Wheat Microspore Culture Research Articles

Doubled haploidy methodology for three forage grasses [crested wheatgrass (Agropyron cristatum (L.) Gaertn.), hybrid bromegrass (Bromus riparius x B. inermis), and meadow bromegrass (Bromus riparius Rehm.)

Doubled haploidy (DH) methodology is used in many plant species to accelerate crop improvement and cultivar development; however not all species are amenable to the tissue culture technique. Experiments were undertaken to develop DH protocols for three perennial grasses [crested wheatgrass (Agropyron cristatum (L.) Gaertn.), hybrid bromegrass (Bromus riparius x B. inermis), and meadow bromegrass (Bromus riparius Rehm.)]. The initial experiment screened these forage grass species to established wheat (Triticum aestivum L.) microspore culture protocols. Following the initial screen, several factors influencing microspore embryogenesis were evaluated. These included genotype, donor plant conditions, developmental stage of the microspore, pretreatments, media composition, and culture conditions. For regeneration of the embryos to plants, media composition and culture conditions were assessed. Microspore-derived embryos/calli as well as green haploid/doubled haploid plants were regenerated from all three forage grasses. Differences were observed between species and genotypes within species in terms of embryogenic response. Modifications to the initial wheat DH protocol included the donor plant conditions, developmental stage of the microspore to late uninucleate to early binucleate and media composition. Regenerated plants were grown in the greenhouse.

Improving the efficiency of wheat microspore culture methodology: evaluation of pretreatments, gradients, and epigenetic chemicals

The objective of the study was to develop efficient widely adaptable microspore culture methodology for spring and winter wheat genotypes. Current wheat microspore culture methods are still inadequate for routine application, especially for winter genotypes. The present study involved increasing the frequency of microspore embryogenesis and improving the conversion of microspore-derived embryos to fertile green plants. A systematic evaluation was conducted testing numerous factors at all phases of the microspore embryogenesis process. Results from pretreatments, maltose gradients, and histone deacetylase inhibitors are presented. A 28 days cold pretreatment of spikes was optimal for winter wheat genotypes whereas a 21 days cold pretreatment was best for spring genotypes. Microspore suspensions containing a high frequency of embryogenic microspores were collected by resuspension in a 23% maltose gradient with subsequent pelleting with a mannitol containing solution. The application of trichostatin A (TSA) enhanced embryogenesis and/or green plant regeneration. Other epigenetic chemicals like scriptaid, BIX-01294, and sodium butyrate were also evaluated. Scriptaid enhanced embryogenesis but regenerated plantlets showed abnormal shoot and root development. The improved methodology was validated using a range of spring and winter wheat genotypes. Enhanced microspore embryogenesis and green plant regeneration in spring and winter wheat was observed with 21 or 28 days cold pretreatments, a 23% maltose gradient, and the application of trichostatin A (TSA).

Nuclear Fusion during Early Stage of Microspore Embryogenesis Indicates Chromosome Doubling in Wheat (Triticum aestivum)

Studies of barley and maize indicate that chromosome doubling occurs via nuclear fusion during an early stage of microspore embryogenesis, but the time and mechanism by which chromosome doubling occurs in bread wheat (Triticum aestivum) remains undetermined. The purpose of this study was to determine the relative time during induction culture when chromosome doubling may occur in wheat, and to identify early indicators for doubled haploid microspores. Microspore nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) and observed under a fluorescent microscope on the day of isolation, three days after isolation, and six days after isolation. The change in the percentage of microspores containing a single small nucleus, two small nuclei, a single enlarged nucleus, and three or more nuclei was then tracked throughout the six-day period. Ploidy levels were estimated by determining the cross-sectional area and number of nucleoli in microspores containing small and large nuclei then comparing the results of each respective cell-type. The percentage of microspores containing enlarged nuclei increased throughout the six-day test period, and the percentage of binucleated microspores containing small nuclei decreased. Comparison of the changes in average percentage of microspores containing a single small nucleus, binucleated microspores, microspores containing a single large nucleus, and multinucleate microspores on days 0, 3, and 6 indicates that nuclei classified as “small” are likely haploids and nuclei classified as “large” are doubled haploids. The percentage of microspores with enlarged nucleus (nuclei) during the first six days of induction culture could be used as an early indicator for the frequency of chromosome doubling in wheat microspore culture.

