Callus development observed from different culture time periods. (A) At 30 d, the callus was green with a high differentiation ability. (B) At 60 d, the callus was turning yellowish brown. (C) At 80 d, the callus turned to nodular tissue. (D) At 100 d, the callus became atrophied or brown without differentiation ability. Red bars indicate the scale = 1 cm.
Effects of the culture time period on the callus proliferation of Ananas tricolor. Culture condition: Murashige and Skoog medium added to 3 mg⋅L–1 6-benzylaminopurine and 2 mg⋅L–1 1-naphthylacetic acid under 2000 μmol⋅m–2⋅s–1 of light for 16 h per day at a temperature of 20 °C.
Red buds are observed on the callus (A, B) after 35 d of subculture. (A) Arrow indicates the adventitious buds. (B) Three red buds are noted. The arrow indicates the growth point, which is light green, which means the red buds and adventitious buds are not integrated. (C, D) Callus after 45 d of subculture. f1, f2, and f3 refer to the three flower buds. The pistils of f1 and f3 are clearly visible. b, sepals; g, pistil; white arrow, green growing point. The orange bars indicate the scale = 1 cm.
Flower development after 70 d of subculture. (A) Three flower buds were identified. (B) The adventitious buds on the other side grew obviously within 25 d, and there were new adventitious buds at the base. (C) Similar things as pollen and stamen on flower 2 (f2). (D) Part of flower 1 (f1). The petals at the lower end become dark and wilt gradually. (E) f1 and f2 bloomed; flower 3 (f3) did not. b, sepal; g, pistil. The orange bars indicate the scale as shown.
The flower bud sepals are open and fleshy after 5 months of subculture. The orange bar indicates the scale = 0.5 cm.
Other buds induced from the callus of Ananas tricolor. (A–F) The bud developed into adventitious buds. (G) Red buds on the callus, which has withered. (H, I) The purplish red bud induced from the callus, which has vitrified and died. The orange bars indicate the scale as shown.
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In natural conditions, it takes more than 3 years to complete the Ananas juvenile phase, and another 2 years for adult vegetative growth of the plantlet from in vitro buds. Ethylene has often been used to shorten the juvenile and vegetative phases to produce earlier flowering. It is important to induce in vitro flowering of Ananas plants to understand the flowering mechanism more completely, which is also related to flower organ differentiation and development as well as the pineapple fruit eye development. In this study, Murashige and Skoog (MS) basal medium was used to select the best combination for adventitious bud induction from the callus of Ananas bracteatus var. tricolor (A. tricolor). Flower induction from the callus was studied using 6-benzyladenine (6-BA) and 1-naphthylacetic acid (NAA) at four different concentrations (0, 1.0, 2.0, and 3.0 mg⋅L–1). Our results showed that when MS was added with 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA under 2000 μmol⋅m–2⋅s–1 of light for 16 hours per day at a temperature of 20 °C, the callus of A. tricolor grew quickly, and adventitious buds were induced. After more than four successive subcultures (at day 80), differentiation of flower buds was observed on the aging callus tissue before a complete floral organ developed. This research could be used for the flowering regulation of Ananas plants in the future. Inducing flowers directly from the callus has important scientific significance for the differentiation and morphogenesis of Ananas plants.
Ananas L. belongs to the family Bromeliaceae and originated in the American tropics. It is a perennial and monocotyledonous herb (Gómez-Lim and Litz, 2004). In natural conditions, it takes nearly 5 years to complete its life cycle from plantlets to fruiting using buds as explants. Ethylene has often been used to shorten the juvenile and vegetative phases and produce earlier flowering (Wang et al., 2017), which promote earlier fruiting. The pineapple flower inflorescence is infinite, and the inflorescence is composed of 60 to 200 small flowers. Usually, the florets open spirally from the base to the top (Lu et al., 2013).
