Establishing the phenological development stages of Sophora moorcroftiana using the BBCH scale

Sophora moorcroftiana (Benth.) Baker (Leguminosae: Sophora) is a deciduous dwarf shrub widely distributed along riverbanks, valleys, and hillsides in the Yarlung Tsangpo, Nyenchu, and Lhasa Rivers in Tibet¹. This species serves as an important pioneer species for windbreak, sand fixation and soil conservation in the region. It exhibits drought tolerance, soil adaptation and robust environmental adaptability, playing a key role in the plateau ecosystem^2,3. Current research predominantly focuses on its medicinal properties and drought resistance mechanisms. Yin, X. et al.^4,5 found that S. moorcroftiana seeds are rich in flavonoids, matrine-type alkaloids, terpenoids, steroids, and their derivatives. Matrine-type alkaloids exhibit a wide spectrum of pharmacological activities, including antiviral, antifibrotic, antitumor, leukocytosis-promoting, and immunomodulatory effects^6,7. The tender branches, leaves, and seeds of S. moorcroftiana also have high nutritional value, with the seeds containing substantial protein. This makes the species an important source of nutrition in the protein-deficient regions of Tibet and a high-quality forage resource for plateau livestock⁸.

Phenology is the study of periodic biological events in relation to environmental changes. The BBCH scale (Biologische Bundesanstalt, Bundessortenamt, and Chemische Industrie) provides a standardized coding system that describes comparable phenological stages in both monocotyledonous and dicotyledonous plants. It was developed through the collaboration among scientists from the German Federal Biological Research Centre for Agriculture and Forestry (BBA), the Federal Plant Variety Office (BSA), and the German Agricultural Chemical Industry (IVA)^9,10. BBCH scale is now widely used for crops, forestry species, and medicinal plants, and has standardized descriptions of phenological periods and their time periods for all flowering plants¹¹. Many woody plants are described by the BBCH, such as mango¹², longan¹¹, sugar apple¹³, sweet cherry¹⁴, and persimmon¹⁵. The BBCH scale can be used to characterize the different phases in a hierarchical manner, which makes it more suitable for reflecting the phenology of shrubs at high altitudes and facilitates direct comparisons with other species or taxonomic groups. In 1997, the BBCH scale was officially recommended for use by the European and Mediterranean Plant Protection Organization (EPPO), and the Global Phenological Monitoring Programme also adopt this scale as a standard for phenological observations⁹. To complete certain developmental stages, plants require a specific amount of thermal energy¹⁶, with effective accumulated temperature being the most effective method for estimating plant maturity¹⁷. Predicting plant maturity can reduce agricultural costs and guide optimal cultivation timing¹⁸.

Although substantial progress in understanding the ecological, medicinal and forage values of S. moorcroftiana has been done, the species continues to face several challenges, including declining populations, underutilization of its resources, outdated cultivation techniques, and absence of a standardized framework for phenological characterization. To address these issues, we conducted a two-year systematic observation of the growth stages of S. moorcroftiana. A comprehensive phenology framework was also established for the plateau shrub S. moorcroftiana. Each developmental stage was described in detail, and the corresponding temperature requirements for each phenophases were analyzed. These analyses enabled the identification of the optimal seeds harvesting period, the growth stages most susceptible to insect infestation, as well as the appropriate temperature conditions for the cultivation of S. moorcroftiana. Collectively, these findings provide a scientific basis for conservation, breeding, introductions and biopharmaceutical exploration of S. moorcroftiana, and establish a robust phenology model for this ecologically and economically important species.

Experimental materials

The experiment was conducted at the nursery base of the Xizang Agricultural and Animal Husbandry University in Nyingchi, Tibet Autonomous Region (94°20′40″E; 29°40′24″N). Nyingchi, located in southeastern Tibet along the middle reaches of the Yarlung Tsangpo River, has an average altitude of 2800 m. It has unique topographic features such as high mountains and valleys, and great diversity of landforms with huge vertical drop. Nyingchi is one of the main distribution areas of S. moorcroftiana. Climate data from the past 30 years, sourced from the Xizang Meteorological Administration, indicates that the region exhibits a plateau temperate humid-subhumid monsoon climate. The annual average temperature is 9.48 °C, with the highest temperature recorded at 16.49 °C (July) and the lowest at 1.15 °C (January). Its soil is sandy loam with high year-round surface temperatures, regular watering and irrigation, no towering irrigation or trees around, and excellent sun exposure conditions, providing favorable conditions for S. moorcroftiana growth.

