having nearly 60 species (Xiang & Boufford, 2005;Murrell & Poindexter, 2016), this genus includes only a few known species producing hybrids. Both in natural conditions and botanical gardens, C. florida, C. kousa, and C. nuttallii from the subgenus Kraniopsis Raf. (Molnar, 2018) can hybridize with each other. Some natural hybrids were also described between C. racemosa Lam. and C. rugosa Lam. (Wagner, 1990). Prospective natural hybrids were also reported between C. mas and C. officinalis (Morozowska et al., 2013) and between C. controversa Hemsl. and C. alternifolia L. f. (Gawrońska et al., 2019). Thus, the data we have obtained about the cultivar Etude is important not only for clarifying the characteristics of industrial use, but also to investigate the peculiarities of hybridization and to characterize the inheritance of traits in the genus Cornus in general.

Conclusions
The value of biometric parameters of fruits and leaves in the adaptation strategy of C. officinalis plants and its hybrid under the conditions of introduction were evaluated. Important and indifferent signs for breeding work to obtain promising productive varieties for industrial cultivation were determined. Among the cultivated at the NBG, the cultivar Etude and genotypes G-03 and G-04 of C. officinalis were the most noteworthy. Cultivar Etude is promising for large-fruited forms with fruits that do not fade long after ripening. The data obtained indicate the strong potential of C. officinalis for commercial use in Ukraine. The results of this study are also important for the investigation of hybridization in the genus Cornus. Biometric characteristics of the fruit, endocarp, and leaf were found determining the degree of adaptation of C. officinalis to the current climate conditions of Ukraine (in particular, the Right-Bank Forest Steppe) and essential for further breeding work.