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Agrobacterium Mediated Genetic Transformation in Lentil (lens Culinaris Medik.) Followed by in Vitro Flowering and Seed Formation

Subroto K. Das
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka

Kishwar Jahan Shethi
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka

M. I. Hoque
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka

R. H. Sarker
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka

Abstract/ Executive Summary:

Genetic transformation system was developed for two microsperma varieties of lentil (Lens culinaris Medik.), namely Bari Masur‐4 (BM‐4) and Bari Masur‐5 (BM‐ 5) using Agrobacterium tumefaciens strain LBA4404 harbouring binary plasmid pBI121, containing GUS and nptII genes. Three different types of embryo explants, namely cotyledonary node (CN), decapitated embryo (DE) and cotyledone attached decapitated embryo (CADE) were used. Highest GUS positive expression was found in DE followed by CADE as detected by transient assays. Following Agrobacterium infection CADE showed better response in developing multiple shoots on MS supplemented with 2.22 μM BAP, 2.32 μM Kn, 0.29 μM GA₃ and 30.35 μM tyrosine. Selection of the transformed shoots was carried out by gradually increasing the concentration of kanamycin up to 200 mg/l. Transgenic lentil shoots were produced with an overall frequency of 1.009%. In vitro rooting appeared to have a limitation in obtaining complete plantlets in lentil, therefore in vitro flowering and seed formation were induced in transformed shoots of lentil with a view to recovering of the transgenic progenies. GUS positive shoots were found to produce in vitro flowers and pods on half‐strength MS containing 98.4 μM IBA and 2.69 μM NAA. Expression of gene was detected in various tissues of the transformed shoots. Stable integration of GUS gene was also confirmed through PCR analysis.

Keywords: Lens culinaris, Transformation, In vitro flowering, Seed formation

Location: Dhaka

Start Date:

End Date:

Program Area: Knowledge Management

Commodity: Lentil

Objectives:

The main objective of this study was to develop a protocol for Agrobacterium mediated genetic transformation for local microsperma varieties of lentil for their improvement. Moreover, attempts were also made to recover transgenic plantlets of lentil through in vitro flowering and seed formation.

Methodology:

Two microsperma varieties of lentil (Lens culinaris Medik.), namely Bari Masur‐4 (BM‐4) and Bari Masur‐5 (BM‐5) cultivated in Bangladesh were used as plant materials for this investigation. Seeds of these two varieties of lentil were collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur and maintained in the Plant Breeding and Biotechnology Laboratory of the Department of Botany, University of Dhaka. Three different types of embryo explants, namely cotyledonary node (CN), cotyledon attached decapitated embryo (CADE), and decapitated embryo (DE) were used for the development of in vitro shoots. For seed germination, the seeds were first washed in 70% ethanol (v/v) for 1 min, and then surface sterilized with 0.1% HgCl₂(w/v) solution for 15 min. Seeds were then washed three or four times with sterilized distilled water. The surface sterilized seeds were then cultured on 0.8% (w/v) water agar medium or cotton bed for germination. CN explants were prepared by removing the root and shoot tip as well as half of the cotyledon from three‐day‐old germinated seeds. CADE and DE explants were prepared from overnight soaked sterilized seeds by splitting them open and removing the root and shoot tips from each embryo. All the three types of explants, CN, CADE and DE were cultured on multiple shoot regeneration medium containing MS salts supplemented with 2.22 μM BAP, 2.32 μM Kn, 0.29 μM GA₃_, 30.35 μM tyrosine and 3% sucrose (w/v) in 250 ml Erlenmeyer flask containing 60 ml of medium (Sarker et al. 2003b). The pH of the medium was adjusted to 5.8 with 1M NaOH prior to adding 0.8% agar (w/v), and autoclaved at 121ºC for 20 min. The culture vessels were incubated in the growth room under 16/8 h light/dark cycle at 25 ± 2ºC.

Source of Information: Plant Tissue Cult. & Biotech. 22(1): 13-26, 2012 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

It was observed that in higher concentration of kanamycin (100 mg/l) about 50% of flower bud failed to open and in 200 mg/l of kanamycin no flower bud formation was observed. For these, shoots that survived in the selection medium were separated and transferred to half‐strength of MS containing 98.4 μM IBA and 2.69 μM NAA and 50 mg/l kanamycin. After 2 ‐ 3 weeks in vitro flower formation was observed in the healthy shoots recovered through kanamycin selection. It was found that out of 43 transformed shoots only 11 responded to flowering and the maximum number of flower per shoot was 3. It was also found that after 12 ‐ 15 days 5 out of these 11 flowering shoots produced viable and healthy pods under in vitro condition. The transgenic nature of the transformed shoots was confirmed by amplification of GUS gene present within the genomic DNA of randomly selected tranformants. Specific primers were used for this purpose as mentioned in the materials and methods. For the amplification of DNA through PCR 30 cycles were maintained. After these 30 cycles the amplified DNA was visualized through agarose gel electrophoresis. The results obtained following PCR analysis. The development of effective rooting from in vitro derived shoots in lentil was found to be extremely difficult. Therefore development of in vitro flower and seed formation using the selected shoots was considered to be an effective method in recovering transgenic lentil plantlets. Using this protocol, future study can be conducted to transfer useful genes conferring disease, and pest resistance in microsperma varieties of lentil

Knowledge Management: Journal