- Email: [email protected]
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
Agricultural Sciences in China
October 2011
2011, 10(10): 1489-1500
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis FU Xue-lin, LU Yong-gen, LIU Xiang-dong, LI Jin-quan and ZHAO Xing-juan Key Laboratory of Plant Molecular Breeding, Guangdong Province/College of Agriculture, South China Agricultural University, Guangzhou 510642, P.R.China
Abstract Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid (AA, 2n=2x=24) and autotetraploid (AAAA, 2n=4x=48) cultivated rice were crossed as maternal parents with O. officinalis (CC, 2n=2x=24), none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations (DAP). By rescuing hybrid embryos, in vitro F 1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globular- or pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes. Key words: breeding barriers, interspecific hybridization, crossability, hybrid sterility, Oryza sativa, Oryza officinalis
INTRODUCTION Oryza officinalis Wall ex Watt (CC, 2n=2x=24) is one of the important wild species in Oryza genus and one of the three wild species indigenous to China. It has a number of beneficial genes for rice breeding such as resistance genes for brown planthopper (BPH), whitebacked planthopper (WBPH), and bacterial blight (BB). It also tolerates to drought, cold, and other abiotic stresses (Bellon et al. 1999; He 2002; Barclay 2004; Qin et al. 2007). Through interspecific hybridization and backcrossing between O. officinalis and Oryza Received 21 August, 2010
sativa, some of the resistance genes have been transferred into cultivars and mapped on chromosomes. Thus some new germplasms have been innovated including monosomic alien addition lines (MAALs) (Shin and Katayama 1979; Jena and Khush 1989, 1990; Tan et al. 2004a, b, 2005), introgression lines (Jena et al. 1992), and advanced backcross generations (Yan et al. 1996; Zhong et al. 1997; Huang et al. 2001). Nevertheless, previous studies showed that the crossability between O. sativa and O. officinalis was less than 3.5% (Jena and Khush 1989, 1990; Yan et al. 1996). The hybrids were completely male sterile (Jena and Khush 1989; Yu et al. 1993). Because of severe
Accepted 14 December, 2010
FU Xue-lin, Associate Professor, Ph D, E-mail: [email protected]; Correspondence LU Yong-gen, Professor, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60143-0
1490
reproductive isolation, obtained hybrids between O. sativa and O. officinalis were fewer, and it is even more difficult to produce BC1F1 plants. However, there was little information of the mechanisms of the breeding barriers in O. sativa and O. officinalis. Especially, there was still none direct evidences on the precise mechanisms for the low crossability and hybrid female sterility even though that is very important for probing into the method to overcome the barriers effectively. So, it is indispensable to detect the evidences and clarify the mechanisms of breeding barriers between O. sativa and O. officinalis in order to effectively mine and utilize the beneficial genes of O. officinalis in a large scale in rice breeding. By using whole-mount eosin B-staining confocal laser scanning microscopy and other methods, this study was undertaken to observe the pollen germination, hybrid embryo, and endosperm development in the crosses of O. sativa and O. officinalis, young embryo culturing in vitro to produce hybrid plantlets, development of hybrid embryo sacs and pollen grains and the sterilities. The objective was to clarify, at the cytological level, the mechanisms of interspecific hybridization barriers and hybrid sterilities between O. sativa and O. officinalis.
FU Xue-lin et al.
sterilized with 75% ethanol and 0.1% HgCl2 solution, then young embryos were inoculated on 1/4 MS medium supplemented with 30 g L-1 sugar and 7 g L-1 agar in a laminar flow cabinet. When the plantlets attained 3-4 leaf stages, they were hardened and transplanted. After 40 d of embryo culturing in vitro, the rates of plantlet production and the crossability were analyzed. Plantlet production rate (%)=Number of plantlets/Number of young embryos cultured×100. Crossability (%)= Number of plantlets/Number of spikelets pollinated×100. All the hybrid plantlets were identified by main traits and SSR markers. SSR assay was carried out according to the method described by Panaud et al. (1996).
Pollen germination, fertilization, hybrid embryo, and endosperm development in the interspecific hybridization
In this experiment, indica cultivar Guangluai 4 (2n=2x=24), japonica cultivar Liaojing 944 (2n=2x=24) and their corresponding autotetraploids (2n=4x=48) were used as maternal parents. One accession of O. officinalis indigenous to Guangxi Zhuang Autonomous Region in China was used as paternal parent. The cultivars were planted at the experimental farm of South China Agricultural University (SCAU). O. officinalis and the interspecific hybrids between O. sativa and O. officinalis were planted at the Wild Rice Core Collection Nursery of SCAU.
Pollen germination was observed by aniline blue staining method described by Hu (1994). When Guangluai 4 (2x and 4x) was crossed with O. officinalis, more than 15 spikelets at 30 min after pollination for each cross combination were fixed in Carnoy’s solution for more than 12 h, then the spikelets were transferred to graded ethanol series of 90, 80, 70, 50, 30, and 10% and distilled water to hydrate, 20-30 min for each grade. After that, the lemma of spikelet was cut out and the pistil with palea was cleared in 5 mol L-1 NaOH solution for about 2-3 h, and then rinsed with distilled water for three times. Before observation, the pistil was dissected carefully on a slide and blocked out the background with 0.01% toluidine blue solution and was rinsed with distilled water for three times, then the pistil was stained with 0.01% aniline blue solution for several minutes and pollen germination was observed under a Leica DMRXA (Heidelberg, Germany) fluorescenct microscope. WE-CLSM (whole-mount eosin B staining confocal laser scanning microscopy, Leica, Germany) was used to observe fertilization and hybrid embryo and endosperm development according to the method described by Fu et al. (2009a).
Interspecific hybrid plantlets production and crossability analyses
Cytological observations on interspecific hybrid sterility
At 13-16 DAP, swollen hybrid ovaries were surface
Hybrid spikelets of Guangluai 4 2x/O. officinalis at all
MATERIALS AND METHODS Cross combinations
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
development stages were collected and fixed in Carnoy’s solution for more than 2 d. Embryo sacs and anthers were dissected. Structures and development of embryo sacs were observed using WE-CLSM. Matured pollen grains were stained with 1% I2-KI and observed under light microscope. Pollen mother cells at meioses of interspecific hybrid were observed with 1% acetic acid carmine squash technique. Microsporogenesis was observed by semi-thin section light microscopy according to the method described by Feng et al. (2001). Slides were observed and photographs were obtained under a Leica DMRXA microscope. All the images were processed with Microsoft Adobe Photoshop 7.0 software.
RESULTS Seed set of the interspecific hybridizations and the crossabilities In the cross combinations, rates of swollen hybrid ovaries on the maternal plants at 13-16 DAP were low, but the rates of swollen hybrid ovaries in Liaojing 944 2x/ O. officinalis and Liaojing 944 4x/O. officinalis were higher than those of Guangluai 4 2x/O. officinalis and Guangluai 4 4x/O. officinalis (Table 1). Hybrid ovaries shrunk gradually and aborted finally (Fig. 1-A-D). Nevertheless, by rescuing hybrid embryos in vitro hybrid plantlets were produced with the rates of 16.67 and 7.41% in Guangluai 4 2x/O. officinalis and Liaojing 944 2x/O. officinalis, respectively. Crossabilities of the two combinations were only 0.42 and 0.46%. None hybrid plantlets were produced in Guangluai 4 4x/ O. officinalis and Liaojing 944 4x/O. officinalis. It meant that the crossability barrier was severe when both diploid and autotetraploid cultivated rice were crossed with O. officinalis which was more severe when using the autotetraploids as maternal parents.
1491
Pollen germination, fertilization and development of hybrid embryo and endosperm in the interspecific hybridizations In order to detect the pre- and post-fertilization barriers between O. sativa and O. officinalis, the processes of pollen germination, double-fertilization, and hybrid embryo and endosperm development in the two cross combinations, Guangluai 4 2x/O. officinalis and Guangluai 4 4x/O. officinalis, were observed. Almost all of the normal pollen grains of O. officinalis germinated on the stigma of Guangluai 4 2x and Guangluai 4 4x and pollen tubes elongated into the styles of them (Fig. 1-E and F). However, rates of double-fertilization and egg cell single-fertilization in Guangluai 4 2x/ O. officinalis were 72.50 and 17.50%, and those in Guangluai 4 4x/O. officinalis were only 27.12 and 33.90%, respectively. Furthermore, as much as 68.19% of non-fertilized embryo sacs were structurally abnormal in Guangluai 4 4x/O. officinalis (Table 2). According to above results and the previous results (Fu et al. 2009b), it can be concluded that fertilization rates at diploid-diploid level (2x×2x) were higher than that at tetraploid-diploid level (4x×2x) in the crosses between O. sativa and O. officinalis, especially for the doublefertilization rate. At 1, 3, and 7 d after anthesis, embryo and endosperm development of Guangluai 4 2x was in a routine way (Fu et al. 2009a). Comparatively, development of hybrid embryo and endosperm in Guangluai 4 2x/ O. officinalis and Guangluai 4 4x/O. officinalis were much more abnormal. In Guangluai 4 2x/O. officinalis, both fertilization traces (with a rate of 9.09%) and globular-shaped embryos (with a rate of 90.91%) were observed at 1 DAP. In double-fertilized embryo sacs, some of the free endosperm nuclei developed normally (Fig. 2-A) while others degenerated early (Fig. 2-B). Whereas, in singlefertilized embryo sacs, some globular-shaped embryos
Table 1 Results of hybridizations and hybrid plantlets production by embryo rescuing Cross combinations GLA 2x/O. officinalis GLA 4x/O. officinalis LJ 2x/O. officinalis LJ 4x/O. officinalis
Spikelets pollinated
No. of swollen ovaries
3 577 2 067 433 937
120 31 47 62
Rate of swollen ovaries (%) 3.35 1.50 10.85 6.62
No. of young embryos cultured
No. of plantlets produced
90 11 27 12
15 0 2 0
Rate of plantlets production (%) 16.67 0 7.41 0
Crossability (%) 0.42 0.46 -
GLA, Guangluai 4; LJ, Liaojing 944. The same as below.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1492
FU Xue-lin et al.
