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Effects of abscisic acid on callus induction and regeneration of different wheat cultivars to mature embryo culture

Bahman Fazelienasab1, Mansour Omidi2 and Mehdi Amiritokaldani3

1 M.Sc student biotechnology at Tehran University Email fazelinsb@ut.ac.ir
2
Assistant professor at Tehran University
3
M.Sc student biotechnology at Tehran University Email mamirit@yahoo.com

Abstract

Response of five cultivars of Iranian bread wheat to mature embryo culture and different concentrations of ABA was examined. Mature embryos from five cultivars (Tabasi, Bolani, Shiraz, Shoaleh and Azar) were placed in MS medium supplemented with 10 mg/l 2-4-D and 30 g/l sucrose and under different levels of ABA (0, 2, 4, 6 and 8 mg /l). Callus growth was measured by volume using a hoker scale. There were significant differences among cultivars in callus growth rates. Tabasi and Bolani showed the best and the least callus growth, respectively. In addition, ABA levels had significant effects in callus induction. The best and the least callus volume were observed in control and 8 mg/l ABA treatment, respectively. In fact, the more hormone used, the less callus gained. The interaction between ABA levels and cultivars was not significant, indicating this hormone in all levels affected callus induction of all cultivars in the same manner. In control treatment calluses had the brightest colour and with increase in ABA concentration calluses became darker; in 8 mg/l ABA calluses became dark-brown. In this manner, with increase in ABA concentration in mediums calluses compacted and the most friable calluses were observed in control medium. When regeneration rates were compared, there were significant differences among cultivars and ABA levels. The best and the least regeneration percentage were observed in control and 8 mg/l ABA level, respectively. Tabasi showed the maximum regeneration rate. In contrast with callus induction, the interaction between cultivars and ABA levels was significant and suggested that different ABA levels had different effects on cultivars.

Media summary

ABA, because of its preventive effects on cell cycle cause decrease in growth factors such as callus induction, regeneration percentage, callus volume, callus fresh weight in vitro culture.

Key words

ABA, wheat, callus induction, regeneration, MS medium

Introduction

Auxins and cytokinins have their chief effects on callus induction and regeneration; varying their concentration in the medium, causes differences in amount, rate and growth pattern of explants (5, 17). Other hormones also have effects on callus induction and regeneration but most effects are attributed to auxins and cytokinins because of a lack of information regarding other hormone types and their interactions (21). ABA is one of the five classical plant hormones that have not been extensively studied for its application in callus induction and regeneration but recent investigation has shown that this hormone prevents callus induction phase in tissue culture of different plant species (16). ABA acts as inducer of somatic embryogenesis and maturation of these embryos in somatic embryogenesis in plants such as carrot, coconut, and date (16, 19). In tissue culture of coconut the concentration of this hormone and its interaction with an appropriate auxin was critical in increasing callus induction and regeneration (19). As well this hormone causes induction of different responses of explants to tissue culture in different concentration of auxin (19). Decrease of 1PCD induced with cytokinins in vitro is one ABA effect that is becoming a field for study of PCD mechanism (4). The main aim of this research is studying effects of ABA to factors such as callus induction, regeneration, callus volume, callus colour in wheat.

Material and methods

Seeds of five wheat cultivars, Tabasi, Azar, Shiraz, Bolani and Shoaleh, were obtained from seed the Plant Research Institute of Karaj, Iran. These seeds were placed in containers containing sterilized distilled water at room temperature for 6 hours. Then the seeds were rinsed three times , disinfected with alcohol (70%) for two minutes and washing with distilled water three times. They were then exposed to commercial sodium hypochlorite (50%) for twenty minutes and washed with distilled water for three times. Embryos were separated from seeds with scalpel and forceps and were planted in Petri dish with 10 cm diameter containing about 25-30 millilitre MS medium supplemented with 10 mg.l-1 2,4-dichlorophenoxy acetic acid (2,4-D), different concentrations of ABA (0, 2, 4, 6, 8 mg.l-1), 100 mg.l-1 myo-inositol, 0.1 mg.l-1 thiamine-HCL and 3% (w/v) sucrose; pH was adjusted to 5.8 before adding 7 mg/l-1 agar. Autoclaving was done for 20 minute at 120C and 150kPa (22). Ten mature embryos were placed in every container. Special procedure of in vitro culture was in the manner that embryos were downwards (1, 5, 13, 17). Experiments were done in triplicate and the dishes were placed in growth chamber in 25C (7). After one month some characteristics were noted such as callus induction percentage, regeneration percentage, callus volume, callus colour (14) and the calli of control treatment were transferred to regeneration medium, MS medium supplemented with 0.5 mg.l-1 indole acetic acid and 1mg.l-1 benzylaminopurine and after one month the number of regenerated calli was noted (3).

