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High Root Biomass Production in Anchored <i>Arabidopsis</i> Plants Grown in Axenic Sucrose Supplemented Liquid Culture

High Root Biomass Production in Anchored Arabidopsis Plants Grown... SHORT TECHNICAL REPORTS separately. Furthermore, it is possible High root biomass production in anchored to supplement the system with sugars Arabidopsis plants grown in axenic sucrose to promote root growth. supplemented liquid culture MATERIALS AND METHODS Marie-France Hétu, Linda J. Tremblay, and Daniel D. Lefebvre This technique for sterile culture Queen’s University, Kingston, ON, Canada of intact plants simply requires that there be a support matrix such as a BioTechniques 39:345-349 (September 2005) stainless steel screen for roots to take hold of during germination. Culturing There are many benefits to growing Arabidopsis in solution-based media, especially when is carried out in conventional 125-mL large amounts of root tissue are required for molecular and biochemical studies. Roots grown wide-mouth Erlenmeyer flasks on a in soil are brittle and tend to break easily when removed from their substrate. We have devel- shaker under fluorescent lights. This oped an axenic liquid culture system that simplifies growing large amounts of roots from in- technique, inspired by our preliminary tact plants. This technique consists of germinating 15 seeds on 2.5 cm stainless steel screens studies (11), has been optimized for placed on half-strength semisolid Murashige and Skoog medium containing 1% or 2% su- maximal biomass production and crose. The screens anchor and support the plantlets in an upright position while keeping the experimental manipulation. roots and shoots separate. The seedlings are transferred with forceps to 125-mL wide-mouth A. thaliana-type Columbia seeds Erlenmeyer flasks containing 10 mL of half-strength Murashige and Skoog liquid medium were surface-sterilized in 1 mL of 30% and 1% sucrose. The flasks are placed onto a floor rotary shaker under fluorescent lights. ® domestic bleach and 0.03% Triton After 3 days, the sucrose is increased to 3% and the volume to 15 mL for 7 days. During any X-100 for 8 min at room temperature. further experimental manipulations, sucrose is not supplied. The media is changed every 3–4 The seeds were rinsed three times days to replenish the nutrients. The presence of sucrose in the media dramatically increases with 1 mL sterile double-distilled the biomass, and large amounts of root tissue can easily be harvested. water and resuspended in 50 μL sterile double-distilled water. Using aseptic techniques, 20 seeds were pipeted and spread out on screens using sterile pipet tips cut to have wider openings. INTRODUCTION explants that require the addition of The seeds were sown on 2.5 cm exogenous plant growth regulators or, stainless steel screens (type 304 woven The small size of the model plant as in the case of hairy root cultures, wire mesh, 40 × 40 holes per linear in., species, Arabidopsis thaliana (L.) genetic transformation (5,7). The 0.01 in. wire diameter, 0.015 in. clear Heynh., presents difficulties for inves- validity of studying tissue not obtained opening; Ferrier Wire Goods, Toronto, tigations of gene products expressed at from intact plants is a serious concern. ON, Canada), placed on 1% or 2% minimal levels and for the study of fine As such, numerous investigations sucrose Murashige and Skoog (6) physiological responses. A common would benefit by growing whole nutrient agar plates (0.5× Murashige complaint among molecular biologists plants axenically. However, and biochemists already coping with the sterile culture of intact low cytoplasmic content in plants is the Arabidopsis plants generally inability to obtain large quantities of involves growing seedlings on tissue, particularly roots. In addition, it semisolid media (0.7% agar) is often desirable to obtain tissue from or throwing seeds directly into plants free of bacteria, fungi, algae, liquid media with or without 0.1% agar to help prevent and other contaminating organisms. Although hydroponic culture has plant aggregation (8–10). been used with limited success in These techniques may involve Arabidopsis plant production (1–3), oxygen deprivation and present germination is low, and solution-based difficulties when separating cultures encourage other organisms to root from shoot tissue. inhabit the root system. Furthermore, In this paper, we describe an easily implemented axenic even in the best hydroponic system, Figure 1. Technique for producing anchored Arabidopsis some of the root tissue grows inside flask culturing system in which plants grown in axenic sucrose-supplemented liquid cul- rockwool plugs and is inaccessible (3). Arabidopsis seeds germinate ture. (A) Pipeting seeds on stainless steel screens placed on The sterile culture of plants has and become anchored by Murashige and Skoog semisolid agar plates. (B) Lifting 7- usually been limited to cell, tissue, or their roots. This maintains the day-old seedlings with long metal forceps. (C) Transferring seedlings to a 125-mL wide-mouth Erlenmeyer glass flask. callus cultures grown on semisolid plants in an upright position, (D) Placing the flasks onto a floor rotary shaker under fluo- media or in liquid suspension (4–6). thereby enabling researchers to rescent lighting conditions. harvest root and shoot material These are started from sterilized Vol. 39, No. 3 (2005) BioTechniques 345 SHORT TECHNICAL REPORTS Table 1. Axenic Liquid Culture Treatments of Arabidopsis Plants Agar Plates Nutrient and Suc Treatments (Day 8–17) 0% Suc Liquid Media (Day 1–7) Liquid