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Male and female gamete abortions, and reduced affinity between the uniting gametes as the causes for sterility in an indica/japonica hybrid in rice

C.G. Xu, H.Y. Liu and Qifa Zhang

National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research, Huazhong Agricultural
University, Wuhan 430070, China. http://croplab.hzau.edu.cn. Email croplab@mail.hzau.edu.cn; qifazh@mail.hzau.edu.cn

Abstract

Hybrid sterility frequently occurs in crosses between indica and japonica subspecies of Asian cultivated rice. In this study, we investigated the cytological processes involved in formation and development of male and female gametes as well as their interactions in fertilization, using an indica/japonica hybrid in comparison with an indica/indica hybrid. It was found that more than 50% of the microspores generated in the indica/japonica hybrid could not develop into functional pollen. The abortion rate of microspores in the indica/japonica hybrid was much higher than that in the indica/indica hybrid. Abortive embryosacs made up roughly 70% of the embryosacs examined in the indica/japonica hybrid, which was also much higher than that detected in the indica/indica hybrid. Moreover, the amount of pollen adherence on stigmas of the indica variety by hand-pollination of pollen from the japonica variety was much smaller than the indica/indica pollination, and the number of pollen grains adhered on the stigmas by natural self-pollination was much larger in the indica/indica hybrid than in the indica/japonica hybrid. The indica/japonica hybrid also encountered difficulties in pollen tube growth after pollination, and the fertilization rate of indica/japonica hybrid was much lower than indica/indica hybrid. These results clearly illustrated the complexity of the mechanisms underlying the inter-subspecific hybrid sterility in rice involving both pre- and post-zygotic reproductive isolation mechanisms.

Media summary

The hybrid sterility in rice sub-specific hybrid involved both pre- and post-zygotic reproductive isolation mechanisms.

Key words

Hybrid sterility indica/japonica cross microspore megaspore Oryza sativa L.

Introduction

Hybrid sterility frequently occurs in crosses between indica and japonica subspecies of Asian cultivated rice (Oryza sativa L.) (Oka 1988). Cytological mechanisms of the inter-subspecific sterility have been investigated, which produced variable results depending on the interests of the investigators, such as male gamete abortions (Teng et al. 1996; Zhu et al. 1996), female gamete abortions (Wang 1992; Liu et al. 1993, 1997; Zhu et al. 1996), and reduced dehiscence of the anthers (Maeka et al. 1991; Liu et al. 1993). Despite the large number of studies conducted thus far, the mechanism of the indica-japonica hybrid sterility is still a debating issue. In the study reported in this paper, we investigated the cytological processes underlying the formation and development of both microspores and megaspores, and fertilization, in an indica/japonica hybrid in comparison with an indica/indica hybrid. The objective was to identify, at the cytological level, the causal mechanism for the partial sterility in the indica/japonica hybrids.

Methods

Genetic materials

The genetic materials used in this study were two rice cultivars (Nanjing 11 and IR36) of O. sativa ssp. indica, and one cultivar (Akihikari) of O. sativa ssp. japonica. These three varieties are regarded as typical indica or japonica, and were recommended and used as testers for compatibility analyses (Liu et al. 1996). Nanjing 11 was crossed with Akihikari to produce an inter-subspecific hybrid, and with IR36 to produce an intra-subspecific hybrid. The parents and the hybrids were grown in 2001 and 2002 in the Experimental Farm of Huazhong Agricultural University, Wuhan, China.

Observing abortions during male and female gamete formation

The processes of stamen and pistil development were examined using a microscope. The young panicles development to one or two days before flowering were fixed in a solution containing an 18:1:1 mixture of 70% ethanol, formalin and acetylic acid. Anthers and ovary were collected from the fixed floret tissue, stained with Ehrlich’s haematoxylin and dehydrated with ethanol. A microscopic slide was prepared from each section following the procedures described by Sun (1987) and Wang (1992).

Observation of affinity between pollen and stigmas

The affinity between pollen and stigmas was examined by observing the behavior of the pollen on the stigma after pollination. The emasculated panicles of maternal parents were bagged and pollinated with pollen from the respective paternal parents. Twenty to 30 florets were collected from each hybrid 30 min after pollination, fixed in 3% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.1), and stored at 0-4C for 6-7 days. Adherence and germination of the pollen grains, and growth of the pollen tubes on stigmas were examined by observing the fixed tissues of pollinated stigmas under a scanning electron microscope.

Results

The processes of male and female gamete formation and development were examined in two crosses, Nanjing 11/IR36 and Ninjiang 11/Akihikari, presumably representing typical inter- and intra-subspecific hybrids, respectively. During the process of microspore formation and development, the inter-subspecific hybrid was mostly normal up to the stage of meiosis and tetrad formation, with the abortion occurring largely at the one-nucleus stage, primarily due to the incorrect timing in the degeneration of the tapetum cells. Consequently, more than 50% of the microspores that were generated in the inter-subspecific cross cannot develop into functional pollen. This rate is 3-4 times higher than the abnormality observed in the intra-subspecific cross. Pollen activity tests further confirmed that the proportion of fertile pollen produced by the intra-subspecific hybrid was approximately three times higher than that by the inter-subspecific hybrid. Similarly, during the formation and development of female gametes, both intra- and inter-subspecific crosses appeared to be normal in meiosis, and also up to the point of formation of functional megaspores. Our observation suggested that the abnormality starts at or after the first cell-division during the process of embryosac formation and development. The abortive embryosacs made up roughly 70% of the embryosacs examined in the inter-subspecific cross, a rate that is also about 4 times higher than the abnormality detected in the intra-subspecific cross.

