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Keywords:

  • Oryza rufipogon;
  • O. sativa;
  • pollen competition;
  • sequential pollination;
  • reproductive isolation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  •  Post-pollination competition is reported here in cultivated rice (Oryza sativa) and a perennial wild rice (O. rufipogon) to investigate the occurrence of crop-to-wild gene flow.
  •  Wild and cultivated rice (variety Minghui-63) were grown in a common garden in Hunan province, China, and crop-specific genetic markers were used to detect hybridization following hand-pollinations. Using 11 sequential pollination treatments, the effects of the relative timing of pollination on the success of foreign pollen was investigated.
  •  Foreign pollen from the crop resulted in lower pollen germination, fewer pollen tubes per style, and a significant reduction of seed set, demonstrating a disadvantage of foreign pollen even in the absence of pollen competition. When 1 : 1 pollen mixtures were applied, only 2% of the resulting seeds were hybrids, revealing a much stronger disadvantage of foreign pollen when competing with conspecific pollen. Testing the effects of the relative timing of pollination on the success of foreign pollen suggested that conspecific pollen is often more successful than foreign pollen. Nonetheless, hybridization is possible following the deposition of pollen mixtures, especially when foreign pollen arrives earlier than conspecific pollen.
  •  Pollen competition between wild and cultivated rice could slow the rate of crop-to-wild gene flow, but even if pollen competition was ubiquitous it would not prevent gene flow from the crop.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reproductive isolation either at the pre- or postzygotic stage is the main mechanism for species differentiation, which restricts hybrid formation and gene exchange between species (Arnold et al., 1993). During interspecific hybridization, foreign and conspecific pollen will compete on stigmas and in styles. Such interspecific pollen competition can play a major role in prezygotic isolation for certain plant groups (Baker & Shore, 1995; Rieseberg et al., 1995; Diaz & Macnair, 1999). Study of interspecific pollen competition is therefore of importance in determining the degree of hybridization (Arnold et al., 1993; Carney et al., 1994; Baker & Shore, 1995). However, other studies also show that pollen competition is of moderate intensity for reproductive isolation among species (Schlichting et al., 1990; Klips, 1999). The controversy indicates that the intensity of interspecific pollen competition varies among species.

A number of studies of pollen competition focused on the occurrence of competition when pollen grains were deposited simultaneously on stigmas, these studies investigated the performance of pollen donors in mixed pollination, such as pollen tube growth rates and the degree of pollen tube attrition in styles. However, we believe that attention should also be paid to the study of competition between early and late-arriving pollen resulting in sequential pollination. Sequential pollination is usually defined as foreign and conspecific pollen grains being deposited on stigmas at different times (Snow et al., 2000) and it frequently occurs under natural conditions (Arnold et al., 1993). Sequential pollination is thought to have a strong effect on the siring ability of the different pollen types (Smith, 1970; Cruzan, 1998; Snow et al., 2000). Studies further indicate that conspecific pollen preference often occurs both in mixed and in sequential pollination, and this phenomenon was actually observed by Darwin (for review see Arnold, 1997). Nevertheless, our knowledge on the effects that influence pollen performance in sequential pollination have is still limited. Studies using modern technology, particularly molecular markers, can help us to understand more fully the effect of sequential pollination on siring ability (Cruzan, 1998).

With the fast development of biotechnology, more genetically modified organisms (GMO) have become available. While these have the potential to benefit humankind, they also have raised great biosafety concerns. Possible ecological risk caused by transgenes escaping through introgression between transgenic crops and their wild relatives has become one of the serious biosafety concerns. Many transgenic rice varieties and lines have been produced and are ready to be released to the environment for field test in China. If transgenes such as those conferring herbicide tolerance, pest resistance, and drought tolerance are transferred to wild and weedy rice through outcrossing, these wild relatives may significantly enhance their fitness and reproduce rapidly. Eventually these introgressed wild relatives may become super weeds out of human control and cause serious ecological risk (Gray & Raybould, 1998). It is therefore important to understand pollination biology and breeding systems of related rice species, and the extent of introgression between rice and its wild relatives. This knowledge will facilitate the design of a proper farming system to minimize the possibility of gene escape from rice to its wild relatives and to avoid ecological risks caused by such introgression in which transgenes are involved.

