Introgression of salt-tolerance from somatic hybrids between common wheat and Thinopyrum ponticum

Plant Science 167 (2004) 773–779

Introgression of salt-tolerance from somatic hybrids between common wheat and Thinopyrum ponticum Chen Suiyun, Xia Guangmin∗ , Quan Taiyong, Xiang Fengnin, Jin Yan, Chen Huimin School of Life Sciences, Shandong Unversity, Jinan 250100, PR China Received 24 February 2004; received in revised form 28 April 2004; accepted 10 May 2004 Available online 4 June 2004

Abstract Introgression lines derived from somatic hybrids between Thinopyrum ponticum Podp. (Agropyron elongatum (Host) Nevishi) and Triticum aestivum L. cv. Jinan 177 were screened for salt-tolerance in hydroponic experiments. Their growth rates, salt-tolerance index and the content of free proline, Na+ , K+ and Na+ /K+ in the leaf were compared with those of wheat cv. Jinan 177. The lines were tested under natural saline conditions in two locations. One line expressed higher salt-tolerance than its parental wheat and a check salt-tolerant cultivar. Karyotype analysis showed that the two lines possessed 42 chromosomes of wheat introgressed with small chromosome segments from Th. ponticum. We suggest that these two lines show stable inheritance of salt tolerance, and that this salt-tolerance has been transferred from Th. ponticum into wheat. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Somatic hybrid lines; Triticum aestivum L.; Thinopyrum ponticum (Agropyron elongatum); Salt-tolerance; GISH

1. Introduction About 7% of the world’s arable land is affected by salinity [1,2], as is a similar proportion of China’s land area [3]. As a major contributor to wheat production in China, a priority in Shandong province is to breed salt-tolerance into wheat. The complexity of salt stress tolerance as a trait, and its polygenic inheritance are important factors contributing to the difficulty of this task [4]. Wide hybridization is a potential route for introducing genes for complex traits, since unlike genetic transformation; a large number of genes are simultaneously transferred. This can be achieved via either sexual or somatic hybridization; the latter allows for the incorporation of both nuclear and non-nuclear (plastome and mitochondriome) genes from the exotic parent, which is impossible using the sexual route. This is of particular interest, given that cytoplasmic genes are known to play an important role in determining stress resistance. In our early work, salt-tolerant genes of Aeleuropus littoralis was averted into wheat cells by asymmetric somatic hybridization of wheat with A. littoralis treated by UV. But ∗

Corresponding author. Tel.: +86 531 8364525; fax: +86 531 8565610. E-mail address: [email protected] (X. Guangmin).

the hybrids did not regenerate plant [5]. None successful example has been reported about salt-tolerance transferred via somatic hybridization. Thinopyrum ponticum Podp. (2n = 70) is a perennial grass characterised by enhanced levels of tolerance to abiotic factors, notably salinity stress, as well as for resistance to a number of important wheat diseases. It also has high seed protein content. We have previously reported the generation of some highly asymmetric somatic hybrid plants between wheat and Th. ponticum treated by UV and fertile progenies and many hybrid lines have been obtained via culture of the hybrid ovaries [6]. In this paper, we report on the transfer of some salt-tolerance traits of Th. ponticum into the hybrid lines, and the extent to which Th. ponticum chromatin is stably inherited.

2. Materials and methods 2.1. Plant material Grains of Th. ponticum, common wheat (Triticum aestivum L. cv. Jinan 177) were provided by Shandong Academy of Agriculture Science, and salt-tolerant wheat cultivars Dekang 961 from Dezhou Academy of Agriculture

