SpeedFlower: a comprehensive speed breeding protocol for indica and japonica rice

Summary

To increase rice yields and feed billions of people, it is essential to enhance genetic gains. However, the development of new varieties is hindered by longer generation times and seasonal constraints. To address these limitations, a speed breeding facility has been established and a robust speed breeding protocol, SpeedFlower is developed that allows growing 4–5 generations of indica and/or japonica rice in a year. Our findings reveal that a high red‐to‐blue (2R > 1B) spectrum ratio, followed by green, yellow and far‐red (FR) light, along with a 24‐h long day (LD) photoperiod for the initial 15 days of the vegetative phase, facilitated early flowering. This is further enhanced by 10‐h short day (SD) photoperiod in the later stage and day and night temperatures of 32/30 °C, along with 65% humidity facilitated early flowering ranging from 52 to 60 days at high light intensity (800 μmol m⁻² s⁻¹). Additionally, the use of prematurely harvested seeds and gibberellic acid treatment reduced the maturity duration by 50%. Further, SpeedFlower was validated on a diverse subset of 198 rice accessions from 3K RGP panel encompassing all 12 distinct groups of Oryza sativa L. classes. Our results confirmed that using SpeedFlower one generation can be achieved within 58–71 days resulting in 5.1–6.3 generations per year across the 12 sub‐groups. This breakthrough enables us to enhance genetic gain, which could feed half of the world's population dependent on rice.

Short abstract

We've developed a robust SpeedFlower protocol for rapid rice breeding, enabling 4–5 generations of indica and/or japonica rice annually. The SpeedFlower protocol was validated on 198 diverse rice accessions, representing all 12 sub‐groups of Oryza sativa L. With a timeframe of 58–71 days per generation, we achieved 5.1–6.3 generations per year. Therefore, SpeedFlower has shown a transformative potential in enhancing genetic gain with the pace of climate change.

Highlights.

Breakthrough in rice breeding: The introduction of a speed breeding facility and the innovative SpeedFlower protocol marks a significant leap in rice breeding, addressing generation time and seasonal constraints.

SpeedFlower protocol emphasis: The SpeedFlower protocol focuses on optimizing light spectrum, intensity, photoperiod, temperature, humidity, nutrient levels and hormonal regulation to expedite growth, flowering and maturity in rice.

Remarkable acceleration in flowering and maturity: It has demonstrated flowering within just 60 days for tested rice varieties and achieved a 50% reduction in seed maturity time, irrespective of their natural flowering durations. Accelerated flowering has been observed in 12 diverse groups of Oryza sativa L. under the SpeedFlower protocol.

Rapid trait mapping: The SpeedFlower protocol significantly expedites the development of mapping populations, enabling rapid mapping of crucial traits.

Fast forward breeding: The SpeedFlower protocol revolutionizes breeding programs, allowing completion of breeding cycle in about 1.5 years.

Global impact and promise: This transformative breakthrough holds immense promise for significantly boosting genetic gain in rice through breeding with the speed of climate change.

Introduction

The rate of genetic gain attained in major agricultural crops thus far is inadequate to satisfy the necessary food demands (Cooper et al., 2020). The average estimated rate of genetic gain in rice drought breeding programs is 0.13% (2.29 kg/ha/year), while in irrigated breeding programs, it is 0.23% (8.75 kg/ha/year) (Juma et al., 2021; Khanna et al., 2022). However, these rates are insufficient to meet the increasing future demands for rice with a growing population. The Breeder's equation suggests that genetic gain can be enhanced by improving additive genetic variation within the population (σa), increasing selection intensity (i) and improving selection accuracy (r), while decreasing the generation time (l) required to achieve the desired outcome (ΔG = (σa) (i)(r)/l) (Eberhart, 1970; Sinha et al., 2021). It is generally considered that one of the simplest ways to increase genetic gain is to reduce the time of the breeding cycle (Atlin et al., 2017). Over time, various breeding techniques such as pedigree, bulk, modified bulk, single seed descent (SSD), doubled haploid (DH) and rapid generation advancement (RGA) have been utilized for self‐pollinated crops to decrease generation times and facilitate the development of new varieties (Grafius, 1965; Mackill, 1996; Poehlman and Sleper, 1995; Stoskopf et al., 1993). The Speed Breeding (SB) technique has been introduced to effectively manage environmental factors and is suitable for short‐ and long‐day crops, allowing it to be utilized throughout the year. As a result, SB has gained popularity as a method for advancing multiple generations per year.

The first detailed results on SB, which accelerates flowering and generation time through controlled environmental conditions such as extended photoperiod, light intensity, temperature and early harvest of premature seeds in long‐day crops, were jointly reported by Ghosh et al. (2018) and Watson et al. (2018). Watson et al. (2018) utilized an SB protocol to advance four generations of canola and six generations of spring and durum wheat, barley, peas and chickpeas, achieving a faster rate compared to the two to three generations possible in a glasshouse. Additionally, SB allowed crossing and inbreeding (8–12 generations) to be completed within 2 years rather than 6–7 years in field conditions, as reported by Ghosh et al. (2018). SB also enabled the development of one of the world's first wheat varieties with high protein milling features, DS Faraday (Schwager, 2017). Short‐day crops such as soybean, rice and amaranthus have also been optimized using SB protocols (Jähne et al., 2020; Rana et al., 2019).

A biotron speed‐breeding technique involving optimized photoperiod, day and night temperatures and humidity was used to introduce the salinity tolerance gene (hst1) in the japonica rice variety Yukinko‐ma (Rana et al., 2019). The resulting BC₃F₃ population was developed over 17 months. Its field performance was evaluated, with the plants exhibiting performance similar to their counterparts grown under field conditions after being shifted from SB conditions. However, this protocol involves tiller removal and embryo rescue, which may not be practical for large populations. Meanwhile, Jähne et al. (2020) developed an SB protocol for soybean that can accommodate up to five generations using optimized light spectrum, intensity and photoperiod. They also induced early flowering in two japonica rice varieties by using far‐red (FR) light, resulting in a 20‐day reduction in flowering time. However, despite early flowering in two japonica rice accessions, the remaining accession did not flower early under the same conditions and displayed genotype‐specific responses. The utilization of SB in glasshouses and controlled chambers has the potential to enhance breeding outputs for both short‐ and long‐day crops. This is achieved by providing more controlled growth conditions, which work more uniformly for diverse germplasm and breeding materials compared to field conditions. One example is the acceleration of flowering in some genotypes of rice and amaranth using different spectrum ratios and FR light. Moreover, a speed vernalization protocol for winter crops like wheat and barley that allowed five generations per year was developed using SB conditions (Cha et al., 2022). Recently, SB protocols with up to six generations were also optimized with different light conditions in the case of Cannabis sativa (Schilling et al., 2023) and in cassava (Rodrmguez et al., 2023).

The success of SB in various crops has led us to establish a state‐of‐the‐art SB facility (SpeedBreed), at the International Rice Research Institute, South Asia Regional Centre (ISARC), Varanasi, India. This facility aims to optimize the SB protocol for all types of rice varieties without the need for tedious embryo rescue techniques and tiller removal. The optimized protocol works for all the maturity durations of indica and japonica rice (early, medium and late) and enables synchronous flowering. Further validation of the developed protocol on a 3000 rice genomes project (3K RGP) panel representing different groups of Oryza sativa L. confirmed the results. The study emphasizes the importance of understanding plant physiological responses to photobiological entities such as light spectrums, light intensity, photoperiod, and other growth parameters such as temperature, humidity and foliar fertilizer management for successful SB. The research also provides insights into rice growth stages and the factors affecting early flowering and maturation. This can help develop stage‐specific interventions and comprehensive SB protocols in rice breeding. Overall, the SpeedBreed facility and protocol have the potential to significantly speed up rice breeding and improve the efficiency of the process, which could be extended across crops to enhance genetic gains.

