Journal of Experimental Botany, Vol. 52, No. 359, pp. 1227-1238,
June 1, 2001
© 2001 Oxford University Press
Original Papers |
Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores
Department of Plant Agriculture, Biotechnology Division, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Received 19 September 2000; Accepted 15 February 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 °C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 °C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55–60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 °C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 °C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85–97% of these ELS regenerated green shoots. The frequency of doubled haploids (74–83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 °C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.
Key words: Barley, pretreatment, microspore, embryogenesis, mitosis, mannitol, nuclear fusion.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
High frequency regeneration of fertile plants (doubled haploids) from isolated microspores is an important tool for different plant breeding and biotechnological applications. Microspores can be isolated in large numbers providing a relatively uniform population of haploid, single cells capable of developing directly into embryos and plants. Thus, they provide excellent tools for studying embryogenesis, in vitro selection in culture and cell cycles relative to transformation.
Chromosome doubling during culture is an advantage of anther or isolated microspore culture that is lacking during haploid production by wide hybridization in cereal crops. In barley and wheat, doubling frequencies of 75–85% of regenerated plants have been observed, resulting in completely fertile doubled haploids (Höekstra et al
., 1992, 1993; Hu and Kasha, 1999; Kasha et al., 2001). This frequency is higher than most previous reports in these crops (Jähne and Lörz, 1995). The high frequency of completely fertile plants indicates that chromosome doubling must occur very early, most likely at the time of induction.
The mechanism of chromosome doubling has been one of much speculation and the relationship to the influence of pretreatments is obscure, with endoreduplication and nuclear fusion as the most likely methods. A C-mitosis, such as occurs during colchicine treatment, may result in a simple restitution nucleus with a doubled chromosome number. In Datura, it was proposed that both endoreduplication and nuclear fusion were involved in chromosome doubling and that the combination of both methods could explain the ploidy levels obtained that were higher than diploid (Sunderland, 1974; Sunderland et al., 1974). Nuclear fusion was described as occurring when two nuclei synchronously entered into division, formed a common metaphase plate and spindle and resulted in two nuclei, each with more than one set of chromosomes (Sunderland, 1974). If one or both of the nuclei had undergone endoreduplication prior to nuclear fusion, triploid or higher ploidy level plants could be formed. Sunderland also showed clear evidence of endoreduplication from the generative nucleus and chromosomes from different nuclei on a common metaphase plate. However, a different form of nuclear fusion using EM was observed whereby the nuclear membranes of interphase nuclei coalesced (Chen et al., 1984). While highly speculative with unknown frequencies, this method of nuclear fusion could be tested. It would form a single nucleus in previously binucleate microspores, whereas nuclear fusion via a common spindle would result in binucleate microspores.
Both the stage of the microspore when collected for pretreatment and the pathway of nuclear development have also been considered to influence the frequency of doubling (Sunderland, 1974). He concluded that microspores collected at uninucleate stages 1–3 (early, mid and late, respectively) resulted in mostly haploid and doubled haploid plants while those collected at later stages (4–6, mitosis and binucleate) resulted in mostly doubled haploids as well as some triploid and tetraploid plants. The highest frequency of doubled haploids (67%) came from microspores collected at stage 3. It has also been demonstrated in wheat that the pretreatment method will influence the pathway along which the nuclei will develop (Hu and Kasha, 1999).
A cellular signal or the disruption of signals has been hypothesized as responsible for the switch of microspore development from the normal gametophytic to an embryogenic (sporophytic) pathway (Twell et al., 1998). This developmental switch can be induced by the pretreatment of anthers or spikes. Commonly used pretreatments are temperature shock and nutrient starvation and have led to the concept that induction is by various forms of stress (Sunderland et al., 1979; Touraev et al., 1997). For Brassica napus and Nicotiana species, heat stress (Westecott and Huang, 1995; Touraev et al., 1996) and/or colchicine (Zhao et al., 1996) can be particularly effective pretreatments. For barley and wheat, a 28 d cold pretreatment at 4 °C has been most commonly used (Jähne and Lörz, 1995). However, a mannitol pretreatment (initiated by Roberts-Oehlschlager and Dunwell, 1990) has now become the method of choice in cereals (Kasha et al., 2001). A 3–4 d pretreatment of fresh anthers in 0.3–1.5 M mannitol at 25 °C (replacing the 28 d cold pretreatment) significantly increased embryogenesis and plant regeneration in barley isolated microspore culture (Ziauddin et al., 1990; Höekstra et al., 1992, 1996; Caredda et al., 1999; Cistué et al., 1999). Hoekstra et al., using barley anther culture, evaluated osmotic levels in pretreatments using mannitol and concluded that at the optimum level, mannitol improved ELS and plant production compared to a long cold period (Hoekstra et al., 1997). Caredda et al. found that organelle structure was preserved better with mannitol pretreatment compared to cold pretreatment and resulted in higher ratios of green plants (Caredda et al., 1999). It was observed (Hu et al., 1995) that a 6 d or 7 d cold pretreatment, combined with a solution of 0.4 M mannitol with the macro nutrients of FHG media (Hunter, 1988), had beneficial effects on embryogenesis and green plant regeneration in wheat microspore culture. Hoekstra et al. found that calcium in the mannitol pretreatment solution was similarly beneficial (Hoekstra et al., 1997). Indrianto et al. reported the combination of high temperature shock while in mannitol to be effective in wheat (Indrianto et al., 1999).