0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.05.010

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Science. Lines II-I-8 and II-2 were bred from the somatic hybrid Jinan 177/Th. ponticum. 2.2. Hydroponic experiment Grains of lines II-I-8 and II-2 and their parents were surface-sterilized by 0.2% HgCl2 supplemented with a drop of liquid detergent (GLOD FISH, China) for 5 min. The grains were rinsed four times with sterilized distilled water, and then germinated in distilled water at room temperature (20–25 ◦ C) for about 24 h. About 40 uniformly germinated grains were removed to a hydroponic tank, and grown in 1/4 strength Hoagland’s solution in a green house with natural light and natural temperature (15–20 ◦ C). 500 ml 1/2 strength Hoagland’s solution containing half of Ca2+ supplemented with various concentrations of NaCl was added every day from emergence of leaf 3. To avoid salt shock, NaCl was added stepwise at a rate of 25% of the final concentration (0, 100, 150, 200, 250, 350 mM) every three days until the final concentration was reached. The appearance of chlorosis was taken as a symptom of salt injury. A salt-tolerance index was calculated by multiplying the sum of the growth duration (days) at different concentration of NaCl before the onset of chlorosis, by the respective NaCl concentration in millimole. Seedlings remained at the final salt concentration for five days before being harvested. At harvest, seedling fresh weight (FW) was measured after removing any surface water by blotting with filter paper, and dry weight (DW) was measured after baking at 80 ◦ C for 2–3 h. The water content (WC) was measured according to the following formula WC (%) = ((FW − DW)/FW) × 100. 2.3. Measurement of the free proline content and the inorganic ion content Proline content of seedling leaf was measured as described by Bates et al. [7]. The Na+ and K+ contents were analyzed spectrophotometrically after mineralizing the dry samples with nitric acid, using a 180–80 HITACH polarized zeeman atomic absorption spectrophotometer.

1:3 acetic acid–ethanol for two days. For chromosome counting, the root meristem was squashed under a cover slip in Carbol Fucshin solution. More than 500 cells were counted for each genotype. For GISH analysis, the root meristem was squashed in 45% acetic acid. Cover slips were removed with a razor blade after freezing in liquid nitrogen, and the slides were air dried. Total genomic DNA of Th. ponticum was labelled as a probe, and the GISH procedure was as described by Xiang et al. [8]. Fluorescent images were captured using a fluorescence microscope (Nikon Eclipse E600, Japan).

3. Results 3.1. Growth rate and salt-tolerance index in F3 Seedlings of Th. ponticum grew healthily and exhibited no evidence of salt injury at any NaCl concentration. The salt-tolerance index of these plants was 3500 (Table 1). Some of the leaves of the parental wheat cv. Jinan 177 wilted in the 250 mM NaCl treatment before reaching the target concentration. Over 10% of leaves exhibited chlorosis at the tips after one day in 250 mM NaCl, two days in 200 mM NaCl, and four days in 150 mM NaCl. The minimum salt-tolerance index was 1750. After five days, 30% of the leaves in 150 mM NaCl, and 100% of leaves in 200 mM NaCl exhibited salt injury, and about 50% of the plants in the latter treatment were wilted and dehydrated. DW and WC of the two hybrid lines decreased little in 100–150 mM, but both were markedly lower in the 250 mM treatment (Table 1). No visible salt injury occurred in the hybrids at 100–150 mM NaCl. Both hybrids exhibited salt injury after three days at 250 mM NaCl and after four days at 200 mM NaCl. After five days of treatment, about 15% and 10% of lines II-I-8 and II-2, respectively, showed visible damage at 250 mM NaCl. The salt-tolerance index of both hybrid lines was intermediate between the parents. Thus, the order of sensitivity to 200–250 mM NaCl was Jinan 177 > II-I-8 > II-2 > Th. ponticum. 3.2. Measurement of the free proline content in the leaves of F3 plants

2.4. Cultivation tests in naturally saline soils F4 lines II-I-8 and II-2 were grown at a coastal site (with about 0.35–0.55% salt concentration) in Dongying area, Shandong province in 2000; and F5 lines and a local salt-tolerant wheat variety Dekang 961 at both a coastal site (with about 0.35–0.55% salt concentration) in Dongying, and on an inland site (with about 0.4–0.6% salt concentration) in Dezhou area in 2001. 2.5. Cytological analysis and GISH analysis For cytological analysis, root tips of 0.5–1 cm in length were excised and placed in ice–water for 24 h, then fixed in

As shown in Fig. 1, the free proline content of Th. ponticum increased as the salt concentration was raised. It increased rapidly at a low salt concentration (100 mM), and steadily but more slowly as the concentration was raised to 150 mM. At all levels of salt treatment, the free proline content of Th. ponticum was higher than that of any other genotype tested. The free proline content of the parental wheat also increased with salt concentration. However, above 150 mM, tested leaves exhibited salt injury and the free proline content decreased. Although the salt-response curves of proline for hybrids were somewhat similar to those of wheat, their proline content was always higher than that of wheat.

