A novel source of cytoplasmic male sterility in Calibrachoa pubescens

Calibrachoa pubescens is a species native from the south of Brazil, Uruguay and northeast Argentina. An accession identified as 7.3.1.1 was collected at San Martín Department in Corrientes Province, Argentina and is included in the calibrachoa breeding program at the Institute of Floriculture, INTA. This accession is male sterile and produces male-sterile progeny, characterized by the lack of pollen production. Male sterility may be controlled by nuclear or cytoplasmic genes with each type presenting a different mode of inheritance. The objective of this research was to present a novel source of cytoplasmic male sterility in Calibrachoa pubescens. Crosses were made in the greenhouse between the male-sterile line 7.3.1.1 as the female parent and seven male-fertile lines of diverse origin. F1s were backcrossed and self-pollinated. Individual plants of the progenies were classified as male-fertile or male-sterile according to pollen viability. Analyses of observed segregations showed that male sterility observed in 7.3.1.1 results from the interaction of a male sterile cytoplasm and nuclear restorer genes. A novel male sterile cytoplasm is now available for breeders. Main advantages of cytoplasmic male sterility in ornamentals breeding are: efficient hybrid production, increased flower longevity, avoidance of pollen allergens and control of plant invasiveness.


INTRODUCTION
The cultivation of calibrachoa (million bells) as an ornamental plant is relatively recent.It begins with the launching of the first cultivars in the 1990s.However, today it is one of the most important bedding and balcony plants with global economic importance (KANAYA et al., 2010;JĘDRZEJUK et al., 2017).Calibrachoa cultivars are bred by interspecific hybridization using different wild species of Calibrachoa spp.Cerv.(Solanaceae) (KANAYA et al., 2010).At present Argentina grows 12 native species of Calibrachoa (GREPPI et al., 2013).The Institute of Floriculture of INTA carries out a breeding program of calibrachoa using native species of Argentina to create new vigorous and freely-flowering calibrachoa plants with unique and attractive flower coloration and good garden and summer performance.
C. pubescens (Spreng.)Stehmann is a species native from the south of Brazil, Uruguay and northeast Argentina (GREPPI et al., 2013).An accession identified as 7.3.1.1 was collected from a small population located at Tres Cerros, San Martín Department in Corrientes Province, Argentina, and it was included in the calibrachoa breeding program (Figure 1).This accession is male sterile and produces male-sterile progeny, characterized by the lack of pollen production.Female fertility, on the other hand is normal.Male sterility may be controlled by nuclear or cytoplasmic genes, with each type presenting a different mode of inheritance.Moreover, cytoplasmic male sterility can be restored by the effect of nuclear genes, representing a good example of the interaction between the mitochondrial and nuclear genomes (CHEN and LIU, 2014).Male sterility is a desirable trait for breeding purposes in ornamental crops to produce hybrid seed, to increase flowering duration (SMITH et al., 2004), to avoid pollen allergens (SINGH et al., 2012) and to control invasiveness (ANDERSON et al., 2006).The objective of this research was to present a novel source of cytoplasmic male sterility in Calibrachoa pubescens.
Table 3. List of male-fertile lines of calibrachoa used in this study and their origins.

Male fertile line
Origin The backcross (BC1) between F1 (7.3.1.1 x Dark Blue) x Dark Blue was also obtained.An intraspecific cross was made in the greenhouse between C. pubescens 7.3.1.1 and the male-fertile accession D2 collected from the same population (Table 2).F1s were backcrossed (BC1s) to the male fertile parent and self-pollinated to produce F2s.
Frequencies distributions of pollen viability were determined for each analyzed generation.

