IN VITRO PROPAGATION OF GARLIC (ALLIUM SATIVUM L) FROM MERISTEM CULTURE��

Food and Agricultural Research and Extension Institute (FAREI)

�Pratima Greedharry

Research Scientist

rajpratimah@hotmail.com

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Van

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Personal details:

• Pratima Greedharry
• Research Scientist (FAREI)

Academic background:

BSc in Horticulture with Business Management (Mauritius)

MSc in Biotechnology (Australia)

Email: rajpratimah@hotmail.com

Telephone Number: +23058276586

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Research Aim:

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The aim of this study is to establish and optimize a reliable protocol for the production of virus-free garlic propagules that have high yield with improved quality for Mauritian farmers.

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Introduction

Garlic

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Introduction (Cont.)

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• Used as spice and condiment globally
• Beneficial effect on human health due to presence of allicin and other organosulphur
• Major garlic growing countries are USA, Egypt, China, Korea and India

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Introduction (Cont.)

• Production of garlic in Mauritius is very low
• Most garlic is produced by the small planters (mainly for local market and home consumption)
• Limited supply of clean planting materials

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Introduction (Cont.)

• Consequently importation of garlic is very high

Data obtained from Biometry Division (FAREI)

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Introduction (Cont.):Propagation

• Vegetatively propagated by cloves as most garlic varieties have low flowering ability and are sterile
• Cloves are likely sources of viral contamination and main cause of reduction in yield
• Use of tissue culture techniques as an alternative method for the production of virus-free garlic bulbs

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Materials and methods

Plant material:

One imported garlic accession namely VFG158 introduced from the World Vegetable Centre (WVC) and eleven local garlic accessions namely, Beeharry, Boodnah, Bondah, Gooniah, Haulkhory, Ramdhuny, Ramjee, Rampall, Sujeebun, Sujeebun2 and Unuth received from Agronomy Division of FAREI was tested

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Materials and methods (cont.) : Culture media

Different media formulations tested in this study for shoot proliferation and hyperhydricity were:

• G (MS supplemented with 0.25mg/L NAA+0.5mg/L 2iP)
• G1 (MS supplemented with 1.5mg/L BAP+0.5mg/L NAA)
• G2 (MS supplemented with 3mg/L 2iP+0.3mg/L NAA),
• G3 (cultures are placed on MS supplemented with 0.5mg/L BAP+0.5mg/L NAA {G3A}, then transferred on MS supplemented with 2mg/L BAP+2mg/L NAA{G3B})
• G4 (MS supplemented with 2mg/L BAP+0.5mg/L IBA)
• G5 (cultures are placed on LS supplemented with 0.25mg/L BAP+0.23mg/L IAA {G5A}, then transferred on LS supplemented with 2.3mg/L BAP+0.93mg/L NAA{G5B})
• G6 (MS supplemented with 0.5mg/L NAA+2mg/L 2iP)
• G7 (MS supplemented with 2mg/L BAP+0.1mg/L NAA)
• G8 (of hormone-free MS medium),
• G9 (Gamborg B5 supplemented with 0.5mg/L BAP+0.1mg/L NAA)
• G10 (cultures are placed on MS supplemented with 0.5mg/L 2iP+0.25mg/L NAA {G10A}, then transferred on MS supplemented with 1.5mg/L BAP+0.5mg/L NAA{G10B})
• G11 (MS supplemented with 0.5mg/L BAP+1mg/L 2iP),
• G14 (MS supplemented with 2mg/L BAP+2mg/L NAA)
• G15 (MS supplemented with 1mg/L BAP+0.5mg/L NAA), B2 (MS enriched with 12% sucrose)

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Materials and methods (Cont.) : Culture Media

Different media formulations tested in this study for bulbification were:

• B2 (MS enriched with 12% sucrose)
• B4 (MS supplemented with 2mg/L Kin+1mg/L GA3 and enriched with 3% sucrose)
• B5 MS supplemented with 2mg/L BAP+1mg/L GA3 and enriched with 90% sucrose)

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Materials and methods (Cont.) : Culture Conditions

• Media were supplemented with 3% (w/v) sucrose and 0.24% (w/v) Sigma phytagel
• The pH of the medium was adjusted to 5.86 before autoclaving for 15 min at 121°C
• Cultures were placed in growth room at 25°C ±1.0°C with a 16-h photoperiod and light intensity of 2000 – 5000 lux

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Materials and methods (Cont.)