Table 1 Growth rates and salt-tolerance index of F3 generation in different concentrations of NaCl

DW(%)

WC(%)

FW(%)

DW(%)

WC(%)

FW(%)

DW(%)

WC(%)

FW(%)

DW(%)

WC(%) 51.8 52.1 50.8 48.2 49.6

6.8 6.8 6.7 6.4 6.6

86.9% 86.9% 86.8% 86.7% 86.7%

226.7 218.4 194.2 164.4 127.0

39.8 28.8 26.8 24.5 20.9

86.9% 86.8% 86.2% 85.1% 83.6%

302 (100) 301.2 (99.7) 288.7 (95.6) 267.4 (88.5) 229.6 (76.3)

41.1 40.7 39.6 37.7 34.9

86.4% 86.5% 86.3% 85.9% 84.8%

133.3 (100) – 129.1 (96.8) 118.7 (89.0) 96.1 (72.1)

21.7 – 21.1 20.3 17.3

83.7% (100) – 83.7% (100) 82.9% (99.0) 82% (98.0)

Note: FW: fresh weight (mg per plant); DW: dry weight; WC: water content; (%): percentage of the sample related to the control.

(97.2) (93.5) (79.7)

775

Fig. 2. Na+ content of somatic hybrid lines II-2, II-I-8 and their parents at various salt concentrations.

Figs. 2 and 3 show the accumulation of Na+ and K+ in the leaves of the hybrid lines II-I-8 and II-2 and their parents under salt stress. It is clear that the Na+ content in both parents and the two hybrids increased continuously as the salinity rose in the range of 0–250 mM (Fig. 2). The absolute concentration of Na+ in the leaves varied between genotypes, such that Th. ponticum < II-2 < II-I-8 < Jinan 177 at the higher NaCl concentration of 250 mM. With regard to K+ , leaf concentrations decreased continuously as the salinity increased. However, among the various genotypes, K+ content in Th. ponticum decreased at the slowest rate across the various treatments. However, it exhibited a clear decreasing trend as NaCl concentration increased in wheat (Fig. 3). In the range 150–250 mM NaCl, Na+ content increased in the two hybrid lines less than it did in the parental

3.3. Measurement of the inorganic ion content in the leaves of F3 plants

Fig. 1. Proline content of somatic hybrid lines II-2, II-I-8 and their parents at various salt concentrations.

C. Suiyun et al. / Plant Science 167 (2004) 773–779

5d × 100 + 5d × 150 + 4d × 200 + 3d × 250 = 2800 5d × 100 + 5d × 150 + 4d × 200 + 3d × 250 = 2800

(100) (100) (100.1) (99.9) (99.4) (98.1) (100) (99.0) (96.4) (91.7) (84.9) (100) (99.9) (99.2) (97.9) (96.2) (100) (72.4) (67.3) (61.6) (52.5) (100) (96.2) (85.7) (72.5) (56.0) (100) (100) (99.9) (99.8) (99.8) (100) (100) (98.5) (94.1) (97.1)

5d × 100 + 4d × 150 + 2d × 200 + 1d × 250 = 1750 5d × 100 + 5d × 150 + 5d × 200 + 5d × 250 = 3500 Index of salt tolerance

FW(%)

(100) (100.6) (98.1) (93.1) (95.8)

II-I-8 II-2 Wheat cv. Jinan 177 Th. Ponticum

NaCl concentration (mM) 0 (control) 100 150 200 250

776 C. Suiyun et al. / Plant Science 167 (2004) 773–779

Fig. 3. K+ content of somatic hybrid lines II-2, II-I-8 and their parents at various salt concentrations.

wheat. The Na+ /K+ ratio changed in a similar fashion (Fig. 4).