Fig. 1 Hybrid ovaries and pollen germination of O. officinalis from the hybridized spikelets in maternal plants. A, hybrid ovaries of Guangluai 4 2x/O. officinalis at 13-16 DAP. B, hybrid ovaries of Guangluai 4 4x/O. officinalis at 8-10 DAP. C, hybrid ovaries of Liaojing 944 2x/O. officinalis at 14 DAP, showing the basal parts with embryos cut away for embryo rescue. D, hybrid ovaries of Liaojing 944 4x/O. officinalis at 8-14 DAP. E, pollen germination and pollen tube growth of O. officinalis on the stigma of Guangluai 4 2x. F, pollen germination and pollen tube growth of O. officinalis on the stigma of Guangluai 4 4x. Bar=1 cm in A-D, bar=10 μm in E and F.
with incompact structures degenerated and unfertilized polar nuclei left the egg apparatus (Fig. 2-C). At 3 DAP, 13.60% embryos developed into pear-shaped ones, while 77.27% embryos were still in globular-shaped stage, fertilization traces were observed in 9.09% of the ovaries in hybridized spikelets. Normal pear-shaped embryos were formed both in double-fertilized embryo sacs (Fig. 2-D and G) and in single-fertilized ones (Fig. 2-H and I), however, degeneration of embryo (Fig. 2-E and F) and free endosperm nuclei (Fig. 2-D, E, and G) were in a large scale. Till 7 DAP, only 9.5% embryos began to differentiate organs (Fig. 3-F), 47.6% embryos were still in normal pear-shaped state (Fig. 3-D, E, H, and I), 38.20% of the globular-shaped embryos were degenerating (Fig. 3-A and B) as well as 4.80% pearshaped embryos were degenerated (Fig. 3-C and G). Similarly, some free endosperm nuclei degenerated before forming endosperm cells (Fig. 3-A and D), and some endosperm cells formed abnormally (Fig. 3-B, E, and F) or degenerated ahead of time (Fig. 3-C). Furthermore, some embryo sacs distorted (Fig. 3-D, G, and H) along with the degeneration of embryo and endosperm.
Table 2 Fertilization results of the cross combinations at 1 DAP Cross combinations GLA 2x/O. officinalis GLA 4x/O. officinalis
Number of Embryo sacs observed
Rate of single egg fertilization (%)
Rate of double fertilization (%)
Total fertilization rate (%)
40 59
17.50 33.90
72.50 27.12
90.00 61.02
As shown in Table 3 and Fig. 4, fertilization traces (Fig. 4-A) (with a rate of 76.47%) and globular-shaped embryos (with a rate of 23.53%) were observed in Guangluai 4 4x/O. officinalis, showing the rate of fertilization trace was much higher than that in Guangluai 4 2x/O. officinalis at 1 DAP. The rate of pear-shaped embryos (5.56%) at 3 DAP was also lower than that in Guangluai 4 2x/O. officinalis (13.60%), and 88.89% embryos ceased at globular-shaped stage. None embryo was observed to differentiate organs at 7 DAP, among which 25% embryos were still globular-shaped and 75% were pear-shaped. In Guangluai 4 4x/ O. officinalis, not only hybrid embryos but also endosperms degenerated early. That is, at 1 DAP, small embryos developed slowly and were incompact multicells (Fig. 4-B and C), or without embryos but degeneration trace of synergid cells (Fig. 4-D). At the same
Rate of nonfertilized Abnormal embryo sacs/ embryo sacs (%) Non-fertilized embryo sacs (%) 10.00 39.98
0.00 68.19
time, only several free endosperm nuclei formed but trended to degenerate in double-fertilized embryo sacs (Fig. 4-A and B). At 3 DAP, hybrid embryos either stagnated in globular-shaped stage (Fig. 4-E, F, and I) or began to degenerate (Fig. 4-G, H, and K) even if a few of them developed into pear-shaped ones (Fig. 4L). Whatever, free endosperm nuclei degenerated in a large scale before forming endosperm cells in doublefertilized embryo sacs (Fig. 4-E, F, and G), or formed endosperm cells abnormally (Fig. 4-H), normal cellular endosperm seldom filled in the embryo sac cavity (Fig. 4-J). Till 7 DAP, pear-shaped embryos did not differentiate primary organs accordingly (Fig. 5-B), moreover a number of embryos degenerated (Fig. 5-A, C, and D). Degradation of some embryo sacs were observed in abnormal ovaries (Fig. 5-E) with female-germ unit degenerating (Fig. 5-F).
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
Fig. 2 Development of hybrid embryo and endosperm in Guangluai 4 2x/O. officinalis at 1 and 3 DAP. A-C, development of hybrid embryos and endosperms at 1 DAP. A, normal development of embryo and free endosperm nuclei, showing a small globular-shaped embryo (arrow) and several free endosperm nuclei (arrowhead). B, a small globular-shaped embryo (arrow) and degenerating free endosperm nuclei. C, a single-fertilized embryo sac showing a globular-shaped embryo with incompact structure (arrow) and polar nuclei (arrowhead). D-I, development of hybrid embryos and endosperms at 3 DAP. D, a normal pear-shaped embryo (arrow) and degenerated free endosperm nuclei. E, degenerating embryo and degenerated free endosperm nuclei. F, residue of degenerated embryo (arrow) and abnormal formation of endosperm cells. G, a normal pear-shaped embryo (arrow) and degenerated endosperms. H, a normal pear-shaped embryo (arrow) and polar nuclei (arrowhead) in a single-fertilized embryo sac. I, a normal pearshaped embryo (arrow) and polar nuclei (arrowhead) left, degenerated tissues in chalazal end in a single-fertilized embryo sac. Bars=80 μm. The same as below.
Development and characterization of interspecific hybrid plantlets By rescuing young embryos, plantlets of hybrid F1 were obtained in both Guangluai 4 2x/O. officinalis and Liaojing 944 2x/O. officinalis. The production rates of hybrid plantlets in the two combinations were 16.67 and 7.41%, respectively. As shown in Fig. 6, these interspecific hybrids had obvious heterosis in some traits, such as tillering ability, plant height, and number of
1493
Fig. 3 Development of hybrid embryo and endosperm in Guangluai 4 2x/O. officinalis at 7 DAP. A, a degenerating embryo (arrow) and some free endosperm nuclei without forming cellular endosperms. B, a degenerating embryo (arrow) and abnormal formation of cellular endosperms. C, a small pear-shaped embryo (arrow) and degenerated endosperm. D, a large pear-shaped embryo (arrow) and residue of degenerated endosperm nuclei in a distorted embryo sac. E, a large pear-shaped embryo (arrow) and abnormal cellular endosperm. F, an embryo differentiating organs (arrow) and abnormal cellular endosperms. G, a degenerating embryo (arrow) in a distorted embryo sac. H, a pear-shaped embryo (arrow) in a distorted single-fertilized embryo sac. I, a pear-shaped embryo (arrow) and unfertilized polar nuclei (arrowhead) in a singlefertilized embryo sac.
spikelets per panicle. Some traits of hybrids, such as long awns, exoteric large purple stigma, grain shattering, and dispersed panicles, resembled that of O. officinalis. Some other traits of hybrids, such as length and width of flag leaf, length and width of grain, were intermediated between maternal and paternal parents. Besides, hybrids were sensitive to photoperiod in Guangzhou, Guangdong Province, China (23°08´N) like O . officinalis. SSR assay with primer RM253 showed that the main bands of the hybrid plants exhibited heterozygous bands of biparents (Fig. 7). The hybrids’ root tip cells had 24 chromosomes (Fig. 8), which further illuminated that these hybrids were true diploid plants with A and C genomes.
Table 3 Results of hybrid embryo development at 1, 3, and 7 DAP1) Days after pollination (DAP) 1 3 7
GLA 2x/O. officinalis RFT (%) 9.09 9.09 0
RGE (%) 90.91 77.27 47.62
RPE (%) 0 13.60 47.60
GLA 4x/O. officinalis RED (%) 0 0 4.76
RFT (%) 76.47 5.56 0
RGE (%) 23.53 88.89 25.00
RPE (%) 0 5.56 75.00
RED (%) 0 0 0
RFT, rate of fertilization trace; RGE, rate of globular-shaped embryos; RPE, rate of pear-shaped embryos; RED, rate of differentiated embryos.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1494
FU Xue-lin et al.
Fig. 5 Development of hybrid embryo and endosperm in Guangluai 4 4x/O. officinalis at 7 DAP. A, a degenerating embryo (arrow) and abnormal cellular endosperms. B, a pear-shaped embryo (arrow) and abnormal cellular endosperms. C, a degenerating embryo (arrow) in a abnormal embryo sac. D, a small pear-shaped embryo (arrow) and residues of degenerated endosperm. E, a degenerated embryo sac in a abnormal ovary. F, an embryo sac with degenerated female-germ unit (arrow) in a abortive ovary.
Fig. 4 Development of hybrid embryo and endosperm in Guangluai 4 4x/O. officinalis at 1 and 3 DAP. A-D, development of hybrid embryo and endosperm at 1 DAP. A, egg cell fertilization trace (arrow) and several free endosperm nuclei. B, an multi-cell embryo (arrow) and several free endosperm nuclei (arrowhead). C, a small multi-cell embryo (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac. D, degeneration trace of synergid cells (long arrow) and unfertilized polar nuclei left (arrowhead) in a single-fertilized embryo sac. E-L, development of hybrid embryo and endosperm at 3 DAP. E, a globular-shaped embryo (arrow) and degenarating free endosperm nuclei. F, a globular-shaped embryo (arrow) and residues of degenerated endosperm nuclei. G, a degenerating embryo (arrow) and residues of degenerated endosperm nuclei. H, a degenerating embryo (arrow) and abnormal formation of endosperm cells. I, a globular-shaped embryo (arrow) and formation of cellular endosperms. J, the same embryo sac as in I showing cellular endosperm filled in the embryo sac cavity. K, a small globular-shaped embryo degenerating (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac. L, a pear-shaped embryo (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac.
