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Enhanced Regeneration Through Ex Vitro Rooting and Agrobacterium-mediated Genetic Transformation of Eggplant (solanum Melongena L.)

Sabina Yesmin
Plant Biotechnology Division, National Institute of Biotechnology, Ganak Bari, Ashulia, Savar, Dhaka-1349, Bangladesh

MI Hoque
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

RH Sarker
Plant Breeding and Biotechnology Laboratory, Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

Regeneration of in vitro multiple shoots was achieved through organogenesis on MS supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn from cotyledonary leaf explants of two local varieties of eggplant (Solanum melongena L.). Elongation of regenerated shoots was obtained on growth regulator free MS. In vitro root induction from excised regenerated shoots was less effective on MS with or without plant growth regulators. On the other hand regenerated shoots treated with 10 mM IBA for 5 min were found to be effective for ex vitro rooting in sterilized soil. Following sufficient development of roots, the ex vitro rooted plantlets were acclimatized in growth room condition, and were transferred to the field having 100% survival rate. The regeneration system developed was utilized for Agrobacterium-mediated genetic transformation using Agrobacterium tumefaciens strain LBA4404/pBI121 containing GUS and nptII genes. Adequate transformation response was obtained from cotyledonary leaf segments with bacterial suspension having an optical density of 0.50 at 600 nm with 30 min incubation followed by co-cultivation period of 72 hrs in Nayantara (BARI Begun-5) variety of eggplant. Selection of transformed shoots was carried out on MS supplemented with 2.0 mg/l BAP, 0.5 mg/l Kn, 300 mg/l carbenicillin and 100 mg/l kanamycin. Stable integration of GUS and nptII genes in Nayantara were confirmed through PCR analysis using the genomic DNA isolated from transformed shoots.

Keywords: Eggplant, Ex vitro rooting, Transformation, Agrobacterium, nptII and GUS gene

Location: Vegetable Seed Division of Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Eggplant

Objectives:

To facilitate Agrobacterium-mediated genetic transformation in popular eggplant varieties of Bangladesh.

Methodology:

Two local varieties of eggplant (Solanum melongena L.) such as Nayantara (BARI begun-5) and Kazla (BARI begun-4) were used as experimental materials for this investigation. Seeds of these varieties were obtained from the Vegetable Seed Division of Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur. The procedures for the sterilization of seeds, germination of seeds, and preparation of explants, in vitro regeneration as well as rooting were carried out following protocols developed by Sarker et al. (2006). The cotyledonary leaf explants were cultured on MS with various concentrations and combinations of BAP and Kn for in vitro regeneration of shoots through organogenesis. The pH of the regeneration medium was adjusted to 5.8 before autoclaving. Cultures were incubated and maintained in a growth room with a fixed photoperiod of 16/8 hrs dark/light cycle at 25 ± 1°C. For ex vitro rooting, the in vitro raised shoots were separated out from the culture vessels and cut obliquely at the base. Then the base of excised shoots was dipped into 10 mM IBA solution for five minutes and transferred to small plastic pots containing moist sterilized soil. These pots were then covered with transparent perforated polythene bags and were kept in the growth room for two weeks. After two weeks the cover was removed and the plantlets were hardened during the next week. The genetic transformation was performed using Agrobacterium tumefaciens strain LBA4404 harboring binary plasmid pBI121 containing a scoreable reporter gene GUS (βglucuronidase) driven by CaMV promoter and a selectable marker gene, nptII encoding for the enzyme neomycin phosphotransferase. This reporter gene was used in assessing the efficiency of transformation. A small amount of glycerol stock (preserved in -80⁰ refrigerator) of Agrobacterium-strain was streaked on solid YEP medium and incubated at 28ºC for 48 hrs. After 48 hrs a single colony of Agrobacterium tumefaciens was picked from the plate and again streaked on a new YEP medium. After 48 hrs the bacteria were taken from the plate and dissolved in 30 ml of liquid MS containing 9% (w/v) sucrose. Bacterial suspension having optical density (OD) of 0.3, 0.5 and 0.8 were chosen for transformation. The wavelength of optical density (OD) of this suspension was determined at 600 nm with the help of a spectrophotometer (Shimadzu, Japan). 100 μmol/l acetosyringone was added to the Agrobacterium suspension before infection. The cotyledonary leaf explants were precultured on regeneration medium (RM) containing 2.0 mg/l BAP + 0.5 mg/l Kn for 48 hrs. After 48 hrs the explants were dipped in the bacterial suspension and incubated for variable time periods (20, 30 and 40 min).

