Studies on In Vitro Response to Callus Induction and Regeneration of Five High Yielding Indica rice Varieties

Due to growing population, there is an increasing demand of rice production but the productivity of rice is lessened day by day. To overcome this problem various biotechnological tools can be used for developing various rice varieties. However, the lack of a simple and efficient protocol for callus induction, embryogenic callus formation and quick plant regeneration in this cereal crop. In this study embryogenic calli from mature seeds of five indica rice varieties viz. Binadhan-5, Binadhan-6, BRRI dhan-48, BRRI dhan-58 and IR-64 were observed that is done in four different types of media composition. The highest callus induction were observed in media containing Sucrose as a carbon source. Among those varieties Binadhan-6 and BRRI dhan-48 showed highest rate of callus induction respectively. This study will be useful for selecting suitable callus induction medium for callus induction in future that will be useful for not only national but also international plant breeders for producing new variety and so on. Introduction Rice (Oryza sativa L.) is a monocot plant that belongs to the genus Oryza, family Gramineae (Poaceae) and tribe Oryzeae. It also comprised two subspecies, Indica and Japonica [16]. It is being cultivated for more than 10,000 years [14]. It is cultivated in 10.1 million ha and produces 95 % of the total food grain of Bangladesh. Out of the all crops that are using as feed in all over the world rice provide 23 % of the calories consumed by the whole population [9]. In Asia, two main subspecies named indica and japonica are grown among the three species. Indica rice encompasses 80% of the world rice cultivation [15] and this types of rice is mainly grown in South and SouthEast Asian countries of the world [18]. Larger than half of the population of the world using rice as a staple food [7] that serves more than 90% of the Asian population and is occupy the second position among all cereal crops cultivated in the world, after wheat [12]. For coping with the high rate of increasing population of the world the total rice production has to be intensified 50% within 2025 [8]. This goal cannot be accomplished by increasing the cultivation area under rice cultivation because of limitation of proper lands. On the other hands the farm areas are being lessen by residential areas in the developing countries. Previous experiments showed that the production rate of rice is unpleasantly affected by abiotic stress and that is highly defenseless with the change of weather [5, 6, 19, 3]. Various Modern biotechnological tools requires the optimization of an efficient regeneration protocol for specific cultivar(s) is an essential for the composition of the nutrient media is a major factor that influencing the regeneration frequency of rice [2]. Various researchers used different types of media for callus induction and regeneration [20],[21]. This experiment considering the objectives of identifying a suitable medium for callus induction from mature dehusked seeds. We hope that this study will be useful for local as well as for international plant breeders for improving the callus induction and regeneration efficiency in Rice in future. Journal of Horticulture and Plant Research Submitted: 2019-02-25 ISSN: 2624-814X, Vol. 7, pp 97-104 Revised: 2019-04-04 doi:10.18052/www.scipress.com/JHPR.7.97 Accepted: 2019-08-07 2019 SciPress Ltd, Switzerland Online: 2019-08-28 SciPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/ Materials and Methods This study was conducted in the Laboratory of Biotechnology division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Plant material Five different healthy and dehusked seeds of indica rice (Oryza sativa L.) named Binadhan-5, Binadhan-6, BRRI dhan 48, BRRI dhan 58, and IR 64 were used as experimental material to study different parameters associated with callus induction of plant [4]. Surface sterilization of seeds Seeds were surface sterilized by dipped in 70% ethyl alcohol for 1 minute with vigorous shaking after washing 5 to 6 times with distilled water. After that it were washed with double distilled water for 3 to 4 times and Surface disinfection was done by using of 50% NaOCl and 1 drop Tween-20 for 15 minutes at 300 rpm in a shaker. Then the seeds were subsequently washed 7 to 10 times with sterile distilled water to remove traces of sterilant that would be toxic to the seed materials if kept for longer duration. The surface sterilized seeds were then dried for 5-6 min on a sterilized filter paper. Callus induction Sterilized seeds were cultured directly in MS medium supplemented with different carbon source and media composition required as per treatment. In this experiment we used four types of media composition. MS medium containing 30g/L sucrose (T1) [23]; 30g/L Maltose (T2) [5]; 30 g/L Sucrose, 0.5 g/L Proline and 0.5 g/L Glutamine (T3) [24] and 30 g/L Maltose, 0.3 g/L Casein hydrolysate, 0.6 g/L Lproline and 0.25 mg/L BAP (T4). The culture media were autoclaved at 1.16 kgcm-2 pressure and 1210 C temperature for 30 minutes. Sterilized Seeds were cultured horizontally on to the media with different treatments under aseptic conditions and then incubated at 270 C in dark conditions. Every treatment contained three replications. The callus induction frequency was recorded after 2 weeks of culturing. The following Equation was used for determining the callus induction frequency as given below. Percent of embryogenic callus were calculated on the basis of the number of induced callus and the total number of embryogenic callus. Subculture Two weeks after the inoculation of explants, the embryogenic callus attained convenient size. Then they were removed aseptically and the root and shoot are discarded from the callus on a sterilized glass plate inside the laminar airflow cabinet and were placed again on a freshly prepared sterilized medium containing appropriate hormonal supplements. Then the subcultured callus were incubated in growth chamber at controlled temperature (27±10 C) in dark. Regeneration from embryogenic calli After 2 weeks the subcultured calli were transferred onto regeneration media containing either two or three growth regulators comprised of MS salts, 30 g/l maltose, 2 mg/l kinetin, 0.2 mg/l naphthalene acetic acid (NAA), PH 5.8. The media was supplemented with 8 g/l agar during the first phase of regeneration and 10 g/l agar during the second phase of regeneration. These microcalli were incubated at 27±10 C in dark for 7 days for the first phase of regeneration. During the second phase of regeneration these microcalli were transferred to fresh regeneration medium with 10 g/l agar concentration and incubated in light at 27±10 C temperature [13]. 98 JHPR Volume 7


