吉田｜Genotypic Difference of Sorghum bicolor in the Callus Formation

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本頁は，ＰＰＳ5(3)(2002)に掲載されたものです．図表一部省略．

Genotypic Difference of Sorghum bicolor in the Callus Formation and Callus Growth on Aluminum-Containing Medium

Anas* and Tomohiko Yoshida**
(*United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology;
**Faculty of Agriculture, Utsunomiya University 321-8505, Japan)

Abstract: We examined the genetic variation in the callus performance of the Al-tolerant and Al-susceptible genotypes of sorghum (Sorghum bicolor L.) on Al-containing medium. Addition of Al to the medium with a low pH, low Ca and low phosphate resulted in a severe decrease in the percentage of callus formation (CF) in both Al-tolerant and Al-susceptible genotypes. However, the percentage of callus formation in the explants from Al-tolerant genotypes was higher than that from susceptible genotypes on Al-containing medium. The genotypic difference in the relative growth of calli on Al-containing medium and in the suppression of callus formation by Al was in agreement with that in the hematoxylin staining score. Al might have been considered as a limiting factor that affected the callus growth. There was a significant correlation (r = -0.664*) between the percent relative suppression of callus formation by Al and the percentage of callus formation on Al-containing medium. The callus growth in fresh weight and size on Al-containing medium relative to that on Al-free medium (relative growth on Al-containing medium) was greater in Al-tolerant genotypes than in the other genotypes. The relative growth of callus in fresh weight and size on Al-containing medium significantly correlated (r = -0.820** and r = -0.956**, respectively) with the percent relative suppression of callus formation by Al. The tissue culture is considered to be a useful tool for selecting Al-tolerant haploids at the callus stage.

Key words: Aluminum, Sorghum bicolor, Tissue culture.
Receive 20 June 2001. Accepted 9 February 2002. Corresponding author: T. Yoshida
*Address: Plant Breeding Program Study, Agriculture Faculty, Padjadjaran Univ. Bandung 40600, Indonesia.
Abbreviation: CF, callus formation; GS, growth size; GW, growth weight; MS, Murashige and Skoog; RGS, relative growth size; RGW, relative growth weight; RSC, relative suppression of callus formation.

The use of tissue culture for selecting Al-tolerant genotypes has been reported in carrot (Daucus carota L.) (Ojima and Ohira, 1983), sorghum (Smith et al., 1983; Duncan et al., 1995), Nicotiana plumbaginifolia (Conner and Meredith, 1985a), and wheat (Barnabas et al., 2000). Previous screening by tissue culture was generally made based on the growth of calli derived from the explants of each genotype on Al-containing medium. However, even when the variation in Al tolerance was not detected in the original explants it appeared during the culture process.
Parrot and Bouton (1990) reported that alfalfa plants from the Al tolerant germplasm rapidly expressed Al tolerance at the callus stage. If the performance of Al tolerance in tissue culture is similar to that at the plant level, tissue culture may be more useful for breeding programs because selection can be earlier and faster than that in the field. Moreover, the selection by tissue culture can be applied to identify Al-tolerant plants in a segregating population.
Although this approach has not been applied to sorghum, previous work with alfalfa showed that Al tolerance of plants was also expressed at the cellular level (Parrot and Bouton, 1990). This approach should be very useful to detect the tolerant callus derived from anther culture for the production of double haploid plants. In the subsequent generations, the Al-tolerant character should be expressed as an important agricultural trait in the field experiment.
The effectiveness of the selection for Al-tolerance by tissue culture is influenced by the availability of Al3+ in the culture medium that can suppress callus growth (Conner and Meredith, 1985b; Taylor, 1995). Therefore, the availability of Al3+ as the most toxic form of Al for the plants in the media during culture must be considered.
The present study aims to study the genetic variation of Al-tolerance in sorghum and to elucidate the callus performance of the Al-tolerant and Al-susceptible genotypes on the Al-containing medium with high availability Al3+ for the callus.

