FEATURES OF PARENTS ’ SELECTION AND EFFICIENCY OF ARTIFICIAL HYBRIDIZATION OF CANNA

Розглянуті можливості штучної гібридизації з використанням сортів з різних груп канни та перспективність залучення інтродукованих видів для генетично-селекційного збагачення сучасного сортового різноманіття канни. Метою роботи був підбір батьківських пар за визначенням індексу фертильності пилку інтродукованих видів і сортів різних груп канни для отримання селекційного різноманіття форм стійких при вирощуванні в південному посушливому регіоні України (на прикладі м. Кривий Ріг). Матеріали та методи. Дослідження проводились на 7 інтродукованих видах і варіаціях (C. tuerckheimii Kraenzi.; C. indica var. warscewiczii Nob. Tanaka; C. iridiflora RuizPav.; C. flacida Salisd.; C. indica var. edulis Ker Gawl.; C. indica var. coccinea Mill.; C. indica L.) та 28 сортах канни (з них 67,8 % закордонної селекції). Цитологічний аналіз пилку проводили згідно прийнятої методики(за йодною реакцією крохмалю). Гібридизацію здійснювали з використанням існуючих розробок для канни. Строки гібридизації та час проведення штучного запилення були встановлені з урахуванням екологокліматичних умов промислового регіону. Результати. На основі цитологічного аналізу пилку інтродукованих видів і перспективних сортів канни підібрані батьківські пари для штучної гібридизації з метою генетично-селекційного збагачення її різноманіття та отримання зразків, придатних для вирощування в степовій зоні України, зокрема в екологічно-дестабілізованому регіоні. Розроблений підхід до відбору вихідних форм канни для штучної гібридизації на основі цитогенетичного аналізу пилку. Висновки. За показниками якості пилку (індекс фертильності) в період завершення бутонізації виділені перспективні інтродуценти канни для подальшого використання батьківськими формами в гібридизаційних схемах. На основі проведеної штучної гібридизації перспективних для степової зони і промислового регіону України інтродуцентів роду Сanna визначені найбільш результативні комбінації схрещувань. При цьому при схрещуванні сортів групи Крозі між собою отримано найбільше результативних комбінацій. З отриманого фонду гібридних рослин канни виділені селекційні форми за комплексом декоративно-цінних ознак рослин і введені до колекційного фонду та подальшого їх впровадження в озеленення Ключові слова: види та сорти роду Canna, пилок, стерильність, індекс фертильності, гібридизація, батьківські форми, селекція


Introduction
Genus Canna (Canna L.) unites about 50 species of permanent grass plants of the Cannaceae family order of the ginger family [1,2]. The centre of origin of most species of this genus is tropical and subtropical areas of Central America, Islands of the Caribbean Basin, the northern equatorial Andes and the north Brazilian provinces. Now this culture is introduced in Asia, Africa, India and China [3,4]. Since the 17th century, the garden Canna culture has taken a prominent place in the decorative landscaping of Europe [5,6].
Very few varieties from the modern decorative forms of Cannes are used in landscape design. During the last decades a part of modern foreign varieties turned out to successfully be introduced in Ukraine, even in its steppe zone. The varied fund of the introduced plant of Canna, created in the Kryvyi Rih Botanical Garden of NAS of Ukraine (farther -KBG), is a confirmation of this [7]. It is the need to get new Canna varieties, adapted to different ecological and climatic conditions of the regions of Ukraine, became a prerequisite for this work.
There are 7 species and forms and 37 varieties of Canna of foreign and domestic breeding (foreign varieties make up 67 %) in this collection. Of these, 17.0 % of varieties of group I (C. foliosa-parviflora hort.), 57 % of varieties of group II (Crozy group) and 26 %group III (C. orchiodes Bailey. or Italian group) [7,8].

