Phenological Growth Stages of Abelmoschus manihot: Codification and Description According to the BBCH Scale

Abstract

Abelmoschus manihot L. (A. manihot) has received more and more attention due to its potential edible and medicinal value. It shows higher yield and related fine agronomic traits suitable for disadvantaged areas and low-input planting. However, a systemic description of the phenological growth stages of A. manihot, an alternative, multipurpose crop of worldwide interest, does not exist. This study aims to detail the phenological growth stages of A. manihot based on the BBCH scale. Nine principal growth stages were described from seed germination to senescence, along with 69 secondary growth stages, including germination, leaf development, formation of side shoots, primary stem elongation, inflorescence emergence, flowering, fruit development, maturation of fruit and seed, and senescence. However, the morphology and structure of A. manihot become complex with growth, and some growth stages, like inflorescence development, flowering, fruit development, and fruit maturation, overlap totally or partially with each other. Thus, the three-digit scale is considered necessary for a complementary description of these growth stages and illustrations for clarification. Moreover, the unique morphology and structure features of the pistil, stamen, and ovary and the development process of fruits and seeds were described in detail at different stages. The basic and extended BBCH scales will add new information on defining and identifying A. manihot phenological growth stages. They will help farmers efficiently schedule and manage A. manihot cultivation and improve knowledge dissemination among growers and researchers.

1. Introduction

Phenology is one of the reliable tools for better understanding plant responses to environmental changes. It reveals the changes in timing and duration of different life cycle events during the whole growing period in plants, which will help to select the scientific and reasonable management strategy for their yield and quality [1]. In plants, phenological growth stages include vegetative and reproductive growth stages, which had already been described based on a series of standard scales, such as the Fleckinger scale, basic Biologische Bundesantalt, Bundessortenamt and Chemische Industrie (BBCH) scale, etc. [2,3,4]. The BBCH scales represent a unified coding system for describing phenological similar growth stages in mono- and dicotyledonous plants. The basic BBCH scale, using a two-digit code, is designed with ten distinguishable principal growth stages using numbers from 0 to 9, each subdivided into ten secondary growth stages numbered from 0 to 9 in ascending order [4]. However, the basic BBCH scale cannot be precise enough in all growth stages to describe a specific stage. Thus, an extended BBCH scale, using a third-digit code, was established for some specific stages of plants. The so-called third-digit code, mesostage, allows further subdivisions of these secondary growth stages [3,4,5]. The basic and extended BBCH scales have been widely considered reliable for describing the phenological growth stages of horticultural crops, medicinal plants, aromatic plants, etc. [6,7,8,9,10]. These reports showed that the BBCH scale provides a standardized definition and detailed description of plant growth stages and will contribute to scientific communications between researchers and farmers for field management [4,11,12].

Abelmoschus manihot L. (A. manihot) belongs to the Malvaceae family and is commonly known as Huang Shu Kui (in China), Aibika (in Papua New Guinea and Indonesia), and Dakpul (in Korea). It is an annual or perennial plant for various uses, including food, pharmaceutical, chemical, fodder, timber, etc. [13]. Native to Asia and eastern Europe, it is mainly cultivated in China, Papua New Guinea, Eastern Indonesia, Nepal, Fiji, India, Sri Lanka, Vanuatu, New Caledonia, and Northern Australia [13,14,15,16]. A. manihot has greater productivity on infertile soils, easy to breed and develop, low-input planting, which make it an attractive potential crop [15,16,17]. The plant resembles okra but shows excellent variability in the shape and size of leaf, flower, fruit, branching and flowering [15,18,19]. More than 128 phytochemical ingredients, including flavonoids, polysaccharides, nucleosides, etc., have been isolated and identified from different parts of A. manihot [17,20,21]. These chemicals possess various biological activities, such asanti-diabetic nephropathy, antioxidant, antiadipogenic, anti-inflammatory, anticonvulsant, antidepressant, antitumor, immunomodulatory, and hepatoprotective activities, etc. However, they also affect cerebral infarction, bone loss, etc. [17,22,23]. In China, numerous health foods have been commercially developed using the flowers, roots, stems, and leaves of A. manihot [13]. Moreover, the tender leaves and flowers of A. manihot are consumed as traditional vegetables in China, South Pacific Islands, Papua New Guinea, and eastern Indonesia [14,17]. These examples highlight the greater nutritious and medicinal value of A. Manihot.

Despite the availability of these various uses in A. manihot, it remains a minor and underutilized crop. Therefore, the description of the phenological growth stages is relevant for agronomic and botanical studies, which is needed as a starting point to facilitate field management and research. Unfortunately, there are no reports on the phenological growth stages of A. manihot. Thus, this work aims to describe the principal and critical growth stages of A. manihot according to the BBCH scale, from germination/bud development to senescence under controlled and/or field conditions.

2. Materials and Methods

2.1. Study Site

The study site was located at the Village Jing-shan, Town Yong-an, District Shuangliu, Chengdu, China, at latitude 30°365954′ N, longitude 104°004114′ W and altitude 411–425 m above sea level. Climatically, the site has a typical subtropical humid monsoon climate, and the mean monthly temperature was 13.3 °C with a daily range of 13–17 °C, average maximum temperatures of 25.6 °C in July and an average minimum temperature of 5.5 °C in January. The annual total precipitation of 985.1 mm was recorded, mostly falling in summer with an average relative humidity of 81%. Soils were humic Umbrisols, acid (pH 5.5–6.0), with sandy-loam texture and high organic matter content (7–10%). Climatic data were collected from the China Meteorological Administration from 2021–2022 [24].

2.2. Seed Germination and Seedling Establishment

A. manihot seeds were harvested in December 2020 and December 2021 at the Village Jing-shan, Town Yong-an, District Shuangliu, Chengdu, China. Seeds were soaked in distilled water for 24 h at room temperature, and the water was changed two or three times. Petri dishes containing seeds were incubated in a daily photoperiod (12 h light/12 h dark) at 25 °C temperature. Different germinated seeds were recorded daily and selected for observing the initial growth stages during the germination stage. The germination experiment was performed in three repetitions with 100 seeds. Moreover, five seeds were planted in the 10 kg soil pots (28 cm × 21 cm). After germination, only one seedling per pot was maintained up to Stage 2, and 10 pots were established. The soil was kept moderately moist during the culture process, and unified weeding, insect control and disease prevention management were carried out.