Pretreatments also influence the stage of microspores. Hu and Kasha found that uninucleate microspores of wheat completed the first mitotic division during both the 28 d cold pretreatment and the 6–7 d 0.4 M mannitol pretreatment at 28 °C (Hu and Kasha, 1999). Thus, most microspores isolated for culture from heat pretreated spikes or anthers would also be expected to be at the binucleate stage or older. It was also reported that a spike pretreatment combining 0.4 M mannitol solution and cold pretreatment for 4 d in wheat essentially blocked the mitotic division of the nucleus, keeping all microspores at the same stage during pretreatment, and also resulted in the formation of large numbers of true embryo-like structures (ELS) (Hu and Kasha, 1999).
Since species and genotypes within species often differ in their response in culture, the original objective of this study was to find the best pretreatment method that would induce good microspore culture response across genotypes. This report presents cytological data on the effects of four pretreatment methods on six quite different barley genotypes. During these studies, clear evidence for the predominance of nuclear membrane fusion as the method of chromosome doubling following mannitol pretreatment of barley isolated microspores was obtained for the first time. The pathways of nuclear development in microspores with different pretreatments are cytologically described for barley.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Materials
Six barley genotypes, consisting of four winter barleys (GBC 781, GBC 778, GBC 777, and Igri) and two spring barleys (Lina and Manley), were used to investigate microspore and nuclear stage progression during different pretreatments prior to microspore culture. Igri and Lina are cultivars (cv.) of European origin while GBC 777, GBC 778, and GBC 781 are advanced lines from Thompson and Sons Ltd., Ontario, winter barley breeding programme. Manley is a 2-rowed malting spring barley widely grown on the Canadian Prairies. The response in isolated microspore culture after different pretreatments was evaluated for cv. Igri and to a lesser extent in Lina.
Growth conditions of donor plants
The growth conditions of winter genotypes were the same as described previously (Ziauddin et al., 1992). Growth conditions of the spring genotypes Lina and Manley were similar to those for winter genotypes except that no vernalization was required. Immature spikes were collected when the majority of microspores were at the mid-uninucleate stage and the leaf sheath was then surface-sterilized with 70% ethanol before pretreatment. The system of staging microspores described previously was used (Kasha et al., 2001).
Pretreatment
Four pretreatments were investigated for their effects on the mitotic divisions of microspores during pretreatment and subsequent culture:
- (a) Spikes in sheath, wrapped with aluminium foil and stored at 4 °C for 28 d.
- (b) Isolated spikes, in 100x15 mm Petri dishes and stored at 4 °C for 28 d in the dark.
- (c) Isolated spikes, in 100x15 mm Petri dishes with 20 ml of 0.3 M mannitol and stored at 4 °C for 4 d in the dark.
- (d) Fresh anthers, in 60x15 mm Petri dishes with 3 ml of 0.3 M mannitol and stored at 25 °C for 4 d in the dark.
- (b) Isolated spikes, in 100x15 mm Petri dishes and stored at 4 °C for 28 d in the dark.
Pretreatments (a) and (b) were similar except for the presence or absence of the leaf sheath. All spikes used for a replication of all pretreatments came from the same batch of plants grown at the same time. Spikes were collected at four different times to provide four replications. Data were analysed using ANOVA and means were compared using an LSD test.
Isolation of microspores
Two methods of microspore isolation were used, depending upon the pretreatment. Following pretreatments (a), (b) and (c), blender isolation was used. Pretreated spikes were cut into 2–3 cm fragments, and placed in a blender with cold 0.3 M mannitol solution. The fragments of spikes were blended at low speed for 5 s. The homogenate was strained through four layers of cheesecloth to remove the debris, then through 100 µm mesh nylon filter. The microspores were collected from supernatant by centrifugation for 4 min at 500 rpm. Following pretreatment (d), microspores were isolated by vortexing the senescent anthers in culture media and collected by centrifugation (Hu et al., 1995).