Results

Establishment of SpeedBreed facility

The SpeedBreed facility is equipped with six optimization and two multiplication growth chambers that allow precise control of various growth parameters such as light spectrums, intensity, photoperiod, temperature, relative humidity, CO₂ levels, as well as an automated irrigation system (Table S1; Figure S1; Supplementary note). Full‐spectrum light contains four tuneable LED channels at 450 nm (blue), 660 nm (red), 735 nm (FR) and a white 5700 K LED channel, which are distributed uniformly using primary and secondary optic plates to achieve optimal light distribution over a growing area of 1.2 × 1.2 m (Figure S1a–c). The bench area was designed considering the light footprint, and the distance between the plant canopy and the light surface can be adjusted to control light intensity. Temperature is controlled using hot and cold air conditioning units (Figure S1d). The chamber's interior is made up of grade 316 stainless steel (SS) and exterior with polyurethane foam (PUF) panels to ensure precise control over humidity and temperature levels (Figure S1b). The chambers also feature perforated 316 SS floors that spread fresh air evenly throughout the chambers coming from air dampers and spreading all over the chamber through vertical airflow (Figure S1d–f). Ultrasonic humidifiers were used to maintain humidity levels precisely inside the chambers (Figure S1g). CO₂ is supplied through ducts that provide controlled CO₂ levels in each chamber. An automatic irrigation system provides sufficient amount of water to the plants and speeds up the generation time. All growth parameters are controlled by a customized climate control master (CCM) software (Figure S2), which integrates with light software helioCONNECT. The facility is equipped with UPS units and an electrical generator to ensure an uninterrupted power supply throughout the experiment.

Optimization of robust SB protocol (SpeedFlower)

We have conducted three distinct experiments to optimize the SB protocol. Each experiment focused on a different parameter while keeping other factors constant (Figure S3). First, we have studied the growth period of rice which consists of three distinct sequential growth stages: vegetative, reproductive and maturation. The vegetative growth stage is divided into the basic vegetative phase (BVP) and the photoperiod‐sensitive phase (PSP). The very young plants are insensitive to photoperiod in BVP. After the BVP, the plant enters the PSP, during which floral initiation can be triggered by short days. The optimum photoperiod in PSP for most varieties is about 9–10 h. The vegetative stage is also characterized by active tillering. The reproductive and maturation stages range from 30 to 35 days in most of the varieties. The reproductive phase starts with panicle primordia, followed by the emergence of a flag leaf, booting, heading and anthesis. Maturation stages include the milky, dough and maturation stages (Figure S4).

We examined the effects of varying light spectrum and intensity, photoperiod and early germination on flowering and maturity duration of rice (Figures 1, 2, 3, 4). By integrating the results obtained from the three experiments, we optimized a robust SB protocol called SpeedFlower (Table 1). SpeedFlower incorporates the findings from the three experiments involving different light spectrum/intensity, photoperiods and early germination conditions (Figures 1, 2, 3 and Figure S3). SpeedFlower proved to be successful across all durations of indica rice, spanning from early‐duration varieties such as CO‐51 and IR64 to medium‐duration varieties like Sarjoo‐52 and DRR Dhan 44, as well as late‐duration varieties including Swarna and Samba Mahsuri. The protocol has also been optimized for photosensitive landraces (Kalanamak, Black rice) of indica and two genotypes (Betagomblin and Gedonzipetan) of the japonica subspecies (Table 2; Figure 4k). To validate the efficacy of SpeedFlower, we conducted additional tests using 198 diverse genotypes representing all 12 groups of Oryza sativa L.

Figure 1.

Effect of spectrum and intensity on days to flowering. These graphs (a–j) illustrate the effects of six spectrum and light intensity combinations on the days to flowering of 10 different varieties. This experiment was performed using different ratios of the light spectrum and intensities while keeping photoperiod, temperature and humidity constant. The mean days to flowering were evaluated on the Y‐axis. While six different combinations of spectrum and intensity, viz., 2R > 1B and 400 PPFD; 2R > 1B and 800 PPFD; 2B > 1R and 400 PPFD; 2B > 1R and 800 PPFD; B = R and 400 PPFD; B = R and 800 PPFD, are plotted on the X‐axis using box plots. For the box plots, boxes denote the 25^th–75^th percentile, whiskers denote the full data range, cross symbols denote the mean and centre lines denote the median. The alphabets above the boxes (a, b and c) in panels (a–j) represent statistical significance between the groups of spectrum and intensity ratios computed using Tukey's test (P < 0.05).

Figure 2.

Mean days to flowering in response to different photoperiods under SB. These graphs (a–j) show how the initial long photoperiod (20, 22, or 24 h) for 6, 9, 12, or 15 days affected the length of flowering in five different groups (early, medium, late and landraces of indica and two genotypes of japonica). This study was conducted using different photoperiods while keeping spectrum (2R > 1B), intensity (800 μmol m⁻² s⁻¹), temperature (32/30 °C Day/night) and humidity (65%) constant. Out of 12 different combinations of long‐day photoperiod, all varieties almost flowered early within 60 days in the 24 h long‐day photoperiod for the initial 15 days. The days to flowering are shown on the Y‐axis. The alphabet D on the X‐axis designates the effect of the initial long‐day photoperiod for 6, 9, 12 and 15 days with a 20, 22 and 24 h photoperiod, respectively. Bar graphs represent the mean value of days to flowering. The alphabets above the bar graphs (a, b, c, d and e) in the panels (a–j) designate statistical significance between groups of photoperiod treatments computed using Tukey's test (P < 0.05).

Figure 3.

Early germination experiment to hasten the maturity period. (a) Panicles were tagged based on the percentage of anthesis. In rice, anthesis in the whole panicle requires approximately 1 week in the field and approximately 2 days in SB conditions. P1 was assigned a 50% anthesis observed panicle, and P2 was assigned to a 100% anthesis observed panicle. (b) Premature panicles were harvested 9, 11, 13 and 15 days after anthesis. (c) Harvested seeds were kept in an incubator for conversion from the milky to the maturation stage at 38 °C for 16–24 h. (d) Dry seeds were treated with 20, 40, 60 and 80 ppm of GA3 at 16–18 °C for 12 h. (e) GA3‐treated seeds were washed 2–3 times with distilled water and transferred onto blotting paper on a petri plate. (f) Petri plates were shifted into SB for a 10‐h photoperiod with a 2R > 1B ratio at 32 °C Day and night temperature. The results demonstrate >80% germination in seeds (at 13 and 15 days) treated with 80 ppm of GA3 in tested rice genotypes in 1 week.

Figure 4.

Overview of SB in rice. (a) The presented illustration shows the development of plants from the seedling stage to the maturation stage under an optimized SB protocol. (b) The illustration depicts the effect of varying intensities of the same spectrum on flowering duration. CO‐51 flowered early in the 800 PPFD or μmol m⁻² s⁻¹ compared to the 400 μmol m⁻² s⁻¹ of the 2R > 1B spectrum. (c) The picture illustrates the provision of an initial long day (24 h for 15 days) to short‐day photoperiod that induces early flowering in CO‐51 as compared to a glass house and a complete short‐day photoperiod (10 h of light). (d) The picture shows the difference between plant growth observed at two intensities (400 and 800 μmol m⁻² s⁻¹) with three different spectrums (2R > 1B, 2B > 1R and B = R) in Swarna. (e) The graph presents information about spectrums present in sunlight at a PPFD of 462 μmol m⁻² s⁻¹. (f–h) These graphs give information about the three major spectrums (2R > 1B, 2B > 1R, R = B) used for the optimization of the spectrum with yellow, green and far‐red light spectrum in the background (i) The presented illustration shows an optimized shade avoidance response (SAR) spectrum for the induction of flowering using a high FR > R > B > G > Y ratio at PPFD: 60 μmol m⁻² s⁻¹. This spectrum ratio was provided 15 days after sowing. (j) This graph shows the optimized end‐of‐the‐day (EOD) spectrum with only far‐red at PPFD: 30 μmol m⁻² s⁻¹ far‐red (FR) light (k) A statistical comparison shows the difference between mean days to flowering in the field and SB in 10 verities, which was used for protocol optimization in SB. Error bars, SE; statistical significance was determined by a two‐sided t‐test: ***P < 0.001.