2.3. Field Experiment and Phenological Observations

Data on measurement and phenological observations were collected from two consecutive growing seasons (2021–2022) under field conditions and in the greenhouse on the campus of Sichuan Agricultural University and Village Jing-shan, Town Yong-an, Chengdu, China. In the field, A. manihot seeds were sown in mid-February 2021 and 2022. After seedling emergence, a single seedling per pot was retained to a final density of 1 m × 2 m. Fifth healthy and uniform plants were randomly selected, labeled, and monitored throughout the growth cycle. Watering, fertilizers, fungicides, and insecticides were applied as needed. Weeds were hand removed at the early growth stages of the seedling. Measurements and observations of the phenological phases of each labeled plant were recorded two to three times per week (germination, leaf development, formation of side shoots, main stem elongation, inflorescence emergence, flowering, fruit development, maturation of fruit and seed, and senescence) or once per week. For a detailed description of the BBCH scale in A. manihot, several traits of agronomic importance (flower development, fruit length, fruit width, fruit length/width ratio, and fruit development) were observed at specific stages. To characterize flower bud development and establish a numeric scale, buds were collected at different stages. When the first floral bud on the main stem was visible (about 4 mm in length, 3 mm in width), we defined it as Stage 500 and began to collect it. At each observation, five representative plants were selected, and five flower buds were tagged and selected on the main stem of each plant. The external morphology of each bud was measured using an electronic vernier caliper and dissected and photographed with a Leica L2 stereomicroscope equipped with Nikon D7500.When mentioned, the date corresponding to a given growth stage was defined as when at least 50% of these plants reached a given stage. After flowering, 15 fruits from 15 plants were collected every three days until the date of harvest. Each fruit’s length, width, fresh weight, and dry weight were estimated with an electronic calliper and a precision balance, respectively. The morphological characters of different developmental fruits were observed using a binocular microscope at ×6 or ×10.

2.4. Statistical Analyses

All treatments were arranged in a completely randomized design with three replicates. Data were expressed as means ± SD. One-way analysis of variance (ANOVA) and Tukey’s test were performed using Waller-Duncan multi-interval test using SPSS 26.0 software (IBM^® Corporation, Armonk, NY, USA). The minimum significant difference (LSD) in the ANOVA test was applied to analyze the significant difference. Statistical significance was set 95% confidence level (p < 0.05).

3. Results

3.1. Principal Growth Stages

In the present study, the BBCH phenological scale specific to A. manihot was established and characterized with nine of ten principal growth stages (Table 1 and Figure 1). These principal stages begin with stage 0 for seed germination and ends with stage 9 for senescence and are divided into the vegetative and reproductive growth stages. The vegetative growth stages included stage 0 for seed germination and bud development, stage 1 for leaf development, stage 2 for primary stem growth, formation of a jorquette, stage 3 for formation and growth of side shoots, and stage 9 for senescence. The following reproductive growth stages contained stage 5 for inflorescence development, stage 6 for flowering, stage 7 for fruit development, and stage 8 for maturity of fruit and seed. In the BBCH scale, the principal growth stages are described using numbers from 0 to 9 in ascending order, while certain stages may be omitted depending on the plant species. For example, stage 4, representing harvestable vegetative parts or propagated organs, was omitted in this study. Several principal phases, such as leaf development, main stem growth, side shoots growth, flowering, fruit development, and fruit maturity, partially or completely overlap and/or parallel (Figure 1). In A. manihot, the two-digit code is sufficiently precise for seed germination and bud development, formation of side shoots, primary stem elongation, maturity of fruit and seed, and senescence and rest. However, for inflorescence development (Stage 5), flowering (Stage 6), and development of fruit (Stage 7), the third digit scale is necessary to further subdivide the secondary stage into more sub-stages [4]. Thus, 69 secondary growth stages are described with the nine principal growth stages in A. manihot.

Table 1. Description of phenological growth stages of A. manihot according to the basic and extended BBCH scale.

Phenophase	Two-Digit Code	Three-Digit Code	Description	Period
0	00	000	Dry/inactive seed or seed dressing (Figure 2a)	Late February
	01	001	Beginning of seed imbibition (Figure 2b)
	03	003	Seed imbibition complete (Figure 2c)
	05	005	Radicle emerged from the seed (Figure 2d)
	06a	006a	Elongation of the radicle, no root hair (Figure 2e)
	06b	006b	Formation of root hairs (Figure 2f)
	07	007	Hypocotyl with cotyledons breaking through seed coat (Figure 2g)
	08	008	Hypocotyl with cotyledons outside seed coat (Figure 2h)
	09a	009a	Emergence: Cotyledons break through the soil surface, folded cotyledons (Figure 2i)
	09b	009b	Emergence: elongation of hypocotyl, folded cotyledons (Figure 2j)
	09c	009c	Emergence: cotyledons unfold slightly (Figure 2k)
1	10	100	Cotyledons completely unfolded (Figure 3a)	Early March
	11a	101a	First leaf on main stem emergence (Figure 3b)
	11b	101b	The first leaf starts to grow or elongate (Figure 3c)
	11c	101c	The first leaf on the main stem unfolded (Figure 3d)
	12	102	The second leaf on the main stem unfolded (Figure 3e)
	13	103	The third leaf on the main stem unfolded (Figure 3f)
	14	104	The fourth leaf on the main stem unfolded (Figure 3g)
	15	105	The fifth leaf on the main stem unfolded (Figure 3h)	Late-March
	19	109	Ninth or more leaves on the main stem unfolded
		119	19th or more leaves on the main stem unfolded (Figure 3j)	Mid-April
		121	The first leaf on the side shoots unfolded
		122	The second leaf on the side shoots unfolded
		123	The third leaf on the side shoots unfolded
		129	Ninth or more leaves on side shoots unfolded (Figure 3j)	Late-April
2	21	201	The first side shoot is visible (Figure 3h)	Late-March
	22	202	The second side shoots visible
	23	203	The third side shoots visible (Figure 3i)
	25	205	The fifth side shoots visible
	27	207	The seventh side shoots visible (Figure 3j)
	29	209	Ninth or more side shoots are visible (Figure 3m).	Late April
3	31	301	Main stem up to 20 cm long (Figure 3i).	Late April
	32	302	Main stem up to 50 cm long (Figure 3j).
	33	303	Main stem up to 70 cm long (Figure 3k)	Mid-June
	34	304	Main stem up to 90 cm long (Figure 3m)
	35	305	Main stem up to 120 cm long (Figure 3n).	Late June
	37	307	Main stem up to 200 cm long (Figure 3o).	Late August
	39	309	Maximum main stem length reached up to 240 cm long.	Late October
5	50	500	The first floral buds on the main stem are visible (Figure 4a).	Mid-May
	51	501	Flower bud swelling (Figure 4b).
	53	503	The flower bud continues to swell, stigma visible inside the petals (Figure 4c).
	54	504	Flower bud and flower pedicel elongate (Figure 4d).
	55	505	First individual flowers visible until close (Figure 4e).	Late May
	56	506	The petals begin to turn yellow (Figure 4f).
	57	507	The petals break through the sepals (Figure 4g).
	59	509	The first flower petals visible, the petals elongated quickly, flower bud began to open. The first flower will blossom the next day (Figure 4h).	Early June
		520	First floral buds on side shoots visible	Late June
		529	The first flower on side shoots will blossom in the next day.
6	61	601	First flower opening and fruit set on the main stem (Figure 5a).	Early June
	62	602	Second flower opening and fruit set on the main stem.
	63	605	Fifth flower opening and fruit set on the main stem.
	69	609	Ninth or more flowers opening and fruit set on the main stem (Figure 5b).
		619	19th flower opening and fruit set on the main stem (Figure 5d).
		621	First flower opening and fruit set on the side shoots.
		629	Ninth or more flowers opening and fruit set on the side shoots (Figure 5e).
7	70	700	First flower on the main stem drops and fruit expose.	Early June
	71	701	First fruit on the main stem reach the final size (Figure 5b).	Early July
	72	702	Second fruit on the main stem reach the final size.
	73	703	Third fruit on the main stem reaches the final size.
	79	709	Ninth or more fruits on the main stem reach the final size.
		719	19th fruit on the main stem reach the final size.	Mid-July
		721	The first fruit on the side shoots reach the final size.
		722	The second fruit on the side shoots reach the final size.
		723	The third fruit on the side shoots reach the final size.
		729	Ninth or more fruits on the side shoots reach the final size (Figure 5e)
8	81	801	10% of fruits have a yellow pod. The seed coat becomes brown and hard.	Early August
	83	803	30% of fruits have a yellow pod. The seed coat becomes brown and hard.
	85	805	50% of fruits have a yellow pod. Seed coat becomes brown and hard (Figure 5g)
	87	807	90% of fruits have yellow pods. The seed coat becomes brown and hard.
	89	809	All of the fruit pod seed coat becomes brown and hard.
9	91	901	All fruits open, the fruit pod becomes brown and hard, and the leaf turns yellow (Figure 5g).	Midder December
	95	905	50% of leaves fall.	Late December
	96	906	All leaves are brown.
	97	907	All leaves fall, and aboveground parts dead (Figure 5h)	Late January