Microspore culture
The microspores collected from pretreatments (b), (c) and (d) were resuspended in liquid FHG medium (Hunter, 1988) that included 62 g l-1 maltose monohydrate, 730 mg l-1 glutamine, 1 mg l-1 BAP, and 10 mg l-1 PAA. The suspension was then filtered through two layers of Whatman No. 2 filter paper (42.5 mm) under vacuum, resulting in a well-spread mat of microspores on the top of filter paper. Approximately 800 000 viable microspores were applied to each filter paper. This corresponded to the number of microspores isolated from 10 spikes of 2-rowed barley or 4 spikes from 6-rowed barley. The top filter paper with the microspores was then placed on the top of 20 ml FHG solid medium (solidified with 3.0 g l-1 GelRite) in 100x15 mm Petri dishes. The dishes were incubated at 25 °C in the dark for 3 weeks. At this stage, embryo-like-structures (ELS) were transferred to modified MS solid medium (Kasha et al., 1997 ). After 10–14 d, the plantlets were transferred to MS regeneration medium in magenta boxes.
Fixation and DAPI staining of microspores
Microspores at different pretreatment and culture stages were collected, fixed with Carnoy I solution (3 ethanol:1 glacial acetic acid) at 4 °C for 24 h, and transferred into 70% ethanol for further storage. The stages collected were: fresh spikes; day 1 to day 4 in mannitol pretreatment at both 4 °C and 25 °C; days 0, 7, 14, 21, and 28 in cold pretreatment; and day 1 to day 15 in induction culture media. DAPI (4'-6-diamidino-2-phenylindole.HCl), a type of DNA-specific fluorochrome, was stored at -20 °C as a 1 mg ml-1 stock solution in distilled water in the dark. The stock solution was diluted 1:40 in McIlvaines citrate-phosphate buffer (pH 4.5) and stored at 4 °C. This solution could be maintained and used for 3 d. Microspores were put on the top of a slide, stained with one drop of DAPI for 3–5 min and then observed under a Zeiss fluorescent microscope. Cells were photographed under both UV and visible light.
Relative densitometry measurement of nuclear DNA
During pretreatment of spikes in 0.3 M mannitol for 4 d at 25 °C, microspores rapidly entered into mitosis and showed the various types of nuclear events such as nuclear fusion that are described under Results. Therefore, additional spikes of Igri were collected and fixed in Carnoy I solution at days 0–4 during the pretreatment and used to measure the nuclear DNA content of nuclei. These microspores were stained in Feulgen and observed under visible light with a Zeiss microscope and staged as uninucleate, binucleate or large uninucleate. A few of the freshly collected microspores, considered to be in the G1 stage, and some entering the first mitotic division at day 1 (expected to have the G2 level of DNA), were measured to establish the relative DNA levels for G1 and G2 stages of the nuclei. Video camera images of microspores were captured on the screen of a connected computer using the Northern Eclipse (NE) program. The Densitometry Function in NE was used to measure the relative DNA content in different nuclei. The density data accumulated by line scanning through whole nuclear area was logged into an Excel program. Various numbers of nuclei were measured at the culture stages. Mean relative DNA density values and the range of values are presented for these stages.
Chromosome doubling
In the European barley Lina, a random sample of embryos from various pretreatments were grown to maturity to determine the fertility of the plants. The pretreatments sampled were slightly different from the earlier experiment, namely 3 d or 7 d pretreatment of spikes in 0.3 M mannitol at 4 °C and spikes in Petri dishes at 4 °C for 21 d. Completely fertile plants in barley were considered to have been chromosomally doubled as a result of the pretreatment and/or subsequent procedures.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Nuclear development of microspores during different pretreatments
Pretreatment at 4 °C for 28 d was handled in two ways, namely pretreatments (a) and (b). For (a), tillers with spikes containing microspores at the mid-uninucleate stage (Fig. la
) were placed in water in a refrigerator. Spikes of collected tillers were examined after 7, 14, 21, and 28 d pretreatment. Some microspores had entered into the first mitotic division after 7 d in the cold (Fig. 1b, c); At day 4, from 38% to 74% of microspores had two nuclei in genotypes GBC 781, GBC 778, GBC 777, and Igri (Fig. 1d; Table 1), while most of the microspores in spring genotypes Lina and Manley were still in uninucleate stages. By day 28, most microspores of all genotypes, except Manley, were binucleate. In pretreatment (b), spikes were removed from the leaf sheath at the time of collection and placed in a Petri dish with a couple of drops of sterile water and placed in the dark in the refrigerator. The progression of the microspores through mitosis was slower with this pretreatment (b) compared to pretreatment (a) (Table 1). At day 28, most of microspores had become binucleate in genotypes GBC 781, GBC 778, GBC 777, and Lina. However, most of the microspores were still uninucleate in cvs Igri and Manley. Comparing the results of pretreatment (a) and (b) across different genotypes at day 28, cv. Igri was the only one which showed a significant decrease in the frequency of binuclear microspores (70% versus 1%) using method (b). The first mitotic division of the nucleus of microspores was nearly all asymmetric (94%) under cold pretreatment without mannitol, resulting in two nuclei of different sizes (vegetative and generative nuclei) and different DAPI fluorescent intensity (Fig. 1d).