Literature review
The culture of Canna has taken an important place in ornamental plant beautification in Europe since 17 th century. The species or varieties of Canna were used in landscape design of the time. All modern polyploid species of Canna, in fact, are made on basis of 5 parents' species, which were involved in hybridization from 1848 to 1892. They are now united under name of Canna hybrida hort. ex Backer. So, for 45 years' period the fast transformation of the uncultivated plant into the orna-mental plant has taken place mostly because of distant hybridization and poliploidy. The first stage took place in the middle of the ХІХ century, when the French plant breeder Theodore Anne created the first hybrids of Cannes from crossing species C. indica and C. glaucа. These hybrids became known as С. аnnaei hort. [7]. The second stage of hybridization involves the work of Lyon plant breeder Pierre Antoine Crozier. He got the first large-flowered hybrids by crossing C. indica and C. indica var. warszewiczii. From further hybridization of these forms with C. ehemannii plants, this breeder obtained a garden form, which was later called "Canna Crozier" or "French Canna" [9]. This form has become the prototype and standard of the modern garden form "Cannes Crozier" [10]. Hybrids, called iris-like or orchid-shaped Italian Cannes (C. orchioides Bailey) are the result of the third stage of breeding cultivation of these plants. They were obtained by crossing Crozier's hybrids (as the maternal form) with different North American species, in particular, C. flaccida Salisb.
Nowadays there are some varieties (Queen of Italy), which were gotten as a result of vegetative mutation, but creation of better varieties was connected with repetitive cycles of hybridization and polyploidy [11,12]. Te breeding potential of this plant is high because the genofond of Canna genus is little used [13]. Despite the variety of modern species of Canna (about 1000 according to some informational resources), only about 200 species are used in practice. In decorative floriculture of Ukraine species of foreign selections prevail.
T. Theophilova developed a classification of Cannes by flower shape. On this basis, all the representatives of the collection of Canna can be divided into three groups: I -Canna foliosa-parviflora hort.small-flowered species; II -C. Сrozy hort.
However, these varieties of Canna are little adapted to the soil and climatic conditions of Ukraine. The leading center of introduction and breeding work for the creation of native varieties of Canna is located in the Crimea (Nikita). Weather and climatic conditions of this territory are more suitable for growing the plants of the Cannaceae family [14,15]. Therefore, the selection of domestic varieties, most adapted to the new conditions of growing, is very important [16].
The Kryvyi Rih region is situated in the southern agroclimatic arid region of Ukraine. The sum of active air temperatures of atmospheric is from 3000 ° C in the north of the region to 3200 °C in the south [17,18]. The territory of this region receives an average of 400-450 mm of precipitation per year. Two thirds of the rainfall account for the warm part of a year (300-320 mm). Atmospheric precipitation in the winter months falls less -100-130 mm. During all summer months there is a deficit in the balance of humidity, although 60-70 % of annual rainfall falls during the growing season. This is due to high summer air temperatures and a significant predominance of evaporation over the sum of precipitation. The moisture deficit in June is -76 mm, in July -141 mm, in August -142 mm. The total annual deficit of moisture is equal 350-420 mm [19].
The city of Kryvyi Rih is reported to have the high complex index of atmosphere pollution [20].
The complex of hard climatic and ecological conditions of the region has an impact on the existing variety of plants in landscape gardening and peculiarities of their growth and development [21]. The inventory of green areas of Kryvyi Rih showed that only 7 species and 12 varieties of the perennial herbage plants are used in greening the city. There are only 2 species of Cannes among them, 4 varieties of II group (Crozy group) and 2 varieties from III group (orchid-like group or Italian group). This confirms the importance of breeding work and introduction of promising taxa of this genus and their continued use in planting of greenery of the region [22,23].
According to the literature, artificial hybridization, using introduced varieties and species of Canna, is rarely successful [24]. A different number of chromosomes in cultural forms and species plays a large role [25]. Matveeva (1980) found out that the ideal valence for Canna is likely to be a triploidy which allows valuable decorative characters to appear.
Triploid forms were widely used in artificial hybridization. Tetraploids received spontaneously became useful only as an interim stage for creation of more effective triploids [9].
It is well known, that the quality control of pollen of species and varieties determines the possibility of using them in breeding work [26,27]. In a range of works certain records about comparative study of pollen of some species genes and varieties of Canna L. on the basis of morphological characters are given [28,29]. An average pollen size of Canna samples is from 50 to 110 micron, fertility varies within a broad rangefrom 0 to 93 % [30,31]. Mature pollen grains are two-celled; spermiogenesis takes place in a pollen tube [32,33]. About 20-25 thousand of pollen grains are formed in an anther of Canna [27].

The aim and objectives of the study
The aim of this work was the need to create new varieties of Canna, which will be maximally adapted to the different ecological and climatic conditions of Kryvyi Rih regions, as well as the selection of parental pairs by determining the pollen fertility index of introduced species and varieties of different groups of Canna. The growing need in homegrown varieties of ornamental plants is based on economical practicability of the development of own selection database too.
To accomplish the aim, the following tasks have been set: 1. To determine the pollen quality index (fertility index) of introduced species and varieties of Canna.
2. To identify the promising parental forms by the fertility index.
3. To reveal the most effective combinations of parental forms of introduced species and varieties of Canna.
4. To create the selection fund of samples of Canna, which can be suitable for growing in the steppe zone of Ukraine.

