Ionocidium ‘ Cerrado 101 ’ : intergeneric orchid hybrid with high quality of blooming ( 1 )

Orchids are considered one of the most important potted-flowering in the world. Oncidium genus, as well their hybrids group (OHGs) has used for the flower market as pot or cut flower. However, some horticultural characteristics require improvements, e.g. the production of precocious cultivars, independent-season blooming or easy flowering induction, high quality and durability of flowers and variations in color of flowers, to compete with Phalaenopsis and Dendrobium orchids, as other flower groups. Aiming this purpose there were used hand-crossing pollination between the Oncidium ‘Sweet Sugar’ and Ionopsis utricularioides, a Brazilian wild species, for evaluate the capacity of crossing and to select progenies with interest of use in floriculture as new cultivar. The seeds obtained from this cross were seeded on in vitro conditions, followed by acclimatization and cultivation on greenhouse conditions until de flowering time. One of the plants obtained presented interesting characteristics, as good and rapid vegetative development and high quality of blooming. This hybrid obtained from an intergeneric crossing resulted in a plant with vegetative and flower type characteristics and color similar of Oncidium female parent, but with larger number of ramifications in inflorescence and number of flowers (60% and 219,4%, respectively) than Oncidium parent (♀), and with more similarity with Ionopsis (♂). This hybrid cultivar, called Ionocidium ‘Cerrado 101’ is one more option of OHGs in this competitive market and can be used either for pot and also for cut orchid flowers production, main because it longer inflorescence (83 cm).


INTRODUCTION
Orchidaceae family is the largest family in number of species of flowering plants, with more than 26,500 species (KEW, 2011) with one of the most geographical distribution in the world with species in all continents, with most part concentrated in tropical regions (CHASE et al., 2003).
Oncidium is one of the most important genus of orchids for flower market, together with Dendrobium, Phalaenopsis and Cattleya hybrid groups, and represents one of the genus with most ornamental applications, with several number of species and a large number of interespecific hybrids registered in Royal Horticultural Society and used in floriculture for pot and cut flowers production (FARIA et al., 2015;RHS, 2017).Two Oncidium Hybrid Groups (OHGs) deserve to be highlighted in the Brazilian flower market, the OHGs called 'Golden Rain Orchids' (including Oncidium Aloha 'Ywanaga', O. 'Aloha Case', O. 'Sweet Sugar') with large inflorescences with yellow canary flower color and the 'Chocolate Orchid' (O.'Sharry Baby'), with red-brown flowers distributed in inflorescences and with a sweet smell that remember chocolate.Ionopsis is another genus from subtribe Oncidiiniae with occurrence in Cerrado vegetation areas and riparian forests, and their ornamental potential and it use for OHGs breeding was previous reported (CARDOSO and ISRAEL, 2005;CARDOSO, 2014).
Breeding of orchids, including Oncidium hybridization are large explored in horticulture, but few papers described the methods of breeding and the segregation of morphological characteristics, as showed for Cattleya Hybrid Group (Cardoso et al. 2016;Cardoso, 2010), for Dendrobium (CARDOSO, 2012;FARIA et al., 2009FARIA et al., , 2011FARIA et al., , 2013) ) and for Oncidium (FARIA et al., 2015).
Among interesting characteristics for developing new Oncidium varieties are the resistance to pests and diseases, compact size of plants, season-independently flowering induction or easy control of blooming induction, high quality of blooming and flowers, e.g.size, number and new colors.Among the characteristics that difficult the breeding programs and selection of new cultivars by specific methods of breeding for orchids are the long period for blooming, ranging from 2 to 4 years after seeding, and the limited knowledge about hereditability of characteristics (PAN et al., 2012).The absence of this type of information for orchids difficult the prediction of morphologic characteristic transmission to the progenies, in breeding programs which uses hybridization.
The high genetic variability and the capacity of crossings between species makes orchid breeding very exciting.The use of controlled crossings followed by the selection of superior progenies is the wide used method to obtain new cultivars in orchid industry (CARDOSO et al., 2016).In addition, the establishment of breeding programs of orchids and other species used in floriculture associated with technologies of propagation are required for the sustainability and self-sufficiency of the floriculture in developing countries, which is now, in most part of these countries, are extremely dependent of international breeders located in developed countries (CARDOSO, 2013), which cover royalties for use it protected cultivars.
Oncidium has different commercial hybrids used in floriculture (FARIA et al., 2015) and Ionopsis is a correlated genus wild from Cerrado and Atlantic Forest from southest region of Brazil.Some interesting characteristics of Ionopsis utricularioides is the presence of flowers with rose color, associated with the production of more than one inflorescence/blooming/plant and inflorescences with very large number of flowers (CARDOSO and ISRAEL, 2005;CARDOSO, 2014), characteristics that partially are not common in Oncidium species and hybrids, requiring intergeneric crossings for this purpose.
Although Oncidium and Ionopsis are near botanically and morphologically (Subtribe Oncidiniiae) and some hybrids were registered in Royal Horticultural Society (RHS, 2017), the inheritance of horticultural and ornamental characteristics from this intergeneric hybrids were not well reported.Also, breeding methods using commercial orchid hybrids (OHGs) in crossing with wild species could be resulted in hybrids which combine commercial characteristics with more adapted plants to local climatic conditions.
The aim of this experiment is to determine the capacity of crossing between these two species by hand crossing pollination, follow by the cultivation, evaluation and selection of superior progenies for breeding and use in floriculture as new cultivar.