|
|
Two variations were also used with 0.3 M mannitol pretreatment, namely (c) and (d). First, isolated spikes were kept for 4 d at 4 °C in the dark in a refrigerator. All microspores stayed at the uninucleate stage for all six genotypes (Table 1
). A more detailed classification of stages was conducted on cv. Igri as shown in Table 2. All microspores remained at the uninucleate stage and the classification of microspores as early (E), mid (M) or late (L) uninucleate showed very little change over the 4 d.
|
In the second mannitol variation, the anthers containing mid-uninucleate microspores (Fig. 2a
) were extracted from the spikes and partially submerged in mannitol for 4 d pretreatment at 25 °C, as previously utilized for microspore transformation (Yao et al., 1997). Sufficient spikes were only available for three winter genotypes in this treatment. The first mitotic nuclear division was completed during the pretreatment process in most microspores. After day 1, microspores could be seen in the various stages of mitosis such as premitosis (Fig. 2b), prophase (Fig. 2c), metaphase (Fig. 2d), anaphase (Fig. 2e) and telophase (Fig. 2f). The first mitotic division of the microspore nucleus could be either asymmetric or symmetric. By day 2, about 41–68% of the microspores were binucleate in the three genotypes (Table 1). However, by day 3 the frequency of binucleate microspores had decreased and based on nuclear size, the frequencies of microspores that appeared to contain a fused nucleus averaged 72%, 39% and 32%, respectively, across three genotypes GBC 778, GBC 777 and Igri, respectively. At day 4, about 87%, 54% and 75% of microspores of these genotypes contained the large and apparently fused single nucleus.
|
The relative frequencies of symmetric and asymmetric microspores were determined in a repeated study with cv. Igri. By day 2 about 55% of the binucleate microspores were symmetric (Table 3
). This pretreatment tended to result in the two nuclei being closely associated in the microspore (Fig. 3a). About 63% were binucleate by day 2. At the end of the first division, the two nuclei in microspores appeared to gradually coalesce (Fig. 3b, c), resulting in the formation of fused and doubled haploid microspores containing a single, large and bright nucleus (Fig. 3d). The frequencies of potentially fused nuclei was 38, 80 and 85% by days 2, 3 and 4, respectively. The concept that the nuclei had coalesced was supported by the decrease in frequencies of binucleates and the increase of uninucleate (including large ‘fused’) microspores with time in pretreatment (Table 1, 3). As seen in Table 3, the frequencies of binucleate microspores decreased from 62.4% at day 2 to 19.0% and 14.3% by days 3 and 4, respectively.
|
|
Relative DNA content at different stages of microspore development in cv. Igri
To confirm spontaneous chromosome doubling during the barley microspore culture process, densitometry measurements were conducted of relative DNA content in different microspore stages and in nuclei of different sizes before and following the spike pretreatment in 0.3 M mannitol at 4 °C and 25 °C for 4 d. The relative nuclear DNA levels of 4 d cold pretreated microspores were similar to the DNA contents in freshly harvested microspores (data not given). During the spike pretreatment in 0.3 M mannitol at 25 °C for 4 d, microspores entered into first division, followed by apparent fusion of two daughter nuclei (c. 85% of the time), resulting in a large single nucleus (Table 3). Only about 15% of viable microspores stayed at the haploid uninucleate or binucleate stages. Relative DNA densities of nuclei at various stages of pretreatment are shown in Table 4. The values for freshly harvested microspores at the mid-uninucleate stage (considered to be at the G1 stage of the haploid mitotic cell cycle) averaged 306 units. For those nuclei that were in first mitosis at day 1, the DNA level should be equivalent to that of G2, and the average was 550 units or roughly twice that of those in G1. The mean total DNA level in binucleate microspores was 781 units with quite a wide range of values that could represent microspores with both nuclei at the G1 phase up to those microspores with both nuclei at the G2 phase of the cell cycle. The relative mean DNA level in the large unicucleate microspores observed from days 1 to 4 was 1164 units, roughly four times the level observed in the day 0 haploid G1 stage. This indicates that the microspores with a large single nucleus have at least doubled their chromosome number and are likely fused nuclei that were at the G2 stage. It is possible that the category of small uninucleate microspores observed from day 1 to day 4 could include some fused microspores that had not undergone DNA synthesis to reach the G2 phase or that they represented fusion of asymmetric nuclei where the one nucleus had undergone DNA synthesis while the other remained at the G1 stage prior to fusion. The high proportion of fused nuclei by day 4 (Table 3) would indicate that both symmetric and asymmetric nuclei can fuse since it is much higher than the frequency (55%) of symmetric divisions in the binucleates only.