The plant material of S. moorcroftiana used in this study was identified by Prof. Fumei Xin from Xizang Agricultural and Animal Husbandry University. The identification was based on the herbarium (PE 02330018) provided by Xiangyun Zhu and deposited in the Chinese Virtual Herbarium (https://www.cvh.ac.cn). Our S. moorcroftiana plants (id:19944579) have been preserved and shared in the Plant Photo Bank of China (http://ppbc.iplant.cn/). This study used all S. moorcroftiana plants at the nursery to measure effective cumulative temperature, and selected twelve 6-year-old plants with the same height and crown width for phenological observations. Maintained by irrigation and weeding. Traits were recorded and photographed weekly or bi-weekly, and dormancy lasted for two years, from bud dormancy to pre-germination to flowering and fruiting. Climate data is sourced from the Xizang Meteorological Administration, with daily temperature monitoring conducted between 2023 and 2024. During this period, no abnormal climate conditions were observed, ensuring the data’s strong representativeness. Representative photographs of each phenological stage were selected, describing traits from bud dormancy to flowering, fruiting, and the next dormancy period, spanning two years. During the study period, the plants were kept healthy with consistent water and fertilizer management and no use of pesticides, external hormones.

BBCH scale

The BBCH scale was employed to classify the phenological stages of S. moorcroftiana. It uses a two-digit code: one digit for the primary growth stage (0–9) and another for secondary stages (0–9)¹². This study identified eight of the ten primary BBCH stages, starting from bud development (0), leaf development (1), stem elongation (3), inflorescence emergence (5), flowering (6), fruit development (7), seed maturation (8), to senescence and dormancy (9). The coding system facilitates comparisons, with higher numbers indicating further progression within the same primary stage. Separate overlapping stages with diagonal strokes⁹.

Effective accumulated temperature

Plant threshold temperature varies according to species and phenological period, and the threshold temperature of S. moorcroftiana has not been reported yet. For S. moorcroftiana, a threshold of 7 °C was established based on the average temperature of initial developmental stages and literature references¹⁹. We calculated the effective accumulated temperature using the meteorological dataset recorded by Weather Station No. 1 at the experimental site from 1991 to 2020. Phenological observations and photography documented the morphology at each stage, calculating the heat accumulation needed for each phase.

where: GDD ( °C·d) denotes growing cumulative temperature; n denotes the number of days elapsed during the development period; T_i denotes the average air temperature on day i; B = 7 ℃ denotes the biological zero degree of the development stage²⁰.

Description of S. moorcroftiana phenological stages using BBCH Codes

Through observations and recordings of the phenological stages of S. moorcroftiana, this study identified eight major growth stages and their durations (Table 1, Fig. 1). The plant morphology of S. moorcroftiana is not amenable to description in terms of the second stage (formation of lateral buds or tillers) and the fourth stage (development of harvestable vegetative parts or vegetative reproductive organs/bud formation). The study documented changes in S. moorcroftiana phenology through photography, irrigation, and weeding, recording the status at regular intervals from bud dormancy until the next dormancy, lasting two years. We described the morphological characteristics of individual plants using the BBCH scale and found no phenological differences between them.

Primary growth stage 0: bud development

Bud dormancy at the observation site lasted until early March, with bud swelling beginning in mid-March, and flowering occurring at the end of bud development (Fig. 2).

00. Bud dormancy: Leaf buds are grayish-white and closed in a dormant state.

01. Bud swelling starts: The leaf buds begin to expand again in mid-March, and the buds are ready to unfold.

03. Bud swelling ends: Buds reach full swelling and slightly open.

07. Bud scale separation: Bud scales separate, revealing purple leaf tips.

09. Visible purple bud tip: Purple tips become visible, protruding 5–10 mm above the grayish-white scales.

Primary growth stage 1: leaf development

Leaf development occurs from mid-April to mid-August, taking about four months for complete leaf maturation (Fig. 2).

10. First leaf separation: Leaves turn from purple to gray-green, with the outermost leaf separating.

11. First leaf unfolds: The leaflets of the first compound leaf open, with petioles extending to 10% of their final length.

