FABRICATION OF PLANT ARRAY CHIP FOR HIGH-THROUGHPUT GERMINATION

AND GROWTH RATE SCREENING OF ARABIDOPSIS THALIANA

Youn-Hee Park*, Nayoung Lee**, Giltsu Choi** and Je-Kyun Park*

*Department of Bio and Brain Engineering, KAIST, REPUBLIC OF KOREA and **Department of Biological Sciences, KAIST, REPUBLIC OF KOREA

ABSTRACT

This paper presents a novel approach to develop a plant array chip for high-throughput germination

screening against various environmental factors applied to solid media. The plant array chip was con-

structed from poly(dimethylsiloxane) (PDMS), which was consisted of 20 vertical channels and 20 20

seed arrays. For plant growth, a precursor of phyto agar containing nutrients and hormones was embed-

ded into the arrays prior to seeding. In this study, we investigated the effects of two different precursor

conditions, which contained different concentrations of Murashige and Skoog (MS) media or abscisic ac-

id (ABA), on the rate of plant growth. As expected, the MS media enhanced germination and cell growth,

while ABA inhibited both.

KEYWORDS: Arabidopsis thaliana, Plant array chip, Germination

INTRODUCTION

Recently, µTAS researchers have attempted to access a new way of studying plant science.

Particularly, a vertical plant chip was developed for high-throughput phenotyping of Arabidopsis

thaliana plants [1]. This approach was based on continuous flow system for water-culture environment.

However, a closed water-culture system is not compatible with conventional germination method using

solid media. In this study, we present a plant array chip, which reduces temporal and spatial constraints,

for high throughput screening based on the germination and growth rates of Arabidopsis thaliana on a

single array chip. This solid media-based plant chip is more familiar with transplant for post-observation

than the dynamic flow-based device.

EXPERIMENTAL

Figure 1. (a) Schematic illustration of a plant array chip for screening through a variety of germination

and growth conditions. A magnified picture of (b) Arabidopsis thaliana seed and (c) germinated seed. (d)

After germination, a radicle transformed to primary root system. Scale bars = 500 µm. (e) Fabrication

and seeding process of PDMS-based plant array chip. Two PDMS array layers were casted from

polycarbonate molds. A media precursor was used for agarose gelation at room temperature prior to cell

seeding.

209978-0-9798064-8-3/µTAS 2015/$20©15CBMS-0001 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 25-29, 2015, Gyeongju, KOREA

A plant array chip consists of 20 20 wells, which contains each column with a single media

condition (Figure 1a). It can be seeded up to 400 seeds and applied to 20 different environmental

conditions on a single chip. To provide reproducible results, we set the different condition every three

column and one colored dividing column. Consequently, the effects of five experimental conditions on

the rate of growth can be observed at a single chip simultaneously.

Figure 1b-d shows the growth of the seed, representing the germination process of Arabidopsis.

Germination is known as the phenomenon that radicle comes through the seed coat. Thus, the growth rate

was investigated by measuring the length of radicle few days after seeding. The plant array chip was

fabricated with poly(dimethylsiloxane) (PDMS) using polycarbonate molds (Figure 1e). After the two

PDMS layers were bonded completely with a PDMS precursor, sol-state agarose based medium was

injected to each column and induced agarose gelation at room temperature.

RESULTS AND DISCUSSION

The fabricated plant array chip was placed in a petri dish (Figure 2a), and cultured in an incubator

maintained at constant temperature (~21.5 °C). The white light LED was used as a stimulus for the ger-

mination. For the demonstration of plant array chip, we tested the effects of various concentration of Mu-

rashige and Skoog (MS) medium or abscisic acid (ABA) on the germination and growth of Arabidopsis

seeds. To verify homogeneous microenvironment on the plant array chip, seven-day growth of Arabidop-

sis thaliana can be successfully observed on entire plant array chip (15 20 seed arrays) with the same

condition (Figure 2b,c).

Figure 2. (a) A plant array chip was stored at petri dish for humidity and anti-contamination. (b) 300

seeds of Arabidopsis thaliana were initially planted on the array. (c) After 7 days, the plants uniformly

grown under conventional MS media condition. Scale bars = 4 mm.

Five different concentrations (0.0011% ~ 0.0176%) of MS media were loaded into the plant array

chip to determine the germination rate according to the MS concentration. As a result, the germination

rate increased according to the MS concentration during five days (Figure 3). However, ABA shows a

severe effect on the germination and post-germination growth. The higher concentration of ABA (~1000

nM) induced dormancy, the lower germination rate was observed (Figure 4).

Figure 3. Effect of MS concentration on the plant germination. (a) Magnified pictures for actual obser-

vation at 5 days after seeding. Scale bar = 4 mm. (b) A germination rate increased according to the MS

concentration during five days (limited time span).

(b) n = 12

210

Furthermore, post-germination growth arrest was observed by the difference of radicle length (~ 800

µm) at 10 days after seeding. These results indicate that our device is expected to be available for the ef-

ficient screening based on the complex mechanism of germination and plant growth. In addition, simulta-

neous screening of multiple plant hormones may be adapted to the plant array chip.

Figure 4. Effect of ABA concentration on the germination and growth rates. (a) Top view of the plant ar-

ray chip at 10 days after seeding. Scale bar: 4 mm. (b) Both a germination rate and (c) radicle length

decreased according to the ABA concentration.

CONCLUSION

We have developed a new plant array chip for high-throughput germination screening of Arabidopsis

thaliana. Using a single plant array chip, the effects of germination and plant growth against several me-

dia conditions were investigated by measuring the length change of radicle after seeding. As expected,

the concentration of MS media affected the growth rate and ABA hormone interfered with germination in

each chip.

Using the same plant chip, vertical channel configurations can be used for providing 20 different me-

dia conditions on single plant array chip. More importantly, the array configuration reduces the spatial

limitation to compare several environmental factors on the germination screening. In addition, temporal

screening as well as long-term tracking of plant cell is very useful for the characterization of genetically

engineered seeds.

ACKNOWLEDGEMENTS

This research was supported by a National Leading Research Laboratory Program (Grant NRF-

2013R1A2A1A05006378) and a Converging Research Center Program (Grant 2011K000864) through

the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning.

REFERENCES

[1] H. Jiang, Z. Xu, M.R. Aluru, L. Dong, “Plant chip for high-throughput phenotyping of Arabidopsis,”

Lab Chip, 14, 1281–1293, 2014.

[2] J.D. Bewley, “Seed germination and dormancy,” Plant Cell, 9, 1055–1066, 1997.

CONTACT

Y.-H. Park, TEL +82-42-350-4355; younhee.park26@kaist.ac.kr

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