Speed Breeding Scheme of Hot Pepper through Light Environment Modification

Abstract

Crop breeding for high yields and quality is an important measure to ensure food security. In conventional breeding, a long generation time is required. Speed breeding could accelerate the flowering and fruiting of crops by providing suitable environmental conditions in order to reduce the generation times. This study aimed to determine a speed breeding scheme for hot peppers. Two hot pepper varieties, ‘Xiangyan 55’ and ‘Xiangla 712’, were investigated for their growth and development under different light intensities, photoperiods, and red-to-far-red ratios. Hot pepper plants bloomed at 39.88 ± 0.74 days after sowing under photosynthetic photon flux density (PPFD) 420 µmol·m⁻²·s⁻¹ and a 12-h photoperiod and had seed with acceptable germination rates at 82 days after sowing. Blooming was 2–3 days earlier when the photoperiod was extended to 20 h, but the fruit and seed development were not significantly improved. Supplementation of far-red light (R:FR = 2.1) significantly accelerated the red ripening of pepper fruit and improved seed germination rates. The modification of the light environment accelerated hot pepper growth and development, reduced breeding cycles, and could produce up to four generations per year.

1. Introduction

Developing a crop variety requires a long generation time because it takes time for homozygous lines to form after hybridization. If only one generation of crops is produced each year, a homozygous line needs 7–9 years to emerge [1]. Global population growth has increased the demand for foods and vegetables; in order to meet these challenges, crop breeding needs to be faster. Speed breeding controls the environmental conditions to accelerate the flowering and fruiting, with shortened reproduction cycles [2]. Speed breeding techniques have been applied to crops with important economic value, including Poaceae, Fabaceae, and Brassicaceae [3].

The light environment plays a significant role in speed breeding. Three components of the light environment (light quality, light intensity, and photoperiod) affect plant photosynthesis and photomorphogenesis. In general, high light intensities are able to provide the necessary photosynthetic energy needed to produce flower-promoting substances and support the growth of flower primordia [4]. When compared to 200 µmol·m⁻²·s⁻¹ light intensity, 500 µmol·m⁻²·s⁻¹ PPFD will produce a high dose of irradiation per plant, thereby increasing the amount of light assimilation substances in the plant and accelerating the formation and growth of fruit [5]. However, the symptoms of leaf damage were evident with prolonged exposure to high light intensities (more than 500 µmol·m⁻²·s⁻¹). The extended photoperiod induced earlier flowering in long-day crops, such as spring wheat, barley, canola, and chickpeas. Under artificial greenhouse conditions with a 22-h photoperiod, the average reduction was 22 ± 2 days (wheat), 64 ± 8 days (barley), 73 ± 9 days (canola), and 33 ± 2 days (chickpea) [2]. Light quality changes in the environment can be detected by plants through photoreceptors, such as phytochromes [6]. Phytochromes can exist as either inactive or active forms. The red-to-far-red ratio of the incident radiation is closely related to the balance between these two morphologies [7]. Low R:FR ratios accelerate flowering in many crops, including Arabidopsis thaliana, Campanula carpatica, and Gypsophila paniculate [8,9,10]. In addition, 80% far-red light (R:FR = 2) significantly accelerates fruit ripening in tomato but does not affect the flowering time [11].

Hot pepper (Capsicum spp.) fruit provides a rich source of capsaicin, carotene, vitamin C, and mineral elements. Hot pepper is cultivated in at least 44 countries, and it is a very important vegetable in the world [12]. Global pepper production shows a steady growth trend, and the total global production was about 40 million tons in 2020 [13]. Using conventional breeding methods, one generation of peppers needs 148 to 184 days in autumn–winter and 117 to 154 days in spring–summer; hence, peppers do not have more than two generations per year [14]. Therefore, it is crucial to use technologies that shorten the generation time and accelerate breeding. In this study, we aim to explore a light-emitting diodes (LEDs)-based light environment modification scheme (including light intensity, photoperiod, and red-to-far-red ratio) to speed up the growth and development of hot peppers and obtain more generations per year.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

