BACKGROUND
doubled haploid  technology is a valuable tool in modern breeding programs since it allows for the production of completely homozygous lines within a few months and dramatically reduces the time required to establish new cultivars  <cit> .
in addition, many genomic approaches such as association or qtl mapping benefit greatly from the use of dh populations  <cit> .
dh plants are routinely generated in crops such as barley  <cit> .
however, due to high costs and low efficiency, dh production is yet to be established in applied breeding programs for other cereals such as triticale.
of the different methods available for dh production, microspore embryogenesis  shows the greatest potential due to the abundance of microspores per spike and consequently the higher frequency of dh output as compared to other approaches such as wide crosses.
factors limiting the application of microspore culture at a commercial level include the rate of embryogenesis and regeneration, the frequency of albinism among regenerants and the frequency of chromosome doubling required to obtain fertile dh plants  <cit> .
in barley, the rate of spontaneous chromosome doubling is high and ranges from 70-90 %  <cit> .
by contrast, the rate of spontaneous doubled haploids reported for triticale ranges from 6-38 %  <cit> , which similar to most crops is too low and variable to omit a specific step for induced chromosome doubling.
the most common doubling agent is colchicine which is traditionally applied in vivo <cit> .
this application of colchicine at the seedling stage, however, has several disadvantages: large amounts of colchicine solution are required which imparts high costs and increased exposure to this toxic substance, a high rate of mortality of seedlings owing to the treatment, and production of mixoploid or chimeric plants which results in low seed set and consequently the requirement of additional seed multiplication before field testing  <cit> .
the application of colchicine during early stages of androgenesis could alleviate some of these problems  <cit>  and has been applied to different cereals including wheat  <cit> , durum wheat  <cit>  and rice  <cit> .
in wheat, the incorporation of colchicine during the first hours of anther or microspore culture improved the doubling rate  <cit> .
the application of doubling agents, however, not only increases the percentage of chromosome doubling, but also affects the whole androgenetic process.
the optimum concentration and time of application for chromosome doubling may have negative effects on embryogenesis and regeneration rate, as well as on the percentage of green plants  <cit> .
thus, in vitro chromosome doubling must be investigated and optimized in light of the entire androgenetic process.
in triticale, in vitro application of colchicine has been applied in anther culture approaches  <cit>  but not for microspore culture which is emerging as the method of choice.
the objective of this work was to investigate the suitability of in vitro chromosome doubling during microspore culture for efficient doubled haploid production in triticale.
more specifically, the aim was to determine the optimum concentration and incubation period of colchicine for in vitro chromosome doubling while considering the androgenetic response as a whole.
RESULTS
visual scoring of petri dishes containing microspores in embryo induction revealed no obvious effect of the colchicine incubation on the number of regenerated embryos 4 weeks after microspore isolation .
in addition, the embryos were of similar high quality and almost no calli were formed.
the regeneration of plants from the transferred embryos averaged  <dig> % for the negative controls without colchicine  and similar results were obtained for most combinations of colchicine concentration and incubation duration.
lower values were found for the 5 mm concentration at 48 h and 72 h and for 1 mm at 72 h. no clear trend could be observed relating to the percentage of green plants.
whereas colchicine increased the percentage of green plants for the 24 h and 72 h periods, it decreased green plant formation for the 48 h colchicine treatments.
in comparison, the in vivo approach showed a higher regeneration rate .
survival rate refers to the rate of plants that survived the transfer from culture medium to soil and grew to maturity.
means of columns followed by different letters indicate significant differences at the α =  <dig>  level.
the survival rate for plants derived from colchicine-treated seedlings was 44% i.e.
approximately half of the green plantlets that were transferred from culture medium could be grown to maturity .
the remainder died, predominantly within 4 weeks of the colchicine treatment and subsequent transfer to soil.
compared to this in vivo chromosome doubling treatment, the survival rate was generally higher for the in vitro colchicine approach with an average survival rate of  <dig> %.
