Introduction

The species of quinoa (Chenopodium quinoa Willd.) is an annual herb in the genus Chenopodium of the family Chenopodiaceae1. It is native to Peru, Bolivia, Ecuador, Colombia, and other medium and high elevation mountainous areas in the Andes of South America2. Quinoa is a pseudocereal, known as the ‘mother of all foods’, and it is not only rich in protein, dietary fibre, minerals, etc., but also contains a variety of phytochemicals such as saponins, phytosterols, and flavonoids3,4,5,6. Moreover, the plant is also suitable for people with celiac disease owing to its gluten-free properties7. It also has excellent resistance and wide adaptability to salinity, drought, and frost6,8,9. As a comprehensive economic crop with excellent functional characteristics and included by the United Nations Food and Agriculture Organization (FAO) among the important crops for grain sustenance in the coming century, quinoa is a superfood with high market demand and broad prospects for development and utilization10,11. However, despite the large cultivation area of quinoa in South America, its total yield is low due to limited planting environment, large yield fluctuations, insufficient processing technology, and political factors10,12,13. As the global population continues to increase, the contradiction between supply and demand between natural resources and food demand is becoming more and more obvious14. Therefore, the world is paying more attention to the research on quinoa molecular breeding technology. However, the molecular breeding techniques require transgenic systems and gene editing systems, which in turn require the building of efficient systems for in vitro regeneration.

However, the basic research on quinoa in China mainly focuses on the chemical composition of quinoa, antiretroviral properties, cultivation methods, which is in the initial stage15. Yu et al.16 optimized the induction and proliferation system of quinoa stem segment callus, and screened out the most suitable type of induction medium. Reports of other studies on quinoa tissue culture are rare. Studies on in vitro regeneration in quinoa, mainly focusing on direct organogenesis and callus induction17,18. For example, Regalado et al.17 obtained 7.96 ± 2.92 and 4.10 ± 3.00 shoots/outgrowth in Murashige and Skoog (MS) medium added at 2 mg/L 6-benzyl adenine (BA) using apical segments of two coastal species (Cahuil and Villa) as explants, respectively. Hesami et al.19 revealed that maximum shoot induction (93.33%) was obtained from cotyledonary node segments on MS + 2 mg/L BA, averaging 4.96 shoots induced per node explant. In the study by Gong et al.20, a direct organ regeneration system for quinoa ‘Faro’ was established by using seedling cotyledons as explants. In their study, double macroelements (2MS) + 1.0 mg/L BA + 0.1 mg/L 1-naphthaleneacetic acid (NAA) was the most suitable medium for induction of adventitious shoots (12.14%), and the most suitable medium for proliferation in subculture was 2MS + 0.5 mg/L BA (3.6 shoots/explant). Duan et al.21 used quinoa stem segments with axillary buds as explants, and the optimal medium was MS + 1.5 mg/L BA + 0.5 mg/L NAA for axillary bud induction, with a sprouting rate of 90.0% and full buds; it was MS + 1.0 mg/L BA + 0.2 mg/L NAA that was most suitable for adventitious buds to proliferate, with a proliferation coefficient of 4.9. Eisa et al.18 placed quinoa hypocotyls on MS medium modified with 0.45 µM 2,4-Dichlorophenoxyacetic acid (2,4-D), induced callus within two weeks of culture, transferred them to hormone-free MS medium for subculture, and obtained cytosolic embryogenesis sprouts, yet the seedling success rate was only 5%. Hesami et al.22 induced callus using quinoa hypocotyl as a starting explant and induced 6.33 adventitious shoots with the highest shoot regeneration frequency of 83.33% with the addition of 1.0 mg/L BA, 1.0 mg/L kinetin (KT), and 0.2 mg/L indole-3-butyric acid (IBA) to the MS medium. Cao et al.23 used stem segments of Taiwan red quinoa for callus induction as explants, which indicated that MS + 1.5 mg/L BA + 0.2 mg/L NAA was the optimal culture for callus induction, with an induction rate of 84.33%. In the above reports, a certain number of adventitious shoots were obtained by the direct organogenesis route, yet there were still problems such as low efficiency of intact regenerated plants and low proliferation efficiency. Callus induction is unsuitable for transgenic and genetic engineering studies and the induced callus suffer from poor induction rates, low multiplication coefficients, and premature senescence of bottle seedlings24,25. Obviously, the indirect pathway of adventitious shoots via callus is rarely reported, and the highly effective in vitro regeneration system based on quinoa hypocotyls as the starting explants is lacking. Therefore, it is imperative to establish an efficient in vitro regeneration system for quinoa.