|
Nuclear development of microspores in induction culture on the top of filter paper
Microspores of cv. Igri that had been pretreated by methods (b), (c) and (d) were cultured on filter paper on top of solidified media at 25 °C in the dark and samples were collected at various times to examine microspore development. Following pretreatment (c), most cells had entered premitosis after 24 h of culture while some cells were in division and others were binucleate. The first mitotic division of microspores following 4 d of cold pretreatment in 0.3 M mannitol could be either symmetric or asymmetric, with about 60% being symmetric, the same as observed during the 0.3 M mannitol pretreatment at 25 °C (treatment (d) in Table 3). By day 2, microspores had continued to divide. At same time, the closely associated nuclei from the first division had begun to undergo fusion. At day 3, 85% of viable microspores appeared to have a fused single nucleus while 15% were binucleate microspores. This development pattern of microspores during induction culture was the same as during 0. 3 M mannitol pretreatment of anthers at 25 °C (Figs 2a–f, 3a–d). Many of the fused microspores also underwent a further mitotic division (Fig. 4a, prophase; Fig. 4b, metaphase), resulting in two similar doubled haploid nuclei (Fig. 4c). The continued division in these microspores resulted in four nuclei in microspores (Fig. 4d, e) and subsequently in the formation of embryos (Fig. 4f).
|
At the end of pretreatment (d) (mannitol at 25 °C), the majority of viable microspores contained a single large fused nucleus while a few were binucleate. After 24 h in induction culture there was little change in stages but, subsequently, most fused nuclei underwent mitotic divisions, resulting in the formation of embryos. The embryogenesis process was similar to that described in Fig. 4
.
Microspore culture responses following different pretreatments
Subsequent to pretreatments (b), (c) and (d), microspores of cv. Igri were cultured to determine their response in embryo and plant regeneration. It was observed that about 12 000 embryo-like structures (ELS) per plate could be formed and, upon transfer of samples of 1000 embryos to regeneration media, between 85–95% of ELSs could regenerate green shoots, regardless of the pretreatment used (Table 5). Microspore culture response following pretreatment (a) was not conducted because it was very similar to (b). The differences in the average numbers of ELS formed and green plants developing per plate following pretreatments (b), (c) and (d) were not significant, even though the nucleate stages at the beginning of induction culture and the times required from spike harvest to initiation of green plantlet regeneration were quite different among the three investigated pretreatment methods (Table 5).
|
Chromosome doubling
Chromosome doubling was high following either cold or cold plus mannitol pretreatments in the cultivar Lina. Based on 89 and 79 plants, respectively, the percentage of completely fertile (doubled haploid) progeny following 3 d and 7 d pretreatment in 0.3 M mannitol at 4 °C was 74% and 80%. Based on 52 progeny from spike pretreatment at 4 °C for 21 d, 83% were fertile doubled haploids. Thus, even though the pathways following first mitosis are different (predominantly asymmetric following cold versus the majority being symmetric after mannitol pretreatment), the frequencies of early microspore doubling are similar based on plants regenerated. These frequencies are consistent with the 79% fertile plants observed for microspore culture of wheat following pretreatment (d) (Hu and Kasha, 1997). Most of the remaining plants regenerated are sterile haploid plants while a few partially sterile plants (less than 5%) could be polyploid or aneuploid. Sunderland concluded that collection and pretreatment of later stages (mitosis and binucleate) resulted in higher frequencies of polyploids (Sunderland, 1974).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
A number of significant observations were made in this study of barley microspores. First, the pathway of nuclear development from microspores varies depending upon the pretreatment method employed. This was similar to findings in wheat (Hu and Kasha, 1999
). The 4 d 0.3 M mannitol pretreatments led to a majority of symmetrical first nuclear divisions (B-pathway), either during the 25 °C pretreatment or in culture after pretreatment at 4 °C. In contrast, the 28 d cold pretreatment at 4 °C resulted in over 90% asymmetrical first divisions (A-pathway). Genotypes influenced the time or frequency of nuclear divisions, but not the type or pathway of nuclear divisions. Second, the combination of both mannitol and cold pretreatment for 4 d can delay the first nuclear division during pretreatment, apparently keeping the microspores at the G1 stage or other stages of the cell cycle as evidenced from cell stage and the DNA measurements. Once removed from the pretreatment, the nuclei enter into division in induction culture. This has implications for chromosome doubling, the timing of transformation procedures in order to obtain plants homozygous for transgenes from bombarded microspores, and for synchronization of microspore cultures.
Perhaps the most important finding is the indication that the combined pretreatment may be a key to overcoming genotype differences in the induction of embryogenesis from microspores of barley and possibly other species. In addition, the cytological observations clarify the mode of chromosome doubling during microspore culture in plants, which in turn has implications for transformation of haploid microspores.