Plant tissue culture could help rapid plant reproduction by providing nutrients within a sterile environment, which is especially important for the production of perennial plants. Cell, tissue, and plant growth can be controlled by altering the composition of nutrients and adding different plant growth regulator or bacteriostatic agents (Huang et al., 2019; Teixeira et al., 2006; Thomas, 2008). Organogenesis and somatic embryogenesis are two ways of performing in vitro plant regeneration (Gong and Shen, 2013; Liu et al., 2010), but a flower organ must eventually be formed from the regenerated plants. Scientists have tried to induce flower buds from callus or explants directly, as reported in Nicotianatabacum, Dracaena fragrans cv. Massangeana (Li and Chang, 1999), Citrus reticulata cv. Kinnow (Singh et al., 2006), and Triticum sativum (Lu, 1992).
Plant growth regulators play key regulatory roles in plant growth and development. High concentrations of 6-BA and NAA are favorable for callus proliferation. NAA is an ingredient in many commercial postharvest horticultural products, both in vegetables and fruit (Nikolelis et al., 2008). It is also a rooting agent and is used for vegetative propagation of plants from stem and leaf cuttings (Saifuddin et al., 2009). 6-BA was shown to have a significant physiological function during tissue regeneration, production, quality, and accumulation of secondary metabolites during a stress response (Chen and Yang, 2012; He and Shi, 2014; Singh et al., 2015).
Although the parts of Ananas plants during in vitro studies include stem tips, axillary buds, seeds, and leaves, axillary buds and stem tips are the most commonly used materials (Fu et al., 2016). In addition, one study was conducted successfully to obtain a ‘Smooth-Cayenne’ pineapple transgenic plant by somatic embryo regeneration (Firoozabady et al., 2006). With ‘Shenwan’ pineapple, the addition of a high 2,4-dichlorophenoxy acetic acid concentration was considered more conducive to somatic embryo induction, whereas a low concentration of 6-BA causes browning in the callus (He et al., 2007).
There is little known about in vitro flower induction from the callus of Ananas plants. Ananas fruit is a type of berry. At the end of flowering, the ovary expands while the perianth persists, and the fruit is polymerized from multiple small fruit to form a compound fruit. It is important to induce in vitro flowering of Ananas plants to understand the flowering mechanism more completely, which is also related to flower organ differentiation and development as well as pineapple fruit eye development.
It is also important to study direct flower induction and formation from callus subsequent flower bud differentiation and morphogenesis. This study would shorten the development period, and is a good way to study the in vitro induction of flowers from the callus of A. tricolor using growth hormones (NAA, 6-BA) in a controlled environment, including temperature (12, 16, and 20 °C), nutrient deficiency (culture for a long time to cause the nutrient deficiency), and so on.
The axillary buds of A. bracteatus var. tricolor were collected from the garden of South China Agricultural University, Guangzhou, China.
According to He et al. (2007), senescent leaves were removed and the axillary buds washed with tap water before the buds were cut into 0.5 cm above the growing point. The buds were then presterilized with 7% NaClO for 7 min and washed with sterile water three times, and disinfected with 0.1% HgCl for 8 min and washed with sterile water three times. Excess leaves and roots were removed, and cut into four pieces of axillary bud tissue with leaf base growth points, and disinfected with 0.1% HgCl for 5 min, and then washed with sterile water three times. The samples were inoculated in MS medium (Murashige and Skoog, 1962) containing 2.0 mg⋅L–1 BA and 2.5 mg⋅L–1 NAA to induce the callus for 5 weeks. Samples were kept at 20 ± 2 °C for 15 d in the dark and were then transferred to 16 h of light per day to incubate 20 d.
