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Relationship between tissue culture and agronomic traits of spring wheat
Plant Science 164 (2003) 1079 /1085 www.elsevier.com/locate/plantsci
Relationship between tissue culture and agronomic traits of spring wheat Wei Li, Chen-Hui Ding, Zhong Hu, Wei Lu, Guang-Qin Guo * Institute of Cell Biology, Lanzhou University, Lanzhou 730000, China Received 8 November 2002; received in revised form 11 February 2003; accepted 13 February 2003
Abstract Plant regeneration was achieved from mature and immature embryos of ten spring wheat cultivars (cv.s), which showed strong genotype effect on both callus growth rate and the frequency of plant regeneration. Relationship between tissue culture and agronomic traits was surveyed by correlation and regression analysis, revealing credible relationship between the two traits, especially in mature embryo cultures. It was found that the frequency of regeneration from callus derived from mature embryos could be estimated from kernel number per spikelet, effective tillering and longevity; which was less significant in immature embryo culture. Such relationships may be due to the fact that both traits are influenced by the endogenous hormone levels. This result suggests that it is possible to screen genotypes with good tissue culture traits directly at the agronomic trait level, a practice rather simple and straightforward. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Triticum aestivum ; Tissue culture; Agronomic traits; Correlation analysis
1. Introduction Wheat is one of the most important crops in the world. With the development of plant molecular biology and genetic engineering, its transformation has become one of the central issues in molecular breeding, which depends largely on reliable system of plant regeneration via tissue culture [1]. Many factors can affect wheat regeneration, such as explant source [2 /5], donor plant growth condition [5,6], culture medium [7 /9], and genotype [9 /13], among which genotype is often the dominant one. Unfortunately, however, for a given cultivar, the cultural response determined by its unique genotype cannot be known before being tested in experiment, because we know very little about the related mechanisms. In several cases, in vitro regeneration is shown to be a quantitative trait [14,15], and the hexaploid nature of wheat makes the situation even more complex. However, if the critical tissue culture traits could be correlated to one or more agronomic
* Corresponding author. E-mail address: [email protected] (G.-Q. Guo).
traits that are much easier to measure, we will have a rather simple way to predict the outcome of tissue culture in advance, avoiding the need to test large number of genotypes at the tissue culture step, which is very expensive, laborious and time consuming. However, such works are scarce [16]. In tissue culture of cereals including wheat, 2,4dichlorophenoxyacetic acid (2,4-D) is often used for callus induction, and different combinations of auxin such as napthaleneactic acid (NAA) and indoleacetic acid (IAA) and cytokinin (including zeatin, kinetin and 6-benzyladenine (6-BA) etc.) were used for stimulating plant regeneration from calli [14]. Recently, a substituted phenylurea named thidiazuron (TDZ) is shown to be an efficacious regulator of in vitro morphogenesis of many dicot plant species [17], and a few reports also demonstrate its positive effects on wheat [13,18] and other cereal species [18,19], showing that comparison with other commonly used hormones, TDZ is among the best for wheat regeneration [13,18]. So this growth regulator was incorporated into our present tissue culture regime to promote plant regeneration from wheat calli induced by 2,4-D from ten different spring
0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00113-4
W. Li et al. / Plant Science 164 (2003) 1079 /1085
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cultivars (cv.s), and the relationship between their tissue culture and agronomic traits was tested.
2. Materials and methods 2.1. Plant materials Ten cv.s of spring wheat (Triticum aestivum L.) (Tables 1 and 2), kindly provided by Jia at the State Key laboratory of Arid Agroecology, Lanzhou, were used as sources of immature and mature embryos for callus induction and plant regeneration. They were cultivated in the northwest regions of China that have very similar semi-arid climate of plateau. 2.2. Plant growth and agronomic traits measurements Seeds were sown in the garden field at Lanzhou University in mid-March of 1999 and 2000. In all the experiments, rows were 5 m long with a density of 20 seeds m 1 and separated by 0.25-m from each other. Plants were irrigated properly throughout the growing seasons to avoid any serious water stress and fertilized with urea at sowing and tillering stages to give sufficient nutrition. Weeds were periodically removed by hand. Although the growth environment was not artificially controllable, all the cv.s grew in the same field and thus subjected to parallel conditions, so variation in their agronomic traits is primarily caused by the genotype effects. For agronomic traits analysis, 30/40 randomly selected plants of each cv. were used. At anthesis, total tillers and effective tillers per plant were counted. Effective tillering was defined as the ratio of effective
to total tillers per plant. At physiological maturity, plant height was measured from the soil surface to the top of the spike on the main shoot. The total and effective (with kernels) spikelets per spike, effective spikeleting (percent effective in total spikeletes), and longevity were recorded at harvesting. The spikes of these measured plants were oven-dried, and their kernel numbers per spikelet and spike were determined. The average kernel weight for each cv. was calculated as the total kernel weight divided by their numbers. 2.3. Tissue culture and plant regeneration 2.3.1. Medium The basic medium used both for callus induction and plant regeneration in this experiment was MB, which contained MS salts [20], B5 vitamins [21], 2 mg l 1 glycine, 300 mg l 1 glutamin, 500 mg l 1 casein hydrolysate, 3% sucrose and 0.7% agar at pH 5.8 [22]. 2.3.2. Immature embryo culture Two weeks after anthesis, spikes with immature embryos about 0.5 /1.0 mm in size were harvested from the fields and surface-sterilized for 1 min in 70% ethanol. The immature embryos were aseptically excised from caryopses and placed with the scutellum upwards on the MB medium supplemented with 2 mg l 1 2,4-D and 1 mg l 1 abscisic acid (ABA) at 259/1 8C in darkness to induce callus. To initiate shoots, the calli were transferred onto the MB medium with TDZ and cultured for 2 weeks at 259/1 8C in a 16-h light:8-h dark cycle provided by 40-W white fluorescent tubes (1500 lx). The calli with one or more buds were then transferred onto 1/2 MB medium free of growth regulators to induce root formation.