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Result & Discussion: Growth regulators and their functions

•  Auxin- promote roots growth and cell division

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• Cytokinin- promote shoots growth and cell division 

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• Gibberellin- promote cell enlargement and shoot elongation

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• Different media formulations were prepared based on the different functions of the growth regulators

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• Different concentrations of the growth regulators were also tested (low and high concentrations)

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Result & Discussion (Cont.)

Effect of different media formulations on shoot regeneration and proliferation

• Regeneration of meristem-tip culture on hormone-free Murashige Skoog medium (MS)
• Optimal culture media for shoot proliferation are G, G1, G2, G6, G14 and G15 as shown in the Fig 1
• Genotypic variations observed in the garlic accessions

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Result & Discussion:Effect of different media formulations on shoot proliferation (Cont.)

Fig. 1. Frequencies of multiple shoot formation recorded in garlic cultures. (A) Frequencies of multiple shoot formation on different shoot multiplication media during three generations. (B) Frequencies of multiple shoot formation per of different garlic or group of garlic accessions on different shoot multiplication media during three generations.

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Result & Discussion (Cont.): Effect of different media formulations on shoot proliferation

Fig. 2. Frequencies of multiple shoot formation recorded in garlic cultures onto six selected media. (A) Frequencies of multiple shoot formation per garlic accession onto six selected shoot multiplication media. (B) Frequencies of multiple shoot formation of garlic accessions onto six selected shoot multiplication media during three generations. (C) Multiplication rate of garlic accessions onto six selected shoot multiplication media during three generation

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Result & Discussion (Cont.):Effect of different media formulations on hyperhydricity

• Hyperhydricity formerly termed vitrification is a morphological abnormality
• Vitrified cultures have a glassy appearance with enlarged, thick and water-soaked translucent stems and leaves
• Multiplication factor and vigour of in vitro culture are affected
• Hyperhydricity is suppressed on six media namely G, G1, G2, G6, G14 and G15 as shown in Fig 3
• Lower concentration of BAP or 2ip was used to avoid vitrified shoots

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Result & Discussion (Cont.):Effect of different media formulations on hyperhydricity

Fig. 3. Percentage of vitrification recorded in garlic cultures. (A) Percentage of vitrification for each garlic accession on different shoot multiplication media during three generation. (B) Percentage of vitrification different garlic or group of garlic accessions on different shoot multiplication media during three generation.

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Result & Discussion (Cont.):

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Bulblet formation

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• No swelling of multiple shoots in B4 and B5 media during first six weeks while high frequency of swelling in B2 media

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• Higher number of bulblets formed in B5 media as shown in Fig.4.

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Result & Discussion: Bulblet formation(Cont.)

Fig. 5. Bulblet formation in different bubification media when cultured at 25±2°C and 16L/8D

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Conclusion:

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• Six shoot multiplication media (G, G1, G2, G6, G14 and G15) was established for shoot proliferation and hyperhydricity suppression
• Two bulbification media (B2 and B5) optimal for bulblets formation
• A reliable protocol for rapid shoot regeneration and multiplication from meristem-tip culture and bulblet formation from multiple shoots clumps was optimised

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Reference:

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Asha Devi, A., Khar, A. and Lawande, K., 2007. Genotypic response of short day garlic (Allium sativum L.) accessions to shoot multiplication. . Journal of Spices and Aromatic Crops, 16, pp.15-21.

Barandiaran, X., Martín, N., Alba, C., Rodríguez-Conde, M., Di Pietro, A. and Martín, J., 1999. An efficient method for the in vitro management of multiple garlic accessions. In Vitro Cellular & Developmental Biology - Plant, 35(6), pp.466-469.

Barandiaran, X., Martín, N., Rodríguez-Conde, M., Di Pietro, A. and Martín, J., 1999. Genetic variability in callus formation and regeneration of garlic ( Allium sativum L.). Plant Cell Reports, 18(5), pp.434-437.

Bhojwani, S., 1980. In vitro propagation of garlic by shoot proliferation. Scientia Horticulturae, 13(1), pp.47-52.

Bhojwani S S and Datu P K 2013 Production of Virus Free Plants by Tissue Culture Plant Tissue Culture ed. S S Bhojwani and P K Datu (India: Springer India) pp 227–243

Cafrune, E., Perotto, M. and Conci, V., 2006. Effect of Two Allexivirus Isolates on Garlic Yield. Plant Disease, 90(7), pp.898-904.

Conci, V., Canavelli, A., Lunello, P., Di Rienzo, J., Nome, S., Zumelzu, G. and Italia, R., 2003. Yield Losses Associated with Virus-Infected Garlic Plants During Five Successive Years. Plant Disease, 87(12), pp.1411-1415.

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Thank You

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