In the present study, a highly efficient and stabilized system for in vitro regeneration of quinoa hypocotyls has been established by exploring the medium formulations and technical parameters for different steps of callus induction, adventitious shoot induction, adventitious shoot elongation, overcoming the premature senescence, and adventitious root formation using the hypocotyls of quinoa aseptic solid seedlings as the explants. Overcoming premature senescence is one of the key elements to establish a system for efficient in vitro regeneration for quinoa, which provides technological supports for the genetic improvement and biological breeding in this species, as well as theoretical references for overcoming premature senescence of shoots in the regeneration process of other species.

Results

Effect of PGRs on the induction of callus

Distinct combinations of plant growth regulators (PGRs) on induction of callus were investigated. The combinations of BA and NAA at different concentrations induced callus, yet the callus induction rates varied significantly (Table 1). The callus induction rate presented a tendency of increasing followed by decreasing with increasing NAA with same BA concentrations was a constant value. Similarly, the callus induction rate suggested a tendency of increasing followed by decreasing with increase in the concentration of BA when the value of NAA was held unchanged. At 0.2 mg/L of BA and 2 mg/L of NAA, the maximum 97.78 ± 1.92% callus induction was observed (Fig. 1C). This was followed by callus induction rate (92.22 ± 1.92%) in presence of BA (0.2 mg/L) and NAA (3 mg/L). However, the least induction rate of callus was only 48.89 ± 1.92% when BA was 0.4 mg/L, and NAA was 4 mg/L. Therefore, it had been chosen as the best medium for the induction of callus and MS medium containing 0.2 mg/L BA and 2 mg/L NAA was employed for further adventitious shoot induction experiments.

Table 1 Effect of PGRs on the induction of callus in Quinoa hypocotyls after 42 D.
Full size table
Fig. 1
figure 1

Efficient in vitro regeneration of shoots by using hypocotyls explants of quinoa. (A) Seeds germinated aseptically in MS medium without hormones after 3 d (bar = 2.00 cm); (B) Callus induced in MS medium with 0.2 mg/L BA and 2 mg/L NAA after 21 d (bar = 1.00 cm); (C) Callus of hypocotyl induction in MS medium with 0.2 mg/L BA and 2 mg/L NAA after 21 d (bar = 1.00 cm); (D) Callus were subcultured in MS medium supplemented with 1 mg/L BA, 0.1 mg/L NAA and 0.2 g/L proline after 28 d (bar = 0.70 cm); (E) Differentiation of callus in presence of MS medium with 1 mg/L BA, 0.1 mg/L NAA and 0.2 g/L proline after 28 d (bar = 0.85 cm); (F) Adventitious shoots differentiated in MS supplemented with 1 mg/L BA, 0.1 mg/L NAA and 0.2 g/L proline after 28 d (bar = 1.00 cm).

Full size image

Effect of proline concentration on the texture of callus

The callus with the addition of proline had a highest callus density compared to those without proline (Table 2). The highest callus density (0.42 ± 0.006 g/cm3) was observed for the callus in MS medium (with 1 mg/L BA and 0.1 mg/L NAA) added with 0.2 g/L proline, which were yellowish green, dense, and exhibited granularity, making them embryonic callus with high differentiation potential (Fig. 1D). The callus was gradually loose and yellowish in colour when the proline concentration was below 0.2 g/L. The callus wounds were gradually turned dark yellow in colour when the proline concentration was above 0.2 g/L, with a gradual increase in brittleness and a decrease in the potential for subsequent differentiation. Therefore, callus subsculture medium was optimal with the addition of 0.2 g/L proline.

Table 2 Effect of proline concentration on the texture of callus after 28 D.
Full size table

Effect of PGRs on the induction of adventitious shoots

To determine the optimal combination of PGRs for adventitious shoots induction, all the callus were transferred to MS medium of different BA and NAA combinations.

