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Rapid customization of Solanaceae fruit crops for urban agriculture

Nature Biotechnology volume 38, pages 182–188 (2020)Cite this article

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Abstract

Cultivation of crops in urban environments might reduce the environmental impact of food production1,2,3,4. However, lack of available land in cities and a need for rapid crop cycling, to yield quickly and continuously, mean that so far only lettuce and related ‘leafy green’ vegetables are cultivated in urban farms5. New fruit varieties with architectures and yields suitable for urban farming have proven difficult to breed1,5. We identified a regulator of tomato stem length (SlER) and devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) and precocious growth termination (SP). Application of our strategy using one-step CRISPR–Cas9 genome editing restructured vine-like tomato plants into compact, early yielding plants suitable for urban agriculture. Field data confirmed that yields were maintained, and we demonstrated cultivation in indoor farming systems. Targeting the same stem length regulator alone in groundcherry, another Solanaceae plant, also enabled engineering to a compact stature. Our approach can expand the repertoire of crops for urban agriculture.

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Fig. 1: Condensed shoots of the tomato si mutant and identification of the underlying gene.
Fig. 2: Creating highly compact, rapid-flowering tomatoes by genome editing.
Fig. 3: CRISPR–Cas9 generation of a rapid-cycling, highly compact cherry tomato variety.
Fig. 4: CRISPR–Cas9-generated compact groundcherry.

Data availability

Raw data for all quantifications and primer sequences are in Supplementary Dataset 1. Sequences that confirmed CRISPR–Cas9 edits are in Supplementary Dataset 2 and the raw Sanger sequence traces for those edited sequences are in Supplementary Dataset 3. Seeds may be requested by contacting Z.B.L.

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Acknowledgements

We thank members of the Lippman laboratory for valuable comments and discussions. We thank G. Robitaille, J. Kim, A. Krainer, J. Dalrymple, M. Strahl and J. Wong for technical support. We thank K. Swartwood, M. Tjahjadi and B. N. Williams for assistance with tomato and groundcherry transformations. We thank T. Mulligan, K. Schlecht, B. Hendrick, A. Krainer and S. Qiao, and staff from Cornell University’s Long Island Horticultural Research and Extension Center, for assistance with plant care. We thank M. E. Bartlett for assistance with the phylogenetic tree. We thank N. Van Eck for assistance with the LED growth chamber experiment. We thank D. Harris, D. Lucas and J. Friedman from Freight Farms for assistance with the vertical farm experiment. We thank D. Zamir (Hebrew University), N. Ori (Hebrew University), Y. Eshed (Weizmann Institute) and K. Hoshikawa (University of Tsukuba) for providing seed. This research was supported by the Howard Hughes Medical Institute; by the Next-Generation BioGreen 21 Program SSAC (grant no. PJ0134212019) from the Rural Development Administration, Republic of Korea to S.J.P. and Z.B.L.; by the National Research Foundation of Korea (grant nos. 2017R1A4A1015594 and 2016R1C1B2015877) funded by the Ministry of Science, ICT and Future Planning to S.J.P.; by an Agriculture and Food Research Initiative competitive grant from the USDA National Institute of Food and Agriculture (grant no. 2016-67013-24452) to S.H. and Z.B.L.; and by the National Science Foundation Plant Genome Research Program (grant no. IOS-1732253 to J.V.E. and Z.B.L., and grant no. IOS-1546837 to Z.B.L.).

Author information

Author notes

  1. Zachary H. Lemmon

    Present address: Inari Agriculture, Cambridge, MA, USA

  2. Yossi Capua

    Present address: Centre for Organismal Studies, Department Stem Cell Biology, Ruprecht-Karls-University Heidelberg, Heidelberg, Germany

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Choon-Tak Kwon, Zachary H. Lemmon & Zachary B. Lippman

  2. Division of Biological Sciences and Research Institute for Basic Science, Wonkwang University, Iksan, Republic of Korea

    Jung Heo & Soon Ju Park

  3. Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel

    Yossi Capua

  4. Gulf Coast Research and Education Center, University of Florida, Wimauma, FL, USA

    Samuel F. Hutton

  5. Boyce Thompson Institute for Plant Science, Ithaca, NY, USA

    Joyce Van Eck

  6. Plant Breeding and Genetics Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, USA

    Joyce Van Eck

  7. Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Zachary B. Lippman

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Contributions

C.-T.K. and Z.B.L. designed the research and performed the experiments. J.H. and S.J.P. performed the MicroTom experiments and tomato transformation. Z.H.L. performed mapping analysis. Y.C. generated tomato CRISPR mutants. S.F.H. contributed to the tomato yield trial. J.V.E. performed tomato and groundcherry transformations. C.-T.K. and Z.B.L. wrote the manuscript, with editing contributed by all authors.