Table 1.

Optimal growth conditions for the speed breeding protocol 'SpeedFlower' in rice.

Growth stages→			Vegetative phase↓		Reproduction phase↓	Maturation phase↓
			BVP	PSP
	Time of application↓	Duration of growth stage→	1–15 days	PSP and Reproductive phase (16–60 days)		Maturation Phase (up to 15 days from anthesis)
Photoperiod (day light)→			24 h	10 h	10 h	10 h
Light spectrum and intensity→	First spectrum and intensity of day		R > B > G > Y > FR, 800 PPFD (24 h)	R > B > G > Y > FR, 800 PPFD (9 h)	R > B > G > Y > FR, 800 PPFD (9 h)	R > B > G > Y > FR, 800 PPFD (9 h)
	Second spectrum and intensity of day		NA	FR > R > B > G > Y, 60 PPFD (30 min)	FR > R > B > G > Y, 60 PPFD (30 min)	FR > R > B > G > Y, 60 PPFD (30 min)
	EOD‐spectrum and intensity		NA	FR (30 min), 30 PPFD	FR (30 min), 30 PPFD	FR (30 min), 30 PPFD
Temperature→	Day		32–33 °C	32 °C	32 °C	30‐32 °C
	Night		30 °C	30 °C	30 °C	30 °C
Humidity→	Day and night		65%	65%	65%	65%

→, different arrows are showing different parameters; B, blue spectrum; BVP, Basic vegetative phase; EOD, end of the day; FR, far‐red spectrum; G, green spectrum; h, hours; NA, Not applicable in the case of a 24 h photoperiod; PPFD, PPFD measures light intensity within the PAR spectrum in micromoles per square meter per second (μmol m⁻² s⁻¹); PSP, Photoperiod sensitive phase; R, red spectrum; Y, yellow spectrum.

Table 2.

Comparing generation advancement time for rice: speed breeding vs. field conditions across all varietal durations.

Genotypes	Subspecies	Duration	DFF in field	Generation time in field †	DTF in SBF	Early flowering ‡	Maturation time in SBF	Generation in SBF	Generation reduction §	Generation per year ¶
CO‐51	Indica	Early	87.0	122.0	55.2	31.8	15.0	70.2	42.5	5.2
IR64			84.0	119.0	55.9	28.1	15.0	70.9	40.4	5.1
DRR Dhan 44		Medium	93.0	128.0	53.0	40.0	15.0	68.0	46.9	5.4
Sarjoo‐52			93.0	128.0	54.6	38.4	15.0	69.6	45.7	5.2
Swarna		Late	114.0	149.0	56.5	57.5	15.0	71.5	52.0	5.1
Samba Mahsuri			114.0	149.0	59.9	54.1	15.0	74.9	49.8	4.9
Black rice		Landrace (Late)	108.0	143.0	51.6	56.4	15.0	66.6	53.4	5.5
Kalanamak			130.0	165.0	52.6	77.4	15.0	67.6	59.0	5.4
Gedonzipetan	Japonica	Early	84.0	119.0	57.3	26.7	15.0	72.3	39.2	5.0
Betagomblin		Medium	91.0	126.0	52.4	38.6	15.0	67.4	46.5	5.4

DFF, days to 50% flowering; DTF, days to flowering; SBF, SB facility.

^† One generation time.

^‡ Early flowering in SB as compared to field.

^§ Percentage reduction in one generation time under SB as compared to field.

^¶ Generation per year in SB.

Ratios of spectrum and intensity affect the flowering duration

Different combinations of the light spectrum (Figure 4f–h) and intensity had varying effects on the flowering time of plants. The box plot analysis suggests that recorded flowering data are normally distributed. The groups of light treatments (spectrum and intensity) had a significant effect (P < 0.05) on reducing the mean flowering time among rice varieties as compared to the field or sunlight spectrum (Figure 1). For early‐duration varieties such as CO‐51 and IR64, early flowering was observed at 60 and 62 days in the SB facility with a spectrum ratio of 2R > 1B and an intensity of 800 μmol m⁻² s⁻¹, compared to 87 and 84 days, respectively, in the field (Figure 1a,b). Similarly, DRR Dhan 44 flowered early at 63 days in the SB facility with the same light combination, compared to 93 days in the field (Figure 1c). The medium‐duration variety, Sarjoo‐52, exhibited flowering between 60 and 64 days under all three spectrum ratios and at an intensity of 800 μmol m⁻² s⁻¹, compared to 93 days in the field (Figure 1d). Late‐duration varieties showed varying responses. Swarna exhibited early flowering at approximately 74–75 days under both 400 and 800 μmol m⁻² s⁻¹ intensities with a 2R > 1B spectrum ratio (Figures 1e and 4d). Samba Mahsuri, on the contrary, showed a slightly different response, with flowering occurring within a similar range of 66–70 days across the entire spectrum and at both low and high intensities (Figure 1f). Late‐duration varieties, such as Swarna and Samba Mahsuri, were found to be insensitive to intensity, as no significant difference was observed in inducing early flowering.

Landraces such as Black rice exhibit flowering at 49 days with a 2R > 1B spectrum ratio and an intensity of 800 μmol m⁻² s⁻¹. Under field conditions, Black rice took 108 days to flower (Figure 1g). Kalanamak, which typically takes over 130 days to flower, showed early flowering ranging from 76 to 81 days under all three spectrums at an intensity of 800 μmol m⁻² s⁻¹ (Figure 1h). Regarding japonica rice, Gedonzipetan, under the 2B > 1R/1R = 1B spectrum flowered at 69–72 days and in 76 days under 2R > 1B spectrum at both intensities (Figure 1i). Another japonica rice, Betagomblin exhibited comparative early flowering (58 days) under a 2R > 1B spectrum ratio at an 800 μmol m⁻² s⁻¹ intensity (Figure 1j). In field conditions, Gedonzipetan and Betagomblin took 84 and 91 days, respectively, to flower. Overall, the results suggest that a high red‐to‐blue spectrum ratio (2R > 1B) and high light intensity (800 μmol m⁻² s⁻¹) are optimal for growing a heterogeneous population in the SB facility.