Introduction
Rice (Oryza sativa L.) is a monocot plant that belongs to the genus Oryza, family Gramineae (Poaceae) and tribe Oryzeae. It also comprised two subspecies, Indica and Japonica [16]. It is being cultivated for more than 10,000 years [14]. It is cultivated in 10.1 million ha and produces 95 % of the total food grain of Bangladesh. Out of the all crops that are using as feed in all over the world rice provide 23 % of the calories consumed by the whole population [9]. In Asia, two main subspecies named indica and japonica are grown among the three species. Indica rice encompasses 80% of the world rice cultivation [15] and this types of rice is mainly grown in South and South-East Asian countries of the world [18]. Larger than half of the population of the world using rice as a staple food [7] that serves more than 90% of the Asian population and is occupy the second position among all cereal crops cultivated in the world, after wheat [12]. For coping with the high rate of increasing population of the world the total rice production has to be intensified 50% within 2025 [8]. This goal cannot be accomplished by increasing the cultivation area under rice cultivation because of limitation of proper lands. On the other hands the farm areas are being lessen by residential areas in the developing countries. Previous experiments showed that the production rate of rice is unpleasantly affected by abiotic stress and that is highly defenseless with the change of weather [5,6,19,3]. Various Modern biotechnological tools requires the optimization of an efficient regeneration protocol for specific cultivar(s) is an essential for the composition of the nutrient media is a major factor that influencing the regeneration frequency of rice [2]. Various researchers used different types of media for callus induction and regeneration [20], [21]. This experiment considering the objectives of identifying a suitable medium for callus induction from mature dehusked seeds. We hope that this study will be useful for local as well as for international plant breeders for improving the callus induction and regeneration efficiency in Rice in future.

Materials and Methods
This study was conducted in the Laboratory of Biotechnology division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh.

Surface sterilization of seeds
Seeds were surface sterilized by dipped in 70% ethyl alcohol for 1 minute with vigorous shaking after washing 5 to 6 times with distilled water. After that it were washed with double distilled water for 3 to 4 times and Surface disinfection was done by using of 50% NaOCl and 1 drop Tween-20 for 15 minutes at 300 rpm in a shaker. Then the seeds were subsequently washed 7 to 10 times with sterile distilled water to remove traces of sterilant that would be toxic to the seed materials if kept for longer duration. The surface sterilized seeds were then dried for 5-6 min on a sterilized filter paper.