no negative effect was observed for the different colchicine concentrations as compared to the respective controls .
the rate of fertile doubled haploid plants from the colchicine treatment at the seedling stage was  <dig> % , while the percentage of fertile plants in the negative controls, which is equivalent to the spontaneous doubling rate, averaged  <dig> %.
there was a clear effect of the in vitro colchicine treatment on the proportion of fertile plants .
the highest values, ranging from  <dig> % to  <dig> %, were obtained for 1 mm and 5 mm concentrations and incubation times of 48 h and 72 h. these values roughly doubled that of the spontaneous doubling rate and were even higher than that obtained by the in vivo procedure.
consistent with this, we observed significant variance components of colchicine concentration and incubation time for the proportion of fertile plants .
the plants produced mainly fertile tillers as compared to the plants from the in vivo approach for which, on average, half of the tillers were sterile .
there appeared to be no effect on the number of seeds per fertile tiller.
<dig> mean number of sterile and  <dig> fertile tillers per seed-setting plant, and  <dig> mean number of seeds per fertile or partially fertile tiller.
means of columns followed by different letters indicate significant differences at the α =  <dig>  level.
discussion
for scientists as well as for commercial breeders, the establishment of a routinely applicable doubled haploid  protocol is of significant interest.
for triticale, microspore culture has been shown to provide a high frequency of dh plants, and thus is becoming the method of choice for dh production  <cit> .
key issues remain, however, that prevent the widespread application of this method.
a major limitation relates to the low frequency of spontaneous dh plants produced in triticale, which necessitates chemically-induced doubling of the chromosomes to obtain fertile plants.
in this study we compared the traditionally employed in vivo approach to those where colchicine is applied in vitro during the first stage of microspore culture.
the in vitro application of colchicine has been reported to induce positive as well as negative effects on the whole androgenetic pathway, influencing the events of embryogenesis, regeneration rate and the percentage of green plants  <cit> .
in contrast to zhou et al.
<cit> , who found an increased frequency of embryogenesis in brassica by the application of colchicine, we observed no effect of the in vitro colchicine treatment on embryo formation in the experiments described here  nor in our routine dh program .
we did, however, observe a reduced regeneration rate for the treatments with high colchicine concentrations and extended incubation time .
a similar trend was observed by soriano et al.
<cit>  for the wheat cultivar chris, but not for two other cultivars, indicating that this process is likely genotype dependent.
a critical point for a dh procedure is the survival rate, which gives a measure of how many green plants regenerated on culture medium survive until maturity.
we found that this survival rate was only 44% for the in vivo approach mainly due to a high mortality after the colchicine treatment of the seedlings.
colchicine is an anti-microtubule agent that not only doubles the chromosomes in the meristem but also has severe effects on the health status of the seedlings, possibly explaining the high mortality  <cit> .
by contrast, the in vitro approach resulted in much higher survival rates  as the seedlings are not subjected to the deleterious colchicine treatment.
in this approach, the survival rate of seedlings appeared to be largely dependent upon the transfer from culture medium to soil.
we found that the rate of spontaneous doubled haploids was  <dig>  %, which is consistent with previous reports for triticale  <cit> , and confirms that an additional chemically-induced chromosome doubling step is required for triticale dh programs.
the in vitro colchicine method tested here  resulted in improved frequency, with the best treatments more than doubling the rate of fertile plants as compared to the spontaneous doubled haploid rate.
more importantly, this method also outperformed the in vivo approach.
the highest rates of fertile plants were observed for the 1 mm and 5 mm colchicine concentrations applied for  <dig> or 72 h .
the increasing proportion of fertile plants among the regenerated green plants produced after in vitro treatment can be seen in the mosaic plot .
our results on the number of sterile and fertile tillers per regenerated green plant suggest that the in vivo approach mainly produces chimeric plants with sectors that are still haploid .
by contrast, the plants derived from the in vitro approach produced predominantly fertile tillers suggesting that the doubling occurred during an early stage of embryogenesis, and that the plants are entirely doubled haploid.