An improved wheat microspore culture technique for the production of doubled haploid plants

An improved wheat microspore culture technique for the production of doubled haploid plants

Effect of Cold-Mediated Pretreatment on Microspore Culture in Winter and Spring Wheat

Microspore culture of wheat generates completely homozygous (doubled haploid) plants in a single generation thereby reducing the time required for wheat variety development. Success of microspore culture in spring wheat is relatively higher than that in winter wheat. Cold mediated pretreatment was reported to improve response of microspore culture in wheat. The objective of the study was to determine and compare the influence of cold pretreatment on microspore culture in spring and winter wheat. Three spring (“Chris”, “Express”, and “Macon”) and three winter (“Anton”, “Antelope”, and “Camelot”) wheat cultivars were used. In cold pretreatment, excised anthers were incubated in solution B at 25°C-28°C for 4-5 days followed by cold treatment at 4°C for 5 days and were compared with the no-cold pretreatment at 25°C-28°C for 4-5 days. Isolated microspores were cultured in induction medium (MMS4) at 27°C-28°C for 25-30 days in the dark. Embryos (1-2 mm size) were transferred to regeneration medium (MMS5). Numbers of multicellular structures, transferable embryos and green plants were counted and data were used for analysis of variance using a generalized linear model. It was observed that cold pretreatment increased multicellular structures, transferable embryos and green plants in both spring and winter wheat. However, the degree of improvement was higher in spring wheat compared to winter wheat. The cultivars within spring and winter wheat responded differently. Development of embryos from pro-embryos was 4-5 folds lower in winter wheat than that in spring wheat, indicating requirement of a possibly different hormonal composition in induction medium for improving embryo induction in winter wheat. This report may provide future direction of research to improve microspore culture response in winter wheat.

Assessment of Polyamines and Trehalose in Wheat Microspores Culture for Embryogenesis and Green Regenerated Plants

Most aspects of microspore culture protocol have the capacity to cause stress to microspores, hence, less stressful treatments might be required to avoid deleterious effects. In stressed plants, polyamines and trehalose can act as compatible solutes or osmoprotectants by stabilizing proteins and biological membranes. To improve green plant regeneration in wheat microspore culture, this study assessed the effects of polyamines (putrecine, spermidine, spermine) and trehalose on androgenic response namely embryogenesis, green plant regeneration and ploidy of green plants regenerated in three spring wheat genotypes. Microspores of the genotypes produced significant numbers of embryos and green plants among polyamine treatments but trehalose had no effect (P ≤ 0.05). Polyamine treatments for 30 min generally produced more green plants per 100 microspores than the 60 min treatments in all three genotypes. At least three out of twelve polyamine treatments in each genotype improved the production of double haploid plants and seed setting in regenerants. Wheat genotype, concentration and duration of polyamine treatment had significant impact on embryogenesis and regeneration of green plants in this study. The study also showed that polyamines could be used to accelerate cultivar development in wheat breeding.

An Improved Wheat Microspore Culture Technique for the Production of Doubled Haploid Plants

ABSTRACTMicrospore culture is used to generate completely homozygous plants in a single generation, thereby reducing cost and time required for the production of doubled haploid (DH) plants for breeding and genetic studies. Many factors are known to influence green plant recovery including composition of regeneration media, donor plant genotype, microspore developmental stage, and pretreatment conditions. The objectives of this study were to: (i) develop an improved regeneration medium for wheat microspore culture and (ii) determine the optimal pretreatment conditions and regeneration media combination for increasing green plant recovery rates. Four wheat cultivars, two pretreatment methods (0.4 M mannitol at 4°C and solution B containing 0.3 M mannitol with inorganic components at room temperature), and five regeneration media were tested. Green plant recovery rates from each treatment combination were analyzed using the Proc Logistic model in SAS. Regeneration medium fortified with ascorbic acid produced the highest number of green plants across the different pretreatment conditions, regeneration media, and cultivars tested. Pretreating wheat (Triticum aestivum L.) spikes with 0.4 M mannitol at 4°C followed by embryoid induction and regeneration in a medium fortified with ascorbic acid resulted in the recovery of the highest number of green plants. This high‐efficiency method may prove useful for producing DH populations for wheat improvement efforts.

Arabinogalactans and arabinogalactan-proteins induce embryogenesis in wheat (Triticum aestivum L.) microspore culture

The objective of this study was to improve induction of embryogenesis in wheat microspore culture in order to obtain a high number of regenerable embryos. The arabinogalactan (AG) Larcoll and the arabinogalactan-protein (AGP) from gum arabic were tested on two spring genotypes to see if they could increase microspore viability and induce embryogenesis in the microspore culture. Adding Larcoll significantly decreased microspore mortality in both genotypes regardless of the presence or absence of ovaries in the culture. Similarly, gum arabic had a strong effect on the number of embryos produced and regenerated green plants. In fact, by using only gum arabic we were able to obtain green plants from wheat microspore cultures without the presence of ovaries. In addition to preventing a high mortality rate of the cells, our results show that the induction of embryogenesis in wheat microspore cultures is strongly affected by the use of both AG or AGP.