Initial long‐day photoperiod controls plant growth and flowering duration

Different durations of a long photoperiod (20, 22 and 24 h) for the initial 6, 9, 12 and 15 days were found to influence flowering duration in rice. The analysis revealed a significant difference among treatment groups in terms of reducing the mean flowering time (P < 0.05) (Figure 2). Among the 12 combinations of photoperiods and day lengths tested (Figure S3), CO‐51 exhibited early flowering at 55 days when exposed to a 24‐h long‐day photoperiod provided either until the 12^th or 15^th day after sowing, followed by a constant 10 h photoperiod (Figures 2a and 4c). Like CO‐51, early flowering was observed at 53 and 55 days in a 24 h LD photoperiod for the initial 15 days in DRR Dhan 44 and Sarjoo‐52, respectively (Figure 2c,d). As for IR64, early flowering occurred in 50 days at a 22 h LD photoperiod for the initial 15 days, followed by 54–56 days in the remaining 10 combinations, except for a 20 h LD photoperiod for the initial 6 days (Figure 2b). Swarna showed early flowering between 55 and 56 days when exposed to both 22 and 24‐h long‐day photoperiods for the initial 15 days (Figure 2e). In the case of Samba Mahsuri, early flowering was observed at 58 days with a 20‐h long‐day photoperiod for the initial 9 and 15 days, while flowering occurred at 59–63 days in the remaining combinations, which is relatively longer compared to other varieties (Figure 2f). This experiment also investigated the induction of early flowering in the widely acclaimed late‐duration Basmati rice variety, Pusa Basmati 1121. This variety exhibited early flowering, ranging from 59.50 to 62.75 days, under the extended 20, 22 and 24‐h long‐day photoperiods during the initial 6, 9 and 12 days (Figure S8). Conversely, a minor delay in flowering was observed, occurring between 64 and 68.80 days under the extended 20‐h, 22‐h and 24‐h photoperiods during an initial 15‐day period. Notably, in this variety, an extended photoperiod of 15 days led to delayed panicle initiation and eventual flowering. In field conditions, Pusa Basmati 1121 required 105 days to reach the flowering stage (Table S6). Early flowering was observed in Kalanamak at 50–51 days with a 24 h LD photoperiod for the initial 9 and 12 days and similarly with a 20 h LD photoperiod for the initial 12 and 15 days (Figure 2h). It was fascinating to note that black rice flowered between 45 and 52 days in all possible combinations (Figure 2g). In the case of Betagomblin, early flowering was observed in 50 days at a 24 h LD photoperiod with an initial period of 12 days, followed by 52 days at the same photoperiod with an initial period of 15 days (Figure 2i). Unlike the above genotype, Gedonzipetan flowered between 56 and 61 days with all photoperiod and initial day treatments. Overall results suggest that a 24 h LD photoperiod for the initial 12–15 days is superior for inducing early flowering in a heterogeneous population in an SB facility (Figure 2a–j).

Initial long‐day photoperiods provide one‐two productive tillers per plant

To effectively utilize the SB facility for generation advancement in the breeding program, we optimized our protocol with the aim of producing not more than one productive tiller per plant during the initial days of plant growth. We examined the effects of different long‐day photoperiods (20, 22 and 24 h) on the number of productive tillers at various timepoints (15, 12, 9 and 6 days) (Table S2; Figure S5a–c). These photoperiod variations were combined with the previously optimized conditions of light spectrum, intensity, temperature and humidity. Our result revealed that the number of productive tillers increased as the duration of exposure to the photoperiod increased. Under the optimized SB conditions, the average number of tillers ranged from one to three. Based on the results, we have chosen 20 h photoperiod and 6 days of exposure to achieve one productive tiller per plant for generation advancement. The mean tiller numbers of data showed that the 20 h photoperiod provided for the initial 12 days showed two tillers in nine genotypes out of 10 (Table S2; Figure S5a–c). In contrast, the 22 and 24‐h photoperiods have on average less than two tillers on all 4 days (15, 12, 9 and 6) of initial LD photoperiod exposure.

Premature seed harvesting and hormonal treatment reduce maturity time

Minimizing the duration of maturity in rice crops can greatly reduce the overall generation time, as they typically take around 30–45 days to mature after flowering. To expedite the seed‐to‐seed duration, we treated prematurely harvested seeds (after 15, 13, 11 and 9 days of anthesis) with different concentrations of GA3 (ranging from 20, 40, 60 and 80 ppm). The germination performance of the selected genotypes (representing different maturity durations and landraces of indica and japonica) was initially low without GA3 treatment, as observed in the control group (Table S3). Among the different harvesting durations, the seeds harvested on the 15^th day exhibited the highest germination percentage across all GA3 concentrations, followed by those harvested on the 13^th, 11^th and 9^th days. The overall germination percentages varied from 12.5% to 100% across different GA3 concentrations and harvesting durations. Among the GA3 concentrations tested, 80 ppm was found to generate the best germination results for most harvested seed durations. In contrast, the lowest germination percentages were observed at a concentration of 20 ppm. We have also observed sub‐optimal levels of germination at 40 ppm (50%–100%) and 60 ppm of GA3 (65.5%–100%). Based on the results, we observed that the lowest concentration of GA3 treatment (60 ppm) triggers good germination within just 9 days of premature seed harvesting (Figure S6a–d). This optimized premature seed maturity and early germination approach significantly reduces the 50% time required for the maturity of seeds, demonstrating its efficacy in accelerating the breeding process.

SpeedFlower advances 4–5 generations of rice per year

After a comprehensive analysis of each parameter pertaining to early flowering and maturation duration, a final protocol, SpeedFlower (Figure 5) was formulated. SpeedFlower proved to be effective and rapid for all the tested varieties, allowing for accelerated generational advancement (Tables 1 and 2). Notably, SB‐grown plants exhibited a significant reduction in days to flowering compared to their field‐grown counterparts (P < 0.001) (Figure 4e,k; Figure S7; Table 2). SpeedFlower includes a 24‐h long‐day photoperiod for the initial 15 days with a superior 2R > 1B light spectrum with an intensity of 800 μmol m⁻² s⁻¹, followed by a short day (10 h) photoperiod until panicle harvesting. Throughout the experiments, temperatures (32/30 °C, day and night) and humidity (65%) were kept constant. After flowering, premature seeds are harvested between 13 and 15 days after anthesis, treated with 80 ppm GA3 and demonstrate germination rates exceeding 85% (Figure 3; Table S3). SpeedFlower shortens the vegetative phase, reduces flowering duration and hastens the maturity period by harvesting premature seeds. The premature seeds germinated and the next generation was advanced with an early germination experiment. SpeedFlower was also tested in landraces and japonica rice accessions, and accelerated flowering was observed. We developed SpeedFlower intending to finish each rice generation within a time frame of 67–75 days, which includes a 15‐day maturity period. SpeedFlower has enabled 4.9–5.5 generations across all tested rice varieties with varying maturity durations (early, medium and late), including landraces, indica and japonica rice (Table 2). The protocol produced a range of 24–83 seeds per panicle (Table S4).

Figure 5.

Optimization of speed breeding protocol ‘SpeedFlower’ for indica and japonica rice. (a) Selection of Growth Parameters: We carefully selected various growth parameters aligned with rice's physiological and growth requirements to expedite growth and flowering. Enclosed chambers were established to provide short‐day conditions during the reproductive phase, considering rice's short‐day plant nature. (b) Selection of diverse rice varieties: Multiple durations of indica (early, medium, late and landraces) and japonica (early and medium) rice varieties were considered to refine the ‘SpeedFlower’ protocol for rice, ensuring its adaptability across all Oryza sativa L. types. (c) Optimization of spectrum and intensity: Three spectrum ratios (R > B, B > R and R = B) were optimized in conjunction with two light intensities (400 and 800 μmol m⁻² s⁻¹). In this experiment, in continuation of these three spectrum ratios, supplementary SAR and EOD far‐red light spectrums were provided for the induction of early flowering in rice. The experiments demonstrated that an R > B spectrum ratio with supplementary SAR and EOD far spectrum, along with 800 μmol m⁻² s⁻¹ light intensity, facilitated the induction of early flowering in rice. (d) Optimization of photoperiod: Three distinct photoperiods (20, 22 and 24 h) were implemented for 6, 9, 12 and 15 days to examine the impact of extended photoperiod on rice. Among these treatments, a 24 h photoperiod for the initial 15 days significantly accelerated the flowering duration. (e) Optimization of growth stage‐specific foliar spray to accelerate flowering: In the vegetative stage, foliar sprays containing urea, N:P:K, micronutrients, CaNO₃ and MgSO₄ were applied based on fertilizer deficiency symptoms. During the reproductive stage, foliar sprays comprising N:P:K, micronutrients, CaNO₃ and MgSO4 were used to expedite flowering. In the absence of nitrogen deficiency, N:P:K was used instead of urea to prevent delays in panicle and flowering initiation. During the maturation stage, foliar sprays containing P, K, CaNO₃ and micronutrients with iron and boron were utilized to promote seed setting and early seed development. (f) Early germination experiment to reduce maturity duration: Immature seeds harvested at various days (9, 11, 13 and 15 days) after flowering were incubated at 38 °C to transition from the milky or dough stage to the maturation stage. Different concentrations of GA3 (20, 40, 60 and 80 ppm) were employed to assess germination in the immature harvested seeds. (g) Using a–f experiments, ‘SpeedFlower’ was optimized. (h) Out of 198, 157 are indica (ind1A, ind1B, ind2, ind3 and indx), 19 are japonica (subtrop, temp, trop and japx), 14 are aus, 6 are aromatic (subpopulation aro) and 2 are admix were used to validate the ‘SpeedFlower’ protocol and observed flowering in all genotypes under 60 days.