the few sterile tillers may be attributable to inadvertent stress applied to the plants , or to doubling taking place at a later stage of embryogenesis such that a minority of the recovered plants are also mosaic for haploid and doubled haploid sectors.
whereas the enhancement of the proportion of fertile plants is an important goal, the number of fertile plants derived from a defined number of transferred embryos is the ultimate measure of dh procedure success.
in this study we found that this is maximized by three treatment combinations: 1 mm colchicine applied for 24 h, or the lower concentration of  <dig>  mm colchicine for  <dig> or 72 h. the variation in optimal incubation time is advantageous because it gives the experimenter more flexibility, especially in applied high-throughput dh programs.
with regard to the rate of fertile plants produced from a standard number of transferred embryos, the in vivo approach was only slightly less effective than the in vitro approach.
this similar rate, however, is misleading since a fair comparison must consider the effort required to reach this value.
culturing of seedlings and fostering of treated plants to maturity, including the bagging required to prevent cross-pollination, are resource and time consuming steps.
for the in vivo approach, many plants are maintained throughout the entire method but do not yield any dh seeds.
by contrast, the higher rate of fertile plants and the higher survival rate observed for the in vitro approach more than compensate for the reduced regeneration rate as compared to the in vivo approach.
CONCLUSIONS
in conclusion, we have shown that an in vitro approach for chromosome doubling performs well for microspore culture of triticale.
the higher rate of fertile plants produced per number of transferred embryos and the more economical employment of resources make the in vitro approach an attractive alternative to the traditionally used in vivo colchicine treatment of seedlings.

 doubled haploid technology is a valuable tool in modern breeding programs since it allows for the production of completely homozygous lines within a few months and dramatically reduces the time required to establish new cultivars. in addition, many genomic approaches such as association or qtl mapping benefit greatly from the use of dh populations. dh plants are routinely generated in crops such as barley. however, due to high costs and low efficiency, dh production is yet to be established in applied breeding programs for other cereals such as triticale. of the different methods available for dh production, microspore embryogenesis shows the greatest potential due to the abundance of microspores per spike and consequently the higher frequency of dh output as compared to other approaches such as wide crosses. factors limiting the application of microspore culture at a commercial level include the rate of embryogenesis and regeneration, the frequency of albinism among regenerants and the frequency of chromosome doubling required to obtain fertile dh plants. in barley, the rate of spontaneous chromosome doubling is high and ranges from 70-90 %. by contrast, the rate of spontaneous doubled haploids reported for triticale ranges from 6-38 %, which similar to most crops is too low and variable to omit a specific step for induced chromosome doubling. the most common doubling agent is colchicine which is traditionally applied in vivo. this application of colchicine at the seedling stage, however, has several disadvantages: large amounts of colchicine solution are required which imparts high costs and increased exposure to this toxic substance, a high rate of mortality of seedlings owing to the treatment, and production of mixoploid or chimeric plants which results in low seed set and consequently the requirement of additional seed multiplication before field testing. the application of colchicine during early stages of androgenesis could alleviate some of these problems and has been applied to different cereals including wheat, durum wheat and rice. in wheat, the incorporation of colchicine during the first hours of anther or microspore culture improved the doubling rate. the application of doubling agents, however, not only increases the percentage of chromosome doubling, but also affects the whole androgenetic process. the optimum concentration and time of application for chromosome doubling may have negative effects on embryogenesis and regeneration rate, as well as on the percentage of green plants. thus, in vitro chromosome doubling must be investigated and optimized in light of the entire androgenetic process. in triticale, in vitro application of colchicine has been applied in anther culture approaches but not for microspore culture which is emerging as the method of choice. the objective of this work was to investigate the suitability of in vitro chromosome doubling during microspore culture for efficient doubled haploid production in triticale. more specifically, the aim was to determine the optimum concentration and incubation period of colchicine for in vitro chromosome doubling while considering the androgenetic response as a whole.  