Annals of the Sri Lanka Department of Agriculture. 2008.10:09-17.
OPTIMIZATION OF IN VITRO PLANT REGENERATION OF SEVERAL INDICA RICE CULTIVARS TO FACILITATE Agrobacterium-MEDIATED TRANSFORMATION A.S.M.T. ABAYAWICKRAMA1 and TOYOAKI ANAI2 Rice Research and Development Institute, Batalagoda, Ibbagamuwa, Sri Lanka 2 Laboratory of Genetics and Biotechnology, Faculty of Agriculture, Saga University, Japan 1
ABSTRACT Genetic transformation is an essential aspect of gene manipulation in plants which can break the species barriers and make it possible to transfer any gene to the desired crop. Agrobacterium-mediated transformation has greatly facilitated the widespread application of transformation into the rice genome for the purpose of varietal improvement. Prior to genetic transformation, regeneration techniques have to be perfected since the high regeneration ability is an important prerequisite for a successful transformation. As such, the present study was conducted with the objective of optimizing the tissue culture conditions and identification of suitable cultivars for efficient transformation among 15 different Indica cultivars. Callus induction ability was tested for different basal media (N6 or MS salts and vitamins) with different proline and 2, 4-D levels. Cultures were incubated at two levels of illumination (light and dark) at two different temperature levels (25 째C and 30째C). To study the transformation efficiency, Enhanced Green Fluorescent Protein (EGFP) was used as a marker for transient expression. Co-incubation was done with Agrobacterium tumefaciens strain EHA 101 harboring the plasmid pBI-AT1-EGFP. Results revealed that the efficient callus induction was observed when MS was used as the basal medium, whereas, regeneration ability of Indica rice was greatly improved when cultures were incubated under light at 30째C. Both callus induction and regeneration ability were significantly high in IR24, K-13, K-15 and Tetep. Transformation efficiency of these Indica cultivars improved when co-incubation was done with Agrobacterium tumefaciens at 22-25째C for 3 days. KEYWORDS: Indica rice, Regeneration, Transformation.
INTRODUCTION Establishment of high efficiency Agrobacterium-mediated transformation techniques for Indica rice (Oryza sativa L.), offers the potential for the introduction of specific genes into existing elite lines, for the purpose of varietal improvement (Abdullahel et al., 2004). However, Agrobacteriummediated transformation of rice has been limited to japonica and javanica varieties and there are only a few reports on Indica rice (Zhang, 2005). These studies have revealed that the transformation efficiency is either low or highly specific to certain genotypes. Prior to genetic transformation, regeneration techniques have to be perfected since the high regeneration ability is an important prerequisite for successful transformation via Agrobacteriummediated transformation or any other transformation technique (Toki, 1997).
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As such, the present study was conducted with the objective of optimizing the tissue culture conditions and identification of suitable varieties for efficient transformation among several Indica cultivars. To study the transformation efficiency of Indica rice via Agrobacterium, Enhanced Green Fluorescent Protein (EGFP) was used as a marker for transient expression. MATERIALS AND METHODS Seed materials Indica rice cultivars, Blue bonnet, Dvdh rai, Hod sodri, K-13, K-14, K-15, K-16, Ladywright, Lemont, M202, M302, IR24, Simanoce, Star Bonnet and Tetep were obtained from the Crop Science laboratory of Saga University, Japan. Japonica cultivar, Reihou was obtained from the Genetics and Plant Breeding laboratory of Saga University. Medium and culture conditions The following culture media were tested (Table 1). Cultures were incubated at two levels of illumination (light and dark) at two different temperature levels (25째C and 30째C). Table 1. Culture media used for tissue culture and transformation. Medium
Composition
CI
N6 (or MS) salts and vitamins, 30 mg/l sucrose, 1 g/l casamino acid, 2800 mg/l (or 500 mg/l) proline, 2 mg/l (or 3 mg/l) 2,4-D, 2 g/l gelrite, pH 5.7
RE
MS salts and vitamins, 30 mg/l sucrose, 30 g/l sorbitol, 2 g/l casamino acid, 0.02 mg/l NAA, 2 mg/l kinetin, 4 g/l gelrite, pH 5.7
CO