Results and discussion

Percentage of callus induction

In this experiment callus was inducted in the control treatment of all cultivars at 100%. The more ABA present, the less percentage of callus induction was obtained. At 8 mg.l-1, some cultivars showed only 70% callus induction due to the negative effects of ABA in decreasing auxin action. This affect was evident on two factors, cell division and cell expansion, causing reduced callus induction in the presence of auxin (6, 18, 20) (not data shown).

Callus volume

Callus volume was estimated using a hoker volume scale and results showed that different cultivars had significant differences with regard to callus volume (10) (table 3). The effects of different levels ABA to callus induction were defined relative to the control treatment. All cultivars had the greatest volume of callus at zero ABA (Fig 2 and 5). With increase in ABA concentration in mediums we observed decrease in callus volume as in 8 mg.l-1, some cultivars had callus more or less 80% callus volume in comparison of control treatment because of preventative effects of ABA upon cell cycle (6). In addition, we observed that the interaction between cultivar and hormone level was non-significant. It means that ABA affected all cultivars in the same manner and decreased volume of callus in all cultivars.

Callus colour

The calli of control treatment had the lightest colour. With an increase in ABA concentration, the calli became darker. In 8 mg.l-1calli turned brown, probably indicating that ABA can cause callus necrosis and as well prevent from their growth (16, 18) (Fig 6).

Callus compression

ABA showed no significant difference on callus compression even though it was observed that control treatment calli of all cultivars represented the most friable calli and seemed to be comparatively watery. In other treatments, no significant difference was observed.

Percentage of Regeneration

The calli of the control treatment of all cultivars were transferred to regeneration medium supplemented with 0.5 mg.l-1indole acetic acid (IAA) and 1mg.l-1 benzyl amino purine (BAP) and under different levels of ABA (0, 2, 4, 6 and 8 mg.l-1). After one month it was observed that cultivars showed different rates and different abilities of regeneration (10) (Fig 3). In addition, levels of hormone represented significant differences in regeneration. The interaction between hormone and cultivar was significant. The control treatment showed the greatest ability of regeneration and these represented the longest shoot at approximately 2-3 centimetre. The more ABA concentration used, the less regenerated plants were obtained. At 8 mg.l-1 some very short shoots (about 2-3 millimetre) were observed.

General discussion

Assessment of abscisic acid effects have been limited in tissue culture even though it had became evident that this hormone induces in vitro some oxidases such as catalase (CAT). In fact, with increases in oxidase activity, the anti-oxidative action of these enzyme were induced which could facilitate the culture of tissues that excrete phenols into medium from their excised regions (17). ABA frustrates the negative effects of phenols and facilitates or induces callus induction in these tissues (17). In cereals such as wheat, ABA restored the ability of embryogenesis to embryos that have passed the optimum phase of embryogenesis (19). Study of ABA effects on plant cell cycle have showed that this hormone induces CDK inhibitory protein (ICK) and a reduced rate of cell cycle (6). Cyclin-dependent kinases (CDK) are the chief regulators in the cell cycle and may be the target of ABA resulting in reduction in callus induction in some tissue culture. What has obtained from this study is that ABA is a preventive factor of callus induction and regeneration in some cereals such wheat. This hormone interacts with other classes of hormones such auxins, cytokinin and gibberellins. The location of this interaction is in G2–M transition with induction of encoding of ICK proteins or prevention of expression of genes that are responsible for CDK (5, 18). The reasons of interference among hormone action are because of association and commonality of molecules and elements in their signalling pathways. Hormones have common receptors in some points of cell cycle especially in checkpoint. Reception of all mechanisms of hormone action in tissue culture and assessment of interaction between this hormone with other hormones especially auxins and cytokinins needs more experimentation.

Figure1: The comparison of callus volume among cultivars

Figure 2: comparison of callus volume among ABA levels

Figure 3: The comparison of percentage of regeneration among cultivars

Figure 4: The comparison of percentage of regeneration among ABA levels

Table 1: Variance analysis of callus volume

Source

Degrees of freedom

Sum of squares

Mean square

F value

Probability

Cultivar A
Hormone levels B
AB
Error

4
4
16
50

53.172
36.461
26.417
44.983

13.293
9.116
1.651
0.9

14.7755**
10.81319**
1.8352n.s

0.0000
0.0000
0.0523

Total

74

161.034

     

** Significant in 1% * significant in 5% n.s non significant

Table 1: Variance analysis of regeneration

Source

Degrees of freedom

Sum of squares

Mean square

F value

Probability

Cultivar A
Hormone levels B
AB
Error

4
4
16
50

8429.547
6621.547
5671.520
292.667

2107.387
1655.387
354.470
5.853

360.0319**
282.8109**
60.5587**

0.0000
0.0000
0.0000

Total

74

21015.280

     

** Significant in 1% *significant in 5% n.s non significant

Fig 5: Difference in Regeneration percentage in control and 8m treatment(Azar)

Fig 6: Difference in Callus colour in control and 8m treatment (Tabasi)

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1 . PCD: Programmed cell death

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