This study used two hot pepper cultivars (Xiangyan55 and Xiangla712) provided by Hunan Xiangyan Seed Industry Co., Ltd.(Changsha, China) The seeds of the hot pepper were sown into damp sponge blocks and placed in a dark environment. Four days later, the germinated seeds were transplanted into a deep-flow technology system using 1/2 strength of Hoagland’s solution (EC ≈ 1.5 mS cm², pH ≈ 6.5). The light environment was 250 µmol·m⁻²·s⁻¹ PPFD white LED (Chenghui Equipment Co., Ltd., Guangzhou, China) from 6:00 to 18:00 until the two cotyledons were fully developed; then, they were transferred to different conditions until harvest. All the experiments were conducted in an artificial lighting plant factory (temperature 21 ± 2 °C, relative humidity 60 ± 10%) at South China Agricultural University.

The light quality of white: red: blue = 3:2:1 is conducive to cultivating robust hot pepper plants (unpublished data). On this basis, the specific light treatments were designed for this experiment as follows:

Experiment I: 240, 300, 360, and 420 µmol·m⁻²·s⁻¹ PPFD, respectively, with a 12 h photoperiod.

Experiment II: a 14, 16, 18, and 20 h photoperiod, respectively, with 420 µmol·m⁻²·s⁻¹ PPFD.

Experiment III: under PPFD of 420 µmol·m⁻²·s⁻¹ and a 12 h photoperiod, the additional FR light intensity was set to 30, 50, 70, and 90 µmol·m⁻²·s⁻¹, and the corresponding R:FR ratio was: 6.3, 3.8, 2.7, and 2.1.

2.2. Growth and Morphology Evaluation

The plants were observed daily, and the number of days at which the plants reached different growth stages was recorded: the first flower bud, initiation of the anthesis (when 50% of flowers reached anthesis), breaker stage (incipient red color formation), and red ripened stage (fruit completely red in color). Three biological replicates were analyzed for all 36 fruits, each of which came from twelve plants.

2.3. Sampling and Germination Ability Test

In order to examine the germination capacity of the seeds at various maturities, the fruit was picked between 73 and 91 days after sowing before the test. The fruit’s seeds were removed and dried for two days at 30 °C in an oven. The seeds were then placed on moist filter paper, immersed in 50–55 °C water for four hours, and stored in a constant temperature box at 25–30 °C. On the seventh day, the germinated seeds were counted. The seed germination rate = the number of germinated seed/ total seed.

2.4. Statistical Analysis

Analysis of variance (one-way ANOVA) and comparison of means using Duncan’s test at p < 0.05 level were performed by SPSS 26.0 (Chicago, IL, USA). Graphs were created using Origin 2018 (Origin Lab, Northampton, MA, USA).

3. Results

3.1. Effects of Light Intensity on the Growth and Development of Hot Peppers

The times to reach the growth stages were different for the hot peppers under different light intensities (PPFD). The effect of light intensity on the flowering stage, breaker stage, and red ripened stage of peppers are shown in Figure 1.

In Xiangyan55, 300 to 360 µmol·m⁻²·s⁻¹ PPFD reduced the flowering time, and the shortest day from sowing to flowering was 38 ± 0.19 days. The time to the fruit entering the breaker stage was shorter under ≤420 µmol·m⁻²·s⁻¹. The fruit turned completely red earliest under 360 µmol·m⁻²·s⁻¹; it was only 86 ± 0.20 days from sowing to full maturity (Figure 1b).

For Xiangla712, the earliest to reach each physiological stage was under the highest light intensity (420 µmol·m⁻²·s⁻¹). The plants under 420 µmol·m⁻²·s⁻¹ reached the flowering stage at 43 ± 0.48 days, the breaker stage at 83 ± 0.25 days, and the red ripened stage at 86 ± 0.25 days (Figure 1f).