Several pathways (A, B, and C) have been proposed that can lead to the formation of multicellular structures in barley based on anther culture (Sunderland, 1974, 1979; Sunderland and Evans, 1980; Chen et al., 1984; Huang, 1986). These pathways often vary with the species being studied but sometimes more than one pathway has been observed within a culture (Sangwan and Sangwan-Norreel, 1996; Hu and Kasha, 1999). Zaki and Dickinson reported that only the B-pathway was observed in Canola and they proposed that the symmetric first mitotic division of microspores was a key factor resulting in embryogenesis (Zaki and Dickinson. 1991). The present study has demonstrated that there are at least two pathways (A and B) in barley microspore culture and they are influenced by the pretreatment methods. The first nuclear mitotic division could be symmetric (c. 60%) or asymmetric (c. 40%) (Table 3) following the use of 0.3 M mannitol, whereas the 28 d cold pretreatment of spikes resulted in over 90% asymmetrical first divisions (Fig. 1). This provides evidence that the induction of embryogenesis is not coupled to the type of first mitotic division, since both symmetric and asymmetric division can result in high frequencies of embryo induction and chromosome doubling. Studies with mutants affecting microspores in Arabidopsis have led to the hypothesis that disruption of polarity and microtubules associated with membrane or cell wall formation may be related to pollen embryogenesis (Twell et al., 1989; Souter and Lindsey, 2000). This is compatible with the observations of the failure of cell wall formation in early divisions (Reynolds, 1993; Eady et al., 1995; Hu and Kasha, 1999). Additional information on mannitol pretreatment and subsequently on the induction culture is presented in this study.
The production of completely fertile doubled haploid plants would indicate that chromosome doubling must occur early in microspore culture. This is supported by the observations of two closely associated nuclei appearing to fuse gradually (Fig. 3a, b, c) resulting in the formation of fused and doubled haploid microspores containing a single, large and bright nucleus (Fig. 3d). Microspores with a single nucleus entering division with 14 chromosomes (Fig. 4b) is also consistent with the concept that doubling occurs by nuclear fusion at this stage of culture. However, the 14 chromosomes could also arise by endomitosis or C-mitosis. Sunderland's figures of chromosomes of Datura innoxia Hill. from different nuclei on a common metaphase plate might be more logically interpreted as the metaphase following nuclear fusion (Sunderland, 1974). The haplo and diplo chromosomes originating from two different nuclei are well mixed, which would not be expected if two adjacent nuclei were entering division on a common plate. Nevertheless, his observations of diplo chromosomes do clearly demonstrate the occurrence of endoreduplication which, in this instance, he had suggested occurred in the generative nucleus. Alternatively, nuclear fusion of such nuclei could also lead to triploids or to higher ploidy levels from fusion in multinucleate microspores such as observed here in barley or elsewhere in wheat (Hu and Kasha, 1999).
Results of densitometry measurements of relative DNA content of different types of microspores of cv. Igri (Table 4) support the nuclear fusion pathway as the main mechanism of spontaneous chromosome doubling following mannitol pretreatment of barley microspores. Since both mannitol and cold pretreatment of microspores collected at mid- to late-uninucleate stage could result in the high frequencies of green plant regeneration and chromosome doubling (74–83%, Kasha et al., 2001), the main mechanism of high frequency chromosome doubling appears to be the disruption of microtubules leading to the failure of membrane or cell wall formation, thus enabling nuclear fusion to occur between closely associated nuclei. The observations of Sunderland (Sunderland, 1974) as well as those of the authors indicate that nuclear fusion can occur between symmetric or asymmetric nuclei. Fusion in multinucleate microspores could also result in higher ploidy levels of plants, but evidence for this was not seen in the limited samples examined here.
Genotype is one of the major factors affecting plant tissue culture response. Studies in barley microspore culture (Kasha et al., 2001) showed an overall good response among different genotypes but that the frequencies of ELSs formation and green plant regeneration varied significantly. Looking into the results of cytological studies in Table 1, the percentage of microspores that had become binucleate could vary from 1% in cv. Igri. to 93% in cv. GBC 777 at the end of the 28 d cold pretreatment of spikes. The frequencies of fused uninucleate microspores at the end of mannitol pretreatment of anthers also varied from 54–87% among the three genotypes investigated here. These results are an indication that genotype effects in microspore culture could start as early as the staging of microspores which may influence the first nuclear division.
Microspores are ideal targets for gene transformation. Regeneration of homozygous, diploid transgenic plants could be achieved in one step by chromosome doubling if the bombarding microspores were at the uninuclear stage. When binuclear microspores were used as the target for transformation, the resulting transgenic plants would be expected to be heterozygous or chimeric. Isolated microspores of barley have been successfully used as the target for microprojectile bombardment to produce fertile transgenic plants (Jähne et al., 1994; Yao et al., 1997; Carlson, 1998). Homozygous transgenic plants were obtained using a long cold pretreatment (Jähne et al., 1994), while both haploid and heterozygous transgenic plants were produced with mannitol pretreatment at 25 °C (Yao et al., 1997). Microspores were pretreated in mannitol at 4 °C and heterozygous transgenic plants were also obtained (Carlson, 1998). Results of a cytological study of barley microspore culture in this paper may help to explain reasons for obtaining homozygous plants (Jähne et al., 1994) or heterozygous plants (Yao et al., 1997). It is possible that microspores that had become binucleate during the long cold pretreatment were bombarded in G1 and the subsequent division of the vegetative cell and fusion of the resulting nuclei could lead to homozygous transgenics. Following the mannitol pretreatment (Yao et al., 1997) the microspores would have a single fused nucleus and bombardment would lead to a heterozygous transgenic. Although Carlson used both cold and mannitol (Carlson, 1998), it is likely that the microspores had rapidly entered into the S phase following removal from the pretreatment and were at G2 at the time of bombardment. Further analysis of the timing of DNA synthesis and the development of procedures (Kumlehn and Lörz, 1999) to monitor development of individual microspores would help to clarify this situation.
