locus, regenerating a 10-kb Sal I fragmentof the CHLI gene, the same size found inwild-type plants (Fig. 3A). Sequence anal-ysis of the CHL1 gene in four of therevertants verified that the element hadexcised, leaving behind a small insertion(Fig. 2). In addition, new restriction frag-ments that hybridized with radiolabeledTagl sequences were evident in the rever-tants (Fig. 3B). Thus, in the revertants,Tagl or Tag) -related elements had insertedinto new loci. We conclude that Tagl is amobile transposable element.

To confirm that Tagl is an endogenouselement of Arabidopsis, genomic DNA wasisolated from the untransformed parentused to construct the transgenic Ac lines.The parent originated from the ecotypeLandsberg and carries the morphologicalmutation erecta. Southern blot analysiswith radiolabeled Tagl DNA indicated thatthe Landsberg erecta parent contains Tagland two additional Tagl-related elements,each present in only one copy per haploidgenome (Fig. 4). No Tagl or related se-quences were found in two other ecotypesof Arabidopsis, Columbia and Wassilewskija(Fig. 4).

By selecting for chlorate-resistant mu-tants of Arabidopsis from a population car-rying an active Ac element, we havetrapped a new mobile Arabidopsis transpo-son. Tagl transposition may have beenstimulated in the Landsberg plants by theDNA breakage or genomic stress caused bythe integration of T-DNA into the Arabi-dopsis genome, by the transposition of Ac(13), or by the propagation of the plantcells in tissue culture (14). Upon activa-tion, the element transposed to the chlllocus and, when homozygous, producedchll mutant progeny. We think it unlikelythat the Ac transposase directly mobilizesTagl, as no Ac transposase binding site(AAACGG) is found adjacent to the in-verted repeats of Tagl as it is in Ac (15).Whatever the mechanism of activation, thenow mobile Tag) should be useful for tag-ging plant genes.

REFERENCES AND NOTES

1. B. McClintock, Science 226, 792 (1984); 0. Nel-son, Ed., Plant Transposable Elements (Plenum,New York, 1988); V. Walbot, Annu. Rev. PlantPhysiol. Plant Mol. Biol. 43, 49 (1992); D. E. Bergand M. M. Howe, Eds., Mobile DNA (AmericanSociety of Microbiology, Washington, DC, 1989).

2. E. M. Meyerowitz, Ce1/56, 263 (1989); R. Shields,Nature 337, 308 (1989).

3. K. A. Feldmann, M. D. Marks, M. L. Christianson,R. S. Quatrano, Science 243, 1351 (1989); M. D.Marks and K. A. Feldmann, Plant Cell 1, 1043(1989); C. Koncz et al., EMBO J. 9, 1337 (1990);M. F. Yanofsky, H. Ma, J. L. Bowman, G. N.Drews, E. M. Meyerowitz, Nature 346, 35 (1990);K. A. Feldmann, Plant J. 1, 1 (1991).

4. V. Arondel et al., Science 258, 1353 (1992).5. D. F. Voytas and F. M. Ausubel, Nature 336, 242

(1988); D. F. Voytas, A. Konieczny, M. P. Cum-

mings, F. M. Ausubel, Genetics 126, 713 (1990);A. Konieczny, D. F. Voytas, M. P. Cummings, F. M.Ausubel, ibid. 127, 801 (1991).

6. J. Peleman, B. Cottyn, W. V. Camp, M. V. Mon-tagu, D. Inze, Proc. Natl. Acad. Sci. U.S.A. 88,3618 (1991).

7. M. A. Van Sluys, J. Tempe, N. Federoff, EMBO J.6, 3881 (1987); R. Schmidt and L. Wilmitzer, Mol.Gen. Genet. 220,17(1989); C. Dean, C. Sjodin, T.Page, J. Jones, C. Lister, Plant J. 2, 69 (1992); J.Swinburne, L Balcells, S. R. Scofield, J. D. G.Jones, G. Coupland, Plant Cell 4, 583 (1992); C.Grevelding etal., Proc. Natl. Acad. Sci. U.S.A. 89,6085 (1992).