visual scoring of petri dishes containing microspores in embryo induction revealed no obvious effect of the colchicine incubation on the number of regenerated embryos 4 weeks after microspore isolation. in addition, the embryos were of similar high quality and almost no calli were formed. the regeneration of plants from the transferred embryos averaged % for the negative controls without colchicine and similar results were obtained for most combinations of colchicine concentration and incubation duration. lower values were found for the 5 mm concentration at 48 h and 72 h and for 1 mm at 72 h. no clear trend could be observed relating to the percentage of green plants. whereas colchicine increased the percentage of green plants for the 24 h and 72 h periods, it decreased green plant formation for the 48 h colchicine treatments. in comparison, the in vivo approach showed a higher regeneration rate. survival rate refers to the rate of plants that survived the transfer from culture medium to soil and grew to maturity. means of columns followed by different letters indicate significant differences at the α =  level. the survival rate for plants derived from colchicine-treated seedlings was 44% i.e. approximately half of the green plantlets that were transferred from culture medium could be grown to maturity. the remainder died, predominantly within 4 weeks of the colchicine treatment and subsequent transfer to soil. compared to this in vivo chromosome doubling treatment, the survival rate was generally higher for the in vitro colchicine approach with an average survival rate of %. no negative effect was observed for the different colchicine concentrations as compared to the respective controls. the rate of fertile doubled haploid plants from the colchicine treatment at the seedling stage was %, while the percentage of fertile plants in the negative controls, which is equivalent to the spontaneous doubling rate, averaged %. there was a clear effect of the in vitro colchicine treatment on the proportion of fertile plants. the highest values, ranging from % to %, were obtained for 1 mm and 5 mm concentrations and incubation times of 48 h and 72 h. these values roughly doubled that of the spontaneous doubling rate and were even higher than that obtained by the in vivo procedure. consistent with this, we observed significant variance components of colchicine concentration and incubation time for the proportion of fertile plants. the plants produced mainly fertile tillers as compared to the plants from the in vivo approach for which, on average, half of the tillers were sterile. there appeared to be no effect on the number of seeds per fertile tiller. mean number of sterile and fertile tillers per seed-setting plant, and mean number of seeds per fertile or partially fertile tiller. means of columns followed by different letters indicate significant differences at the α =  level. discussion for scientists as well as for commercial breeders, the establishment of a routinely applicable doubled haploid protocol is of significant interest. for triticale, microspore culture has been shown to provide a high frequency of dh plants, and thus is becoming the method of choice for dh production. key issues remain, however, that prevent the widespread application of this method. a major limitation relates to the low frequency of spontaneous dh plants produced in triticale, which necessitates chemically-induced doubling of the chromosomes to obtain fertile plants. in this study we compared the traditionally employed in vivo approach to those where colchicine is applied in vitro during the first stage of microspore culture. the in vitro application of colchicine has been reported to induce positive as well as negative effects on the whole androgenetic pathway, influencing the events of embryogenesis, regeneration rate and the percentage of green plants. in contrast to zhou et al., who found an increased frequency of embryogenesis in brassica by the application of colchicine, we observed no effect of the in vitro colchicine treatment on embryo formation in the experiments described here nor in our routine dh program. we did, however, observe a reduced regeneration rate for the treatments with high colchicine concentrations and extended incubation time. a similar trend was observed by soriano et al. for the wheat cultivar chris, but not for two other cultivars, indicating that this process is likely genotype dependent. a critical point for a dh procedure is the survival rate, which gives a measure of how many green plants regenerated on culture medium survive until maturity. we found that this survival rate was only 44% for the in vivo approach mainly due to a high mortality after the colchicine treatment of the seedlings. colchicine is an anti-microtubule agent that not only doubles the chromosomes in the meristem but also has severe effects on the health status of the seedlings, possibly explaining the high mortality. by contrast, the in vitro approach resulted in much higher