The resulting callus was cut into 5 × 5-mm pieces, weighed and cultured on the callus proliferation medium (MS containing 0, 1.0, 2.0, and 3.0 mg⋅L–1 6-BA and NAA) in culture bottles at 20 ± 2 °C, under 16 h of light per day (2000 lx). Four to five pieces of callus tissue were placed in each bottle and then incubated in the dark for 30 d. Callus weight changes were recorded every 7 d using a scale (ME104E; Mettler Toledo Instruments Co. Ltd., Greifensee, Switzerland) by removing the callus from the culture bottle on clean bench (KS18; Thermo Fisher Scientific Inc., Waltham, MA). The experiments were repeated five times.
The callus was cut into 5 × 5-mm pieces and cultured on MS medium containing 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA. Six to seven pieces of the cut callus tissue were placed in each bottle and were cultured under 16 h of light per day (2000 lx). The culture time of callus tissues was arranged into five groups (30, 40, 60, 80, and 100 d) for subculture at 12, 16, and 20 °C. Three replicates (three bottles) were set for each of the culture groups. During the culture period, the culture medium was not renewed. The growth and differentiation of the callus culture were recorded using a digital camera (Nikon D5000) every 7 d.
All the data were processed and analyzed (Duncan’s test for significance; significance level, P < 0.05; Pearson’s correlation analysis) using Excel (Microsoft Corp., Redmond, WA) and SPSS 2 (SPSS Inc., Chicago, IL).
Our results show that the media of callus proliferation had different effects under different environmental conditions (Table 1). All treatments had a significant weight increase over the control, which means the treatments could help to induce the callus. Among the treatments, the MS medium containing 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA was the best. Therefore, it can be concluded that this treatment under a constant temperature (20 ± 2 °C) with 24 h of lighting, and a culture time period of 30 to 40 d was suitable and can differentiate adventitious buds significantly.
Callus grew well in culture for 30 and 40 d, and most callus differentiated adventitious buds. After 60 d, the callus turned yellowish brown and showed little differentiation. After 80 and 100 d, callus growth almost stopped. The longer culture periods caused the callus to be hard, atrophied, or brown (Fig. 1). The results also show that callus weight decreased with an increasing culture time, and the callus began to atrophy and lose water after 80 d (Fig. 2).
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
Although the culture media provides nutrients and water for explant growth, resources became limiting over time, and after 45 d the cultures generally consumed most of the nutrients and water. Over long periods, the media volume became smaller until it was finally absorbed completely.
The callus that was cultured for 80 d was then subcultured after 35 d in MS medium with 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA. At this point, one of the calluses showed some red buds, which looked like flower differentiation; adventitious buds were differentiated on the other side of the callus (Fig. 3). On the 45th day of subculture, tissues with flower-like buds were transferred to a separate culture bottle containing MS with 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA maintain nutrients supply and easy observation. It was found that flower organs, such as pistil and stamen, extended out from the callus (Fig. 3). The red part of the surface should be sepals, and the color was lighter than observed initially. However, the adventitious buds on the other side of the callus had changed little.
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
On the outside of each floret, there was a green growing point on the callus with three red florets and three sepals. There were three petals in the sepals that were reddish, with white at the top, and had a spiral distribution, and one pistil inside, which was slightly higher than the sepals and petals; three stamens could be seen. The petals withered after bloom, and the sepals and the pistil maintained their structure, but their overall color was dim.
Twenty-five days after the second subculture, the color of the pistil and sepals became lighter, and three flower buds appeared on the same growing point, with a bloom sequence of flower 1, flower 2, and flower 3 (f1, f2, and f3) (Fig. 4). Seventy days after subculture, the flowering period lasted about 2 months. At the same time, adventitious buds on the other side of the aging tissue grew significantly within 25 d of the second subculture. New callus and adventitious buds were formed at the growth point, whereas the callus that differentiated the flower buds gradually shrunk.
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
The flower buds and adventitious buds were separated with a scalpel. The adventitious buds were kept in the same culture bottle for nutrient absorption in Mar. 2017, and the flower buds were chosen as the reference material. The culture medium was replaced with fresh medium every 30 d. It was observed that the growing point at the base of the flower buds began to expand, and adventitious buds grew well. After 5 months, it was found that sepals of flower 1 were completely spread out and become fleshy, whereas flowers 2 and 3 had no obvious changes (Fig. 5). After that, the flowering time was over.
