Obtaining Triploid Hybrids by Means of Open Pollinations and Controlled Crosses Involving Diploids Parental

The creation of triploid hybrids is an important genetic improvement strategy for the development of new commercial citrus scion varieties. The objective of this work was to quantify the frequency of triploids obtained from natural and controlled crosses of different mandarin varieties under varying environmental conditions in the state of Bahia. The experiments were conducted in the municipalities of Cruz das Almas (Recôncavo Baiano region) and Mucugê (Chapada Diamantina region). The first experiment was based on fruits from open pollinations of the varieties ‘Page’, ‘Ortanique’, ‘Ellendale’, ‘Clemenules’, ‘Swatow’, ‘Piemonte’, ‘Fortune’, ‘South Africa’, ‘Montenegrina’, ‘Kincy’, ‘Span Americana’, ‘Fremont, ‘Nova’, ‘Dancy’ and ‘Murcott’, and the second involved fruits from controlled crosses of female parents ‘Nova’, ‘Fortune’ and ‘Ortanique’ and male parents ‘Page’, ‘Montenegrina’, ‘Swatow’, ‘Fremont’ and ‘Kincy’. The seeds selected were inoculated in test tubes containing approximately 10 mL of Woody Plant Medium (WPM). When the plants reached circa 60 days of age, leaf samples were removed for quantification of the DNA by the flow cytometry technique. In Mucugê, three triploids were identified from open pollinations, ‘Clemenules’ (1) and ‘Ortanique’ (2), while in Cruz das Almas, no triploids were obtained. In the controlled crosses, triploids were only obtained in Mucugê: ‘Ortanique’ × ‘Montenegrina’ (4), ‘Ortanique’ × ‘Kincy’ (1) and ‘Ortanique’ × ‘Swatow’ (2). Based on the data, it is suggested that the environmental conditions of Mucugê favored the formation of triploids.


Introduction
Citrus are among the most important group of fruit trees in Brazil, due to the nutritive value of the fruits and the socioeconomic role they play in the export (Knudsen, Almeida, Langer, Abreu, & Halberg, 2011).In this aspect, the expansion of the genetic base of Brazilian orchards, especially related to the varieties used for scion and rootstocks, including those with capacity to adapt to abiotic stresses, tends to contribute significantly to the achievement of economically superior incomes in the country (Syvertsen & Sanchez, 2014).
Although citrus presents great importance in the economy, Brazil has little expressive participation in the world exports of fresh orange and mandarin.In addition to phytosanitary issues, one of the reasons for the low Brazilian participation in this market is the lack of cultivars that meet the minimum quality requirements demanded by the external market of fresh fruit, being one of the main ones, the absence of seeds in the fruits (Latado, Carvalho, & Machado, 2007).
Parthenocarpy is a fundamental peculiarity for the production of seedless fruits, and is a characteristic that is present in citrus germplasm (Aleza, Cuenca, Juárez, Pina, & Navarro, 2010a).Thus, the creation of triploid hybrids is an important genetic improvement strategy for the development of new commercial scion varieties (Ollitrault, Dambier, Luro, & Froelicher, 2008).
The objective of this study is to quantify the frequency of triploids obtained from natural crosses (open pollinations) and controlled crosses of different mandarin varieties in varying environmental conditions in the state of Bahia, in Brazil.

Agroclimatic Conditions of the Study Area
The experiments were conducted in two municipalities in the state of Bahia in Brazil.The first was Cruz das Almas, located in the Recôncavo Baiano region, with geographic coordinates 12°40′19″ southem latitude and 39°06′23″ eastem longitude, at an elevation of 226 m.The climate is hot and tropical with an average annual rainfall of 1170 mm, varying from 900 mm to 1300 mm, with the rainy season lasting from March to August and the dry season was from September to February.The average annual temperature is 24.5 °C and the relative humidity is 80%.
The second municipality was Mucugê, in the Chapada Diamantina region, with geographic coordinates 13°00′19″ southem latitude, 41°22′15″ westem longitude and at an altitude of 983 m.The climate is characterized as semi-humid, with the rainy season lasting from October to March and the dry season from April to September.The average annual rainfall is about 1100 mm, with an average of 600 mm in the dry season and 1500 mm in the wet season, while the average yearly temperature is 20 °C.

Experimental Design
Two experiments were conducted, one involving fruits collected from open pollinated trees of two farms and the second one involving fruits collected from controlled pollinations.