Media (Day 8–10) Liquid Media (Day 11–17) (Day 18–24) Strength [Pi] Suc Strength [Pi] Suc Strength [Pi] Suc Strength [Pi] Suc No. [MS] (mM) (%) [MS] (mM) (%) [MS] (mM) (%) [MS] (mM) (%) 1 0.5 0.625 1 0.5 0.625 0 0.5 0.625 0 0.5 0.625 0 2 0.5 0.625 1 0.5 0.625 1 0.5 0.625 1 0.5 0.625 0 3 0.5 0.625 1 0.5 0.625 2 0.5 0.625 2 0.5 0.625 0 4 0.5 0.625 1 0.5 0.625 3 0.5 0.625 3 0.5 0.625 0 5 0.5 1.25 1 0.5 1.25 0 0.5 1.25 0 0.5 1.25 0 6 0.5 1.25 1 0.5 1.25 1 0.5 1.25 1 0.5 1.25 0 7 0.5 1.25 1 0.5 1.25 2 0.5 1.25 2 0.5 1.25 0 8 0.5 1.25 1 0.5 1.25 3 0.5 1.25 3 0.5 1.25 0 9 0.5 1.25 1 1 1.25 0 1 1.25 0 1 1.25 0 10 0.5 1.25 1 1 1.25 1 1 1.25 1 1 1.25 0 11 0.5 1.25 1 1 1.25 2 1 1.25 2 1 1.25 0 12 0.5 1.25 1 1 1.25 3 1 1.25 3 1 1.25 0 13 1 1.25 1 1 1.25 0 1 1.25 0 1 1.25 0 14 1 1.25 1 1 1.25 1 1 1.25 1 1 1.25 0 15 1 1.25 1 1 1.25 2 1 1.25 2 1 1.25 0 16 1 1.25 1 1 1.25 3 1 1.25 3 1 1.25 0 17 0.5 0.625 1 0.5 0.625 1 0.5 0.625 2 0.5 0.625 0 18 0.5 0.625 1 0.5 0.625 1 0.5 0.625 3 0.5 0.625 0 19 0.5 1.25 1 0.5 1.25 1 0.5 1.25 2 0.5 1.25 0 20 0.5 1.25 1 0.5 1.25 1 0.5 1.25 3 0.5 1.25 0 21 0.5 1.25 1 1 1.25 1 1 1.25 2 1 1.25 0 22 0.5 1.25 1 1 1.25 1 1 1.25 3 1 1.25 0 23 1 1.25 1 1 1.25 1 1 1.25 2 1 1.25 0 24 1 1.25 1 1 1.25 1 1 1.25 3 1 1.25 0 Numbers at the left of the table represent the various treatments. Abbreviations: [ ] = concentration; MS = Murashige and Skoog media; P = phosphate; and Suc = sucrose. and Skoog with B5 vitamins, 25 mg/L Screens holding the seedlings flaming the flask’s lip using a Bunsen MES [2-(N-morpholino) ethanesul- were transferred with 20-cm metal burner, flaming forceps after dipping fonic acid], 0.7% agar, pH 5.8) (Figure forceps into four pre-autoclaved 125- into ethanol before lifting one screen 1A). Two percent sucrose increases mL wide-mouth Erlenmeyer glass from a plate and placing it root-side root length, permitting easier transfer flasks (Fisher Scientific, Nepean, ON, down into the flask, then flaming the to liquid medium. Screens were Canada) containing 10 mL sterile 1% flask’s lip once more, and replacing sterilized by autoclaving and placed on sucrose Murashige and Skoog with the foil. The flasks were placed onto the Murashige and Skoog plates using liquid medium (0.5× Murashige and a floor rotary shaker set at 70–80 rpm ethanol-flamed forceps. All manipula- Skoog with B5 vitamins, 25 mg/L and in 16 h day fluorescent lighting tions were performed in a sterile laminar MES, 1% sucrose, pH 5.8) with the conditions (100–150 μmol quanta -2 -1 flow hood. Nutrients and sucrose opening covered with a double layer PAR m s ) for 3 days at 22°–24°C concentrations were altered for growth of aluminum foil (Figure 1, B and (Figure 1D). The anchored plantlets comparisons (Table 1). The plates were C). To avoid submerging the plants in were counted within the first 2 weeks sealed with 3M Micropore™ surgical liquid, the volume was increased to 15 of growth (16 ± 3/flask; mean ± sd, n = tape and placed in the dark for 48 h at mL after 3 more days of growth (day 4). To avoid overcrowding, a maximum 4°C. The plates were then placed under 11). Nutrients and sucrose concentra- of 20 plantlets per flask should be used 16 h day fluorescent lighting conditions tions were also altered for growth (15 is optimal). In all of the following -2 -1 (80–120 μmol quanta PAR m s ) for 7 comparisons (Table 1). Care was taken steps, the media was replaced every days at 22°C. to maintain sterility. The procedure 3–4 days, taking care to maintain sterile involved removing the foil cover, conditions as described above. 346 BioTechniques Vol. 39, No. 3 (2005) On day 11, sucrose was increased salinity, hormone, or light regime treat- decreased or removed altogether to 3% for a 7-day period to promote ments. Short days may be adopted to when enough root biomass has been root growth. When roots became prevent flowering. produced. The plants will return to their restrained by flask walls, rotary Anthocyanin production gives a natural color within 3 days. Lighting shaking was increased to 80–90 rpm. purple pigmentation to leaves and is and temperature conditions should be The plants must gently swirl to be produced in response to environmental closely monitored. oxygenated; however, excess agitation stresses such as high light intensity, Harvesting of tissue was performed may cause damage. Growth then low temperature, nutrient deprivation, by removing the plants from their flasks proceeded for an additional 7 days in and exposure to high sugar concentra- using 30 cm forceps, briefly blotting Murashige and Skoog without sucrose tions (12). Purple pigmentation is not them on paper towels, and separating to avoid potential sugar effects on gene induced in media containing 0% to 2% the roots and shoots using scissors and expression. If desired, plants can be sucrose, but may occur in 3% or higher. tweezers. Fresh and dry weights of the grown for 3–7 days without sucrose and This is a reversible phenomenon if not samples were obtained. then given additional treatments for up exposed to more than 3% sucrose. If to 14 days without sucrose. These may leaves become purple, the sucrose level include mineral nutrient, heat shock, or exposure time to sucrose may be RESULTS AND DISCUSSION In general, increased sucrose caused increased root and shoot biomass. The plants appeared to be morphologically normal and the roots produced primary and secondary structures. However, high nutrient concentrations (phosphate or total Murashige and Skoog) had a negative effect on growth in the presence of sucrose. To determine which conditions gave the healthiest plants with the most biomass, Arabidopsis was grown on