Materials and Methods
1. Plant materials and explant preparation
Ten selected sorghum genotypes were used in this experiment (Table 1). All of these genotypes except G1 were previously screened for Al tolerance by hematoxylin staining and the growth on the soil in pots with Al added (Anas and Yoshida, 2000). G4, SPA 2, and SPAD were considered as tolerant genotypes and G2, G5, C9 x H11, C9 x H13 were considered as susceptible genotypes (Table 1). G3 and G7 showed intermediate tolerance by the hematoxylin staining method. The G1 genotype was chosen because of its high cell proliferation rate in tissue culture (Can and Yoshida, 1999).
Surface-sterilized seeds were soaked in water with a few drops of TWEEN added for 20 min and then in a calcium hypochlorite (60%) solution for 10 min. The seeds were then rinsed three times with sterile water, and soaked in sterile water in a petri dish at room temperature. The water was renewed every day. After small radicles had emerged, seeds were rinsed three times with sterile water and allowed to grow on a solidified hormone-free MS medium. The seeds were then kept in an incubator in darkness at 25oC. After six or ten days, explants for callus induction were obtained from the sorghum plantlets.
Calli were induced from the cut pieces of mesocotyl at zones III and IV, which were specified by Gendy et al. (1996) (Fig. 1). Zone III is the sub-apical zone and zone IV is the base of the apical meristem. The transverse thin cell layers, 0.3 - 1 mm thickness, of the mesocotyl (called explants, hereafter) were made by cutting mesocotyl transversely at these zones.

2. Media
Calli were induced on the medium containing Murashige and Skoog (MS) inorganic salts, 2.0 mg L-1 kinetin, 1.0 mg L-1 IAA and 2.5 mg L-1 2,4-D. The culture medium was adjusted to pH 5.8. This medium was referred to as Al-free medium.
To examine the Al toxicity, we used the standard MS medium modified according to the report by Conner and Meredith (1985b). It is known that Al3+ is the most toxic form of Al to the plant and that the availability of Al3+ depends on the solution pH (Koyama et al., 1990). The pH of the culture medium was adjusted to pH 4, at which Al3+ was considered as the dominant form of Al in the solution.
The activity of Al3+ in culture medium is also limited by the formation of a solid phase complex with phosphate in the medium (Ojima and Ohira, 1983). The precipitation of Al in the standard MS culture medium might be eliminated by reducing the phosphate concentration and the pH (Conner and Meredith, 1985b). In addition, the calcium concentration should also be reduced because the calcium ion is well known to partially alleviate Al toxicity. In this experiment, therefore, the phosphate concentration was reduced from 1250 (M to 10 (M and calcium was reduced from 3000 (M to 100 (M. Callus was transferred to fresh medium periodically to eliminate micronutrient deficiency.
The Al concentration of the Al-containing medium was adjusted to 400 (M by adding AlCl3･6H2O to the medium, after autoclaving but immediately prior to gelling of the medium.

3. Culture condition
The explants were cultured on 20 mL of the media with and without Al added for the observation of the callus formation. For each treatment, at least 1050 explants were planted over three replications. Petri dishes with explants were placed in an incubator in darkness at 30oC. Observations were made at two, five and eight weeks after planting.
To examine the effect of Al on the callus growth, we subcultured the calli twice on Al-free medium at one- or two-month intervals before the treatment with Al. After subculture on Al-free medium twice, the calli were cut into small pieces as uniform as possible and planted on the medium with or without Al added in petri dishes. Two or three calli were planted in each petri dish. At least 30 calli per treatment were used for one replication. The initial weight and size of each callus were measured individually.
Since we observed in a preliminary study that calli did not show visible growth during the first one or two weeks after planting, the weight and size of each callus was measured at three-week intervals up to the 11th week. Thus, all calli were transferred to fresh medium at three-week intervals to prevent micronutrient deficiency.

4. Calculation and Experimental design
The percentage of callus formation (CF) is calculated as,
CF = (number of explants with calli)(total number of explants)-1 x 100.
The relative suppression of CF by Al (RSC) is calculated as,
RSC = 100 - {(CF on Al-containing medium)(CF on Al-free medium)-1( x 100.
Fresh callus weight was determined as the difference between the weight of a petri dish with callus and that after removal of the callus. The percent growth of callus in fresh weight (GW) is calculated as (Conner and Meredith 1985a),
GW = {100 x (Wf - Wi)}(Wi)-1
where, Wi and Wf are the initial and final fresh weight of callus, respectively. GW on Al-containing medium relative to that on Al-free medium is expressed by relative growth in weight (RGW) and calculated as,
RGW = {(GW on Al-containing medium)(GW on Al-free medium)-1} x 100.
A multiplication of the longest and the widest axes of callus was used to show callus size. The percent growth in size of callus (GS) is calculated as,
GS = {100 x (Sf - Si)}(Si)-1
where Si and Sf are the initial and final size of callus, respectively. GS on Al-containing medium relative to that on Al-free medium was shown by relative growth in size (RGS) and calculated as,
RGS = {(GS on Al-containing medium)(GS on Al-free medium)-1} x 100.
The CF, RSC, RGW and RGS of each genotype were then analyzed in randomized complete block design. Three replications were designed both in Al-free medium and Al-containing medium. Those with a similar planting time were regarded as one replication in this experiment.

