Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.
Doubled haploid production via microspore culture is a technique known to accelerate crop breeding by shortening the breeding cycle through achieving homozygosity in one generation. Prior research observed that some embryogenic microspores aborted their development before reaching the embryoid stage. Such embryogenic abortion reduces embryoid yield, making microspore cultures less efficient. The present research aims at identifying stages during which microspore development is susceptible to embryogenic abortion. Information gained through delineation of the developmental dynamics of microspores in culture could be used to improve the efficiency of microspore culture. Embryogenic microspores were isolated from stress-treated wheat (Triticum aestivum L.) tillers and cultured in liquid medium. The development of embryogenic microspores was monitored over a 35 day period. At day 7, 10, 14, 21, 28, and 35, the developing microspores were counted and categorized into multicellular structures, pre-embryoids, immature embryoids and mature embryoids. The results showed that 44% - 62% of embryogenic microspores halted their development before the mature embryoid stage. Of these aborted embryogenic microspores, 21% - 33% terminated as multicellular structures, 16% - 19% arrested their development as pre-embryoids, and 7% - 10% halted development as immature embryoids. Identifying factors that are responsible for embryogenic abortion and finding remedy to the issue will help improve the efficiency of doubled haploid production.
Keywords:
Microspore Culture, Embryogenic Abortion, Doubled Haploids, Triticum aestivum L.
Triticum aestivum was once considered as a recalcitrant species for androgenesis―a process through which plants are regenerated from anthers or immature pollen. Although the first success in androgenesis was achieved half a century ago with Datura inoxia [
Embryogenesis in vivo results in development of an embryo, a reproductive process that requires pollination and ensuing fertilization. However, in vitro embryogenesis, also called somatic embryogenesis, is the production of a pseudo-embryo (or embryoid) from a cell without involving fertilization. This embryoid so obtained is functionally equivalent to an embryo and able to germinate into a plant under adequate conditions. To that end, microspores must first be developmentally reprogrammed to produce embryoids [
Various forms of pretreatment have been used to induce microspore embryogenesis. These include, but are not limited to, temperature shock, high osmolarity, reduction in sugar or nutrients, and exposure to ultraviolet light. Water stress, anaerobic conditions, and inducer chemicals were also employed for reprogramming (as reviewed in [
Various components are required to sustain the development towards mature embryoids from embryogenic microspores. These include balanced nutrients, optimal pH, adequate osmolarity and temperature. The inclusion of ovaries in culture was found to increase the number of pre-embryoids, embryoids, and regenerated plants [
While induced microspore embryogenesis seems to be the method of choice for doubled haploid production, challenges for generating a sufficient number of plants for practical breeding still remain. One of the challenges in wheat is the developmental abortion of initially embryogenic microspores during culture. A significant number of embryogenic microspores failed to evolve into mature embryoids, thus reducing the number of double haploids. Some embryogenic microspores started cell divisions but terminated at various points, as multicellular structures, pre-embryoids or immature embryoids. Although such embryogenic abortion has been observed in multiple species, a stage-wise quantitative analysis of embryogenic abortion is lacking. A better understanding of the developmental dynamics among embryogenic microspores is probably the first step towards finding a remedy for embryogenic abortion, hence allowing a wider use of this technology in wheat breeding. Thus, the present study is designed to answer two related questions: 1) how frequently do embryogenic abortions occur in culture? 2) at which stage of the embryogenic development do abortions occur the most? Once these two questions are answered, we can then move on to test if any of the developmental halts can be alleviated. The ultimate goal is to improve the efficiency of doubled haploid production through reducing the abortion frequency among embryogenic microspores.
Three genotypes were used for these experiments: Chris, Pavon 76 and WED202-16-2. Donor plants were grown in a plant growth chamber programmed on a daily cycle of 16 hours of light at 25˚C, followed by 8 hours of dark period at 17˚C. Plants were watered as needed. This controlled environment minimized physiological stress on the developing microspores within anthers of the donor plants. Once microspores had reached the mid to late uninucleate stage, they were suitable for embryogenic induction. The tillers of each genotype were excised and prepared for stress treatment. Details for raising donor plants, sampling, and isolating microspores can be found elsewhere [
After isolation, embryogenic microspores were plated to a liquid medium for embryogenesis. The medium contained balanced nutrients, adequate osmolarity and wheat ovaries to allow cell divisions to proceed through a series of stages defined as multicellular structure, pre-embryoid, immature embryoid and mature embryoid. All microspores were cultured according to earlier publication [
Embryogenic microspores, with a defined “star-like” structure reported earlier [
The developmental dynamics of microspores isolated from Chris are shown in
representing the portion that was unlikely to advance beyond this stage. At day 28, immature embryoids declined sharply to 5% while mature embryoids rose to 17.5%. At day 35, only 2.5% remained as immature embryoids, while 21.7% have progressed to mature embryoids.
At day 35, total microspores consisted of 7.9% MCS, 7.1% pre-embryoids, 2.5% immature embryoids, 21.7% mature embryoids and 60.8% non-dividing microspores. An average of 17.5% dividing microspores failed to reach the finishing line as mature embryoids. In other words, 44.6% of dividing microspores aborted their development as MCS, pre-embryoids or immature embryoids. Among the 60.8% of microspores that failed to evolve into MCS, most never initiated cell division, and only less than 10% completed one cell division (visual estimate, not accurate counting). This largest fraction of microspores are unlikely the target for any medium manipulation for improving the efficiency of microspore culture. On the other hand, once embryogenic microspores advanced to immature embryoids, very few (<2.5%) aborted their development, as even more immature embryoids will grow into mature ones beyond day 35.