8. B. Aberg, Ann. R. Agric. Coil. Swed. 15, 37(1947).

9. F. J. Braaksma and W. J. Feenstra, Theor. Appl.Genet. 64, 83 (1982); M. Caboche and P. Rouze,Trends Genet. 6, 187 (1990); J. L. Wray and J. R.Kinghorn, Eds., Molecular and Genetic Aspects ofNitrate Assimilation (Oxford Univ. Press, Oxford,1989); N. M. Crawford, in Genetic Engineering:Principles and Methods, J. K. Setlow, Ed. (Ple-num, New York, 1992), pp. 89-98.

10. H. Doddema, J. J. Hofstra, W. J. Feenstra, Phys-

11.

12.

13.14.

15.

16.

17.

iol. Plant. 43, 343 (1978); H. Doddema and G. P.Telkamp, ibid. 45, 332 (1979); H. J. Scholten andW. J. Feenstra, ibid. 66, 265 (1986).C. Dean, C. Sjodin, T. Page, J. Jones, C. Lister,Plant J. 2, 69 (1992).Y.-F. Tsay, J. I. Schroeder, K. A. Feldmann, N. M.Crawford, Cell 72, 705 (1993).B. McClintock, in (1).V. M. Peschke, R. L. Phillips, B. G. Gengenbach,Science 238, 804 (1987).R. Kunze and P. Starlinger, EMBO J. 8, 3177(1989); S. Feldmar and R. Kunze, ibid. 10, 4003(1991).J. Wilkinson and N. Crawford, Plant Cell 3, 461(1991).We thank E. Johnson and K. Long for technicalhelp and R. Schmidt and M. Yanofsky for discus-sion. Supported by the Powell Foundation and theNational Institutes of Health (GM 40672 to N.M.C.and 5T32CA09345-12 for M.J.F.) and the AFRCPMB Programme to C.D. This paper is dedicatedto the memory of Barbara McClintock (1902-1992).5 November 1992; accepted 22 February 1993

Soil Quality and Financial Performance ofBiodynamic and Conventional Farms in New Zealand

John P. Reganold,* Alan S. Palmer, James C. Lockhart,A. Neil Macgregor

Biodynamic farming practices and systems show promise in mitigating some of the det-rimental effects of chemical-dependent, conventional agriculture on the environment. Thephysical, biological, and chemical soil properties and economic profitability of adjacent,commercial biodynamic and conventional farms (16 total) in New Zealand were compared.The biodynamic farms in the study had better soil quality than the neighboring conventionalfarms and were just as financially viable on a per hectare basis.

Concerns about environmental, econom-ic, and social impacts of chemical or con-ventional agriculture have led many farmersand consumers to seek alternative practicesthat will make agriculture more sustainable.Both organic and biodynamic farmers useno synthetic chemical fertilizers or pesti-cides, use compost additions and manuresto improve soil quality, control pests natu-rally, rotate crops, and diversify crops andlivestock. Unlike organic farmers, biody-namic farmers add eight specific prepara-tions, made from cow manure, silica, andvarious plants, to enhance soil quality andplant life (1).We examined soil properties and finan-

cial performance on pairs or sets of biody-namic and conventional systems over a4-year period (1987 to 1991) on the NorthIsland of New Zealand (Table 1). We alsoJ. P. Reganold, Department of Crop and Soil Sciences,Washington State University, Pullman, WA 99164.A. S. Palmer and A. N. Macgregor, Department of SoilScience, Massey University, Palmerston North, NewZealand.J. C. Lockhart, Department of Agricultural and Horti-cultural Systems Management, Massey University,Palmerston North, New Zealand.

*To whom correspondence should be addressed.

made financial comparisons between thesefarms and representative conventionalfarms in each study region on the basis ofmodels used by the New Zealand Ministryof Agriculture and Fisheries (MAF) (2). Afarm pair consisted of two side-by-sidefarms, one biodynamic and one conven-tional; a farm set consisted of three adjacentfarms, one biodynamic and two conven-tional. The choice of five farm pairs andtwo farm sets (totaling 16 farms) was madeon the basis of surveys, interviews, andon-farm soil examinations of more than 60farms to ensure that all soil-forming factors,except management (3), were the same ineach farm pair or set.