Table 1 Differences in callus formation and plant regeneration between ten spring wheat cv.s from immature and mature embryos Genotype
Tableland 602 8797-21 Yong 3263 98j51 021-128 92jian46 Monk-head 8139 8275 Dingxi 24 Mean LSD (0.05) CV (%) a b c
Fc (%)
Average FW of callus/explanta (mg)
Frequency of regeneration from callus (%)
Ib
Mc
I
M
I
M
100 98.9 100 100 100 100 98.8 97.7 100 100 99.5 2.6 1.5
100 100 100 100 100 100 100 100 100 100 100 0 0
48.4 45.0 40.4 53.6 60.0 63.5 64.2 72.7 62.6 59.8 57.0 19.3 23.6
70.0 56.7 65.6 127.7 74.4 63.0 153.4 44.2 74.9 105.4 83.5 77.7 63.1
38.1 34.3 52.5 30.0 60.3 45.3 42.7 44.7 57.1 28.1 43.3 7.7 26.0
51.5 50.7 82.0 58.9 56.7 33.7 58.9 37.5 46.1 58.8 53.5 6.9 25.0
Recorded on the 20th day of callus induction. I, immature embryos. M, mature embryos.
81.6 74.8 65.1 88.3 56.4 79.8 104.2 83.3 101.5 96.7 83.2 6.1 18.2 Tableland 602 8797-21 Yong 3263 98j51 021-128 92jian46 Monk-head 8139 8275 Dingxi 24 Mean LSD (0.05) CV (%)
64.4 49.8 75.5 56.6 56.5 56.5 43.1 43.7 51.9 70.9 56.9 8.3 19.6
43.8 40.1 37.6 27.2 30.0 35.1 35.6 43.5 36.3 34.8 36.4 1.1 14.1
111.7 110.3 110.3 115.7 113.3 106.7 115.7 113.3 119.7 118.7 113.5 2.0 0.9
Height (cm) Tillers/plant Effective Effective Spikelets/spike Effective Effective Kernel number/spikelet Kernel number/spike Average weight Longevity tillers/plant tillering (%) spikelets/spike spikeleting (%) of kernel (mg) Genotype
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2.3.3. Mature embryo culture Mature seeds were surface sterilized with 70% ethanol for 30 s followed by 0.1% mercuric chloride for 20 min, then washed three times with sterile water. They were inoculated on 1/2 MB basal medium at 259/1 8C for germination. After 20 /24 h, the germinated embryos were aseptically picked out with a scalpel and transferred onto callus induction media. The media and the conditions used for callus induction, shoot initiation and rooting were all the same as those described above for the immature embryos. In both mature and immature embryo cultures, the frequency of callus induction (Fc) and the average fresh weight (FW) of callus per explant were determined 4 weeks after callus induction; and their regeneration frequency (Fr) (percent callus forming shoots) were calculated after 2 weeks on the regeneration medium.
About 30 explants per cv. were used in each treatment of tissue culture; and the agronomic traits measurement was done with 30/40 plants for each cv. Every treatment/measurement in this experiment had at least three replicas in parallel. Genotype effects on tissue culture of mature and immature embryos and agronomic traits were tested by analysis of variance (ANOVA) and leastsignificant-difference tests. Relationships between every possible pairs of various agronomic and tissue culture traits were determined by Pearson correlation analysis. Stepwise multiple regression analysis was used to evaluate the correlation of various agronomic traits to the critical tissue culture traits, and regression coefficients were described when significant (P /0.05). All statistical analyses were performed with the statistical package of SYSTAT 7.0 [23].
52.0 47.6 61.4 22.1 27.3 37.2 35.0 31.3 20.5 17.2 35.2 17.6 46.9
19.1 17.4 19.4 25.8 18.6 19.9 16.7 18.8 18.8 21.0 19.5 1.0 12.6
18.6 15.9 19.3 18.7 18.0 18.2 15.9 16.3 18.0 20.3 17.9 1.4 8.8
97.0 91.1 99.3 72.7 96.7 91.1 94.6 86.5 95.8 96.5 92.1 3.4 8.4
3.4 3.2 3.9 3.0 3.1 3.0 2.7 2.7 2.9 3.5 3.1 0.3 12.6
2.4. Statistical analysis
1.7 1.9 2.6 1.7 2.2 2.2 3.1 1.6 1.2 2.1 2.0 0.6 29.8
3. Results
3.8 5.1 4.5 9.8 9.4 7.0 9.5 5.9 7.9 13.2 7.6 2.5 40.4
Table 2 Differences in agronomic traits between ten spring wheat cv.s
W. Li et al. / Plant Science 164 (2003) 1079 /1085
3.1. Effect of TDZ on plant regeneration Since TDZ is a relatively new regulator used for wheat regeneration [13,18], we did a preliminary experiment to determine its optimal concentration, which was shown to be 1.0 mg l 1 and was the same as that reported in [13]. So it was used for all cv.s in this experiment. When the calli forming buds were transferred onto rooting media, new shoots continued to form, and after about 2 weeks, roots appeared from the base of many shoots. However, some shoots formed no root even after 6 weeks. Higher concentration of TDZ tended to suppress root formation from shoots (data not shown).
