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An Efficient Protocol for Somatic Embryogenesis of Garlic (alliumsativum L.) Using Root Tip as Explant

M. N. Hassan
Patuakhali Science and Technology University, Dumki, Patuakhali-

M. S. Haque
Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali-8602

M. M. Hassan
Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali-8602

M. S. Haque
Department of Post-harvest Technology and Marketing, Patuakhali Science and Technology University, Dumki, Patuakhali-8602

Abstract/ Executive Summary:

Genetic improvement of garlic through conventional breeding is very difficult due to sterile nature of its flower. Hence, an alternative system is desirable to induce genetic variation. Tissue culture could be a good opportunities and somatic embryo genesis is one of the potential techniques of tissue culture for in vitro regeneration of garlic plant. The successes and production of somatic embryo depends on several factors such as optimization of media components, genotypes and explant type. Therefore, in the present investigation, garlic root tips were used as explant for callus and somatic embryo induction under different plant growth regulator combination. It was found that MS+1.0 mg l-1 2,4-D was the most favorable (86.10% regeneration with 2.19 cm callus diameter) for callus induction. This concentration also induced and produced good quality somatic embryo. In addition, MS+2.0 mg l-1 Kinetin gave better regeneration of somatic embryo and yielded the highest number (4.670) and longest length (7.0 cm) of shoots per callus. The procedure used a single hormonal signal for callus and somatic embryo induction as well as hormone free medium for further development of plantlet. Besides, maximum duration for callus induction and somatic embryo production was 17 and 10.67 days respectively. Thus, it appears that the protocol is cheap and time bound and particularly useful for conducting experiment for genetic improvement of garlic. Furthermore, as the protocol is cost effective, it can be further tested for commercial feasibility.

Keywords: Root tip, Explant, In vitro culture, Somatic embryogenesis, Plantlet regeneration

Location: Department of Biotechnology of BAU, Mymensingh

Start Date: 00-04-2010

End Date: 00-12-2011

Program Area: Variety and Species

Commodity: Garlic

Objectives:

To optimize the media components for the development of an efficient protocol for somatic embryo genesis.

Methodology:

The study was conducted in the USDA-BIOTECH Laboratory of the Department of Biotechnology of Bangladesh Agricultural University from April, 2010 to December, 2011. Explants were taken from sprouted garlic cloves cultured on water agar. Garlic variety namely Japanese cv. white roppen and Bangladeshi garlic were taken to investigate there in vitro regeneration potentiality. Garlic cloves were surface sterilized with 70% alcohol for 30 sec and then 0.1% HgCl₂ solution with 2 drops Tween–20 per 100 ml for 5 minutes subsequently washed by autoclaved distilled water for three times to remove trace of HgCl₂ which would be toxic to the explant. The cloves were then ready for placement into the media. Culture methods: Sterilized cloves were placed into sterilized sprouting medium in culture vessels and then incubated in dark till the sprouting of the cloves. After that it was transferred to 12 h light period. Within 3-5 days roots were initiated and ready for use as explants. Explant culture: The aseptically grown seedlings were placed on a sterile vial. Young root tips measuring 2-3 mm in length were separated and placed in the sterile culture media containing different concentrations of 2,4-D. Two or three root tips were inoculated in each vial and covered with lid and sealed with parafilm. Subculture or transfer: When the calli attained a convenient size they were removed aseptically from the culture vessels and placed on a sterile vial, inside the airflow cabinet. The calli were cut into small pieces and were placed into freshly prepared sterilized media with appropriate concentration of growth regulators. These were again sub-cultured to freshly prepared medium containing different hormone supplements for the maintenance of callus or for shoot-root differentiation. The culture vessels showing signs of contamination were discarded. Repeated sub-culturing was done at an interval of 15 days for the maintenance of calli. Incubation: Culture vessels with inoculated explants were incubated both in dark and light condition under controlled temperature of 28ºC. About 16 h photo periods with a light intensity of 2000-3000 lux was maintained. Observation was carried out daily to note the response. Preparation of pot: Potting mixture containing properly mixed ground soil and cowdung in the ratio of 1:1 and was placed into earthen pots for growing the plant lets in vivo condition after autoclaving. Transfer of plant lets on to soil: When the plant lets became 3-5 cm in length with 2-3 well developed leaves and roots, then the plant lets were removed from the vials. Medium attached to roots was gently washed out with tap water. Plant lets were then transplanted to pots containing the above mentioned potting mixture. Analysis of data: The data were analyzed using Complete Randomized Design (CRD) and the treatment mean is compared with the LSD (Least significant difference) test.

Source of Information: J. Bangladesh Agril. Univ. 12(1): 1–6, 2014 ISSN 1810-3030

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The study demonstrated good success of root tip as explants for in vitro plant regeneration of garlic through somatic embryo genesis. The investigation also found some growth regulator combination which gave excellent performances regarding callus induction and somatic embryo production. The protocol described here used a single hormonal signal to induce callus and somatic embryo and hormone free medium for plant regeneration from somatic embryo. Thus the protocol is cost effective. Besides, a high success rate was achieved comparatively within short period of time. Therefore, the protocol can be widely used for rapid in vitro plant regeneration of garlic in commercially feasible way. Moreover, genetic improvement of garlic requires an efficient tissue culture protocol and the protocol stated here can be a good supplement in this respect.

Knowledge Management: Journal