After incubation the explants were transferred to co-cultivation medium (MS + 2.0 mg/l BAP + 0.5 mg/l Kn + 100 μmol/l acetosyringone) and incubated for 2, 3 and 4 days at 25 ± 2ºC in an incubator in the dark. After co-cultivation, the explants were thoroughly washed with sterilized distilled water 4 - 5 times and finally washed for 10 mins with distilled water containing 300 mg/l carbenicillin. After that, the explants were transferred to a selection medium containing MS supplemented with 2.0 mg/l BAP, 0.5 mg/l Kn, 100 mg/l Kanamycin and 300 mg/l carbenicillin. Explants were sub-cultured on the same selection medium at a regular interval of three to four weeks. After 45 to 50 days of infection, the green compact callus was found to develop at the cut ends of explants. These green calli were further sub-cultured and maintained on the same selection medium until the shoots were developed. After 100 -110 days small shoots and shoot buds were subcultured to MS basal medium for proliferation and elongation of shoots. Elongated (2.5 - 4 cm long) shoots were excised and used for ex vitro root induction. After 3 to 4 weeks, plantlets with a well-developed root system were transplanted to large earthen pots containing soil and organic manure (5: 1) for further growth and development in the greenhouse condition. Following each transformation experiment, randomly selected ten co-cultured explants were exposed for GUS histochemical assay to determine the efficiency of transformation using X-gluc (5-bromo-4-chloro-3-indolyl-β-D glucuronide) solution and incubated at 37ºC for 24 - 48 hrs. After treatment, explants and plant parts were bleached with 70% ethanol to remove chlorophyll before scoring GUS expression. Leaves, shoots, roots, flower, anther, pollen grain and germinated seedlings were assayed from randomly selected transformed and control (wild) plants. The genomic DNA was extracted from the young leaves of matured transformed and one non-transformed (control) plants using CTAB method (Doyle and Doyle 1990). Plasmid DNA of pBI121 was used as a positive control. The presence of introduced GUS and nptII genes were detected by PCR analysis (eppendorf Master Cycler Gradient, Germany). The gene-specific primers for GUS gene 5´CATGAAGATGCGGACTTACG-3´ and 3´-ATCCACGCCGTATTCGGCGT-5´ were used as forward and reverse at a concentration of 10 pmol/μl. For the confirmation of the nptII gene, DNA was subjected to PCR analysis using 5´TAGCTTCTTGGG TATCTTTAAAATA-3´and 3´-CCAGTTA CCTTCGGAAAAAGAGTT5´, as forward and reverse primer respectively. For 25 µl PCR reaction 0.2 μl Taq DNA polymerase, 10 × 2.5 μl buffer and 0.5 μl dNTPs were used. For GUS gene DNA was denatured at 95ºC for 5.0 min, followed by 30 amplification cycles. Each cycle was programmed with three different thermal periods: at 95ºC for 1.0 min to denature DNA, at 56ºC for 30 sec to anneal the primer and at 72ºC for 1.0 min for the extension or elongation of DNA by Taq DNA polymerase. For nptII gene DNA was denatured at 95ºC for 5.0 min, followed by 30 amplification cycles using 95ºC for 1.0 min (denaturation), 55ºC for 1.0 min (annealing) and 72ºC for 1.0 min (extension). The final extension lasted for 10 min at 72ºC to allow complete extension of all amplified fragments. After completion of the cycling program, the reaction was held at 4ºC. The amplified

Source of Information: Plant Tissue Cult. & Biotech. 31(1): 97-108, 2021 (June)

Article Link (Online Journal): DOI: https://doi.org/10.3329/ptcb.v31i1.54115

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, it may be mentioned that an efficient and reproducible Agrobacterium tumefaciens- mediated transformation protocol was established for eggplant varieties of Bangladesh using the cotyledonary leaf as explants using screenable (GUS) and selectable (nptll) marker genes. Following this standardized protocol various other agronomically important genes/genes can be transferred to the locally grown eggplant varieties. Particularly this technique of transformation can be utilized for the development of salinity and drought tolerant eggplant varieties which will certainly contribute significantly in the future agriculture of Bangladesh.

Knowledge Management: Journal