The developmental dynamics of microspores from Pavon 76 and WED202-16-2 are summarized in
The results indicate that of the total number of microspores initially cultured, about 40% proceeded to the multicellular structure phase while the remaining 60% failed to divide or divide beyond the first two cell cycles (Figures 7-9). The 60% non-dividing fraction likely represented microspores which were either non-embryo- genic ones incapable of dividing, or embryogenic ones that had incurred substantial damage during blending, filtration and centrifugation. These damages to microspores were not visible through observation using an inverted microscope. Of the embryogenic microspores that proceeded to multicellular structures, 54% - 58% developed into mature embryoids for the genotypes studied. The remaining 42% - 46% halted their development at the multicellular stage (18% - 20%), pre-embryoid (11.5% - 18.6%), or immature embryoid (6.4% - 9.5%).
As more dividing microspores were observed to arrest their development as MCS or pre-embryoids (29.5% - 38.6%), the first 10 to 14 days in induction culture should be the target period for reducing the embryogenic abortion. Regardless of genotypes, the percentages of the multicellular structure and the pre-embryoid remained relatively constant for the day 21 onward. This consistency was also observed in all four Petri dishes of each genotype. These results indicate that after three weeks in induction culture, a vast majority of the dividing microspores that remained as multicellular structures and pre-embryoids will not develop any further, thus failing to reach the mature embryoid stage. By day 35 in induction culture, an average of 20% of the multicellular structures failed to develop into a pre-embryoid and 17% of pre-embryoids could not develop into immature embryoids. Therefore, after 21 days in induction culture a total of 37% of the multicellular structures observed on day 10 halted their development. By day 28, microspores that developed into immature embryoids had a very high success rate of developing into mature embryoids. Of all microspores proceeded to immature embryoids, only 7% failed to evolve into mature embryoid, likely due to intensified competition for space and/or nutrients among emerging embryoids. So the goal for any future remedy for embryogenic abortion should be set to increase the number or percentage of immature embryoids, which corresponds quite well with the yield of mature embryoids.
Knowing at what stage in induction culture embryogenic microspores abort their development is valuable for several reasons. First, if abortion is caused by damage during pretreatment and isolation or other undue stress before the embryogenic culture, we can work to refine the protocol and minimize such damage and stress. The fact that at least 60% of microspores failed to divide and form MCS suggests that physical damage and other stresses before induction culture are significant impediments to microspore cultures. Further refinement of protocols in isolating and handling microspores might lower the embryogenic abortion. Secondly, knowing the detriment of wounding and/or stress damages to microspores may lead to a remedy that minimizes its impact. Nursing factors released into the ovary conditioned medium [
Our results seemed to suggest that embryogenic abortion could result from both pre-culture damage and initial culture stresses, as many seemingly embryogenic microspores failed to form MCS, and a considerable faction of MCS stopped developing further along embryogenesis. To minimize the pre-culture damages, one might consider shedding microspores from excised anthers [
Recent research found that Arabinogalactan Larcoll, a sugar, was useful for decreasing the mortality rate of developing microspores. Results suggest that Arabinogalactan Larcoll aids in microspore recovery from pretreatment and isolation stress [
There are several possible causes for the embryogenic abortions observed during microspore culture. One particular cause could be a competition for nutrients. At day 14 in induction culture, the number of cells that aborted was very high. This is also the time period where the immature embryoid increases in frequency. The immature embryoid is much larger than the multicellular structure and pre-embryoid. Not only is it made up of a larger number of cells, but these cells also begin to differentiate. The immature embryoids are increasing in size at a more rapid rate than the multicellular structures and pre-embryoids. Since they are larger and more specialized, it is possible that the immature embryoids are pooling more of the resources, leaving an insufficient amount for the other smaller structures. One possible solution to this problem could be to remove the immature embryoids from the induction culture at approximately day 21. They can then be placed in a separate Petri dish with growth medium, thus leaving the multicellular structures and pre-embryoids free to develop without having to compete with immature embryoids for nutrients.
Another possible cause resulting in spontaneous abortion is a competition for space. As the microspores progress through the various phases and cellular divisions occur, the population density within the Petri dish drastically increases. This puts added strain on the developing microspores and may halt development. As the cell frequency within the Petri dish increases, the larger structures also have a greater advantage over the less developed structures. These structures have already begun to differentiate and thus have a selective advantage over the smaller and less developed structures.
One possible solution to reduce culture stress could be to refresh the medium and add fresh ovaries to each Petri dish at day 21 in induction culture, as suggested by earlier reports [
In conclusion, through analysis of the development dynamics of wheat microspores in culture, we find that many embryogenic microspores that initially divided into multicellular structures spontaneously aborted before completing their development into mature embryoids. These abortions occurred most frequently in the phases of multicellular structure and pre-embryoid before day 21 in induction culture. While prevention of embryogenic abortions is beyond the scope of this paper, there are many implications for future research. One future study could be to remove immature embryoids from the induction culture at approximately day 21 and test if more of the remaining multicellular structures/pre-embryoids will develop forward. The removal may leave the multicellular structures and pre-embryoids free to develop without having to compete with immature embryoids for nutrients and/or space. Another area of research could be to test the effects of refreshing the medium and adding fresh ovaries to each Petri dish at day 21 in induction culture. Furthermore, it might be worthwhile to explore the combination of refreshing and transferring of immature embryoids to a separate Petri dish. If the addition of ovary-conditioned medium and/or Arabinoglacta Larcoll to induction culture can reduce the minimal cell density required for embryogenic division, the competition for nutrients and/or space should not be an issue in the first place.
Ming Y.Zheng,KierstenBieren,RolandGriggs, (2015) Developmental Dynamics of Wheat (Triticum aestivumL.) Microspores under Culture. Advances in Bioscience and Biotechnology,06,693-701. doi: 10.4236/abb.2015.612072
MCS: multicellular structure.