Oryza rufipogon Griff. is the putative ancestor of the Asian cultivated rice, O. sativa L. (Oka, 1988), and is the most important genetic resource for rice improvement (Yuan et al., 1989; Xiao et al., 1996). Natural hybridization and gene flow between O. rufipogon and O. sativa has been reported in many locations (Oka & Chang, 1961; Langevin et al., 1990; Majumder et al., 1997; Suh et al., 1997; Lu, 1999). Differences in daily flowering time between O. rufipogon and O. sativa provide opportunities for the cultivated rice to pollinate O. rufipogon earlier than conspecific pollen when the two species grow nearby. No study of pollen competition between rice and its wild species has been reported, although it is necessary to understand hybridization and the isolation mechanisms between them. The objectives of this study were to investigate: the intensity of interspecific pollen competition between O. rufipogon and O. sativa and its consequence on hybrid formation; the siring ability of different pollen donors in sequential pollination by using molecular markers; and the frequency of gene exchange between O. rufipogon and O. sativa under controlled conditions.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant materials

Tillers of O. rufipogon were collected from a Chaling population located in a mountainous area of Hunan province in China (26°50′ N, 11°340′ E). O. rufipogon was used as both pollen recipient and donor in this study. The cultivated rice variety Minghui-63 (donated by Prof. S. M. Mu, Hubei Academy of Agricultural Science of China) was used as a foreign pollen donor. Minghui-63, which has a 90–95 day maturation period, is a universal paternal line for hybrid rice breeding and produces a relatively large number of pollen grains. The study site was located in rice fields approx. 3 Km from the Chaling O. rufipogon population and isolated by surrounding hills. The experimental population (5 × 5 m2) was planted with O. rufipogon and Minghui-63 by rows in turn with 0.5 m between the rows in 1999 and 2000 in the middle of April for O. rufipogon and middle of June for Minghui-36.

Experiment design

The experiment included 15 treatments defined as follows, with sequential pollination in A1~A11 treatments (except for A6 treatments) and simultaneous pollination in B, C, and D treatments.

  •  A1~A5, A7~A11 treatments: sequential pollination in which the left branch of the O. rufipogon stigma against the lemma was pollinated by Minghui-63 and the right branch by the conspecific pollen, respectively, at 10 different time intervals (A1~A5, A7~A11) from 50 min earlier to 50 min later than the simultaneous pollination (A6 treatment) with Minghui-63 or O. rufipogon pollen to each stigma branch, respectively (Table 1).
  •  B treatment: mixed pollination, O. rufipogon stigmas were pollinated by a pollen mixture of Minghui-63 (50%) and O. rufipogon (50%). Both stigma branches were pollinated by the same type of mixed pollen.
  •  C treatment: foreign pollination, O. rufipogon stigmas were pollinated by Minghui-63.
  •  D treatment: conspecific pollination, O. rufipogon stigmas were pollinated by the conspecific pollen.
Table 1.  The design of the A1~A11 treatments (sequential pollination) and comparisons of Oryza rufipogon and Minghui-63 on pollen germination and the numbers of pollen tube (Pt) across mid-styles and at the base of styles in O. rufipogon in the sequential pollination experiment by t-tests
Code of treatmentsPollen donor1Pollinated branches of stigma2Pollination time intervals (min)3Sample sizeComparisons of Oryza rufipogon and Minghui-634
Pollen germinationPt across mid-stylesPt at the base of styles
  • 1

    Os, Minghui-63; Or, O. rufipogon.

  • 2

    L, the left branches; R, the right branches of O. rufipogon stigma.