In contrast to the optimal medium for induction of callus, higher levels of BA and lower levels of NAA in the media were more favourable for inducing adventitious shoots (Table 3). As the concentration of BA increased, the induction rate and the mean number of adventitious shoots per explant slightly increased and then decreased. At BA concentrations below 0.7 mg/L, formation of few adventitious shoots was observed which failed to survive and hence could not generate healthy shoots. At 1 mg/L BA and 0.1 mg/L NAA hormone concentrations, the callus gradually proliferated after 14 d of culture, many green shoots appeared after transferring to the same medium and subculture for 14 d (Fig. 1E), differentiation into numerous adventitious shoots after 28 d (Fig. 1F), with 89.63 ± 2.67% and 40.5 ± 0.74 adventitious shoots for induction and the mean number of adventitious shoots per explant, respectively. A significant decline in adventitious shoot induction was observed at BA concentrations higher than 1 mg/L due to persistent callus of young cultures. As a result, the optimal medium was MS + 1 mg/L BA + 0.1 mg/L NAA + 0.2 g/L proline for adventitious shoot induction in quinoa hypocotyls.

Table 3 Effects of PGRs on the induction of adventitious shoots in Quinoa hypocotyls after 56 D.
Full size table

Effect of PGRs on the elongation of adventitious shoots

Reduced concentrations of both BA and NAA were used in the elongation medium as compared to the adventitious shoot induction medium (Table 4). Maximum shoot elongation was 70.86 ± 1.31% and the mean height of shoots per explant 3.07 ± 0.03 cm was determined for BA and NAA at 0.1 mg/L and 0.01 mg/L, respectively. However, the premature senescence of these elongated shoots was observed as most of them growing slowly and yellowing in colour. In next set of experiment we tried to overcome this problem with a simple strategy-adjustment of macroelement intensity.

Table 4 Effect of PGRs on the elongation of adventitious shoots in Quinoa hypocotyls after 35 D.
Full size table

Effect of MS macronutrient intensity on overcoming premature senescence of shoots

Premature senescence of shoots is detrimental to research on commercial production of seedlings, gene editing and transgenics. In this study, we showed that increasing the intensity of macroelements in MS basal medium had a significant effect on the subsequent elongation and overcoming the premature senescence of adventitious shoots. The percentage of indeterminate shoot elongation and shoot height significantly increased, and the rate of premature senescence of shoots was significantly reduced with the ongoing fortification of the MS basal medium with macroelements (Table 5). In MS medium, premature senescence of adventitious shoots amounted to 42.77 ± 1.06%. Yellowing of elongated leaves and early flowering of shoots in test tube were premature senescence (Fig. 2A). In double macroelements (2MS) medium, the leaves were partially tender and green with a small amount of premature senescence after elongation of adventitious shoots (Fig. 2B). In triple macroelements (3MS) medium, the adventitious shoot elongation rate reached 88.76 ± 1.14%, the height of the shoots was 6.85 ± 0.02 cm, and the premature senescence rate was only 8.15 ± 1.09%, with the leaves being tender and green, and the shoots were robust and well established (Figs. 2C and 3A). Compared to 3MS medium, the shoots in quadruple macroelements (4MS) medium were equally tender and green, yet the shoots were shorter with significantly lower adventitious shoot induction and shoot height, and premature senescence of 15.56% (Fig. 2D).

Therefore, the most suitable medium for overcoming premature senescence and promoting the elongation of adventitious shoots was 3MS medium containing 0.1 mg/L BA and 0.01 mg/L NAA, that not only overcome the premature senescence but also elongated the adventitious shoots.

Table 5 Effect of MS macroelement concentrations on overcoming premature senescence of adventitious shoots after 35 D.
Full size table
Fig. 2
figure 2

Effect of MS macroelement concentrations on overcoming premature senescence of adventitious shoots after 35 D. (A) Elongated adventitious shoots in MS medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA (bar = 1.00 cm); (B) Elongated adventitious shoots in double macroelements (2MS) medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA (bar = 1.00 cm); (C) Elongated adventitious shoots in triple macroelements (3MS) medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA (bar = 1.50 cm); (D) Elongated adventitious shoots in quadruple macroelements (4MS) medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA (bar = 1.50 cm).