The biodynamic farms had been man-aged biodynamically for at least 8 years,with the oldest for 18 years, to provide timefor the biodynamic farming practices toinfluence soil properties. The farm pairs orsets included a range of representative farm-ing enterprises in New Zealand: marketgarden (vegetables), pip fruit (apples andpears), citrus, grain, livestock (sheep andbeef), and dairy. Farms in each pair or sethad the same crop and livestock enterprise.Paddocks (fields) chosen for study in each

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farm pair or set had soils in a single soilprofile class and were located at the junc-ture of adjoining farms. The soil of eachpaddock was sampled at numerous locations(4). In total, 130 soil samples from 22paddocks were taken and analyzed (5).

In six of the seven farm sets (Table 2),the biodynamically farmed soils had betterstructure and broke down more readily to agood seedbed than did the conventionallyfarmed soils. The crumb and nut structuresfound predominantly on the biodynamicfarms provide better aeration and drainagefor crop or grass growth compared with theblocky and clod structures found mostly onthe conventional farms (6). Soil was morefriable, which makes it more easily tilled by

farm machinery, on four of the seven bio-dynamic farms compared with that of theirconventional neighbors.

The surface soil bulk density was signif-icantly less on four of the biodynamic farmsthan on their conventional counterparts(Table 2); when all data were aggregated,bulk density was significantly lower on thebiodynamic farms (Table 3). Bulk density isrelated to mechanical impedance and soilstructure, both of which affect root growth.Penetration or cone resistance is anotherindicator of mechanical impedance. Two ofthe three biodynamic farms in pasture hadsignificantly lower penetration resistancesin the upper 20 cm than their conventionalcounterparts had. The results were variable

for the horticultural and mixed farms (Ta-ble 2). Overall (Table 3), the biodynamicfarms had a significantly lower penetrationresistance in the upper 20 cm; there was no

difference between farming systems in soil20 to 40 cm below the surface.

Organic matter content, soil respiration,mineralizable nitrogen, and the ratio ofmineralizable nitrogen to organic carbonwere significantly higher on almost all thebiodynamically farmed soils than on theconventionally farmed soils (Table 2). Theaggregated data (Table 3) indicate signifi-cantly higher values for these four parame-ters on the biodynamic farms. The higheramounts of organic matter on the biody-namic farms have contributed to better soil

Table 1. General farm characteristics. Abbreviations: bio, biodynamic; veg, vegetables; con, conventional; pip, pip fruit; cit, citrus; and org, organic.

Number Farm Pad-

Farm Main of years size docks* Fertilizerst Pesticides and pestenterprise (1966 to (ha) per (1983 to 1991) management (1983 to 1991)1991) farm

Bio veg Market 13 con; 1 1 1 Manures, composts, Cultural controls,* biologicalgarden 4 org; bonemeal, fishwastes, controls,§ copper and sulfur spraysil

8 bio biodynamic preparationsCon veg Market 25 con 45 1 12-5-14 and 12-10-10 of Propyzamide, alachlor, maneb, propineb,

garden N-P-K vinclozolin, methamidophosBio pip Pip fruit 10 con; 5 2 Composts, fish manures, Cultural controlst

15 bio biodynamic preparationsCon pip 1 Pip fruit 25 con 7 1 12-10-10 of N-P-K, Terbacil, simazine, glyphosate,

potassium chloropyrifos, guthion, azocyclotin,superphosphate polyram, captan, triadimefon,

dodine, bitertanolCon pip 2 Pip fruit 25 con 24 1 12-10-10 of N-P-K Amitrole, simazine, terbacil, glyphosate,

guthion, azocyclotin, chloropyrifos,polyram, captan, dodine, bitertanol,myclobutanil, fruit-fed ANA

Bio cit Citrus 17 con; 10 3 Composts, fish fertilizer, Copper sprayll biological controls§8 bio biodynamic preparations

Con cit 1 Citrus 25 con 12 2 Nitraphoska (N-P-K Glyphosate, paraquat, acephate,fertilizer), urea, copper oxychloridesuperphosphate with traceelements