Source of Planting Material
The planting material (seed) used for raising seedlings was collected from fruits raised in two experimental locations.The fruits from the open pollinations were collected in different months of the year 2015, the fruits of all the varieties were collected when they were ripe (with the coloration of the yellowish bark), the same collection criteria were used, both for fruits from open pollinations and from controlled crosses.The experiment was conducted in three locations: at the Citrus Active Germplasm Bank of the Embrapa Cassava and Fruits, located at the municipality of Cruz das Almas; and in two farms: (Alpercata farm and Lavoura and Pecuária Igarashi Ltda farm) both located in the municipality of Mucugê.The controlled pollinations were performed at the Citrus Active Germplasm Bank of the Embrapa Cassava and Fruits, located at the municipality of Cruz das Almas; and in two farms: (Alpercata farm and Lavoura and Pecuária Igarashi Ltda farm) both located in the municipality of Mucugê.The controlled crosses were carried out in the month of September of the year of 2015, involving the varieties Nova, Fortune and Ortanique as female parental, and the male parents 'Page', 'Montenegrina', 'Swatow', 'Fremont' and 'Kincy'.The number of controlled pollinations varied according to the availability of flowers at the time of their realization.They were carried out in the period of greatest solar intensity and temperature, on dry days, between 10:00 a.m. and 4:00 p.m., to facilitate release and use of the pollen.With respect to the environmental effect on the formation of seeds with potential to generate triploid plants, the conditions in Mucugê were favorable.Three triploids were identified from this location, one of the 'Clemenules' variety and two 'Ortanique' (Table 1).For the locality of Cruz das Almas, triploids were not obtained.
Though the environment in Mucugê favored the formation of triploid in comparation to Cruz das Almas, mention should be made of the low seed germination rate in both environments and, consequently, the small number of plants obtained.But, all the plants produced by in vitro culture, regardless of the seed type and local, were evaluated by flow cytometry.

Ploidy of Citrus Plants Obtained From Controlled Crosses, Measured by Flow Cytometry
We performed 197 pollinations in Cruz das Almas (  In the controlled crosses performed in both Cruz das Almas and Mucugê, we used as female parents the varieties 'Nova', 'Ortanique' and 'Fortune', and as male parents 'Page', 'Swatow', 'Montenegrina', 'Fremont' and   Regarding seed type, all the triploids came from small seeds, and of the 13 plants obtained from these seeds, seven were triploids (54% frequency).However, no triploid plants were obtained in Cruz das Almas (Table 4).
In general, although the environment conditions in Mucugê promoted a higher frequency of triploids than in Cruz das Almas, where no plants were obtained with this ploidy from the controlled crosses, the number of plants obtained in both environments was low and directly proportional to the seed germination rate, which was also low in the majority of the varieties evaluated.