Full size image

Effect of IBA concentration on induction of rooting

The results showed that relatively small doses of IBA induced adventitious root formation in in vitro shoots (Table 6). The rooting rate tended to increase rapidly and then decreased slightly with increased concentrations of IBA within a certain range. The highest rooting percentage was 98.52 ± 0.75% after 21 d, the number of d to rooting was 9.0 ± 0.3 d, and the mean number of roots per explant was 16.90 ± 0.38 when IBA was 0.6 mg/L (Fig. 3B).

Table 6 Effect of IBA concentration on induction of rooting after 21 D.
Full size table

Acclimatization

The survival rate of quinoa regenerated plantlets was as high as 95% after 30 d of acclimatization, and it was robust with new leaf growth and tender green leaves (Fig. 3C).

Fig. 3
figure 3

Regeneration shoots elongation, rooting and acclimatization of hypocotyl explants in quinoa. (A) Elongated adventitious shoots were cultured in triple macroelement (3MS) medium supplemented with 0.1 mg/L BA and 0.01 mg/L NAA (bar = 1.18 cm) after 21 d; (B) Rooted of in vitro shoots in presence of 0.6 mg/L IBA after 21 d (bar = 1.50 cm); (C) Acclimatization regenerated plantlets transferred to nutrient soil after 30 d. (bar = 2.50 cm).

Full size image

Discussion

In past years, quinoa (Chenopodium quinoa Willd.) is gradually attracting widespread awareness for being a nutritious crop. In vitro regeneration techniques have been explored by several studies as an important tool for the conservation and improvement of quinoa germplasm resources.

Proline, which is an important amino acid, played a significant part for establishing an efficient regeneration system for the plants26. Research has shown that proline not only performs a critical function during plant salt stress, heat stress, drought and low temperature, but also participates in antioxidant protection27,28,29,30,31. For example, application of exogenous proline alleviated the inhibition of radish seedling growth under salt stress and further increased the activity of antioxidant enzyme systems and the content of organic osmoregulatory substances32. Similar effects also existed in cucumber, melon33 and other species, such as under high-temperature stress, exogenous proline promoted the accumulation of free proline, soluble sugars and soluble proteins in cucumber leaves, which improved the osmoregulatory capacity of the plant, maintained the water balance in the plant, and thus improved heat tolerance34. For the present study, we examined the impact of proline concentration for texture of callus. As the proline concentration increased, the densification of the callus increased significantly. This indicates that the application of certain concentration of proline had a significant effect on the callus. Pawar et al. (2023) found that exogenous addition of proline can positively affect the frequency of callus and regeneration35. The addition of L-proline and L-tryptophan to rice healing tissue induction medium greatly increased the frequency of embryonic callus formation by Chowdhry et al.36. Pawar et al.37 found that healing tissues obtained in medium supplemented with proline and glutamine had a higher frequency of plant regeneration. These studies are in agreement with our study38.

In addition, premature senescence is a specific phenomenon in plant regeneration. Premature senescence occurs in plants causing chlorophyll and other macromolecules to be degraded, resulting in decreased photosynthetic activity of leaves and nutrient transport from senescent tissues to young tissues and reproductive organs39. In in vitro culture of quinoa hypocotyls, the occurrence of premature senescence will prevent in vitro fruiting and directly block the molecular breeding process. Premature senescence is a problem of slow growth and yellowing of functional leaves caused by insufficient fertilization and nutritional imbalance. In our study, 3MS medium added at 0.1 mg/L BA and 0.01 mg/L NAA plants were found to have tender green leaves, robust growth, good plant shape, and high number of plants, which is an optimal medium for overcoming premature senescence of shoots. Potassium (K), calcium (Ca), magnesium (Mg), hydrogen (H), oxygen (O), nitrogen (N), and phosphorus (P) are macroelements that work in concert with each other in the plant to support growth and development40,41. As the concentration of macroelements increases, nitrogen in the form of nitrate and ammonium ions increases along with phosphate ions, accumulating nutrients thereby increasing biomass helping to overcome senescence42. It is noteworthy that yellowing of the shoot tips occurred during the later stages of rooting culture, but did not affect rooting or transplant survival of plantlets. We hypothesize that the yellowing of leaves is triggered by normal plant metabolism, and we recognize that conducting physiological studies in the future will help to test these hypotheses. In this paper, we report for the first time that premature senescence of quinoa hypocotyls is overcome, which is conducive to improving the yield and quality of the crop, and also provides a theoretical reference for overcoming the premature senescence of sprouts during regeneration in other species.