3.4. Measurement of biomass and calculation of salt-tolerance index of F4 plants

Seedlings with fully expanded third leaves were tested. Because the F4 leaves were bigger than the F3 ones, both dry and fresh F4 weights were much greater than those representing the F3 (Table 2). However, there was some consistency between the generations. While Th. ponticum grew normally in the various treatments, the parental wheat (cv. Jinan 177) plants all completely wilted and dried at 350 mM NaCl treatment. On the whole, the two hybrid lines grew much better than the parental wheat—although some plants exhibited severe salt injury, only few plants died at 350 mM NaCl. The hybrid II-2 grew better than II-I-8, and more than 80% plants did not exhibited wilt for whole plant,

Fig. 4. Na+ /K+ ratio of somatic hybrid lines II-2, II-I-8 and their parents at various salt concentrations.

Table 2 Growth rates and salt-tolerance index of F4 generation in different concentrations of NaCl NaCl concentration

Th. Ponticum FW(%)

DW(%)

WC(%)

FW(%)

DW(%)

WC(%)

FW(%)

DW(%)

WC(%)

FW(%)

DW(%)

WC(%)

0 (control) 100 200 250 350

60.1 59.9 57.9 57.8 56.0

7.6 7.5 7.4 7.7 7.6

87.4% 87.5% 87.2% 86.6% 86.4%

498 511 495 353 242

41.4 46.2 39.4 37.7 32.2

91.7% 90.9% 90.0% 89.3% 86.7%

478.8 464.2 457.4 393.5 345.5

56.5 55.7 55.8 48.8 46.3

88.2% 88.0% 87.8% 87.6% 86.6%

328.2 323.9 291.4 239.9 222.5

40 (100) 39.2 (98) 39.7 (99.3) 32.4 (81.0) 32.6 (81.5)

87.8% 87.9% 87.4% 86.5% 85.3%

Index of salt tolerance

5d × 100 + 5d × 200 +5d × 250 + 5d × 350 = 4500

(100) (99.7) (96.3) (96.2) (93.2)

Wheat cv. Jinan 177

(100) (98.7) (97.4) (101.3) (100)

(100) (100.1) (99.8) (99.1) (98.9)

(100) (102.6) (99.4) (70.9) (48.6)

(100) (111.6) (95.2) (91.1) (77.8)

5d × 100 + 3d × 200 +2d × 250 + 0d × 350 = 1600

II-2

(100) (99.1) (98.1) (97.4) (94.5)

II-I-8

(100) (96.9) (95.5) (82.2) (72.2)

(100) (98.6) (98.8) (86.4) (81.9)

5d × 100 + 4d × 200 +3d × 250 + 3d × 350 = 3100

Note: FW: fresh weight (mg per plant); DW: dry weight; WC: water content; (%): percentage of the sample related to the control.

(100) (99.8) (99.5) (99.3) (98.2)

(100) (98.7) (89.1) (73.1) (67.8)

5d × 100 + 4d × 200 +3d × 250 + 1d × 350 = 2450

(100) (100.1) (99.5) (98.5) (97.2)

C. Suiyun et al. / Plant Science 167 (2004) 773–779 Table 3 Growth parameters of F4 hybrid lines in the natural salinity field conditions Material

Number of grains/spike

1000 grains weight (g)

Yield (kg/666.7 m2 )

II-2 II-I-8 Jinan 177

50 46 –

55 45 –

451.0 303.6 –

777

in line II-2. This establishes that the introgression events in these two lines were distinct.