N6 salts and vitamins, 30 mg/l sucrose, 1 g/l casamino acid, 10 g/l glucose, 200 mg/l acetosyringone, 2 g/l gelrite, pH 5.7
SE
N6 salts and vitamins, 30 mg/l sucrose, 2 mg/l 2,4-D, 2800 mg/l proline, 1 g/l casamino acid, 10 g/l glucose, 2 g/l gelrite, pH 5.7, 40 mg/l hygromicin B, and 500 mg/l carbenicilin CI=callus induction, RE= regeneration, CO=co-incubation and SE=selection medium MS=Murashige and Skoog
Callus induction Cultivars Tetep, K-13 and Ladywright were selected to represent the Indica group and cultivar Reihou was selected to represent the japonica group. Dehusked seeds were sterilized by 10% sodium hypochlorite and tween 20. After 20 min., the solution was removed and seeds were thoroughly washed with sterilized water. Sterilized seeds were transferred into
AGROBACTERIUM-MEDIATED TRANSFORMATION OF INDICA RICE 11
petri dishes (20 seeds per petri dish) containing eight different callus induction media (CI). Cultures were tested at two levels of temperature (25 °C and 30°C) with two levels of illumination (light and dark). Two replicates per treatment combination were maintained. Seeds were incubated for 14 days under different culture conditions and the callus induction ability was recorded according to the 1-9 standard scale where, 1=no callus induction, 3=poor callus induction, 5=moderate callus induction, 7=good callus induction and 9=very good callus induction. Regeneration Optimized callus induction conditions were used to test the regeneration ability. Seeds of fifteen Indica cultivars and one japonica cultivar were incubated on CI medium containing MS salts and vitamins (Murashige and Skoog, 1962), 2800 mg/l proline and 2 mg/l 2, 4-D. Cultures were incubated under light at 30°C. After 14 days intact calli were separately transferred to regeneration medium, RE (Table 1). Regeneration ability was tested under light at 30°C. Regeneration ability was scored according to 1-9 scale at 14 and 21 days after transferring to RE medium. After 28 days, seedlings were transferred into fresh RE medium without plant hormones. Analysis of variance was performed using SAS statistical software to analyse the data. Mean separation was done by DMRT. Transformation Enhanced Green Fluorescent Protein (EGFP) was used as a marker for transient expression in this experiment. Agrobacterium tumefaciens strain EHA 101 harboring the plasmid pBI-AT1-EGFP was grown on Polypepton medium containing 50 mg/l hygromycin B and 50 mg/l kanamycin for 3 days at 22-25°C. Agrobacterium was scraped from the AB plate and resuspended in liquid co-incubation medium supplemented with 200 mg/l acetosyringone. Optical density of bacterial suspension was 0.05-0.1 at 600 nm. Three days prior to co-incubation, calli were sub cultured into fresh N6 medium (Chu, 1975). Embryogenic calli were immersed in Agrobacterum suspension for 10 minutes. Then the calli were blotted dry and transferred into co-incubation medium, CO (Table 1). Co-incubation was done for 3-4 days under dark at 22°C. Agrobacterium infected calli were first washed with sterile distilled water followed by N6 liquid medium supplemented with 500 mg/l carbenicilin and 1% plant preservative mixture (Plant Cell Technology, Japan). The washed calli were transferred into the selection medium, SE (Table 1). Cultures were incubated under light at 30 °C for 14-21 days. Transformed calli were observed under fluorescence steremicroscope. Only growing calli were
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transferred to regeneration medium, RE (Table 1).
AGROBACTERIUM-MEDIATED TRANSFORMATION OF INDICA RICE 13
RESULTS Callus induction Callus induction ability of three Indica cultivars and one japonica cultivar were tested on CI medium. The callus induction ability of three Indica cultivars was higher than that of japonica cultivar (Fig. 1). Particularly, Tetep and K-13 showed high callus induction ability. When MS was used as the basal medium, Indica cultivars showed efficient callus induction ability. Increasing proline content (2800 mg/l) also enhanced the callus induction. Irrespective of variety groups (Indica or japonica), incubating cultures at 30째C resulted in a significant increase in callus induction. There was no significant difference in callusing with the 2 different levels of 2, 4-D tested. Cultures which were kept under light showed efficient callus induction (Table 2). Table 2.