Results and Discussion
All genotypes used in this experiment formed calli on Al-free medium, but the percentage of the explants that formed calli (CF) significantly varied with the genotype (p < 0.01) either on Al-containing or Al-free medium (Fig. 2). Genotype SPA2, G1 and G3 formed more calli on Al-free medium than the other genotypes, and the genotype SPAD formed the fewest calli on Al-free medium, suggesting the poor callus formation in tissue culture. There were two types of calli, friable and compact calli (Fig. 3). Gendy et al. (1996) also reported that they observed the formation of both friable and compact callus on sorghum mesocotyl tissue. In all cases, the genetic variance was higher than the individual variance (Table 2), suggesting that the difference in callus performance among genotypes was genetic. Although Gendy et al. (1996) reported that the explant size had a significant influence on callus formation, the size of explants was not the main factor that caused a different callus performance among genotypes in this study, because we used explants with a relatively uniform size for callus induction in this experiment.
Addition of Al to the medium with low pH, low Ca and low phosphate resulted in a severe decrease in the percentage of callus formation (CF) in both Al-tolerant and Al-susceptible genotypes (Fig. 4.). The direct effect of the lack of these major inorganic nutrients (Ca and P) was eliminated by transferring the calli to the fresh medium at three-week intervals.
Parrot and Bouton (1990) reported that medium modification other than of Al (low pH, low Ca, low phosphate) resulted in a depression of callus growth relative to that on standard medium for both Al-tolerant and Al-susceptible genotypes. However the addition of Al to this modified medium resulted in an additional decrease in callus growth of the Al-susceptible population of Alfalfa, but not in the Al-tolerant population. In this experiment Al was added at a concentration of 400 (M. Taylor (1995) reported that Al at a concentration above 200 (M inhibited the callus growth in Phaseolus vulgaris. However, an excessively high Al concentration was required for the complete inhibition of the cell growth in the experiment by Conner and Meridith (1985b) with Nicotiana plumbaginifolia.
There was a significant variation among genotypes in callus performance in the present experiment. The relative suppression of callus formation by Al (RSC) showed a significant difference (p < 0.01) among genotypes (Table 2). The RSC in the Al-tolerant genotypes was lower than that in the susceptible genotypes. This suggested that the Al-tolerant genotypes were more capable of forming callus than susceptible genotypes on Al-containing medium. Some of the susceptible genotypes showed 100% RSC (Table 2 and Fig. 2). This means that they did not form calli on Al-containing medium.
The RSC data were also consistent with the CF data on Al-containing medium in all genotypes (Fig.2). There was a significant negative correlation (r = -0.664*) between RSC and CF on Al-containing medium. Genotypes that showed low RSC also showed high capability of callus formation on Al-containing medium. There was a significant variation (p < 0.01) among genotypes in RGW. The addition of Al to the medium resulted in decrease of callus weight in Al-tolerant and Al-susceptible genotypes. However, the RGW of tolerant genotypes was still higher than that of the other genotypes with exception of SPAD (Table 2). The genotype SPAD, classified as Al-tolerant by the previous screening method (Table 1), showed a response different from other tolerant genotypes, probably because of its low callus formation even in Al-free medium as was mentioned above and shown in Fig. 2. This suggested that the fresh callus weight of tolerant genotypes were significantly heavier than that of the susceptible genotypes on Al-containing medium. Al-susceptible population of Alfalfa also showed an additional decrease in callus growth when it was planted in the modification medium supplemented with Al (Parrot and Bouton, 1990).
There was a significant variation (p < 0.01) among genotypes in RGS. Generally, RGS of Al-tolerant genotypes was higher than that of the other genotypes with the exception of SPAD as was shown in Table 2. The RGS did not show a significant correlation with the RGW at the 4th week after planting (data not shown), but it showed a significant correlation (r = 0.824**) at the 11th week (Table 3). RGW and RGS also showed a significant negative correlation (r = -0.820** and r = -0.956**) with RSC. Genotypes that showed a high RGW and RGS also showed low RSC, as was shown for Al-tolerant genotypes such as G4 and SPA2 (Table 2). The variation in Al-tolerance among these genotypes has been observed by hematoxylin staining and the growth on the soil with Al in pots (Anas and Yoshida, 2000). Variation in Al tolerance was also expressed at the callus stage in the present study. In all cases, the callus performance showed a significant correlation with the Al tolerance score in hematoxylin staining (Table 3), which suggests that Al might be the major limiting factor that affected the callus growth in Al-containing medium in this experiment. RSC showed a significant correlation (r = 0.852**) with the hematoxylin score. Genotypes that showed a low RSC (G4 and SPA2) showed high tolerance score 1 by the hematoxylin staining screening method. A significant correlation was also observed between the hematoxylin staining and either RGS or RGW. Genotypes that had been classified as tolerant by the hematoxylin staining method generally showed a good performance on Al-containing medium.
Callus performance was not significantly correlated with the score of growth response to Al in the soil in pot (Table 3). The evaluation of tolerant genotype by a short-term (15 d) growth response to the Al by the pot method might curtail the accuracy of the screening method. Detection systems not dependent on the rate of seedling or root development, would improve the success of screening procedure (Konzak et al., 1976). However, the growth response to the Al in pot method showed a significant correlation with the score of hematoxylin staining (Anas and Yoshida, 2000).