There were no significant differences in the average numbers of formed ELSs and regenerated green plants per plate following pretreatments (b), (c) and (d) (Table 5). However, the nucleate stages of microspores at the beginning of induction culture and the time frames from spike harvesting to green plantlet regeneration were very different (Table 5). The 4 d anther pretreatment in 0.3 M mannitol at 25 °C could produce green plantlets by 30–40 d so that this procedure would be good for producing doubled haploids for plant breeding programmes (Kasha et al., 2000). Mannitol at 4 °C for 4 d, which kept the microspores in a haploid uninucleate stage during the pretreatment, needs further evaluation for its potential in genetic transformation. It could rapidly produce more than 12 000 ELS with high green plant regeneration per plate within a short time frame (37–47 d) and has good potential for applications in barley and other species.
|
Acknowledgments |
|---|
It is a pleasure to acknowledge L Shugar, Thompson and Sons Seeds, for providing the three winter barley lines labelled GBC. Financial support for the research from the Natural Sciences and Engineering Research Council of Canada and Ontario Ministry of Agriculture, Food and Rural Affairs are most gratefully acknowledged.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +1 519 763 8933. E-mail: kkasha{at}plant.uoguelph.ca
2 Present address: Monsanto Co., 700, Chesterfield Parkway N., St Louis, MO 63198, USA.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Caredda S, Devaux P, Sangwan RS, Clément C. 1999. Differential development of plastids during microspore embryogenesis in barley. Protoplasma 208, 248–256.
Chen CC, Howarth MR, Peterson RL, Kasha KJ. 1984. Ultrastructure of androgenic microspores of barley during the early stages of anther culture. Canadian Journal of Genetics and Cytology 26, 484–491.
Carlson AR. 1998. Visual selection of transgenic barley (Hordeum vulgare L.) structures and their regeneration into green plants. MSc thesis, University of Guelph.
Cistué L, Ramos A, Castillo AM. 1999. Influence of anther pretreatment and culture medium composition on the production of barley doubled haploids from model and low responding cultivars. Plant Cell, Tissue and Organ Culture 55, 159–166.
Eady C, Lindsey K, Twell D. 1995. The significance of microspore division and division symmetry for vegetative cell-specific transcription and generative cell differentiation. The Plant Cell 7, 65–74.[Abstract]
Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F. 1992. Anther and microspore culture of Hordeum vulgare L. cv. Igri. Plant Science 86, 89–96.
Hoekstra S, van Zijderveld MH, Heidekamp F, van der Mark F. 1993. Microspore culture of Hordeum vulgare L.: the influence of density and osmolarity. Plant Cell Reports 12, 661–665.
Hoekstra S, van Bergen S, van Brouwershaven IR, Schilperoot RA, Heidekamp F. 1996. The interaction of 2,4-D application and mannitol pretreatment in anther and microspore culture of Hordeum vulgare L. cv. Igri. Journal of Plant Physiology 148, 696–700.
Hoekstra S, van Bergen S, van Brouwershaven IR, Schilperoot RA, Wang M. 1997. Androgenesis in Hordeum vulgare L.: effects of mannitol, calcium and abscisic acid on anther pretreatment. Plant Science 126, 211–218.
Hu TC, Kasha KJ. 1997. Improvement of isolated microspore culture of wheat Triticum aestivum L. through ovary co-culture. Plant Cell Reports 16, 520–525.
Hu TC, Kasha KJ. 1999. A cytological study of pretreatment effects on isolated microspore culture of wheat Triticum aestivum cv. Chris. Genome 42, 432–441.
Hu TC, Ziauddin A, Simion E, Kasha KJ. 1995. Isolated microspore culture of wheat Triticum aestivum L. in a defined media. I. Effects of pretreatment, isolation methods and hormones. In Vitro Cellular and Developmental Biology31, 79–83.
Huang B. 1986. Ultrastructural aspects of pollen embryogenesis in Hordeum, Triticum and Paeonia. In: Hu H, Yang H, eds. Haploids of higher plants in vitro. Beijing, 91–117.
Hunter CP. 1988. Plant regeneration from microspores of barley, Hordeum vulgare. PhD thesis. Wye College, University of London.
Indrianto A, Herbele-Bors E, Touraev A. 1999. Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspores. Plant Science 143, 71–79.
Jähne A, Becker D, Brettschneider R, Lörz H. 1994. Regeneration of transgenic, microspore-derived, fertile barley. Theoretical and Applied Genetics 89, 525–533.
