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JOURNAL OF BIOSCIENCE AND BIOENGINEERING
Vol.96, No.5, 503-505. 2003
A Facile Tranformation of Arabidopsis thaliana
Using Ceramic Supported Propagation System


EI-ICHIRO FUKUSAKI,1 TATSUHIKO IKEDA,1 DAISUKE SUZUMURA,2
AND AKIO KOBAYASHI

Department of Bitechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka,
Suta, Osaka 565-0871, Japan1 and Technical Depertment, Phytoculture Control Co., Ttd.,
5-18-14 Sekime, joto-ku, Osaka 536-0008, Japan


Recieved 17 April 2003/Accepted 2 September 2003
@Arabidpsis thaliana can be a useful model plant in the field of plant molecular biology because it is a sole dicotyledonous plant whose genome sequence has been completely determined at the present time. To develop functional genomics of A. thaliana, a large number of transgenic plants must be produced. However, transformation of the plants requires wide space and long time. For these reaoson, a highly efficient method has been desired to be developed. In order to save apace, ceramic tubes. The new method using ceramics tubes facilitated to prepare plants with a uniform growth level. In addition, the colander procedure after infiltration, which is one of tedious procedures, could be omitted. The new system without soil usage could cut down the cleaning up after expariment.

[Key words: Arabidpsis thaliana, transformation, vacuum infiltration, ceramic]

@Arabidpsis, which is a sole dicotylendonous plant whose genome sewuence has been completely determined, is thought to be a useful model plant in the field of plant morecular biology (1). To obtain transgenic Arabidpsis, an Agrobacterium mediated gene tranfer by vacuum infiltration method is widely emplayed (2). However, the conventional method is not suitable for standard protocol to perform a large number of transformation which is necessary for functional genemics. The conventional method has problems as follows: (i) space requirement, (ii) special techniques for cultivation of Arabidpsis before infiltration (the timing of disbudding, the state of the plants and the numbers of applicable plants), (iii) the tedious work after infiltration caused by soil application (autclave and disposal), (iv) the time required for propagation before infiltration about 50d; we need about 100d from seeding to getting T1 seeds, but the half of period is for propagation before infiltration. The problem of space is especially seriou in Japan because most of the institutes are restricted in their laboratory space. We attempted to reduce space for transformation expariments using a ceramic tube with pore size of around 3m, instead of soil, as a supporting matrix. Water was delivered to the surface of the porous ceramics from a water tank by capillary action and taken by the plant roots via gas-exchange through the porous ceramics (3). Water supply could be easily controlled by the ceramic cultivation, wheweas it is difficult to keep the water level by the conventional hydroponics culture system. The ceramic cultivation system would be suitable for plants that are weak both in desiccation and over moisture. These ceramic tubes, which are prepared from homogeneous material by high temperature sintering have superior durability and uniformity. We succeeded in repeatable cultivation of Arabidpsis with the reproducibilty in growth rate. Plants of Arabidpsis thaliana (Columbia) for transformation were propageted according to the reported method (4) (Fig.1). First, ceramic tubes (14mm in outer diameter, 9mm in innerdiameter, 45mm in length) were immersed in water containing 0.1% hyponex (Hyponex Japan, Osaka) and seeds were applied on the inner top end of ceramic tubes. After maintaining the seeds for 4 weeks at 23 under the light condition of 30mol quants/m2Es, seedlings were transferred to the inner top of the second ceramic tubes (14mm in outer diameter, 9mm in inner diameter, and 85mm in length) that were immersed in luquid fertilizer (1.5mM NaH2PO4E2H2O, 0.25mM Na2HPO4E12H2O, 1.5mM MgSO4E7H2O, 2mM Ca(NO3)2E4H2O, 3mM KNO3, 67M Na2EEDTA, 8.6M FeSO4E7H2O, 10.3M MnSO4E4H2O, 30M H3BO3, 1.0M ZnSO4E7H2O, 1.0M CuSO4E5H2O, 0.024M (NH4)6EMo7O24E4H2O, 0.13M CoCl2E6H2O) (Fig. 2A, left and center). These plants were cultivated for another 4 weeks (23, 30mol quants/m2Es) (Fig. 2A, right).
@We performed transformation according to the general vacuum infiltration method (2), except that ceramic tubes were employed. Agrobacterium tumefaciens (EHA105) harboring the binary vector pBI121 (Clontech, Palo Alto, CA, USA) was fully grown in LB medium (500ml) containing kanamycin at 100mg/l for 48h at 28. After centrifugation, the bacterial pellet was resuspended in 1l of the infiltration medium (IM; 1/2 Gamborg's B-5 Medium containing 0.1% Hyponex 5-10-5, 1% Sucrose, 0.02% Silwet L-77 and 0.044M 6-benzylaminopurine). Ten of the ceramic tubes were immersed upside down in 300ml of Agrobacterium containing IM medium in a 500-ml beaker (Fig. 2B).And three beakers were put in vacuo (50kPa) for 5 min in a vacuum chamber. The infiltrated plants were placed on a tray as before (Fig. 2C). All the treatments were performed with latex gloves. The same procedure was repeated six times to infect 60 plants in the ceramic tubes in total. Four weeks after planting (23, 30mol quants/m2Es), about10,000T1 seeds were harvested. A half of the harvested seeds was sown on selection medium (1/2 Murashige and Skoog medium containing kanamycin 100mg/l). Two weeks later, seven transformed plants were selected. These experiments were repeated three times in a similar manner.
@The transformation efficiency of the new method using ceramic tubes was almost the same that of the conventional method using soil (Table 1). The space required for this new method was less than three-fourth of the space of the conventional method (Table 1). In the conventional system, five plants are usually grown in one pot. Among them only four or three plants are applicable for infiltration due to the variation of growth condition. In other words, it is very difficult to achieve the uniform growth level of Arabidpsis. The new method using ceramic tubes facilitates to prepare plants with uniform grouwth level, which means that the emergence of useless plants can be minimized and thereby it is more effective in space saving. In addition, the colarnder procedure after infiltration, which is one of tedious procedure, could be ommited. And the new system without soil usage can cut down the cleaning up after experiment. In the near future, we will set up an outsouring business to provide Arabidpsis using ceramic supported propagation system. More than half of the space in climate chambers would be saved and utilized for the more effective application. We believe that many institutes can reduce time and space by the outsourcing business.