Conclusion
The percentage of callus formation and the callus growth on Al-containing medium varied with the genotypes, indicating that the tissue culture could be used to screen Al-tolerant genotypes. The differences in callus growth and the percentage of callus formation among genotypes were in agreement with those in the hematoxylin staining.
Tissue culture is considered as a useful tool for selecting Al-tolerant haploid plants at the callus stage.

References


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Table 1.  Ten sorghum genotypes used and their aluminum tolerance score. 

Score 1, tolerant; score 5, susceptible; RRL, relative root length in growth response method. G2, Hegari; G5, RTx403. 1)
Data were copied from Anas and Yoshida (2000). 2)Data was from Duncan et al. (1995)

Table 2.  Percent relative suppression of callus formation by Al (RSC), relative growth in fresh weight of callus on 
Al-containing medium (RGW) and relative growth in size of callus on Al-containing medium (RGS) at 11th week after
 planting in various sorghum genotypes.

Values followed by a common letter in the column are not significantly different at the 0.05 level by Duncan's Multiple
 Range Test. Genotype G2, G5, C9xH13 were excluded from the analysis because of no variation over replications.

Table 3.  Correlation among relative suppression of callus formation (RSC), relative growth in fresh weight of calli on 
Al-containing medium (RGW), relative growth in size of calli on Al-containing medium (RGS), hematoxylin staining 
score on Al medium (HS) and score of growth response to Al added in the soil in pot (ASP).

*, **; Significant at 0.05 and 0.01 level, respectively; 1) Data were copied from Anas and Yoshida (2000). CF was 
measured at eight week after planting; GS and GW were measured at 11th week after planting. 

Fig. 1. Sorghum mesocotyl.  The transverse thin cell layer (1 - 0.3 mm) was cut from the mesocotyl at zones III and IV.

Fig. 2. Percentage of callus formation (CF) on Al-free medium, that on Al-containing medium and percent relative
 suppression of callus formation by Al (RSC) at eight weeks after planting.
 
A common letter on a graph is not significantly different at the 0.05 level by Duncan's multiple range test. Genotype 
G2, G5 and C9xH13 were excluded from the analysis because no callus variation over replications was observed.

Fig. 3. Two types of calli. A; compact callus. B; friable callus.

Fig. 4.  Differences in callus formation and callus growth of the same genotype (G1) between Al-free medium and 
Al-containing medium. A, callus formation on Al-free medium (formed callus are enclosed by circle); B, callus 
formation on Al-containing medium; C, callus growth on Al-free medium; D, callus growth on Al-containing medium.

Anas and Yoshida - Al Tolerance of the Callus of Sorghum Genotype