Cytological evidences of development and sterility of hybrid embryo sacs By method of WE-CLSM, embryo sac development process and matured embryo sac structure in hybrid F1 of Guangluai 4 2x/O. officinalis were observed. All of 78 matured hybrid embryo sacs observed were completely sterile. There were multiple types of sterile embryo sacs, such as embryo sac degeneration (about 96.2%) (Fig. 9-G), small embryo sac cavity without female germ unit and antipodal cells (Fig. 9-H), no differentiation of
Fig. 6 Hybrid F 1 plants and their panicles in Guangluai 4 2x/ O. officinalis and Liaojing 944 2x/O. officinalis. A, hybrid F1 plant of Guangluai 4 2x/O. officinalis. B, hybrid F1 plant of Liaojing 944 2x/O. officinalis. C, panicles from left to right, Guangluai 4 2x, hybrid F1, and O. officinalis. D, panicles from left to right, Liaojing 944 2x, hybrid F1, and O. officinalis.
a normal embryo sac cavity (Fig. 9-I), abnormal ovule without embryo sac (Fig. 9-J), abnormal nucellus tissue without embryo sac in ovary (Fig. 9-K), differentiation of a normal embryo sac cavity without normal female germ unit and antipodal cells (Fig. 9-L) and so on. However, hybrid embryo sac was the polygonum type as Guangluai 4. During its development process,
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
1495
Fig. 7 Amplified products of hybrids and their parents at the locus of RM253. M, 50 bp DNA ladder (MBI company GeneRulerTM); lane 1, Guangluai 4 2x; lane 2, hybrid F 1 of Guangluai 4 2x/ O. officinalis; lanes 3 and 6, O. officinalis; lane 4, Liaojing 944 2x; lane 5, hybrid F1 of Liaojing 944 2x/O. officinalis.
Fig. 9 Embryo sac development and matured embryo sac structure of hybrid Guangluai 4 2x/O. officinalis. A-F, embryo sac formation stage. A, an archesporial cell. B, a normal megasporocyte. C, an abnormal megasporocyte with the abnormal nucleus position. D, tetrad formation stage showing a tetrad. E, four sister-cells of a tetrad degenerated abnormally. F, a functional megaspore degenerated along with nucellus tissues degeneration. G-L, various types of abnormal embryo sacs at embryo sac matured stage. G, embryo sac degeneration trace. H, a small embryo sac cavity without female germ unit and antipodal cells. I, no differentiation of a normal embryo sac cavity. J, an abnormal ovule without embryo sac. K, abnormal nucellus tissues without embryo sac. L, differentiation of a normal embryo sac cavity but without normal female germ unit and antipodal cells.
Fig. 8 Chromosomes of root tip cells in hybrid F1 of Guangluai 4 2x/O. officinalis (2n=2x=24). Bar=5 μm.
archesporial cell was formed at archesporial cell formation stage (Fig. 9-A), normal or abnormal megasporocytes formed at megasporocyte formation stage (Fig. 9-B and C), tetrads formed at megasporocyte meiosis stage (Fig. 9-D) but soon degenerated abnormally (Fig. 9-E), and few normal functional megaspore formed, degeneration of functional megaspore and nucellus tissues occurred in a large scale at functional megaspore formation stage (Fig. 9-F). Therefore, it might be concluded that the next stages such as mononucleate embryo sacs formation stage could not continue normally.
Cytological evidences of development and sterility of hybrid pollen grains Pollen grains in hybrid of Guangluai 4 2x/O. officinalis at pollen matured stage were all typical abortive ones (Fig. 10-A) and the anthers were slim with a few of pollen grains, which meant that the hybrid F1 was highly male sterile. Observations on hybrid PMC meiosis I and II showed that PMC meiosis was abnormal in all the processes (Table 4). The chromosome configuration at diakinesis and metaphase I was 2n=24=17.5 I× (13-22)+2.6 II×(1-4)+0.4 III×(0-1). In details, univalents were the main type of chromosome configuration at diakinesis and metaphase I, while only 1-4 bivalents and 1 trivalent were observed in several PMCs (Fig. 10-B, C, and D). Similarly, univalents distributed
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1496
disorderly at metaphase I, some of which were stickiness (Fig. 10-E) or asynchronous in movement (Fig. 10-F) such as some straggling chromosomes (Fig. 10G). Therefore, migration of univalents at anaphase I were disorderly (Fig. 10-H). At telophase I, abnormal chromatin masses were formed (Fig. 10-J) and abnormal cytokinesis occurred (Fig. 10-K), so abnormal dyad such as dyad with micronuclei and abnormal nuclei were formed (Fig. 10-L). At metaphase II, chromosome straggling was still observed (Fig. 11-A) and spindle misorientation occurred popularly (Fig. 11-B and C) inducing chromosomes separated irregularly (Fig. 11-D), such as all chromosomes were in one of the sister cells in some dyads (Fig. 11-E) at anaphase II. Then at telophase II more abnormalities were observed, i.e., micronucleus (Fig. 11-G), disjunction of chromosomes only in one sister cell of a dyad (Fig. 11-G), and asynchronous disjunction of chromosomes and absence of cytokinesis in one sister cell (Fig. 11-H). In this case, a number of abnormal tetrads were formed, i.e., tetrads with multi-nuclei (Fig. 11-I), tetrads with two microcytes (Fig. 11-J), tetrad with abnormal nuclei and sister cells in disorder arrangement (Fig. 11-L), triad with a binucleate microspore (Fig. 11-K) and triad with abnormal nuclei (Fig. 11-M). Therefore, many microspores with abnormal nucleus were formed (Fig. 11-N) and finally became abortive pollen grains (Fig. 11-O). Furthermore, by semi-thin section observations it showed that some PMCs degenerated early at PMC formation stage (Fig. 12-A) and prophase I (Fig. 12B). Uninucleate microspores degenerated soon at early uninucleate microspore stage (Fig. 12-C) or at middle uninucleate microspore stage (Fig. 12-D), and abnormal huge microspores were sometimes formed (Fig. 12-E). At pollen maturity stage, only remnants of abortive pollen grains remained in anther chambers (Fig. 12-F).
DISCUSSION Cross barriers and hybrid plantlets obtaining in the interspecific hybridization In this study, we performed cytological observations on pre- and post-fertilization barriers between O. sa-
FU Xue-lin et al.
Fig. 10 Meiosis I of the hybrid F 1 PMCs in Guangluai 4 2x/ O. officinalis (×1 000). A, pollen matured stage, showing typical abortive pollen grains by I2-KI staining (×100). B, a PMC at diakinesis stage showing 22 I+1 II (arrow). C, a PMC at prometaphase I showing 13 I+4 II+1 III (arrow). D, a PMC at metaphase I showing 18 I+3 II (arrow). E, a PMC at metaphase I showing chromosome stickiness. F, a PMC at metaphase I showing asynchronous chromosomes. G, a PMC at metaphase I showing chromosome straggling (arrow). H, a PMC at anaphase I showing migration of univalents in disorder. I, A PMC at telophase I showing micronuclei (arrow). J, a PMC at telophase I showing abnormal chromatin masses. K, a PMC at telophase I showing abnormal cytokinesis. L, a dyad with micronuclei and abnormal nucleoli.
tiva and O. officinalis. To our knowledge, this is the first report to describe systemically the characteristics of pollen germination, fertilization, hybrid embryo and endosperm development in the wide cross between O. sativa and O. officinalis. Results indicated that incrossability and hybrid sterilities were the main reproductive isolations in the interspecific hybridizations. Abnormal development of hybrid embryo and endosperm in maternal plants instead of abnormal pollen germination and fertilization is the main cause of incrossability inducing non-seed set in the interspecific hybridizations between O. sativa with O. officinalis. Additionally, incrossability barrier was more severe when using the tetraploid as maternal parent. In our previous studies (Fu et al. 2007, 2009a), we found that incrossabilities between O. sativa and O. alta (CCDD genome) as well as O. sativa, but development of hybrid embryos delayed and ceased in the globular-shaped stage at 6-12 DAP, and the en© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
1497
Table 4 Abnormal cells and abnormalities of hybrid PMC meioses Phases
No. of observed cells
No. of abnormal cells (%)
Metaphase I
36
36 (100)
Anaphsae I
34
34 (100)
Telophase I
40
40 (100)
Dyad
52
52 (100)
Metaphase II
53
53 (100)
Anaphsae II
39
Telophase II
38
Tetrad
56
Abnormalities Chromosomes distributed disorderly around the equatorial plate Straggling chromosomes Chromosome stickines Laggards Precocious migration Chromosomebridges and breakage fragments Chromosomes imbalanced in two poles Chromatin masses in two poles Nucleolus with multi-nucleoli and in different sizes Cytoplasm divided unequally Abnormal spindle orientation Sister cells of a dyad in different sizes Dyad with multi-micronuclei Triads Chromosomes distributed disorderly around theequatorial plate Straggling chromosomes Abnormal spindle orientation
27 (75.0) 7 (19.4) 2 (5.6) 17 (50.0) 7 (20.6) 7 (20.6) 3 (8.8) 24 (60.0) 8 (20.0) 6 (15.0) 2 (5.0) 28 (53.8) 22 (42.3) 2 (3.8) 31 (58.5) 10 (18.9) 8 (15.1)
39 (100)
Chromosomes imbalanced in sister cells of a dyad Chromosome migration asynchronously
4 (7.5) 31 (79.5)
38 (100)
Abnormal spindle orientation Meiosis asynchrony in sister cells
8 (20.5) 20 (52.6)
Chromatin masses in poles, micronuclei formed by laggards Unequal chromosomes distributed in sister cells
13 (34.2) 3 (7.9)
Abnormal spindle orientation Micronuclei
2 (5.3) 25 (44.6)
Triads Meiosis asynchrony in sister cells
14 (25.0) 9 (16.1)
56 (100)
Pentads Sister cells of a tetrad in disorder arrangement Early Uninucleate Microspore
40
No. of abnormal cells (%)
40 (100)
Micronuclei Double nucleoli Three nucleoli
dosperm started degenerating at 4-5 DAP (Suputtitada et al. 2000). It was thought that embryo disaggregation was induced by earlier endosperm degeneration. Based on all above studies, it shows that a few of hybrid embryos could develop into pear-shaped stage, and furthermore, several of them could differentiate primary organs. It is possible to produce hybrid plantlets by embryo rescuing if only an embryo matured enough to continue developing in vitro. Therefore, it could be concluded that hybrid invalidity between O. sativa and non-AA genome wild species was popular, it is an important way to rescue embryo timely before its abortion to produce hybrid plantlets.
