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Genetic Transformation in White Jute Through Agrobacterium and Salinity Screening of Transgenic Plant

M. N. AMIN
Scientific Officer
Plant Breeding Division, Bangladesh Agricultural Research Institute (BARI), Gazipur-1701

ASMA KHATUN
Chief Scientific Officer
Genetic Resources and Seed Division, Bangladesh Jute Research Institute (BJRI), Manik Miah Avenue, Dhaka

M. S. R. BHUIYAN
Professor
Department of Genetics and Plant Breeding, Sher-e-Bangla Agricultural University (SAU), Sher-e-Bangla Nagar, Dhaka-1207

M. A. SAYED
Scientific Officer
Plant Breeding Division, Bangladesh Rice Research Institute (BRRI), Gazipur-1701

S. R. KHANDKER5
MS S

Abstract/ Executive Summary:

The experiment was conducted to establish an efficient and reproducible protocol for the plant regeneration and genetic transformation in white Jute (Corchorus capsularis L.). The regeneration and transformation processes depend on optimum growth conditions, suitable explants and varieties. An attempt was made for Agrobacterium mediated genetic transformation in white jute varieties using gene construct conferring both salt and drought tolerance (CIPK and Gly-1) along with the marker genes. Interestingly the two varieties (CVL-1 and Tricap-1) showed the response of both callus induction and plant regeneration on a single formulation i.e. MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l IAA. Explants were dipped to liquid culture of bacteria for one minute and then transferred to co-cultivation media for 24 hours. Shoot regeneration from Agrobacterium infected cotyledon was found highest in variety CVL-1 (43%) than Tri cap (38%). After co-cultivation and selection histochemical GUS assay was performed in different varieties (vars. Tricap-1, CVE-3 & CVL-1). In the transformed explants, GUS reporter gene was expressed showing blue colour in the explants tissues. Among the varieties CVE-3 showed the highest expression blue colour in the explants tissues. Those transgenic plants are transferred to salt medium and soil for evaluation.

Keywords: Media, Agrobacterium tumefaciens, Corchorus olitorius, Transformation

Location: BARI, Gazipur

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Jute

Objectives:

To establish an efficient and reproducible protocol for the plant regeneration and genetic transformation in white Jute (Corchorus capsularis L.).

Methodology:

