Effect of carbohydrate content on the embryoid and plant production in triticale anther culture. Cereal Res. Effect of induction medium pH amd maltose concentration on in vitro androgenesis oh hexaploid winter triticale and wheat. Plant Cell Tiss.
Kasha K. Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley Hordeum vulgare L. Marciniak K. Effect of genotype, medium and sugar on triticale x Triticosecale Wittm. Murashige T. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Oleszczuk S. Direct embryogenesis and green plant regeneration from isolated microspores of hexaploid triticales x Triticosecale Wittmack cv. Plant Cell Rep.
Pauk J. Androgenesis in hexaploid spring wheat F2 populations and their parents using a multiple-step regeneration system. Ponitka A. The influence of various in vitro culture conditions on androgenetic embryo induction and plant regeneration from hexaploid triticale x Triticocecale Wittm. Ry ppy P. Haploidy in triticale, In: In vitro haploid production in higher plants, ed. Jain, S. Sopory, R. Veilleux, Kluwer Acad.
Schumann G. In vitro production of haploids in triticale, In: Biotechnology in Agriculture and Forestry, ed. Bajaj, Springer-Verlag Berlin, — Iiwi ska E. Effect of harvest time and soaking treatment on cell cycle activity in sugarbeet seeds.
Seed Sci Res 9: 91— Iusarkiewicz-Jarzina A. Effect of genotype and media composition on embryo induction and plant regeneration from anther culture in triticale. Lusarkiewicz-Jarzina A. Efficient production of spontaneous and induced doubled haploid triticale plants derived from anther culture. Cereal Res Commun 31 3—4 : — Tuvesson S. Large-scale production of wheat and triticale doubled haploids through the use of a single-anther culture method. Wang Y.
Y, Sun C. The induction of pollen plantlets of Triticale and Capsicum annuum for anther culture. Warzechac R. Hybrid triticale—prospects for research and breeding — Part II: Development of male sterile lines, Proceed. B Progeny from this ear were germinated on media, sampled, and identified by PCR as either haploid or diploid. Based on the PCR calls, a few putative haploid and diploid plants were then transplanted to soil and grown until the diploid plants were shedding pollen.
C,D Adult leaf samples were tested to confirm ploidy status. The diploid 2N peak was set at units X-axis by adjusting the gain while running cells extracted from a known diploid plant through the flow cube. The image in D is representative of the histograms found for all putative haploids. In the Arabidopsis study, a high level of aneuploidy was found among the progeny, likely a result of partial chromosome elimination during haploid induction. In contrast, all 60 of the maize haploids we identified by PCR assay were confirmed as true haploids via ploidy analysis.
We also screened 96 putative diploids in the ploidy analyzer, specifically sampling them from ears that had haploid induction occurring. Of these 96 putative diploids, 95 were scored as true diploids via ploidy analysis, while one embryo was scored as an aneuploid. Several other putative diploids sampled from the same ear were confirmed as diploids. Doubled haploid generations allow breeders to bypass years of inbred line generation by single seed decent. For crops that do not have reliable haploid production methods, the Ravi and Chan study suggested that engineered haploid induction could be available for all crops.
In an attempt to demonstrate the validity of that theory, we tested several biotechnological approaches toward engineering CENH3- based haploid induction in maize. However, from individual to individual, the haploid induction rate varied widely and on average, the male, hemizygous induction crosses induced haploids at a frequency of 0.
While this rate was much higher than the other reproductive strategies tested in this study Table 5 , it is still relatively low compared to the native Stock6 induction system Coe, It is unclear why the hemizygous trasngenics consistently performed better than their homozygous counterparts. One might imagine that the less transgenic CENH3 available, the higher the frequency of faulty centromere construction.
Table 5. Summary of haploid induction rate data following outcrosses with CENH3 -altered transgenic maize lines. There were significant differences between the outcomes of the Arabidopsis work and the present study. While more events and crosses were tested here, the highest HIR was significantly lower than in Arabidopsis Ravi and Chan, In addition, the most frequent type of haploid induction seen in maize was gynogenetic induction, in which male inducer chromosomes are lost and female chromosomes are retained.
Only rarely was the converse seen—when the female line was the inducer and the male chromosomes were retained. It is not clear what accounts for the difference between the species, or if a lack of androgenetic haploidy will be a common feature for other monocotyledonous crops.
There is a natural androgenetic mutation in maize called indeterminate gametophyte1 ig1 Evans, It would be interesting to determine if induction rates increase when CENH3-tailswap technology is tested in an ig1 background.
