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In Vitro Regeneration and Genetic Transformation of Peanut (arachis Hypogaea L.)

R. H. Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Aparna Islam
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

A regeneration protocol for a locally grown peanut (Arachcs hypogaea L.) strain, DM-1 via organogenesis was developed prior to transformation. Maximum number of shoots developed via callus when leaflet explants from DM-1 were cultured on MS medium supplemented with 5.0 mg/1 BAP. maximum number of shoots were obtained directly from leaflet explants on the same medium when supplemented with 5.0 mg/1 BAP and 0.5 mg/1 Kn. For the induction of roots 0.2 mg/1 NAA in half-strength of MS medium was found to be the most suitable. Agrobacterium tumefaciens strain containing binary plasmid pBI 121 with GUS (β-glucuronidase) and NPT II kanamycin resistance gene, neomycin phosphotransferase) genes were used for co-cultivvtion of leaflet, hypocotyl and epicotyl explants from DM-1. Numerou GUS positive regions were visualized within the various co-cultivated explant tissues following GUS histochemical assay indicating the transformation ability of such explants of peanut.

Keywords: Arachis hypogaea, Regeneration, Genetic transformation

Location: Oil Seed Division, Bangladesh Agricultural Research Institute ( BARI), Joydebpur, Gazipur

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Ground nut

Objectives:

To study the in vitro regenerating ability of this selected strain of peanut before performing genetic transformation experiments.

Methodology:

Seeds of peanut ( Arachi s hypogaea L.) strain DM-1 were obtained from the Oil Seed Division of Bangladesh Agricultural Research Institute ( BARI), Joydebpur, Gazipur were used in the present investigation. To get explants from in vitro grown plants, seeds were germinated on sterile water soaked cotton following their surface sterilization in 0.1% HgCl₂ for 5 minutes and then rinsing with sterile distilled water three to four times. Different explants like leaflet, epicotyl and hypocotyl were collected from these germinated seeds. Plantlet regeneration : Experiments were conducted on MS medium having different concentrations of benzylamin opurin e (BAP), singly or in combination with kinetin (Kn) for multiple shoot regeneration with or without the intermediate state of callus. For all media, pH was adjusted to 5.8 before autoclaving. The cultures were maintained under fluorescent light on a 16 h photoperiod at 25 ± 2°C. For rooting different types of auxins including indole-3-acetic acid (IAA), napthalene acetic acid (NAA) and indole-3-butyric acid (IBA) were used in half strength of MS medium. Regenerated shoots of 2 - 3 em long were dissected and placed vertically on the medium for root induction. Agrobacterium strain and vector plasmid: Agrobacte rium tumefaciens strain LBA 4404 used in this study obtained by the courtesy of Professor Zeba Islam Seraj of the Department of Biochemistry, University of Dhaka. This Agrobacterium strain with the binary plasmid pBI 121, contains the GUS reporter gene (β-glucuronidase) and the NPT II (Neomycin Phosphotr a nsferase) kanamycin resistant gene. Stocks of this Agrobacterium harbouring the binary plasmid was grown and maintained in YMB (Yeast extract mannitol broth) agar medium having 50.0 mg/l kanamycin with pH adjusted at 7.0 - 7.2. Agrobacterium suspension was made using the same component of YMB medium except agar used for co-cultivation purposes. This Agrobacterium su pension culture was prepared, by inoculating a single colony from the stock of Agrobacterium in the YMB medium and maintained for 19 hours in a shaking incubator. Co-cultivation : Expalnts of epicotyl, hypocotyl and leaflet obtained from in vitro germinated seeds were infected by Agrobacterium strain. The cut explants were dipped in the Agrobacterium suspension for 60 minutes and transferred to Petri dishes containing co-cultivation medium. The co-cultivation medium is composed of normal regeneration medium as described above. Petri dishes were incubated under flourescent light having 16 h photoperiod. GUS histochemical assay : Following co-cultivation explants were subjected to histochemical assay to determine the efficiency of infection and the possible transient expression of GUS. For this purpose the co-cultivated explants were incubated in phosphate buffered solution containing 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc; 0.16 mg/ml) at 37°C overnight and the tissues were bleached by soaking in 70% ethanol before observation under microscope. When required such explant tissues were used to prepare thin section. A characteristic blue colour inside the explant cells indicated the presence of the expression of GUS gene.

Source of Information: The Dhaka University Journal of Biological Science, Vol-08, No. - 02, 1999, ISSN 1021-2484

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The hormonal combination was found to be most effective regarding the frequency of multiple shoot bud formation as well as the formation of multiple shoots/explant. These findings did not show any similarity with a number of reports regarding multiple shoot regeneration in peanut. Mroginski et at. obtained maximum regeneration through callus culture on MS medium supplemented with 1.0 m g/l BAP and 1.0 m g/l NAA. Pittman et al. also reported maximum frequency of shoot bud regeneration from leaflet derived callus using above concentration of BAP and NAA. Such variable response regarding regeneration served in the present investigation m ay be due to the use of different genotypes of peanut from other investigators. Genotypes has been considered as an important factor in determining regeneration potential in peanut. A significant number of epicotyl, hypocotyl and leaflet explants co-cultured with Agrobacterium showed positive GUS staining. GUS positive regions were detected at the cut end as well as within the internal tissues of the various explants. Results of such investigations have clearly revealed the incorporation of GUS gene through Agrobacterium mediated transformation with the explants of peanut growing in Bangladesh. The explants so far studied leaflet was found to be more susceptible to Agrobacterium infection and moreover, successful regeneration was also obtained from this explant. Therefore, further transformation experiments can be carried out using leaflet explant for obtaining transgenic peanut plant.

Knowledge Management: Journal