/0.183 0.665** 0.625** /0.223 0.428* 0.152 0.271 0.535** 0.126 0.342 0.176 0.075 0.246 0.341 0.394* /0.386* /0.247 0.003 0.028 0.020 0.000 0.098 0.436* /0.204 0.324 0.256 0.216 /0.011 0.330 0.421* /0.233 0.557** 0.300 0.168 0.113 0.199 0.150 0.057 0.202 /0.310 0.002 0.004 /0.004 0.011 /0.358 0.473* 0.154 /0.110 0.227 /0.013 0.644** /0.158 /0.014 /0.24 0.220 0.196 /0.003 0.113 0.647** /0.490** /0.332 0.543** 0.366* 0.498** 0.068 /0.239 0.382* 0.451* /0.493** 0.434* /0.090 0.118 /0.589** 0.393* 0.370* 0.074 0.285 /0.022 0.266 0.232 /0.115 0.076 /0.219 0.385* /0.112 0.454* 0.376* 0.392* 0.200 /0.656**
*, **, Significantly different at P B/0.05 and P B/0.01, respectively.
0.085 0.159 0.367* /0.548** 0.600**
14 13 12 11 10 9 8 7 6 5
/0.022 /0.374* /0.249 0.050 0.938** 0.121 0.675** /0.056 0.873** /0.266
In immature embryo cultures, Fc was related to kernel numbers per spike (r /0.421, P B/0.05) and effective spikelets per spike (r /0.557, P B/0.01), but Fr was only related to effective spikeleting (r /0.473, P B/0.05). Weight of callus from immature embryos was positively related to shoot length (r /0.394, P B/0.05) and tillers per plant (r /0.436, P B/0.05), but negatively to kernel numbers per spikelet (r / /0.386, P B/0.05). The Fr in mature embryo cultures was related to effective spikelets per spike (r /0.428, P B/0.05) and significantly to kernel numbers per spikelet (r /0.665, P B/0.01), spike (r /0.625, P B/0.01) and effective tillers per plant (r /0.535, P B/0.01). The weight of callus showed correlation to effective tillers/plant (r /0.367, P B/0.05), and significantly to shoot length (r /0.734, P B/0.01), tillers per plant (r /0.655, P B/0.01) and longevity (r /0.596, P B/0.01). Multiple regression analysis of each tissue culture trait against agronomic traits (Table 4) revealed that total
4
3.4. Correlation and regression analysis between agronomic and tissue cultural traits
3
The agronomic traits measured in this experiment are listed in Table 2. They represent major agronomic traits of wheat and exhibited a wide range of coefficient of variation (CV) from 0.9 (longevity) to 46.9 (effective tillering), indicating that they were influenced to very different degrees by genotype.
1 2
3.3. Agronomic traits
Table 3 Pearson correlation coefficients among investigated agronomic and tissue culture traits of ten spring wheat cv.s
In both immature and mature embryo culture, the Fc was extremely high, nearly (cv.s 8797-21, 8139 and Monk-head) or equal to 100%, with no or very little variations between different genotypes. However, genotype effect was striking on callus growth rate (expressed as FW of callus per explant on the 28th day) and plant regeneration (Table 1). The mean Fr was higher in mature embryos (53.5%) than immature embryos (43.3%). In mature embryo cultures, the highest Fr was obtained with cv. Yong 3263 (82%), significantly higher than that of 60.3% with cv. 021-128 in immature embryo cultures. The lowest Fr showed less difference between mature (cv. 92jian46, 33.7%) and immature (cv. Dingxi 24, 28.1%) cultures. Pearson correlation analyses showed that the FW of callus per explant was closely correlated between immature and mature embryo cultures (r /0.572, P B/ 0.01), but their Fc and Fr showed no significant relations to each other or to the weight of callus among different cv.s within each culture. It is noteworthy that no significant correlation existed between the Fr from immature and mature embryos (Table 3).
15
16
3.2. Effect of genotypes on tissue culture
0.734** /0.129 0.010 /0.178 0.227 0.219 /0.011 0.367* 0.655** /0.084 0.596** /0.131 0.272 0.572** 0.160
W. Li et al. / Plant Science 164 (2003) 1079 /1085
1 Shoot length 2 Kernel number/spikelet 3 Kernel number/spike 4 Weight of kernel 5 Effective spikelets/spike 6 Spikelets/spike 7 Effective spikeleting 8 Effective tillers/plant 9 Tillers/plant 10 Effective tillering 11 Longevity (days) 12 Fr of immature embryos 13 Fc of immature embryos 14 FW of callus/immature embryo 15 Fr of mature embryos 16 FW of callus/mature embryo
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Table 4 Regression of tissue cultural traits against agronomic traits Tissue culture traits
R2
Agronomic traits
Coefficient
Standard coefficient
P
Fr of mature embryos
0.679
FW of callus/mature embryo
0.804
Fr of immature embryos
0.224
Fc of immature embryos
0.311
FW of callus /immature embryo
0.436
Constant Kernel number/spikelet Effective tillering Longevity Constant Shoot length Effective tillers/plant Effective tillering Constant Effective spikeleting Constant Effective spikeletes/spike Constant Tillers/plant Kernel number/spikelet Weight of kernel
/150.709 20.672 9.062 1.066 /57.325 0.823 3.892 0.505 /20.213 0.689 90.090 0.528 33.801 2.892 /13.004 11.512
/ 0.606 0.410 0.318 / 0.614 0.591 0.411 / 0.473 / 0.557 / 0.660 /0.380 /0.438
/ 0.000 0.002 0.009 / 0.000 0.000 0.000 / 0.008 / 0.001 / 0.001 0.017 0.024
variances accounted for were ranged from 22.4 to 80.4% of tissue culture traits. The Fr in immature embryo cultures against agronomic traits has the least R2 (0.224), and only effective spikeleting was included in the regression equation. On the other hand, the R2 of Fr in mature embryo cultures against agronomic traits was 0.679, and three agronomic traits were included in the equation, among them, kernel number per spikelet contributed most, followed by effective tillering and longevity. Though the correlation coefficient between the Fr in mature embryo cultures and kernel numbers per spike was statistical significant (r /0.625, P B/0.01), due to its closed partial correlation with kernel numbers per spikelet (r /0.938, P B/0.01), the kernel numbers per spike was not included in the regression equation. For weight of callus in mature embryo cultures, it has good regression relationship with the agronomic traits (R2 /0.804), which were effective tillers per plant, shoot length and effective tillering; in immature embryo culture, this figure was R2 /0.436, which was contributed by tillers/plant (2.892), kernel number per spikelet ( /13.004) and weight of kernel (11.512).
