Abstract
‘Speed breeding’ (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants’ daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber–based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.
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Acknowledgements
We acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC) strategic programmes Designing Future Wheat (BB/P016855/1), Molecules from Nature (BB/P012523/1), Understanding and Exploiting Plant and Microbial Metabolism (BB/J004561/1), Food and Health (BB/J004545/1) and Food Innovation and Health (BB/R012512/1), and also support from the Gatsby Charitable Foundation. Development of the benchtop cabinet was supported by an OpenPlant Fund grant from the joint Engineering and Physical Sciences Research Council and BBSRC-funded OpenPlant Synthetic Biology Research Centre grant BB/L014130/1. S.G. was supported by a Monsanto Beachell-Borlaug International Scholarship and the 2Blades Foundation, A.Sarkar by the BBSRC Detox Grasspea project (BB/L011719/1) and the John Innes Foundation, A.W. by an Australian Post-graduate Award and the Grains Research and Development Corporation (GRDC) Industry Top-up Scholarship (project code GRS11008), M.M.-S. by CONACYT-I2T2 Nuevo León (grant code 266954/399852), and L.T.H. by an Australian Research Council Early Career Discovery Research Award (project code DE170101296). We acknowledge M. Grantham and D. Napier from Heliospectra for their help in the choice of LED lights; L. Hernan and C. Ramírez from Newcastle University for their support and advice in the design of the benchtop cabinet; C. Moreau from the John Innes Centre and J. Ghosh from the University of Bedfordshire for help with the pea and grass pea experiments, respectively; and the JIC and UQ horticulture services for plant husbandry and their support in scaling up SB in glasshouses.
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Contributions
S.G. and A.W. drafted the manuscript and oversaw many of the experiments. L.T.H. and B.B.H.W. contributed to the design of experiments and manuscript writing. A.W. designed and implemented the SSD approach for wheat and barley in the LED-supplemented glasshouse at UQ. S.G., O.E.G.-N., R.H.R.-G., L.Y. and M.M.-S. designed, constructed, programmed and tested the benchtop growth cabinet. J.C. performed the energy consumption calculations for the LED-supplemented glasshouse at JIC. For the LED-supplemented glasshouse at JIC, J.S. performed the experiments for wheat, including the SSDs; R.W. for brassicas; R.E.M. for oats; S.H. for additional wheat cultivars; P.G. for barley; T.R. for pea; A.H. for quinoa; A. Sarkar for grass pea; and A. Steed for Brachypodium. J.L., L.P., C.D., M.J.M., W.H., A.O., C.M., C.U., B.H., M.T., P.N., B.B.H.W. and L.T.H. contributed intellectually to the experiments and/or the writing of the manuscript. All authors reviewed and approved the final manuscript before submission.
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Key references using this protocol
Watson, A. & Ghosh, S. et al. Nat. Plants 4, 23–29 (2018): https://www.nature.com/articles/s41477-017-0083-8
Hickey, L. T. et al. Euphytica 168, 303–310 (2009): https://link.springer.com/article/10.1007/s10681-009-9929-0
O’Connor, D. J. et al. Peanut Sci. 40, 107–114 (2013): http://www.peanutscience.com/doi/abs/10.3146/PS12-12.1
Integrated supplementary information
Supplementary Figure 1 Mature eight-week-old pea plants grown in limited media and nutrition (“flask method”) in order to achieve rapid generation advancement.
Pisum sativum (a) accession JI 2822 and (b) cv. Frisson. Dry seeds were sterilised in 10% sodium hypochlorite, rinsed in sterile water, chipped and left to germinate in the dark for 3 days on sterile, wet filter paper. Germinating seeds were transferred to flasks containing 250 mL fine perlite and silver sand (mixed 50:50) and FP nutrient media which had been sterilised (composition described in Supplementary Table 49). Flasks were placed in the dark for a further 5 days. The seedlings were inoculated with Rhizobium, and the elongated shoot passed through the neck of the flask and held in place with a bung. The base of the flask was covered with a black plastic bag. Plants were grown in a Controlled Environment Room at constant 22 °C with a 16-hour photoperiod. After 3 weeks, flasks were watered with 50 mL FP media once a week. After 8 weeks post germination, plants had mature dry seed ready to harvest as shown (indicated by red arrows). JI 2822 plants grown in the glasshouse under lights required 12 weeks post sowing before mature dry seed were ready for harvest.
Supplementary Figure 2 Symptoms of calcium deficiency in wheat grown under speed breeding conditions.
Right: Small, circular depressions on the leaf blade; Left: Tip leaf necrosis.
Supplementary Figure 3
Circuit diagram of the monitoring and control system of the benchtop growth cabinet.