Callus induction
Sterilized seeds were cultured directly in MS medium supplemented with different carbon source and media composition required as per treatment. In this experiment we used four types of media composition. MS medium containing 30g/L sucrose (T1) [23]; 30g/L Maltose (T2) [5]; 30 g/L Sucrose, 0.5 g/L Proline and 0.5 g/L Glutamine (T3) [24] and 30 g/L Maltose, 0.3 g/L Casein hydrolysate, 0.6 g/L L-proline and 0.25 mg/L BAP (T4). The culture media were autoclaved at 1.16 kgcm-2 pressure and 1210 C temperature for 30 minutes. Sterilized Seeds were cultured horizontally on to the media with different treatments under aseptic conditions and then incubated at 27 0 C in dark conditions. Every treatment contained three replications. The callus induction frequency was recorded after 2 weeks of culturing. The following Equation was used for determining the callus induction frequency as given below.
Percent of embryogenic callus were calculated on the basis of the number of induced callus and the total number of embryogenic callus.

Subculture
Two weeks after the inoculation of explants, the embryogenic callus attained convenient size. Then they were removed aseptically and the root and shoot are discarded from the callus on a sterilized glass plate inside the laminar airflow cabinet and were placed again on a freshly prepared sterilized medium containing appropriate hormonal supplements. Then the subcultured callus were incubated in growth chamber at controlled temperature (27±1 0 C) in dark.

Regeneration from embryogenic calli
After 2 weeks the subcultured calli were transferred onto regeneration media containing either two or three growth regulators comprised of MS salts, 30 g/l maltose, 2 mg/l kinetin, 0.2 mg/l naphthalene acetic acid (NAA), P H 5.8. The media was supplemented with 8 g/l agar during the first phase of regeneration and 10 g/l agar during the second phase of regeneration. These microcalli were incubated at 27±1 0 C in dark for 7 days for the first phase of regeneration. During the second phase of regeneration these microcalli were transferred to fresh regeneration medium with 10 g/l agar concentration and incubated in light at 27±1 0 C temperature [13].

Results
Plant regeneration is a system that is essential for establishing an efficient application of biotechnology that can be utilized for crop improvement in future. Therefore, in vitro regeneration from induced callus from mature embryos of five indica rice (Oryza sativa L.) varieties was investigated in this study.
The experiment was accompanied using five varieties of indica rice named Binadhan-5, Binadhan-6, BRRI dhan-48, BRRI dhan-58 and IR-64. Exploration on in vitro regeneration of these varieties was accomplished with callus induction, desiccation of callus, embryogenic callus, and finally plantlet regeneration. Results of different steps of the experiments are described under the following heads.

Callus induction
The first step of this experiment was callus induction from mature embryo. To meet this purpose, callus induction potentiality of mature embryo on different MS media composition was investigated.

Effects of different treatments on callus induction
Mean square values of four different combinations of treatments were found statistically significant for the callus such as percent of callus induction and percent of embryogenic callus. Among those treatments MS + Sucrose + 2 mg/L 2,4-D was found to be the best of all variety ( Table 2)   In the column figures followed by same same letter (s) in a do not statistically significant In the column figures followed by same same letter (s) in a do not statistically significant

Effects of different varieties on callus characters
The genotypic mean square values of rice were found statistically significant for all the characters of callus induction such as percent of callus induction and percent of embryogenic callus under those treatment (Table 3).

Regeneration from embryogenic calli
Two different media containing either two or three growth regulators are used. One (R1) comprised of MS salts, 30 g/l maltose, 2 mg/l kinetin, 0.2 mg/l naphthalene acetic acid (NAA), pH 5.8. The media was supplemented with 8 g/l agar during the first phase of regeneration and 10 g/l agar during the second phase of regeneration. While other media (R2) consisted of MS salts, 30 g/l maltose, 2.7 mg/l BAP, 1.

Discussion
In previous study it were showed that the callus induction frequency were between 60% and 90% [22], in the present study the callus induction rate reached up to 72% that support the previous study. However, high callus induction and regeneration may differ in different media composition and varieties [4], [5]. In this study, whitish and globular calli were found in media containing maltose and sucrose treated media formed brownish calli. In parallel to the above observations, Visarada et al. and Abiri et al. [1], [17] reported that nodular, compact and whitish calli were highly 102 JHPR Volume 7 embryogenic while watery, yellowish, rhizogenic and necrotic calli were stated to be nonembryogenic. Non-embryogenic calli are not competent for regeneration [12]. Based on color and structural morphology, the calli of the present study were mostly embryogenic in maltose treated media where they reached up to 38 %. Previous studies illustrated that maltose gave high embryo formation in indica rice [11]. It has been reported that Callus induction is highly genotype specific [4]. In this experiment there is a huge difference of callus induction among the five rice varieties.