4. Discussion Genotype was one of the most important factors for successful regeneration from callus of wheat [24], and has been investigated intensively [9 /13,25]. But little is known about the nature of such genetic control. In the present study, all the cv.s were spring genotypes that had developed for cultivation in the northwest regions of China, which have very similar semi-arid climate of plateau. In addition, we grew all the cv.s in the same field provided with parallel conditions to minimize any
environment-induced physiological variation in their agronomic traits. Although TDZ was shown to be the best for wheat regeneration among the growth regulators used so far [13,18], in our present results different cv.s still gave varied Fr from 27.1 to 82%, suggesting that the strong genotype effect can not be overcome by simply manipulating the medium compositions or other experimental parameters. Ozgen et al. showed that the correlations between callus induction rate, weight of callus and regeneration capacity in both mature and immature embryo cultures of wheat were insignificant, suggesting that these culture traits are independent from each other and likely to be controlled by different genes [25]. Our present results support this conclusion, since different tissue culture traits are related to different agronomic traits (Tables 1 and 4). Genetic control of regeneration from callus has been investigated in other cereal species. It is shown that several genes control the regeneration from embryoderived callus of barley [26,27]. Peng and Hodges also presented evidence in rice tissue culture that regeneration ability is under control of both nuclear and cytoplasmic genes [28]. In sorghum, Ma et al. found that the ability to form regenerable callus varied from genotype to genotype, was heritable, and acted as a dominant trait [29]. They thought that at least two gene pairs were involved. Willman et al. suggested at least one gene or a block of genes control the expression of somatic embryogenesis from maize [30]. Taken together, these studies strongly support the view that regeneration from callus is a quantitative trait controlled by multiple genes. Recently, Bregiter and Campbell identified new quantitative trait loci for plant regeneration of barley [15]. Due to its hexaploid nature, such work in wheat is
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more difficult. With the rapidly increasing number of important agronomic traits being mapped to their chromosomal loci, from which finding those closely correlated with the in vitro regeneration capacity may be helpful to decipher the genetic control of the latter. Our results tend to support this assumption, and the tissue culture traits in mature embryo culture appeared more reliable in correlation with the agronomic traits. The frequency of regeneration from callus of mature embryo can be regressed against kernel number per spikelet, effective tilllers and longevity (Table 4). The weight of callus has correlation with shoot length, tillers per plant and effective tillering (Table 4). At the molecular level, these imply that genes controlling the correlated agronomic and tissue culture traits are overlap or interact to each other. It has been proposed that genotype effect may be related to variations in endogenous hormone levels [31]. Hess and Carman showed that genotype and environment could influence the response of explant through the level or ratio of endogenous hormone [6]. The application of exogenous growth regulators, in combination with those of endogenous origin produced under the specific internal genetic control and environmental influence, can alter the hormone balance in favor of organogenesis or embryogenesis in lieu of their normal mode of development, eliciting a specific response [14]. Likewise, hormones can also influence agronomic traits. For example, cytokinins can enhance tillering in sorghum [32]. So probably it is hormones that link tissue culture traits to their correlated agronomic traits, such as those studied in this experiment. Such hormonemediated mechanism can also explain the phenomenon why such correlation between the two kinds of trait in immature embryo culture was less significant than that in mature embryo culture. It is known that endogenous hormone levels change greatly in embryo development [6]. In immature embryo culture, it is difficult to get the uniform size of explants, and this may distort the outcome of analysis. Based on these discussions, we get the following theme:
which implies that the relationship between the two traits might also be hold even there exist some nongenotype variations. In conclusion, tissue culture traits, especially those from mature embryos, have credible correlation with major agronomic traits. These results may facilitate the study of the genetic control of regeneration from wheat callus and can be used as an easy way to screen genotypes with good tissue culture traits. This conclu-
sion needs to be tested with more wheat cv.s or cereal species in the future.