Interspecific hybrid sterility barriers and the overcoming methods between O. sativa and nonAA genome wild species Interspecific hybrid sterility limited further backcross
5 (8.9) 3 (5.4) 33 (82.5) 4 (10.0) 3 (7.50)
between hybrid and its parents which became the bottleneck for transferring elite genes from wild species to cultivars. In the present study, we found embryo sac and pollen were all highly sterile in hybrid F1 between O. sativa and O. officinalis. The sterile embryo sac was mainly caused by abnormal functional megaspore formation and degeneration. As far as we know, it is WE-CLSM method that easy to provide direct evidences of embryo sac sterility of the interspecific hybrids between O. sativa and non-AA genome wild species. Observation of PMC meioses indicated that univalents, irregular chromosome segregation, abnormal cytokinesis, and so on were the main cause for hybrid pollen sterility. Besides, PMC degeneration before meiosis or during the meiosis process also reduced the formation of pollen grains. In previous studies, only chromosome parings were observed in the meiosis I of the interspecific hybrid F1 between O. sativa and O. officinalis (Jena and Khush 1989; Yan et al.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1498
FU Xue-lin et al.
Fig. 12 Abnormalities in microspore development process of hybrid F 1 in Guangluai 4 2x/O. officinalis by semi-thin section process (×400). A, PMC formation stage, showing some PMC degenerating. B, prophase I at PMC meiosis stage, showing one of PMCs degenerated. C, early mono-nucleate microspore stage, showing degenerating microspores in an anther chamber. D, middle uninucleate microspore stage, showing abortive microspores. E, middle mono-nucleate microspore stage, showing an abnormal huge microspore. F, pollen matured stage, showing remnants of abortive pollen grains.
Fig. 11 Meiosis II and the abortive microspores of the hybrid F1 in Guangluai 4 2x/O. officinalis (×1 000). A, metaphase II showing chromosome straggling in one sister-cell (arrow). B, metaphase II showing spindle misorientation. C, metaphase II showing spindle misorientation and a small microcyte (arrow). D, anaphase II showing disorder disjunction of chromosomes. E, anaphase II showing chromosomes gathered in one of the sister cells. F, telophase II showing a micronucleus (arrow). G, telophase II showing disjunction of chromosomes only in one sister cell of a dyad. H, telophase II showing asynchronous disjunction of chromosomes and absence of cytokinesis in one sister cell. I, a tetrad with multinuclei. J, a tetrad along with two microcytes (arrow). K, a triad with a binucleate microspore (arrow). L, a tetrad with abnormal nuclei and sister cells in disorder arrangement. M, a triad with abnormal nuclei. N, a released microspore in early uninucleate microspore stage with abnormal nucleus. O, abortive pollen grains (×400).
1996) or other non-AA genome wild species, such as O. punctata (Yasui and Iwata 1991), O. minuta (AmanteBordeos et al. 1992; Brar et al. 1996; Mariam et al. 1996), O. latifolia (Multani et al. 2003; Yi et al. 2008), O. australiensis (Multani et al. 1994), O. brachyantha, and O. granulata (Brar et al. 1996). But, there is none further reports on hybrids’ microspore development. The present study was more important for providing
the more evidences. Above hybrids were male sterile which were thought to be caused by univalents. Therefore, irregular behaves of chromosomes from different genomes of parents in hybrid PMCs induced the meiosis process disorderly and produced of abortive microspores and sterile pollens. In order to produce the interspecific BC1F1 population, intensive backcrossing in which interspecific F 1 hybrids were used as maternal parent and O. sativa as paternal parent, was usually applied to overcome the F1 sterility barriers although it was too difficult. In our opinions, it is necessary to explore new ways to overcome interspecific hybrid sterility and utilize the elite genes within wild rice in non-AA genome. The method of doubling chromosomes of O. sativa or hybrid (Multani et al. 1994; Mariam et al. 1996) may increase the fertility of hybrid F1 and obtain backcross progenies.
Acknowledgements The authors gratefully acknowledge the guidance of Prof. Xu Xuebin, Agriculture College of South China Agricultural University, and the assistance of Ms. Yu Shuhong in the experimental work. Confocal laser scanning microscope was provided by the Testing Center of South China Agricultural University. This study was financially supported by the Key Project of the
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
Natural Science Foundation of Guangdong Province, China (021037), the Natural Science Foundation of Guangdong Province, China (9151064201000067), the Special Fund for Agro-Scientific Research in the Public Interest, China (201003021), and the Joint Fund of the National Natural Science Foundation of ChinaGuangdong Province (U0631003).
References Amante-Bordeos A, Sitch L A, Nelson R, Oliva N P, Aswidinnoor H. 1992. Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice, Oryza sativa. Theoretical and Applied Genetics, 84, 345-354. Barclay A. 2004. Cultivated rice receives help from wild cousins. Approach Technology, 31, 10-12. Bellon M R, Brar D S, Lu B R, Pham J L.1999. Rice genetic resources. In: Dowling N G, Greenfield S M, Fischer K S, eds., Sustainability of Rice in the Global Food System. International Rice Research Institute, Manila. pp. 251-283. Brar D S, Dalmacio R, Elloran R, Aggarwal R, Angeles R, Khush G S. 1996. Gene transfer and molecular characterization of introgression from wild Oryza species into rice. In: Khush G S, ed., Rice Genetics III. International Rice Research Institute, Manila. pp. 477-485. Feng J H, Lu Y G, Liu X D, Xu X B. 2001. Pollen development and its stages in rice (Oryza sativa L). Chinese Journal of Rice Sciences, 15, 21-28. (in Chinese) Fu X L, Lu Y G, Liu X D, Li J Q. 2009a. Crossability barriers in the interspecific hybridization between Oryza sativa and O. meyeriana. Journal of Integrative Plant Biology, 51, 21-28. Fu X L, Lu Y G, Liu X D, Li J Q, Feng J H. 2007. Cytological mechanisms of interspecific incrossability and hybrid sterility between Oryza sativa L. and O. alta Swallen. Chinese Science Bulletin, 52, 755-765. Fu X L, Lu Y G, Liu X D, Li J Q, Zhao X J. 2009b. Comparative embryological studies on infertility of interspecific hybridizations between Oryza sativa with different ploidy levels and O. officinalis. Rice Science, 16, 58-64. He G C. 2002. Mining and transfer of elite genes of wild rice. In: Luo L J, Ying C S, Tang S X, eds., Rice Germplasm. Hubei Science and Technology Press, Wuhan. p. 279. (in Chinese) Hu S Y. 1994. Method of preparation of slides used to examine the pollen germination on the stigma and pollen tube growth in the style. Chinese Bulletin of Botany, 11, 58-60. (in Chinese) Huang Z, He G, Shu L, Li X H, Zhang Q F. 2001. Identification and mapping of two brown planthopper resistance genes in rice. Theoretical and Applied Genetics, 102, 929-934. Jena K K, Khush G S. 1989. Monosomic alien addition lines of rice: Production, morphology, cytology and breeding
1499
behavior. Genome, 32, 449-455. Jena K K, Khush G S. 1990. Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice, Oryza sativa L. Theoretical and Applied Genetics, 80, 737-745. Jena K K, Khush G S, Kochert G. 1992. RFLP analysis of rice (Oryza sativa) introgression lines. Theoretical and Applied Genetics, 84, 608-616. Mariam A L, Zakri A H, Mahani M C. 1996. Interspecific hybridization of cultivated rice, Oryza sativa L., with the wild rice, O. minuta Presl. Theoretical and Applied Genetics, 93, 664-671. Multani D S, Jena K K, Brar D S, Reyes B G, Angeles E R, Khush G S. 1994. Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin to cultivated rice of O. sativa L. Theoretical and Applied Genetics, 88, 102-109. Multani D S, Khush G S, Reyes B G, Brar D S. 2003. Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv to rice, Oryza sativa L. Theoretical and Applied Genetics, 107, 395-405. Panaud O, Chen X, McCouch S R. 1996. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L). Molecular and General Genetics, 252, 597-607. Qin X Y, Zhu R C, Tang J H, Li W K, Li D Y, Wei S M, Huang F K. 2007. Genetic analysis and utilization of brown planthopper (BPH)-resistant genes in Oryza officinalis Wall. Journal of Plant Genetic Resources, 8, 41-45. (in Chinese) Shin Y B, Katayama T. 1979. Cytological studies on the genus Oryza: XI. Alien addition lines of O. sativa with single chromosomes of O. officinalis. Japanese Journal of Genetics, 54, 1-10. Suputtitada S, Adachi T, Pongtongkam P, Peyachoknagul S, Apisitwanich S, Thongpradistha J. 2000. Breeding barriers in the interspecific cross of Oryza sativa L. and Oryza minuta Presl. Breeding Science, 50, 29-35. Tan G X, Jin H J, Li G, He R F, Zhu L L, He G C. 2005. Production and characterization of a complete set of individual chromosome additions from Oryza officinalis to Oryza sativa using RFLP and GISH analyses. Theoretical and Applied Genetics, 111, 1585-1595. Tan G X, Ren X, Weng Q M, Shi Z Y, Zhu L L, He G C. 2004a. Mapping of a new resistance gene to bacterial blight in rice line introgressed from Oryza officinalis. Acta Genetica Sinica, 31, 724-729. (in Chinese) Tan G X, Weng Q M, Ren X, Huang Z, Zhu L L, He G C. 2004b. Two whitebacked planthopper resistance genes in rice share the same loci with those for brown planthopper resistance. Heredity, 92, 212-217. Yan H H, Hu H Y, Fu Q, Yu H Y, Tang S X, Xiong Z M, Min S
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1500
FU Xue-lin et al.
K. 1996. Morphological and cytogenetical studies of the hybrids between Oryza sativa and Oryza officinalis. Chinese
between Oryza sativa and O. latifolia by in situ hybridization. Chinese Science Bulletin, 53, 2973-2980.
