Fruit set and cross compatibility of B. semperflorens × B. ‘Orange Rubra’
When 26 cultivars in five series of B. semperflorens were used for inter-sectional crosses of B. semperflorens × B. ‘Orange Rubra’, the fruit set ratio of single flower type cultivars including triploids ranged from 70–100% (94.3% in average), which was higher than that of double flower type cultivars (Queen series), which gave 60–70% fruit set ration with the average of 63.3% ().
Despite of the high fruit set ratios, most of the harvested seeds at maturity 30 DAP were crimpy and failed to germinate, whereas remaining plumpy seeds germinated 2 weeks after inoculation on ER medium. Among the 26 cultivars of B. semperflorens used as female parent, 10 cultivars yielded progenies with the plantlet yield efficiency (PYE) of 0.11 to 1.70, whereas ‘Ambassador’ series containing 8 triploid and 1 tetraploid cultivars was unable to obtain any progenies. In terms of single flower type cultivars, ‘Sprint-Pink’ gave the highest PYE of 1.70, followed by ‘Varsity-White’ and ‘Monza-White’. On the other hand, PYE of a double flower type cultivar ‘Queen-Pink’ (1.14) showed almost the same as single flower type cultivars. Unfortunately, the other 2 double flower type cultivars did not yield any progenies.
In embryo rescue experiment (), four out of five cultivars of B. semperflorens examined yielded progenies, whereas ‘Ambassador-Coral’ (4x) failed to yield plantlets. In the successful cultivars, the highest PYE was obtained when cultured at 12–16 DAP, whereas cultures with earlier or later DAP resulted in reduced PYE (). Furthermore, the best hybrid efficiencies of 12–16 DAP were obviously higher than 30 DAP in the same cultivar (, ). For instance, the highest PYE of embryo rescue method in B. ‘Sprint-Pink’ × B. ‘Orange Rubra’ was 9.7, which was evidently better than the value (1.7) in the culture of mature seeds.
In both mature and immature seed cultured on ER medium, zygotic embryos germinated as abnormal embryoids without apparent cotyledons after 15 days of culture () and only root elongation was observed till 30 days of culture. Then, shoots were formed at the top of abnormal embryo 45 days after culture (). Although those rosette shoots did not grow well without development of healthy root system, they developed into normal plantlets after excision and transfer onto root induction medium (). They were then transferred to pots in soil, acclimatized for at least 3 weeks under sunshade conditions, and successfully grown in greenhouse () till flowering to compare the difference of plant characteristics ().
Fig. 1 Regeneration of hybrid plants after reciprocal crosses between B. semperflorens and B. ‘Orange Rubra’. (A) Germinated embryos from immature seeds 15 days after inoculation onto ER medium. (B) Shoot regeneration from the top of abnormal (more ...)
The effect of different culture medium on the embryo rescue
For evaluating the influence of different culture media on the embryo rescue, the immature fruits were harvested from the cross of B. ‘Sprint-Pink’ × B. ‘Orange Rubra’ at 12 DAP. The result indicated that PC medium had an ability to obtain slightly more progenies than ER medium (). However, all the progenies on PC medium emerged as calluses and later needed to transfer onto shoot induction medium for regenerating shoots (). Consequently, PC medium required more time and labor for obtaining normal in vitro plantlets than ER medium.
The influence of different culture medium on the embryo rescue efficiency in the inter-sectional hybridization between B. semperflorens ‘Sprint-Pink’ and B. ‘Orange Rubra’
Estimation of hybridity, ploidy and genomic composition by using flow cytometry
Totally 142 progenies, which were obtained from 9 cross combinations between B. semperflorens and B. ‘Orange Rubra’, were subjected to flow cytometry (FCM) analysis to evaluate the relative DNA contents of both parental donors and their hybrids ( and ). The relative DNA contents of B. semperflorens diploid cultivars, tetraploid cultivars and B. ‘Orange Rubra’ were 2, 4 and 4.74 units, respectively. In the case of cross combination between B. semperflorens diploid cultivar (SS genome) and B. ‘Orange Rubra’ (RR genome), more than half (59%, 72/122) of the hybrids showed expected normal genome combination (RS) with relative DNA content of 3.37 unit (). Some unexpected DNA contents that might correspond to the different genomic combinations of both parents such as RRS (), RSS, RRSS () and RRRRSS were also obtained as 4.37, 5.74, 6.78 and 11.48 units, respectively. A noteworthy fact is that the RRRRSS genome appeared only in the cross combination between diploid B. semperflorens ‘Monza-Pink’ and B. ‘Orange Rubra’ ().
Flow cytometric analysis of the ploidy level and genome combination of hybrids in inter-sectional cross between B. semperflorens and B. ‘Orange Rubra’
Fig. 2 Relative DNA content of hybrids between B. semperflorens and B. ‘Orange Rubra’ were estimated by flow cytometric analysis. (A) 1: B. semperflorens ‘Queen-Pink’ (SS). 2: Hybrid (RS). 3: B. ‘Orange Rubra’ (more ...)
