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In Vitro Bulblet Formation from Shoot Apex in Garlic (allium Sativum L.)

R. Roksana
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

M. F. Alam
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

R. Islam
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

M. M. Hossain
Department of Botany, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Multiple bulblet formation was induced by repeated (fourth) subculture of regenerated plantlets on both liquid and semi-solid MS medium supplemented with 0.5 mg/l 2ip + 0.25 mg/l NAA. The regenerated bulblets were successfully established in soil.

Keywords: Shoot apex, Bulblet formation, Allium sativum

Location: Department of Botany, University of Rajshahi, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Garlic

Objectives:

Standarization of a protocol for primary establishment of apical meristems and subsequently for rapid shoot proliferation,
Standarization of a protocol for bulblet formation following root induction and the elongation and
Acclimatization of in vitro bulblets/plantlets into soil under field condition.

Methodology:

The cloves were sterilized with 95% ethyl alcohol for 30 seconds for getting contamination free explants to isolate shoot apex. Isolated shoot apecies (0.2 - 0.3 mm) were inoculated aseptically on MS medium (with or without growth regulators) in both liquid and semi-solid conditions. Three kinds of cytokinins 2ip, BAP and Kn with different concentrations (0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 5.0 mg/l) were used singly in MS basal medium. In addition three cytokinins, two kinds of auxins NAA and IBA were also used. Three concentrations of 2ip (0.25, 0.5 and 1.5 mg/l) and five concentrations of NAA (0.05, 0.1, 0.2, 0.25 and 0.5 mg/l) were tested for initial shoot apex culture. Similar combinations were used for BAP + NAA, BAP + IBA and Kn + NAA. After 21 days of inoculation the shoot apecies were transferred from liquid to semi-solid medium was same as in liquid culture. Proliferated shoot apex with multiple clumps were repeatedly subcultured after every three weeks on fresh medium to initiate in vitro bulblets formation. The developed bulblets were separated as individual clove, and cultured on MS medium having different combinations of 2ip, BAP, Kn and 2ip + NAA, BAP + NAA, BAP + IBA, Kn + NAA to observe further bulblets formation. In all cases 3% sucrose and 1 g/l agar were used with pH 5.8. The medium was autoclaved at 15 Ib/sq. inch pressure and at 121_C for 20 mins. The culture tubes were placed in a growth chamber at 25 ± 1⁰C and light period 14 - 16 hrs (2000 - 3000 lux). The data were analyzed using mean ( X ) and standard error (SE) for shoot multiplication and bulblet formation. Lastly the regenerated plantlets/ bulblets were transplanted on sterile soil (using formalin) with 1 : 1 soil and sand. A single plantlet was transplanted in a (9 ∇ 6 cm) poly bag in net house covered by large polythene sheet for maintaining shade and humidity. Necessary cultural practices were done for good growth of the plants. Field evaluation of the regenerated plants is under way.

Source of Information: Plant Tissue Cult. 12(1) : 11-17, 2002 (June)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

From primary shoot apex shoot initiation was observed in all treatments on both liquid and semi-solid conditions. However, in all cases liquid medium was found suitable for early shoot initiation. Similar results were also observed in others, viz. tomato, carnation, potato, and strawberries. Among the different liquid media earliest shoot initiation (three - four days) was observed in the combination of 0.5 mg/l 2ip + 0.25 mg/l NAA followed by in treatment 1.5 mg/l BAP + 0.5 mg/l NAA and 0.5 mg/l 2ip alone. On the other hand late (eight - ten days) initiation was noticed in 5.0 mg/l of Kn. From this result it is revealed that combination of cytokinins and auxins is better for early establishment of shoot apex than using cytokinins alone. Data for elongation and multiple proliferation of shoot were taken after 21 and 42 days from first subculture of primary established shoot apex. At 21 days interval highest mean (9.8 ± 1.2) shoot proliferation was observed in treatment of 0.5 mg/l 2ip + 0.25 mg/l NAA, followed by 7.9 ± 0.87 in 1.5 mg/l BAP + 0.5 mg/l NAA. However, longest (9.8 cm ± 0.63) shoot was measured in treatment of 1.0 mg/l 2ip + 0.5 mg/l NAA. Similar observation was recorded after 42 days of interval. These results showed that the use of low concentration of 2ip + NAA was found suitable for high percentage of shoot-root proliferation rather than use of high concentration and combinations of phytohormones. Beside 2ip + NAA combination, BAP + NAA was also suitable. The acclimatized plantlets were transplanted in formaline sterile soil to observe their survival rate. About 98% transplanted plants survived at the field condition and initially no abnormalities were observed among the regenerated plants.

Knowledge Management: Journal