semisolid Murashige and Skoog agar plates containing different concen- trations of nutrients and sucrose. The healthiest shoots were observed when plants were grown with 0% or 1% sucrose, but 1% was required for the roots to penetrate and become anchored to the stainless steel screens. The root biomass increased with the level of sucrose, but the shoot biomass decreased (as based on leaf size). Purple stems and dark green or purple leaves were observed after 21 days of growth when 3% sucrose was used, indicating anthocyanin synthesis. Nutrient concentrations of either half- or full-strength Murashige and Skoog did not seem to have any visually quali- tative effect on growth. Various nutrient concentrations were also employed to determine their effects on biomass accumulation. Because 1% sucrose is the concen- tration normally used to screen geneti- cally transformed plants (13), this was used in the semisolid media (Murashige and Skoog plates). The treatments used Figure 2. Biomass analysis of root and shoot tissue of Arabidopsis thaliana under various liquid to determine conditions for optimal culture treatments. (A) Fresh weight biomass per individual plants for selected treatments. (B) Dry biomass production are described weight biomass per individual plants for selected treatments. (C) Root-to-shoot ratios of fresh and dry weights for selected treatments. Values are the means ± sd (n = 4); all samples were composed of 16 ± 3 in Table 1, and selected results are plants. Treatments are described in Table 1. presented in Figure 2. Vol. 39, No. 3 (2005) BioTechniques 347 SHORT TECHNICAL REPORTS All growth treatments consisted of increasing P by itself in the presence of biomass (Figure 2). Treatment 18 was sowing seeds on stainless steel screens sucrose lessened growth. determined to be optimal for maximum placed on semisolid Murashige and When all nutrients were doubled to biomass production with a relatively Skoog media for 7 days and then trans- full strength in the liquid media subse- low standard deviation among samples. ferring the grown seedlings to liquid quent to growth on plates containing Root-to-shoot ratios were calculated media for an additional 17 days (Figure high P (treatments 9–12), the increased for both fresh and dry weights (Figure 1). Because high sugar (5%) has been biomass in response to sucrose was 2C). Interestingly, without sucrose, the shown to inhibit plant growth (14), even lower than in treatments 5–8. fresh weight root-to-shoot ratio was we did not subject plants to more than This indicates that the other nutrients significantly lower than the dry weight 3% sucrose. Sucrose was not supplied had an additional inhibitory effect root-to-shoot ratio by an average of in the media in the final 7 days as a on growth in the presence of sucrose. 88% (treatments 1, 5, 9, and 13). This precaution, should any altered gene The use of full-strength Murashige could be due to higher carbohydrate expression or regulation be caused by and Skoog medium has been found storage occurring in sucrose-fed roots. its presence. The total growth period to reduce the effectiveness of sucrose Half-strength Murashige and Skoog was 24 days. in initiating adventitious roots (14); nutrients with 2% or 3% sucrose in the Treatments 1–4 contained half- CaCl and MgSO were the inhibitory liquid media gave the highest root-to- 2 4 strength Murashige and Skoog nutrients. We have found that elevated shoot ratios. nutrients in all media with a range of P inhibited the production of roots, To determine if the plants were 0% to 3% sucrose in the liquid media. but it would be interesting to analyze affected by nutrient and sucrose An average 20-fold increase in both the effects of other nutrients present in concentrations in the semisolid media root and shoot biomass occurred in the Murashige and Skoog with respect to prior to the transfer to axenic liquid presence of sucrose (Figure 2). Figure growth of primary, secondary, lateral, culture, Arabidopsis was grown on 3 illustrates root growth in the presence and adventitious roots. This contradicts semisolid media containing various of 0%, 1%, 2%, and 3% sucrose at 3 the findings of Williamson et al. (9), concentrations of Murashige and time points (treatments 1–4). The root- where it was found that the ability of Skoog nutrients and sucrose. Because to-shoot ratio also increased. When the root system to respond to phosphate 3% sucrose induced the largest root the phosphate (P ) concentration was availability was independent of sucrose biomass production in the previous doubled to 1.25 mM (concentration supply. In their study, however, only treatments, this concentration was in 1× Murashige and Skoog) with the 1% sucrose was assessed on semisolid used in the liquid media. The change remaining nutrients kept at the same media. in nutrients and sucrose concentrations level throughout the culturing period The nutrient concentrations were in the semisolid media did not have a (treatments 5–8), a less pronounced at full strength throughout the culture significant effect on the final plants’ increase in biomass was observed period in treatments 13–16. Consistency biomass (data not shown). as sucrose levels increased. As such, in nutrient levels throughout the growth To assess if growing multiple plants period appears to help obtain a in a limited amount of space caused maximum biomass. A higher an overcrowding effect on biomass, biomass especially for roots four replicates of 1, 2, 3, 4, 5, 10, 15, was produced in half- versus 20, 30, 50, and 60 plants per screen on full-strength Murashige and half-strength Murashige and Skoog Skoog (treatments 1–4 versus semisolid medium containing 1% and 13–16). 