Table 1. Description of phenological growth stages of A. manihot according to the basic and extended BBCH scale.

Phenophase	Two-Digit Code	Three-Digit Code	Description	Period
0	00	000	Dry/inactive seed or seed dressing (Figure 2a)	Late February
	01	001	Beginning of seed imbibition (Figure 2b)
	03	003	Seed imbibition complete (Figure 2c)
	05	005	Radicle emerged from the seed (Figure 2d)
	06a	006a	Elongation of the radicle, no root hair (Figure 2e)
	06b	006b	Formation of root hairs (Figure 2f)
	07	007	Hypocotyl with cotyledons breaking through seed coat (Figure 2g)
	08	008	Hypocotyl with cotyledons outside seed coat (Figure 2h)
	09a	009a	Emergence: Cotyledons break through the soil surface, folded cotyledons (Figure 2i)
	09b	009b	Emergence: elongation of hypocotyl, folded cotyledons (Figure 2j)
	09c	009c	Emergence: cotyledons unfold slightly (Figure 2k)
1	10	100	Cotyledons completely unfolded (Figure 3a)	Early March
	11a	101a	First leaf on main stem emergence (Figure 3b)
	11b	101b	The first leaf starts to grow or elongate (Figure 3c)
	11c	101c	The first leaf on the main stem unfolded (Figure 3d)
	12	102	The second leaf on the main stem unfolded (Figure 3e)
	13	103	The third leaf on the main stem unfolded (Figure 3f)
	14	104	The fourth leaf on the main stem unfolded (Figure 3g)
	15	105	The fifth leaf on the main stem unfolded (Figure 3h)	Late-March
	19	109	Ninth or more leaves on the main stem unfolded
		119	19th or more leaves on the main stem unfolded (Figure 3j)	Mid-April
		121	The first leaf on the side shoots unfolded
		122	The second leaf on the side shoots unfolded
		123	The third leaf on the side shoots unfolded
		129	Ninth or more leaves on side shoots unfolded (Figure 3j)	Late-April
2	21	201	The first side shoot is visible (Figure 3h)	Late-March
	22	202	The second side shoots visible
	23	203	The third side shoots visible (Figure 3i)
	25	205	The fifth side shoots visible
	27	207	The seventh side shoots visible (Figure 3j)
	29	209	Ninth or more side shoots are visible (Figure 3m).	Late April
3	31	301	Main stem up to 20 cm long (Figure 3i).	Late April
	32	302	Main stem up to 50 cm long (Figure 3j).
	33	303	Main stem up to 70 cm long (Figure 3k)	Mid-June
	34	304	Main stem up to 90 cm long (Figure 3m)
	35	305	Main stem up to 120 cm long (Figure 3n).	Late June
	37	307	Main stem up to 200 cm long (Figure 3o).	Late August
	39	309	Maximum main stem length reached up to 240 cm long.	Late October
5	50	500	The first floral buds on the main stem are visible (Figure 4a).	Mid-May
	51	501	Flower bud swelling (Figure 4b).
	53	503	The flower bud continues to swell, stigma visible inside the petals (Figure 4c).
	54	504	Flower bud and flower pedicel elongate (Figure 4d).
	55	505	First individual flowers visible until close (Figure 4e).	Late May
	56	506	The petals begin to turn yellow (Figure 4f).
	57	507	The petals break through the sepals (Figure 4g).
	59	509	The first flower petals visible, the petals elongated quickly, flower bud began to open. The first flower will blossom the next day (Figure 4h).	Early June
		520	First floral buds on side shoots visible	Late June
		529	The first flower on side shoots will blossom in the next day.
6	61	601	First flower opening and fruit set on the main stem (Figure 5a).	Early June
	62	602	Second flower opening and fruit set on the main stem.
	63	605	Fifth flower opening and fruit set on the main stem.
	69	609	Ninth or more flowers opening and fruit set on the main stem (Figure 5b).
		619	19th flower opening and fruit set on the main stem (Figure 5d).
		621	First flower opening and fruit set on the side shoots.
		629	Ninth or more flowers opening and fruit set on the side shoots (Figure 5e).
7	70	700	First flower on the main stem drops and fruit expose.	Early June
	71	701	First fruit on the main stem reach the final size (Figure 5b).	Early July
	72	702	Second fruit on the main stem reach the final size.
	73	703	Third fruit on the main stem reaches the final size.
	79	709	Ninth or more fruits on the main stem reach the final size.
		719	19th fruit on the main stem reach the final size.	Mid-July
		721	The first fruit on the side shoots reach the final size.
		722	The second fruit on the side shoots reach the final size.
		723	The third fruit on the side shoots reach the final size.
		729	Ninth or more fruits on the side shoots reach the final size (Figure 5e)
8	81	801	10% of fruits have a yellow pod. The seed coat becomes brown and hard.	Early August
	83	803	30% of fruits have a yellow pod. The seed coat becomes brown and hard.
	85	805	50% of fruits have a yellow pod. Seed coat becomes brown and hard (Figure 5g)
	87	807	90% of fruits have yellow pods. The seed coat becomes brown and hard.
	89	809	All of the fruit pod seed coat becomes brown and hard.
9	91	901	All fruits open, the fruit pod becomes brown and hard, and the leaf turns yellow (Figure 5g).	Midder December
	95	905	50% of leaves fall.	Late December
	96	906	All leaves are brown.
	97	907	All leaves fall, and aboveground parts dead (Figure 5h)	Late January

Figure 1. Principal growth stages of A. manihot and their association with temperature and precipitation. Bar represents the elapsed time of different growth stages.