Validation of SpeedFlower in 3K RGP subset of rice

In order to validate SpeedFlower, a subset of 198 genotypes from the 3K RGP was chosen based on their molecular diversity, different flowering durations and geographic locations. In field conditions, the flowering time of these genotypes ranged from 58 to 127 days. However, when grown under the optimized SpeedFlower, all 198 genotypes successfully flowered within a shorter period of 58 days. These genotypes encompassed all 12 distinct groups of Oryza sativa L. classes, ensuring representation of the entire rice population (Table S5). Out of 198, 157 are indica (ind1A, ind1B, ind2, ind3 and indx), 19 japonica (subtrop, temp, trop and japx), 14 aus, 6 aromatic (subpopulation aro) and 2 admix. The average flowering times of genotypes grown under SpeedFlower varied across different groups. Admix genotypes took around 51.5–54.5 days, equivalent to 5.3–5.5 generations/year, while aus genotypes ranged from 46 to 56 days (5.1–6 generations/year). Aro genotypes showed flowering times between 46 and 54.5 days (5.3–6 generations/year), and japx genotypes exhibited flowering at around 51 days (5.5 generations/year). Subtrop genotypes took 51–56.5 days (5.1–5.5 generations/year), while temp genotypes ranged from 47.5 to 54 days (5.3–5.8 generations/year). Trop genotypes showed flowering times between 48 and 56.5 days (5.1–5.8 generations/year) and ind1A genotypes took 43–57 days (5.1–6.3 generations). Ind1B genotypes ranged from 48 to 55 days (5.2–5.8 generations/year), ind2 genotypes from 45 to 56 days (5.1–6.1 generations/year), ind3 genotypes from 43 to 56 days (5.1–6.3 generations/year) and indx genotypes from 44 to 55.5 days (5.2–6.2 generations/year). Overall, all the groups completed one generation within a timeframe of 58–71 days (Table S5). These variations in flowering times across the different groups highlight the diverse genetic and physiological characteristics observed within each group and the corresponding generation cycles achieved under SpeedFlower.

Discussion

Developing an effective SB protocol for any crop requires a comprehensive understanding of its physiological growth stages and the application of stage‐specific growth parameters. Like other crops, rice has three main growth stages: vegetative, reproductive and grain filling (maturation), which take 3–6 months to complete a generation. In different rice varieties, the vegetative stage lasts 30–90 days, while the reproductive and maturation stages last 30–35 days (Yoshida, 1981). These differences in vegetative stage define the duration of rice and are responsible for longer generation times in rice. To accelerate rice generation advancement and breeding, each growth stage must be shortened and all varieties must flower at almost the same time. Along with an understanding of physiological interventions, precise environmental conditions are also important for generational advancement.

Achieving rapid generation advancements in SB requires not only a deep understanding of physiological interventions but also the careful management of precise environmental conditions and optimal timing to leverage the plasticity of plants in inducing early flowering. Although there are numerous reports on SB protocols for long‐day crops (Cha et al., 2022; Ghosh et al., 2018; Watson et al., 2018), research on short‐day crops is limited. For short‐day plants, protocols have been optimized for crops such as soybean, amaranth and specific japonica subspecies of rice. In contrast to SB in long‐day plants, these optimizations involve growth stage‐specific interventions by utilizing specific ratios of growth parameters to achieve rapid growth, early flowering and maturation (Jähne et al., 2020; Rana et al., 2019).

Light, including its spectrum, intensity and photoperiod, is the primary energy source, playing a critical role in determining plant growth rates. Our study found that a high red‐to‐blue (2R > 1B) spectrum ratio and a light intensity of 800 μmol m⁻² s⁻¹ were the most effective in inducing early flowering across most rice varieties. Additionally, we observed that a high blue‐to‐red ratio (2B > 1R) at the same intensity slightly outperformed an equal ratio of blue and red (B = R) in terms of inducing early flowering. These findings highlight the importance of specific spectral ratios and light intensities in optimizing the SB process. Previous studies also suggest that the most effective and efficient wavelengths for driving photosynthesis occur in the red region of the spectrum (600–700 nm), followed by blue (400–500 nm) and green (500–600 nm) (Evans, 1987; Inada, 1976; Muneer et al., 2014).

In our current study, when we refer to a high red‐to‐blue spectrum ratio (2R > 1B), it should not be interpreted as the exclusion of other spectrums. In fact, we provided the green, yellow and FR spectrums in decreasing order along with the red and blue spectrums. The combined spectrum ratios of red, blue, green, yellow and FR were considered to determine the induction of flowering duration (Table 1; Figures 1 and 4). This comprehensive approach to providing multiple spectral combinations allows us to assess the overall impact of different spectral combinations on flowering responses. We also observed that the provision of a high shade avoidance ratio (a higher FR‐to‐other‐spectrum ratio) for 30 min (Figure 4i), together with exclusive FR light for 30 min at the end‐of‐the‐day (EOD), contributed to the induction of early flowering (Figure 4j; Table 1). These findings are consistent with a study conducted on Eustoma plants, which demonstrated that providing FR light for 3 h at the end‐of‐the‐day resulted in early flowering, longer main stems and higher node numbers compared to untreated plants (Takemura et al., 2014). Similarly, Jähne et al. (2020) also observed that a FR‐enriched spectrum induced early flowering in two japonica rice genotypes.

Our experimental results also demonstrated that among the two light intensities tested (400 and 800 μmol m⁻² s⁻¹), the higher intensity of 800 μmol m⁻² s⁻¹ significantly enhanced flowering induction across the different spectrum combinations (Figure 1). This indicates that a higher/optimal light intensity is essential for promoting photosynthesis and overall plant growth, leading to early flowering. Our study revealed genotype‐level differences in response to different light intensities. Early and medium‐duration varieties exhibited distinct flowering responses, while late‐duration varieties displayed a similar flowering pattern under high light intensity (800 μmol m⁻² s⁻¹) across all spectrums. This suggests that flowering in late‐duration varieties is more influenced by light intensity than spectrum. Light intensity is also known as photosynthetic photon flux density (PPFD), and the unit is μmol m⁻² s⁻¹ (Kusuma et al., 2021). PPFD is universally used to quantify light intensity in the region of photosynthetic active radiation (PAR) in the visible spectrum (400–700 nm) of light. In previous studies, the terms ‘photosynthetic photon flux’ (PPF) and ‘photosynthetic photon flux density’ (PPFD) were utilized interchangeably. However, the presence of redundancy denoted by ‘area’ in PPF restricts its applicability. Furthermore, photosynthetic flux density (PFD) measures light intensity in the region from 350 nm to 780 nm, including UV light and far‐red light (Zhen and Bugbee, 2020). Light intensity, including PAR (400–700 nm) and the far‐red light (701–750 nm) region, is considered an extended PPFD (ePPFD). The daily light integral (DLI) is a measure of the amount of PAR received in a specific area over a 24‐h period. It is typically expressed in units like mol/m²/day. DLI is calculated by multiplying the average PAR intensity in μmol m⁻² s⁻¹ by the number of seconds in a day (86 400 s) (Faust et al., 2005; Ficht et al., 2023). In the SpeedFlower protocol, a 24‐h photoperiod and a light intensity of 800 μmol m⁻² s⁻¹ were employed for the initial 15 days, yielding a DLI of 69.12 mol m⁻² d⁻¹ per day. Subsequently, during the reproductive phase in the second photoperiod, a light intensity of 800 μmol m⁻² s⁻¹ was utilized for a duration of 9 h, resulting in a DLI of 25.92 mol m⁻²d⁻¹ per day. In this second photoperiod, the light intensity of 800 μmol m⁻² s⁻¹ of R > B spectrum was maintained for 9 h.