14. 40% of juvenile leaves unfolded: Juvenile leaves spread out, turning light green, and petioles lengthen.

17. 70% of leaves unfolded: Leaf unfolds to final 70%, leaf is light green, leaf thickens, petiole hardens.

19. All leaves fully unfolded: Leaf blades reach full size, petioles are elongated to their final size, and leaflets are mostly thickened to a dark green color.

Primary growth stage 3: shoot development

Shoot development often occurs concurrently with leaf development. S. moorcroftiana shoot development over an extended period, ceasing during fruit development. Non-reproductive branch tips form spines (Fig. 2).

30. Shoot begin to elongate: The bud axis becomes visible and starts developing as the first compound leaf separates. This stage coincides with stage 10.

31. 10% of the final shoot length: The stem thickens and appears dark green, covered with white trichomes.

32. 20% of the final shoot length: The stem thickens further, coinciding with stage 11.

34. 40% of the final shoot length: The stem lengthens and turns gray-green as trichomes decrease.

36. 60% of the final shoot length: The shoot continues to extend, with most branches forming spikes at the end, non-reproductive branch tips form spines.

39. 90% or more of the final shoot length: The shoot hardens, completing development, turning green with almost no trichomes.

Primary growth stage 5: inflorescence emergence

The inflorescence development stage occurs from early April to mid-May, with flower buds and leaves developing simultaneously (Fig. 3).

50. Flower buds begin to swell: The bracts are close to the buds, and the surface is densely covered with gray-white tomentum.

51. Continued bud swelling: Bracts unfold, and purple flower buds reach 50% of their final size.

52. Bud axis starts elongating: The bud axis extends to 20% of its final length, with small flowers densely arranged and dark purple flower buds covered with gray-white trichomes.

54. Bud axis reaches 40% of final length: Small flowers slightly separate, and the color lightens.

56. Bud axis reaches 60% of final length: Small flowers become purple and increase in size.

59. Bud axis reaches full length: The inflorescence develops to its final size, with some small flowers emerging from the bracts and revealing purple petals.

Primary growth stage 6: flowering

The flowering stage occurs from mid-May to early June. The flowers are purple, and the pods appear as the petals begin to fade (Fig. 3).

62. Anthesis: Approximately 20% of flowers are open.

64. Further flowering: More flowers open, reaching 40% of the final flowering stage.

65. Full flowering: All flowers are fully open.

67. Start of wilting: Petals turn yellow and begin to drop or dry out.

69. End of flowering: Most of the petals fall off and dry out, the undersides of most petals are grayish-purple, and fruit pods appear.

Primary growth stage 7: fruit development

Fruit development for S. moorcroftiana occurs from early June to early August. Each pod typically contains 1–5 seeds (Fig. 3).

70. Seeds begin to form: Wilted petals encase 1–2 cm long, slightly flattened pods covered in white trichomes.

74. Pods elongate: Pods grow longer, with wilted petals falling off completely, reaching 30% of their final length. The pods are grayish-white, and trichomes decrease.

75. Pods further elongate: Pods reach 90% of their final length, turning light green, with seeds swelling and the remaining areas still covered with white trichomes.

77. Seeds swell further: Pods reach full length, and seeds are dark green, swollen and oval, with reduced trichome density.

79. Seed development complete: Pods turn gray-green, with brown spots on the swollen parts, and slightly wrinkle.

Primary growth stage 8: seed maturation

Seed maturation of S. moorcroftiana occurs from early August to mid-September (Fig. 3).

81. Early seed maturation: Pods are gray-green, with fewer trichomes on the swollen seed areas, and seeds are tender and light green.

82. Pods begin to change color: Pods turn yellow-green with brown spots appearing on the surface, and peduncles turn brown.

85. Seeds mature further: Pods become yellow–brown, with reduced trichomes, and seeds turn light yellow.

89. Full development and maturity: Pods dry out, turn brown, wrinkle slightly, and the tips of the pods crack, revealing yellow seeds.

Primary growth stage 9: senescence and beginning of dormancy

The onset of senescence and dormancy occurs in tandem with the process of fruit ripening, and October to December is the stage of defoliation of S. moorcroftiana, with little variation among individual plants (Fig. 4).

