Journal of Horticultural Sciences

D. P. Prakash ¹, Y. L. Ramachandra ², Vageeshbabu S. Hanur ³

Affiliations
1 College of Horticulture, University of Horticultural Sciences, Munirabad - 583 233, Koppal, Karnataka, IN
2 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga - 577 451, IN
3 Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bengaluru - 560 089, IN

Abstract

The present study purports to assess the effect of Agrobacterium infection time, co-cultivation and cell density on in vitro response in hypocotyl explants of eggplant (brinjal) cv. Manjarigota. Agrobacterium (OD₆₀₀ 0.3-0.5) infection for 10-15 minutes (24.44±2.34%) was found to be optimum, while, higher or lower infection-time resulted in reduced callus initiation, shoot regeneration and explant survival. Explants with no (only Agrobacterium infection) or short (1 day) co-cultivation, showed reduced callus-initiation response and turned yellow, with no regeneration. Callus-initiation response increased from Day 1 (96.66±03.33%), and reached a maximum on Day 2 and Day 3 (100±00.00%). It decreased on further increase in co-cultivation time. Explants co-cultivated for three days showed highest regeneration response (30.00±02.96%) which thereafter reduced with further increase in co-cultivation time. Explants infected with Agrobacterium culture at 0.05 OD₆₀₀ showed hardly any regeneration, and turned yellow and necrotic on the selection medium. Highest regeneration response (28.33±02.33%) was obtained in explants infected with 0.1 OD₆₀₀ culture, and this gradually reduced as celldensity increased (upto 1.0 OD₆₀₀), becoming zero in explants treated with cultures at 1.5 OD₆₀₀ or above. Agrobacterium overgrowth was noticed on explants infected with cultures of 0.5 OD₆₀₀ and above. Exposure of hypocotyl explants to higher cell-density, longer infection-time and prolonged co-cultivation regime resulted in severe necrosis of explants; time taken for development of Agrobacterium overgrowth was less with increase in the level of these factors. Regenerated shoots were healthy, green, elongated and showed ischolar_main induction on culture medium containing Kanamycin.

Keywords

Eggplant, Manjarigota, Regeneration, Agrobacterium, PCR.

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D. P. Prakash ¹, Y. L. Ramachandra ², Vageeshbabu S. Hanur ³

Affiliations
1 College of Horticulture, Munirabad-583 233, IN
2 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga-577 451, IN
3 Division of Biotechnology, ICAR-Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bengaluru - 560 089, IN

Abstract

An attempt was made to assess the effect of size, age and position of the explant, pre-culture and high cytokinin concentration in the pre-culture medium on shoot regeneration in brinjal hypocotyls co-cultivated with Agrobacterium. The study was carried out using hypocotyl explants of brinjal cv. Manjarigota, Agrobacterium strain A208 and shoot regeneration medium (full-strength basal MS medium, 2μM BAP + 0.05μM NAA, 3% sucrose and 0.8% agar) containing Cefotaxime (250-500mg l^-1) and Kanamycin (100mg l^-1). Hypocotyl explants showed callus initiation and shoot regeneration response after 10-12 and 20-22 days of culture, respectively. Five-day-old explants did not survive Agrobacterium infection, and ten-day-old explants showed higher shoot regeneration (29±1.91%) than older explants. Explants of medium size (1cm long; 32±2.62%) from the apical region (38.57±2.61%) showed better shootregeneration ability than explants of any other size or region. A period of four days of pre-culture (33.33±3.76) was optimal best for best shoot-regeneration in hypocotyl explants. No regeneration was seen in hypocotyl explants at shorter or longer pre-culture period. High cytokinin (10μM) in shoot regeneration medium during pre-culture enhanced shoot regeneration response (47.27±2.98%) in explants co-cultivated with Agrobacterium. Effects of various factors documented in this study will be useful in developing an efficient Agrobacterium-mediated transformation protocol in brinjal cv. Manjarigota.

Keywords

Eggplant, Pre-Culture on High BAP, Explant Characters, PCR.