Among the various parameters investigated in our study, photoperiod emerged as a critical factor for achieving synchronized and early flowering. Previous research on SB protocols has emphasized the significance of extended photoperiods in promoting early flowering in both short‐day and long‐day plants (Cha et al., 2022; Ghosh et al., 2018; Rana et al., 2019; Watson et al., 2018). However, in the case of rice, it becomes critical to determine the appropriate duration of the long photoperiod provision stage, considering that a short day is required for inducing early flowering. This necessitates an understanding of the photoperiod‐insensitive and photoperiod‐sensitive phases. The vegetative stage in rice is divided into the basic vegetative phase (BVP) and the photoperiod‐sensitive phase (PSP). The BVP is photoperiod‐insensitive, as is the PSP, which requires a short photoperiod for panicle initiation of flowering (Yoshida, 1981). To identify the duration of the BVP, we exposed the plants to long‐day photoperiods of 20 h, 22 h and 24 h for the initial 6, 9, 12 and 15 days, followed by an immediate shift to short‐day conditions. By testing these 12 combinations (Figure 2), our study demonstrated that providing long daylight during the BVP significantly influenced the number of tillers and flowering in nearly all rice varieties (Figure 2, Figure S5). Importantly, our optimized parameters also yielded positive results in a strongly photoperiod‐sensitive variety, Kalanamak, which typically takes 130 days to flower in the field but only 60 days in the SB facility (Table 2). The late duration Basmati rice variety, Pusa Basmati 1121, demonstrated an acceleration of flowering onset, manifesting at approximately 59.50–62.75 days when exposed to extended photoperiods of 20, 22 and 24 h of light during the initial 6, 9 and 12 days (Table S6). Conversely, a marginal delay in the flowering induction, ranging between 64 and 68.80 days, was observed when subjected to prolonged photoperiods during the initial 15‐day period. Our study suggests that photosensitive genotypes exhibit a strong response to optimized growth parameters, followed by late, medium and early duration genotypes, respectively.

Overall, exposing the plants to a 24‐h light period for the initial 12–15 days proved beneficial in terms of plant growth and tillering ability. These findings underscore the importance of manipulating photoperiod duration during specific vegetative stages to achieve efficient and accelerated flowering in rice. Additionally, gaining precise knowledge about the transition from the photoperiod‐insensitive to the photoperiod‐sensitive phase can inform the provision of extended daylight exposure, leading to significant time savings in SB protocols.

Temperature plays a crucial role in regulating plant growth, particularly during the flowering and grain‐filling stages. The interaction between photoperiod signalling pathways and temperature has been well‐documented (Halliday et al., 2003). For rice, the optimal temperature range for growth and maturation is typically around 30–32 °C. Heading date and yield traits in rice production are strongly influenced by temperature variations. Higher temperatures can lead to early heading and yield losses (Yoshida, 1981). In our study, we maintained a consistently high temperature of 32/30 °C (day/night) throughout the experiments, promoting robust plant growth and early flowering under different treatment conditions. To create favourable growth conditions, we also maintained a humidity level of 65% throughout the plant's growth stages, striking a balance between temperature and humidity. However, it is worth noting that while the optimized temperature regime facilitated early flowering, we observed the presence of both filled and chaffy grains in the panicles (Table S4). This suggests that while the optimized temperature and other parameters promoted early flowering, there was an impact on seed development and grain quality.

Nutrients are crucial for plant growth, flowering and maturation. In our study, we found that the controlled application of nutrients (macronutrients and micronutrients) had a significant impact on the timing of flowering. Specifically, we observed that restricting nitrogen (N) application after the booting stage and ensuring sufficient foliar application of phosphorus (P) and potassium (K) at a concentration of 2 g/L promoted early flowering in rice. These nutrients are known to play essential roles in plant development, including flowering induction. Conversely, we also observed negative effects associated with nutrient deficiencies and imbalances. In the cases of low water availability, high temperatures and inadequate fertilizer application, we observed deficiencies in calcium, nitrogen and zinc, as well as symptoms such as panicle apical spikelet abortion, leaf tip burning and stunted growth. These nutrient deficiencies and imbalances can adversely affect plant health and development, including reproductive processes.

The previous SB experiments have demonstrated the effectiveness of premature seed harvesting and hormonal treatments in reducing seed maturation time and promoting early germination (Watson et al., 2018). Non‐deep physiological dormancy is caused by physiological factors in the embryo and surrounding layers and is common in crops such as rice. Treatments such as GA3, scarification, stratification, or dry storage can be utilized to break this dormancy (Atwell et al., 1999; Linkies et al., 2010; Willis et al., 2014). In our study, prematurely harvested seeds at 13 and 15 days after anthesis exhibited a germination potential of over 80% after treatment with 80 ppm of GA3 at 16–18 °C. This finding highlights the significance of premature seed harvesting and the effectiveness of hormonal treatment using GA3 in promoting germination in rice seeds. By harvesting premature seeds, we can potentially reduce the seed maturation time and facilitate early germination, which could have significant implications for crop management and production.

Our optimized protocol, SpeedFlower, demonstrates a remarkable impact of SB on crop research. With SpeedFlower, we can expedite crossing and inbreeding activities, completing them within 1.5–2 years instead of the usual 6–7 years required in the field (Figure S7). To check the universality of SpeedFlower, 198 diverse genotypes from 12 groups of Oryza sativa L. were grown, and all genotypes flowered within 58 days. Therefore, SpeedFlower has universal applicability. This substantial reduction in breeding time offers tremendous benefits in rice breeding. By incorporating SpeedFlower into breeding programs such as the development of mapping populations, genomic selection and genome editing, we can achieve a two‐fold increase in genetic gain, reducing the time required for line fixation by over half. Additionally, we can use SB conditions to phenotype traits that are stable in such environments and targeted field conditions. For instance, we can combine SpeedFlower with phenotyping plants for disease and pest resistance, as demonstrated in different studies (Dinglasan et al., 2022; Riaz and Hickey, 2017). Integration of SB and genomic selection into rice breeding programs can address the time‐consuming process of trait pyramiding, the limitations of breeding less heritable traits and the dearth of high‐value genes and donors.

The SB technique is still in its early stages, and there are both advantages and disadvantages associated with its implementation that require further research. One important aspect to consider is the impact of stress on plants, which can induce various changes, including epigenetic modifications. This raises the intriguing possibility that plants can acquire adaptive traits through these modifications, which can be passed on to their offspring. Understanding how plants adapt to stress is crucial for the development of resilient crop varieties that can thrive in challenging growing conditions. In summary, SB has the potential to make a significant impact on global food security by rapidly developing improved rice varieties. This technique can play a crucial role in feeding a large proportion of the world's rice‐dependent population by accelerating genetic gains in the breeding process.