90. Bud and leaf development cease: The buds stop growing and the leaves remain dark green in color.

91. Leaves begin to discolor: Leaflet tips yellowed with dark brown dots.

95. Half of the leaves fall off: 50% of the leaves fall off, most of the leaves are yellow and some are still green.

97. Easily or completely dislodged: Leaf blades are almost entirely lost, petioles remain on the tree but are easily shed.

99. Dormancy: The trees completely enter into dormant period.

Primary growth stage 2 and 4

The BBCH standard is predicated on the developmental stage of the main stem, and given the high degree of uniformity in the development of the plant’s main stem, the growth stage of lateral branches is not usually described specifically. The temporal dynamics, quantitative development, and spatial distribution of lateral branches are found to be profoundly influenced by light, nutrients, and hormones. Furthermore, it is important to note that different lateral branches of the same plant may be at different stages of development. The incorporation of the developmental stage of lateral branches may serve to reduce the generalizability of the scale. Stage 4 of the BBCH generally describes asexual reproductive organs and harvestable nutrient parts such as root tillers, stolons and root systems, whereas asexual reproduction in S. moorcroftiana tends to be at the root tiller level, and harvestable root systems belong to the stage of nutrient growth that is not applicable to Stage 4 of the BBCH. Similar to Xanthoceras sorbifolium¹¹, Sapindus mukorossi²¹, Spondias dulcis²² and shea tree²³.

Effective accumulated temperature

The primary objective of phenological research is to link climatic events with specific phenological stages, providing an approximate prediction of these stages²⁴. Temperature is a critical factor affecting plant development. Different growth stages of plants require varying effective cumulative temperature and durations. This study calculated the duration (days) and effective cumulative temperature (degree-days) required for each phenological stage of S. moorcroftiana from 2023 to 2024 using Eq. 1 (Table 2). The mean and standard deviation of the duration and effective temperature were calculated. The duration of the bud stage was 30 ± 1 d with an effective cumulative temperature of 48.95 ± 10.61 °C·d. The leaf development stage had the longest duration of 119 ± 6 d with an effective cumulative temperature of 1063.13 ± 112.32 °C·d. The inflorescence development stage lasted for 33 ± 1 d with an effective cumulative temperature of 147.98 ± 3.64 °C·d. The flowering stage lasted 21 ± 1 d with an effective temperature of 190.50 ± 23.69 °C·d. Fruit development was longer at 49 ± 2 d with an effective temperature of 534.00 ± 42.21 °C·d. And seed maturation lasted 38 ± 1 d with an effective temperature of 455.13 ± 10.92 °C·d.

As a cold-tolerant, drought-tolerant and barren-tolerant pioneer shrub, S. moorcroftiana thrives under harsh conditions in Tibet, with its development closely linked to the local ecological environment. Using the BBCH scale, we systematically examined the growth and developmental stages of S. moorcroftiana, filling the gap in previous research on its phenological characteristics and effective accumulated temperature requirements. This research contributes to a better understanding of the growth patterns of S. moorcroftiana and provides a solid foundation for ecological conservation, breeding of superior varieties, and resource utilization. We observed and documented the complete developmental loop of S. moorcroftiana, from bud dormancy to flowering, fruiting, and seed maturation, identifying eight major growth stages and 41 secondary growth stages. The BBCH scale offers a standardized tool for describing and comparing the growth stages of various plant species. The results indicated that, due to the low temperature in the growing environment, the buds of S. moorcroftiana remained dormant from November to early-March of the following year, with germination occurring approximately one month later than for other tree species^11,21,23. S. moorcroftiana leaf buds develop at the same time, but their development time is longer, stopping at the early stage of fruit maturation, with new buds developing during fruit development^12,25. At stage 34, the sterile branch ends of S. moorcroftiana developed into spikes. The systematic description of phenology provides comprehensive insights into S. moorcroftiana development, and the scale’s comparability enhances the utility of phenological observations globally. The use of the BBCH coding system in phenological studies is crucial.