Seeds of white Jute (vars. Tri cap, CVL-1, CVE-3) were surface sterilized by immersion in 0.1% (w/v) Mercuric chloride for 20 min, followed by 4-5 washes with deionized water. All seeds were placed on the surface of the 50 ml aliquots of hormone free agar solidified (0.8%, w/v) MS basal medium (Murashige and skoog, 1962) in 100 ml conical flasks. In another set of experiment, surgical cotton (1 gm approx. in each flask) was used instead of agar in association with MS basal liquid medium to obtained optimum seedling production. Each flask contained 20 ml of hormone free MS liquid medium. Cultures were placed in a growth room with 20°C under 1.0 Wm2 of daylight fluorescent tubes with 12 h photoperiod. Seed germination percent and number of healthy seedling was recorded. Cotyledons (with attached petiole) of varieties of C. capsularis were cultured on MS medium supplemented with 2 mg/l BAP and 0.5 mg/l IAA. The ultimate goal of in vitro technique was to production of free-living plantlets via shoot and root formation from the explants. The responses of different varieties towards shoot regeneration were found varied. When the shoot was 2-3 cm length, they were rescued aseptically from the cultured flasks and was separated from each other and again cultured individually on 250 ml conical flask with freshly prepared MSO (hormone free MS medium) medium for root production. Conical flask containing plantlets was incubated at 28°C under a 1 Wm2 of daylight florescent tube with a 12 h photoperiod. Agrobactarium tumefaciens LBA4404 harbouring the binary vector pBI121 containing selectable marker CIPK gene and screenable marker GUS gene were grown on YMB (Yeast Mannitol Broth) medium (1.0% Mannitol, 0.04% Yeast extract, 0.01% NaCl, 0.02% MgSO₄_.7H₂O, 0.05% K2HPO₄) containing Kanamycin as the selective agent at 200 rpm in a shaker at 28^oC for overnight. Bacterial concentration was determined by visual observation. Agrobacterium from these cultures were used for infection of cotyledonary petioles of young jute plants. Cotyledonary petioles from germinated seedlings were used as explants. After seven days, cotyledons were excised from the seedlings. This was carried out by gently holding the hypocotyls with forceps, and cutting between the joint just below the shoot tip using sterilized surgical blades. Two kinds of culture media were needed for the Agrobacterium strain. One for maintaining Agrobacterium stock and the other for the infection of explants. For maintenance, one single colony from Agrobacterium stocks was streaked into freshly prepared Petri dish containing YMB medium having Kanamycin. The Petri dish was sealed with parafilm and kept in room temperature for 48 h. For infection, Agrobacterium stock single streak was taken in an inoculation loop and was inoculated in a conical flask containing LB medium with 50 mg/L Kanamycin .The cultures were allowed to grow at 28° C to get optimum population of Agrobacterium for infection and co cultivation of explants. The Agrobacterium grown in liquid LB media was used for infection and incubation. To get suitable and sufficient infection of the explants, freshly excised explants were dipped into bacterial suspension for 1 min before transferring them to co-cultivation medium. Following infection and incubation, the explants were co-cultured on plant regeneration medium in Petri dishes containing 2mg BAP and 0.5mg IAA. Prior to transfer of all explants to regeneration medium, they were blotted dry with sterile filter papers for a short period to remove excess bacterial suspension. All the explants were maintained in co-cultivation medium for 24 hours. Petri dishes containing explants were placed under fluorescent illumination with 12 hours dark cycle at 280 C. The intensity of light was maintained at 1000 lux. Following 24 hours co-cultivation, the explants were transferred to regeneration medium consisting of MS medium supplemented with 2.0 mg/1 BAP, 0.5 mg/1 IAA and 500 µg/ml Cefotaxime. After 6-7 weeks, the regenerated shoots were transferred to hormone free MS medium with 250µg/ml Cefotaxime. Amount of Cefotaxime was gradually reduce in every sub culturing. Putatively transformed shoots were transferred to 25mM/L, 50mM/L, 75mM/L, 100mM/L; 125mM/L salt containing MS media. Plantlet was rescued aseptically from the cultured flask and washed to remove the chemical associated with it and placed normal environment for hardening. Seedlings were transferred to plastic pots containing autoclaved sterilized soil mix (peat moss, perlite, and vermiculite, 5:3:2, v/v/v. Young, pampered seedlings that were grown either indoors or in a greenhouse need a period to adjust and acclimate to outdoor conditions, prior to planting in the garden. After hardening, regenerated shoots were transferred to normal soil. GUS activity was detected as described (Jefferson et al., 1998). Randomly selected co-cultivated cotyledons cultured on selective medium were used for GUS assays. Immediately after inoculation on selection medium, cotyledons were incubated in GUS staining solution at 37^oC for 24 h in dark. The X-glu was broken down by the activity of GUS gene, which was transferred with TDNA in the cotyledonary tissue and produced a characteristic blue colour.

Source of Information: Bangladesh J. Agril. Res. 37(1): 97-107, March 2012, ISSN 0258-7122

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Interestingly the two varieties (CVL-1 and Tricap-1) showed the response of both callus induction and plant regeneration on a single formulation i.e. MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l IAA. Explants were dipped to liquid culture of bacteria for one minute and then transferred to co-cultivation media for 24 hours. Shoot regeneration from Agrobacterium infected cotyledon was found highest in variety CVL-1 (43%) than Tri cap (38%). After co-cultivation and selection histochemical GUS assay was performed in different varieties (vars. Tricap-1, CVE-3 & CVL-1). In the transformed explants, GUS reporter gene was expressed showing blue colour in the explants tissues. Among the varieties CVE-3 showed the highest expression blue colour in the explants tissues. Those transgenic plants are transferred to salt medium and soil for evaluation.

Knowledge Management: Journal