The selection of plant growth regulators has an essential function in establishing an efficient regeneration system for plants. The two most important hormones, auxin and cytokinins, have a key role in regulating cell division, differentiation and organ formation in vitro regeneration studies43,44. The cytokinin BA and auxin NAA are often used in combination for the induction of adventitious shoots in callus45,46. In the study of Dang et al. regeneration shoots were induced successfully from Dioscorea nipponica Makino explants with a combination of BA and NAA at certain concentrations47. Shwe et al. (2019) achieved regeneration of Eucalyptus bosistoana by successfully induction of shoots through the combined use of BA and NAA48. For quinoa hypocotyls, Hesami et al.22 used IBA in combination with KT and BA to induce indeterminate shoots from hypocotyls with a regeneration rate of 83.33% and number of regenerated shoots of 6.33. Our preliminary study showed that this is not the optimum auxin to be used for induction of indeterminate shoots in hypocotyl callus. Therefore we used NAA in our experiment. For rooting, the growth hormone IBA is a commonly used phytohormone, which has a significant effect on induction of rooting in the histoculture process. In our study, the addition of IBA at a concentration of 0.6 mg/L resulted in the formation of adventitious roots after 9.0 ± 0.3 d, and the rate of adventitious root induction was 98.52 ± 0.75% after 21 d. After 30 d of acclimatization, quinoa regenerated plantlets showed a survival rate of 95%. Similar observations were made in Nepenthes mirabilis, where Miguel et al. (2020) cut regenerated shoots and transferred them to rooting culture in medium containing IBA, and found that rooting was possible after 28 d, and the survival rate of rooted plantlets was 100%49. This shows that IBA alone promotes rooting efficiency and root acclimation.

The choice of explants is a critical factor in the regeneration system. In some studies, the quinoa cotyledonary nodes were used as exosomes for induction of shoots, yet their results were not satisfactory19,50. In Regalado’s experiment17 with the apical segments of quinoa, it was found that 1 mg/L KT and 1 mg/L BA resulted in 93 ± 5% shoot regeneration in the coastal variety (Cahuil), yet only 2.14 ± 0.93 of shoots were regenerated. Duan et al. (2020) used quinoa stem segments as explants to induce axillary buds, which resulted in a sprouting rate of 90.0% and an adventitious shoot proliferation coefficient of 4.921. In the study of Hesami et al. using hypocotyls as explants found that the rate of indeterminate shoot regeneration and the number of regenerated shoots were 83.33% and 6.33, respectively22. Although the induced rate of adventitious shoots was more than 90% using apical nodes and stem segments as explants, the mean number of adventitious shoots was less than 4.9. In our study, using 1 mg/L BA and 0.1 mg/L NAA for induction of hypocotyls, the induction rate was 89.63 ± 2.67% induction and adventitious shoots up to 40.50 ± 0.74. This was an improvement of 84.37% over the optimum level of number of adventitious shoots regenerated (6.33) reported in the literature. This may be due to the enhancement of polar transport of cell fate determinants by the addition of proline upfront. The outcomes indicate that organogenesis capacity varies greatly among different tissues of plants and that a variety of factors, such as hormone combinations and concentrations, can influence plant regeneration. The findings are consistent with previous research51,52,53,54.

Conclusion

This study has overcome the problem of premature senescence of quinoa for the first time and established a new regeneration system using quinoa hypocotyls as explants. Experiments were conducted with materials easily available and markedly enhanced the efficiency of regeneration from adventitious shoots, providing technological supports for the protection of quinoa’s excellent germplasm and breeding for industrialization. In addition, this efficient regeneration strategy provides technical support for the genetic improvement of this species, as well as a reference for overcoming premature senescence of shoots during regeneration in other species.