Journal of Rice Sciences, 10, 138-142. (in Chinese) Yasui H, Iwata N. 1991. Production of monosomic alien addition
Yu W J, Luo K, Guo X X. 1993. Obtaining the hybrids of Oryza sativa × O. officinalis without embryo culture by using male-
lines of Oryza sativa having a single O. punctata chromosome. In: Khush G S, ed., Rice Genetics II. International Rice
sterile lines. Acta Genetica Sinica, 20, 348-353. (in Chinese) Zhong D B, Luo L J, Guo L B, Ying C S. 1997. Studies on
Research Institute, Manila. pp. 147-155. Yi C D, Tang S Z, Zhou Y, Liang G H, Gong Z Y, Gu M H. 2008.
resistance to brown planthopper (BPH) of hybrid between Oryza sativa and Oryza officinalis. Southwest China Journal
Development and characterization of interspecific hybrids
of Agricultural Sciences, 10, 5-9. (in Chinese) (Managing editor WANG Ning)
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
October 2011
2011, 10(10): 1489-1500
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis FU Xue-lin, LU Yong-gen, LIU Xiang-dong, LI Jin-quan and ZHAO Xing-juan Key Laboratory of Plant Molecular Breeding, Guangdong Province/College of Agriculture, South China Agricultural University, Guangzhou 510642, P.R.China
Abstract Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid (AA, 2n=2x=24) and autotetraploid (AAAA, 2n=4x=48) cultivated rice were crossed as maternal parents with O. officinalis (CC, 2n=2x=24), none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations (DAP). By rescuing hybrid embryos, in vitro F 1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globular- or pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes. Key words: breeding barriers, interspecific hybridization, crossability, hybrid sterility, Oryza sativa, Oryza officinalis
INTRODUCTION Oryza officinalis Wall ex Watt (CC, 2n=2x=24) is one of the important wild species in Oryza genus and one of the three wild species indigenous to China. It has a number of beneficial genes for rice breeding such as resistance genes for brown planthopper (BPH), whitebacked planthopper (WBPH), and bacterial blight (BB). It also tolerates to drought, cold, and other abiotic stresses (Bellon et al. 1999; He 2002; Barclay 2004; Qin et al. 2007). Through interspecific hybridization and backcrossing between O. officinalis and Oryza Received 21 August, 2010
sativa, some of the resistance genes have been transferred into cultivars and mapped on chromosomes. Thus some new germplasms have been innovated including monosomic alien addition lines (MAALs) (Shin and Katayama 1979; Jena and Khush 1989, 1990; Tan et al. 2004a, b, 2005), introgression lines (Jena et al. 1992), and advanced backcross generations (Yan et al. 1996; Zhong et al. 1997; Huang et al. 2001). Nevertheless, previous studies showed that the crossability between O. sativa and O. officinalis was less than 3.5% (Jena and Khush 1989, 1990; Yan et al. 1996). The hybrids were completely male sterile (Jena and Khush 1989; Yu et al. 1993). Because of severe
Accepted 14 December, 2010
FU Xue-lin, Associate Professor, Ph D, E-mail: [email protected]; Correspondence LU Yong-gen, Professor, E-mail: [email protected]
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60143-0
1490
reproductive isolation, obtained hybrids between O. sativa and O. officinalis were fewer, and it is even more difficult to produce BC1F1 plants. However, there was little information of the mechanisms of the breeding barriers in O. sativa and O. officinalis. Especially, there was still none direct evidences on the precise mechanisms for the low crossability and hybrid female sterility even though that is very important for probing into the method to overcome the barriers effectively. So, it is indispensable to detect the evidences and clarify the mechanisms of breeding barriers between O. sativa and O. officinalis in order to effectively mine and utilize the beneficial genes of O. officinalis in a large scale in rice breeding. By using whole-mount eosin B-staining confocal laser scanning microscopy and other methods, this study was undertaken to observe the pollen germination, hybrid embryo, and endosperm development in the crosses of O. sativa and O. officinalis, young embryo culturing in vitro to produce hybrid plantlets, development of hybrid embryo sacs and pollen grains and the sterilities. The objective was to clarify, at the cytological level, the mechanisms of interspecific hybridization barriers and hybrid sterilities between O. sativa and O. officinalis.
FU Xue-lin et al.
sterilized with 75% ethanol and 0.1% HgCl2 solution, then young embryos were inoculated on 1/4 MS medium supplemented with 30 g L-1 sugar and 7 g L-1 agar in a laminar flow cabinet. When the plantlets attained 3-4 leaf stages, they were hardened and transplanted. After 40 d of embryo culturing in vitro, the rates of plantlet production and the crossability were analyzed. Plantlet production rate (%)=Number of plantlets/Number of young embryos cultured×100. Crossability (%)= Number of plantlets/Number of spikelets pollinated×100. All the hybrid plantlets were identified by main traits and SSR markers. SSR assay was carried out according to the method described by Panaud et al. (1996).
Pollen germination, fertilization, hybrid embryo, and endosperm development in the interspecific hybridization
In this experiment, indica cultivar Guangluai 4 (2n=2x=24), japonica cultivar Liaojing 944 (2n=2x=24) and their corresponding autotetraploids (2n=4x=48) were used as maternal parents. One accession of O. officinalis indigenous to Guangxi Zhuang Autonomous Region in China was used as paternal parent. The cultivars were planted at the experimental farm of South China Agricultural University (SCAU). O. officinalis and the interspecific hybrids between O. sativa and O. officinalis were planted at the Wild Rice Core Collection Nursery of SCAU.
Pollen germination was observed by aniline blue staining method described by Hu (1994). When Guangluai 4 (2x and 4x) was crossed with O. officinalis, more than 15 spikelets at 30 min after pollination for each cross combination were fixed in Carnoy’s solution for more than 12 h, then the spikelets were transferred to graded ethanol series of 90, 80, 70, 50, 30, and 10% and distilled water to hydrate, 20-30 min for each grade. After that, the lemma of spikelet was cut out and the pistil with palea was cleared in 5 mol L-1 NaOH solution for about 2-3 h, and then rinsed with distilled water for three times. Before observation, the pistil was dissected carefully on a slide and blocked out the background with 0.01% toluidine blue solution and was rinsed with distilled water for three times, then the pistil was stained with 0.01% aniline blue solution for several minutes and pollen germination was observed under a Leica DMRXA (Heidelberg, Germany) fluorescenct microscope. WE-CLSM (whole-mount eosin B staining confocal laser scanning microscopy, Leica, Germany) was used to observe fertilization and hybrid embryo and endosperm development according to the method described by Fu et al. (2009a).
Interspecific hybrid plantlets production and crossability analyses
Cytological observations on interspecific hybrid sterility
At 13-16 DAP, swollen hybrid ovaries were surface
Hybrid spikelets of Guangluai 4 2x/O. officinalis at all
MATERIALS AND METHODS Cross combinations
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
development stages were collected and fixed in Carnoy’s solution for more than 2 d. Embryo sacs and anthers were dissected. Structures and development of embryo sacs were observed using WE-CLSM. Matured pollen grains were stained with 1% I2-KI and observed under light microscope. Pollen mother cells at meioses of interspecific hybrid were observed with 1% acetic acid carmine squash technique. Microsporogenesis was observed by semi-thin section light microscopy according to the method described by Feng et al. (2001). Slides were observed and photographs were obtained under a Leica DMRXA microscope. All the images were processed with Microsoft Adobe Photoshop 7.0 software.
RESULTS Seed set of the interspecific hybridizations and the crossabilities In the cross combinations, rates of swollen hybrid ovaries on the maternal plants at 13-16 DAP were low, but the rates of swollen hybrid ovaries in Liaojing 944 2x/ O. officinalis and Liaojing 944 4x/O. officinalis were higher than those of Guangluai 4 2x/O. officinalis and Guangluai 4 4x/O. officinalis (Table 1). Hybrid ovaries shrunk gradually and aborted finally (Fig. 1-A-D). Nevertheless, by rescuing hybrid embryos in vitro hybrid plantlets were produced with the rates of 16.67 and 7.41% in Guangluai 4 2x/O. officinalis and Liaojing 944 2x/O. officinalis, respectively. Crossabilities of the two combinations were only 0.42 and 0.46%. None hybrid plantlets were produced in Guangluai 4 4x/ O. officinalis and Liaojing 944 4x/O. officinalis. It meant that the crossability barrier was severe when both diploid and autotetraploid cultivated rice were crossed with O. officinalis which was more severe when using the autotetraploids as maternal parents.
1491
Pollen germination, fertilization and development of hybrid embryo and endosperm in the interspecific hybridizations In order to detect the pre- and post-fertilization barriers between O. sativa and O. officinalis, the processes of pollen germination, double-fertilization, and hybrid embryo and endosperm development in the two cross combinations, Guangluai 4 2x/O. officinalis and Guangluai 4 4x/O. officinalis, were observed. Almost all of the normal pollen grains of O. officinalis germinated on the stigma of Guangluai 4 2x and Guangluai 4 4x and pollen tubes elongated into the styles of them (Fig. 1-E and F). However, rates of double-fertilization and egg cell single-fertilization in Guangluai 4 2x/ O. officinalis were 72.50 and 17.50%, and those in Guangluai 4 4x/O. officinalis were only 27.12 and 33.90%, respectively. Furthermore, as much as 68.19% of non-fertilized embryo sacs were structurally abnormal in Guangluai 4 4x/O. officinalis (Table 2). According to above results and the previous results (Fu et al. 2009b), it can be concluded that fertilization rates at diploid-diploid level (2x×2x) were higher than that at tetraploid-diploid level (4x×2x) in the crosses between O. sativa and O. officinalis, especially for the doublefertilization rate. At 1, 3, and 7 d after anthesis, embryo and endosperm development of Guangluai 4 2x was in a routine way (Fu et al. 2009a). Comparatively, development of hybrid embryo and endosperm in Guangluai 4 2x/ O. officinalis and Guangluai 4 4x/O. officinalis were much more abnormal. In Guangluai 4 2x/O. officinalis, both fertilization traces (with a rate of 9.09%) and globular-shaped embryos (with a rate of 90.91%) were observed at 1 DAP. In double-fertilized embryo sacs, some of the free endosperm nuclei developed normally (Fig. 2-A) while others degenerated early (Fig. 2-B). Whereas, in singlefertilized embryo sacs, some globular-shaped embryos
Table 1 Results of hybridizations and hybrid plantlets production by embryo rescuing Cross combinations GLA 2x/O. officinalis GLA 4x/O. officinalis LJ 2x/O. officinalis LJ 4x/O. officinalis
Spikelets pollinated
No. of swollen ovaries
3 577 2 067 433 937
120 31 47 62
Rate of swollen ovaries (%) 3.35 1.50 10.85 6.62
No. of young embryos cultured
No. of plantlets produced
90 11 27 12
15 0 2 0
Rate of plantlets production (%) 16.67 0 7.41 0
Crossability (%) 0.42 0.46 -
GLA, Guangluai 4; LJ, Liaojing 944. The same as below.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1492
FU Xue-lin et al.