  • 3

    +, foreign pollen was applied to stigma earlier than conspecific pollen; −, foreign pollen was applied later than conspecific pollen.

  • 4

    *p < 0.05, **p < 0.01. NS, not significant.

A1OsL+5016NSNSNS
 OrR     
A2OsL+4021NSNSNS
 OrR     
A3OsL+3029NSNSNS
 OrR     
A4OsL+2012NSNSNS
 OrR     
A5OsL+1026NSNSNS
 OrR     
A6OsL 032***
 OrR     
A7OsL−1010*NS*
 OrR     
A8OsL−2020*****
 OrR     
A9OsL−3030*****
 OrR     
A10OsL−4040*****
 OrR     
A11OsL−5050******
 OrR     

Pollination

All pollinations were carried out in the field during September–October in 1999 and 2000. Panicles of O. rufipogon were covered with bags before flowering from 06 : 00 to 09 : 00 h. Spikelets opened 20–30 min after bagging, stamens stretched out and anthers were removed before their dehiscence. Mature anthers were collected separately from Minghui-63 and O. rufipogon and placed in sealed vials to maintain pollen vigor. Pollination was carried out for each treatment using fine-tipped forceps loaded with fresh pollen grains between 09 : 30 and 12 : 30 h. Attempts were made to apply approximately the same amount of pollen grains to the two branches of O. rufipogon stigma to avoid possible pollen density effects (Niesenbaum & Schueller, 1997). Panicles were covered with bags after pollination. Some of the pollinated spikelets were collected and fixed in 70% ethanol 2 h after pollination. The fixed samples were brought back to the laboratory to examine pollen germination and pollen tube growth under a microscope. The other pollinated spikelets remained on the plants until seed collection.

Examination of pollen germination and pollen tube growth

The sampled spikelets were placed on a slide after being cleaned in 8 M NaOH for 8 h and rinsed in distilled water twice. Lemmas were carefully removed, and the pistils were stained with 0.1% aniline blue (in 0.1 M K3PO4) for approx. 30 min, covered with a cover-glass, and squashed slightly for examination under a fluorescence microscope. Pollen germination on stigmas, the number of pollen tubes across mid-styles, and the number of pollen tubes at the base of styles was recorded. The positions of callous pollen tube plugs were used as markers for pollen tubes (Weller & Ornduff, 1989).

Determination of seed set and paternity

Mature seeds were collected from pollinated panicles and seed set was calculated in all treatments. After storage at 4°C for 1 month and heating at 50°C for 24 h to break dormancy, the mature seeds were germinated in Petri dishes at an alternating temperature of 30°C day/25°C night. Seedlings were planted in a glasshouse. Leaf samples were collected from individuals approx. 50 d after seed germination for both isozyme and SSR (Simple Sequence Repeat DNA) examination to confirm hybrid origin. Isozyme extraction and electrophoresis was performed following the description by Wang (1996). DNA extraction used the protocol of Doyle & Doyle (1987) and PCR reactions were performed according to Wu & Tanksley (1993). Hybridization rate was obtained by examining isozyme and SSR variation patterns in parents and hybrids, where seed paternity was identified by Minghui-63-specific markers that consistently appeared in the cultivated rice only.

Data analysis

A one-way ANOVA procedure was used in the A1~A11 treatments to examine the effects of the sequential pollination on pollen germination, the number of pollen tubes across mid-styles, and the number of pollen tubes at the base of styles. Regression analysis was conducted to variants when significant correlation between pollen germination or pollen tube growth and pollination time was observed. t-tests were made to compare pollen germination, pollen tubes across mid-styles and at the base of styles between Minghui-63 and O. rufipogon in the A1~A11 treatments. The data from the B, C, and D treatments were analyzed using a one-way ANOVA to test the effects of pollen donor on pollen germination and number of pollen tubes. All the data obtained from the B, C, and D treatments were also compared by t-tests. To demonstrate relationships between interspecific pollen competibility in sequential pollination, the mean values of the data from left and right branches in the A1~A11 treatments were transformed into ratios that were further changed into square roots. The square-root transformed data were analyzed by a regression module combined with pollination time. All statistics analyses were performed using the STATISTICA for Windows soft package (single user version (5.0), StatSoft Inc. (1995).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pollen germination on stigmas of O. rufipogon

