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Embryogenic callus induction and efficient plant regeneration in three varieties of soybean (glycine max)

Nazrul Islam
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Touhidul Islam
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Munir Hossain
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Bakul Bhattacharjee
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

M. Monzur Hossain
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

S. M. Shahinul Islam
Plant Genetic Engineering Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

An efficient callus induction and plant regeneration system has been developed using three local soybean (Glycine max L.) varieties. All the varieties showed good callusing (78.30 - 88.80%) from shoot tip (ST) in MS + 3.0 mg/l 2,4-D + 1.0 mg/l BAP. Best callus induction and embryo formation were recorded in T5 for BS-6 as 88.80 and 81.20%, respectively from ST. In case of cotyledonary node (CN) the maximum callus induction (82.40%) and embryo formation (74.20%) were recorded also in T5 for BS?6. Highest frequency (79.40%) of plant regeneration was recorded where ST were used and cultured in MS supplemented with 2.5 mg/l BAP + 1.0 mg/l NAA as well as 76.30% from CN in BS-6. The length of shoot was observed 5.32 and 4.62 cm, respectively from ST and CN for BS-6 with the same medium composition. It was observed that half strength of MS + 2.0 mg/l IBA showed best rooting (9.04). Among the genotypes BS?6 proved to be best explants than ST that exhibited better performance on callus induction and green plant regeneration for all parameters.

Keywords: Cotyledonary node, Embryogenic calli, Regeneration, Somatic embryogenesis, Glycine max

Location: Gazipur, Bangladesh

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Soybean

Objectives:

To develop an efficient callus induction and regeneration systems using shoot tip and cotyledonary nodal explants for further advance biotechnological research of soybean in Bangladesh.

Methodology:

Mature seeds of three soybean genotypes viz., BARI Soybean-5 (BS?5), BARI Soybean-6 (BS?6) and Shohag were surface sterilized with 70% (v/v) ethanol and then completed the sterilization procedure by 40% Clorex (NaOCl; ROTH, Germany) + 1 - 2 drops Tween 20 + 1 - 2 drops savlon. Surface sterilized seeds were then plated in culture vessels that contained 20 ml MS0 and incubated them at 25 ± 2°C in dark. The shoot tip and cotyledonary nodal segments of 14 days old seedlings were cut into small pieces and inoculated them in MS that supplemented with six different concentrations of PGRs either singly or in combination for callus induction. Then culture vessels were incubated at 25 ± 2°C in dark for 12 hrs photoperiod (30 μ mol/m/2s) provided by white florescent tubes. The treatments were as follows T₁ = 2.0 mg/l 2,4-D; T₂ = 3.0 mg/l 2,4-D; T₃ = 4.0 mg/l 2,4-D; T₄ = 2.0 mg/l 2,4-D + 0.5 mg/l BAP; T₅ = 3.0 mg/l 2,4-D + 1.0 mg/l BAP and T₆ = 4.0 mg/l 2,4-D + 1.5 mg/l BAP. After 10 - 14 days of inoculation, calli were formed and data were recorded on the basis of callus induction frequencies. Fourteen days old calli were sub-cultured in the same fresh medium for embryo formation. After four weeks of culture, data were recorded on the basis of somatic embryos formation (SEmF). For embryo maturation (EM) four weeks old embryos were transferred to MS supplemented with different concentration of BAP (1.5, 2.0, 2.5, 3.0 and 3.5 mg/l) + 2,4-D (0.5 mg/l). Afterwards four weeks of culture embryo maturation frequencies were recorded. After maturation the somatic embryos were transferred to regeneration medium (RM) that was supplemented with different concentrations of BAP (1.5, 2.0, 2.5, 3.0 and 3.5 mg/l) + 1.0 mg/l NAA. After 4 weeks the lengths of shoots were measured and plant regeneration frequencies were recorded. When length of shoot was around 3- 5 cm transferred them to rooting medium (RIM). Then well rooted plants were transferred to pots and after acclimation plants were transferred to the field. Data were recorded on the basis of callus induction, embryo formation and its maturation, plant regeneration, shoot length, roots per plants, length of root. The average or mean values were computed from three replications with standard error (SE). The experiment was arranged CRD and data were statistically analyzed by the statistical package software SPSS (version ?20) and Microsoft Excel. The analysis of variance (ANOVA) was performed and means were compared by DMRT at 5% level of probability for interpretation of results (Gomez and Gomez 1976).

Source of Information: Plant Tissue Cult. & Biotech. 27(1): 41?50, 2017 (June)

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Present results indicated that higher concentration of BAP (2.5 mg/l) with lower doses of 2,4-D (0.5 mg/l) increased embryo maturation. Lazzeri et al. (1987) and Tian et al. (1994) were reported that positive effect of the cytokinins on somatic embryogenesis in soybean. McKently (1991) reported that when the auxin concentration was increased, the probability of a normal shaped embryo was decreased. Higher concentration of auxin not only decreased the number of embryos but also delayed embryogenesis (Reddy and Reddy 1993). Kim et al. (2001) observed that 8.0 roots/shoot when 1.5 mg/l IBA used singly. Yuan et al. (2001) reported that when as PGRs, IBA added in MS the root formation was enhanced. Liu et al. (1998) observed that suitable root formation in MS with 2.0 mg/l IBA. Bhojwani and Razdan (1983) reported that using auxins in the medium enhanced root formation in plants.

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