Con cit 2 Citrus 25 con 9 1 Urea, superphosphate, Glyphosate, terbuthylazinefertigation with ammonium plus terbumeton, dimethoate,nitrate, calcium nitrate, clofentezine, thiazolidone, diazinon,sulfate of potash copper oxychloride, maneb plus

zinc and manganese, benomylBio mixed Grain, sheep, 15 con; 202 1 Rock phosphate, seaweed, Cultural controls,f biological controls§

and beef 10 bio composts, biodynamicpreparations

Con mixed Grain, sheep, 25 con 280 1 Superphosphate, urea, MCPA + triazine, MCPB 2,4-D,and beef chlormequat chloride¶ chlorsulfuron, pirimicarb,

terbuconazole, cultural controls*Bio livestock Sheep and 12 con; 180 1 Fish fertilizer, biodynamic Cultural controls,f biological controls§

beef 13 bio preparationsCon livestock Sheep and 25 con 445 1 Fish fertilizer, rock MCPA, glyphosate, picloram,

beef phosphate, chicken dimethyl carbatemanure

Bio dairy 1 Dairy 1 con; 6 org; 25 1 Rock phosphate, seaweed, Cultural controls,f biological controls§18 bio fish fertilizer,

biodynamic preparationsCon dairy 1 Dairy 25 con 51 1 Potassium superphosphate MCPABio dairy 2 Dairy 15 con; 235 2 Biodynamic preparations Cultural controls,* biological controls§

10 bioCon dairy 2 Dairy 25 con 150 2 15-10-10-8 of N-P-K-S, urea 2,4-D*Number of paddocks or fields where soil was sampled on a particular farm. tIncludes organic and synthetic chemical fertilizers. *Cultural pest controls includephysical and mechanical practices such as rotating and diversifying crops, green manuring, clearing weeds from field borders, and altering the timing or way ofplanting. §Biological pest controls involve the introduction or buildup of natural predators, parasites, and pathogens that keep pest populations below injuriousnumbers. BApproved chemical spray by the New Zealand biodynamic and organic certification boards. ¶A plant growth regulator.

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Table 3. Mean values of aggData were analyzed with Avariation due to different efwas absorbed or removed (

Soil property

Bulk density (Mg m-3)Penetration resistance

(0 to 20 cm) (MPa)Penetration resistance

(20 to 40 cm) (MPa)Carbon (%)Respiration (p1 02

hour-1 g-1)Mineralizable N(mg kg-1)

Ratio of mineralizableN to C (mg g-1)

Topsoil thickness (cm)tCEC (cmol kg-1)!:Total N (mg kg-1)Total P (mg kg-1)Extractable P(mg kg-1)

Extractable S(mg kg-1)

Extractable Ca(cmol kg-1)§

Extractable Mg(cmol kg-1)§

Extractable K(cmol kg-1)§

pH*P < 0.01. tTopsoil thicknEand subsurface (A) horizons.capacity in centimoles of cationgram of soil. §Centimole ch<ion per kilogram of soil.

structure and consistencesity and cone resistance ththose of their conventionsrespiration and the rationitrogen to organic carbotion of the microbial actwhich accounts for the t

nutrients such as nitrogen,sulfur for plant growth (7

Earthworms were couimarket gardens to give atof biological activity. Froncm in diameter by 15 cneach paddock, we found tifarmed soil to average 17.square meter compared wiper square meter on thfarmed soil. By mass, thfarmed soil had 86.3 g osquare meter, whereas thfarmed soil had 3.4 g olsquare meter. These diffelikely due to the use of pe,reduce earthworm populaconventional farm.

Topsoil was significantbiodynamic farms than (tional neighbors (Table 2)more topsoil was present ofarms (Table 3). These

regated soils data. partly due to the significantly lower soilNOVA so that the bulk densities on the biodynamic farms.nterprises or soils Greater organic matter content and biolog-~~13). ~ ical activity contributed to the formation of

bio All con topsoil at a faster rate on the biodynamic\AllO ~i con *i c

Farms farms farms. Soil erosion was not significant onany of the paddocks in this study.

1.07 1.15* Cation exchange capacity and total ni-2.84 3.18* trogen were more often higher on the indi-

vidual biodynamic farms, whereas total and3.55 3.52 available phosphorus, available sulfur, and4.84* 4.27 soil pH were more often higher on the

73.7* 55.4 individual conventional farms (Table 2).This relation, except for total phosphorus,

140.0* 105.9 holds true when the aggregated nutrientdata were compared (Table 3). Aggregated2.99* 2.59 amounts of calcium, magnesium, and potas-

22.8* 20.6 slum were similar in the two systems. There21.5* 19.6 were a number of statistically significant340* 4260 differences in the amounts of phosphorus,560 1640 sulfur, potassium, calcium, and magnesium45.7 66.2 between individual farms, although few dif-10.5 21.5* ferences were of biological significance

(that is, almost all soils were of adequate12.8 13.5 fertility for their respective crops) (9).