The ANOVA analysis showed that germination of Minghui-63 pollen was significantly affected by pollination time, but that of O. rufipogon pollen was not affected by pollination time (Table 2). Germination of Minghui-63 pollen was not significantly different from that of O. rufipogon pollen in the A1~A5 treatments, when Minghui-63 pollen grains were deposited on O. rufipogon stigma 10–50 min earlier than the conspecific pollen grains (Table 1). When the left and right stigma branches were simultaneously pollinated with Minghui-63 and O. rufipogon pollen, germination rates were 67.5% and 74.9%, respectively, showing significant differences (P = 0.05). When Minghui-63 pollen grains were deposited 10–50 min later than the conspecific pollen grains, pollen germination of Minghui-63 was significantly lower than that of O. rufipogon (Table 1). The linear regression of the square-root transformed ratios of pollen germination between Minghui-63 and O. rufipogon in the A1~A11 treatments was y = 1.082–0.025* : (r2 = 0.95, P < 0.001)(Fig. 1).

Table 2.  One way ANOVA for the effects of pollination time on pollen germination, pollen tube (Pt) growth in the A1~A11 treatments
Sourcedf1Msdf2MsFP-level
  1. Sample size is 16, 21, 29, 12, 26, 32, 10, 20, 30, 40, 50 panicles in the A1~A11 treatments, respectively.

Minghui-63 pollen germination100.0531520.0143.8280.000
Minghui-63 Pt across mid-styles105.0861521.4623.4780.000
Minghui-63 Pt at the base of styles101.5861520.5073.1300.001
O. rufipogon pollen germination100.0781490.0371.3570.204
O. rufipogon Pt across mid-styles105.3651493.5641.7330.078
O. rufipogon Pt at the base of styles102.1481490.6913.1100.001
image

Figure 1. Comparison of mean pollen germination and pollen tube (Pt) growth in sequential pollination (the A1~A11 treatments) between Minghui-63 and Oryza rufipogon. The vertical axis indicates the square root of ratios of mean pollen germination or pollen tube (Pt) number between Minghui-63 and O. rufipogon in sequential pollination. The horizontal axis indicates the pollination time. The three lines represent the competitive ability of foreign pollen decreases with the increase of pollination time delays, although the data of pollen tube at the base of styles does not show perfect regression. Circles, pollen germination; squares, Pt across mid-style; diamonds, Pt at the base of the style. Sample size is 16, 21, 29, 12, 26, 32, 10, 20, 30, 40, 50 panicles in the A1~A11 treatments, respectively.

Download figure to PowerPoint

The ANOVA analysis of pollen germination in the B, C, and D treatments suggested that the pollen donor had significant effects on pollen germination (F = 10.347, P < 0.001). t-tests further showed that mixed pollen (B treatment) had the lowest germination, the conspecific pollen (D treatment) had the highest, and Minghui-63 pollen had intermediate germination (C treatment) (Table 3).

Table 3.  The means of pollen germination, pollen tube (Pt) across mid-styles and at the base of styles from mixed pollination, foreign pollination, and conspecific pollination
PollinationSample sizeGerminationPt across mid-stylesPt at the base of styles
  1. The means followed by the different letters are significantly differences at the 5% level by Duncan’s Multiple Range Test (DMRT).