Fig. 1 Hybrid ovaries and pollen germination of O. officinalis from the hybridized spikelets in maternal plants. A, hybrid ovaries of Guangluai 4 2x/O. officinalis at 13-16 DAP. B, hybrid ovaries of Guangluai 4 4x/O. officinalis at 8-10 DAP. C, hybrid ovaries of Liaojing 944 2x/O. officinalis at 14 DAP, showing the basal parts with embryos cut away for embryo rescue. D, hybrid ovaries of Liaojing 944 4x/O. officinalis at 8-14 DAP. E, pollen germination and pollen tube growth of O. officinalis on the stigma of Guangluai 4 2x. F, pollen germination and pollen tube growth of O. officinalis on the stigma of Guangluai 4 4x. Bar=1 cm in A-D, bar=10 μm in E and F.
with incompact structures degenerated and unfertilized polar nuclei left the egg apparatus (Fig. 2-C). At 3 DAP, 13.60% embryos developed into pear-shaped ones, while 77.27% embryos were still in globular-shaped stage, fertilization traces were observed in 9.09% of the ovaries in hybridized spikelets. Normal pear-shaped embryos were formed both in double-fertilized embryo sacs (Fig. 2-D and G) and in single-fertilized ones (Fig. 2-H and I), however, degeneration of embryo (Fig. 2-E and F) and free endosperm nuclei (Fig. 2-D, E, and G) were in a large scale. Till 7 DAP, only 9.5% embryos began to differentiate organs (Fig. 3-F), 47.6% embryos were still in normal pear-shaped state (Fig. 3-D, E, H, and I), 38.20% of the globular-shaped embryos were degenerating (Fig. 3-A and B) as well as 4.80% pearshaped embryos were degenerated (Fig. 3-C and G). Similarly, some free endosperm nuclei degenerated before forming endosperm cells (Fig. 3-A and D), and some endosperm cells formed abnormally (Fig. 3-B, E, and F) or degenerated ahead of time (Fig. 3-C). Furthermore, some embryo sacs distorted (Fig. 3-D, G, and H) along with the degeneration of embryo and endosperm.
Table 2 Fertilization results of the cross combinations at 1 DAP Cross combinations GLA 2x/O. officinalis GLA 4x/O. officinalis
Number of Embryo sacs observed
Rate of single egg fertilization (%)
Rate of double fertilization (%)
Total fertilization rate (%)
40 59
17.50 33.90
72.50 27.12
90.00 61.02
As shown in Table 3 and Fig. 4, fertilization traces (Fig. 4-A) (with a rate of 76.47%) and globular-shaped embryos (with a rate of 23.53%) were observed in Guangluai 4 4x/O. officinalis, showing the rate of fertilization trace was much higher than that in Guangluai 4 2x/O. officinalis at 1 DAP. The rate of pear-shaped embryos (5.56%) at 3 DAP was also lower than that in Guangluai 4 2x/O. officinalis (13.60%), and 88.89% embryos ceased at globular-shaped stage. None embryo was observed to differentiate organs at 7 DAP, among which 25% embryos were still globular-shaped and 75% were pear-shaped. In Guangluai 4 4x/ O. officinalis, not only hybrid embryos but also endosperms degenerated early. That is, at 1 DAP, small embryos developed slowly and were incompact multicells (Fig. 4-B and C), or without embryos but degeneration trace of synergid cells (Fig. 4-D). At the same
Rate of nonfertilized Abnormal embryo sacs/ embryo sacs (%) Non-fertilized embryo sacs (%) 10.00 39.98
0.00 68.19
time, only several free endosperm nuclei formed but trended to degenerate in double-fertilized embryo sacs (Fig. 4-A and B). At 3 DAP, hybrid embryos either stagnated in globular-shaped stage (Fig. 4-E, F, and I) or began to degenerate (Fig. 4-G, H, and K) even if a few of them developed into pear-shaped ones (Fig. 4L). Whatever, free endosperm nuclei degenerated in a large scale before forming endosperm cells in doublefertilized embryo sacs (Fig. 4-E, F, and G), or formed endosperm cells abnormally (Fig. 4-H), normal cellular endosperm seldom filled in the embryo sac cavity (Fig. 4-J). Till 7 DAP, pear-shaped embryos did not differentiate primary organs accordingly (Fig. 5-B), moreover a number of embryos degenerated (Fig. 5-A, C, and D). Degradation of some embryo sacs were observed in abnormal ovaries (Fig. 5-E) with female-germ unit degenerating (Fig. 5-F).
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
Fig. 2 Development of hybrid embryo and endosperm in Guangluai 4 2x/O. officinalis at 1 and 3 DAP. A-C, development of hybrid embryos and endosperms at 1 DAP. A, normal development of embryo and free endosperm nuclei, showing a small globular-shaped embryo (arrow) and several free endosperm nuclei (arrowhead). B, a small globular-shaped embryo (arrow) and degenerating free endosperm nuclei. C, a single-fertilized embryo sac showing a globular-shaped embryo with incompact structure (arrow) and polar nuclei (arrowhead). D-I, development of hybrid embryos and endosperms at 3 DAP. D, a normal pear-shaped embryo (arrow) and degenerated free endosperm nuclei. E, degenerating embryo and degenerated free endosperm nuclei. F, residue of degenerated embryo (arrow) and abnormal formation of endosperm cells. G, a normal pear-shaped embryo (arrow) and degenerated endosperms. H, a normal pear-shaped embryo (arrow) and polar nuclei (arrowhead) in a single-fertilized embryo sac. I, a normal pearshaped embryo (arrow) and polar nuclei (arrowhead) left, degenerated tissues in chalazal end in a single-fertilized embryo sac. Bars=80 μm. The same as below.
Development and characterization of interspecific hybrid plantlets By rescuing young embryos, plantlets of hybrid F1 were obtained in both Guangluai 4 2x/O. officinalis and Liaojing 944 2x/O. officinalis. The production rates of hybrid plantlets in the two combinations were 16.67 and 7.41%, respectively. As shown in Fig. 6, these interspecific hybrids had obvious heterosis in some traits, such as tillering ability, plant height, and number of
1493
Fig. 3 Development of hybrid embryo and endosperm in Guangluai 4 2x/O. officinalis at 7 DAP. A, a degenerating embryo (arrow) and some free endosperm nuclei without forming cellular endosperms. B, a degenerating embryo (arrow) and abnormal formation of cellular endosperms. C, a small pear-shaped embryo (arrow) and degenerated endosperm. D, a large pear-shaped embryo (arrow) and residue of degenerated endosperm nuclei in a distorted embryo sac. E, a large pear-shaped embryo (arrow) and abnormal cellular endosperm. F, an embryo differentiating organs (arrow) and abnormal cellular endosperms. G, a degenerating embryo (arrow) in a distorted embryo sac. H, a pear-shaped embryo (arrow) in a distorted single-fertilized embryo sac. I, a pear-shaped embryo (arrow) and unfertilized polar nuclei (arrowhead) in a singlefertilized embryo sac.
spikelets per panicle. Some traits of hybrids, such as long awns, exoteric large purple stigma, grain shattering, and dispersed panicles, resembled that of O. officinalis. Some other traits of hybrids, such as length and width of flag leaf, length and width of grain, were intermediated between maternal and paternal parents. Besides, hybrids were sensitive to photoperiod in Guangzhou, Guangdong Province, China (23°08´N) like O . officinalis. SSR assay with primer RM253 showed that the main bands of the hybrid plants exhibited heterozygous bands of biparents (Fig. 7). The hybrids’ root tip cells had 24 chromosomes (Fig. 8), which further illuminated that these hybrids were true diploid plants with A and C genomes.
Table 3 Results of hybrid embryo development at 1, 3, and 7 DAP1) Days after pollination (DAP) 1 3 7
GLA 2x/O. officinalis RFT (%) 9.09 9.09 0
RGE (%) 90.91 77.27 47.62
RPE (%) 0 13.60 47.60
GLA 4x/O. officinalis RED (%) 0 0 4.76
RFT (%) 76.47 5.56 0
RGE (%) 23.53 88.89 25.00
RPE (%) 0 5.56 75.00
RED (%) 0 0 0
RFT, rate of fertilization trace; RGE, rate of globular-shaped embryos; RPE, rate of pear-shaped embryos; RED, rate of differentiated embryos.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1494
FU Xue-lin et al.
Fig. 5 Development of hybrid embryo and endosperm in Guangluai 4 4x/O. officinalis at 7 DAP. A, a degenerating embryo (arrow) and abnormal cellular endosperms. B, a pear-shaped embryo (arrow) and abnormal cellular endosperms. C, a degenerating embryo (arrow) in a abnormal embryo sac. D, a small pear-shaped embryo (arrow) and residues of degenerated endosperm. E, a degenerated embryo sac in a abnormal ovary. F, an embryo sac with degenerated female-germ unit (arrow) in a abortive ovary.
Fig. 4 Development of hybrid embryo and endosperm in Guangluai 4 4x/O. officinalis at 1 and 3 DAP. A-D, development of hybrid embryo and endosperm at 1 DAP. A, egg cell fertilization trace (arrow) and several free endosperm nuclei. B, an multi-cell embryo (arrow) and several free endosperm nuclei (arrowhead). C, a small multi-cell embryo (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac. D, degeneration trace of synergid cells (long arrow) and unfertilized polar nuclei left (arrowhead) in a single-fertilized embryo sac. E-L, development of hybrid embryo and endosperm at 3 DAP. E, a globular-shaped embryo (arrow) and degenarating free endosperm nuclei. F, a globular-shaped embryo (arrow) and residues of degenerated endosperm nuclei. G, a degenerating embryo (arrow) and residues of degenerated endosperm nuclei. H, a degenerating embryo (arrow) and abnormal formation of endosperm cells. I, a globular-shaped embryo (arrow) and formation of cellular endosperms. J, the same embryo sac as in I showing cellular endosperm filled in the embryo sac cavity. K, a small globular-shaped embryo degenerating (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac. L, a pear-shaped embryo (arrow) and unfertilized polar nuclei (arrowhead) in a single-fertilized embryo sac.