4. Discussion

Note: The salt concentration of the field was 0.35–0.55%.

therefore, its salt tolerance was much superior to parental wheat. 3.5. Cultivation tests in naturally saline soils Parental wheats grew slowly and weakly, produced few ears and could not be counted normally. However, the hybrids grew normally, especially II-2, which exhibited strong salt-tolerance, grew uniformly and strongly, and had high yield properties in saline soil (Table 3). At the F5 generation, a new salt-tolerant wheat cv. Dekang 961 was selected as control. In the saline soil with the lower salt content, this control grew better than the hybrid lines. However, in the saline soil with higher salt content (0.6%), hybrid line II-2 exhibited better agronomic traits than cv. Dekang 961 (Table 4). 3.6. Chromosome composition of F5 hybrid lines In lines II-I-8 and II-2, respectively, 78.2 and 75.6% of cells showed 2n = 40–42 (Table 5). No clear chromosome morphological variants were observed. GISH karyotypes were obtained with total DNA of Th. ponticum as probe. Small segments of labelled chromosome, rather than entire chromosome(s) or chromosome arm(s) were present in both somatic hybrids. However the two hybrids could be distinguished from one another. For example, a small insert is present in the long arm of a pair of short chromosomes in line II-I-8 (Fig. 5D), but not in the equivalent chromosome

Variation in levels of salt tolerance can be difficult to quantify, as growth reduction depends heavily on the length of time when the plants are exposed to salinity stress. Salinity lowers the water potential of roots, and this rapidly causes reductions in growth rate, along with a suite of metabolic changes identical to those caused by water stress [9]. Later, there may be salinity-specific effects that have an impact on growth and/or senescence. The first phase of growth reduction—referred to as the ‘osmotic’ phase—is quickly apparent, and is due to the presence of salt outside the roots. There appears to be surprisingly little genotypic variation for this trait. Following this is the second phase of growth reduction, which takes some time to develop, and is associated with advanced senescence of older leaves [10]. A high level of salt (0–350 mM) together with a 50% reduction in Ca2+ content of the culture medium was used in order to generated our results rapidly. As noted by Munnus [10], the length of time required before growth differences between genotypes can be seen depended on the salinity and the degree of salt tolerance of the species. The second phase starts earlier when salinities are higher [10]. Our tests showed that it is possible to measure growth difference among different salt–tolerant samples both under the conditions mentioned above and over a short period. We show that the parental wheat (cv. Jinan 177), at the three-leaf stage, grew well in 100 mM NaCl, although it did not grow well in saline soils. However both hybrid lines II-1-8 and II-2 grew well in saline soils, with the latter being superior to the former. However, in short hydroponic experiments, they did not differ in growth rate in the range 100-200 mM NaCl. Certainly, many other factors influencing crop productivity differ between the field and laboratory conditions [11,12]. We con-

Table 4 Growth parameters of F5 hybrid lines in field salinity conditions Sites

Concentration of salt

Material

Numbers of grains/spike

1000 grains weight (g)

Yield (kg/666.7 m2 )

Dongying

0.55%

II-2 II-I-8

38.7 28.8

42.5 50.4

320 261.3

Dongying

0.35%

II-2 II-I-8

40.8 –

44.6 –

455 –

Dezhou

0.6%

II-2 II-I-8 Dekang 961(ck)

54.3 40.4 26.5

38.0 35.5 24.0

287.0 261.5 281.5

Dezhou

0.5%

II-2 II-I-8 Dekang 961(ck)

56.8 – 34.2

37.0 – 27.6

269.5 – 302.7

Dezhou

0.4%

II-2 II-I-8 Dekang 961(ck)

58.5 47.7 33.5

44.5 40.0 26.5

315.0 297.5 387.0

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C. Suiyun et al. / Plant Science 167 (2004) 773–779

Table 5 Chromosome numbers of F5 hybrids from root tip preparations Plant

No. of cells

Average no. of chromosome

Distribution of chromosome number (2n) 38

39

40

41

42

43

II-I-8 II-2 Jianan 177

605 542 503

40.76 40.82 (7.2%) 41.34

58 (9.6%) 39 (16.1%) 12 (2.4%)

67 (11.1%) 87 (17.5%) 2 (0.4%)

102 (16.9%) 95 (7.56%) 98 (19.5%)

121 (20%) 41 (50.6%) 84 (16.7%)