2% sucrose were grown for comparison Treatments 17–24 were purposes. Significant differences performed to determine if a 3- were not found in root biomass using day adaptation period in liquid any number of plants. Significant media containing 1% sucrose differences were not found in shoot (as in the semisolid media) biomass when sowing up to 15 plants prior to increasing the sucrose on 1% sucrose and up to 20 plants on concentration to 2% or 3% for 2% sucrose. Higher numbers of plants an additional 7 days would showed significant decreases in shoot enhance biomass. It did not, biomass (data not shown). Using a although standard deviations maximum of 20 plants per screen is of the means were decreased in recommended (15 plants is optimal). many cases (data not shown). The seeds should be spread out on the A period of adaptation from screens as uniformly as possible. semisolid to liquid media in To summarize, a simple procedure the same sucrose concen- for axenic plant culture has been Figure 3. Photographs of liquid cultures of Arabidopsis tration benefits the plantlets described. Stainless steel screens thaliana plants. Flasks are angled to show root growth over and increases reproducibility. provided excellent support for the time at days 14, 17, and 24 in increasing sucrose concentra- Treatments 4, 18, and 24 had plants as well as ease of transfer tions. Numbers at the top of the panels correspond to treat- ments 1–4 as described in Table 1. the highest root and shoot between media and ease of separation 348 BioTechniques Vol. 39, No. 3 (2005) 8. Malamy, J.E. and K.S. Ryan. 2001. COMPETING INTERESTS of root tissue from shoot tissue upon Environmental regulation of lateral root initia- STATEMENT harvest. Plant growth was assessed to tion in Arabidopsis. Plant Physiol. 127:899- determine the best culturing conditions. High shoot biomass was produced The authors declare no competing 9. Williamson, L.C., S.P.C.P Ribrioux, A.H. Fitter, and H.M.O. Leyser. 2001. Phosphate under several treatments using 1% interests. availability regulates root system architecture sucrose in the semisolid media, but the in Arabidopsis. Plant Physiol. 126:875-882. best root production with the lowest 10.Nichols, K.W., G.R. Heck, and D.E. REFERENCES variation occurred when half-strength Fernandez. 1997. Simplified selection of Murashige and Skoog was used in all transgenic Arabidopsis thaliana seed in liquid 1. Tocquin, P., L. Corbesier, A. Havelange, culture. BioTechniques 22:62-63. media with 1% or 2% sucrose during A. Pieltain, E. Kurtem, G. Bernier, and C. 11.Malboobi, M.A. and D.D. Lefebvre. 1997. A germination, then a 3-day incubation in Périlleux. 2003. A novel high efficiency, low phosphate-starvation inducible β-glucosidase 1% sucrose liquid medium, followed by maintenance, hydroponic system for synchro- gene (psr3.2) isolated from Arabidopsis thali- nous growth and flowering of Arabidopsis 7 days in 3% sucrose. Using this axenic ana is a member of a distinct subfamily of the thaliana. BMC Plant Biol. 3:2. BGA family. Plant Mol. Biol. 34:57-68. flask-culture system and described 2. Gibeaut, D.M., J. Hulett, G.R. Cramer, 12.Nagira, Y. and Y. Ozeki. 2004. A system growth conditions, an average 50% and J.R. Seemann. 1997. Maximal biomass in which anthocyanin synthesis is induced increase in root biomass was obtained of Arabidopsis thaliana using a simple, low- in regenerated torenia shoots. J. Plant Res. maintenance hydroponic method and favor- when compared with hydroponically 117:377-383. able environmental conditions. Plant Physiol. grown Arabidopsis (2). On average, 1.1 13.Weigel, D. and J. Glazebrook. 2002. 115:317-319. Arabidopsis-A Laboratory Manual. CSH g of fresh root tissue can be obtained 3. Huttner, D. and D. Bar-Zvi. 2003. An im- Laboratory Press, Cold Spring Harbor, NY. per flask when using 15 Arabidopsis proved, simple, hydroponic method for grow- 14.Takahashi, F., K. Sato-Nara, K. Kobayashi, seedlings. These growth conditions ing Arabidopsis thaliana. Plant Mol. Biol. M. Suzuki, and H. Suzuki. 2003. Sugar-in- Rep. 21:59-63. can be easily modified to accom- duced adventitious roots in Arabidopsis seed- 4. Yamada, H., N. Koizumi, N. Nakamichi, lings. J. Plant Res. 116:83-91. modate smaller or greater numbers of T. Kiba, T. Yamashino, and T. Mizuno. Arabidopsis plants as well as larger 2004. Rapid response of Arabidopsis T87 plant species as required. Lighting cultured cells to cytokinin through His-to- Received 19 October 2004; accepted Asp phosphorelay signal transduction. Biosci. conditions will need to be addressed 9 March 2005. Biotechnol. Biochem. 68:1966-1976. in future experiments to determine if 5. Shanks, J.V. and J. Morgan. 1999. Plant these have an impact on plant growth ‘hairy root’ culture. Curr. Opin. Biotechnol. Address correspondence to Daniel D. and biomass production. 10:151-155. Lefebvre, Queen’s University, Department of 6. Murashige, T. and F. Skoog. 1962. A revised Biology, Kingston, ON, K7L 3N6, Canada. medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15:473- e-mail: [email protected] ACKNOWLEDGMENTS 7. Klok, E.J., I.W. Wilson, D. Wilson, S.C. This research was supported by Chapman, R.M. Ewing, S.C. Somerville, To purchase reprints W.J. Peacock, R. Dolferus, and E.S. Dennis. funding from the Natural Sciences of this article, contact 2002. Expression profile analysis of the low- and Engineering Research Council of oxygen response in Arabidopsis root cultures. [email protected] Canada to D.D.L. Plant Cell 14:2481-2494. Vol. 39, No. 3 (2005) BioTechniques 349 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BioTechniques Unpaywall