Figure 1. Principal growth stages of A. manihot and their association with temperature and precipitation. Bar represents the elapsed time of different growth stages.

Figure 2. Illustrations of seed germination of A. manihot according to BBCH scale. (a), dry seed. (b), beginning of seed imbibition. (c), seed imbibition complete. (d), radicle emerged from seed. (e), elongation of radicle without root hair. (f), formation of root hairs. (g), hypocotyl with cotyledons breaking through seed coat. (h), hypocotyl with cotyledons outside seed coat. (i), cotyledons break through soil surface, and cotyledons folded. (j), continuous elongation of hypocotyl, folded cotyledons. (k), cotyledons unfold slightly. I, root hair. II, hypocotyl and cotyledons.

Figure 2. Illustrations of seed germination of A. manihot according to BBCH scale. (a), dry seed. (b), beginning of seed imbibition. (c), seed imbibition complete. (d), radicle emerged from seed. (e), elongation of radicle without root hair. (f), formation of root hairs. (g), hypocotyl with cotyledons breaking through seed coat. (h), hypocotyl with cotyledons outside seed coat. (i), cotyledons break through soil surface, and cotyledons folded. (j), continuous elongation of hypocotyl, folded cotyledons. (k), cotyledons unfold slightly. I, root hair. II, hypocotyl and cotyledons.

Figure 3. Illustrations of main vegetative growth stages of A. manihot according to the BBCH scale. (a), Cotyledons completely unfolded. (b), First leaf on main shoot emergence. (c), The first leaf starts to grow and develop. (d), The first leaf on the main shoot unfolded. (e), The second leaf on the main shoot unfolded. (f), The third leaf on the main shoot unfolded. (g), The fourth leaf on the main shoot unfolded. (h), The fifth leaf on the main shoot unfolded. (i), The seventh leaf on the main shoot unfolded, and the third side shoots visible and main stem grown up to the length of 20 cm. (j), The seventh side shoots visible, and main shoot grown up to the length of 50 cm. (k), The 19th or more leaves on the main shoot unfolded, and main shoot grown up to the length of 70 cm. (l), The ninth or more leaves on the side shoots unfolded, and main shoot grown up to the length of 170 cm. (m), The ninth or more side shoots are visible, and main shoot grown up to the length of 90 cm. (n), Main shoot grown up to the length of 120 cm. (o), Main shoot grown up to the length of 120 cm.

Figure 3. Illustrations of main vegetative growth stages of A. manihot according to the BBCH scale. (a), Cotyledons completely unfolded. (b), First leaf on main shoot emergence. (c), The first leaf starts to grow and develop. (d), The first leaf on the main shoot unfolded. (e), The second leaf on the main shoot unfolded. (f), The third leaf on the main shoot unfolded. (g), The fourth leaf on the main shoot unfolded. (h), The fifth leaf on the main shoot unfolded. (i), The seventh leaf on the main shoot unfolded, and the third side shoots visible and main stem grown up to the length of 20 cm. (j), The seventh side shoots visible, and main shoot grown up to the length of 50 cm. (k), The 19th or more leaves on the main shoot unfolded, and main shoot grown up to the length of 70 cm. (l), The ninth or more leaves on the side shoots unfolded, and main shoot grown up to the length of 170 cm. (m), The ninth or more side shoots are visible, and main shoot grown up to the length of 90 cm. (n), Main shoot grown up to the length of 120 cm. (o), Main shoot grown up to the length of 120 cm.

Figure 4. Inflorescence development of A. manihot according to the BBCH scale. (a), The first floral bud on the main shoot was visible. (b), Flower bud started to swell. (c), The flower bud continued to swell, stigma was visible inside the petals. (d), Flower bud and flower pedicel began elongation. (e), First flower was visible until close. (f), The petals started to turn yellow. (g), The petals break through the sepals. (h), The petals of first flower was visible, the petals quickly elongated, and the first flower will blossom the next day. I–VIII, anatomy of flower bud structure at different development stages. Scale bar = 5 mm.

Figure 4. Inflorescence development of A. manihot according to the BBCH scale. (a), The first floral bud on the main shoot was visible. (b), Flower bud started to swell. (c), The flower bud continued to swell, stigma was visible inside the petals. (d), Flower bud and flower pedicel began elongation. (e), First flower was visible until close. (f), The petals started to turn yellow. (g), The petals break through the sepals. (h), The petals of first flower was visible, the petals quickly elongated, and the first flower will blossom the next day. I–VIII, anatomy of flower bud structure at different development stages. Scale bar = 5 mm.

Figure 5. Illustrations of main reproductive growth stages in A. manihot according to the BBCH scale. (a), First flower blossomed, and fruit set on the main shoot. (b), Ninth or more flowers blossomed and fruit set on the main shoot. (c), Seventh flower blossomed on the side shoots. (d), 19th flower blossomed, and fruit set on the main shoot. (e), Ninth or more flowers blossomed, and fruit set on the side shoots. (f), 40% of fruits have yellow pods. The seed coat became brown and hard. (g), All fruits open, the fruit pod became brown and hard, and the leaves turn yellow. (h), All leaves fall, and aboveground parts dead.

Figure 5. Illustrations of main reproductive growth stages in A. manihot according to the BBCH scale. (a), First flower blossomed, and fruit set on the main shoot. (b), Ninth or more flowers blossomed and fruit set on the main shoot. (c), Seventh flower blossomed on the side shoots. (d), 19th flower blossomed, and fruit set on the main shoot. (e), Ninth or more flowers blossomed, and fruit set on the side shoots. (f), 40% of fruits have yellow pods. The seed coat became brown and hard. (g), All fruits open, the fruit pod became brown and hard, and the leaves turn yellow. (h), All leaves fall, and aboveground parts dead.