To evaluate financial viability, we ex-1.71 1.68 amined farmers' annual accounts from 1987

to 1991. These accounts are the only com-0.97 1.00 mon source of farm financial data in New6.10 6.29* Zealand because few New Zealand farmers--ssinldekeep financial records of individual farmess includes surface*Cation exchange enterprises beyond annual accounts (10).

charge (+) per kilo- Reliable economic data from annual ac-arge of specified cat- counts were available for 11 of the 16 farms.

We compared the financial performance ofthe biodynamic farms both with that of

and to bulk den- their conventional neighbors and with thatLat are lower than of the average, representative conventionalal neighbors. Soil farm (2) in the region of each farm pair orof mineralizable set. Most of the products from the biody-n give an indica- namic farms were sold as certified organic orivity of the soil, biodynamic at a premium price up to 25%recycling of vital higher than the market price of a similar,phosphorus, and conventional product.). Profits can be different from one farm tonted on the two another because of the ownership structurenother indication or the amount of fixed costs such as debtn 30 soil cores (15 servicing. To compensate for these differ-i deep) taken on ences, we excluded fixed costs from ourie biodynamically calculations and used an analysis of enter-5 earthworms per prise gross margins as a measure of financialth 21 earthworms performance (11). Gross margin is the dif-e conventionally ference between total farm income pere biodynamically hectare and variable or operating expensesf earthworms per per hectare. Examples of variable costs in-le conventionally elude those of fertilizers, pesticides, biody-f earthworms per namic preparations, fuel, and labor. Werences were most only examined farming enterprises requir-sticides, shown to ing similar commitments of owner-operatortions (8), on the resources per hectare, except for dairy farm

pair 2, where the biodynamic farm wasly thicker on two selling yogurt and the conventional farmn their conven- milk. Here, the additional direct costs ofi. Overall, 2.2 cm yogurt production were included in them the biodynamic gross margin analysis of the biodynamicdifferences were farm.

SCIENCE * VOL. 260 · 16 APRIL 1993

One biodynamic farm (livestock) hadgreater, two biodynamic farms (mixed anddairy 2) had lower, and two biodynamicfarms (market gardens and citrus) had sim-ilar gross margins compared to those of theirconventional neighbors (Table 4). Com-pared with the representative conventionalfarms (2) in their regions, three biodynamicfarms (citrus, livestock, and dairy 1) andthree conventional farms (mixed, live-stock, and dairy 2) were more prosperous,two biodynamic (mixed and dairy 2) wereless prosperous, and one conventional farm(citrus) was comparable. In the majority ofcases, the biodynamic farms had less year-to-year variability in gross margin than didthe conventional farms. Economic stabilityis one of the most significant characteristicsof sustainable farming systems. Total in-come and variable costs were not consis-tently lower or higher on the biodynamicfarms than on their adjacent conventionalneighbors or the MAF representative (2)conventional farms.

From farmer interviews and their annualaccounts, we determined that the biody-namic citrus, livestock, and dairy 1 farmshave been able to secure reliable marketsfor their products, which is an importantfactor for economic stability. Gross marginsfor the biodynamic market garden were lessthan for the conventional counterpart in1988 and 1989 but greater in 1990 and1991. Annual returns per hectare for thebiodynamic market garden have increasedconsistently over this 4-year period becauseof the development ofbiodynamic or organ-ic markets and improved productivity andfarm management practices. The biody-namic mixed farm (except in 1991) and thebiodynamic dairy farm 2 have not matchedthe annual gross margins representative ofconventional farms in the same region.

Although gross margins provide a com-parison of financial performance of twofarms under different management ap-proaches, total gross margins illustrate thefinancial return to each whole farm or tothe major farm enterprise. Total gross mar-gin is simply the gross margin times theeffective enterprise area of each farm oreach MAF model. The biodynamic farmshad lower total gross margins than theirconventional neighbors and most of theMAF conventional farms (Table 4). Muchof this difference was due to the smaller sizeand greater enterprise diversity of the bio-dynamic farms.

The biodynamic farms proved in mostenterprises to have soils of higher biologicaland physical quality: significantly greaterorganic matter content and microbial activ-ity, more earthworms, better soil structure,lower bulk density, easier penetrability, andthicker topsoil. The results of the soilchemical analyses were variable. On a per

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'Sl~ahectare basis, the biodynamic farms werejust as often financially viable as theirneighboring conventional farms and repre-sentative conventional farms.