Jähne A, Lörz H. 1995. Cereal microspore culture. Plant Science 109, 1–12.
Kasha KJ, Ziauddin A, Cho U-H, Simion E, Petroski R, Cistué L. 1997. Anther and microspore culture in barley and wheat. Journal of Applied Genetics38, 373–380.
Kasha KJ, Simion E, Oro R, Yao QA, Hu TC, Carlson AR. 2001. An improved in vitro technique for isolated microspore culture of barley. Euphytica (in press).
Kumlehn J, Lörz H. 1999. Monitoring sporophytic development of individual microspores of barley Hordeum vulgare L. In: Clément C, Pacini J, Audran J-C, eds. Anther and pollen: from biology to biotechnology. Berlin: Springer-Verlag, 183–190.
Reynolds TL. 1993. A cytological analysis of microspores of Triticum aestivum Poaceae during normal ontogeny and induced embryogenic development. American Journal of Botany 805, 569–576.
Roberts-Oehlschlager SL, Dunwell JM. 1990. Barley anther culture: pretreatment on mannitol stimulates production of microspore-derived embryos. Plant Cell, Tissue and Organ Culture 20, 235–240.[Web of Science]
Sangwan RS, Sangwan-Norreel BS. 1996. Cytological and biochemical aspects of in vitro androgenesis in higher plants. In: Jain SM, Sopory SK, Veilleux RE, eds. In vitro production of higher plants, Vol. 1. Kluwer Academic Press, 95–109.
Souter M, Lindsey K. 2000. Polarity and signaling in plant embryogenesis. Journal of Experimental Botany 51, 971–983.
Sunderland N. 1974. Anther culture as a means of haploid induction. In: Kasha KJ, ed. Haploids in higher plants: advances and potential. Guelph, Canada: Univiversity of Guelph, 91–122.
Sunderland N. 1979. Anther and pollen culture 1974–1979. In: Davis DR, ed. The plant genome. Norwich: The John Innes Charity, 171–181.
Sunderland N, Collins GB, Dunwell JM. 1974. The role of nuclear fusion in pollen embryogenesis of Datura innoxia Mill. Planta 117, 227–241.[Web of Science]
Sunderland N, Evans LJ. 1980. Multicellular pollen formation in cultured barley anthers. II. The A, B and C pathways. Journal of Experimental Botany 31, 501–540.
Sunderland N, Roberts M, Evans LJ, Wildon DC. 1979. Multicellular pollen formation in cultured barley anthers. Journal of Experimental Botany 30, 1133–1144.
Touraev A, Indrianto A, Wratschko I, Viente 0, Heberle-Bors E. 1996. Efficient microspore embryogenesis in wheat Triticum aestivum L. induced by starvation at high temperature. Sexual Plant Reproduction 9, 209–215.
Touraev A, Vicente O, Herbele-Bors E. 1997. Initiation of microspore embryogenesis by stress. Trends in Plant Science 2, 285–323.
Twell D, Park SK, Lalanne E. 1998. Asymmetric division and cell-fate determination in developing pollen. Trends in Plant Science 3, 305–310.
Twell D, Wing R, Yamaguchi J, McCormik S. 1989. Isolation and expression of an anther-specific gene from tomato. Molecular and General Genetics 217, 240–245.
Westecott MB, Huang B. 1995. Application of haploidy in genetic manipulation of canola. In: Terzi M, ed. Current issues in plant molecular and cellular biology. Kluwer Academic Publishers, 155–160.
Yao QA, Simion E, William M, Krochko J, Kasha KJ. 1997. Biolistic transformation of haploid isolated microspores of barley Hordeum vulgare L. Genome 40, 570–581.
Zaki MAM, Dickinson HG. 1991. Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis 1 to embryogenic development. Sexual Plant Reproduction 4, 48–55.
Zhao J-P, Simmonds DH, Newcomb W. 1996. Induction of embryogenesis with colchicine instead of heat in microspores of Brassica napus L. cv. Topas. Planta 198, 433–439.[Web of Science]
Ziauddin A, Simion E, Kasha KJ. 1990. Improved plant regeneration from shed microspore culture in barley Hordeum vulgare L. Plant Cell Reports 9, 69–72.
Ziauddin A, Marsolais A, Simion E, Kasha KJ. 1992. Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid PAA. Plant Cell Reports 11, 489–498.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Murovec, N. Stajner, J. Jakse, and B. Javornik Microsatellite Marker for Homozygosity Testing of Putative Doubled Haploids and Characterization of Mimulus Species Derived by a Cross-genera Approach J. Amer. Soc. Hort. Sci., September 1, 2007; 132(5): 659 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Segui-Simarro and F. Nuez Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers J. Exp. Bot., March 1, 2007; 58(5): 1119 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Maraschin, W. de Priester, H. P. Spaink, and M. Wang Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective J. Exp. Bot., July 1, 2005; 56(417): 1711 - 1726. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||