Plants are able to complete their growth and development under suitable temperature conditions, and cumulative temperature is an important parameter for measuring the rate of development and maturity of plants²⁶. Based on the effective cumulative temperature data for 2023–2024, the duration and standard deviation of effective cumulative temperature (°C·d) for bud development, inflorescence formation, flowering, and fruit development stages are relatively small. In contrast, the leaf development stage requires the longest duration and the highest effective cumulative temperature. It is possible to provide better economic and ecological value by calculating the effective temperature and heat required by different species at different growth stages to select suitable places for introduction, such as grapes and olives^17,25. Effective accumulated temperature aids in predicting developmental stages and optimizing management practices, such as fertilization, pruning, and pest control, to enhance growth efficiency and yield²⁷. Additionally, S. moorcroftiana growth varies with altitude and climate, implying that local accumulated temperature conditions should be considered to increase survival rates and adaptability during introduction and cultivation²⁸.

As an important sand-fixing and wind-preventing plant, S. moorcroftiana has a positive impact on the ecological environment of the Tibetan region, and also shows great potential in the field of biomedicine. Studies have shown that its seeds and young leaves contain abundant active compounds, such as flavonoids, alkaloids, and terpenoids, with notable antiviral, antitumor, and immunomodulatory properties^4,7. This study’s phenological observations provide a clear time frame for harvesting these medicinal components. Harvesting seeds at full maturity (BBCH stage 89) ensures maximum extraction efficiency of active ingredients²⁹. The study also emphasised the challenges of biotic stress during seed maturity. Observations revealed severe pest infestation at stage 77, with more than 70% of the seeds being damaged by insects, such as Robinia pseudoacacia bee, which exhibits asynchronous emergence. These bees spend most of their developmental period inside the seeds, consuming the majority of the kernel while the seeds are still immature. Therefore, preventive measures are of primary importance. Chemical control should be applied during the seed ripening stage, typically from late August to early September of the preceding year. Additionally, fumigation should be carried out at stages 70 to 73 during the second half of June of the following year². Accurate phenological monitoring enables early pest control, reducing damage and increasing seed yield. This phenology-based pest management strategy has proven effective in other crops, significantly reducing pesticide use and protecting the environment³⁰.

Although this study provided systematic data on the phenological characteristics of S. moorcroftiana, the observations lasted only two years and were not sufficient to fully reveal the effects of all growth environment variables on its development. Therefore, follow-up studies should extend the observation period to cover the precise phenological stage. Multi-point data from various distribution areas and altitudes of S. moorcroftiana should be incorporated to validate and supplement the phenological model and heat demand specified in this study. It is evident that observations pertaining to insect pests were not meticulously documented, thereby precluding the possibility of proposing specific control measures. In addition, the establishment of the phenological period of S. moorcroftiana should be combined with long-term meteorological data, especially with the comprehensive analysis of other local climatic characteristics, in order to improve the accuracy of its acclimatization study and phenological prediction in the plateau region. Future research should concentrate on investigating the dynamics of alkaloid content in S. moorcroftiana plants at differing developmental stages. Furthermore, the optimal harvesting period to enhance medicinal value should be ascertained. Additionally, the genetic diversity of S. moorcroftiana and its influence on phenology should be examined. Finally, molecular biology techniques should be utilised to study the relationship between its growth and environmental adaptability. Given intensifying global climate change, the applicability of these results to field conditions requires verification. Continuous monitoring of S. moorcroftiana and other plateau plants’ phenology will provide crucial insights for ecological conservation and resource management.

This study systematically describes eight primary and 41 secondary phenological stages of S. moorcroftiana using the BBCH scale, filling a significant gap in phenological research for this species. Following two years of uninterrupted observation and temperature analysis, the effective cumulative temperatures required for each developmental stage were determined, and the critical role of temperature in growth was demonstrated. The results of the study demonstrate the applicability and comparability of the BBCH scale in the context of highland plant research. This standardized climatic description and effective cumulative temperature can predict the growth and development process of S. moorcroftiana, thus guiding the appropriate harvesting period, determining suitable areas for planting, and improving the accuracy of pest control through BBCH. Furthermore, it provides a scientific basis for field management, collection of medicinal components, and selection of superior varieties of S. moorcroftiana. Additionally, as an important plant for ecological restoration and biomedical resources, recognizing key phenological stages in S. moorcroftiana’s life cycle will enhance ecological and economic benefits. In the future, there is still a need to combine long-term observations and broader regional studies to further improve the phenology model of S. moorcroftiana and optimize resource use and conservation strategies.