Factors Affecting the Infection of Vesicular Arbuscular Mycorrhizal Fungi in Transformed Root Culture

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1 Factors Affecting the Infection of Vesicular Arbuscular Mycorrhizal Fungi in Transformed Root Culture Poonpilai Suwanaritl, Savitri Ascharakul2, Omsub Nopamornbodi3 and Malee Suwana-adth4 I Department of Microbiology, Faculty of Science, Kasetsart University 2 Department of Agro-industrial Technology. Faculty of Applied Science. King Mongkut's Institute of Technology 3 Soil Science Division, Department of Agriculture. Ministry of Agriculture and Cooperative 4. National Science and Technology Development Agency (NSTDA) Induction of hairy roots by Agrobacterium rhizogenes ATCC was investigated on bulbs of 4 kinds of plants. The induction was successful oniy in the cases of potato and carrot. The transformed hairy roots could be re-propagated without any limitation via roots culturing. Growth rate of the carrot roots was highest at 14 days and still good when transferred to M2 medium (low in Nand P). Growth rate of the potato roots was highest at 10 days but growing them in M2 medium was impossible because of bacterial contamination. In a study on sodium hypochlorite and chloramine- T as surface disinfectants, the most effective concentration and time of chloramine- T to reduce contamination and induce spore germination were 5% for 5 minutes for Acaulospora spinosa and 5% for 10 minutes for Glomus mosseae. Factors affecting the infection of G. mosseae in transformed carrot roots were studied. Studies were conducted with six levels of ph, three levels of sucrose, five levels of KH2P04, two levels of DNA, five levels of KNO) and four levels of KNOJ supplemented with NH4NO), two levels of myo-inositol, two levels of nicotinic acid, two levels of pyridoxine-hci, two levels of thiamine-hci, and two levels of glycine. Vesicle-like structures were formed when the fungus was cultured in the medium with KH2P04 at 4.8 mg/i and DNA at 60 and 100 mg/i as sources of P and in the medium with KNO) at 50 or 80 mg/i without NH4NO) as a source of N. Suitable ph's were 5-7. All of the growth factors had positive effects on the infection. Roots that formed vesicle-like structures were inoculated to corn plants in pots. After two months, spores of Glomus mosseae were repropagated. INTRODUCTION. Mycorrhiza is a symbiotic association of plant roots and a fungus. Vesicular asbuscular mycorrhiza (VAM) is a special type of l11ycorrhiza which produces fungal structures, vesicles and arbuscules, in the cortex region of the roots. VAM are formed by non septate fungi belonging to the genera Acaulospora. Elltrophaspora. Glomus, Gigaspora. Scutellospora and Sclerocystis in the order

2 Glomales (Morton and Benny, 1990). These fungi are obligate symbionts and can not be cultured on any artificial medium. VAM fungi are not host specific. One of the major impediment to use of, and research into, VAM fungi is our current inability to grow the organisms in pure culture (Powell and Bagyaraj, 1984). Propagation of VAM propagules have to be done in sterilized potted medium (of soil, sand, vermiculite) with a suitable host plant. Anyhow, the inoculum is still not pure. The use of culture of roots transformed by Agrobacterium rhizogenes as a substrate for pure culture propagation of VAM fungi was therefore studied. MATERIALS AND METHODS Preparation of transformed roots 'Transformed roots were" prepared by induction of hairy roots with Agrobacterium rhizogenes according to the following procedures. Four kinds of plants, namely, potato, carrot, sweet potato and taro were used as test plants. Bulbs of the test plants were treated with 70 % alcohol before cutting into pieces of about 1.8 cm x 1.8 cm x 1.8 em. Surface sterilization of the test plant bulbs were made by treating with % clorox added with 20 drops of Tween 20 for 15 minutes (Moore et al., 1979) followed by washing with sterilized water for 4-5 times. The pre-sterilized test bulbs were cut into small pieces by sterilized cork borer (11 em in diameter). The two ends of the obtained cylindrical pieces of bulbs were then removed to obtained the sterile inner part of bulbs. The obtained cuts were then placed on 1 % water agar in petri dishes. About 0.1 ml of two days old suspension of Agrobacterium rhizogenes ATCC (approximately 109 cells/ml) was dropped on each of the cuts on water agar. The petri dishes were then sealed with parafilm paper to prevent evaporation of water and contamination. Incubation of the cuts was made in the dark at 23-2S0C until transformed roots emerged from the bulb cut. Elimination of Agrobacterium rhizogenes. One centimeter of the transformed root was transferred to Linsmair and Skoog Q 965) medium added with amphicillin at 0.5 gll. After days of cultivation, elongated roots were cut and transferred to medium added with amphicillin. This procedure was repeated for 4-5 times on the obtained c~ltures, in order to make sure that all inoculated bacteria were eliminated.

3 Propagation of transformed roots Measurement of tile growtll of roots

4 Cultivation of VAM fungi on transformed root culture

6 Factors affecting infection ofvamfungi on transformed root

11 CONCLUSION ACKNOWLEDGMENT REFERENCES

Effect of host plant, cultivation media and inoculants sources on propagation of mycorrhizal fungus Glomus Mossae

EUROPEAN ACADEMIC RESEARCH Vol. V, Issue 12/ March 2018 ISSN 2286-4822 www.euacademic.org Impact Factor: 3.4546 (UIF) DRJI Value: 5.9 (B+) Effect of host plant, cultivation and inoculants sources on propagation