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W. Li et al. / Plant Science 164 (2003) 1079 /1085 [19] M. Gallo-Meagher, R.G. English, A. Abouzid, Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus, In Vitro Cell. Dev. Biol.-Plant 36 (2000) 37 /40. [20] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassay with tobacco tissue cultures, Physiol. Plant. 15 (1962) 473 /497. [21] O.L. Gamborg, R.A. Miller, L. Ojima, Nutrient requirements of suspension cultures of soybean root cells, Exp. Cell Res. 50 (1968) 150 /158. [22] G.Q. Guo, F. Maiwald, P. Lorenzen, H.-H. Steinbiss, Factors influencing T-DNA transfer into wheat and barley cells by Agrobacterium tumefaciens , Cereal Res. Commun. 26 (1998) 15 /22. [23] SPSS Inc., SYSTAT for WINDOWS: statistics, version 6.0 edition, SPSS Inc., Chicago, IL, 1996. [24] R.J. Mathias, Factors affecting the establishment of callus culture in wheat, in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry, vol. 13, Springer, Berlin, 1990, pp. 24 /45. [25] M. Ozgen, M. Turet, S. Altmok, C. Sancak, Efficient callus induction and plant regeneration from mature embryo culture of
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Relationship between tissue culture and agronomic traits of spring wheat Wei Li, Chen-Hui Ding, Zhong Hu, Wei Lu, Guang-Qin Guo * Institute of Cell Biology, Lanzhou University, Lanzhou 730000, China Received 8 November 2002; received in revised form 11 February 2003; accepted 13 February 2003
Abstract Plant regeneration was achieved from mature and immature embryos of ten spring wheat cultivars (cv.s), which showed strong genotype effect on both callus growth rate and the frequency of plant regeneration. Relationship between tissue culture and agronomic traits was surveyed by correlation and regression analysis, revealing credible relationship between the two traits, especially in mature embryo cultures. It was found that the frequency of regeneration from callus derived from mature embryos could be estimated from kernel number per spikelet, effective tillering and longevity; which was less significant in immature embryo culture. Such relationships may be due to the fact that both traits are influenced by the endogenous hormone levels. This result suggests that it is possible to screen genotypes with good tissue culture traits directly at the agronomic trait level, a practice rather simple and straightforward. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Triticum aestivum ; Tissue culture; Agronomic traits; Correlation analysis
1. Introduction Wheat is one of the most important crops in the world. With the development of plant molecular biology and genetic engineering, its transformation has become one of the central issues in molecular breeding, which depends largely on reliable system of plant regeneration via tissue culture [1]. Many factors can affect wheat regeneration, such as explant source [2 /5], donor plant growth condition [5,6], culture medium [7 /9], and genotype [9 /13], among which genotype is often the dominant one. Unfortunately, however, for a given cultivar, the cultural response determined by its unique genotype cannot be known before being tested in experiment, because we know very little about the related mechanisms. In several cases, in vitro regeneration is shown to be a quantitative trait [14,15], and the hexaploid nature of wheat makes the situation even more complex. However, if the critical tissue culture traits could be correlated to one or more agronomic
* Corresponding author. E-mail address: [email protected] (G.-Q. Guo).
traits that are much easier to measure, we will have a rather simple way to predict the outcome of tissue culture in advance, avoiding the need to test large number of genotypes at the tissue culture step, which is very expensive, laborious and time consuming. However, such works are scarce [16]. In tissue culture of cereals including wheat, 2,4dichlorophenoxyacetic acid (2,4-D) is often used for callus induction, and different combinations of auxin such as napthaleneactic acid (NAA) and indoleacetic acid (IAA) and cytokinin (including zeatin, kinetin and 6-benzyladenine (6-BA) etc.) were used for stimulating plant regeneration from calli [14]. Recently, a substituted phenylurea named thidiazuron (TDZ) is shown to be an efficacious regulator of in vitro morphogenesis of many dicot plant species [17], and a few reports also demonstrate its positive effects on wheat [13,18] and other cereal species [18,19], showing that comparison with other commonly used hormones, TDZ is among the best for wheat regeneration [13,18]. So this growth regulator was incorporated into our present tissue culture regime to promote plant regeneration from wheat calli induced by 2,4-D from ten different spring
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cultivars (cv.s), and the relationship between their tissue culture and agronomic traits was tested.
2. Materials and methods 2.1. Plant materials Ten cv.s of spring wheat (Triticum aestivum L.) (Tables 1 and 2), kindly provided by Jia at the State Key laboratory of Arid Agroecology, Lanzhou, were used as sources of immature and mature embryos for callus induction and plant regeneration. They were cultivated in the northwest regions of China that have very similar semi-arid climate of plateau. 2.2. Plant growth and agronomic traits measurements Seeds were sown in the garden field at Lanzhou University in mid-March of 1999 and 2000. In all the experiments, rows were 5 m long with a density of 20 seeds m 1 and separated by 0.25-m from each other. Plants were irrigated properly throughout the growing seasons to avoid any serious water stress and fertilized with urea at sowing and tillering stages to give sufficient nutrition. Weeds were periodically removed by hand. Although the growth environment was not artificially controllable, all the cv.s grew in the same field and thus subjected to parallel conditions, so variation in their agronomic traits is primarily caused by the genotype effects. For agronomic traits analysis, 30/40 randomly selected plants of each cv. were used. At anthesis, total tillers and effective tillers per plant were counted. Effective tillering was defined as the ratio of effective
to total tillers per plant. At physiological maturity, plant height was measured from the soil surface to the top of the spike on the main shoot. The total and effective (with kernels) spikelets per spike, effective spikeleting (percent effective in total spikeletes), and longevity were recorded at harvesting. The spikes of these measured plants were oven-dried, and their kernel numbers per spikelet and spike were determined. The average kernel weight for each cv. was calculated as the total kernel weight divided by their numbers. 2.3. Tissue culture and plant regeneration 2.3.1. Medium The basic medium used both for callus induction and plant regeneration in this experiment was MB, which contained MS salts [20], B5 vitamins [21], 2 mg l 1 glycine, 300 mg l 1 glutamin, 500 mg l 1 casein hydrolysate, 3% sucrose and 0.7% agar at pH 5.8 [22]. 2.3.2. Immature embryo culture Two weeks after anthesis, spikes with immature embryos about 0.5 /1.0 mm in size were harvested from the fields and surface-sterilized for 1 min in 70% ethanol. The immature embryos were aseptically excised from caryopses and placed with the scutellum upwards on the MB medium supplemented with 2 mg l 1 2,4-D and 1 mg l 1 abscisic acid (ABA) at 259/1 8C in darkness to induce callus. To initiate shoots, the calli were transferred onto the MB medium with TDZ and cultured for 2 weeks at 259/1 8C in a 16-h light:8-h dark cycle provided by 40-W white fluorescent tubes (1500 lx). The calli with one or more buds were then transferred onto 1/2 MB medium free of growth regulators to induce root formation.