In the 3 cross combinations between tetraploid cultivars of B. semperflorens (SSSS genome) and B. ‘Orange Rubra’ (RR genome), 83% progenies (25/30) showed the expected triploid genome combination (RSS) with relative DNA content of ca. 4.37 unit. However, the plants with unexpected genome combinations, RRS and RRSS, were also produced in the crosses using B. ‘Monza-Coral’ (4x) in the former and both ‘Monza-Coral’ and ‘Varsity-White’ in the latter, respectively.
In addition, the relative DNA content of the hybrids obtained from reverse cross combination, B. ‘Orange Rubra’ (RR) × tetraploid B. semperflorens ‘CU1’ (SSSS), was also investigated (). Among 12 progenies totally analyzed, only 2 were revealed to be expected RSS hybrids () with 4.37 units, whereas remaining 10 progenies (83%) displayed completely equal DNA content to that of B. ‘Orange Rubra’ (4.74 units, RR genome) ().
Flow cytometric analysis of the ploidy level and genome combination of hybrids in inter-sectional cross between B. ‘Orange Rubra’ and B. semperflorens ‘CU1’
Hybridity analysis by using RAPD markers
DNA bands specific to B. semperflorens cultivars and B. ‘Orange Rubra’ were detected by RAPD analysis using the primers OPE-4 and OPE-3, among the 10 random primers tested. The putative hybrids 2R, 6R 19R, 29R and QR, obtained from the different cross combinations between B. semperflorens and B. ‘Orange Rubra’, showed the specific bands for both parents using OPE-4 (). Although the putative hybrids 23R and 28R did not show the specific band of B. semperflorens by OPE-4 primer, the hybridity was confirmed by another primer OPA-2 (data not shown). Moreover, hybridity of the putative hybrids obtained from the reverse crosses, i.e., B. ‘Orange Rubra’ × B. semperflorens ‘CU1’ was also confirmed by using the primer OPE-3 even in the plants that showed RR genome type (). Thus, all the progenies obtained from both reciprocal crosses were confirmed to be hybrids.
Fig. 3 RAPD analysis for confirming the hybridity of the palnts obtained from the crosses between B. semperflorens and B. ‘Orange Rubra’ (A) and from the reverse cross (B). (A) The primer OPE-04 was used. R: ‘Orange Rubra’. 2: (more ...)
Characteristics of the hybrids
In the crosses where B. semperflorens was used as female parents, two triploid hybrids with different genomic types (RRS and RSS) already attained to flowering stage (). Both types of hybrids showed almost intermediate plant height but differed in flower color depending on the genomic combinations; RRS plant had more Orange Rubra-like flowers whereas RSS showed more B. semperflorens-like ones (). In the reverse crosses where tetraploid B. semperflorens was used as male parent, hybrid plants with RSS genome showed almost the same dwarf plant height as B. semperflorens (). Although RR genome type plants were also obtained in this cross combination, they also showed the same dwarf phenotype as RSS hybrids (). In these two genome types of hybrids, RSS plants bore almost the same size of flowers to B. semperflorens, whereas RR plants produced comparable size of flowers to B. ‘Orange Rubra’. One of the interesting results found in the hybrid phenotypes was the leaf variegation recognized as silver spots on leaves (). Although the character of silver spots is originally found in B. ‘Orange Rubra’ to some extent but not in any cultivars of B. semperflorens, it was expressed more strongly in the hybrids than B. ‘Orange Rubra’ (). Moreover, we also observed the flower fragrance in triploid (RRS) hybrid (), which had stronger expression than the other hybrids and B. ‘Orange Rubra’.
Fig. 4 Characteristics of hybrid plants obtained from reciprocal crosses between B. semperflorens and B. ‘Orange Rubra’. (A) 1: Female parent: B. semperflorens ‘Sprint-Pink’. 2: Hybrid ‘SO1’ (RRS). 3: Male parent: (more ...)
Pollen fertility of hybrids
The pollen fertilities of parental plants examined were as follows: 72.2% in ‘Sprint-Pink’ (2x), 20.4% in ‘Varsity-Pink imp’ (4x), 39.2% in ‘CU1’ and 74.1% in ‘Orange Rubra’, respectively (), whereas triploid cultivars of B. semperflorens had almost no pollen fertility. Among the hybrids which attained to the flowering stage, all the triploid hybrids including RRS genome (SO1–SO3) obtained from B. semperflorens ‘Sprint-Pink’ × B. ‘Orange Rubra’ and RSS genome (VO1–VO5 and OC1–OC2) obtained from B. semperflorens ‘Varsity-Pink imp’ × B. ‘Orange Rubra’ and reverse cross, B. ‘Orange Rubra’ × B. semperflorens ‘CU1’ showed almost no pollen fertility. However, the diploid hybrids possessing almost equal amount of DNA to ‘Orange Rubra’ (RR type) obtained from the cross of B. ‘Orange Rubra’ × B. semperflorens ‘CU1’ showed relatively high pollen fertility (24–45%) which was comparable to B. ‘Orange Rubra’.
Pollen fertility of B. semperflorens, B. ‘Orange Rubra’ and their hybrids with different genomic compositionsa