The germination ability of the seeds was significantly different at different maturity stages (Figure 1c,g). For Xiangyan55, the average germination increased from 6 ± 1.04% at 73 days after sowing (DAS) to 91 ± 3.75% at 87 DAS (Figure 1c). For Xiangla712, the average germination increased from 7 ± 1.88% at 76 DAS to 87 ± 2.03% at 91 DAS (Figure 1g). More than 60% of the seeds harvested at 82 DAS (Xiangyan55) and 87 DAS (Xiangla712) germinated, and the proportion of germinated seeds did not show a significant difference between light intensity treatments (Figure 1d,h).

3.2. Effects of the Photoperiod on the Growth and Development of Hot Peppers

The growth stages of the hot peppers were significantly affected by different photoperiods (Figure 2).

The earliest to reach each physiological stage was under the extended photoperiod. For Xiangyan55, the 20 h photoperiod reduced flowering time, and the shortest day from sowing to flowering was 37 ± 0.15 days. The breaker stage was reached at 82 ± 0.58 DAS, and the red ripening stage was reached at 86 ± 0.24 DAS under a 20 h photoperiod (Figure 2b). For Xiangla712, the earliest to reach each physiological stage was under the extended photoperiod (≥18 h). Plants under the 20 h photoperiod reached the flowering stage at 43 ± 0.29 DAS, the breaker stage at 90 ± 0.24 DAS, and the red ripened stage at 95 ± 0.24 DAS (Figure 2d).

The effects of increasing light time on the growth stages of different cultivars were consistent.

There was no discernible difference in the germination ability of seeds with different photoperiod treatments for fruits harvested at 86 DAS (Xiangyan55) and 89 DAS (Xiangla712), and the germination rate was over 80% (Figure 2c,e).

3.3. Effects of the Red-to-Far-Red Ratio on the Growth and Development of Hot Peppers

The growth stages of hot peppers were significantly affected by the red-to-far-red ratio (Figure 3).

A low R:FR ratio (2.1) achieved an early breaker stage and red ripening stage. For Xiangyan55, the flowering time of the plants was advanced under the higher R:FR ratios (≥3.80), and the shortest day from sowing to flowering was 44 ± 0.38 days. However, the best response to accelerate the breaker stage and red ripening stage was under the R: FR ratio of 2.1, which required 83 ± 0.25 and 88 ± 0.95 DAS, respectively (Figure 3c). The effects of a low R:FR ratio on ‘Xiangla712′ were consistent. The time for the fruit to enter the breaker stage and red ripened stage was shorter under a low R:FR ratio, which was 90 ± 0.33 and 96 ± 0.33 DAS, respectively (Figure 3e).

4. Discussion

Light has an impact on plant architecture by influencing stem lengthening, branch emission, and leaf expansion, which in turn promotes blooming, fruit setting, and seed generation [15]. Flowering is a complex process, and plants need enough leaves (and corresponding leaf area) to respond to a complex network of endogenous signaling to induce flowering [16,17]. In this study, the flowering time of Xiangyan55 and Xiangla712 were shortened under the light intensities of 300 and 420 µmol·m⁻²·s⁻¹ PPFD, respectively (Figure 1b,f). Under this light intensity, the number of leaves increased significantly, which might induce the earlier flowering time. Similarly, the number of leaves of Eustoma grandiflorum increased with increasing light intensity, and the average time to first flowering dropped by 7 to 10 days when the light intensity rose from 100 to 400 µmol·m⁻²·s⁻¹ [18]. Petunia x hybrida flower bud formation occurred two weeks earlier under high light intensity (360 µmol·m⁻²·s⁻¹) than under low light intensity (40 µmol·m⁻²·s⁻¹) [19]. A previous study found that direct germination of immature seeds can significantly reduce sorghum’s breeding cycle [20]. To determine whether hot pepper plants could be harvested earlier for speed breeding, the germination rate of immature seeds harvested at four various times were tested (Figure 1c,g). The seeds harvested at 77 DAS had a germination rate of 28 ± 3.12%, while seeds harvested at 82 DAS (43 days after flowering) had a germination rate of 82 ± 3.13%. Therefore, for speed breeding, hot pepper can obtain germinable seed at 77 DAS under 420 µmol·m⁻²·s⁻¹ and a 12 h photoperiod.