Conclusion

In conclusion, establishing a fully controlled SB facility with an optimized comprehensive protocol, SpeedFlower for indica as well as japonica rice offers a promising solution for addressing the limitations of longer generation times and seasonal constraints. The reduction in generation times by almost half can result in fasten the varietal release process ultimately increase in genetic gain. Therefore, SpeedFlower can enable the development of new high‐yielding rice varieties in a much shorter duration, ultimately contributing to global food security.

Materials and methods

Establishment of SB facility

To achieve SB in rice, a speed breeding facility (SpeedBreed) has been customized with controlled growth parameters using fully enclosed walk‐in growth chambers (Table S1; Figure S1; Supplementary note).

Plant material

An SB protocol was developed to rapidly advance rice generations using early (CO‐51 and IR64), medium (Sarjoo‐52 and DRR Dhan 44) and late (Swarna and Samba Mahsuri) duration varieties and landraces (Kalanamak and Black rice) of indica subspecies. Two genotypes (Betagomblin and Gedonzipetan) of the japonica rice subspecies were also used to optimize the SB protocol. These materials were utilised in all four optimization experiments at the SB facility (Figure S3). To validate the current optimized SB protocol, 198 genotypes from 3K RGP were selected to test the robustness of the current protocol (Table S5). Fresh seeds of all varieties were taken from the ISARC germplasm and kept in an incubator at 40–45 °C for 48 h to break the seed dormancy prior to sowing.

Rice growing methods under SB conditions

Ebb and flow (flood and drain) trays were installed beneath the lights in the facility chambers to grow plants in pots. These benches were equipped with automatic irrigation features. Pots (250 mL volume). were filled with autoclaved soil (121 °C for 15–30 min at 15 psi) and decomposed farmyard manure (FYM) in a 3:1 mixture. Two seeds were placed on the wet soil surface of each pot and completely covered with a fine layer of soil to germinate quickly. The same method of pot preparation and sowing was used for all experiments performed at the SB facility.

Agronomic practices for growing rice under SB

At the SB facility, three separate experiments were conducted to optimize stage‐specific plant growth parameters and agronomic practices in different optimization chambers (Figure S3). Foliar sprays of nitrogen, phosphorus, potassium, Ca(NO₃)₂ and MgSO₄ were used to meet macronutrient requirements, along with micronutrients (iron, manganese, boron, molybdenum, copper and zinc). All fertilizers were applied under SB through the foliar spray (2 g/L) in accordance with plant visual observations and deficiency symptoms. The use of nitrogen was limited before the booting stage to prevent a delay in flowering. To shorten the maturity period after anthesis, fertilizer use was discontinued and irrigation was gradually reduced. Reverse osmosis (RO) water was used for spraying fertilizer to increase the efficiency of externally applied fertilizers for plants. Also, RO water was used for irrigation in the tray. A completely randomized design (CRD) was used to arrange pots of different varieties on the bench to ensure that each experimental unit an equal chance of receiving treatment in the SB chambers. In each experiment, five pots of each rice variety with 15 plants were selected for each treatment (Figure S1b).

Optimization of the speed breeding protocol in rice

Optimization of spectrum and intensity for early flowering induction

In the first experiment, three light spectrum combinations (2R > 1B, 2B > 1R and 1R = 1B) and two light intensities (400 and 800 μmol m⁻² s⁻¹) were used to determine the best combination of spectrum and intensities for inducing early flowering. This led to six combinations of light spectrum and intensity. While providing these three major spectrums, green, yellow and FR light spectrums were used in the background. The light spectrum (2R > 1B) along with the SAR (30 min) and EOD FR spectrum (30 min) was provided after 15 days of sowing. Day and night temperatures (32/30 °C) and humidity (65%) were kept constant throughout the experiments (Figure S3).

Optimization of photoperiod to achieve early flowering and different tiller numbers

A second experiment was conducted to see the effect of different photoperiods on early flowering induction. Plants were grown at different photoperiods, viz., 20, 22 and 24 h, for the initial 6, 9, 12 and 15 days at the basic vegetative phase (BVP), followed by shifting into short‐day conditions (10 h) in different growth chambers. This led to 12 combinations of growth conditions (long‐day photoperiod and initial days of long‐day exposure) (Figure S3). The superior spectrum and intensity from the first experiment were chosen for this experiment. The light spectrum (2R > 1B) along with the SAR (30 min) and EOD FR spectrum (30 min), intensity (800 μmol m⁻² s⁻¹), temperature (32/30 °C Day/night) and humidity (65%) were kept constant throughout the experiment. Treatment of SAR and EOD FR spectrum ratios was initiated after 15 days of an initial long photoperiod with a 10‐h photoperiod. This experiment also revealed variation in tiller number in conjunction with early flowering.

Early germination in prematurely harvested seeds using GA3

The third experiment identified the superior GA3 concentration responsible for above 80% germination in prematurely harvested seeds. Rice panicles were tagged on the day of anthesis. Under SB conditions, flowers were tagged on the day of anthesis. Panicles were harvested on days 9, 11, 13 and 15 after anthesis. Harvested panicles were kept in the incubator at 38 °C for 24 h for drying and to attain the suitable moisture (approximately 14%–16%) required for germination. Then chaffy seeds were separated from the normal seeds (filled grains). For GA3 treatment, seeds of selected rice varieties (one each from all groups of indica and japonica) were taken for germination studies. The seeds were kept in different petri plates for 12 h with four GA3 concentrations (20, 40, 60 and 80 ppm) at 18–22 °C, and the control was without hormone treatment. Then the seeds were washed 4–5 times with distilled water and kept in a petri dish containing wet blotting or germinating paper. Then petri plates were transferred in SB conditions at a complete short‐day photoperiod (10 h) with a 2R > 1B spectrum, 800 μmol m⁻² s⁻¹ light intensity, 32 °C day and night temperatures and 65%–70% humidity. Moisture levels in petri dishes were maintained up to germination with distilled water. The germination percentage was recorded for only 7 days. Then the seedlings were shifted, with 1 inch of each plumule and radicle, into the soil in new pots containing soil and FYI (3:1 ratio) for the second generation.

A robust speed breeding protocol (SpeedFlower) for indica and japonica rice

All optimized superior parameters from experiments one to three were combined in the fourth experiment to establish SpeedFlower. This includes a 24‐h initial photoperiod for 15 days, later 10‐h photoperiods, the 2R > 1B spectrum along with the SAR and EOD spectrum, 800 μmol m⁻² s⁻¹ intensity and premature seed harvest on 15 days with 60 and 80 ppm of GA3. In all four experiments, the same day and night temperatures (32 and 30 °C) and humidity (65%) were used.

Validation of the optimized SpeedFlower in 3K RGP subset of rice

A subset from the 3K RGP was selected to validate the optimized SpeedFlower in the SB facility. For this study, 198 random genotypes were taken from a 3K RGP panel and grown in an optimized SB protocol (Table S5). Using CRD, two replications of each genotype were grown. These entries represent all 12 groups of Oryza sativa L. This material was advanced using SpeedFlower, the harvesting of premature seeds on the 15^th day and 80 ppm GA3 treatments.

Author contributions

VKS, PS and UMS conceived the idea and supervised the study. VKS, A Kumar, UMS, PS, PGK, SA and SS helped in the establishment of the SpeedBreed facility. PGK, SD and UMS performed the experiment. PGK, UMS, VKS and PS interpreted the results and wrote the first draft of the manuscript. S Bhosale, S Kumar, SKS, SA, JB, NRG, SC, RS, SKP, S Banerjee, RD, SPS, S Kalia, TRS, A Kohli, HB and A Kumar commented on the manuscript and edited the MS. All authors read and approved the final manuscript.