Materials and Methods
The research was done on 7 species and 28 varieties of Canna of this collectors' fund. Pollen was chosen in the finishing phase of budding for the cytologic analysis and fertility was being defined by the iodine reaction of starch, according to the approved method [34,35].
An attempt to preliminarily evaluate the feasibility of using varieties and species of Canna for artificial hybridization was undertaken. The ratio of fertile and sterile pollen is used ("fertility index" hereinafter, the designation F1is proposed). Hybridization was brought into action using known techniques for Canna [36]. In this case, castration of the flower was carried out in the phase of the ripe bud [14,37]. With the opening of Canna flower, the negative impact of exogenous factors on the pollen state increases. Therefore, the pollination of flowers was carried out in the morning in the sunny weather (from 6:00 to 10:00) to avoid active drying of pollen [38].
We found that in conditions of the Kryvyi Rih region these works are the most effective recommended to do from the third decade of August to the second decade of September in the period of mass Canna budding [37]. The statistical processing of received results was conducted according to the common method (t-Student et. all) [39] using modern software (M. excel, Past).

Results and discussion
The fertility of pollen of Canna was studied in different phases of the development of a plant among varieties and species, which is in condition of the Kryvyi Rih region, when the vegetation finishes by fruit forming. The analysis of the pollen fertility at the different development phases of a flower showed that introduced varieties and species of Canna has the different potential of the reproductive function in condition of the Kryvorizhzhia. Only a small part of the studied varieties formed up to 50 % of viable pollen in the closed bud. These data are almost in agreement with the results of the evaluation of male generative structures in the cultivation of Canna in more favorable conditions [14,31]. At the same time, we observed a clear tendency of loss of the pollen's vital potential, when comparing it in a closed bud and in an open flower [37]. The fertility of pollen of Canna species and varieties can vary greatly depending on the growing conditions or the characteristics of the growing season too [40,41]. In particular, we have found that in the conditions of the Kryvyi Rih region, the highest fertility of Canna pollen was before the plant flowering. It is confirmed, that pollen of Canna has higher fertility in the final period of buddingbefore opening of a flower, that's why it was being selected for pollinating of maternal forms on hybridization in this phase [13,38].
Average pollen sizes of Canna specimens from the Nikita Botanical Gardens' collection range from 50 to 110 microns, fertility ranges widely from 0 to 93 % [14,31]. Earlier, we established a significant loss of biometric and vital parameters of pollen of the variety Otblesk Zakata and the variety Podarok Kryma at the introduction from the Crimea to the Kryvorizhzhia. The average sizes of ripe pollen grains of the variety Otblesk Zakata decreased by 3.6 %, of the variety Podarok Krymaby 8.2-10.3 %, and the fertility decreased by an average of 24 % [6].
The maximum of pollen fertility in inspected species of Canna was 70-72 % during cultivation in the collection of KBG (Fig. 2). This is a much lower figure than the status of the male generative structures of Canna, when grown on the southern coast of Crimea. For example, in C. indica up to 85.42 % of morphologically normal pollen grains are formed there [14]. Оnly a third of the pollen formed was fertile in the final period of budding in the vast majority of introduced species of Canna in the Kryvorizhzhia (Fig. 3, 4).
It is noted, that in the Italian group (orchid flowering) Cannas varieties with the potentially high living index of pollen (FI 0.5) are missing (Fig. 3). Certain prospectivity can be predicted only with using parental forms in the The total number of investigational pollen grains was from 426 to 682 pieces.  The total number of investigational pollen grains is from 425 to 752 pieces.
Among species of the Crozy group variety for the uttermost result it is better to use varieties with the distinct predominance of fertile pollen in the bud (from FI=0.7 to FI= 1.4) as parental forms for artificial cross (See fig. 4). Using varieties with FI 0.5 as parental forms is of little promise (Krymskie Zori, Klara Kurtik, Louis Cayeux and The President). Most varieties of this group have FI=0.7 and in particular they were widely involved in hybridization schemes.
It has been found, that in the population of studied pollen of Canna varieties the part of fertile pollen in a bud exceeds the quantity of sterile seeds, at least in 1.5-2 times, for C. coccinea at 2.5 and C. indica in 3.5 times. It means, at first, that life opportunities of Canna pollen are significantly lower than in other varieties, and, at second, it has a distinction as to a variety belonging to a certain variety group.
In our work the usage of varieties, FI of which 1.0 (or FI 1.0) were the most effective in the hybridi-zation. So, pollination took place quite successfully with the following formation of hybrid seeds in combinations with the usage of varieties Madam Angel, Shedevr, Orange Beauty, Otblesk Zakata.