MATERIAL AND METHODS
This program of breeding of Oncidium and correlated genera have been realized since the year of 2003, with works started at the Fundação Shunji Nishimura de Tecnologia, located at Pompeia City, Sao Paulo State, and after 2013 at Centro de Ciências Agrárias of University Federal of Sao Carlos, in Araras City, SP.
The actual breeding program consisted basically of selection of parents with characteristics of interest, followed by hand cross pollination and selection of superior progenies after in vitro and greenhouse ex vitro cultivation.Within this purpose there were selected as progenitors: the commercial hybrid of Oncidium, called O. 'Sweet Sugar', with general compact size and flowers with big size canary-yellow color, and; a selected plant of Ionopsis utricularioides obtained from a particular collection from author, with good number of inflorescences (3-4/blooming) with typical rose flowers.Both plants were used as female and male parent to improve the chances of successful to obtain fruit set and viable hybrid seeds.
The hand cross pollination was realized after emasculation of flowers used as female parent, in whom the pollineas were previous collected from the female flowers and bagged in an Eppendorf® of 1.5 ml.The pollineas from the male parent were immediately used for pollinate the stigma of female flowers parent, using a scalpel for pollination.Ten pollination of each crossing were realized.All these procedures were realized between 8 and 10:00 a.m and the pollinated flowers have between 7 to 14 days from anthesis.
Fruits obtained were collected approximately 120-140 days after pollination, when the fruits shown yellow color (instead of green) in it distal portion, signalizing the begin of the fruit dehiscence.In laboratory conditions, the fruits were completed opened and the seeds were collected in a Petri dish and immediately were transferred to a flask for asepsis and in vitro germination.Asepsis were realized using sodium hypoclorite at 0.3% of active chlorine for 15 minutes, followed by three washings in distilled autoclaved water (unpublished, tested by author).For all stages of in vitro growth there was used the Murashige and Skoog (1962) culture medium with macronutrients reduced by half concentration (MS½), adding 30 g L -1 of sucrose, 1 g L -1 of activated charcoal, and solidified with 6 g L -1 of Agar-Agar.The culture medium was autoclaved at 121 °C for 20 min.
The selection of progenies was based in a similar methodology described by Cardoso et al. (2016) for Cattleya breeding.After in vitro phases, which have duration of 9-months, only the seedlings with at least 5-cm length were used for acclimatization procedures, which were realized under greenhouse conditions protected with agricultural plastic and net shade of 80% of sunlight break.There was used 98 cells plastic tray and organic substrate consisted of coconut fiber for acclimatization.After three months of growth, survival plants were transplanted to definitive pots with 12 cm of diameter and 1,0 liter capacity, containing as organic substrate coconut chips and pinus bark 1:1 (v/v) (Vida Verde®, Mogi Mirim, Brazil).
The selection of progenies as new cultivar was based on visual good and vigorous vegetative development with absence of pests and diseases, rapid growth.Reproductive development was observed mainly by the early blooming and high quality of flowers and inflorescences, based on: architecture and length of inflorescences, and flower size and color.There were characterized the first and second blooming of the selected progeny as new cultivar and a minimum of ten flowers for progeny or progenitor was measured, and the data were presented by the mean with standard deviation.