Discussion
Triploid citrus hybrids can be obtained by crossing diploid parents through production of unreduced gametes by the female parent, where the frequency of these gametes depends on the genotype (Esen & Soost, 1971;Luro, Maddy, Jacquemond, Froelicher, Morillon, Rist, & Ollitrault, 2004).Triploid embryos are more often produced by small seeds, between 1/6 and 1/3 of normal size.Generally these seeds do not germinate under normal greenhouse conditions, so it is necessary to rescue and culture these embryos in vitro to attain high germination rates (Aleza et al., 2010b).Cameron and Frost (1968) observed that the frequency of obtaining natural triploid plants in interspecific or intraspecific progenies of Citrus originating from pollinations between diploid parents was generally low (near 5%), while according to Essen and Soost (1971), the rate varies in function of the maternal genotype.However, Aleza et al. (2010b), investigating controlled 2x × 2x hybridizations involving monoembryonic and non-apomictic female parents of citrus, and using embryo rescue and in vitro culture techniques, achieved a 92% rate of triploid plants.Esen and Soost (1971, 1973a, 1973b) concluded that triploid plants are formed from small seeds, which can be obtained via 2x × 2x crosses, but the frequency of this event is generally low.In this context, the authors proposed the hypothesis that embryos formed from polyploid endosperm was the factor responsible for the small seed size, because all the seeds containing triploid embryos presented pentaploid endosperm.
In citrus, the endosperm of seeds that contain diploid embryos is naturally triploid.However, histological studies performed by Esen andSoost (1971, 1973a) demonstrated that small seeds contain embryos with pentaploid endosperm, which grows more slowly and develops prematurely, indicating that this is the factor responsible for reducing the size of the seeds that contain triploid embryos.
Cytogenetic analysis carried out by Aleza, Cuenca, Hernández, Juárez, Navarro, and Ollitrault (2015) revealed that triploid citrus embryos are associated with pentaploid endosperm, and there is strong evidence that these embryos result from fertilization of unreduced ovules by normal haploid pollen.Finally, they observed that depending on the genotype, the frequency of duplication between female gametes varied from less than 1% to more than 20%.Aleza, Juárez, Cuenca, and Ollitrault, (2012a), working with 2x × 4x hybridizations of citrus, obtained 1061 plants from normal seeds, of which 279 were triploids (26%), and found that in hybridizations using diploid (2n) female parents with tetraploid (4n) male parents, it is possible to obtain triploid plants from both normal and underdeveloped seeds.
Some studies have shown that the aspects of the male parent and environmental conditions influence the production of triploid citrus plants (Aleza et al., 2010b;Luro et al., 2004;Viloria & Grosser, 2005).The influence of environmental conditions on plants' reproductive success is well documented, by affecting the production and size of pollen and the growth of the pollen tube (Young & Stanton 1990).Besides this, the temperature during the progamic phase is one of the most important environmental factors that affect the performance of pollen.However, the response to temperature during the reproductive phase depends on the genotype (Hedhly, Hormaza, & Herrero, 2005).
Aleza, Froelicher, Schwarz, Agusti, Hernández, Juárez, Luro, Morillon, Navarro, and Ollitrault (2011), Hussain, Curk, Ollitrault, Morillon, and Luro (2011), Otto and Whitton (2000), and Saleh, Allario, Dambier, Ollitrault, and Morillon (2008) all have proposed the hypothesis that different environmental conditions, such as temperature, can have an effect on chromosome duplication events.Barrett and Hutchison (1978) identified polyploidy rates of 0.75% and 0.90% from seeds of citrus plants grown in Florida and California, respectively, and associated this difference with temperature variation.The effect of low temperature on polyploidization events appears to be a general rule, both in plants and animals (Otto & Whitton, 2000;Ramsey & Schemske, 1998).Besides the adaptive advantage of polyploids, the larger rate of polyploidization events in a colder climate could be one of the factors related to the increased frequency of polyploid species in the Northern Hemisphere (Asker & Jerling, 1992).Aleza et al. (2011) reported that the overall frequency of tetraploid seedlings among 30 Citrus varieties cultivated in Valencia (Spain) and Corsica (France) was from 1% to 8% (varying from 0% to 9% according to the cultivar and environmental conditions).Besides this, they confirmed the importance of the environmental conditions on the rates of tetraploids, studying the same genotype ('Carrizo' citrange) harvested in different countries.They found higher rates in the Mediterranean than in tropical areas and significant negative correlations between the rates of tetraploids and mean temperatures during the floral induction and flowering period.Based on this, they suggested that colder growing conditions favor tetraploidization events in citrus nucellar cells.
Temperature affects the efficiency of producing citrus hybrids based on 2x × 4x hybridizations.The production of triploid hybrids is influenced by the fecundation success of the pollen, which is dependent on the pollen quality.In turn, this is determined not only by the male genotype, but also the environmental conditions, level of compatibility between the male and female parents, and environmental effects on the germination and growth of the pollen tube (Viloria & Grosser, 2005;Young & Stanton, 1990).Aleza et al. (2012b) studied the effect of parents and environmental conditions on the production of citrus triploid hybrids in 4x × 2x sexual hybridizations during five years.The hybridization results indicated the existence of an interaction between male parents and environmental conditions on the production of triploids.Besides this, the results of two previous studies indicated that environmental conditions also affect the production of triploid hybrids in 2x × 2x hybridizations (Aleza et al., 2010b;Luro et al., 2004).Aleza et al. (2010b), investigating hybridizations using 2x × 2x crosses over a period of 10 years (1996 to 2006), observed that triploid hybrids were produced by seeds that had between 52% and 62% of the size of normal seeds, and that embryo rescue and flow cytometry are two essential techniques for genetic improvement programs aiming to obtain triploids in citrus plants based on hybridizations between diploid parents.

Conclusions
The frequency of obtaining triploids varied according to the genotype (the Ortanique variety showed the greatest potential to produce triploids); of seed size (small and underdeveloped seeds presented ability to generate triploids); and the environment (triploids were obtained only in the Mucugê environment).
However, further studies must be done, to better understand the effect of environmental conditions on the formation of triploid individuals.