Supplementary Figure 4 Benchtop cabinet for conducting speed breeding.
(a) Front view of the cabinet. (b) Front view of the cabinet with the door open to show the lighting and wheat plants (Triticum aestivum cv. Apogee) growing inside. (c) Apogee wheat plant grown in the cabinet, photographed at 55 DAS (Days after sowing). (d) Pea (Pisum sativum) variety JI 2822 grown in the cabinet, photographed at 50 DAS.
Supplementary Figure 5 Light spectrum measurements in in the benchtop growth cabinet 20 cm below one of the LED bulbs.
The x-axis represents the wavelength of light in nanometres, and y-axis is the normalised spectral power distribution. (Power distribution is measured in mW.m-2, and all values on y-axis are divided by the maximum value in the distribution in order to obtain normalised values). Graph was produced from measurements made by the MK350S LED meter from UPRtek, using the uSpectrum software produced by the same manufacturer.
Supplementary Figure 6 Barley spikes from plants grown under Heliospectra LED lights.
Barley cv. Golden Promise from 22-hour light regime (left) and 16-hour light regime (right). Scale bar is 5 cm.
Supplementary Figure 7 Pods from Brassica rapa R-0-13 grown in LED-supplemented glasshouses at the John Innes Centre, UK.
Plants grown under (a) a 22-hour photoperiod or (b) a 16-hour photoperiod.
Supplementary Figure 8 Pods harvested from Brassica napus RV31 grown in LED-supplemented glasshouses at the John Innes Centre, UK.
Plants grown under (a) a 22-hour photoperiod or (b) a 16-hour photoperiod.
Supplementary Figure 9 Layout of the glasshouse used for speed breeding at the John Innes Centre, UK.
(Left) Photograph with Heliospectra LX60C2 LED supplementary lighting; (Right) Schematic of light positioning within the glasshouse relative to the bench, plants and other light fixtures.
Supplementary Figure 10 Layout of the glasshouse used for speed breeding at The University of Queensland, Australia.
(Left) Photograph with Heliospectra E602G LED supplementary lighting; (Right) Schematic of light positioning within the glasshouse relative to the bench, plants and other light fixtures.
Supplementary Figure 11 Light spectrum measurements under a Heliospectra LX602C LED fixture in JIC glasshouse.
(a) Spectrum measurement in the glasshouse at bench level (244 cm from light fixture) on a clear, sunny day at 12 noon. (b) Spectrum measurement in the glasshouse at bench level (244 cm from light fixture) on a cloudy day at 12 noon. (c) Spectrum measurement in the glasshouse at bench level (244 cm from light fixture) at night. The x-axis of all three graphs represents the wavelength of light in nanometres, and y-axis is the normalised spectral power distribution. (Power distribution is measured in mW.m-2, and all values on y-axis are divided by the maximum value in the distribution in order to obtain normalised values). All graphs were produced from measurements made by the MK350S LED meter from UPRtek, using the uSpectrum software produced by the same manufacturer.
Supplementary Figure 12 Light spectrum measurements under a Heliospectra E602G LED fixture in the UQ glasshouse.
Weighted McCree action spectrum and photosynthetic photon flux density (PPFD; µmol.m-2.s-1) from under a Heliospectra E602G light using the Spectrum Genius Essence Lighting Passport light sensor and associated Spectrum Genius Agricultural Lighting app (AsenseTek Inc., Taiwan). (1) Centre measurement at 12 noon on a clear, sunny day, (2) Centre measurement at 12 noon on an overcast day and, (3) Centre measurement at night; a, bench level (155 cm from light) and b, approximate wheat spike height (95 cm from light). Figures were exported from the software.
Supplementary Figure 13 Symptoms of copper deficiency in wheat grown under speed breeding conditions.
Left (top): Curling and death of young leaf tips and down the leaf blade; Left (bottom): Young leaves becoming stuck as they emerge and forming loops or curling; Right (top and bottom): Spikes wither and turn white at the tips. No seed is produced in these areas and spikes may be twisted.
Supplementary Figure 14 Symptoms of iron deficiency in wheat grown under speed breeding conditions.
Young leaves appear striped with yellowing of the interveinal spaces.
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Supplementary Text and Figures
Supplementary Figures 1–14 and Supplementary Tables 1–49
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Ghosh, S., Watson, A., Gonzalez-Navarro, O.E. et al. Speed breeding in growth chambers and glasshouses for crop breeding and model plant research. Nat Protoc 13, 2944–2963 (2018). https://doi.org/10.1038/s41596-018-0072-z
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DOI: https://doi.org/10.1038/s41596-018-0072-z
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