Optimization of culture conditions for improved plant regeneration efficiency from wheat microspore culture

The production of haploid plants through microspore culture is a very important tool for plant breeding. However, progress in microspore culture for many species has been hampered by a number of factors that have resulted in low recovery of regenerated green plants. In this study, a series of experiments were conducted to increase the regeneration of haploid green plants from isolated wheat microspores. The use of different basal media and variations in media components resulted in the increased recovery (approximately double) of regenerated haploid wheat plants. Our findings demonstrate that CHB medium, in combination with 2,4-d, was a better medium for embryoids induction and plant regeneration than medium MC17 with either 2,4-d or PAA growth hormones. Wheat microspores cultured without ovary co-cultivation did not respond. Furthermore, high efficiency of microspore derived embryoids (up to 296 MDEs per 100 anthers) and green plant regeneration (up to 71 green plants per 100 anthers) were achieved by the use of gelrite instead of agarose as a gelling agent, and by the addition of media additives such as spent medium or MET.

Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis

The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids `germinate' to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research.

Regeneration of fertile wheat (Triticum aestivum) plants from isolated zygotes using wheat microspore culture as nurse cells

A new method for growing isolated wheat zygotes by co-cultivation with microspores isolated from the same species has been developed. Although the mortality of isolated zygotes within 6 h after the transfer into the nurse culture was relatively high (70%) in this method, the majority of the structures which survived developed into fertile plants (61%). Zygotic structures grown in these cultures were morphologically not as regular as embryos grown in planta, but they resembled them closely in cytological properties and developmental pattern.

Improving green plant production via isolated microspore culture in bread wheat (Triticum aestivum L.)

Although a high efficiency of microspore embryogenesis can be achieved for a wide range of genotypes, some genotypes regenerate a high proportion of albino plants. Significant improvements in embryo regeneration and green plant formation were achieved by adding 10% NPB98 medium to a microspore pretreatment solution containing a chemical inducer formulation. For the wheat genotype WED 202-16-2, which is known to produce a moderate frequency of albinos, the plant regeneration rate was 15% higher, and the green plant percentage increased 27% following the addition of this nutrient supplement during embryo initiation. There were no negative effects on the responsive genotype Svilena. Embryo production in both genotypes was not affected. Our results indicate that providing readily available nutrients during microspore embryo initiation is effective in promoting plant regeneration and enhancing green plant development in a wheat genotype with a propensity for albinism.

Assessment and Improvement of Wheat Microspore derived Embryo Induction and Regeneration

Summary In this study we present an efficient protocol for the production of embryos in isolated wheat microspore culture and discuss parameters determining the efficiency of embryo induction and green plant regeneration. Microspores were isolated from the spring wheat genotypes DH83Z118.32 and DHBW3 showing high and low androgenetic response, respectively. The washing (WM) and induction/culture medium (AMC) were optimised and led to an average embryo yield of 1350 embryos per 10 5 microspores with DH83Z118.32. One single culture dish yielding 7250 embryos per 10 5 microspores demonstrated that the androgenetic potential of this genotype is even higher. With DHBW3 an average of 82 embryos per 10 5 microspores was achieved. We developed a non-destructive UV fluorescence image processing system that revealed a correlation between size parameters of microspore populations and their embryo yield. For DH83Z118.32 we found the ability of embryos to regenerate green plants (embryo quality) to be strongly determined by embryo age and size. The highest yield of regenerated green plants was obtained when large embryos (\s>4 mm) were transferred to regeneration medium 25 days after microspore isolation. The formation of albinos was negatively correlated with the embryo size. The early assessment of microspore size and embryo quality combined with optimised culture methods demonstrated that the potential of wheat haploid induction by isolated microspore culture is not yet fully exploited.