Mixed240.467c1.542b0.583c
Foreign140.661b1.214c0.643b
Conspecific320.767a3.406a1.312a

Pollen tube growth in styles of O. rufipogon

Mean number of pollen tubes across mid-styles and at the base of styles was used as a measure of pollen tube growth. The ANOVA analysis showed that the number of Minghui-63 pollen tubes across mid-styles and at the base of styles in O. rufipogon was affected by the relative pollination time. The number of O. rufipogon pollen tubes at the base of styles was also affected by pollination time, and that across mid-styles also can be regarded as being affected by time because it was close to the significant level (P = 0.078) (Table 2). t-tests showed that the numbers of Minghui-63 and O. rufipogon pollen tubes across mid-styles and at the base of styles in the A1~A5 treatments were not significantly different (Table 1). In the A6~A11 treatments, the number of Minghui-63 pollen tubes was significantly lower than that of O. rufipogon pollen tubes (Table 1). The linear regression of square-root transformed ratios of the number of pollen tubes across mid-styles and at the base of styles was y = 1.227–0.089* × (r2 = 0.82, P < 0.001 and y= 1.421–0.145* × (r2 = 0.86, P < 0.001), respectively (Fig. 1).

The ANOVA indicated that pollen donor also had strong effects on pollen tube growth (F1 = 12.920, P1 = 0.000, F2 = 8.795, P2 = 0.000, respectively). t-tests further showed that the D treatment (conspecific) had the highest pollen tube growth rate and the B treatment (mixed) had the lowest (Table 3).

Hybridization rate and its molecular confirmation

Table 4 showed that seeds sets in the A1~A11 treatments (sequential pollination) were nearly the same as in the conspecific pollination. Seed set in the foreign pollination was a half of that in the conspecific pollination (32.3% vs 65.6%). Seed set in the mixed pollination also significantly reduced compared with that in the conspecific pollination (49.1% vs 65.6%). Hybrids were identified by one esterase (EST) locus screened from six enzyme systems and two pairs of SSR primers, RM36 and RM44, were selected from 64 pairs of primers. The three markers showed consistent paternity bands from Minghui-63 in all experiments (Table 4). The hybridization rate in the A1~A11 treatments showed a clear tendency of decrease with delayed pollination of Minghui-63 (Table 4). The mixed pollination had a low hybridization rate (2.0%), and the conspecific pollination did not produce any hybrid as indicated by the EST and SSR markers that showed consistent patterns in the examined samples.

Table 4.  Seed set and hybrid rate obtained from different treatments
CodeSpikelets pollinatedSeed set (%)Hybrid confirmed by EST marker (%)1Hybrid confirmed by SSR marker (%)1
  • 1

    As percentage of seed set.

A1 8863.614.314.3
A2 8153.1 5.9 5.9
A3 9162.6 3.2 3.2
A4 9459.6 3.5 3.5
A5 9363.4 2.6 2.6
A6 8960.8 0 0
A7 9464.9 3.0 3.0
A8 8758.6 0 0
A9 7864.1 0 0
A10 8161.7 0 0
A11 8962.9 0 0
Mixed11249.1 2.0 2.0
Foreign 9732.395.895.8
Conspecific 9365.6 0 0

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The cultivated rice, O. sativa L., is thought to have been domesticated from the wild O. rufipogon in Asia approx. 11 500 yr ago (Normile, 1997), indicating a relatively close relationship between the two species. O. rufipogon is widely distributed in South and South-east Asian countries (with extension to the northern Australia), where O. sativa is the major staple food and is massively cultivated. Natural hybrids between O. sativa and O. rufipogon were commonly found in the areas where the two species occur sympatrically (Oka & Chang, 1961; Langevin et al., 1990; Suh et al., 1997; Lu, 1999). A sound study of compatibility, pollen competition mechanisms and hybridization between O. sativa and O. rufipogon will provide us with background knowledge and data to evaluate the chance of genes escaping to wild relatives from transgenic rice.