Callus induction ability of different rice cultivars under different culture conditions.
Variety
Tetep K-13 Ladywright
Basal Medium MS N6
Proline (mg/l) 500 2800
2, 4-D (mg/l) 2 3
Illumination
Temp (째C)
Light
25
30
7.87* 7.54* 5.87*
7.22 6.69 5.62
7.81* 7.84* 5.72
7.62 7.16 5.75
7.40 7.37 5.59
7.00 6.91 4.97
8.03* 7.62* 6.37*
4.44
4.47
4.37
7.66 7.41 6.00 * 4.62
Dar k 7.37 7.14 5.34 4.22
3.81
5.03*
7.16 7.00 5.47
Reihou 4.59 4.25 4.41 *Significant at 0.05 level probability
Figure 1. Callus induction on CI medium (c.v. Tetep) after 14 days.
Regeneration The regeneration ability of 15 Indica cultivars and one japonica cultivar was tested. To identify the regeneration ability of these cultivars, calli which were grown on optimized CI medium (MS salts and
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vitamins) were transferred into RE medium.
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Table 3 summarizes both callus induction and regeneration ability of different Indica cultivars. The cultivars, Tetep, K-15, IR24, K-13 and K-14 showed efficient callus induction as well as regeneration (Fig. 2). Though Hod sodri and Dvdh rai showed high callus induction, their regeneration ability was poor. In contrast, cultivars, K-16 and Reihou showed poor callus induction while their regeneration ability was very high. Table 3. Comparison between callus induction and regeneration ability of different Indica rice cultivars. Variety Tetep K-15 IR 24 K-13 Hod sodri K-14 Dvdh-Rai Blue bonnet Lady wright Star bonnet M 302 Simanoce Reihou (Japonica) M 202 K-16 Lemont Means followed by common letters DMRT.
Callus induction Regeneration 9.00 a 8.75 a 9.00 a 7.75 b 9.00 a 7.75 b 9.00 a 7.25 b 9.00 a 5.25 b 8.57 b 8.75 a 8.25 b 2.25 d 7.75 c 3.00 d 7.50 c 3.25 d 7.25 c 3.25 d 6.50 d 3.25 d 6.25 d 2.75 d 6.25 d 9.00 a 5.25 e 3.25 d 4.75 e 8.00 b 4.50 e 3.25 d are not significantly different at 0.05 level probability by
Transformation Cultivars IR24, K-13, K-14, K-15 and Tetep were used for transformation as their callus induction and regeneration ability were significantly higher than other cultivars that were tested. Co-incubation of embryonic calli with Agrobacterium suspension under dark at 22째C for 3-4 days facilitated the transformation. Under the experimental conditions used, transformation efficiency of these Indica cultivars was slightly lower than that of Reihou (data not shown). However, this level of transformation efficiency is sufficient to obtain transgenic rice (Fig. 3). Inheritance pattern of the transformed EGFP gene was observed in T1 generation. Figure 4 shows the successful expression of EGFP in the next generation.