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D. P. Prakash ¹, B. S. Deepali ², Y. L. Ramachandra ³, Lalitha Anand ², Vageeshbabu S. Hanur ²

Affiliations
1 College of Horticulture, Munirabad-583 233, Koppal, UHS Bagalkot, Karnataka, IN
2 Division of Biotechnology, Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bangalore 560 089, IN
3 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga 577 451, IN

Abstract

In the present study, effect of size and age of hypocotyl explant on in vitro organogenetic responses was assessed in eggplant cv. Manjarigota. Size and age did not affect callus-initiation response, but showed marked influence on shoot regeneration response. Hypocotyl explants 1.5cm long showed highest shoot regeneration response (77.4%); either increase or decrease in size resulted in reduced response. Five to 15 day old hypocotyl explants showed direct shoot regeneration from cut ends, whereas 20-30 day old hypocotyl explants showed indirect shoot regeneration from callus produced on cut ends. Five day old explants were most responsive, with highest (91.23%) and thirty day old explants least responsive with reference to shoot regeneration response (20.85%). Shoot regeneration frequency decreased with increasing age, whereas shoot regeneration efficiency increased with increasing age of hypocotyl explants.

Keywords

Eggplant, Shoot Regeneration, Age and Size, Hypocotyl Explants.

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D. P. Prakash ¹, B. S. Deepali ¹, R. Asokan ¹, Y. L. Ramachandra ², Lalitha Anand ¹, Vageeshbabu S. Hanur ¹

Affiliations
1 Division of Biotechnology, Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bangalore - 560 089, IN
2 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga, IN

Abstract

Effects of growth regulators and type of explants on transformation and in vitro morphogenetic responses of brinjal cv. Manjarigota were studied. Both hypocotyl and cotyledonary explants showed marked influence on in vitro morphogenetic responses after Agrobacterium co-cultivation. Hypocotyl explants showed callus initiation and regeneration responses earlier than cotyledonary leaves. Hypocotyl explants were found to be better than cotyledonary leaf explants in regenerating shoots after Agrobacterium co-cultivation. There was delay and reduction in both callus and regeneration responses in Agrobacterium co-cultivated explants. Hypocotyl explants showed the highest regeneration response on MS medium containing 2 μM BAP and 0.05 μM NAA while cotyledonary leaves did not show regeneration response after Agrobacterium co-cultivation. However, they showed green buds on MS medium containing 10 μM BAP and 1 μM NAA, which could not differentiate into shoots. Overall, hypocotyl explants were found better in regenerating shoots after Agrobacterium co-cultivation.

Keywords

Growth Regulators, Explant, Brinjal, Transformation.

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D. P. Prakash ¹, B. S. Deepali ¹, R. Asokan ¹, Y. L. Ramachandra ², Lalitha Anand ¹, Vageeshbabu S. Hanur ¹

Affiliations
1 Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore – 560 089, IN
2 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga, IN

Abstract

A study was conducted to find out the effect of antibiotics and gelling agents on Agrobacterium-mediated transformation using hypocotyl explants of brinjal cv. Manjarigota. Hypocotyl explants of brinjal were found to be sensitive even to the lowest level of kanamycin (25 mg/l) tested. Explants that showed increased callus initiation and regeneration response upon cocultivation with Agrobacterium and on kanamycin at 100 mg/l were selected as this indicated a highly effective selection pressure. Cefotaxime did not affect regeneration response and at 500 mg/l, it effectively inhibited Agrobacterium overgrowth completely on Agrobacterium cocultivated hypocotyl explants. There were marked differences in regeneration response in hypocotyl explants cultured on medium solidified with various gelling agents indicating the influence of gelling agent on the activity of kanamycin in culture medium, which indirectly affects selection and recovery of transformants. Antibiotics and gelling agents could therefore affect, directly or indirectly, transformation of brinjal cv. Manjarigota.

Keywords

Solanum melongena, Kanamycin, Cefotaxime, Gelling Agents, Transformation.

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Vageeshbabu S. Hanur ¹, D. P. Prakash ¹, B. S. Deepali ¹, R. Asokan ¹, Y. L. Ramachandra ², Riaz Mahmood ², Lalitha Anand ¹

Affiliations
1 Division of Biotechnology, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore 560 089, IN
2 Department of Biotechnology, Kuvempu University, Shankaraghatta, Shimoga, IN

Abstract

Poor regeneration is one of the limiting factors in the development of transgenic crops since Agrobacterium as a plant pathogen can disturb the fragile in vitro conditions with wounding and infection regimes. We have tried to optimize the transformation system in two important varieties of brinjal after Agrobacterium infection to the explants. The effect of explant was studied and hypocotyls were found to be better than cotyledonary leaves. High BAP during the preconditioning period was found to further enhance the regeneration rate. Therefore, use of hypocotyls and high BAP during preconditioning can improve the regeneration of transformed cells and recovery of transformants in vegetables especially brinjal.

Keywords

Solanum melongena, Transformation, Hypocotyl, BAP, Preconditioning.

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