Having the aim of creation of the selection fund of Canna with wide varieties of samples of the \ great ornamental use, hybridization schemes were made in several ways: 1) in different variations of varieties' combinations from different variety groups; 2) usage of a variety as a maternal or paternal form in artificial crossing with introduced species.
As a result of some stages in hybridization work, 79 selection schemes were researched, from which 63 pairs of intervarietal crossing, 13 variants with the usage of the native kind as a mother form, 3 variants with the usage of the native kind as a father form. The whole number of artificially pollinated buds which were analyzed in different crossing combinations is 3097 (Table 1). It has been noted, that the most promising (30.6 %) combinations were received by crossing among varieties of the Crozy group. From 15 parental pairs, in which variety of the Italian group Canna was a mother form and variety of the Crozy group was a father form, only one variant (♀Liberty×♂America) gave hybrid seeds. Crossing variants among Italian group varieties turned out to be absolutely unfertile.
In general, only 22.2 % variants among variety combinations gave a successful seed formation and 46.2 %variants with the usage of species as a mother form. The combinations of parental pairs, in which mother was a variety and father was introduced species, appeared to be unfertile.
In crossing among varieties only 2 seeds are formed in a formed fruit bud, whereas species have about 6-8 seeds. In our researches the highest results as to the seed formation (from potentially possible) have been stated for variants of intervarietal crossing with the usage of the Crozy varieties group as parental forms (in average -49.5 %). With the usage of varieties of this group as a maternal form (in average -29.7 %) the seed formation was rather successful too.
In fact, only 20 parental forms from all researched combinations of crossing formed fruits (which amounts 25.3 %) ( Table 1). The most successful variants of intervarietal hybridization turned out to be the following: ♀Ai-Petri × ♂Otblesk Zakata, In this case in average one seed was formed in a fruit bud, which is a variety characteristic of Canna.
The further study of phenotypic characters of received hybrid flowers with species usage in selection work showed the absence of forms of the ornamental use in obtained breeding. It is probably connected with the domination of characters of the introduced species, used in crossings. A larger complex of valuable ornamental characteristics was observed in hybrid forms, obtained in variants among variety combinations; especially, where the parental forms used varieties of the Crozy group. According to the results evaluation as to qualitative and quantitative characteristics from pollination of the hybrid breed, 2 selection forms were pointed according to the complex of characteristics of the ornamental use (Fig. 5). The hybrid form 1-1-2011 was marked in the combinations of crossings ♀Podarok Kryma× ×♂Solnechnaia Krasavica. The plant has a bright green leaf color as in the parent variety. The color of staminodias of the resulting hybrid is a distinctive feature. There is a combination of two colors, inherited from the parent varieties with the dominance of carmerinusruber color (45 С) and slavjanskiisolaris color (3 А) (with RHS Colour Chart) [42]. The hybrid form 9-7-2011 have been pointed in the combinations of crossings ♀Madam Angel × ♂Solnechnaia Krasavica. The plant has leaves of a gray-green color and bright yellow flowers as in the par-ent form. Orange dotted all over the flower is a decorative addition (punctuated). The hybrid plant F 1 inherited the shape of the flower as in the mother variety, but the size was smaller. This is especially valuable in accordance with the requirements of modern floriculture for created varieties of Сanna.

Conclusions
1. According to the index of pollen quality (FI), in the final period of bud formation the prospective introduced species Сanna were marked for further usage as parental forms in hybridization schemes.
2. In varieties that had an FI of approximately 1.0 (or more), the results of artificial hybridization were higher. The average FI values for the introduced species were 2.3; for the varieties of the Crozy group -1.1. The prospectivity of using Italian group varieties as parental forms proved to be very low, determined by the FI that did not exceed 0.65.
3. On the basis of performed artificial hybridization the most effective combinations of crossings with the efficiency of seed formation above 50 % were found as promising introduced Canna varieties for the steppe zone and industrial region of Ukraine. It should be noted, that the most effective combinations were obtained when crossing among the varieties of the Crozy group. 4. We matched parental pairs for artificial hybridization of Canna with the aim of genetic and selective enrichment of its varieties and getting samples, which can be suitable for growing in the steppe zone of Ukraine, especially in the ecologically destabilizing region. From the received fund of hybrid Canna flowers 2 selection forms have been pointed out as to the complex of flowers with a characteristic of the great ornamental use and have been introduced to the collection fund for their further implementation in landscape gardening.