RESULTS AND DISCUSSION
The viability of the intergeneric crossing was possible only when Oncidium was used as female and Ionopsis as male parent, which resulted in fruits and seeds.The presence of embryos in seeds was observed using light microscope (data not showed).Using Oncidium 'Sweet Sugar' as female parent, the fruit set was 70% (7/10), instead of no fruit production when Ionopsis utricularioides was used as female.We observed that due to the high difference in flowers size (Table 2) and reproductive parts between these two genotypes (Table 1), the procedure of pollination in Oncidium stigma was not a problem, but the reverse hand-pollination maintained only one of the two pollineas of Oncidium in stigma of the small flower of Ionopsis, being a possible cause of unsuccessful fertilization when Ionopsis was used as female parent.The fruits obtained produced a very small quantity of seeds that was rescued from the fruits and immediately seeded on culture medium on in vitro condition.In the Royal Horticultural Society (RHS, 2017), only five registered intergeneric hybrids between Oncidium and Ionopsis were founded, called: Ionocidium 'Blue Mist' (Ionopsis utricularioides x Oncidium incurvum) and Ionocidium 'Hinazakura' (I.utricularioides x Oncidium Twinkle), were both used Ionopsis as seed parent and; Ionocidium 'Chian-Tzy Catherin' (O.Twinkle x I. paniculata), Ionocidium 'Chian-TzyHoyen' (Oncidium sotoanum x Ionopsis utricularioides) and Ionocidium Seewatloppen (O.bauerii x I. utricularioides), which used Oncidium as seed parent.Other unregistered crossings were founded as Ionocidium 'Popcorn Haruri'.In Brazil, the only registered hybridization with OHGs is obtained from the crossing between Oncidium sarcodes x O. Aloha 'Iwanaga' (FARIA et al., 2015).
On in vitro conditions, after seeding, only 50-100 seedlings were obtained and after 9-months in vitro cultivation, only 30 seedlings with more than 5-cm in length were acclimatized.At greenhouse conditions, 25 progenies were obtained and evaluated for obtaining new cultivar.
The selection of seedlings was started on in vitro conditions, and was realized observing the good vegetative development of progenies.The selection of progenies used a similar methodology described by Cardoso et al. (2016) for Cattleya breeding.On in vitro conditions, there was selected to the next phase only the part of seedlings that effectively differentiated into roots, pseudobulbs and leaves germination, instead of the part that were maintained as protocorm for a long time since after transplantation, which were not used.In this method, only the progenies that differentiated into plantlets were maintained for rooting and acclimatization stages of the micropropagation.The differences among these progenies also was continued observed on greenhouse conditions, selecting for transplantation only the plants with rapid vegetative development, free of pests and diseases and that presented reduced time required for blooming compared among progenies, which range from one to two and half years after acclimatization.
As characteristics, the seedlings obtained and acclimatized in greenhouse conditions showed rapid rooting, vigorous and quickly vegetative development, compared with another common Oncidium cultivars, showing characteristics of high hybrid vigor from this inter-generic crossing.
One selected progeny start it blooming early, approximately one year after acclimatization.However, commercial blooming in this hybrid, with higher number of flowers and longer inflorescences were obtained in the second blooming (plants with approximately 1.5 to 2 years after acclimatization), compared with the first ones.This characteristic of rapid flowering has importance for commercial production of orchids because allow the production of flowers in competitive period compared with other commercial genera as Dendrobium and Phalaenopsis orchids (CARDOSO, 2012;CARDOSO et al., 2016).
Phalaenopsis genus is actually the most commercialized pot flower in the world market and only in Brazil were commercialized 5 million pot plants in 2015, instead of 350 thousand of pot plants of Oncidium (TOGA, 2017).One explanation for these differences in the market is the largest quantity of varieties of Phalaenopsis compared with Oncidium.In Oncidium most of the species have yellow with brown spots or yellow or brown flower colors, including it hybrids (FARIA et al., 2015).Only the crossings among other correlated genera, as Miltonia (Miltonidium), Odontoglossum (Odontocidium) and others were capable to produce new cultivars with more diverse flower colors (LAKSHMAN et al., 2014) for the floriculture market.
The main aims of the actual crossing of yellow-canary predominant flowers from Oncidium 'Sweet Sugar' (♀), and Ionopsis (♂) with rose flowers (Figure 1) were select: 1) progenies with intermediary size between their parents (Table 1); 2) higher number of inflorescences per blooming than Oncidium, commonly with only one (from Ionopsis, Table 1); 3) inflorescences with more number of flowers than Oncidium (from Ionopsis, Table 1) and; 4) progenies with rose flowers (from Ionopsis, Figure 1).From these Values are the result of mean ± standard deviation obtained from the measures of 10 flowers of each genotype four characteristics selected as targets for actual breeding program, only the characteristic of higher number of flowers/inflorescence were obtained in the selected progeny (Table 1).The work with the other targets (1, 2 and 4) will require new crossings using this progeny with another species or back-crossings with Ionopsis.Interestingly, one not aimed characteristic, the longer inflorescence of the hybrid (Table 1), is a characteristic of interest for cut flower production, promoting the double purpose for the selected progenie as new cultivar, called Ionocidium 'Cerrado 101'.This intergeneric hybrid selected, as well as the progenies cultivated from this crossing, maintained most of the vegetative morphology, as size of plants, pseudobulbs and leaves similar to Oncidium-type plant (Table 1; Figure 1).About color, all progenies obtained from this crossing resulted in variations of yellow color flowers, ranging from yellow soft cream, e.g. the new cultivar selected (Figure 1), to yellow predominant color flowers (another progenies), showing that this characteristic is dominant in the crossing with Ionopsis.Also, the format and size of flowers is very similar to Oncidium-type flowers (Table 2; Figure 1).