Table 1 Differences in callus formation and plant regeneration between ten spring wheat cv.s from immature and mature embryos Genotype
Tableland 602 8797-21 Yong 3263 98j51 021-128 92jian46 Monk-head 8139 8275 Dingxi 24 Mean LSD (0.05) CV (%) a b c
Fc (%)
Average FW of callus/explanta (mg)
Frequency of regeneration from callus (%)
Ib
Mc
I
M
I
M
100 98.9 100 100 100 100 98.8 97.7 100 100 99.5 2.6 1.5
100 100 100 100 100 100 100 100 100 100 100 0 0
48.4 45.0 40.4 53.6 60.0 63.5 64.2 72.7 62.6 59.8 57.0 19.3 23.6
70.0 56.7 65.6 127.7 74.4 63.0 153.4 44.2 74.9 105.4 83.5 77.7 63.1
38.1 34.3 52.5 30.0 60.3 45.3 42.7 44.7 57.1 28.1 43.3 7.7 26.0
51.5 50.7 82.0 58.9 56.7 33.7 58.9 37.5 46.1 58.8 53.5 6.9 25.0
Recorded on the 20th day of callus induction. I, immature embryos. M, mature embryos.
81.6 74.8 65.1 88.3 56.4 79.8 104.2 83.3 101.5 96.7 83.2 6.1 18.2 Tableland 602 8797-21 Yong 3263 98j51 021-128 92jian46 Monk-head 8139 8275 Dingxi 24 Mean LSD (0.05) CV (%)
64.4 49.8 75.5 56.6 56.5 56.5 43.1 43.7 51.9 70.9 56.9 8.3 19.6
43.8 40.1 37.6 27.2 30.0 35.1 35.6 43.5 36.3 34.8 36.4 1.1 14.1
111.7 110.3 110.3 115.7 113.3 106.7 115.7 113.3 119.7 118.7 113.5 2.0 0.9
Height (cm) Tillers/plant Effective Effective Spikelets/spike Effective Effective Kernel number/spikelet Kernel number/spike Average weight Longevity tillers/plant tillering (%) spikelets/spike spikeleting (%) of kernel (mg) Genotype
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2.3.3. Mature embryo culture Mature seeds were surface sterilized with 70% ethanol for 30 s followed by 0.1% mercuric chloride for 20 min, then washed three times with sterile water. They were inoculated on 1/2 MB basal medium at 259/1 8C for germination. After 20 /24 h, the germinated embryos were aseptically picked out with a scalpel and transferred onto callus induction media. The media and the conditions used for callus induction, shoot initiation and rooting were all the same as those described above for the immature embryos. In both mature and immature embryo cultures, the frequency of callus induction (Fc) and the average fresh weight (FW) of callus per explant were determined 4 weeks after callus induction; and their regeneration frequency (Fr) (percent callus forming shoots) were calculated after 2 weeks on the regeneration medium.
About 30 explants per cv. were used in each treatment of tissue culture; and the agronomic traits measurement was done with 30/40 plants for each cv. Every treatment/measurement in this experiment had at least three replicas in parallel. Genotype effects on tissue culture of mature and immature embryos and agronomic traits were tested by analysis of variance (ANOVA) and leastsignificant-difference tests. Relationships between every possible pairs of various agronomic and tissue culture traits were determined by Pearson correlation analysis. Stepwise multiple regression analysis was used to evaluate the correlation of various agronomic traits to the critical tissue culture traits, and regression coefficients were described when significant (P /0.05). All statistical analyses were performed with the statistical package of SYSTAT 7.0 [23].
52.0 47.6 61.4 22.1 27.3 37.2 35.0 31.3 20.5 17.2 35.2 17.6 46.9
19.1 17.4 19.4 25.8 18.6 19.9 16.7 18.8 18.8 21.0 19.5 1.0 12.6
18.6 15.9 19.3 18.7 18.0 18.2 15.9 16.3 18.0 20.3 17.9 1.4 8.8
97.0 91.1 99.3 72.7 96.7 91.1 94.6 86.5 95.8 96.5 92.1 3.4 8.4
3.4 3.2 3.9 3.0 3.1 3.0 2.7 2.7 2.9 3.5 3.1 0.3 12.6
2.4. Statistical analysis
1.7 1.9 2.6 1.7 2.2 2.2 3.1 1.6 1.2 2.1 2.0 0.6 29.8
3. Results
3.8 5.1 4.5 9.8 9.4 7.0 9.5 5.9 7.9 13.2 7.6 2.5 40.4
Table 2 Differences in agronomic traits between ten spring wheat cv.s
W. Li et al. / Plant Science 164 (2003) 1079 /1085
3.1. Effect of TDZ on plant regeneration Since TDZ is a relatively new regulator used for wheat regeneration [13,18], we did a preliminary experiment to determine its optimal concentration, which was shown to be 1.0 mg l 1 and was the same as that reported in [13]. So it was used for all cv.s in this experiment. When the calli forming buds were transferred onto rooting media, new shoots continued to form, and after about 2 weeks, roots appeared from the base of many shoots. However, some shoots formed no root even after 6 weeks. Higher concentration of TDZ tended to suppress root formation from shoots (data not shown).