3.2. Specific Growth Stages

3.2.1. Principal Growth Stage 0: Germination

Although A. manihot may be propagated vegetatively using their roots, the most common way of reproduction is through seeds. Mature A. manihot seeds may be harvested from June to December with a hard seed coat and different brown hues (stage 809, Figure 5h). The length and width of the seeds are 0.3–0.4 cm, and 0.2–0.3 cm, respectively (Figure S5). This stage describes the germination period from planted dry seed (stage 000, Figure 2a) to radicle emergence and development of cotyledons (stage 005–009, Figure 2d–k and Table 1). During the seed germination, the 05 secondary growth stage represented the emergence of radicle from the seed coat, which occurred at 3–4 days (Figure 2d). The 06 secondary stages, Phases a and b, were differentiated, and their evolution was different from one day to the next (Figure 2e,f). Phase 06a of this secondary stage lasted about 1–2 days, and Phase 06b lasted about 2–3 days with root hair formation. The 09 secondary stages a, b and c with noticeable differences were evaluated separately (Figure 2i–k). Phase a was represented by the seedling with a seed coat wrapped with semi-expanded cotyledons. Phase b was characterized by the seedlings with unopened cotyledon and slowly elongating hypocotyl. Phase c was characterized by the seedlings with fully developed cotyledons with irregular and slightly opening cotyledons. This sub-stage for each phase lasted between 1–3 days. During the germination stage, the seed imbibition occurred from the 2nd to 4th day (stage 003, Figure 2c and Table 1), and the radicle emergence and elongation occurred between 5 and 6 days (stage 005, Figure 2d). After seven to 8 days, the hypocotyl and cotyledons break through the seed coat (stage 007, Figure 2g), and cotyledons gradually unfold with rising germination time (stage 009, Figure 2i–k). The specific growth stage of A. manihot seeds with high vigor is usually completed in 7–24 days depending on the sowing depth and soil temperature.

3.2.2. Principal Growth Stage 1: Leaf Development

In general, leaves of A. manihot emerged one by one, and the maximum number of leaves on the main stem was observed to be around 80–100 during an annual growing cycle. Stage 1 began with cotyledons completely unfolded (Stage 10, Figure 3a, Table 1). At this time, the cotyledon of A. manihot may be visible but are less than 3 cm long. Under the present conditions, the duration from sowing to stage 10 lasted between 11 and 24 days, depended on the sowing time and depth, management and/or environmental conditions, etc. The next stages of leaves continued with the unfolding of the first true leaf on the main shoot (stage 11 or 101) and finish when at least nine (stage 19) or all leaves of the main shoot had fully unfolded. During this process, the side shoot had formed along with the development of leaves, partially or completely overlapping and/or parallel. This stage began with the first leaf fully unfolding on the side shoot (stage 121) and ends when at least nine or more leaves had been unfolded (stage 129, Figure 3j). At last, the A. manihot canopy is completed when all leaves were unfolded and horizontal in Mid-October and went to senescence until End December or January of the next year (Figure 5).

3.2.3. Principal Growth Stage 2: Formation of Side Shoots

A. manihot is characterized by a development of 4 to 10 and/or more primary axillary side shoots in the shape of a plexiform and continues with the subsequent axillary side shoots with the primary stem elongation, and forms a series of the respective secondary, tertiary, and higher order axillary side shoots (stage 307, Figure 3o). Stage 2 began with the first primary axillary side shoot visible (stage 21 or 201), and ended with the forming of nine or more axillary side shoots, depending on the height of the main shoot (stage 29 or 209, Figure 3m, Table 1). The appearance of side shoots was partially or completely overlap and/or parallel with stages of leaf development (stage 109, 129, Figure 3l), main shoot elongation (stage 309), inflorescence emergence (stage 509, 529, Figure 4h), flowering (stage 609, 629, Figure 4b,e) and fruit development (stage 709, 729, Figure 5e). The first side shoots appeared when the fifth leaves unfolded on the main shoot (Stages 105/201, Figure 3h). The first primary axillary side shoots are typically visible 30–45 days after sowing.

3.2.4. Principal Growth Stage 3: Shoot Development (Main Shoot)

Stage 3 began when the length of the main shoot reached 20~30 cm (stage 301, Table 1), about 10% of the final length of the main shoot (stage 309, Table 1). At stage 309, the length of the main shoot can reach 220–240 cm. This durations and lengths of main shoot varied by sowing time and environmental conditions. The main shoot elongation was until the end of October (Figure 1). Our observations also showed that the durations from one stage to the next are related to cultivation management, growth conditions, etc. In the present study, the elongation of the main shoot in parallel to the leaf development (stage 109, 129, Figure 3), side shoot form (stage 209), inflorescence emergence (stage 500–529), flowering (stage 600–629), fruit development (stage 700–729), and fruit mature (stage 800–809).

3.2.5. Principal Growth Stage 5: Inflorescence Emergence

Stage 5 described the emergence and development of inflorescence of the whole plant. Inflorescence emergence occurred from End May to early June (about 85–100 days after sowing), depending on sowing time and growth condition. At this stage, the primary shoot diameter increased to 1–2 cm and up to the height of 50–150 cm with 4–6 side shoots. As inflorescence remained closed, the morphological character of inflorescence cannot be directly observed. Eight mesostages were used to describe a detailed development process of inflorescence (Table 1 and

Table 2. Major morphological parameters of inflorescence development in A. manihot at different days after emergence (DAE).

Stage	DAE	Bud Length (BUL, mm)	Bud Width (BUW, mm)	BUL/BUW	Pistil Length (PL, mm)	Stamen Length (SL, mm)	PIL/STL	Bracts Length (BRL, mm)	Pedicel Length (PEL, mm)
500	1–2	4.45 ± 0.17 h	3.38 ± 0.11 g	1.32 ± 0.03 e	1.56 ± 0.04 h	2.65 ± 0.03 h	0.59 ± 0.02 e	6.63 ± 0.31 d	Not formed
501	3–4	6.67 ± 0.28 g	4.61 ± 0.18 f	1.45 ± 0.10 d	2.82 ± 0.19 g	3.72 ± 0.39 g	0.76 ± 0.05 d	7.25 ± 0.42 c	2.04 ± 0.19 f
503	5–6	7.67 ± 0.13 f	5.04 ± 0.10 e	1.52 ± 0.01 d	5.16 ± 0.15 f	4.91 ± 0.04 f	1.05 ± 0.02 c	8.14 ± 0.66 c	2.79 ± 0.19 e
504	7–9	18.94 ± 0.13 e	10.08 ± 0.65 d	1.88 ± 0.08 c	8.87 ± 0.11 e	6.91 ± 0.05 e	1.17 ± 0.02 b	13.31 ± 0.88 b	13.71 ± 1.26 d
505	10–11	21.91 ± 0.92 d	11.70 ± 0.57 c	1.88 ± 0.11 c	9.21 ± 0.39 d	7.77 ± 0.41 d	1.19 ± 0.02 a	13.63 ± 1.01 b	13.79 ± 0.46 d
506	12–13	24.65 ± 0.48 c	12.53 ± 0.25 b	1.97 ± 0.04 c	10.64 ± 0.60 c	8.73 ± 0.41 c	1.22 ± 0.03 a	14.51 ± 0.89 b	17.68 ± 0.61 c
507	14–15	35.09 ± 1.95 b	14.08 ± 0.88 a	2.49 ± 0.07 b	12.22 ± 0.14 b	9.65 ± 0.43 b	1.27 ± 0.04 a	17.25 ± 0.52 a	26.28 ± 1.50 b
509	16	51.29 ± 3.84 a	15.03 ± 0.89 a	3.41 ± 0.08 a	20.35 ± 0.31 a	15.09 ± 1.07 a	1.35 ± 0.09 a	18.47 ± 1.20 a	34.01 ± 2.89 a