A B C1 C2
The type of inflorescence has intermediary morphological characteristics between Oncidium and Ionopsis, with architecture similar to Oncidium with good distribution of flowers in inflorescence, but with number of and flowers more similar to Ionopsis (Table 1).Among the main superior characteristics of the progeny selected Ionocidium 'Cerrado 101' is the high quantity of ramifications (8 inflorescence -1 ) and number of flowers (115 flowers plant -1 ), more than three times the number of flowers of Oncidium 'Sweet Sugar' used as female parent (Table 1; Figure 1).These characteristics are of interest for the flower market, due to the greater filling of inflorescence with yellow-cream flowers.
Other characteristics of the Ionocidium 'Cerrado 101' and which still to be improved are the long length of inflorescences (83.0 cm) compared with another OHGs used for pot culture (Table 1) and the maintenance of yellowcream derived color, similar to another cultivars disposable in the market (with few novelties).In the Brazilian market there is no specific classification of characteristics for OHGs until the moment and that describe the size of plants and inflorescences suitable for the orchid market.In Phalaenopsis, one important pot flower orchid in the market, the plants in pots were classified in four classes according the length of inflorescences, being above from 30 cm (class I) to > 75 cm (class IV) and the presence of long inflorescences (Class IV) is not considered a defect for commercialization (VEILING HOLAMBRA, 2017).Similar classification was used for Phalaenopsis cut flowers, with inflorescence length above or below 60 cm in length.
According this classification for Phalaenopsis, the actual cultivar developed for OHGs could be considered appropriated for commercial production, both for pot flower, as well as for cut flower, maintaining characteristics of inflorescences similar to other disposable cultivars in the market, e.g. the Phalaenopsis hybrids.
The tentative of self-pollination of Iononcidium 'Cerrado 101' not resulted in fruits until the moment.Interestingly, the pollineas of this cultivar is white soft cream colored, instead of common yellow observed in both Oncidium and Ionopsis parents, suggesting the possibility of male-sterility in this hybrid.Other tentative of crossings, as example the backcrossing with Ionopsis, using this last as male parent, aiming to obtain rose flowers are in progress by our breeding program.
In vitro micropropagated plantlets of Ionocidium 'Cerrado 101' are maintained in the Laboratory of Plant Tissue Culture and Physiology at Centro de Ciências Agrárias (UFSCar), located in Araras, SP.

Table 1 .
Characteristics of inflorescences from male and female parent and the intergeneric progenie obtained as new cultivar Ionocidium 'Cerrado 101'.

Table 2 .
Size of flower parts (Mean±SD) from male and female parents and the intergeneric progenie obtained as new cultivar Ionocidium 'Cerrado 101'