High Root Biomass Production in Anchored <i>Arabidopsis</i> Plants Grown in Axenic Sucrose Supplemented Liquid Culture

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SHORT TECHNICAL REPORTS separately. Furthermore, it is possible High root biomass production in anchored to supplement the system with sugars Arabidopsis plants grown in axenic sucrose to promote root growth. supplemented liquid culture MATERIALS AND METHODS Marie-France Hétu, Linda J. Tremblay, and Daniel D. Lefebvre This technique for sterile culture Queen’s University, Kingston, ON, Canada of intact plants simply requires that there be a support matrix such as a BioTechniques 39:345-349 (September 2005) stainless steel screen for roots to take hold of during germination. Culturing There are many benefits to growing Arabidopsis in solution-based media, especially when is carried out in conventional 125-mL large amounts of root tissue are required for molecular and biochemical studies. Roots grown wide-mouth Erlenmeyer flasks on a in soil are brittle and tend to break easily when removed from their substrate. We have devel- shaker under fluorescent lights. This oped an axenic liquid culture system that simplifies growing large amounts of roots from in- technique, inspired by our preliminary tact plants. This technique consists of germinating 15 seeds on 2.5 cm stainless steel screens studies (11), has been optimized for placed on half-strength semisolid Murashige and Skoog medium containing 1% or 2% su- maximal biomass production and crose. The screens anchor and support the plantlets in an upright position while keeping the experimental manipulation. roots and shoots separate. The seedlings are transferred with forceps to 125-mL wide-mouth A. thaliana-type Columbia seeds Erlenmeyer flasks containing 10 mL of half-strength Murashige and Skoog liquid medium were surface-sterilized in 1 mL of 30% and 1% sucrose. The flasks are placed onto a floor rotary shaker under fluorescent lights. ® domestic bleach and 0.03% Triton After 3 days, the sucrose is increased to 3% and the volume to 15 mL for 7 days. During any X-100 for 8 min at room temperature. further experimental manipulations, sucrose is not supplied. The media is changed every 3–4 The seeds were rinsed three times days to replenish the nutrients. The presence of sucrose in the media dramatically increases with 1 mL sterile double-distilled the biomass, and large amounts of root tissue can easily be harvested. water and resuspended in 50 μL sterile double-distilled water. Using aseptic techniques, 20 seeds were pipeted and spread out on screens using sterile pipet tips cut to have wider openings. INTRODUCTION explants that require the addition of The seeds were sown on 2.5 cm exogenous plant growth regulators or, stainless steel screens (type 304 woven The small size of the model plant as in the case of hairy root cultures, wire mesh, 40 × 40 holes per linear in., species, Arabidopsis thaliana (L.) genetic transformation (5,7). The 0.01 in. wire diameter, 0.015 in. clear Heynh., presents difficulties for inves- validity of studying tissue not obtained opening; Ferrier Wire Goods, Toronto, tigations of gene products expressed at from intact plants is a serious concern. ON, Canada), placed on 1% or 2% minimal levels and for the study of fine As such, numerous investigations sucrose Murashige and Skoog (6) physiological responses. A common would benefit by growing whole nutrient agar plates (0.5× Murashige complaint among molecular biologists plants axenically. However, and biochemists already coping with the sterile culture of intact low cytoplasmic content in plants is the Arabidopsis plants generally inability to obtain large quantities of involves growing seedlings on tissue, particularly roots. In addition, it semisolid media (0.7% agar) is often desirable to obtain tissue from or throwing seeds directly into plants free of bacteria, fungi, algae, liquid media with or without 0.1% agar to help prevent and other contaminating organisms. Although hydroponic culture has plant aggregation (8–10). been used with limited success in These techniques may involve Arabidopsis plant production (1–3), oxygen deprivation and present germination is low, and solution-based difficulties when separating cultures encourage other organisms to root from shoot tissue. inhabit the root system. Furthermore, In this paper, we describe an easily implemented axenic even in the best hydroponic system, Figure 1. Technique for producing anchored Arabidopsis some of the root tissue grows inside flask culturing system in which plants grown in axenic sucrose-supplemented liquid cul- rockwool plugs and is inaccessible (3). Arabidopsis seeds germinate ture. (A) Pipeting seeds on stainless steel screens placed on The sterile culture of plants has and become anchored by Murashige and Skoog semisolid agar plates. (B) Lifting 7- usually been limited to cell, tissue, or their roots. This maintains the day-old seedlings with long metal forceps. (C) Transferring seedlings to a 125-mL wide-mouth Erlenmeyer glass flask. callus cultures grown on semisolid plants in an upright position, (D) Placing the flasks onto a floor rotary shaker under fluo- media or in liquid suspension (4–6). thereby enabling researchers to rescent lighting conditions. harvest root and shoot material These are started from sterilized Vol. 39, No. 3 (2005) BioTechniques 345 SHORT TECHNICAL REPORTS Table 1. Axenic Liquid Culture Treatments of Arabidopsis Plants Agar Plates Nutrient and Suc Treatments (Day 8–17) 0% Suc Liquid Media (Day 1–7) Liquid Media (Day 8–10) Liquid