Data were expressed as means ± SD (n = 5). Different lowercase letters (a–h) represented significant differences in response to different development stages (p < 0.05). Means with the same letters represented no significant differences (p ≥ 0.05).

and Figure 4). It began when the inflorescence was less than 5 mm and was wrapped by bracts and sepals (stage 500, Figure 4a, I), and then it developed to stage 501, accompanied by sepal differentiation. The peduncle had reached about 10% of its expected growth, and the inflorescence was about 6 mm in diameter (Figure 4b, I). It may take 1–3 days to pass from one stage to the subsequent stage, depending on the growth rate (from stage 51 or 501 to stage 52 or 502 Figure 4b,c, II, III). At stage 503 (Figure 4c, I), the elongation of peduncles and the inflorescences became visible in slightly opening bracts, and a stigma emerged (Figure 4c, III). These inflorescences showed swelling and are fully visible at stage 505 when the peduncle had reached 50% of its final length (Figure 4e, V). The flower bud growth stage ended and fully expands when peduncle elongation and flower bud diameter were maximized. The first petal was visible, but flower buds remain closed and were ready to open (Table 2, stage 509, Figure 4h, VIII). At one and/or two days after emergence (DAE), the corollas were significantly less than those of calyx (<1/2), and the buds were green with a length-width ratio of 1.32. Moreover, the pistils were not yet developed and protruded from the stamens, and the length ratio was less than 1(stage 500, Table 2, Figure 4a, I). At five and/or 6 DAE, the pistils elongated rapidly, and their length was significantly higher than the stamens. The pistils protruded from the stamens, the pistils and stamens length ratio was higher than 1, and the peduncles began to develop (stage 503, Figure 4c, III). From 7 to 9 DAE, the peduncles quickly elongated up to about half of the final length, and the petal anthers began to develop and expanded. Then, the flower buds began to elongate rapidly (stage 504, Figure 4d, IV). At ten and/or 11 DAE, the inflorescences continued to develop until their length was longer than those of the bracts, and the stigma began to swell and becomes darker (stage 505, Figure 4e, V). At 12 and/or 13 DAE, the petals gradually turned yellow, and accelerated growth break through the sepals (stage 506, Figure 4f, VI). At 14–15 DAE, the inflorescences continued to accelerate their development, and then the petals turned yellow (stage 507, Figure 4g, VII). At about 16 DAE, the inflorescences rapidly developed up to their final size with a length of 51.29 mm and a width of 15.03 mm. At the same time, the pistil and stamen also reached a final size, and then these inflorescences will open the following day, namely flowering (stage 509, Figure 4h, VIII and Figure 5). The developmental stages showed statistical significance for the test parameters of inflorescence in A. manihot (Table 2). Moreover, new inflorescences appeared one by one each one and/or two days and last six months from Early June to Mid-November. Depending on the climatic conditions, the inflorescences can continue to develop, accompanied by the elongation of the main shoot, side shoot, and leaf and fruit development (Figure 1).

3.2.6. Principal Growth Stage 6: Flowering

As shown in Figure 5, A. manihot flower includes a pedicel, four sepals, four or five bracks, five petals, an ovary, and a pistil. Its petals are light yellow with a length of 7.8–10.8 cm and a transverse diameter of 12.3–20.0 cm. The principal stage 6 began with the opening of the first flower in the main shoot from 8:00 a.m. to 15:00 and/or to 17:00 p.m. (stage 601, Figure 5a), in which the pistil can be directly observed with five purple stigmas. After pollination, when pollinators were present, five petals were closed and began to fruit set (Figure 5d and Figure 6). Without pollination, it may set parthenocarpic fruits. Stage 602 represented the second flowers opening, and fruits set in plants. Stage 605 represented the fifth flower opening, and fruits set in plants. Stage 609 represented 19th and/or more flowers opening and fruits set in plants. Over the same period, the elongation of main shoot and side shoot development were complete. For the side shoot, stage 621 represented the first flowers opening, and fruits set on the side shoot. Stage 629 represented the 9th and/or more flowers opening, and fruits set on the side shoot. This stage was completed when all fruits set (stage 619, stage 629, Figure 5d,e). This stage has been described during the timing of flowering. However, counting flowers were imprecise, with 20–50 at this stage, due to the uncertain length of the main shoot and side shoots and the number of side shoots, depending on growth conditions and sowing time. Moreover, these flowers unfolded sequentially from the base to the apex of the main shoot and side shoot, and 1–10 flowers unfolded in a single plant daily.

3.2.7. Principal Growth Stage 7: Fruit and Seed Development

Fruit development occurred from Mid-June to Last December, and fruits attained physiological maturity about 21–30 days. When the flower fell, the young seeds started to differentiate, and the growth of the ovaries begins. Stage 7 described concurrent fruit and seed development, morphology, and structure (Table 1 and Figure S3). Fruits changed from green to yellow, green with the development process and turned dark brown with bristles at different maturity stages (Figure 7). Figure S4 represented the transverse and longitudinal diameter of A. manihot fruit at different development stages. Figure S5 showed the transverse, longitudinal and side diameters of A. manihot seeds at different development stages. The dry weight (DW) of single fruit ranged from 0.15 g to 1.8 g (with seed, Figure S6). The fruit development began when the first flower was opening, and the fruit was set on the main shoot, describing the development of first fruits (Stage 700). Stage 701 was observed when the first fruit changed color from green to dark brown and reached its final size (Figure 5b and Figure 7). Stage 702 was described when the color of the second fruit changed from green to dark brown and reached its final size. Stage 719 represented all fruit reached its final size with dark brown. Stage 721 represented the first fruits reaching the final size for the side shoot. Stage 729 represented the 9th and/or more fruits reaching the final size. When all fruits reached the final sizes on the main and side shoot, this stage was completed (stage 719, stage 729, Figure 5e). However, counting fruits were imprecise depending on the number of flowers, pollination, insect attack, etc. Therefore, the stages of fruit development partially or completely overlap and/or parallel with flowering (e.g., stages 619/705 or 629/729/802).

3.2.8. Principal Growth Stage 8: Maturity of Fruit and Seed

Stage 8 described the maturity of the fruit and seed of A. manihot. When fully mature, the fruit changed from brown to black, which bristles may cover. The fruit is parthenocarpic, containing up to 80–100 seeds. Fruit and seed maturity was associated with fruit coloration and relative water content. Stage 81 or 801 began when 10% of fruits were typically fully mature color (Table 1). Stage 85/805 was when 50% of the fruits were typical fully mature color. By growth, stage 89/809, almost all fruits of fruits were typically fully mature in color and ready for harvest.