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DISCUSSION Callus induction ability of Indica rice showed significant increase when MS salts and vitamins were used instead of N6 salts and vitamins (Chu et al., 1975) in CI medium. According to Tahilian and Kothri (2004), callus induction could be improved by CuSO 4 in the MS basal medium. It has been suggested that the copper ions promote callus induction, since they are components or activators of many enzymes involved in electron transport, protein and carbohydrate biosynthesis. Khatun et al. (2003) have demonstrated that, amount of nitrogen given by proline may improve in vitro culture of rice genotype. Different genotypes require very specific nutrient elements at specific stages. Deficiency in any of these nutrients may affect the growth and development of callus. In this experiment both callus induction and regeneration ability were estimated using 1-9 standard scale. The size and the quality of the callus may vary even if the number of calli were similar in different genotypes. Therefore, callus number is not an appropriate parameter to estimate callus induction. As such, use of 1-9 standard scale is more meaningful when estimating the callus induction ability by comparing the size and the quality of calli in different cultivars. Japonica cultivar Reihou was used as the control in this experiment, since it was identified as one of the best performing cultivars among the Japonica group (Koga, 2000). Higher regeneration ability was observed when the calli were induced on optimized CI medium. Various factors such as basal medium, plant growth regulators, age of the explant and genotype have been reported to affect the regeneration frequency of Indica rice. In addition, Saharan et al. (2004) have shown that the regeneration ability of Indica cultivars (HKR-46 and HKR-126) could be increased when calli were subjected to partial desiccation. This finding suggests that some physiological changes during dehydration of callus would enhance the regeneration ability. CONCLUSIONS Results revealed that calli induced on CI medium supplemented with MS salts and vitamins, 30 mg/l sucrose, 1 g/l casamino acid, 2800 mg/l proline, 2 mg/l 2, 4-D has greatly improved the callus induction ability of Indica rice. In addition, cultures which were incubated under light at 30 째C enhanced the callus induction ability. Both callus induction and regeneration ability were shown to be significantly higher in the cultivars IR24, K-13, K-15 and Tetep. Thus, they were identified as suitable cultivars for further transformation experiments. When embryonic calli were co-incubated with Agrobacterium under dark at 22째C for 3-4 days, the transformation efficiency of Indica rice was improved.
AGROBACTERIUM-MEDIATED TRANSFORMATION OF INDICA RICE 17
(a) (b) Figure 2. Regeneration of Tetep calli on (a) RE medium (b) RE medium without plant hormones.
(a) (b) Figure 3. Transgenic calli of Indica rice (c.v. Tetep). (a) Transformed callus with EGFP gene on SE medium and (b) regenerating callus on RE medium under fluorescence steremicroscope.
(a) (b) (c) Figure 4. Expression of EGFP gene in (a) T 1 seedling and (b) T1 seeds of Indica rice (c.v. Tetep) under fluorescence steremicroscope. (c) Control seeds of T 1 generation.
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ACKNOWLEDGEMENTS The financial aid provided by the Ministry of Education, Culture and Sports in Japan is greatly appreciated. REFERENCES Abdullahel, M.A., M.U. Aftab and A. Hossain. 2004. Regeneration study of some Indica rice cultivars followed by Agrobacterium-mediated transformation of highly regenerable cultivar, BR-8. Journal of Biological Science 4(2):207-211. Chu, C.C., C.C. Wang, C.S. Sun, C. Hsu, K.C. Yin, C.Y. Chu and F.Y. Bi. 1975. The establishment of an efficient medium for anther culture of rice through cooperative experiments on the nitrogen sources. Science Sinica 18:659668. Khatun, M.M., M.H. Ali and N.V. Desamero. 2003. Effect of genotype and culture media on callus formation and regeneration from mature seed sctella culture in rice. Plant Tissue Culture 13(2):99-107. Koga, M., H. Tanaka, Y. Takagi and T. Anai. 2000. Improvement of Agrobacterium- mediated transformation method on rice (Oryza sativa L.). Faculty of Agriculture, Saga University, Japan. Bulletin No.85. 155-162 p. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiolology of Plant 15:473-497. Saharan, V., R.C. Yadav and B.P. Chapagain. 2004. High frequency plant regeneration from desiccated calli of Indica rice (Oryza sativa L.). African Journal of Biotechnology 3(5):256-259. Tahiliani, S. and S.L. Kothari. 2004. Increased copper content of the medium improves plant re regeneration from immature embryo derived callus of wheat (Triticum aestivum). Journal of Plant Biochemistry and Biotechnology 13:85-88. Toki, S. 1997. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Molecular Reporter 15 (1):16-21. Yamanaka, S., I. Nakamura, K.N. Watanabe and Y. Sato. 2004. Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice. Theoretical and Applied Genetics 108: 1200-1204. Zhang, Y.J.L.Q. 2005. Optimising the tissue culture conditions for high efficiency transformation of Indica rice. Plant Cell Reporter 23:540-547.