The soybean varieties Heihe27 (HH27, maturity group (MG) 0), Heihe43 (HH43, MG 0), Zigongdongdou (ZGDD, MG VIII), Zhonghuang30 (ZH30, MG III), and Zhonghuang39 (ZH39, MG III) were used in this study. HH27 and HH43 are photoperiod-insensitive and early-maturing soybean varieties. ZH30 and ZH39 are photoperiod-sensitive and medium-maturing soybean varieties. ZGDD is a photoperiod-sensitive and late-maturing soybean variety (Han et al., 1998; Wu et al., 2006).

For the generation of COP, soybean seeds were planted in growth chambers at 26°C. All the organs above the cotyledonary node were removed at VC (unifoliolate leaf stage). Subsequently, the newly-produced leaves or stems at the cotyledonary node were removed soon when they began to emerge ( Supplementary Figure S1 ).

For the generation of COP with only one cotyledon and root, soybean seeds were sterilized with chlorine gas for 16 h and then grown on germination culture medium (Chen et al., 2018). For hydroponic culture, the germinated seeds were cut into two half-seeds ( Supplementary Figure S2 ), and the buds at the cotyledonary node on the half-seeds were removed and then grown in the Hoagland solution.

To analyze the flowering time of COP of different soybean varieties, ZGDD and HH27 COPs were grown in chambers at 26°C under short-day (SD, 12 h light/12 h dark) and long-day (LD, 16 h light/8 h dark) conditions. ZH39, ZH30, HH27, and HH43 COPs were grown in the natural day-length environment in Beijing, China. The sowing dates of HH27, HH43, and ZH39 were 20 May, 21 May, and 30 June 2019, and ZH30 was 20 May, 21 May, and 15 July 2019.

To examine the effect of night break on COP, ZGDD COPs were grown in a chamber at 26°C under SD (12 h light/12 h dark) conditions. A 1-h period of white-light exposure was conducted in the middle of the dark period ( Supplementary Figure S3 ). Samples were harvested after two weeks after the night break treatment.

To test the expression level of flowering-time related genes in COP, ZGDD COPs were grown in chambers at 26°C under SD (12 h light/12 h dark) and LD (16 h light/8 h dark) conditions. The cotyledons were sampled every 3 d from germination.

To test the expression level of GmFT2a in COP with only one cotyledon and root, ZGDD were grown in chambers at 26°C under SD (12 h light/12 h dark) and LD (16 h light/8 h dark) conditions. The cotyledons of 7-d-old COP with only one cotyledon and root were sampled at 4 h after light on.

RNA extraction, reverse transcription, and quantitative real-time PCR were performed as described in our previous study (Wang et al., 2020). The primers for GmActin, GmFT2a, GmFT5a, and E1 were described previously ( Supplementary Table S1 ; Xu et al., 2021). The primers for GmFT1a are as follows: qGmFT1a-F: 5’- ATTCCTGCAACTACAGGGGC-3’, qGmFT1a-R: 5’-AAGTACATGGCCGCTACTGG -3’.

The plant height is the distance from the cotyledonary node to the stem tip. The maximum diameter of the COP is the distance between the ends of the two cotyledons and the maximum diameter of the intact plant is the maximum distance between the two largest leaves in the horizontal plane.

The flowering time was calculated from emergence (VE) to R1 (i.e., beginning bloom: one open flowering at any node on the main stream) (Fehr and Caviness, 1977). The statistical analysis was carried out in Microsoft Excel, and Student’s t-tests were used to assess the significance of differences.

The COPs only contain two cotyledons as the main aboveground organs (Xu et al., 2021). To compare the plant size between the COPs and intact plants, we grew the COPs and intact plants of photoperiod-sensitive and early-maturing soybean variety ZGDD in chambers under SD and LD conditions and investigated the plant height and maximum diameter of 30-d-old plants. Under SD conditions, the plant height for COPs was only 7.3 ± 0.4 cm, which was significantly lower than that of the intact plants (23.2 ± 0.8 cm; Figure 1A ). The maximum diameter for COPs (4.1 ± 0.5 cm) was also smaller than that for the intact plants (10.7 ± 2.6 cm; Figure 1B ). Under LD conditions, we observed similar patterns in which the plant height and maximum diameter for COPs (7.5 ± 0.5 cm and 4.2 ± 0.6 cm, respectively) were significantly smaller than those for the intact plants (37.0 ± 1.9 cm and 11.5 ± 2.2 cm; Figures 1A, B ). Thus, the COPs occupy less growing area than the intact plants.