survival rates as the seedlings are not subjected to the deleterious colchicine treatment. in this approach, the survival rate of seedlings appeared to be largely dependent upon the transfer from culture medium to soil. we found that the rate of spontaneous doubled haploids was  %, which is consistent with previous reports for triticale, and confirms that an additional chemically-induced chromosome doubling step is required for triticale dh programs. the in vitro colchicine method tested here resulted in improved frequency, with the best treatments more than doubling the rate of fertile plants as compared to the spontaneous doubled haploid rate. more importantly, this method also outperformed the in vivo approach. the highest rates of fertile plants were observed for the 1 mm and 5 mm colchicine concentrations applied for or 72 h. the increasing proportion of fertile plants among the regenerated green plants produced after in vitro treatment can be seen in the mosaic plot. our results on the number of sterile and fertile tillers per regenerated green plant suggest that the in vivo approach mainly produces chimeric plants with sectors that are still haploid. by contrast, the plants derived from the in vitro approach produced predominantly fertile tillers suggesting that the doubling occurred during an early stage of embryogenesis, and that the plants are entirely doubled haploid. the few sterile tillers may be attributable to inadvertent stress applied to the plants, or to doubling taking place at a later stage of embryogenesis such that a minority of the recovered plants are also mosaic for haploid and doubled haploid sectors. whereas the enhancement of the proportion of fertile plants is an important goal, the number of fertile plants derived from a defined number of transferred embryos is the ultimate measure of dh procedure success. in this study we found that this is maximized by three treatment combinations: 1 mm colchicine applied for 24 h, or the lower concentration of  mm colchicine for or 72 h. the variation in optimal incubation time is advantageous because it gives the experimenter more flexibility, especially in applied high-throughput dh programs. with regard to the rate of fertile plants produced from a standard number of transferred embryos, the in vivo approach was only slightly less effective than the in vitro approach. this similar rate, however, is misleading since a fair comparison must consider the effort required to reach this value. culturing of seedlings and fostering of treated plants to maturity, including the bagging required to prevent cross-pollination, are resource and time consuming steps. for the in vivo approach, many plants are maintained throughout the entire method but do not yield any dh seeds. by contrast, the higher rate of fertile plants and the higher survival rate observed for the in vitro approach more than compensate for the reduced regeneration rate as compared to the in vivo approach.  in conclusion, we have shown that an in vitro approach for chromosome doubling performs well for microspore culture of triticale. the higher rate of fertile plants produced per number of transferred embryos and the more economical employment of resources make the in vitro approach an attractive alternative to the traditionally used in vivo colchicine treatment of seedlings. 
doubled haploid production is a key technology in triticale research and breeding. our results show a pronounced increase in the proportion of doubled haploid triticale plants compared to the spontaneous doubling rate, but also compared to the doubling obtained by the standard in vivo approach. our results suggest that for triticale, in vitro chromosome doubling is a promising alternative to the in vivo approach. in this study we investigated the applicability of an in vitro approach for chromosome doubling based on microspore culture. in addition, the frequency of plants surviving from culture medium to maturity is also much higher for the in vitro approach. colchicine concentrations of 1 mm for 24 h or  mm applied for or 72 h during the first hours of microspore culture performed best.
 doubled haploid production is a key technology in triticale research and breeding. a critical component of this method depends on chromosome doubling, which is traditionally achieved by in vivo treatment of seedlings with colchicine.  in this study we investigated the applicability of an in vitro approach for chromosome doubling based on microspore culture. our results show a pronounced increase in the proportion of doubled haploid triticale plants compared to the spontaneous doubling rate, but also compared to the doubling obtained by the standard in vivo approach. in addition, the frequency of plants surviving from culture medium to maturity is also much higher for the in vitro approach. colchicine concentrations of 1 mm for 24 h or  mm applied for or 72 h during the first hours of microspore culture performed best.  our results suggest that for triticale, in vitro chromosome doubling is a promising alternative to the in vivo approach. 