Corresponding author

Correspondence to Zachary B. Lippman.

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Competing interests

Z.B.L. is a paid consultant for and a member of the Scientific Strategy Board of Inari Agriculture, and he is also a named inventor on a number of patents and patent applications (Patent Application Publications WO/2017/180474; WO/2014/081730A1; WO/2018/213547) directed to related technology that have been exclusively licensed from CSHL to Inari Agriculture.

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Integrated supplementary information

Supplementary Figure 1 Mapping of the short internode (si) mutant and characterization of multiple loss-of-function alleles in the causative gene SlERECTA (SlER).

a, Length of shoot internodes, distal and proximal section of flower pedicels, peduncles and inflorescence internodes in WT, si and si/+ heterozygotes. 5th, internode between 5th and 6th leaf of primary shoot; 6th, internode between 6th and 7th leaf; 7th, internode between 7th and 8th leaf. n, number of plants and inflorescences. Box plots, 25th–75th percentile; center line, median; whiskers, full data range. The numbers indicate P values (two-tailed, two-sample t-test). b, Mapping-by-sequencing of the si mutant generated by EMS mutagenesis. Differences in SNP index between pools of si and WT individuals derived from a segregating F2 population are shown. Red dashed lines indicate 95% cut-off in SNP index. SlER is located on chromosome 8. c, Genomic DNA and transcript sequences of slerEMS-1. d, Genomic DNA and transcript sequences of slerEMS-2. e, RT-PCR analysis showing an 11bp insertion in the transcript from of slerEMS-1. f, RT-PCR analysis showing a 72bp deletion in the transcript of slerEMS-2. PCR agarose gel images were cropped to accommodate size. g, SlER protein models of WT, slerEMS-1 and slerEMS-2. h, Schematic showing targeting of SlER by CRISPR-Cas9. i, Complementation test between slerEMS-1and the CRISPR-generated null allele slerCR-1. j, Complementation test between slerCR-1 and slerEMS-2. k, Complementation test between slerEMS-1 and slerEMS-2. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel. The experiments were repeated at least twice independently with similar results in e, f, i, j and k.

Supplementary Figure 2 The ultra-compact plant architecture of the classical tomato cultivar ‘MicroTom’ and its enhancement by sler.

a, Shoot of MicroTom and slerMT. DAS, days after sowing. b, Quantification of shoot and internode lengths in MicroTom, slerMT and slerMT/+ heterozygotes. Prim., primary (Length between 1st inflorescence and 1st leaf of primary shoot); Axil., basal axillary (Length between 1st inflorescence and 1st leaf of basal axillary shoot); Symp., sympodial (Length between 1st and 2nd inflorescence of primary shoot). 3rd, internode between 3rd and 4th leaf of primary shoot; 4th, internode between 4th leaf and 5th leaf of primary shoot. n, number of plants. c, Inflorescences of WT and slerMT. d, Length of flower pedicels, peduncles and inflorescence internodes in MicroTom, slerMT and slerMT/+ heterozygotes. n, number of inflorescences. Box plots, 25th–75th percentile; center line, median; whiskers, full data range in b and d. The numbers represent P values (two-tailed, two-sample t-test) in b and d. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel.

Supplementary Figure 3 Mutations in the tomato ortholog of Somatic embryogenesis receptor kinase 1 (SlSERK1) and additional phenotypic characterization of sler, slerl1 and sler slerl1 mutants.