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
During blooming, when cultured for 35 d, although some buds were observed, these buds could not be defined as the flower buds at this stage because they had not yet differentiated. Only after 45 d could these buds be declared to be flower buds, and their flower organs extended out. Under field conditions, a single-flower flowering of Ananas plants is usually as short as 1 or 2 d (Lu et al., 2013); however, flowering was prolonged significantly using the tissue culture method in our study. We observed that the entire flowering process lasted for 10 d or even longer. The flower buds developed on the callus using the tissue culture method did not form bracts, which is different from the common Ananas plants florets.
The callus cultured for 80 d was subcultured on MS medium with 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA; under 16 h of light per day; at 12, 16, or 20 °C; for 1 week before incubating at 20 °C to induce the flower buds in A. tricolor. After the callus was incubated at 20 °C for 30 to 35 d, the callus differentiated into red or purplish red buds. However, subsequent observations revealed that these buds did not differentiate into floral buds, leaf buds, or vitrification (Fig. 6), which may be a result of inadequate induction conditions.
Citation: HortScience 57, 5; 10.21273/HORTSCI15083-21
Tissue age and external environmental factors such as nutrition and drought can promote flowering of fruit trees (Lv and Lin, 1995). The culture medium provides nutrients to the plant during in vitro culture, with different concentrations of hormones having a greater influence on plant development. Kaursawhney et al. (1988) suggested that high concentrations of cytokinin inhibit flowering of tobacco. Ja et al. (2014) reported that genotype, culture medium composition, and plant growth regulators were the main influencing factors in the flowering of orchid tissue cultures.
The results in our study indicated that the surface of the callus turned brown after 80 d of culture, and flower bud differentiation occurred 20 d after subculture. It was speculated that nutrient consumption and water loss in the medium stressed the tissue, forcing the plant developed into the stage of “ripeness-to-flower” and forming flower buds.
The results of our study also confirm that the temperature is a critical factor affecting the growth and development of plants, especially for flower bud differentiation. It was reported previously (Liu et al., 2010) that low temperatures can promote flowering of pineapples, and the pineapple flower rate is high at low temperatures (≤15 °C). In our study, the materials were influenced by the temperature (20 ± 2 °C) for a long time (100 d), under 16 h per day of illumination. The illumination could also be one of the influencing factors to effect flowering. Compared with those cultivated at higher temperatures of 28 ± 2 °C, we assumed that the low temperature with a 16-h photoperiod was some kind of adversity that induced flower bud differentiation in A. tricolor.
In our study, we used a medium (MS with 3 mg⋅L–1 6-BA and 2 mg⋅L–1 NAA) and specific conditions (2000 μmol⋅m–2⋅s–1 light for 16 h per day at a temperature of 20 °C) to induce the callus of A. tricolor to flower. Our results show that flower buds differentiated at 20 ± 2 °C could persist for more than 6 months without wilting in the culture bottle. A further study of inducing flowers from the callus of A. tricolor is being conducted by our research group.
Callus development observed from different culture time periods. (A) At 30 d, the callus was green with a high differentiation ability. (B) At 60 d, the callus was turning yellowish brown. (C) At 80 d, the callus turned to nodular tissue. (D) At 100 d, the callus became atrophied or brown without differentiation ability. Red bars indicate the scale = 1 cm.
Effects of the culture time period on the callus proliferation of Ananas tricolor. Culture condition: Murashige and Skoog medium added to 3 mg⋅L–1 6-benzylaminopurine and 2 mg⋅L–1 1-naphthylacetic acid under 2000 μmol⋅m–2⋅s–1 of light for 16 h per day at a temperature of 20 °C.