The present study revealed that the amount of pollen adherence on the stigmas of the indica variety pollinated using pollen from the japonica variety was much smaller than the indica/indica pollination. In addition, the amount of pollen adherence on stigmas of the Nanjing 11/Akihikari cross was much less than Nanjing 11/IR36 in both the F0 (crossing between the two parents) and F1 (pollination of the hybrid using its own pollen), with artificial pollination (Table 1). Further differences were also detected during the fertilization processes. Majority of the pollen could germinate on the stigmas, and the pollen tubes for most of the germinated pollen could elongate. However, the degrees of the pollen tube elongations differed greatly in the two hybrids. In the Nanjing 11/IR36 hybrid, the pollen tubes were round, straight with a smooth looking. Whereas, various abnormalities of pollen tube growth were observed in the Nanjing 11/Akihikari hybrid (Figure 1).

Table 1. Adherence of pollen on stigmas, fertilization and seed-setting rates in various crosses with artificial emasculation and pollination

 

Cross

No. pollen grains per stigma

Fertilized ovaries (%)

Seed-setting rate (%)

F0

Nanjing 11/Akihikari

2.251.38**

47.6520.23**

24.4115.80ns

 

Nanjing 11/IR36

16.704.11

93.8837.91

34.8315.26

F1

(Nanjing 11/Akihikari)/ (Nanjing 11/Akihikari)

1.751.23**

32.9117.08**

17.9211.30**

 

(Nanjing 11/IR36)/ (Nanjing 11/IR36)

21.409.50

87.4632.62

32.1214.27

ns difference between the intra- and inter-subspecific hybrids was not significant at the 0.05 probability level; The indica/japonica hybrid was lower than the indica/indica hybrid at the 0.01 probability level.

These difficulties in pollen adherence and pollen growth resulted in large differences in the rates of fertilization and seed setting between intra- and inter-subspecific hybrids (Table 1). This clearly suggests a different level of affinity between pollen and stigmas in intra- and inter-subspecific pollinations.

Conclusion

The results clearly demonstrated the complexity of the mechanisms underlying the inter-subspecific hybrid sterility in rice, which can be described as a “syndrome” involving a variety of phenomena including both pre-zygotic reproductive isolation due to the reduced affinity between the pollen and stigmas of the two subspecies, and post-zygotic reproductive isolation or hybrid dysgenesis featured by both male and female gamete abortions, as well as reduced affinity between pollen and stigmas of the F1. Whereas, the relative importance for each of these components in inter-subspecific sterility, and the underlying causes for each of the components remain to be determined in future studies.

Figure 1. Adherence and germination of pollen, and growth of pollen tubes on stigmas observed using a scanning electron microscope. a) Adherence of many pollen grains on stigmas of the Nanjing 11/IR36 hybrid. b, c) No or very few pollen grains adhered on the stigmas of the Nanjing 11/Akihikari hybrid. d) Vigorous pollen grains and pollen tubes growing on stigmas of the Nanjing 11/IR36 hybrid. e-p) Various abnormalities in germination and growth of the pollen tube on stigmas of the Nanjing 11/Akihikari hybrid.

References

Liu KD, Zhou ZQ, Xu CG, Zhang Q, Saghai Maroof MA (1996). An analysis of hybrid sterility in rice using a diallel cross of 21 parents involving indica, japonica and wide compatibility varieties. Euphytica 90:275-280

Liu YS, Zhou KD, Yin GD, Luo WZ (1993). Preliminary cytological observations on female sterility of hybrids between indica and japonica rice. Acta Biologyae Experimentalis Sinica 26:95-99 (In Chinese with English abstract)

Maeka M, Inuka T, Shinbashi N (1991). Spikelet sterility in F1 hybrids between rice varieties Silewah and Hayakogane. Japan J Breed 41:359-363

Oka HI (1988). Origin of Cultivated Rice. Scientific Societies Press, Tokyo, Japan pp181-209

Sun JS and Qian YQ (1987). Methods of Plant Cytology Studies (In Chinese). Scientific Press, Beijing, pp 40-46

Teng JL, Xue QZ, Wang YX (1996). Ultrastructural observations of pollen and anther wall developments between subspecies in rice (Oryza sativa L.). J Zhejiang Agri Univ 22:467-473 (In Chinese with English abstract)

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Zhu XH, Cao XZ, Zhu QS (1998). Investigation on gametophytic sterility and its contribution to spikelet sterility on F1 plants of indicajaponica in rice. Acta Agronomica Sinica 24:421-430

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