REFERENCES AND NOTES

1. H. H. Koepf, The Biodynamic Farm (Anthropo-sophic Press, Hudson, NY, 1989), pp. 94-112.

2. Ministry of Agriculture and Fisheries, Farm Moni-toring Report: North Central Region (MAF, Palm-erston North, New Zealand, 1987-1991); FarmMonitoring Report: North Region (MAF, Hamilton,New Zealand, 1987-1991).

3. H. Jenny, Factors of Soil Formation (McGraw-Hill,New York, 1941), pp. 12-20. Fields not adjacentto the boundary between farms may differ not onlyin soil characteristics but in economic perfor-mance, limiting the economic component of stud-ies with whole farms.

4. Ten pairs of paddocks were directly adjacent toeach other; 5 to 6 soil samples were taken fromeach paddock. Two paddocks were in hill countryand had to be sampled about 300 m apart to getthe same slope and aspect; here 12 soil sampleswere taken from each paddock. Soil sampleswere collected in the spring of 1990 and thesummer of 1990 to 1991 from the upper 10 cm.

5. Soil samples were analyzed for the followingproperties: total carbon, with the use of a Leco(Saint Joseph, MI) high-frequency induction fur-nace; extractable potassium, calcium, and mag-nesium, with the use of a semimicro leachingprocedure; pH in a water suspension; extractablephosphorus and cation exchange capacity asdescribed in L. C. Blakemore, P. L. Searle, and B.K. Daly, New Zealand Soil Bureau Scientific Re-port 80 (Department of Scientific and IndustrialResearch, Lower Hutt, New Zealand, 1987)]; soilrespiration, by manometric measurements of therespiratory uptake of gaseous oxygen by soil [W.W. Umbreit, R. H. Burris, J. F. Stauffer, Manomet-ric and Biochemical Techniques (Burgess, Min-neapolis, 1972)] and modified by A. N. Macgre-gor and L M. Naylor [Plant Soil 65, 149 (1982)];mineralizable soil nitrogen, by incubation [D. R.Keeney and J. M. Bremner, Soil Sci. Soc. Am.Proc. 31, 34 (1967)]; total nitrogen and phospho-rus, with the use of a micro-Kjeldahl digestion ofsoil followed by nitrogen analysis [Technicon,Industrial Method No. 329-74 W/A (Technicon,Tarrytown, NY, 1976)] and phosphorus analysis[J. R. Twine and C. H. Williams, Commun. Soil Sci.Plant Anal. 2, 485 (1971)]; and sulfate, by theautomated Johnson and Nishita technique [B.Heffernan, A Handbook of Methods of InorganicChemical Analysis for Forest Soils, Foliage, andWater (CSIRO Division of Forest Research, Can-berra, Australia, 1985)]. Soil profiles were ana-lyzed in the field for the following properties: soiltexture, structure, and consistence as describedby standard New Zealand Soil Bureau proce-dures [N. H. Taylor and I. J. Pohlen, Soil BureauBulletin 25 (Soil Bureau, Lower Hutt, NewZealand. 1962)]; bulk density with the use ofthin-walled aluminum cores; and penetration re-sistance with the use of a Rimik (Toowoomba,Queensland, Australia) CP10 cone penetrometer.

6. R. G. McLaren and K. C. Cameron, Soil Science:An Introduction to the Properties and Manage-ment of New Zealand Soils (Oxford Univ. Press,Auckland, New Zealand, 1990), p. 132.

7. E. W. Russell, Russell's Soil Conditions and PlantGrowth (Longman, Essex, England, 1988), pp.472-499.

8. J. K. Syers and J. A. Springett, Plant Soil 76, 93(1984).

9. I. S. Cornforth and A. G. Sinclair, Fertiliser Rec-ommendations for Pastures and Crops in NewZealand (MAF, Wellington, New Zealand, 1984);C. J. Clarke, G. S. Smith, M. Prasad, I. S. Corn-forth, Fertilizer Recommendations for HorticulturalCrops (MAF, Wellington, New Zealand, 1986).