Besides cotyledons, the intact plants contain multiple leaves compared to COPs. To identify the longevity of cotyledons in COPs and intact plants, we grew ZGDD in chambers under SD and LD conditions. The results showed that the longevity of cotyledons in intact plants was 16.9 ± 1.7 d and 17.4 ± 0.9 d under SD and LD conditions, respectively. However, the longevity of cotyledons in COPs was significantly increased to 114.7 ± 13.7 d and 136.9 ± 8.9 d under SD and LD conditions, respectively ( Figures 1C, D ), indicating that the COPs have longer cotyledon longevity than the intact plants.

Previous studies have shown that COPs are responsive to photoperiods (Xu et al., 2021). To confirm the photoperiodic response of COPs, we compared the flowering time of COPs and intact plants of ZGDD, and the photoperiod-insensitive and early-maturing variety HH27 grown in chambers under both SD and LD conditions. For the photoperiod-sensitive variety ZGDD, intact plants flowered at 32.0 ± 2.5 d after emergence (DAE) under SD conditions and maintained vegetative growth under LD conditions ( Figures 2A, B ). Similarly, the COPs only flowered 39.4 ± 2.5 DAE under SD conditions and maintained vegetative growth under LD conditions ( Figures 2E, F ). For the photoperiod-insensitive variety HH27, the COPs and intact plants flowered normally both under SD (30.5 ± 5.9 and 28.1 ± 1.7 DAE) and LD (29.8 ± 4.0 and 27.6 ± 1.8 DAE) conditions ( Figures 2C, D, G, H ). The above results indicated that the COPs have the same photoperiodic response as intact plants.

To examine the photoperiodic response of COPs in the natural environment, we grew the COPs and intact plants of HH27 and another early-maturing variety HH43 belonging to MG 0, and medium-maturing varieties ZH30 and ZH39 belonging to MG III under natural day-length conditions in Beijing. Our results indicated that the COPs and intact plants of the above four varieties flowered normally ( Table 1 ). Specifically, the flowering time of COPs flowered early when grown in the short day-length season and flowered late when grown in the long day-length season, which resembles that of intact plants ( Table 1 ). These results showed that the COPs also respond to the natural photoperiod environment.

The night break treatment acts as an important evidence of photoperiodic response and causes delayed flowering time of short-day plants (Xu et al., 2015). To test the effect of night break on the flowering time of COPs, the COPs and intact plants of ZGDD were planted under SD conditions with a night break treatment at midnight with 1-h exposure of white light. We found that both COPs and intact plants of ZGDD failed to flower after the night break treatment and maintained vegetative growth during the experiment ( Figures 3A, B ). Taken together, the COPs have a normal photoperiodic response similar to that of intact plants.

E1, GmFT1a, GmFT2a, GmFT5a are among the key genes in the photoperiodic flowering pathway of soybean (Kong et al., 2010; Sun et al., 2011; Xia et al., 2012; Liu et al., 2018; Cai et al., 2020; Lin et al., 2021). To investigate the molecular mechanism involved in flowering time regulation in COPs, we examined the expression patterns of the above genes in the cotyledons of ZGDD COPs under SD and LD conditions. The results revealed that the floral-inhibiting genes E1 and GmFT1a were significantly upregulated under LD conditions and downregulated under SD conditions, while the floral-promoting genes GmFT2a and GmFT5a were significantly upregulated under SD conditions and downregulated under LD conditions ( Figures 4A–D ). Furthermore, the expression levels of E1, GmFT1a, GmFT2a, and GmFT5a were also analyzed in ZGDD COPs and intact plants treated with a night-break. We found that the expression levels of E1 and GmFT1a were significantly enhanced in the COPs and intact plants under the night-break treatment, while GmFT2a and GmFT5a were significantly repressed ( Figures 4E–H ). Taken together, the COPs present the same expression pattern of key genes in the photoperiodic flowering pathway as intact plants.

Using individual cotyledon, we generated a very simple cotyledon-only plant with only one cotyledon and root ( Figure 5A ). Specifically, a single cotyledon with a 0.2-cm petiole was grown in hydroponic medium. After approximately 7 d, the roots were grown from the petiole. The COP with only one cotyledon and root occupies a very small space size in which 100 plants can be grown in a 17 cm × 17 cm × 5 cm hydroponic tray ( Figures 5B, C ). We analyzed the expression level of GmFT2a in the cotyledon of COP with only one cotyledon and root to identify its photoperiodic response. The results showed that the expression level of GmFT2a was significantly upregulated under SD conditions compared with LD conditions ( Figure 5D ), suggesting that COP with only one cotyledon and root is also sensitive to photoperiod.

Soybean is an important model plant for photoperiodic response studies. Many important discoveries in the photoperiodic response of plants have been interpreted using soybean as the material (Garner and Allard, 1920; Wu et al., 2006). However, intact soybean plants are not suitable for large-scale, rapid, and efficient experimental platforms and basic research due to the large space occupied by the plants and the long growth cycles.