Table 1. Comparison between the conventional (soil) method and the ceramic method in terms
of transformation efficiency and space requirement
conventional (soil) New (ceramics)
Transformation efficiencya 13 14(run 1), 12(run 2), 12(run 3)
Space requirement 19cm2b 14cm2c
a Tranformation efficiency indicates numbers of transformants per 10,000 of T1 seeds sown. The same experiments were repeated three times in the new method (runs 1-3).
b Sixty-four plants could be grown in the space of 35~45cm on soil.
c Sixty-three plants could be grown in the space 46~20cm on ceramic tubes.

Fig. 1. Chart of planting and transformation
A B
C Fig. 2. (A) A.thaliana grown in ceramic tubes.The left is before transfer and the center is after. The right is used for vacuum infiltraton.
(B) Ceramic culture in a vacuum chmber. Ten inverted plants were immersed in 300ml of Agrobacterium culture in the infiltrartion medium.
(C)A.thaliana in ceramic tubes before vacuum infiltration.


REFERENCE
1. The Arabidpsis Genome Initiative: Analysis of the genome sequence of the flowering plant Arabidpsis
thaliana
. Nature, 408, 796-815 (2000)
2. Bechtold, N., Ellis, J., and Pelletier, G.: In planta Agrobacterium mediated gene tranfer by infiltration of adult Arabidpsis thaliana plants. C.R. Acad. Sci. Paris, Scieces de la vie/Life science, 316, 1194-1199 (1993)
3. Akai, T., Honjyou, N., Kuwagaki, M., and Iida, T.: Plants culture methods by microporous ceramics. Shita Rep. (Journal of Society of High Technology in Agriculture), 14, 37-47 (1998). (in Japanese)
4. Inaba, K., Fujiwara, T., Hayashi, H., Chino, M., Komeda, Y., Naito, S.: Isolation of an Arabidpsis thaliana mutant, mto1, that overaccumulates soluble methionine: temporal and spatial patterns of soluble methionine accumulation. Plant Physoil., 104, 881-887 (1994)

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