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Optimization for in Vitro Regeneration in Four Local Cultivars of Indica Rice

Md Taufiqur Rahman
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Md. Hasanuzzaman
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Md. Monirul Islam
Biotechnology Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh.

M.T.R Mondal
Horticulture Division, Bangladesh Institute of Nuclear Agriculture, Mymensingh, Bangladesh.

Md. Shahidul Islam
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Nihar Ranjan Saha
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, Bangladesh.

Abstract/ Executive Summary:

The current investigation was planned and designed for optimizing the concentration of plant growth regulators to generate an efficient in vitro callogenesis and plantlet regeneration in four local cultivars of Indica rice. Effects of six different concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid), kinetin and carbon source, amino acids, and solidifying agents on callus induction were observed using mature seeds as starting material. Two different concentrations of NAA (1-naphthaleneacetic acid) + kinetin and IBA (Indole-3-butyric acid) were used for shoot regeneration and root induction, respectively. The highest frequency (100%) of callus was recorded on the medium containing 2mg/l 2,4-D which was followed by 3 mg/l 2,4-D + 10 mg/l kinetin (95.51%) at the dark condition in cultivar Sadamota. Moreover, MS (Murashige and Skoog) medium supplemented with 2 mg/l 2,4-D, L-proline, and sucrose (as carbon source) increased the callus induction frequency. The highest percentages of callus induction were found when both the gelrite and agar were used as solidifying agents. The maximum shoot regeneration (66.44%) and shoot number (4.49) were obtained at 0.50 mg/l NAA + 10 mg/l kinetin in the cultivar, Sadamota. Moreover, the highest root induction and root number were recorded at 0.50 mg/l IBA in Sadamota. Overall, the highest optimum callus induction, shoot regeneration, and root formation were obtained at 2 mg/l 2,4-D, 0.50 NAA + 10 mg/l kinetin, and 0.50 mg/l IBA, respectively. Sadamota exhibited the best plantlet regeneration followed by Kachamota whereas the lowest was displayed by Dudkalam. This regenerated protocol could be utilized for gene transfer in rice for the development of stress-tolerant and high-yielding lines in the future.

Keywords: Callogenesis, Indica rice, Mature seeds, Plant growth regulators, Plantlet regeneration

Location: Plant Tissue Culture of, Biotechnology Division at Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Rice

Objectives:

To establish a mature embryo-based complete regeneration protocol in local indica rice.

Methodology:

Our total experiments on assessing the callogenesis and regeneration potentials of selected rice cultivars were undertaken in the laboratory of Plant Tissue Culture of, Biotechnology Division at Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. In our study, a total of four local cultivars of rice namely Kachamota, Sadamota, Dudhkalam, and Moulota were used. The mature seeds were used as explant sources which were collected from the Barisal division of Bangladesh. Mature de-husked seeds from each rice cultivar were sterilized aseptically by treating with 70% ethanol (1 min) which was followed by 3.5 % NaOCl + 1-2 drops Tween-20 for 15 min. After that, the sterilized mature seeds were carefully washed thrice with sterile distilled water followed by drying on autoclaved filter paper. Then the completely sterilized rice seeds were transferred to callus culture medium (25 ml in 90 mm petridish) where the seeds scutellum side was up position and plumule slightly embedded into the medium under aseptic condition. Callus induction and subculture To prepare the callus induction medium, we have used MS culture medium which was supplemented with different carbon sources, solidifying agents, and different concentrations of PGRs. The culture medium was autoclaved (121°C, 20 min), after adjusting the pH between 5.6-5.8. Six different combinations of nutrient composition and concentrations of phytohormones supplemented in culture medium for callus induction. Afterward, ten finely sterilized seeds were placed in one petridish on culture medium. Parafilm was used for proper sealing and then the pertidishes were incubated in both dark and light conditions at 25 ± 1°C. After 3 or 4 days, the inoculated seeds swelled at mesocotyl and radicle regions of seeds. For achieving better callus, subcultures were carried out in 2 weeks where the primary calli were incubated on the same medium and condition for another 3 weeks. Plant regeneration Five weeks aged calli were transferred to the regeneration medium having different combinations of phytohormones. The hormonal combination was used for shoot regeneration were as follows: (a) MS + NAA (0.25 mg/l) + Kinetin (10 mg/l); (b) MS + NAA (0.5 mg/l) + Kinetin (10 mg/l). For regeneration, the culture conditions were maintained by adjusting the relative humidity (45–60%), temperature (25 ± 1°C), and cool-fluorescent light (110 mmol/m2/s, 16 h) for 4 weeks. Then, the subculture was performed one time after 2 weeks. Root induction and acclimatization Regenerated shoots were carefully transferred into basal MS medium (half strength) supplemented with 0.25 and 0.5 mg/l IBA for facilitating root induction. The cultures were incubated at 25 ± 1°C and photoperiod (110 mmol/m2/s, 16 h) for 2 weeks. After that, the finely rooted rice plantlets were carefully transferred into plastic pots (peat (1): vermiculite (1), keeping similar incubation conditions. When the rice plants grew 10-15 leaves, they were transferred to the greenhouse. Data collection and statistical analysis After 4 weeks, the callus induction percentages were determined. After 4 weeks in the regeneration medium, the percentages of shoot regeneration and shoots number per callus were determined. Furthermore, incubating for 4 weeks, the root induction percentages and the root number per plantlet were determined. Frequencies of callus formation (%) = (Number of explants producing calli/ Number of explants plated) × 100 Frequencies of shoot regeneration (%) = (Number of calli regenerated plantlets/ Number of calli plated for regeneration) × 100 Frequencies of root induction (%) = (Number of roots generating plantlets/ Number of plantlets for root formation) × 100 The Recorded data were analyzed statistically by following a Completely Randomized Design (CRD). The analysis of variance (ANOVA) was estimated according to Duncan’s Multiple Range Test (DMRT).

Source of Information: Asian J Agric & Biol. 2021(4)

Article Link (Online Journal): DOI: 10.35495/ajab.2021.01.045

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The current investigation describes an efficient and complete regeneration protocol that was generated from mature seeds of indica rice. The optimum callus frequencies concentration were exhibited by culture medium added with 2,4-D (2 mg/l), L-glutamine, Lproline, sucrose (30 mg/l), and gel rite alone (as solidifying agent). Moreover, highly efficient regeneration was optimized using a regeneration medium supplemented with NAA (0.5 mg/l) + Kinetin (10 mg/l). Besides, the addition of IBA (0.50 mg/l) in the rooting medium exhibited the most effective response for root induction and root number. This regenerated protocol could further be used in successful genetic transformation by incorporating improved agronomic traits.

Knowledge Management: Journal