3.2.9. Principal Growth Stage 9: Senescence

Senescence is affected by cultural and climatic conditions. When stressed, the leaves may fall earlier, while in favorable condition, senescence and leaf drop occur from Mid-December to last December or Early January in the next year. Once reproductive development finish, A. manihot plant senescence occurred, and its overground parts completed their life cycle (Figure 5h). Stage 9 described the senescence process of A. manihot and began with the color changes of leaves and stems from green to yellow, which occurred Last Sept. (Table 1, Figure 1 and Figure 5h). The start and duration of this stage can be affected by pests or diseases. Stage 91/901 began with 10% of leaves brownish and basal leaves falling (Figure 5g). Stage 95/905 represented 50% of leaves fall. Stage 97/907 represented all leaves fall, and the withered and dead aboveground parts, indicating the end of the annual growth cycle in A. manihot (Figure 5h). When this stage finished, the roots were ready to harvest and/or used as propagated organs.

4. Discussion

The BBCH scale is a consensual unified tool for standardized definition and detailed description of plant growth stages, which is of guiding comprehensive agronomic practices, like irrigation and nutrient management, fruit harvest, prevention and control of diseases and pests, etc. [4]. The practical use of in 1974 and 1992, the basic and extended BBCH scales were designed to describe growth stages in mono- and dicotyledonous plans [3,6]. To this day, BBCH scale has been generally applied to describe phenological growth stages in both herbaceous and woody plants, such as Arabidopsis [25], Salvia hispanica [26], Artemisia annua [27], Panax ginseng [9], Capsicum annuum, Capsicum chinense, Capsicum baccatum [8], Physalis peruviana [28], Rosa sp. [29], Prunus avium [7], etc. Studies have shown that the specialized metabolites, such as artemisinin in A. annua are related to the phenological phase [30]. Moreover, the correlation between the phenological growth stage and chemical compositions in chipilín leaves was observed, which suggested that the contents of proteins, minerals, phenolic compounds, and total flavonoids are related to the phenological stage of the leaf [30]. These reports showed the BBCH scale to various plants provides a good and uniform reference for growers and scientists and is also of great significance for breeding and production application. As the planting area of A. manihot increases yearly, a detailed and standardized description of the different growth stages for A. manihot is needed. Our report indicated that A. manihot is defined by nine principal growth stages and69 secondary growth stages was described for seed germination, inflorescence emergence, flowering and set, and fruit development (Table 1, Figure 2, Figure 3, Figure 4 and Figure 5). In some plant species, mesostages were used in particular for growth stages of leaf development, shoot development, inflorescence development, flowering, and fruiting [8,9,11,31]. The BBCH scale for various plants has ranged from stages 208 to stages 809 [4]. For example, scales for Physalis peruviana, Salvia hispanica and Panax ginseng have 38, 40 and 58 stages, respectively [9,21,28]. However, the number of stages specific to A. manihot was significantly higher than those in other species (Stage 5). This may be related to the indeterminate and complex growth character of A. manihot (Figure 1, Figure 3 and Figure 6). In Physalis peruviana and Panax ginseng plants, several stages with totally or partially overlap phenomena were observed [9,28]. However, in A. manihot, this phenomenon occurred simultaneously for several months, which still needed further investigation.

Vegetative growth stages of A. manihot includes seed germination and bud development (stage 000–009), leaf development (100–129), formation of side shoots (Stage 200–209) and stem elongation growth (301–309) (Table 1, Figure 2 and Figure 3). As shown in Figure 2, it took 8–15 days from seed to bud. However, it might take about 15–20 days in the field, depending on sowing time, depth, temperature, soil conditions, and seed storage time, etc. Our observations were similar to the previous reports on seed germination and bud development, but there were some differences, especially in the formation of root hairs and cotyledon development [4,32]. These differences may be related to seed characteristics in various plant species [33]. Stage 0 was followed by leaf development (Stage 1, 100–129), formation of side shoots (Stage 2, 200–209) and main shoot elongation(Stage 3, 300–309). The events of seed germination and leaf development completed within about 2–5 weeks from Early March to Early April, whereas the elongation of the main shoots is continuous from Early April to Middle October, and the formation and growth of side shoots took within 6–24 weeks from Middle April to End September, sometimes longer that depend on climatic temperature. Main and side shoot development is strictly linear. The formation and development of leaves also continued during main and side shoot elongation (Figure 3), and its morphological characteristics were related to phenological growth stages (Figure 3b–h, Figures S1 and S2).

At last, A. manihot has an outline that roughly forms a typically plexiform with main shoots and 6–12 primary axillary side shoots. By the end of the season, main shoots can reach from 150 to 300 cm depending on the sowing date, soil nutrition and management, regular watering, irrigation, or fertilization, etc. [15]. The lengths of the main and side shoot directly influence the number of inflorescence and fruit in A. manihot. During field management, more side shoots and protection of vegetative phases will help to improve higher flower yield. Thus, A. manihot growers also regulate the number of primary and side shootsand their proportion based on climate and their growth state.

Moreover, the occurrence and time of these stages vary with climatic conditions and seed treatment, such as temperature, soil moisture, etc. In most plants, like Indian blackberry (Syzygium cumini L.), Cynara (Cynara cardunculus)and dragon fruit (Hylocereus undatus), vegetative growth stages are largely governed by climatic variables and agronomic practices [31,34,35,36]. These factors can directly and/or indirectly affectthe growth and reproduction of plants.A series of findings have shown that atmospheric temperature and soil moistureare the major driving principle for the vegetation growthstages [36]. In A. manihot, in most cases, vegetative phases totally or partially overlap, and the development of vegetative growth proceeds overlap and/or in parallel with the reproductive stages (Table 1 and Figure 1).