Funding information

Authors are thankful to the Department of Biotechnology (DBT), Government of India, under the project ‘Development of superior haplotype based near isogenic lines (Haplo‐NILs) for enhanced genetic gain in rice’ (BT/PR32853/AGill/103/1159/2019). We are also thankful to the Ministry of Agriculture and Farmers' Welfare, Government of India, Indian Council of Agricultural Research (ICAR), Government of India and Bill and Melinda Gates Foundation (BMGF) for providing additional support.

Supporting information

Figure S1 Component of the SpeedBreed facility. (a) SpeedBreed facility. (b) Different optimization chambers containing full spectrum LED lights with greed systems, stainless steel (SS) interiors, an ebb and flow bench for automated irrigation and temperature and humidity sensors. (c) Multiplication chambers with the same specifications as optimization chambers but larger in size. (d) Air conditioning (hot and cold) with dampers to allow the entry of fresh air into the chambers. (e) A perforated stainless‐steel floor for the vertical direction of air conditioning air. (f) A return air system to circulate air uniformly in the chambers. (g) Ultrasonic humidifiers that produce an extremely fine mist with a micron‐sized diameter at a high frequency. The sound vibrations impel moisture into the air to maintain uniform humidity across the chambers.

Figure S2 CCM software settings to attain photoperiod, temperature, humidity and CO₂. This software provides a maximum of eight growth condition schedules in 1 day. These eight schedules run under the line section in this figure; under the HR:MIN section, we provided different timelines from 0 to 24 h in a day to achieve the required photoperiod (for example, 10 h, 20 h, 22 h and 24 h photoperiods used in the current study). These timelines follow the time set in the CCM software. Under the °C section, we attained the temperature requirement according to the day and night photoperiods. Under the Rh% (relative humidity) section, we provided the required relative humidity. This software maintains the CO₂ requirement. This software is integrated with the light software helioCONNECT and can be adjusted by clicking on the right arrow menu of light by opening the helioCONNCET software. This software also provides settings to run one particular program for 15 days. Under control menu, we can change the settings of each parameter, like temperature (ramping settings for increase or decrease temperature), Rh% etc. This CCM software can automatically record parameter data for 6 month.

Figure S3 Workflow of the SB protocol. This diagram depicts the steps involved in optimizing the SB protocol in rice. In this study, five different groups of varieties were selected, including early, medium, late and landraces of indica and two genotypes of japonica. The first experiment was designed to optimize a suitable ratio of spectrum and intensity using other parameters as constants. In the second experiment, photoperiod was optimized using the superior ratio of spectrum and intensity from the first experiment while keeping other parameters constant. The third experiment was designed to reduce maturity duration by harvesting premature seeds and promoting early germination using the GA3 treatment. These three experiments allowed for the standardization of SB protocols for all durations of rice varieties, combining all optimized growth parameters. Thus, this study allowed for 4–5 generations of rice using standardized spectrum, intensity, photoperiod, temperature, humidity and early harvesting of premature seeds.

Figure S4 Rice growth stages. During the growth period, rice completes basically three distinct sequential growth stages: vegetative, reproductive and maturation. The vegetative growth stage is divided into the basic vegetative phase (BVP) and the photoperiod‐sensitive phase (PSP). The very young plants are insensitive to photoperiod in BVP. After the BVP, the plant enters the PSP, during which floral initiation can be triggered by short days. The optimum photoperiod in PSP for most varieties is about 9–10 h. The vegetative stage is also characterized by active tillering. The reproductive and maturation stages range from 30 to 35 days in most of the varieties. The reproductive phase starts with panicle primordia, followed by the emergence of a flag leaf, booting, heading and anthesis. Maturation stages include the milky, dough and maturation stages. Under SB conditions, growth manipulations were mainly done at BVP (providing a 24 h photoperiod) and early harvesting of premature seeds, along with treatment of GA3 to promote early germination. Additionally, all remaining optimized parameters were the same from sowing to harvesting. We also observed different numbers of tillers in different long‐day photoperiods.

Figure S5 Effects of long‐day photoperiods and initial long‐day exposure on productive tillers. This figure illustrates the influence of different photoperiods (20, 22 and 24 h) and exposure durations (15, 12, 9 and 6 days) on the production of number of productive tillers in the ten genotypes selected in the present study. The values presented are the mean ± s.d.

Figure S6 Effect of the application of gibberellic acid‐3 (GA3). Seeds were treated with 0 (control), 20, 40, 60 and 80 ppm of GA3 on premature seeds harvested after 15, 13, 11 and 9 days in rice. Values are mean ± s.d.

Figure S7 Overview of comparison of field and SB protocol. This diagram depicts the difference in growth conditions and stage‐specific responses of rice plants between field and SB conditions. In general, rice flowering duration is highly variable, with most varieties flowering between 90 and 120 days due to a longer vegetative stage duration in the field. However, all varieties have a reproduction and maturation period between 30 and 35 days. In contrast, under SB, all varieties flowered under 60 days and matured in 13–15 days, irrespective of their durations, using stage‐specific optimized photoperiod, spectrum, intensity, temperature, humidity and early germination experiments. Early flowering has been successfully induced using a long‐day photoperiod (24 h) for the initial 15 days of the vegetative phase (basic vegetative phase) and then immediately shifting to short‐day conditions. Under SB, all nutrients were managed through foliar spraying. By integrating the optimal growth conditions, it is possible to advance four to five generations of rice (all durations) instead of one to two generations in the field.

Figure S8 Effect of initial longer photoperiod on flowering duration of Pusa Basmati 1121. This figure illustrates the early flowering observed in Pusa Basmati 1121, with an extended photoperiod of 20 h (61.20 days), 22 h (60.75 days) and 24 h (60.80 days) for the initial 12 days.

Table S1 Specifications of an SB Facility (SpeedBreed). AHU, air handling unit; CCM, climate control master; cfc, chlorofluorocarbon; CFM, cubic feet per minute; dx coil, direct expansion coil; EC, electrical conductivity; HMI, human machine interface; K, kelvin; KW, kilowatt; LED, light emitted diode; LPH, litre per hour; m, meter; MCs, multiplication chambers; mm, millimetre; nm, nanometre; OCs, optimisation chambers; PLC, programmable logic controller; PPGI, pre‐painted galvanized iron; PUF, polyurethane foam; SS, stainless steel; TR, ton of refrigeration.

Table S2 Mean tiller numbers under different initial long‐day photoperiods in SB. D, days; h, hours; All data represents the means of tiller numbers under different photoperiods.

Table S3 Germination percentage in prematurely harvested seeds using early germination experiments. The mean germination percentage was recorded within 7 days of the period after keeping seeds in a petri dish for germination. Late germinated seeds were not considered in this study. This study was performed on six representative varieties from each of the five groups (early, medium, late and landraces of indica, and two genotypes of japonica) of varieties used in the protocol optimization study. This study was performed under 10 h of photoperiod, a 2R > 1B ratio of the spectrum, 800 PPFD, 32/30 °C day and night temperatures and 65% humidity in SB chambers. A zero (0) value in percentage shows that germination was not observed for up to 7 days.

Table S4 Number of seeds per panicle in the optimized SB protocol. LD, long day; Values are mean ± s.d.

Table S5 Validation of the SB protocol in all groups of Oryza Sativa L.

Table S6 Generation advancement time in speed breeding and field for Pusa Basmati 1121. D, days; DFF, days to 50% flowering in the field; DTF, mean days to flowering under SB; h, hours; SBF, SB facility. ^aEarly flowering in days in speed breeding as compared to field; ^bOne generation time under SBF; ^cGeneration per year in speed breeding.

Data availability statement

Data supporting the findings of this work are available within the paper and its supplemental items. The genetic materials that support the findings of this study are available at ISARC, Varanasi, India, and can be obtained from the corresponding authors upon request.