Materials and methods

Plant material and explant preparation

The experiment used seeds of Chenopodium quinoa cultivar ‘Qingqua I’ provided by the Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences (3888 Chenhua Rd, Shanghai, China). The Murashige and Skoog (MS) medium was prepared first, followed by selection of full grown seeds of the year for treatment and inoculation. Seeds were surface sterilized using 75% (v/v) alcohol (Shanghai Titan Technology Co., Ltd.) for 1 min after rinsing under running water for two hours. A 10% sodium hypochlorite solution (Shanghai Titan Technology Co., Ltd.) was then prepared and sterilized for 20 min, washed five times with aseptic water, and finally inoculated onto hormone-free MS medium. After inoculation, the medium was incubated at 25 ± 2 °C under 16 h light conditions (light intensity of 2000–2200 lx) and 70% relative humidity. Adjust base medium pH to 5.8 by using 1.0 N HCl or 1.0 N NaOH (Sartourius, Germany), and its addition of 30 g/L sucrose (Xilong Scientific Co., Ltd.) and 5 g/L agar (Shanghai Shize Biotechnology Co., Ltd.), and the sterilization (SHENAN, Shanghai, China) was operated by autoclave steam at 0.105 MPa pressure and 121 °C for 20 min. Sterile seedling hypocotyls cultured for three to five d were used as explants (Fig. 1A).

Callus induction

Hypocotyls of 3–4 mm sterile seedlings were cut with a sterile scalpel to explore the effects of 6-benzyladenine (BA) and 1-naphthalene acetic acid (NAA) on the induction of callus. The hypocotyls were placed into sterile petri dishes (Shanghai Titan Technology Co., Ltd.) with MS medium containing BA (0.1, 0.2, 0.3, and 0.4 mg/L) and NAA (1, 2, 3, and 4 mg/L) at different concentrations. After 21 d, callus was transferred to triangular flasks [(Sichuan Shubo (Group) Co., Ltd.)] of the same medium (Fig. 1B). For each treatment, three replications were performed and each experiment consisted of 30 explants. After inoculation, the medium was incubated at 25 ± 2 °C under 70% relative humidity and 16 h light conditions (light intensity of 1000–1200 lx). After 42 d of culture, the rate of callus induction was assessed.

Subculture of callus

The impact of proline concentrations on the texture of callus was evaluated. The callus were relayed to MS medium (appended with 1 mg/L BA and 0.1 mg/L NAA) incorporating proline at different concentrations (0, 0.1, 0.2, 0.3, or 0.4 g/L) during subculture. For each treatment, three replications were performed and each experiment consisted of 30 explants. Incubation was carried out at 25 ± 2 °C, light intensity of 2000–2200 lx, 16/8 hours of light/darkness and 70% relative humidity. Callus density and callus morphology were evaluated after 28 d of culture.

Adventitious shoots induction

To induce adventitious shoots using hypocotyl-induced callus, BA (0.5, 0.7, 1.0, 1.5, or 2.0 mg/L) and NAA (0.1, 0.2, or 0.3 mg/L), were added to the MS medium. In order to get a higher density of callus, 0.2 g/L proline was also added in medium. For each treatment, three replications were performed and each experiment consisted of 30 explants. The cultures were incubated at 25 ± 2 °C with a light intensity of 2000–2200 lx, a photoperiod of 16 h of light/8 h of dark, and 70% relative humidity. After 56 d, we measured the number of regenerated shoots and the frequency of regeneration of adventitious shoots.

Adventitious shoots elongation

Adventitious shoots were diced in small clusters and placed in adventitious shoot elongation medium, which was made by adding 0, 0.05, 0.07, 0.1, or 0.3 mg/L BA and 0, 0.01, 0.03, or 0.05 mg/L NAA to the MS medium. For each treatment, three replications were performed and each experiment consisted of 30 explants. Cultivated at 25 ± 2 °C under light intensity of 2000–2200 lx, 16/8 h light/dark and 70% relative humidity. After 35 d of culture, the elongation rate of adventitious shoots and the mean height of shoots per explant were measured.

Overcoming premature senescence

The impact of macroelement of MS basal medium on premature senescence in shoots was carried out by placing adventitious shoots in different intensities of MS medium [MS, double macroelements (2MS), triple macroelements (3MS), or quadruple macroelements (4MS)] in presence of optimized media (0.1 mg/L BA and 0.01 mg/L NAA). For each treatment, three replications were performed and each experiment consisted of 30 explants. Cultivation was carried out at 25 ± 2 °C, 70% relative humidity, light intensity of 2000–2200 lx and 16/8 hours of light/darkness. The elongation rate of adventitious shoots, the mean height of shoots per explant and the premature senescence rate were recorded after 35 d.