Media (Day 11–17) (Day 18–24) Strength [Pi] Suc Strength [Pi] Suc Strength [Pi] Suc Strength [Pi] Suc No. [MS] (mM) (%) [MS] (mM) (%) [MS] (mM) (%) [MS] (mM) (%) 1 0.5 0.625 1 0.5 0.625 0 0.5 0.625 0 0.5 0.625 0 2 0.5 0.625 1 0.5 0.625 1 0.5 0.625 1 0.5 0.625 0 3 0.5 0.625 1 0.5 0.625 2 0.5 0.625 2 0.5 0.625 0 4 0.5 0.625 1 0.5 0.625 3 0.5 0.625 3 0.5 0.625 0 5 0.5 1.25 1 0.5 1.25 0 0.5 1.25 0 0.5 1.25 0 6 0.5 1.25 1 0.5 1.25 1 0.5 1.25 1 0.5 1.25 0 7 0.5 1.25 1 0.5 1.25 2 0.5 1.25 2 0.5 1.25 0 8 0.5 1.25 1 0.5 1.25 3 0.5 1.25 3 0.5 1.25 0 9 0.5 1.25 1 1 1.25 0 1 1.25 0 1 1.25 0 10 0.5 1.25 1 1 1.25 1 1 1.25 1 1 1.25 0 11 0.5 1.25 1 1 1.25 2 1 1.25 2 1 1.25 0 12 0.5 1.25 1 1 1.25 3 1 1.25 3 1 1.25 0 13 1 1.25 1 1 1.25 0 1 1.25 0 1 1.25 0 14 1 1.25 1 1 1.25 1 1 1.25 1 1 1.25 0 15 1 1.25 1 1 1.25 2 1 1.25 2 1 1.25 0 16 1 1.25 1 1 1.25 3 1 1.25 3 1 1.25 0 17 0.5 0.625 1 0.5 0.625 1 0.5 0.625 2 0.5 0.625 0 18 0.5 0.625 1 0.5 0.625 1 0.5 0.625 3 0.5 0.625 0 19 0.5 1.25 1 0.5 1.25 1 0.5 1.25 2 0.5 1.25 0 20 0.5 1.25 1 0.5 1.25 1 0.5 1.25 3 0.5 1.25 0 21 0.5 1.25 1 1 1.25 1 1 1.25 2 1 1.25 0 22 0.5 1.25 1 1 1.25 1 1 1.25 3 1 1.25 0 23 1 1.25 1 1 1.25 1 1 1.25 2 1 1.25 0 24 1 1.25 1 1 1.25 1 1 1.25 3 1 1.25 0 Numbers at the left of the table represent the various treatments. Abbreviations: [ ] = concentration; MS = Murashige and Skoog media; P = phosphate; and Suc = sucrose. and Skoog with B5 vitamins, 25 mg/L Screens holding the seedlings flaming the flask’s lip using a Bunsen MES [2-(N-morpholino) ethanesul- were transferred with 20-cm metal burner, flaming forceps after dipping fonic acid], 0.7% agar, pH 5.8) (Figure forceps into four pre-autoclaved 125- into ethanol before lifting one screen 1A). Two percent sucrose increases mL wide-mouth Erlenmeyer glass from a plate and placing it root-side root length, permitting easier transfer flasks (Fisher Scientific, Nepean, ON, down into the flask, then flaming the to liquid medium. Screens were Canada) containing 10 mL sterile 1% flask’s lip once more, and replacing sterilized by autoclaving and placed on sucrose Murashige and Skoog with the foil. The flasks were placed onto the Murashige and Skoog plates using liquid medium (0.5× Murashige and a floor rotary shaker set at 70–80 rpm ethanol-flamed forceps. All manipula- Skoog with B5 vitamins, 25 mg/L and in 16 h day fluorescent lighting tions were performed in a sterile laminar MES, 1% sucrose, pH 5.8) with the conditions (100–150 μmol quanta -2 -1 flow hood. Nutrients and sucrose opening covered with a double layer PAR m s ) for 3 days at 22°–24°C concentrations were altered for growth of aluminum foil (Figure 1, B and (Figure 1D). The anchored plantlets comparisons (Table 1). The plates were C). To avoid submerging the plants in were counted within the first 2 weeks sealed with 3M Micropore™ surgical liquid, the volume was increased to 15 of growth (16 ± 3/flask; mean ± sd, n = tape and placed in the dark for 48 h at mL after 3 more days of growth (day 4). To avoid overcrowding, a maximum 4°C. The plates were then placed under 11). Nutrients and sucrose concentra- of 20 plantlets per flask should be used 16 h day fluorescent lighting conditions tions were also altered for growth (15 is optimal). In all of the following -2 -1 (80–120 μmol quanta PAR m s ) for 7 comparisons (Table 1). Care was taken steps, the media was replaced every days at 22°C. to maintain sterility. The procedure 3–4 days, taking care to maintain sterile involved removing the foil cover, conditions as described above. 346 BioTechniques Vol. 39, No. 3 (2005) On day 11, sucrose was increased salinity, hormone, or light regime treat- decreased or removed altogether to 3% for a 7-day period to promote ments. Short days may be adopted to when enough root biomass has been root growth. When roots became prevent flowering. produced. The plants will return to their restrained by flask walls, rotary Anthocyanin production gives a natural color within 3 days. Lighting shaking was increased to 80–90 rpm. purple pigmentation to leaves and is and temperature conditions should be The plants must gently swirl to be produced in response to environmental closely monitored. oxygenated; however, excess agitation stresses such as high light intensity, Harvesting of tissue was performed may cause damage. Growth then low temperature, nutrient deprivation, by removing the plants from their flasks proceeded for an additional 7 days in and exposure to high sugar concentra- using 30 cm forceps, briefly blotting Murashige and Skoog without sucrose tions (12). Purple pigmentation is not them on paper towels, and separating to avoid potential sugar effects on gene induced in media containing 0% to 2% the roots and shoots using scissors and expression. If desired, plants can be sucrose, but may occur in 3% or higher. tweezers. Fresh and dry weights of the grown for 3–7 days without sucrose and This is a reversible phenomenon if not samples were obtained. then given additional treatments for up exposed to more than 3% sucrose. If to 14 days without sucrose. These may leaves become purple, the sucrose level include mineral nutrient, heat shock, or exposure time to sucrose may be RESULTS AND DISCUSSION In general, increased sucrose caused increased root and shoot biomass. The plants appeared to be morphologically normal and the roots produced primary and secondary structures. However, high nutrient concentrations (phosphate or total Murashige and Skoog) had a negative effect on growth in the presence of sucrose. To determine which conditions gave the healthiest plants with the most biomass, Arabidopsis was grown on semisolid Murashige and