Interspecific compatibility

Interspecific compatibility is crucial for hybridization (Heslop-Harrison, 1982). Minghui-63 had relatively high pollen germination and pollen tube growth rates in O. rufipogon, which demonstrated that Minghui-63 was highly compatible with O. rufipogon at the stage of prefertilization, although the two species showed obvious differences in their pollen germination and pollen tube growth. Seed set from foreign pollination was 32.3%, of which 95.8% was true hybrid. This further indicated that Minghui-63 was compatible with O. rufipogon at the stage of postfertilization, but the compatibility was weaker than the conspecific pollen. These results indicate that there are relatively limited reproductive barriers between Minghui-63 and O. rufipogon.

Pollen competition

Pollen competition occurs when pollen deposition on stigmas exceed the number of ovules (Snow & Spira, 1991). In rice species, each spikelet has a single ovule, and the average number of pollen grains that are naturally deposited on stigmas of O. rufipogon is approx. eight (Z. P. Song, unpublished). In this study, we applied an average of more than eight pollen grains on stigmas, providing opportunities for pollen competition to occur. In mixed pollination, foreign and conspecific pollen grains compete on stigmas and in styles through pollen germination and pollen tube growth. Interspecific competition has stronger impacts on pollen germination and pollen tube growth rates, compared with conspecific competition. In this study pollen germination and pollen tube growth in the mixed pollination were significantly lower than those in the foreign and conspecific pollinations, indicating the existence of interspecific competition.

However, the low pollen germination and pollen tube growth could also be attributed to pollen tube attrition (inhibitory effect from style tissue) or variation in growth rates (scramble effect) (Cruzan, 1998). The attrition rate of pollen tubes can be determined by comparing the number of pollen tubes at the base of styles with that across mid-styles in the foreign and conspecific pollinations. The attrition rate of pollen tubes was observed to be approx. 47% and 38% in the two treatments, respectively, suggesting that the attrition of foreign pollen tubes (O. sativa) was more significant in the O. rufipogon styles. On the other hand, our results showed a few hybrids were produced in the A8 treatment (delayed pollination) and the mixed pollination, which suggests that the growth rate of foreign and conspecific pollen tubes is not significantly different. Therefore, the scramble effect is neglectable in this experiment. The frequencies of pollen germination on stigmas and pollen tube growth in styles in the mixed pollination were significantly lower than those in the foreign pollination, indicating that competition between pollen grains and pollen tubes from different species did take place, in addition to the existence of inhibitory effect. These results are similar to a previous study of Ipomopsis aggregata, in which the effect of interspecific pollen competition was stronger than that of conspecific pollen competition (Caruso, 1999).

Interspecific competition may affect both normal pollen germination and pollen tubes growth rates (Cruzan, 1990), which can be regarded as an alternative exclusion effect that restricts hybrid formation (Arnold et al., 1993), rather than the rejection effects in incompatible systems (Heslop-Harrison, 1982; Arnold, 1997). It is therefore considered to play a major role as a prezygotic barrier in the reproductive process of many plant groups (Baker & Shore, 1995; Rieseberg et al., 1995; Diaz & Macnair, 1999). It is also revealed by this study that as a prezygotic barrier, interspecific pollen competition plays an important role in the hybridization between O. rufipogon and Minghui-63, although in O. rufipogon the mixed pollination produced a few hybrids (2.0%), indicating that foreign pollen could fertilize the ovule through successful competition with conspecific pollen.

Effects of competition in sequential pollination

Under natural conditions, the daily flowering time among different individuals is not synchronous, especially among such related species as O. rufipogon and the cultivated O. sativa. In other words, sequential pollination should always occur. We have observed that Minghui-63 flowered approx. 1 h earlier than O. rufipogon, although their flowering period overlaps in general, and O. rufipogon is protogynous. This suggests that competition between O. sativa and O. rufipogon through pollination time might occur when they grow together. In sequential pollination, foreign pollen grains have opportunities to occupy stigmas and send pollen tubes to the embryo sac earlier than the conspecific pollen, which could evade the competition with conspecific pollen gains, and therefore enhance the chance of hybridization.