Arabidopsis thaliana is the most-widely used model plant for biological study in plant, especially dicot species, due to its small plant size and short life cycle (Meyerowitz, 1989). For crop plants, the monocot rice (Oryza sativa) is widely used as a model material. In recent years, a new rice variety named ‘Xiaowei’ with small plant size was selected and demonstrated to be grown and screened in large numbers in the laboratory and even in the space station (Hu et al., 2018). This plant expedites the development of the physiology and molecular biology of crops. Similarly, the soybean variety ‘MiniMax’ also has a short plant height which can be used as a model plant (Qu et al., 2010a). Even though Xiaowei and MiniMax are relative smaller than the normal plants, they still have many leaves and big plant size compared to Arabidopsis.

In the current study, we generated a unique tiny plant, COP, with only the cotyledons, root, and hypocotyl that connected the cotyledons and root. Due to the totipotency cotyledons, this COP could complete the entire life processes of growth, development, and reproduction (Xu et al., 2021). It is the smallest version of plant species in nature reported to date. Moreover, COPs retain the photoperiod characteristics of the original variety and are amenable to the study of photoperiodism similar to normal plants. Based on this tiny plant, we established a novel COP experimental system for photoperiodic response studies in short-day plants and other dicot species. In addition, COPs are potential model materials that are applicable to other plant biology studies in addition to photoperiodic responses.

To date, many experimental systems have been established in soybean for photoperiodic response studies, including artificial light control, night-break, grafting experiments, and flowering reversion (Garner and Allard, 1920; Heinze et al., 1942; Downs, 1956; Wu et al., 2006; Qu et al., 2010b). Here, we propose a new COP experimental system that is an important complement to the current systems. Previous experimental systems mainly focus on the physiological and molecular changes of intact plants (Qu et al., 2010b; Zhang et al., 2020; Lin et al., 2021), while the COP experimental system facilitates a use of more accurately and stably controlled environments to trace biological changes in floral induction. Therefore, this novel system has unique characteristics compared to other experimental systems.

The smaller plant size of COP is the superior characteristic. Large numbers of COPs could be grown in a limited space with precise control of environmental factors, which is difficult to perform for intact plants with large plant sizes (Fang and Kong, 2022). COP has a pair of cotyledons as the main aboveground vegetative organ in its entire life cycle, while the intact plant has a longer life cycle and many leaf organs (Friedli and Walter, 2015). In so doing, the COP enables photoperiodic response studies only in the cotyledons and conduction of long-term tracking experiments, eliminating the interference of signaling and nutritional substances that arise from other leaves in the intact plants. The cotyledons of COP rapidly respond to photoperiods and express the floral-promoting gene GmFT2a/5a in SD and the floral-inhibiting genes E1/GmFT1a in LD, which resemble the intact plants (Kong et al., 2010; Sun et al., 2011; Xia et al., 2012; Liu et al., 2018; Cai et al., 2020). Thus, we could monitor the photoperiodic response at the molecular level without waiting for morphological changes, such as flowering, podding, and maturation. The cotyledons of COP are already full-development organs after emergence from soil under dark conditions (Cao et al., 2013), and are rich in nutrients such as protein, oil, and carbohydrates (Han et al., 2013; Wei et al., 2020). Hence, we could explore the beginning photoperiodic response, including light signal transduction and perception, day length discrimination, and gene regulation networks.

Apart from the photoperiodic response, the COP system could be applicable in other research fields. We noticed that the cotyledons could survive for more than 100 d in COPs, while they survived for only 15 d in the intact plants, suggesting that some signals arising from other leaves in the intact plants influence the senescence of cotyledons. It is worth considering the causes of this phenomenon, and it is possible to use this system to study the mechanism of leaf senescence. During the seedling stage, the cotyledons simultaneously undergo a complex process of nutrient degradation, transformation, and synthesis of photosynthetic products (Gonzalez and Vodkin, 2007; Han and Yang, 2015). That is, cotyledons act as both a source and a sink, which can be used to study physiological processes such as source-sink relationships and photosynthesis in plants.

In the tissue culture, regenerated buds are often faced with common problems in the rooting stage, such as no rooting, hardly rooting, and regenerated roots being polluted with bacteria. Due to the stronger root system and rich nutrition in cotyledons, we successfully used the COPs as stocks that are capable of supporting the survival of regenerated buds with problems in rooting as scions in the grafting experiment. Therefore, COPs will significantly increase the survival rate of regenerated buds in tissue culture and in turn elevate the efficiency of genetic transformation. Moreover, the simple COP with one cotyledon and root is cultured in the hydroponic medium. We could easily add hormones, mineral elements, and pesticides to the liquid and investigate the response of COP. In summary, the COP is an ideal experimental system for plant biology studies.