Red buds are observed on the callus (A, B) after 35 d of subculture. (A) Arrow indicates the adventitious buds. (B) Three red buds are noted. The arrow indicates the growth point, which is light green, which means the red buds and adventitious buds are not integrated. (C, D) Callus after 45 d of subculture. f1, f2, and f3 refer to the three flower buds. The pistils of f1 and f3 are clearly visible. b, sepals; g, pistil; white arrow, green growing point. The orange bars indicate the scale = 1 cm.
Flower development after 70 d of subculture. (A) Three flower buds were identified. (B) The adventitious buds on the other side grew obviously within 25 d, and there were new adventitious buds at the base. (C) Similar things as pollen and stamen on flower 2 (f2). (D) Part of flower 1 (f1). The petals at the lower end become dark and wilt gradually. (E) f1 and f2 bloomed; flower 3 (f3) did not. b, sepal; g, pistil. The orange bars indicate the scale as shown.
The flower bud sepals are open and fleshy after 5 months of subculture. The orange bar indicates the scale = 0.5 cm.
Other buds induced from the callus of Ananas tricolor. (A–F) The bud developed into adventitious buds. (G) Red buds on the callus, which has withered. (H, I) The purplish red bud induced from the callus, which has vitrified and died. The orange bars indicate the scale as shown.
Contributor Notes
This study was supported by the National Key R&D Program of China (nos. 2019YFD1000500 and 2018YFD1000500), the Key Areas Research and Development Programs of Guangdong Province (no. 2018B020202011), and the China Scholarship Council (no. 201807630019).
We thank Mark A. Ritenour, Jiuxu Zhang, Liyun Chen, Muhammad Qasim Shahid, and Puhui Ji for linguistic assistance during the preparation of this manuscript.
Z.Z. and Y.H. are corresponding authors. E-mail: poloky2@163.com (Z.Z.) or heyehua@scau.edu.cn (Y.H.).
Callus development observed from different culture time periods. (A) At 30 d, the callus was green with a high differentiation ability. (B) At 60 d, the callus was turning yellowish brown. (C) At 80 d, the callus turned to nodular tissue. (D) At 100 d, the callus became atrophied or brown without differentiation ability. Red bars indicate the scale = 1 cm.
Effects of the culture time period on the callus proliferation of Ananas tricolor. Culture condition: Murashige and Skoog medium added to 3 mg⋅L–1 6-benzylaminopurine and 2 mg⋅L–1 1-naphthylacetic acid under 2000 μmol⋅m–2⋅s–1 of light for 16 h per day at a temperature of 20 °C.
Red buds are observed on the callus (A, B) after 35 d of subculture. (A) Arrow indicates the adventitious buds. (B) Three red buds are noted. The arrow indicates the growth point, which is light green, which means the red buds and adventitious buds are not integrated. (C, D) Callus after 45 d of subculture. f1, f2, and f3 refer to the three flower buds. The pistils of f1 and f3 are clearly visible. b, sepals; g, pistil; white arrow, green growing point. The orange bars indicate the scale = 1 cm.
Flower development after 70 d of subculture. (A) Three flower buds were identified. (B) The adventitious buds on the other side grew obviously within 25 d, and there were new adventitious buds at the base. (C) Similar things as pollen and stamen on flower 2 (f2). (D) Part of flower 1 (f1). The petals at the lower end become dark and wilt gradually. (E) f1 and f2 bloomed; flower 3 (f3) did not. b, sepal; g, pistil. The orange bars indicate the scale as shown.
The flower bud sepals are open and fleshy after 5 months of subculture. The orange bar indicates the scale = 0.5 cm.
Other buds induced from the callus of Ananas tricolor. (A–F) The bud developed into adventitious buds. (G) Red buds on the callus, which has withered. (H, I) The purplish red bud induced from the callus, which has vitrified and died. The orange bars indicate the scale as shown.
