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Detection of Viruses of Bangladeshi and Japanese Garlic and Their Elimination Through Root Meristem Culture

MS Haque
Laboratory of Plant Genetics and Breeding, Graduate School of Bioagricultural Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan

K Hattori
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

Abstract/ Executive Summary:

A number of viruses cause considerable yield loss and quality deterioration in garlic. Root meristems of virus infected plants are known to be free from detectable viruses. This potentiality could be exploited to obtain virus free clones at a high frequency by culturing excised root meristems in vitro. We have developed efficient methods of direct and somatic embryo derived shoot regeneration from root meristems of garlic. The objectives of this work were to detect viruses infecting Bangladeshi and Japanese garlic clones and find an easy and efficient method of eliminating the viruses for the improvement of both yield and quality of garlic. At first, we confirmed the presence of detectable viruses in three Bangladeshi and one Japanese clones. The clones were infected with four different types of viruses: Garlic viruses (GarVs), Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), and Garlic common latent virus (GCLV). To eliminate those viruses, as per our previous method, root meristems were cultured on MS medium supplemented with 1.0 µM NAA and 10.0 µM BA. Shoot primordia developed from the cultured explants within 1 month. The regenerated individual shoot buds (2-5 mm) were separated from the mother explants and transferred to growth regulators free medium. RT-PCR confirmed that the viruses present in the mother garlic plants were absent in the shoots found after two-step culture. The regenerated shoots were rooted on growth regulator free medium and transferred to pots. Results indicated that the plants remained free from LYSV. Virus elimination through root meristem culture emerged as an efficient novel technique for the eradication of multiple viruses as confirmed by RT-PCR in this study. This technique has the potential for the production and supply of virus free propagules (plants/bulblets) for the yield and quality improvement of garlic.

Keywords: Root meristem, Virus, Garlic, Explants, Propagules

Location: Nagoya University, Japan.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Virus, Garlic

Objectives:

The objectives of this study were to detect viruses in Bangladeshi and Japanese garlic using RT-PCR, produce virus free garlic plants by root meristem culture.

Methodology:

Plant material: Garlic (Allium sativum L.) bulbs of a Japanese typical cultivar, white roppen were collected from a seed company. Bulbs of three Bangladeshi garlic clones (G1, G3 and G14) were carried to Nagoya University, Japan. The cloves were separated, outer dry scale leaves removed and surface sterilized first with 70% ethanol for 30 sec and then with 0.1% sodium hypochlorite for 20 min followed by three times washing with sterile distilled water. The cloves were then cultivated on 0.7% agar for sprouting and plantlet formation. Leaf samples were collected from these in vitro grown plantlets. Root tips were excised from theses plantlets after 10 to 15 days. Axenic root- tips were obtained from in vitro garlic shoots cultured following the protocol of Haque et al. (1998). Root tip culture: Root-tips (2-3 mm long) including the meristems were cut off from the plantlets obtained after culture of the sterilized cloves on agar and also from the micropropagated plantlets with a scalpel under sterile conditions. Explants were cultivated in petri dishes containing 25 ml of MS (Murashige and Skoog, 1962) medium supplemented with 1 µM NAA and 10 µM BA (Haque et al., 1997). All media were fortified with 3% sucrose and solidified with 0.8% agar (BA 30) and adjusted to pH 5.8 prior to autoclaving. After 35 to 40 days when the shoot became ca. 5 mm, they were cut off from the mother explants and transferred to growth regulator free MS medium for their further development and rooting. The media were neither changed nor refreshed during the culture period. Cultures were incubated in a growth chamber at 28±2^OC under a constant light condition. Molecular method of virus detection: RT-PCR was used to detect garlic viruses according to the published protocol (Tsuneyoshi et al., 1996). In the RT-PCR analyses, reverse transcription (RT) was performed using a first-strand cDNA Synthesis Kit (Pharmacia) with approximately 1-2 µl of the RT mix was added to 100 µg of a polymerase reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 200 µM each of dNTP, 2.5 U Taq polymerase (GIbco BRL), and 100 ng each of the upstream and downstream primers. Thirty reaction cycles were used, with periods of 30 s for annealing at 50^OC, 45 s for synthesis at 72^OC and 45 s for melting at 94^OC. Following PCR, 10 µl portions of the reaction mixtures were analyzed on 1.5% agarose gels. The primer sequences for allin lyase, used as the positive control of the RT-PCR experiment, are 5-TCTGGTAGTCGATTTGGGTCG- 3´ and 5-GCCGTAGCATTAGGATGTATG-3´. Plantlet establishment: Rooted shoots from virus free culture were washed thoroughly to remove media. Then they were planted in small plastic pots containing vermiculite and kept in an incubation room at 23±2^OC under a constant light condition for acclimatization.

Source of Information: Progressive Agriculture 28 (2): 55-63, 2017

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Like shoot meristems, root meristems are also known to be free from viruses but virus elimination and production of virus free plants through root meristem culture has not been possible due to the recalcitrant nature of root meristem to in vitro regeneration. In this study, we developed an efficient novel technique for the eradication of multiple viruses and confirmed it by RT-PCR. It is simple and easier than the previous methods of virus elimination through shoot meristem culture coupled with thermotherapy. This technique has the potential for the year round production and supply of virus free propagules (plants/bulblets) of garlic for the improvement of its yield, quality and rate of multiplication. This information would be useful for virus eradication in other plants in future.

Knowledge Management: Journal