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Somatic Embryogenesis and Plant Regeneration from Immature Embryo Derived Callus of Inbred Maize (zea Mays L.)

A. M. Shohael
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

M. A. L. Akanda
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

S. Parvez
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

S. Mahfuja
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

M. F. Alam
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

R. Islam
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

N. Joarder
Department of Botany, University of Rajshahi, Rajshshi 6205, Bangladesh.

Abstract/ Executive Summary:

The present study was carried out to develop protocol in which plants could be regenerated with easy and in high numbers from tissue cultures of maize. The entire experiment was conducted at the Plant Breeding and Biotechnology Laboratory of the Department of Botany, Rajshahi University, Bangladesh, during the years 1999-2002. The tissues of immature embryos were tested in three inbred lines (CML-161, CML-323, CML-327) MS and N6 were used. In MS medium the grand mean for plant regeneration was 61.0, 53.91 and 52.83 for inbred lines CML-161, CML-323 and CML-327, respectively. The no. of developed shoots was 13.33, 10.33 and 10.83 for inbred lines CML-161, CML-323 and CML-327, respectively.In N6 medium, the range of embryogenic calli formation was varied from 56.33-72.0%. The results indicated that the highest amount 72.0% was obtained when N6 was supplemented with L-proline 2.3 gm l^-1, Casein Hydrolysate 200 mg l^-1 and 2,4-D 1.0 mg l^-1. Where as in MS medium the embryogenic calli formation was varied from 39.0-68.66%. In this case the highest amount (68.66) was obtained where MS was supplemented with L- Asparagine 150 mg l^-1, Thiamin 50 mg l^-1 and 2, 4-D 1 mg l^-1. Plants were regenerated successfully from embryogenic callus in hormone free MS medium. The results showed that MS (52.83-61.0%) medium was found better than N6 (35.66-42.49%). The mode of somatic embryogenesis was studied using histological technique.

Keywords: Maize inbreeds, Immature embryo, Embryogenic callus, Histology, Additives

Location: Plant Breeding and Biotechnology Laboratory in the Department of Botany, Rajshahi University, Bangladesh.

Start Date: 00-00-1999

End Date: 00-00-2002

Program Area: Variety and Species

Commodity: Maize

Objectives:

To develop a protocol for plant regeneration with ease and in high numbers through tissue culture of maize.

Methodology:

Experimental materials included three maize inbred lines viz., CML-161, CML-323, CML-327. To get genetically uniform explants the purity of inbred lines was maintained through selfing using hand pollination. Initially the dehusked immature cobs were harvested after 10-12 days of post anthesis and the young kernels were used for callus induction. Immature cobs were surface sterilized with 70% ethanol for 2-4 min and rinsed with sterile distilled water. Then the cobs were placed in 30% Clorox bleach solution (1.5% sodium hypochlorite) v/v having a few drops of Tween-80 for 30 min in side the laminar airflow. Embryos were excised aseptically by cutting of the top of the karnel with a sharp scalpel blade. Then the excised embryos were placed as the scutellum up on the medium. About 5-6 explants were inoculated culture-1 bottle containing 20 ml of medium. Initially different concentrations of plant growth regulators such as 2, 4-D (2, 4-D ichlorophenoxy acetic acid) single or incombination with BA (6- Benzyl adenine), Kinetin were used for callus induction. As basal salts both MS and N6 medium containing 3% (w/v) sucrose as source of carbon and 6 gm l^-1 agar (w/v) as solidifying agent (pH 5.7) were used. The media were autoclaved at 120°C for 20 min. Each treatment was repeated five times and was incubated in dark for 21 days at 28±1°C. On the basis of initial results on callus induction, different additives (L- Asparagine, Thiamine, L- proline and Casein Hydrolysate) were used. In MS medium, the following supplements Laspergine and thiamin were used. For N6 the supplements were L- proline and Casein Hydrolysate. Callus induction efficiency was measured, as number of explants induced callus-1 total no. of explants used x 100. The data were recorded after 21 days of inoculation and the results presented as mean ± standard error. For histological studies callus was fixed in FAA (5% formaline, 5% acetic acid, 45% ethanol and 45% alcohol) and dehydrated in a absolute alcohol-chloroform series. Paraffin blocks with materials were prepared and sectioned serially at 12 μm thickness using a rotary microtome. Sections were stained with Safranine-Orange-G, Tannic acid and mounted on glass slides. The picture was taken at 10 to 100X magnification. To promote regeneration, calli were transferred to either hormone free or with hormones (IAA and BA) in MS and N6 medium. Cultures were incubated under fluorescent light (2000 Lux, 16 hour photo period) at 28±2°C. Developed plant lets with primary roots were transferred to ½ strength MS medium for farther root development. After hardening the plant lets with well develop roots were transplanted to small pots containing sterilized soil. The plant lets were developed successfully up to maturity. Like callus induction efficiency plant regeneration efficiency was also measured using similar formula.

Source of Information: Biotechnology, 2(2): 154-161, 2003, ISSN 1682-3974.

Article Link (Online Journal): DOI: 10.3923/biotech.2003.154.161, URL: http://scialert.net/abstract/?doi=biotech.2003.154.161

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Regarding genotype used, CML-161 was found favorable for in vitro culture. In both MS and N6 medium, CML-161 was found better than other inbred lines for plant regeneration and shoot formation per callus. Half strength of MS salt without any growth hormone was found effective for vigorous root development in regenerated plant lets. The plant lets were successfully established in soil. This work sets a starting point for developing more efficient protocol for embryogenic callus production on the treated three maize inbred lines and their further use in biotechnological research for hybrid maize development.

Knowledge Management: Journal