a, Three independent alleles of slserk1 (previously designated spd2) obtained from EMS mutagenesis. Two of the alleles (slserk1S1 and slserk1S2) were missense mutations in the kinase domain and showed identical strong pleiotropic phenotypes. The third allele showed a weaker phenotype and was caused by a missense mutation outside of the kinase domain (slserkW). b, Sequential stages of growth for slserk1S1 plants. c, Normalized RNA-seq expression (RPKM) for SlSERK1 in meristems and major tissues. Sym. inflo., sympodial inflorescence; Sym. shoot; sympodial shoot. d, Seedling stage and flowering plant of sler slserk1S1double mutants. e, Inflorescence of slserk1W. f, PCR analysis of first-generation (T0) CRISPR-Cas9 transgenic plants targeting SlSERK1. PCR agarose gel images were cropped to accommodate size. g, Shoot and inflorescence of slserk1CR T0 plants. h, Sequences of slserk1CR alleles identified from two T0 plants 5 and 7. sgRNA and PAM sequences are represented by red and bold underlined font, respectively. Blue dashes and the numbers in parentheses indicate deletions and sequence gap lengths, respectively. i, Lengths of shoots, shoot internodes, distal and proximal section of pedicels, peduncles and inflorescence internodes in WT plants and slerl1 homozygous mutants. Prim., primary (Length between 1st inflorescence and 1st leaf of the primary shoot); Symp., sympodial (Length between 1st and 2nd inflorescence of primary shoot). n, number of plants and inflorescences. Box plots, 25th–75th percentile; center line, median; whiskers, full data range. The numbers indicate P values (two-tailed, two-sample t-test). j, Early seedling stage of WT, sler and sler slerl1 from plants 16 days after sowing (DAS). k, Plants of WT, sler and sler slerl1 41 DAS. DAT, days after transplanting in b, d and g. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel. The experiments were repeated at least twice independently with similar results in b, d-g, j and k.

Supplementary Figure 4 Comparison of field-grown mature plants of spCR single mutants and spCR slerCR-1 double mutants, and additional comparisons between sp determinate, sp sp5g double-determinate and sp sp5g sler triple-determinate plants.

a, Sequence of a CRISPR-generated null mutation in self pruning (spCR). Red and bold underlined font indicate guide RNA and PAM sequences, respectively. Deletions and sequence gap lengths are indicated by blue dashes and the numbers in parentheses, respectively. b, Representative field-grown mature plants of spCR and spCR slerCR-1. Leaves were removed to show fruit set. DAT, days after transplanting. c, Productivity trial of spCR and spCR slerCR-1. d, Quantification of leaves to first inflorescence, inflorescence numbers for both primary and basal axillary shoots, and flower number per inflorescence in single-, double- and triple-determinate plants. Box plots, 25th–75th percentile; center line, median; whiskers, full data range. The numbers above bars indicate P values (two-tailed, two-sample t-test). n, number of plants. Harvest index, total yield/plant weight. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel.

Supplementary Figure 5 CRISPR-Cas9 mutagenesis of SlER in the cherry tomato cultivar Sweet100 and additional comparisons between Sweet100 sp determinate, sp sp5g double-determinate and sp sp5g sler triple-determinate plants.

a, Sequences of two slerCR alleles of Sweet100. sgRNA and PAM sequences are indicated by red and bold underlined font, respectively. The numbers in parentheses and blue dashes and indicate sequence gap lengths and deletions, respectively. b, Quantification of shoot internode, inflorescence stem sections and peduncle lengths in Sweet100 sp sp5g double mutant and sp sp5g sler triple mutant genotypes. 4th, internode between 4th and 5th leaf of primary shoot; 5th, internode between 5th and 6th leaf of primary shoot. DP, distal section of 2nd pedicel; PP, proximal section of 2nd pedicel; INT, 2nd inflorescence internode. c, Quantification of primary shoot, leaves to first inflorescence, flower number per inflorescence, inflorescence per shoot and sugar content (brix) in Sweet100 sp determinate, sp sp5g double-determinate and sp sp5g sler triple-determinate plants. d, Mature fruits of all three genotypes. e, Quantification of fruit size, fruit height to width ratio, and fruit weight in all three genotypes. n, number of plants, inflorescence and fruits in b, c and e. Box plots, 25th–75th percentile; center line, median; whiskers, full data range in b, c and e. The numbers above bars indicate P values (two-tailed, two-sample t-test) in b, c and e. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel.

Supplementary Figure 6 Yield trials of Sweet100 sp determinate, sp sp5g double-determinate and sp sp5g sler triple-determinate plants in higher-density planting.

a, Representative field-grown plants of Sweet100 single-, double- and triple-determinate plants. DAT, days after transplanting. b, Data on yield components for individual plants. Plant weight, harvest index and percentage of red fruits at harvesting. n, number of plants. c, Yield trial in blocks (eight plants) of Sweet100 single-, double- and triple-determinate plants. Fruit drop per total yield, weight of fruit drop/total yield of a block. n, number of blocks. Box plots, 25th–75th percentile; center line, median; whiskers, full data range. Numbers above bars represent P values (two-tailed, two-sample t-test). Harvest index, total yield/plant weight. Red fruits per total yield, red fruit weight/total fruit weight. All data of yield components were obtained at 65 DAT. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel.