10. A. Wright, in Integrated Systems Analysis and

Climate Impacts, R. W. M. Johnson, Ed. (MAFTech, Wellington, New Zealand, 1989), pp. 55-63.

11. M. D. Boehlje and V. R. Eidman, Farm Manage-ment (Wiley, New York, 1984), pp. 86-91.

12. SAS Institute Inc., JMP User's Guide (SAS Insti-tute, Cary, NC, 1989).

13. SAS/STAT User's Guide, Release 6.03 Edition(SAS Institute, Cary, NC, 1988).

14. We thank the 16 New Zealand farm families for

donating the use of their farms to this study.Supported by the Fertiliser and Lime ResearchCentre at Massey University, a Prince and Prin-cess of Wales Science Award by the Royal Soci-ety of New Zealand, the Massey University Re-search Fund, and International Program Develop-ment at Washington State University.14 September 1992; accepted 4 March 1993

Transient Transfection and Expression in theObligate Intracellular Parasite Toxoplasma gondii

Dominique Soldati and John C. Boothroyd*Toxoplasma gondii is a protozoan pathogen that produces severe disease in humansand animals. This obligate intracellular parasite provides an excellent model forthe studyof how such pathogens are able to invade, survive, and replicate intracellularly. DNAencoding chloramphenicol acetyltransferase was introduced into T. gondii and transientlyexpressed with the use of three vectors based on different Toxoplasma genes. The abilityto introduce genes and have them efficiently and faithfully expressed is an essential toolfor understanding the structure-function relation of genes and their products.

Toxoplasma gondii is a ubiquitous parasitethat can infect almost any warm-bloodedvertebrate. In humans, it has long beenrecognized as a major cause of severe con-genital disease. More recently, it hasemerged as one of the most importantopportunistic pathogens in patients withacquired immunodeficiency syndrome(AIDS) (1). In the laboratory, T. gondii isrelatively easy to handle and maintain andconsequently has become an importantmodel for the study of how obligate intra-cellular parasites function. To date, howev-er, such studies have been hampered by theabsence of a method for introducing DNAinto the parasites. In part, this lack hasbeen due to the difficulty of transfecting onecell inside another: the many membranesthat the transfecting DNA must cross rep-resent a significant barrier, and the depen-dence on the host cell for survival canfurther preclude manipulations of the extra-cellular parasite. As a result, althoughtransfection and stable transformation havebeen achieved for a range of trypanosoma-tids (2-8), such methodologies have notbeen reported for any of the obligate intra-cellular parasites, most notably members ofthe phylum Apicomplexa, which includesToxoplasma, Eimeria, and Plasmodium, thecausative agent of human malaria.

Electroporation has successfully beenused to introduce DNA into many celltypes. It is believed that pores are generatedby reversible electrical breakdown of the

Department of Microbiology and Immunology, Stan-ford University School of Medicine, Stanford, CA94305.*To whom correspondence should be addressed.

cell membrane. Recent studies have shownthat immediately after electroporation,cells are sensitive to the osmolarity andionic composition of the medium and thatthe use of a potassium phosphate-basedelectroporation buffer (cytomix) that re-sembles the cytosol's ionic compositionconsiderably increases cell survival (9). Wechose, therefore, to use such a buffer in ourinitial transfection studies rather than cul-ture medium or phosphate-buffered saline,which contain sodium ions at concentra-tions that are detrimental to the cells. Wefound that electroporation of T. gondii incytomix buffer gives an extremely good rateof cell survival: an average of -80% of theparasites are capable of invading host cellsafter electroporation as compared with thesame population of parasites not subjectedto an electric pulse.

For use as a reporter construct, a plasmid(SAG1/2 CAT) was made containing thechloramphenicol acetyltransferase (CAT)gene (11) and the upstream and down-stream sequences of the T. gondii majorsurface antigen gene, p30 or SAG1 (12)(Fig. 1). This was done by a two-stepmethod. First, reverse polymerase chainreaction (PCR) (13) was performed with anSK+ Bluescript vector (Strategene) con-taining the complete SAG) gene with theuse of primers that generate an Nsi I site atthe second in-frame ATG and a Pac I site atthe stop codon. Then, a CAT cassette witha Nsi I site embracing its ATG and a Pac Isite encompassing its stop codon was gen-erated by PCR and cloned into the corre-sponding Nsi I-Pac I sites of the SAG1expression vector.

Electroporation of this construct into

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