/0.183 0.665** 0.625** /0.223 0.428* 0.152 0.271 0.535** 0.126 0.342 0.176 0.075 0.246 0.341 0.394* /0.386* /0.247 0.003 0.028 0.020 0.000 0.098 0.436* /0.204 0.324 0.256 0.216 /0.011 0.330 0.421* /0.233 0.557** 0.300 0.168 0.113 0.199 0.150 0.057 0.202 /0.310 0.002 0.004 /0.004 0.011 /0.358 0.473* 0.154 /0.110 0.227 /0.013 0.644** /0.158 /0.014 /0.24 0.220 0.196 /0.003 0.113 0.647** /0.490** /0.332 0.543** 0.366* 0.498** 0.068 /0.239 0.382* 0.451* /0.493** 0.434* /0.090 0.118 /0.589** 0.393* 0.370* 0.074 0.285 /0.022 0.266 0.232 /0.115 0.076 /0.219 0.385* /0.112 0.454* 0.376* 0.392* 0.200 /0.656**
*, **, Significantly different at P B/0.05 and P B/0.01, respectively.
0.085 0.159 0.367* /0.548** 0.600**
14 13 12 11 10 9 8 7 6 5
/0.022 /0.374* /0.249 0.050 0.938** 0.121 0.675** /0.056 0.873** /0.266
In immature embryo cultures, Fc was related to kernel numbers per spike (r /0.421, P B/0.05) and effective spikelets per spike (r /0.557, P B/0.01), but Fr was only related to effective spikeleting (r /0.473, P B/0.05). Weight of callus from immature embryos was positively related to shoot length (r /0.394, P B/0.05) and tillers per plant (r /0.436, P B/0.05), but negatively to kernel numbers per spikelet (r / /0.386, P B/0.05). The Fr in mature embryo cultures was related to effective spikelets per spike (r /0.428, P B/0.05) and significantly to kernel numbers per spikelet (r /0.665, P B/0.01), spike (r /0.625, P B/0.01) and effective tillers per plant (r /0.535, P B/0.01). The weight of callus showed correlation to effective tillers/plant (r /0.367, P B/0.05), and significantly to shoot length (r /0.734, P B/0.01), tillers per plant (r /0.655, P B/0.01) and longevity (r /0.596, P B/0.01). Multiple regression analysis of each tissue culture trait against agronomic traits (Table 4) revealed that total
4
3.4. Correlation and regression analysis between agronomic and tissue cultural traits
3
The agronomic traits measured in this experiment are listed in Table 2. They represent major agronomic traits of wheat and exhibited a wide range of coefficient of variation (CV) from 0.9 (longevity) to 46.9 (effective tillering), indicating that they were influenced to very different degrees by genotype.
1 2
3.3. Agronomic traits
Table 3 Pearson correlation coefficients among investigated agronomic and tissue culture traits of ten spring wheat cv.s
In both immature and mature embryo culture, the Fc was extremely high, nearly (cv.s 8797-21, 8139 and Monk-head) or equal to 100%, with no or very little variations between different genotypes. However, genotype effect was striking on callus growth rate (expressed as FW of callus per explant on the 28th day) and plant regeneration (Table 1). The mean Fr was higher in mature embryos (53.5%) than immature embryos (43.3%). In mature embryo cultures, the highest Fr was obtained with cv. Yong 3263 (82%), significantly higher than that of 60.3% with cv. 021-128 in immature embryo cultures. The lowest Fr showed less difference between mature (cv. 92jian46, 33.7%) and immature (cv. Dingxi 24, 28.1%) cultures. Pearson correlation analyses showed that the FW of callus per explant was closely correlated between immature and mature embryo cultures (r /0.572, P B/ 0.01), but their Fc and Fr showed no significant relations to each other or to the weight of callus among different cv.s within each culture. It is noteworthy that no significant correlation existed between the Fr from immature and mature embryos (Table 3).