It is shown in Fig. 1 that the earlier foreign pollen was deposited on the stigmas than the conspecific pollen, the higher the rates of foreign pollen germination and pollen tube growth. Results from our sequential pollination experiment strongly conclude that prior reception of foreign pollen will enhance its competitive ability, which leads to a significant increase of hybrid rates. This result also supports the assumption that frequent sequential pollination has a strong impact on the seed paternity (Smith, 1970; Arnold et al., 1993), and the viewpoint that the prior pollination can increase the siring success of disadvantage pollen type (Arnold, 1997).

Based on Cruzan (1998), siring ability of different pollen types in mixed pollination can be attribute to the effects of differential pollen tube growth rate, pollen tube attrition, and zygote abortion. These effects can be separated by analysis of siring success over a series of sequential pollination using genetic markers such as isozymes and SSR markers. The hybrid rate, as confirmed by EST and SSR markers in our sequential pollination experiment (indicative of siring ability of foreign pollen), was much lower than the seed set in each treatment, which indicates the foreign pollen to be the disadvantaged type. Our data also showed that siring success of the foreign pollen was up to 14% even in the A1 treatment, in which foreign pollen was deposited 50 min earlier than the conspecific pollen, suggesting the possibility of zygote abortion. It is concluded from our results that the siring disadvantage of foreign pollen is most likely attributed to selective postfertilization abortion of ovules (zygote abortion), although prefertilization attrition of pollen tubes (inhibitory effect) might also play certain roles.

In general, O. rufipogon stigmas are receptive for a relatively long period of time, over 20 h (Z. P. Song, unpublished). In this study, all pollinations were performed by hand with fresh pollen grains within the receptive period of this species. Pollen germination of Minghui-63 decreased with delayed pollination, but the germination of O. rufipogon pollen did not show this tendency. One possible explanation for this is that the receptivity of O. rufipogon stigma declines significantly with time for foreign pollen grains, although it can last a relatively long time. Also, the pollen viability of Minghui-63 decreases faster (half-life < 20 min) than that of O. rufipogon (Song et al., 2001), which may partly explain the differences. Numbers of both pollen tubes of Minghui-63 and O. rufipogon in styles decreased with delayed pollination, suggesting that the receptivity of O. rufipogon style tissue also declines with the delay of pollination.

Gene exchange between O. rufipogon and cultivated rice

Gene exchange through introgression is usually mediated by pollen (Arnold et al., 1991), but interspecific reproductive isolation can restrict hybrid formation and consequently limit gene exchange. This study shows that the rice cultivar Minghui-63 is sexually compatible with O. rufipogon, and pollen competition, as a reproductive barrier is not strong enough to avoid hybridization between O. sativa and O. rufipogon, although conspecific pollen will often be more successful than the foreign pollen when interspecific pollen competition occurs in O. rufipogon. Notably, when deposited on O. rufipogon stigmas about 1 h earlier than the conspecific pollen, Minghui-63 pollen grains will have relatively strong competitive ability, and hybrid seeds can result. The earlier pollination of cultivated rice to O. rufipogon than the conspecific pollen could happen naturally due to the differences in daily flowering time between the cultivated rice and O. rufipogon. As was studied earlier, gene exchange between O. sativa and O. rufipogon happens frequently when the two species occur sympatrically, suggesting the possibility of transgene escape from transgenic rice to O. rufipogon. Effective measures are therefore required to prevent pollen flow between the wild and cultivated rice when transgenic rice varieties are released to environments where the wild relatives are available. In addition, assessment of ecological impacts of transgene escaping to wild rice species through outcrossing is urgently needed, particularly in the areas where transgenic rice varieties will be released to the environment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to express our deep appreciation to Prof. Bo Li for his assistance in data analysis, to Dr Yuguo Wang and Guihua Liu for their assistance in the field experiments, and to Prof. A. A. Snow of the Ohio State University for her valuable comments on this manuscript. This work was supported by the National Nature Science Foundation of China (39893360).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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