There are several biotechnological strategies for genetic complementation in plants. In this study we tested two methods to complement native CENH3 with the tail-altered transgenes. In the RNAi approach binary constructs were made containing both the tail-altered transgenes and a dsRNA designed to specifically knock down native Cenh3 transcript.
This method offered the advantage of evaluating haploid induction rates in the T1 generation. We used meiosis-staged qRT-PCR to select homozygous T1 plants with the greatest extent of knockdown for forwarding to haploid induction tests. Unfortunately neither dsRNA construct induced consistent knock down, and overall, the RNAi strategy failed to induce haploids at a significantly higher rate than the background level.
The one plant that induced haploids at a modest rate 2. It is reasonable to infer that haploid induction rates will be low unless the native CENH3 protein product is either significantly reduced or eliminated. A multi-copy RNAi line may perform better than the single-copy line used in this study.
While the transgenic plants in this study did not induce haploids as frequently as in Arabidopsis Ravi and Chan, , neither did they produce nearly as many aneuploid embryos, nor did they experience a dramatic loss of male fertility. As with the native maize Stock6 induction system, it is possible that kernel abortion is a product of partial or complete inducer chromosome elimination in the endosperm, which would produce a ploidy imbalance, a known source of endosperm failure.
Based on these observations, it would be interesting to test in the future whether embryo rescue leads to higher haploid recovery rates. While partial female sterility was observed on some ears, abundant pollen was produced in all events and there were no instances of partial male sterility. In Arabidopsis , high male and female sterility accompanied haploid induction, and pollen was pooled from several individuals just to make a single outcross.
The low pollen load of the transgenics was later associated with the GFP tag, which caused CENH3 depletion in meiotic centromeres resulting in chromosome misalignment and the production of aneuploid microspores Ravi et al. It is unclear why similar levels of sterility did not occur in this study, but it fits with a trend seen in other systems: Lower rates of haploid induction are associated with fewer pleiotropic reproductive defects.
One way in which the data presented here was consistent with the Arabidopsis work was that no haploids were found that retained chromosomes from the transgenic parent. The authors tested this hypothesis with an elegant transgenic experiment, in which native AtCENH3 was complemented by orthologs from Lepidium oleraceum, Brassica napa , and Zea mays.
It appears that in both monocots and dicots, genes with divergent CENH3 tail sequences are complementary in developmental contexts outside of hybridization. Recently, the state of CENH3 technology was advanced by work showing that single nucleotide polymorphisms SNPs in the histone fold domain are sufficient to induce haploids in Arabidopsis Karimi-Ashtiyani et al. Heterologous complementation was then used to show that such SNPs can cause centromere disruption in barley and sugar beet Karimi-Ashtiyani et al.
In the present study, the events with the highest HIR produced multiple haploids in some crosses but none in others. HZ: Producing and analyzing transformation events, manuscript editing.
MN: Experimental design, construct design, manuscript editing. BM: Experimental design, project sponsorship, manuscript editing.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer Dr.
Kumlehn and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.
Thank you for your elegant work, your inspiration and your guidance. Colchicine, an efficient genome-doubling agent for maize Zea mays L. Plant Cell Rep. Chang, M. Kriz and B. Larkins Heidelberg: Springer , — Coe, E. A line of maize with high haploid frequency. Evans, M. The indeterminate gametophyte1 gene of maize encodes a LOB domain protein required for embryo sac and leaf development.
Plant Cell 19, 46— Karimi-Ashtiyani, R. Point mutation impairs centromeric CENH3 loading and induces haploid plants. Kasha, K. Maluszynski, K. Kasha, B. Forster, and I. Kelliher, T. Emergence and patterning of the five cell types of the Zea mays anther locule. This is particularly important given the issues of Food Security facing many countries, and the need to breed more nutritious and resilient crops. Ultimately our work will benefit consumers by making improved varieties of these crops more rapidly available to the public.
This project will provide scientific training and professional development opportunities for postdoctoral associates and students. We will present this activity to the general public via established events such as Saturday Science, the Wisconsin Science Festival, and Wisconsin Science Expeditions. Publications The following are buttons which change the sort order, pressing the active button will toggle the sort order Author Name descending press to sort ascending.
Collaborator Contribution My partners are also working on this joint grant aiming to gene edit genes in onion. Impact One publication to date Start Year Stephen Derek Jackson Principal Investigator.
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