RESULTS AND DISCUSSION
Pollen viability of plants from different generations and from the pollen donors of the crosses was determined using fluorescein-diacetate (FDA) / propidium iodide (PI) and epifluorescence microscopy with UV light (Figure 2).This method allows differentiating bright green viable pollen grains from death pollen grains stained in red.FDA is considered a vital staining dye, which is hydrolyzed by cellular esterases and originates the accumulation and easy detection of fluorescein.On the other hand, PI cannot penetrate intact, living cellular membranes, so the observed red fluorescence is an indicator of non-viable cells or damaged membranes (GREISSL, 1989).A total of 246.764 pollen grains were observed.Plants were classified as fertile, partially fertile or sterile according to the percentage of non-viable pollen.When considering the interspecific crosses, no male fertile plants were observed in the F1s between the male sterile parent 7.3.1.1 and the pollen donors 07-531#4 and Dark Blue.However, fertile plants were found in the other F1s.All the F1 populations showed a reduction in mean pollen viability compared to the male fertile parent (Table 4).In BC1s plants from the crosses F1 (7.3.1.1 x Dark Blue) x Dark Blue, no fertile plants were observed and the mean pollen viability values were drastically reduced (Table 4).The segregation patterns of these BC1s reveal that the male sterility observed in the accession 7.3.1.1 is maternally inherited and determined by cytoplasmic factors.Analysis of the intraspecific cross between 7.3.1.1 and D2 showed no male sterile plants in the F1.The F2 and BC1 were obtained using two different F1 plants as male parents, each of them showing a different degree of male fertility.In both generations, male sterile plants were observed and the mean pollen viability decreased as compared to the F1 generation (Table 4).Frequency distributions of pollen viability in F1s plants from interspecific crosses were represented (Figure 3).In crosses between the male sterile 7.3.1.1 and the pollen donors Dark Blue and 07-531#4 only plants showing high percentages of non-viable pollen were observed.All the other F1s displayed a wider range of categories corresponding to different grades of non-viable pollen.In the BC1s derived from Dark Blue only plants presenting high percentage of non-viable pollen were observed (Figure 4).Interestingly, in the intraspecific cross between 7.3.1.1 and D2 an increase in the frequency of plants with nonviable pollen was evident both in F2 and BC1 generations compared with F1 (Figure 5).Taken together, these data indicate the presence of restorer genes in the pollen donors 08-217#3, 06-53N#1, 9.3.4.6, 08-236#1 and D2, but not in Dark Blue and 07-531#4.Male sterile phenotype in the accession 7.3.3.1 is characterized by the lack of pollen.However, male sterile plants obtained in our crosses presented from complete absence of pollen to variable degree of pollen production.We hypothesize that microsporogenesis seems to be affected at different stages depending on the nuclear background.Similar results were described in cytoplasmic male sterility in petunia, where a wide variation of male sterile phenotypes expressed at different times of the breakdown of microsporogenesis was reported (IZHAR, 1977).Recently, the effect of nuclear background on the expression of male sterile cytoplasms has been reported in chicories (HABARUGIRA et al., 2015) and leaf mustard (WAN et al., 2014).Further research is needed in order to characterize microsporogenesis in 7.3.3.1 and in the male sterile plants originating from different crosses.
It should be mentioned that both male sterile and hermaphrodite plants coexist in the original population of C. pubescens, thus representing a case of gynodioecy (MC CAULEY and BAILEY, 2009).The use of genetic and ecological approaches would be of help to determine the mechanisms involved in the maintenance of gynodioecy in this population.

CONCLUSIONS
To our knowledge this is the first report of a cytoplasmic male sterile cytoplasm in calibrachoa.Due to maternal inheritance of the sterile cytoplasm it will be easy to get its introgression in different backgrounds with breeding purposes.This novel resource will reduce the labor demanded for making crosses by eliminating the need of manual emasculation and will allow hybrid production more efficiently.Besides, cytoplasmic male sterility may be of interest for increasing flower longevity and for controlling the dispersion of invasive species.Although we have not tested the effect of different environmental conditions on cytoplasmic male sterility and fertility restoration, it is likely that they play a role on the expression of these characters, as it has been shown in several species ( VAN MARREWIJK, 1969;IZHAR, 1975;DHALL, 2010;DE STORME and GEELEN, 2014;BUECKMANN et al., 2016).Future research should consider this aspect to better describe the expression of the male sterile cytoplasm of calibrachoa.

Figure 1 .
Figure 1.Accession 7.3.1.1 of Calibrachoa pubescens and geographic localization of the population from which it was collected.

Table 1 .
Accessions list of natural species of calibrachoa used in this study.

Table 2 .
List of calibrachoa cultivars used in this study.