250 (41.3%) 274 6 (1.1%) 305 (60.6%)

7 (1.1%)

clude that longer periods of hydroponic treatments are more predictive of field performance. Th. ponticum has well-developed mechanisms to control the uptake, transport and excretion of salt. In various NaCl treatments, its growth rates decreased only slightly as the salt content increased (Fig. 1). The plants showed little sign of any external injury, and the salt-tolerance index being the most sensitive (Tables 1 and 2). Under stress, free proline was accumulated in the leaves, and K+ content was relatively stable (Figs. 1 and 3). In addition, the Na+ /K+ ratio increased gradually and slowly (Fig. 4), exhibiting consistency with salt-tolerance monocot in the scopes of it tolerant range [13,14]. In wheat, salt tolerance is associated with low rates of transport of Na+ to shoots and with high selectivity for K+ over Na+ [15]. Also, accumulation of solutes such as proline, therefore, is important for adaptation to saline conditions [16–18]. The parental wheat is a glycophyte, under the salinity conditions applied in this experiment; it exhibited

2 (0.4%)

no salt-tolerance either in growth or physiological adaptation. In the higher salinity level treatments, it exhibited severe wilting, chlorosis, grew slowly, and delivered a low salt-tolerance index (Tables 1 and 2). It accumulated the least free proline, the highest Na+ and the Na+ /K+ ratio (Fig. 4). The curves of hybrids II-2 and II-1-8 were similar to that of Jinan 177 (Figs. 1–4), but their salt-tolerance was intermediate between the wheat and Th. ponticum parents. Whether the hybrid lines express salt-tolerance in naturally saline soils, or whether they have any practical breeding value will need to further investigation. Our preliminary data show that they—and especially II-2—do exhibit a level of salt-tolerance (Tables 3 and 4), such that they can outperform the variety Dekang 961 (Table 4), which is considered to be the most salt-tolerant cultivar in China. We believe that the salt-tolerance of hybrid lines is due to the introgression of genetic material from Th. Ponticum. Asymmetric somatic hybridization induced by UV offered a direct and simple method for introgression of small alien

Fig. 5. GISH karyotypes using total Th. ponticum DNA as probe. (A) Th. ponticum; (B) common wheat (cv. Jinan 177); (C) somatic hybrid II-2; (D) somatic hybrid II-I-8. The arrow shows signals present on the long arm of a pair of short chromosomes.

C. Suiyun et al. / Plant Science 167 (2004) 773–779

chromosome segments from donor into the recipient chromosomes [19]. Some hybrid plants of wheat with small alien chromosome segments had been created with this method in our laboratory [8,19–21]. Alien small-segment-translocation benefits stable heredity and localization of the alien chromatin, as well as isolation of the alien target genes. Some of the introgression lines including hybrids II-2 and II-1-8 in this experiment showed some specific traits existed in donor [6]. It was suggested that the introgressed segments in these lines maybe contain target genes. We have not localized the introgressed segments of Th. ponticum in the hybrid lines of this experiment, although the GISH karyotypes show that these are small, and are non-identical in the two selected hybrid lines (Fig. 5), which is confirmed by RAPD-based genotyping [22]. Interestingly we have also detected the presence of cytoplasmic DNA from Th. ponticum in the chloroplast genome of the hybrid lines [22]. Multi-generation tests have suggested that the salt-tolerant traits are stably inherited. Anyway, this work demonstrated that the UV-induced asymmetrical somatic hybridization could be used for introgression of salt-tolerant traits efficiently.

Acknowledgements National Natural Science Foundation of China, No. 30370857, Major Project of Ministry of Education in China and National 863 High Technology Research and Development Project No. 2001AA241032 supported this study. We are grateful to Dr Robert Koebner (Senior scientist of Jone Innes Centre, UK) for language correction and Mrs. Zhang Kun-pu (Dezhou Academy of Agriculture Science) and Shao Qiuling (Dongyun Academy of Agriculture Science) for providing the grains of Dekang 961 and Lumai 23, as well as conducting salt-tolerance experiment in saline soil.

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