Skoog agar plates containing different concen- trations of nutrients and sucrose. The healthiest shoots were observed when plants were grown with 0% or 1% sucrose, but 1% was required for the roots to penetrate and become anchored to the stainless steel screens. The root biomass increased with the level of sucrose, but the shoot biomass decreased (as based on leaf size). Purple stems and dark green or purple leaves were observed after 21 days of growth when 3% sucrose was used, indicating anthocyanin synthesis. Nutrient concentrations of either half- or full-strength Murashige and Skoog did not seem to have any visually quali- tative effect on growth. Various nutrient concentrations were also employed to determine their effects on biomass accumulation. Because 1% sucrose is the concen- tration normally used to screen geneti- cally transformed plants (13), this was used in the semisolid media (Murashige and Skoog plates). The treatments used Figure 2. Biomass analysis of root and shoot tissue of Arabidopsis thaliana under various liquid to determine conditions for optimal culture treatments. (A) Fresh weight biomass per individual plants for selected treatments. (B) Dry biomass production are described weight biomass per individual plants for selected treatments. (C) Root-to-shoot ratios of fresh and dry weights for selected treatments. Values are the means ± sd (n = 4); all samples were composed of 16 ± 3 in Table 1, and selected results are plants. Treatments are described in Table 1. presented in Figure 2. Vol. 39, No. 3 (2005) BioTechniques 347 SHORT TECHNICAL REPORTS All growth treatments consisted of increasing P by itself in the presence of biomass (Figure 2). Treatment 18 was sowing seeds on stainless steel screens sucrose lessened growth. determined to be optimal for maximum placed on semisolid Murashige and When all nutrients were doubled to biomass production with a relatively Skoog media for 7 days and then trans- full strength in the liquid media subse- low standard deviation among samples. ferring the grown seedlings to liquid quent to growth on plates containing Root-to-shoot ratios were calculated media for an additional 17 days (Figure high P (treatments 9–12), the increased for both fresh and dry weights (Figure 1). Because high sugar (5%) has been biomass in response to sucrose was 2C). Interestingly, without sucrose, the shown to inhibit plant growth (14), even lower than in treatments 5–8. fresh weight root-to-shoot ratio was we did not subject plants to more than This indicates that the other nutrients significantly lower than the dry weight 3% sucrose. Sucrose was not supplied had an additional inhibitory effect root-to-shoot ratio by an average of in the media in the final 7 days as a on growth in the presence of sucrose. 88% (treatments 1, 5, 9, and 13). This precaution, should any altered gene The use of full-strength Murashige could be due to higher carbohydrate expression or regulation be caused by and Skoog medium has been found storage occurring in sucrose-fed roots. its presence. The total growth period to reduce the effectiveness of sucrose Half-strength Murashige and Skoog was 24 days. in initiating adventitious roots (14); nutrients with 2% or 3% sucrose in the Treatments 1–4 contained half- CaCl and MgSO were the inhibitory liquid media gave the highest root-to- 2 4 strength Murashige and Skoog nutrients. We have found that elevated shoot ratios. nutrients in all media with a range of P inhibited the production of roots, To determine if the plants were 0% to 3% sucrose in the liquid media. but it would be interesting to analyze affected by nutrient and sucrose An average 20-fold increase in both the effects of other nutrients present in concentrations in the semisolid media root and shoot biomass occurred in the Murashige and Skoog with respect to prior to the transfer to axenic liquid presence of sucrose (Figure 2). Figure growth of primary, secondary, lateral, culture, Arabidopsis was grown on 3 illustrates root growth in the presence and adventitious roots. This contradicts semisolid media containing various of 0%, 1%, 2%, and 3% sucrose at 3 the findings of Williamson et al. (9), concentrations of Murashige and time points (treatments 1–4). The root- where it was found that the ability of Skoog nutrients and sucrose. Because to-shoot ratio also increased. When the root system to respond to phosphate 3% sucrose induced the largest root the phosphate (P ) concentration was availability was independent of sucrose biomass production in the previous doubled to 1.25 mM (concentration supply. In their study, however, only treatments, this concentration was in 1× Murashige and Skoog) with the 1% sucrose was assessed on semisolid used in the liquid media. The change remaining nutrients kept at the same media. in nutrients and sucrose concentrations level throughout the culturing period The nutrient concentrations were in the semisolid media did not have a (treatments 5–8), a less pronounced at full strength throughout the culture significant effect on the final plants’ increase in biomass was observed period in treatments 13–16. Consistency biomass (data not shown). as sucrose levels increased. As such, in nutrient levels throughout the growth To assess if growing multiple plants period appears to help obtain a in a limited amount of space caused maximum biomass. A higher an overcrowding effect on biomass, biomass especially for roots four replicates of 1, 2, 3, 4, 5, 10, 15, was produced in half- versus 20, 30, 50, and 60 plants per screen on full-strength Murashige and half-strength Murashige and Skoog Skoog (treatments 1–4 versus semisolid medium containing 1% and 13–16). 