Cytological evidences of development and sterility of hybrid embryo sacs By method of WE-CLSM, embryo sac development process and matured embryo sac structure in hybrid F1 of Guangluai 4 2x/O. officinalis were observed. All of 78 matured hybrid embryo sacs observed were completely sterile. There were multiple types of sterile embryo sacs, such as embryo sac degeneration (about 96.2%) (Fig. 9-G), small embryo sac cavity without female germ unit and antipodal cells (Fig. 9-H), no differentiation of
Fig. 6 Hybrid F 1 plants and their panicles in Guangluai 4 2x/ O. officinalis and Liaojing 944 2x/O. officinalis. A, hybrid F1 plant of Guangluai 4 2x/O. officinalis. B, hybrid F1 plant of Liaojing 944 2x/O. officinalis. C, panicles from left to right, Guangluai 4 2x, hybrid F1, and O. officinalis. D, panicles from left to right, Liaojing 944 2x, hybrid F1, and O. officinalis.
a normal embryo sac cavity (Fig. 9-I), abnormal ovule without embryo sac (Fig. 9-J), abnormal nucellus tissue without embryo sac in ovary (Fig. 9-K), differentiation of a normal embryo sac cavity without normal female germ unit and antipodal cells (Fig. 9-L) and so on. However, hybrid embryo sac was the polygonum type as Guangluai 4. During its development process,
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
1495
Fig. 7 Amplified products of hybrids and their parents at the locus of RM253. M, 50 bp DNA ladder (MBI company GeneRulerTM); lane 1, Guangluai 4 2x; lane 2, hybrid F 1 of Guangluai 4 2x/ O. officinalis; lanes 3 and 6, O. officinalis; lane 4, Liaojing 944 2x; lane 5, hybrid F1 of Liaojing 944 2x/O. officinalis.
Fig. 9 Embryo sac development and matured embryo sac structure of hybrid Guangluai 4 2x/O. officinalis. A-F, embryo sac formation stage. A, an archesporial cell. B, a normal megasporocyte. C, an abnormal megasporocyte with the abnormal nucleus position. D, tetrad formation stage showing a tetrad. E, four sister-cells of a tetrad degenerated abnormally. F, a functional megaspore degenerated along with nucellus tissues degeneration. G-L, various types of abnormal embryo sacs at embryo sac matured stage. G, embryo sac degeneration trace. H, a small embryo sac cavity without female germ unit and antipodal cells. I, no differentiation of a normal embryo sac cavity. J, an abnormal ovule without embryo sac. K, abnormal nucellus tissues without embryo sac. L, differentiation of a normal embryo sac cavity but without normal female germ unit and antipodal cells.
Fig. 8 Chromosomes of root tip cells in hybrid F1 of Guangluai 4 2x/O. officinalis (2n=2x=24). Bar=5 μm.
archesporial cell was formed at archesporial cell formation stage (Fig. 9-A), normal or abnormal megasporocytes formed at megasporocyte formation stage (Fig. 9-B and C), tetrads formed at megasporocyte meiosis stage (Fig. 9-D) but soon degenerated abnormally (Fig. 9-E), and few normal functional megaspore formed, degeneration of functional megaspore and nucellus tissues occurred in a large scale at functional megaspore formation stage (Fig. 9-F). Therefore, it might be concluded that the next stages such as mononucleate embryo sacs formation stage could not continue normally.
Cytological evidences of development and sterility of hybrid pollen grains Pollen grains in hybrid of Guangluai 4 2x/O. officinalis at pollen matured stage were all typical abortive ones (Fig. 10-A) and the anthers were slim with a few of pollen grains, which meant that the hybrid F1 was highly male sterile. Observations on hybrid PMC meiosis I and II showed that PMC meiosis was abnormal in all the processes (Table 4). The chromosome configuration at diakinesis and metaphase I was 2n=24=17.5 I× (13-22)+2.6 II×(1-4)+0.4 III×(0-1). In details, univalents were the main type of chromosome configuration at diakinesis and metaphase I, while only 1-4 bivalents and 1 trivalent were observed in several PMCs (Fig. 10-B, C, and D). Similarly, univalents distributed
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1496
disorderly at metaphase I, some of which were stickiness (Fig. 10-E) or asynchronous in movement (Fig. 10-F) such as some straggling chromosomes (Fig. 10G). Therefore, migration of univalents at anaphase I were disorderly (Fig. 10-H). At telophase I, abnormal chromatin masses were formed (Fig. 10-J) and abnormal cytokinesis occurred (Fig. 10-K), so abnormal dyad such as dyad with micronuclei and abnormal nuclei were formed (Fig. 10-L). At metaphase II, chromosome straggling was still observed (Fig. 11-A) and spindle misorientation occurred popularly (Fig. 11-B and C) inducing chromosomes separated irregularly (Fig. 11-D), such as all chromosomes were in one of the sister cells in some dyads (Fig. 11-E) at anaphase II. Then at telophase II more abnormalities were observed, i.e., micronucleus (Fig. 11-G), disjunction of chromosomes only in one sister cell of a dyad (Fig. 11-G), and asynchronous disjunction of chromosomes and absence of cytokinesis in one sister cell (Fig. 11-H). In this case, a number of abnormal tetrads were formed, i.e., tetrads with multi-nuclei (Fig. 11-I), tetrads with two microcytes (Fig. 11-J), tetrad with abnormal nuclei and sister cells in disorder arrangement (Fig. 11-L), triad with a binucleate microspore (Fig. 11-K) and triad with abnormal nuclei (Fig. 11-M). Therefore, many microspores with abnormal nucleus were formed (Fig. 11-N) and finally became abortive pollen grains (Fig. 11-O). Furthermore, by semi-thin section observations it showed that some PMCs degenerated early at PMC formation stage (Fig. 12-A) and prophase I (Fig. 12B). Uninucleate microspores degenerated soon at early uninucleate microspore stage (Fig. 12-C) or at middle uninucleate microspore stage (Fig. 12-D), and abnormal huge microspores were sometimes formed (Fig. 12-E). At pollen maturity stage, only remnants of abortive pollen grains remained in anther chambers (Fig. 12-F).
DISCUSSION Cross barriers and hybrid plantlets obtaining in the interspecific hybridization In this study, we performed cytological observations on pre- and post-fertilization barriers between O. sa-
FU Xue-lin et al.
Fig. 10 Meiosis I of the hybrid F 1 PMCs in Guangluai 4 2x/ O. officinalis (×1 000). A, pollen matured stage, showing typical abortive pollen grains by I2-KI staining (×100). B, a PMC at diakinesis stage showing 22 I+1 II (arrow). C, a PMC at prometaphase I showing 13 I+4 II+1 III (arrow). D, a PMC at metaphase I showing 18 I+3 II (arrow). E, a PMC at metaphase I showing chromosome stickiness. F, a PMC at metaphase I showing asynchronous chromosomes. G, a PMC at metaphase I showing chromosome straggling (arrow). H, a PMC at anaphase I showing migration of univalents in disorder. I, A PMC at telophase I showing micronuclei (arrow). J, a PMC at telophase I showing abnormal chromatin masses. K, a PMC at telophase I showing abnormal cytokinesis. L, a dyad with micronuclei and abnormal nucleoli.
tiva and O. officinalis. To our knowledge, this is the first report to describe systemically the characteristics of pollen germination, fertilization, hybrid embryo and endosperm development in the wide cross between O. sativa and O. officinalis. Results indicated that incrossability and hybrid sterilities were the main reproductive isolations in the interspecific hybridizations. Abnormal development of hybrid embryo and endosperm in maternal plants instead of abnormal pollen germination and fertilization is the main cause of incrossability inducing non-seed set in the interspecific hybridizations between O. sativa with O. officinalis. Additionally, incrossability barrier was more severe when using the tetraploid as maternal parent. In our previous studies (Fu et al. 2007, 2009a), we found that incrossabilities between O. sativa and O. alta (CCDD genome) as well as O. sativa, but development of hybrid embryos delayed and ceased in the globular-shaped stage at 6-12 DAP, and the en© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
1497
Table 4 Abnormal cells and abnormalities of hybrid PMC meioses Phases
No. of observed cells
No. of abnormal cells (%)
Metaphase I
36
36 (100)
Anaphsae I
34
34 (100)
Telophase I
40
40 (100)
Dyad
52
52 (100)
Metaphase II
53
53 (100)
Anaphsae II
39
Telophase II
38
Tetrad
56
Abnormalities Chromosomes distributed disorderly around the equatorial plate Straggling chromosomes Chromosome stickines Laggards Precocious migration Chromosomebridges and breakage fragments Chromosomes imbalanced in two poles Chromatin masses in two poles Nucleolus with multi-nucleoli and in different sizes Cytoplasm divided unequally Abnormal spindle orientation Sister cells of a dyad in different sizes Dyad with multi-micronuclei Triads Chromosomes distributed disorderly around theequatorial plate Straggling chromosomes Abnormal spindle orientation
27 (75.0) 7 (19.4) 2 (5.6) 17 (50.0) 7 (20.6) 7 (20.6) 3 (8.8) 24 (60.0) 8 (20.0) 6 (15.0) 2 (5.0) 28 (53.8) 22 (42.3) 2 (3.8) 31 (58.5) 10 (18.9) 8 (15.1)
39 (100)
Chromosomes imbalanced in sister cells of a dyad Chromosome migration asynchronously
4 (7.5) 31 (79.5)
38 (100)
Abnormal spindle orientation Meiosis asynchrony in sister cells
8 (20.5) 20 (52.6)
Chromatin masses in poles, micronuclei formed by laggards Unequal chromosomes distributed in sister cells
13 (34.2) 3 (7.9)
Abnormal spindle orientation Micronuclei
2 (5.3) 25 (44.6)
Triads Meiosis asynchrony in sister cells
14 (25.0) 9 (16.1)
56 (100)
Pentads Sister cells of a tetrad in disorder arrangement Early Uninucleate Microspore
40
No. of abnormal cells (%)
40 (100)
Micronuclei Double nucleoli Three nucleoli
dosperm started degenerating at 4-5 DAP (Suputtitada et al. 2000). It was thought that embryo disaggregation was induced by earlier endosperm degeneration. Based on all above studies, it shows that a few of hybrid embryos could develop into pear-shaped stage, and furthermore, several of them could differentiate primary organs. It is possible to produce hybrid plantlets by embryo rescuing if only an embryo matured enough to continue developing in vitro. Therefore, it could be concluded that hybrid invalidity between O. sativa and non-AA genome wild species was popular, it is an important way to rescue embryo timely before its abortion to produce hybrid plantlets.
Interspecific hybrid sterility barriers and the overcoming methods between O. sativa and nonAA genome wild species Interspecific hybrid sterility limited further backcross
5 (8.9) 3 (5.4) 33 (82.5) 4 (10.0) 3 (7.50)
between hybrid and its parents which became the bottleneck for transferring elite genes from wild species to cultivars. In the present study, we found embryo sac and pollen were all highly sterile in hybrid F1 between O. sativa and O. officinalis. The sterile embryo sac was mainly caused by abnormal functional megaspore formation and degeneration. As far as we know, it is WE-CLSM method that easy to provide direct evidences of embryo sac sterility of the interspecific hybrids between O. sativa and non-AA genome wild species. Observation of PMC meioses indicated that univalents, irregular chromosome segregation, abnormal cytokinesis, and so on were the main cause for hybrid pollen sterility. Besides, PMC degeneration before meiosis or during the meiosis process also reduced the formation of pollen grains. In previous studies, only chromosome parings were observed in the meiosis I of the interspecific hybrid F1 between O. sativa and O. officinalis (Jena and Khush 1989; Yan et al.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1498
FU Xue-lin et al.