15
16
3.2. Effect of genotypes on tissue culture
0.734** /0.129 0.010 /0.178 0.227 0.219 /0.011 0.367* 0.655** /0.084 0.596** /0.131 0.272 0.572** 0.160
W. Li et al. / Plant Science 164 (2003) 1079 /1085
1 Shoot length 2 Kernel number/spikelet 3 Kernel number/spike 4 Weight of kernel 5 Effective spikelets/spike 6 Spikelets/spike 7 Effective spikeleting 8 Effective tillers/plant 9 Tillers/plant 10 Effective tillering 11 Longevity (days) 12 Fr of immature embryos 13 Fc of immature embryos 14 FW of callus/immature embryo 15 Fr of mature embryos 16 FW of callus/mature embryo
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Table 4 Regression of tissue cultural traits against agronomic traits Tissue culture traits
R2
Agronomic traits
Coefficient
Standard coefficient
P
Fr of mature embryos
0.679
FW of callus/mature embryo
0.804
Fr of immature embryos
0.224
Fc of immature embryos
0.311
FW of callus /immature embryo
0.436
Constant Kernel number/spikelet Effective tillering Longevity Constant Shoot length Effective tillers/plant Effective tillering Constant Effective spikeleting Constant Effective spikeletes/spike Constant Tillers/plant Kernel number/spikelet Weight of kernel
/150.709 20.672 9.062 1.066 /57.325 0.823 3.892 0.505 /20.213 0.689 90.090 0.528 33.801 2.892 /13.004 11.512
/ 0.606 0.410 0.318 / 0.614 0.591 0.411 / 0.473 / 0.557 / 0.660 /0.380 /0.438
/ 0.000 0.002 0.009 / 0.000 0.000 0.000 / 0.008 / 0.001 / 0.001 0.017 0.024
variances accounted for were ranged from 22.4 to 80.4% of tissue culture traits. The Fr in immature embryo cultures against agronomic traits has the least R2 (0.224), and only effective spikeleting was included in the regression equation. On the other hand, the R2 of Fr in mature embryo cultures against agronomic traits was 0.679, and three agronomic traits were included in the equation, among them, kernel number per spikelet contributed most, followed by effective tillering and longevity. Though the correlation coefficient between the Fr in mature embryo cultures and kernel numbers per spike was statistical significant (r /0.625, P B/0.01), due to its closed partial correlation with kernel numbers per spikelet (r /0.938, P B/0.01), the kernel numbers per spike was not included in the regression equation. For weight of callus in mature embryo cultures, it has good regression relationship with the agronomic traits (R2 /0.804), which were effective tillers per plant, shoot length and effective tillering; in immature embryo culture, this figure was R2 /0.436, which was contributed by tillers/plant (2.892), kernel number per spikelet ( /13.004) and weight of kernel (11.512).
4. Discussion Genotype was one of the most important factors for successful regeneration from callus of wheat [24], and has been investigated intensively [9 /13,25]. But little is known about the nature of such genetic control. In the present study, all the cv.s were spring genotypes that had developed for cultivation in the northwest regions of China, which have very similar semi-arid climate of plateau. In addition, we grew all the cv.s in the same field provided with parallel conditions to minimize any
environment-induced physiological variation in their agronomic traits. Although TDZ was shown to be the best for wheat regeneration among the growth regulators used so far [13,18], in our present results different cv.s still gave varied Fr from 27.1 to 82%, suggesting that the strong genotype effect can not be overcome by simply manipulating the medium compositions or other experimental parameters. Ozgen et al. showed that the correlations between callus induction rate, weight of callus and regeneration capacity in both mature and immature embryo cultures of wheat were insignificant, suggesting that these culture traits are independent from each other and likely to be controlled by different genes [25]. Our present results support this conclusion, since different tissue culture traits are related to different agronomic traits (Tables 1 and 4). Genetic control of regeneration from callus has been investigated in other cereal species. It is shown that several genes control the regeneration from embryoderived callus of barley [26,27]. Peng and Hodges also presented evidence in rice tissue culture that regeneration ability is under control of both nuclear and cytoplasmic genes [28]. In sorghum, Ma et al. found that the ability to form regenerable callus varied from genotype to genotype, was heritable, and acted as a dominant trait [29]. They thought that at least two gene pairs were involved. Willman et al. suggested at least one gene or a block of genes control the expression of somatic embryogenesis from maize [30]. Taken together, these studies strongly support the view that regeneration from callus is a quantitative trait controlled by multiple genes. Recently, Bregiter and Campbell identified new quantitative trait loci for plant regeneration of barley [15]. Due to its hexaploid nature, such work in wheat is
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more difficult. With the rapidly increasing number of important agronomic traits being mapped to their chromosomal loci, from which finding those closely correlated with the in vitro regeneration capacity may be helpful to decipher the genetic control of the latter. Our results tend to support this assumption, and the tissue culture traits in mature embryo culture appeared more reliable in correlation with the agronomic traits. The frequency of regeneration from callus of mature embryo can be regressed against kernel number per spikelet, effective tilllers and longevity (Table 4). The weight of callus has correlation with shoot length, tillers per plant and effective tillering (Table 4). At the molecular level, these imply that genes controlling the correlated agronomic and tissue culture traits are overlap or interact to each other. It has been proposed that genotype effect may be related to variations in endogenous hormone levels [31]. Hess and Carman showed that genotype and environment could influence the response of explant through the level or ratio of endogenous hormone [6]. The application of exogenous growth regulators, in combination with those of endogenous origin produced under the specific internal genetic control and environmental influence, can alter the hormone balance in favor of organogenesis or embryogenesis in lieu of their normal mode of development, eliciting a specific response [14]. Likewise, hormones can also influence agronomic traits. For example, cytokinins can enhance tillering in sorghum [32]. So probably it is hormones that link tissue culture traits to their correlated agronomic traits, such as those studied in this experiment. Such hormonemediated mechanism can also explain the phenomenon why such correlation between the two kinds of trait in immature embryo culture was less significant than that in mature embryo culture. It is known that endogenous hormone levels change greatly in embryo development [6]. In immature embryo culture, it is difficult to get the uniform size of explants, and this may distort the outcome of analysis. Based on these discussions, we get the following theme:
which implies that the relationship between the two traits might also be hold even there exist some nongenotype variations. In conclusion, tissue culture traits, especially those from mature embryos, have credible correlation with major agronomic traits. These results may facilitate the study of the genetic control of regeneration from wheat callus and can be used as an easy way to screen genotypes with good tissue culture traits. This conclu-
sion needs to be tested with more wheat cv.s or cereal species in the future.
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