Cytoplasmic Effects on Pollen Embryogenesis Induction in Wheat Microspore Culture

Three alloplasmic wheat lines and the donor of nuclear genome have been tested for their ability for inducing pollen embryogenesis in microspore culture. Cultivation with an initial density of 2 x 105 microspores per ml in Cl7 medium conditioned by young anther-derived embryoids was used to induce microspore cell division and the subsequent plant regeneration. Microspore viability after pre-treatment and during cultivation, cytoplasm reorganisation, frequency of microcalli and embiyoids, the number of regenerants have been studied. Cytoplasmic effects during the first stages of pollen embryogenesis in isolated microspore culture were detected. Alloplasmic line (turgidum) x M808 was the best genotype for all parameters studied. One way analysis of variance and determination of the genetic distance also exhibited cytoplasmic influence. It can be concluded that the cytoplasmic effects exist at early stages of pollen embryogenesis induction in wheat microspore culture and they should be taken into account in investigations involving pollen embryogenesis induction.

A cytological study of pretreatments used to improve isolated microspore cultures of wheat (Triticum aestivum L.) cv. Chris

Isolated microspores of wheat can be induced in vitro to switch their development from the gametophytic pollen pathway to a sporophytic pathway, resulting in embryoid or callus formation. The influence of cold or mannitol pretreatment on karyokinesis and cytokinesis in isolated microspore culture responses were investigated. Anthers were pretreated in mannitol for 7 d at 28°C; spikes at 4°C for 28 d. Microspores often completed the 1st mitotic nuclear division during pretreatment while cytokinesis was delayed. During mannitol pretreatments, the 1st mitotic nuclear division was mostly symmetrical while only asymmetric 1st nuclear divisions were seen during or after cold pretreatment. Following the symmetrical division, the two similar nuclei often appeared to fuse to form a diploid nucleus. Subsequently, these nuclei underwent rapid nuclear divisions to form multinucleate, and later, multicellular structures in induction medium. Cold pretreatments also induced muticellular structures but frequencies were lower than after mannitol. A novel pretreatment of spikes, combining 0.4 M mannitol solution at 4°C for 4 d, delayed the 1st nuclear division, keeping all microspores in a haploid uni-nucleate stage and resulted in higher induction frequencies. The proportion of embryos larger than 2 mm that developed into green plants was as high as 70% when transferred to regeneration media. Ninety-five percent of the plantlets transferred from culture to soil survived. The improved pretreatment enhanced the potential of isolated microspore culture in wheat for plant breeding by producing large numbers of plants and for gene transformation by maintaining a uniform population of haploid uni-nucleate stage microspores as targets.Key words: wheat, pretreatment, karyokinesis, embryogenesis, microspore, cold, mannitol.

Anther and isolated microspore culture response of wheat lines from northwestern and eastern Europe

AbstractHexaploid wheat genotypes from north‐western Europe show low responses to current anther culture techniques. This phenomenon was investigated on 145 north‐western European wheat lines. Twenty‐seven lines from eastern Europe were included to observe the response pattern of wheat from an area, where the technique has been used successfully. On average, eastern European wheat lines produced 3.6 green plants per 111 anthers, while only 1.4 green plants per 111 anthers were obtained in north‐western European lines. This difference was due to the high capacity for embryo formation among the eastern European lines, while the ability to regenerate green plants was widespread in both germplasm groups. Isolated wheat microspore culture performed on 85 of these wheat lines gave an average 3.7‐fold increase in green plants per anther compared with the anther culture response. The increased recovery of green plants was due to improved plant regeneration and increased green plant percentage from embryos derived from isolated microspore culture.

In vitro chromosome doubling with colchicine during microspore culture in wheat (Triticum aestivum L.)

Isolated microspores of two DH lines of wheat were treated with 8 different colchicine concentrations up to 3 mM for either 24 h or 48 h during microspore culture. Untreated control cultures produced on average 220 embryos per spike (100,000 microspores), 68% of the regenerated plantlets were green, and 15% of the flowering plants were fertile. The colchicine treatments had a significant effect on chromosome doubling as measured by the percentage of fertile regenerants. Using colchicine concentrations around 1 mM the percentage of fertile plants among the regenerants was increased up to 53%. The highest number of embryos and regeneration rates were observed after 24 h colchicine treatment, while the highest frequencies of green plants and fertile plants were obtained with 48 h colchicine treatments. The highest number of DH plants per spike was found after treatment with colchicine concentrations of 300 to 1000 μM. Such treatments resulted in an estimated average between the two genotypes of 23 doubled haploid plants per spike.

Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 105 microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 105 microspores per ml is recommended for isolated microspore culture.

Utilisation of Microspore Culture in Wheat and Barley Improvement

This review examines some of the recent developments in microspore tissue culture in wheat and barley, and the application of this technology to practical plant breeding. Several new techniques are described as well as areas where additional research is required.