The inflorescence development (stage 500–509) and flowering (stage 600–609) in A. manihot are the most noteworthy stages [15]. Based on our observations, the corroboration with weather parameters, as depicted in Figure 1, the transition of vegetative growth stages to reproductive growth stages (flower bud differentiation) (stage 500) was directly influenced by weather parameters [36,37]. In A. manihot, although inflorescence formation needs a threshold temperature (22–25 °C), breaching this threshold, the inflorescence development stages cannot be controlled by the temperature (Figure 1). Unlike most crops, the reproductive phase of A. manihot exhibit quite long because inflorescence can continuously form along with the growth of main and side shoots from Middle June to Middle December (~180 days). After inflorescence initiation, the development continued for 5–6 months for A. manihot up to Middle Dec. The inflorescence growth indicated slow growth properties and showed rapid growth (Table 2, Figure 4 and Figure 6). This may be due to the elongation of the floral tube and style (Figure 4 and Table 2). In the current study, we found that the floral anthesis of A. manihot lasts for a short lifespan (7–10 h). Moreover, this was a continuous process, and once every day with 1–10 flowers, depending on the length of the main shoot and the number of side shoots. Variations in flowering time were related to various factors, such as temperature, decreasing or increasing photoperiod, significant rain in summer, humidity, etc. [36,37,38]. In Hylocereus undatus, the reports showed that the flowering concurs with the prevalence of long day length (≥13 h), rainfall, higher humidity (>80%) and moderate level of temperature (≈28 °C) [31]. In A. manihot, the length of the flowering period varied from one plant to another plant, which was directly and/or indirectly influenced by the complicated controllable (sowing time, cultivar management, etc.) and uncontrollable variables factors (pests, diseases, climate, and environment, etc.) [15,16,17]. There is a significant difference in the number of flowers among different plants, which is closely related to plant height and branch number, as well as management of nutrition, water, pests, and diseases, etc. This supports the observation that the flowering period is cultivar dependent in cherry and strawberry plants [7,39,40]. In A. manihot, flowering phenology is characterized by some secondary stages like anthesis, pollination and fading of flowers on the same day. Thus, it is difficult to estimate the flowering percentage, which differs from other plant species [4]. There are similar descriptions of flower development for other Malvaceae plants, like Cotton and Okra [18,19,41]. During the flowering period of the A. manihot flower, five stigmas of the pistil are tightly packed together at Stage 509 (Figure 4). Still, the stigmas extend outward and flip over when flowering, and then they are closer to the anther (Figure 5). Moreover, the stigma and stamens are exposed, which is conducive to cross-pollination by bees, ants, etc. After bagging, A. manihot flowers can normally produce pollen, and the pollen has high vitality and can undergo self-pollination (Data not shown). These results indicated that A. manihot flowers are hermaphrodites and may self-fertilize or cross. This is similar to the breeding systems of Gossypium spp. and A. esculentus, and the crossing rate of A. esculentus ranges from 2% to 63% [18,19]. These detailed descriptions of A. manihot flower development allow connecting studies on flower biology and provide a consensus-unified approach to understanding the A. manihot of flower development and its morphological character.

Fruit set begins the ovary swelling, which occurs after anthesis, and this is a continuous phenomenon. A. manihot fruit maturation (stage 801–809) occurs between the Middle of July and the Middle of January the following year. August–December is the significant period of fruit maturity (Stage 7, Figure 1). Almost all flowers may set fruit, indicating that the rate of fruit set in A. manihot is very high. Fruit development of A. manihot showed a sigmoidal growth pattern with three noticeable stages (Early, Middle, and Late stages) (Figure 7). In our case, A. manihot fruit usually takes between 27 and 30 days to reach full size with climatic conditions (Figure 7 and Figures S4–S6). The early stage of fruit development is primarily marked by develop and enlargement of the pericarp, embryo, and seed coat. The endosperm is initially liquid and gradually becomes solid with the developing embryo (Figure 7 and Figure S3). The early stage of fruit development rapidly grew with the gradual formation of cotyledons from 3 to 9 days. Then, the fruit development reached relatively stable growth from 12 to 15 days, when the cotyledon and radicle completely formed, and the seed size reached the maximum. During this phase, the measure remains unchanged, but the synthesis and accumulation of some chemicals take place in the seeds (Figure 7). The last phase is signified with pod lignification, colour change, moisture loss, size change, pod dehiscence, bract abscission, etc., lasting 6–9 days from 18 to 27 days. This phase included the seed coat gradually turning brown to black, the seed surface appearing hairy, the cotyledon gradually turning yellow, and the kernel becoming harder (Figure 7 and Table 2). Change in pod color is the most apparent part of fruit maturation and is caused by the degradation of chlorophyll and synthesis of anthocyanin [42]. Moreover, the present observations showed that the sigmoid curve of fruit growth and kernel growth overlap. In A. manihot fruits, it has five ovules in the ovary. Pods are subsessile, linear, acuminate, compressed, and finely pubescent with 60–80 seed, 4.5–6.5 cm long and 1.5–2.5 cm wide (Figure 7). The weights of single fruit ranged from 3.5to 6.5 g, and the values were related to genetics, growth environment, development period, and fruit position. Moreover, the fruits during early development (July–September) were larger than those of later period development (October–December). The maturation period from July to September (about 24 days) was shorter than from October to December (27–30 days). Certain differences in the number of fruits per plant, length, and width were observed in different plants and seasons. Such as temperature and rainfall, as well as other reasons. Other studies showed that abnormal climate conditions could affect not only inflorescence development, flowering period, and fruit set but also fruit development and maturation period [36,37]. Basnett et al. reported that mature and/or immature fruiting are primarily associated with day length during fruit development, known as late phenological events [43]. However, to what extent the effects of climatic conditions, season and shoot types on the variable maturation period, weight, and size during the A. manihot fruit development needs further study.

Nutrient management, water requirement and pest control are critical field operations for flower and fruit yield enhancement and quality improvement in plants [9,26,31]. Accordingly, fertilization and irrigation management are needed to support better growth during the formation of side shoots (stage 203/301, Figure 3i), before inflorescence emergence and development (stage 501–503, Figure 4), and flowering and fruit development stages (stage 609–712, Figure 6). Moreover, water management during inflorescence (stage 501–503, Figure 4) and flower development is a crucial agronomic operation for maintaining optimum soil moisture levels in A. manihot plants (stage 601–603, Figure 5). In A. manihot, among different phenological growth stages, leaf development (stage 101–109, Figure 3), inflorescence development (stage 501–509, Figure 4) and early fruits development stages are easily influenced by the attacks of biting or sucking insect, like aphids and ants, etc. Furthermore, different reports indicated that the hardness of the fruit pod, decided by the fruit development period, can protect it from pest attacks [9,11]. Thus, appropriate pest management strategies at specific phenological growth stages will help to better the development and yield of flowers and fruit.

5. Conclusions

The present work represents the first detailed and standardized description of the phenological growth stages of A. manihot according to the basic and extended BBCH scale. This scale consisted of nine principal growth stages and69 secondary growth stages from seed germination to senescence. Accordingly, the knowledge of the annual timing of phenological growth stages in A. manihot and their variability will help A. manihot planters adopt essential field management practices according to climatic conditions and growth stages of this crop, such as nutrient management, disease and pest management, flower and fruit drop management and harvest at the optimal times, etc. Furthermore, the standardized BBCH scale of A. manihot will help exchange scientific information between A. manihot breeders and researchers across the world regardless of where they are cultivated. Moreover, it will make it possible to standardize observations and research work in different regions, different countries and compare different cultivars. A. manihot plants are growing under complicated controllable and uncontrollable factors, and there are significant changes in their flower yield and quality. Therefore, future studies will focus on the changes in chemical composition in the A. manihot flower in response to phenological growth stages.