Variations in photoperiod might affect the physiological processes related to plant development, such as flowering and fruiting [21]. Prolonging the photoperiod significantly accelerated oats reaching the flowering stage; the speed breeding (22 h photoperiod) was 45 to 57 d, while 56 to 70 d were needed for normal growing conditions (16 h) [22]. Our results showed that a 20 h photoperiod significantly shortened the time for the flowering, color-breaking, and red-ripening stages in hot peppers (Figure 2b,d). The growth period of peas was shortened to 68 d by extending the photoperiod to 20 h, which was 30–45 d shorter each generation than the traditional single seed descent approach [23]. Anthesis time was significantly shortened for all crop species under an extended photoperiod (22 h light/2 h dark) in a controlled environment compared to 12-h day-neutral conditions; the average reduction was 22 ± 2 days (wheat), 64 ± 8 days (barley), 73 ± 9 days (canola), and 33 ± 2 days (chickpea) [2]. In this study, extending the photoperiod enabled harvesting seeds with germination ability at 86 DAS (Figure 2 c). Under extended photoperiods, plants’ growth rates were higher, primarily related to the increased photosynthesis rates and biomass production, which in turn quickened the reproductive cycle [24]. In this study, when the photoperiod increased from 12 to 20 h, the date of flowering was around 2 days early. Due to the tradeoff between the shortened development stage and energy expense, 420 µmol·m⁻²·s⁻¹ PPFD and a 12-h photoperiod might be suitable for hot pepper speed breeding.

Furthermore, light quality can also influence the flowering process. In plants, phytochromes sense and react to red and far-red light, while cryptochromes and phytotropins sense and respond to blue light. Plant growth and development, including photomorphogenesis, flowering, maturity, and circadian rhythm, are significantly influenced by phytochromes and cryptochromes [25]. Far-red (FR) light encourages phytochromes’ transition from active to inactive states [26]. The R:FR ratio had a substantial impact on lentil floral induction; the R:FR ratio of 3.1 or lower caused flowering to occur 10–11 days earlier than the R:FR ratio of 5.6 [27]. In this study, supplementation of 50 µmol·m⁻²·s⁻¹ FR (R:FR = 3.8) accelerated the flowering of both pepper cultivars (Figure 3c,e). The penultimate leaf of winter canola was RNA sequenced to confirm that additional far-red light significantly increased the expression of several flowering activators, which resulted in earlier flowering than in the controls [28]. Far-red light also promoted fruit growth by increasing the distribution of fruit dry mass [11]. The addition of 90 µmol·m⁻²·s⁻¹ FR (R:FR = 2.1) treatment caused peppers to achieve the red ripening stage earlier and significantly improved the seed germination rate at the same harvest time (Figure 3c–f).

5. Conclusions

The effects of various light settings on the growth and development of two hot pepper cultivars were investigated; specific light regimens accelerated the flowering and maturity of crops and improved seed germination. Under controlled environments, 420 µmol·m⁻²·s⁻¹ PPFD and a 12 h photoperiod accelerated the growth and development of pepper and significantly shortened the breeding cycle (up to more than four generations per year). (Figure 4) Additional low-intensity far-red light sped up the flowering. The seeds harvested at 43 days after flowering already had a higher germination rate. In order to further achieve speed breeding for crop, immature seeds could be harvested earlier, which combined with measures to improve germinating ability, could obtain more generations per year.