Supplementary Figure 7 Selecting for triple-determinate genotypes with different fruit traits from crossbred F2 populations.

a, A selected triple-determinate plant with larger fruits derived from a cross between ‘cocktail’ and Sweet100 sp sp5g sler triple-determinate varieties. b, A selected triple-determinate plant with elongated (ovate) fruits derived from a cross between ‘grape’ and Sweet100 sp sp5g sler triple-determinate varieties. c, Sequences of inherited mutated alleles of sp, sp5g and sler in ‘cocktail’ and ‘grape’ triple-determinate plants.

Supplementary Figure 8 Fine-tuning stem length from an in-frame mutation in the SlER coding sequence and by targeting the SlER promoter region.

a, Concept for generating intermediates between double- and triple-determinate plants by quantitatively modifying shoot and inflorescence internode lengths. b, PCR analysis of T0 transgenic plants targeting promoter region of SlER by CRISPR-Cas9. PCR agarose gel images were cropped to accommodate size. c, Sequences of two SlERCR-pro promoter alleles and one slerCR-3 coding sequence in-frame allele from T2 plants. Red arrows, blue and light blue squares indicate guide RNAs, exons and 5’ UTR, respectively. d, Representative field-grown plants of Sweet100 sp sp5g, sp sp5g SlERCR-pro-4, sp sp5g slerCR-3 and sp sp5g slerCR-1. Leaves were removed to show fruits. DAT, days after transplanting. e, Primary shoot lengths (Length between 1st leaf and 1st inflorescence of the primary shoot) of Sweet100 sp sp5g, sp sp5g SlERCR-pro-14, sp sp5g SlERCR-pro-4, sp sp5g slerCR-3 and sp sp5g slerCR-1plants. n, number of plants. Data of Sweet100 sp sp5g and sp sp5g slerCR-1 are from Fig. 3b. f, Representative first inflorescences of Sweet100 sp sp5g, sp sp5g SlERCR-pro-4, sp sp5g slerCR-3 and sp sp5g slerCR-1 (left) and enlarged photo for of Sweet100 sp sp5g and sp sp5g SlERCR-pro-4 (right) plants. DP3r, 3rd distal pedicel from distal region of the first inflorescence. DP4r, 4th distal pedicel from distal region of the first inflorescence. PP3r, 3rd proximal pedicel from distal region of the first inflorescence. PP4r, 4th proximal pedicel from distal region of the first inflorescence. INT2r, 2nd internode from distal region of the first inflorescence. INT3r, 3rd internode from distal region of the first inflorescence. g, Quantification of pedicels and inflorescence internodes from the proximal region of the first inflorescences in Sweet100 sp sp5g, sp sp5g SlERCR-pro-14, sp sp5g SlERCR-pro-4, sp sp5g slerCR-3 and sp sp5g slerCR-1 plants. h, Quantification of pedicels and inflorescence internodes from distal region of the first inflorescences in Sweet100 sp sp5g, sp sp5g SlERCR-pro-14 and sp sp5g SlERCR-pro-4. n, number of inflorescences in g and h. Box plots, 25th–75th percentile; center line, median; whiskers, full data range in e, g and h. The letters indicate the significance groups at P < 0.01 (One-way ANOVA and Tukey test) in e, g and h. The exact sample sizes (n) for each experimental group/conditions are given as discrete numbers in each panel. The experiment was repeated twice independently with similar results in b.

Supplementary information

Supplementary Materials

Supplementary Figs. 1–8.

Reporting Summary

Supplementary Dataset 1

Supplementary Dataset Tables 1–12. These are the raw data files of all quantifications and the primer sequences.

Supplementary Dataset 2

DNA sequences of CRISPR–Cas9-generated mutations in this study.

Supplementary Dataset 3

Sanger sequence files for Supplementary Dataset 2.

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Kwon, CT., Heo, J., Lemmon, Z.H. et al. Rapid customization of Solanaceae fruit crops for urban agriculture. Nat Biotechnol 38, 182–188 (2020). https://doi.org/10.1038/s41587-019-0361-2

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