Adventitious root induction

Elongated shoots above the 2 cm were intercepted and inoculated on MS medium with: 0.2, 0.4, 0.6, 0.8, or 1.0 mg/L IBA. This transfer was made to encourage further rooting in the presence of various IBA concentrations, and 0 mg/L IBA was used as a control. For each treatment, three replications were performed and each experiment consisted of 30 explants. After inoculation, the medium was incubated at 25 ± 2 °C under 70% relative humidity and 16 h light conditions (light intensity of 2000–2200 lx). Days to the onset of adventitious rooting were recorded and rooting percentage and the mean number of roots per explant were calculated after 30 d.

Acclimatization

Rooted regenerated plantlets were filled with 1 cm of water into the vials and placed at room temperature under natural light. The rooted regenerated plantlets were taken out after 3 d, washed of the medium around their roots, and transplanted into a substrate of peat soil: vermiculite: perlite = 3:1:1. After the substrate was completely moistened, the plantlets were bagged and moisturized, moved to the greenhouse with a temperature of about 25 °C, a photoperiod of 16/8 h light/dark, a light intensity of 1200 lx, and a relative humidity of 70%, and after 14 d the bag was removed for normal maintenance.

Statistical analyses

The following equations were utilized to identify various in vitro regeneration parameters:

Callus induction rate (%) = \(\:\frac{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{c}\text{a}\text{l}\text{l}\text{u}\text{s}\:\text{i}\text{n}\text{d}\text{u}\text{c}\text{e}\text{d}\:\text{e}\text{x}\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}}{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{e}\text{x}\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:}\) × 100%

Shoot regeneration Frequency (%) = \(\:\frac{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{e}\text{x}\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:\text{w}\text{i}\text{t}\text{h}\:\text{a}\text{d}\text{v}\text{e}\text{n}\text{t}\text{i}\text{o}\text{u}\text{s}\:\text{s}\text{h}\text{o}\text{o}\text{t}\text{s}}{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{e}\text{x}\text{p}\text{l}\text{a}\text{n}\text{t}\text{s}\:}\) × 100%

Adventitious shoots elongation rate (%) = \(\:\frac{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\:\text{e}\text{l}\text{o}\text{n}\text{g}\text{a}\text{t}\text{e}\text{d}\:\text{s}\text{h}\text{o}\text{o}\text{t}\text{s}\:}{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{h}\text{o}\text{o}\text{t}\:\text{o}\text{n}\:\text{t}\text{h}\text{e}\:\text{e}\text{l}\text{o}\text{n}\text{g}\text{a}\text{t}\text{i}\text{o}\text{n}\:\text{m}\text{e}\text{d}\text{i}\text{u}\text{m}\:}\) × 100%

Premature senescence rate (%) = \(\:\frac{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\:\text{p}\text{r}\text{e}\text{m}\text{a}\text{t}\text{u}\text{r}\text{e}\:\text{s}\text{e}\text{n}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:}{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{h}\text{o}\text{o}\text{t}\:\text{o}\text{n}\:\text{t}\text{h}\text{e}\:\text{p}\text{r}\text{e}\text{m}\text{a}\text{t}\text{u}\text{r}\text{e}\:\text{s}\text{e}\text{n}\text{e}\text{s}\text{c}\text{e}\text{n}\text{c}\text{e}\:\:\text{m}\text{e}\text{d}\text{i}\text{u}\text{m}\:}\) × 100%

Rooting rate (%) = \(\:\frac{\text{t}\text{h}\text{e}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{r}\text{o}\text{o}\text{t}\text{e}\text{d}\:\text{p}\text{l}\text{a}\text{n}\text{t}\text{l}\text{e}\text{t}\text{s}}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\:\text{s}\text{h}\text{o}\text{o}\text{t}\text{s}\:}\) × 100%

All experimental data were analyzed by ANOVA. Means were compared by Duncan’s Multiple polarity test using SPSS (version 27.0) software.