Fig. 12 Abnormalities in microspore development process of hybrid F 1 in Guangluai 4 2x/O. officinalis by semi-thin section process (×400). A, PMC formation stage, showing some PMC degenerating. B, prophase I at PMC meiosis stage, showing one of PMCs degenerated. C, early mono-nucleate microspore stage, showing degenerating microspores in an anther chamber. D, middle uninucleate microspore stage, showing abortive microspores. E, middle mono-nucleate microspore stage, showing an abnormal huge microspore. F, pollen matured stage, showing remnants of abortive pollen grains.
Fig. 11 Meiosis II and the abortive microspores of the hybrid F1 in Guangluai 4 2x/O. officinalis (×1 000). A, metaphase II showing chromosome straggling in one sister-cell (arrow). B, metaphase II showing spindle misorientation. C, metaphase II showing spindle misorientation and a small microcyte (arrow). D, anaphase II showing disorder disjunction of chromosomes. E, anaphase II showing chromosomes gathered in one of the sister cells. F, telophase II showing a micronucleus (arrow). G, telophase II showing disjunction of chromosomes only in one sister cell of a dyad. H, telophase II showing asynchronous disjunction of chromosomes and absence of cytokinesis in one sister cell. I, a tetrad with multinuclei. J, a tetrad along with two microcytes (arrow). K, a triad with a binucleate microspore (arrow). L, a tetrad with abnormal nuclei and sister cells in disorder arrangement. M, a triad with abnormal nuclei. N, a released microspore in early uninucleate microspore stage with abnormal nucleus. O, abortive pollen grains (×400).
1996) or other non-AA genome wild species, such as O. punctata (Yasui and Iwata 1991), O. minuta (AmanteBordeos et al. 1992; Brar et al. 1996; Mariam et al. 1996), O. latifolia (Multani et al. 2003; Yi et al. 2008), O. australiensis (Multani et al. 1994), O. brachyantha, and O. granulata (Brar et al. 1996). But, there is none further reports on hybrids’ microspore development. The present study was more important for providing
the more evidences. Above hybrids were male sterile which were thought to be caused by univalents. Therefore, irregular behaves of chromosomes from different genomes of parents in hybrid PMCs induced the meiosis process disorderly and produced of abortive microspores and sterile pollens. In order to produce the interspecific BC1F1 population, intensive backcrossing in which interspecific F 1 hybrids were used as maternal parent and O. sativa as paternal parent, was usually applied to overcome the F1 sterility barriers although it was too difficult. In our opinions, it is necessary to explore new ways to overcome interspecific hybrid sterility and utilize the elite genes within wild rice in non-AA genome. The method of doubling chromosomes of O. sativa or hybrid (Multani et al. 1994; Mariam et al. 1996) may increase the fertility of hybrid F1 and obtain backcross progenies.
Acknowledgements The authors gratefully acknowledge the guidance of Prof. Xu Xuebin, Agriculture College of South China Agricultural University, and the assistance of Ms. Yu Shuhong in the experimental work. Confocal laser scanning microscope was provided by the Testing Center of South China Agricultural University. This study was financially supported by the Key Project of the
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis
Natural Science Foundation of Guangdong Province, China (021037), the Natural Science Foundation of Guangdong Province, China (9151064201000067), the Special Fund for Agro-Scientific Research in the Public Interest, China (201003021), and the Joint Fund of the National Natural Science Foundation of ChinaGuangdong Province (U0631003).
References Amante-Bordeos A, Sitch L A, Nelson R, Oliva N P, Aswidinnoor H. 1992. Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice, Oryza sativa. Theoretical and Applied Genetics, 84, 345-354. Barclay A. 2004. Cultivated rice receives help from wild cousins. Approach Technology, 31, 10-12. Bellon M R, Brar D S, Lu B R, Pham J L.1999. Rice genetic resources. In: Dowling N G, Greenfield S M, Fischer K S, eds., Sustainability of Rice in the Global Food System. International Rice Research Institute, Manila. pp. 251-283. Brar D S, Dalmacio R, Elloran R, Aggarwal R, Angeles R, Khush G S. 1996. Gene transfer and molecular characterization of introgression from wild Oryza species into rice. In: Khush G S, ed., Rice Genetics III. International Rice Research Institute, Manila. pp. 477-485. Feng J H, Lu Y G, Liu X D, Xu X B. 2001. Pollen development and its stages in rice (Oryza sativa L). Chinese Journal of Rice Sciences, 15, 21-28. (in Chinese) Fu X L, Lu Y G, Liu X D, Li J Q. 2009a. Crossability barriers in the interspecific hybridization between Oryza sativa and O. meyeriana. Journal of Integrative Plant Biology, 51, 21-28. Fu X L, Lu Y G, Liu X D, Li J Q, Feng J H. 2007. Cytological mechanisms of interspecific incrossability and hybrid sterility between Oryza sativa L. and O. alta Swallen. Chinese Science Bulletin, 52, 755-765. Fu X L, Lu Y G, Liu X D, Li J Q, Zhao X J. 2009b. Comparative embryological studies on infertility of interspecific hybridizations between Oryza sativa with different ploidy levels and O. officinalis. Rice Science, 16, 58-64. He G C. 2002. Mining and transfer of elite genes of wild rice. In: Luo L J, Ying C S, Tang S X, eds., Rice Germplasm. Hubei Science and Technology Press, Wuhan. p. 279. (in Chinese) Hu S Y. 1994. Method of preparation of slides used to examine the pollen germination on the stigma and pollen tube growth in the style. Chinese Bulletin of Botany, 11, 58-60. (in Chinese) Huang Z, He G, Shu L, Li X H, Zhang Q F. 2001. Identification and mapping of two brown planthopper resistance genes in rice. Theoretical and Applied Genetics, 102, 929-934. Jena K K, Khush G S. 1989. Monosomic alien addition lines of rice: Production, morphology, cytology and breeding
1499
behavior. Genome, 32, 449-455. Jena K K, Khush G S. 1990. Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice, Oryza sativa L. Theoretical and Applied Genetics, 80, 737-745. Jena K K, Khush G S, Kochert G. 1992. RFLP analysis of rice (Oryza sativa) introgression lines. Theoretical and Applied Genetics, 84, 608-616. Mariam A L, Zakri A H, Mahani M C. 1996. Interspecific hybridization of cultivated rice, Oryza sativa L., with the wild rice, O. minuta Presl. Theoretical and Applied Genetics, 93, 664-671. Multani D S, Jena K K, Brar D S, Reyes B G, Angeles E R, Khush G S. 1994. Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin to cultivated rice of O. sativa L. Theoretical and Applied Genetics, 88, 102-109. Multani D S, Khush G S, Reyes B G, Brar D S. 2003. Alien genes introgression and development of monosomic alien addition lines from Oryza latifolia Desv to rice, Oryza sativa L. Theoretical and Applied Genetics, 107, 395-405. Panaud O, Chen X, McCouch S R. 1996. Development of microsatellite markers and characterization of simple sequence length polymorphism (SSLP) in rice (Oryza sativa L). Molecular and General Genetics, 252, 597-607. Qin X Y, Zhu R C, Tang J H, Li W K, Li D Y, Wei S M, Huang F K. 2007. Genetic analysis and utilization of brown planthopper (BPH)-resistant genes in Oryza officinalis Wall. Journal of Plant Genetic Resources, 8, 41-45. (in Chinese) Shin Y B, Katayama T. 1979. Cytological studies on the genus Oryza: XI. Alien addition lines of O. sativa with single chromosomes of O. officinalis. Japanese Journal of Genetics, 54, 1-10. Suputtitada S, Adachi T, Pongtongkam P, Peyachoknagul S, Apisitwanich S, Thongpradistha J. 2000. Breeding barriers in the interspecific cross of Oryza sativa L. and Oryza minuta Presl. Breeding Science, 50, 29-35. Tan G X, Jin H J, Li G, He R F, Zhu L L, He G C. 2005. Production and characterization of a complete set of individual chromosome additions from Oryza officinalis to Oryza sativa using RFLP and GISH analyses. Theoretical and Applied Genetics, 111, 1585-1595. Tan G X, Ren X, Weng Q M, Shi Z Y, Zhu L L, He G C. 2004a. Mapping of a new resistance gene to bacterial blight in rice line introgressed from Oryza officinalis. Acta Genetica Sinica, 31, 724-729. (in Chinese) Tan G X, Weng Q M, Ren X, Huang Z, Zhu L L, He G C. 2004b. Two whitebacked planthopper resistance genes in rice share the same loci with those for brown planthopper resistance. Heredity, 92, 212-217. Yan H H, Hu H Y, Fu Q, Yu H Y, Tang S X, Xiong Z M, Min S
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
1500
FU Xue-lin et al.
K. 1996. Morphological and cytogenetical studies of the hybrids between Oryza sativa and Oryza officinalis. Chinese
between Oryza sativa and O. latifolia by in situ hybridization. Chinese Science Bulletin, 53, 2973-2980.
Journal of Rice Sciences, 10, 138-142. (in Chinese) Yasui H, Iwata N. 1991. Production of monosomic alien addition
Yu W J, Luo K, Guo X X. 1993. Obtaining the hybrids of Oryza sativa × O. officinalis without embryo culture by using male-
lines of Oryza sativa having a single O. punctata chromosome. In: Khush G S, ed., Rice Genetics II. International Rice
sterile lines. Acta Genetica Sinica, 20, 348-353. (in Chinese) Zhong D B, Luo L J, Guo L B, Ying C S. 1997. Studies on
Research Institute, Manila. pp. 147-155. Yi C D, Tang S Z, Zhou Y, Liang G H, Gong Z Y, Gu M H. 2008.
resistance to brown planthopper (BPH) of hybrid between Oryza sativa and Oryza officinalis. Southwest China Journal
Development and characterization of interspecific hybrids
of Agricultural Sciences, 10, 5-9. (in Chinese) (Managing editor WANG Ning)
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.