2% sucrose were grown for comparison Treatments 17–24 were purposes. Significant differences performed to determine if a 3- were not found in root biomass using day adaptation period in liquid any number of plants. Significant media containing 1% sucrose differences were not found in shoot (as in the semisolid media) biomass when sowing up to 15 plants prior to increasing the sucrose on 1% sucrose and up to 20 plants on concentration to 2% or 3% for 2% sucrose. Higher numbers of plants an additional 7 days would showed significant decreases in shoot enhance biomass. It did not, biomass (data not shown). Using a although standard deviations maximum of 20 plants per screen is of the means were decreased in recommended (15 plants is optimal). many cases (data not shown). The seeds should be spread out on the A period of adaptation from screens as uniformly as possible. semisolid to liquid media in To summarize, a simple procedure the same sucrose concen- for axenic plant culture has been Figure 3. Photographs of liquid cultures of Arabidopsis tration benefits the plantlets described. Stainless steel screens thaliana plants. Flasks are angled to show root growth over and increases reproducibility. provided excellent support for the time at days 14, 17, and 24 in increasing sucrose concentra- Treatments 4, 18, and 24 had plants as well as ease of transfer tions. Numbers at the top of the panels correspond to treat- ments 1–4 as described in Table 1. the highest root and shoot between media and ease of separation 348 BioTechniques Vol. 39, No. 3 (2005) 8. Malamy, J.E. and K.S. Ryan. 2001. COMPETING INTERESTS of root tissue from shoot tissue upon Environmental regulation of lateral root initia- STATEMENT harvest. Plant growth was assessed to tion in Arabidopsis. Plant Physiol. 127:899- determine the best culturing conditions. High shoot biomass was produced The authors declare no competing 9. Williamson, L.C., S.P.C.P Ribrioux, A.H. Fitter, and H.M.O. Leyser. 2001. Phosphate under several treatments using 1% interests. availability regulates root system architecture sucrose in the semisolid media, but the in Arabidopsis. Plant Physiol. 126:875-882. best root production with the lowest 10.Nichols, K.W., G.R. Heck, and D.E. REFERENCES variation occurred when half-strength Fernandez. 1997. Simplified selection of Murashige and Skoog was used in all transgenic Arabidopsis thaliana seed in liquid 1. Tocquin, P., L. Corbesier, A. Havelange, culture. BioTechniques 22:62-63. media with 1% or 2% sucrose during A. Pieltain, E. Kurtem, G. Bernier, and C. 11.Malboobi, M.A. and D.D. Lefebvre. 1997. A germination, then a 3-day incubation in Périlleux. 2003. A novel high efficiency, low phosphate-starvation inducible β-glucosidase 1% sucrose liquid medium, followed by maintenance, hydroponic system for synchro- gene (psr3.2) isolated from Arabidopsis thali- nous growth and flowering of Arabidopsis 7 days in 3% sucrose. Using this axenic ana is a member of a distinct subfamily of the thaliana. BMC Plant Biol. 3:2. BGA family. Plant Mol. Biol. 34:57-68. flask-culture system and described 2. Gibeaut, D.M., J. Hulett, G.R. Cramer, 12.Nagira, Y. and Y. Ozeki. 2004. A system growth conditions, an average 50% and J.R. Seemann. 1997. Maximal biomass in which anthocyanin synthesis is induced increase in root biomass was obtained of Arabidopsis thaliana using a simple, low- in regenerated torenia shoots. J. Plant Res. maintenance hydroponic method and favor- when compared with hydroponically 117:377-383. able environmental conditions. Plant Physiol. grown Arabidopsis (2). On average, 1.1 13.Weigel, D. and J. Glazebrook. 2002. 115:317-319. Arabidopsis-A Laboratory Manual. CSH g of fresh root tissue can be obtained 3. Huttner, D. and D. Bar-Zvi. 2003. An im- Laboratory Press, Cold Spring Harbor, NY. per flask when using 15 Arabidopsis proved, simple, hydroponic method for grow- 14.Takahashi, F., K. Sato-Nara, K. Kobayashi, seedlings. These growth conditions ing Arabidopsis thaliana. Plant Mol. Biol. M. Suzuki, and H. Suzuki. 2003. Sugar-in- Rep. 21:59-63. can be easily modified to accom- duced adventitious roots in Arabidopsis seed- 4. Yamada, H., N. Koizumi, N. Nakamichi, lings. J. Plant Res. 116:83-91. modate smaller or greater numbers of T. Kiba, T. Yamashino, and T. Mizuno. Arabidopsis plants as well as larger 2004. Rapid response of Arabidopsis T87 plant species as required. Lighting cultured cells to cytokinin through His-to- Received 19 October 2004; accepted Asp phosphorelay signal transduction. Biosci. conditions will need to be addressed 9 March 2005. Biotechnol. Biochem. 68:1966-1976. in future experiments to determine if 5. Shanks, J.V. and J. Morgan. 1999. Plant these have an impact on plant growth ‘hairy root’ culture. Curr. Opin. Biotechnol. Address correspondence to Daniel D. and biomass production. 10:151-155. Lefebvre, Queen’s University, Department of 6. Murashige, T. and F. Skoog. 1962. A revised Biology, Kingston, ON, K7L 3N6, Canada. medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15:473- e-mail: [email protected] ACKNOWLEDGMENTS 7. Klok, E.J., I.W. Wilson, D. Wilson, S.C. This research was supported by Chapman, R.M. Ewing, S.C. Somerville, To purchase reprints W.J. Peacock, R. Dolferus, and E.S. Dennis. funding from the Natural Sciences of this article, contact 2002. Expression profile analysis of the low- and Engineering Research Council of oxygen response in Arabidopsis root cultures. [email protected] Canada to D.D.L. Plant Cell 14:2481-2494. Vol. 39, No. 3 (2005) BioTechniques 349

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