Prediction of the concentration of plant growth regulators for somatic embryogenesis and regeneration of Hyoscyamus niger using Box-Behnken design of response surface methodology

doi:10.21203/rs.3.rs-2430673/v1

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Research Article

Prediction of the concentration of plant growth regulators for somatic embryogenesis and regeneration of Hyoscyamus niger using Box-Behnken design of response surface methodology

https://doi.org/10.21203/rs.3.rs-2430673/v1

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published 06 May, 2023

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Optimizing of the in vitro culture condition, somatic embryogenesis, and plant regeneration is the first step in transformation. In this study, the response surface methodology was used to optimize and predict the concentrations of TDZ, NAA, BAP, and kinetin for callus formation, and regeneration by indirect somatic embryogenesis of Hyoscyamus niger. The results proved that TDZ is more efficient than NAA for the regeneration of H. niger. By using different concentrations of TDZ, BAP, and kinetin, the highest percentage of callusing (100.00%), callus fresh weight (1207.75 mg/explant), percentage of somatic embryogenesis (92.66%) and shooting (93.30%), and the number of the shoot (7.75 per callus) was recorded from petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, leaf explants by using 0.5 mg/L TDZ and 1 mg/L kinetin, petiole explants with addition 0.5 mg/L TDZ, leaf explants by using 0.50 mg/L TDZ and 1 mg/L kinetin, and petiole explants with addition 0.25 mg/L TDZ and 1 mg/L BAP, respectively. Also, the highest percentage of callusing (100.00%), callus fresh weight (3273.64 mg/explant), percentage of somatic embryogenesis (100%), shooting (100.00%), and the number of the shoot (8.61 per callus) were predicted by culturing of petiole explant on the MS medium containing 0.11 mg/L TDZ and 1.253 mg/L BAP, leaf explant by using 0.52 mg/L TDZ, petiole explant by using 0.533 mg/L TDZ and 1 mg/L BAP, leaf explant with addition 0.18 mg/L TDZ and 2.89 mg/L BAP and petiole explant by using 0.1 mg/L TDZ and 3 mg/L BAP.

Explant

Henbane

Plant growth regulators

Regeneration

Somatic embryogenesis

Optimizing and predicting of the effect of plant growth regulators on henbane regeneration

The henbane (Hyoscyamus niger) is an annual or biennial plant belonging to the Solanaceae family. This plant is very important in the pharmaceutical industry due to its high amount of tropane alkaloids such as hyoscyamine and scopolamine. Scopolamine has higher pharmacological activity and fewer side effects than hyoscyamine. Therefore, the demand for scopolamine is about 10 times higher than that for hyoscyamine. These compounds are used to treat stomach aches, earaches, sinusitis, ulcers, kidney, and liver ailments, as well as to relieve pain and as antispasmodics. Despite the therapeutic effects, these compounds also have toxic effects on humans. Scopolamine and hyoscyamine can cause intoxication by paralyzing the nerve endings of the parasympathetic system (Deveci et al. 2022).

Biotechnological methods such as cell and tissue culture are efficient methods for micro-propagation, production of secondary metabolites, and gene transformation (Adedeji et al. 2020). Somatic embryos are obtained through morphological and biochemical changes in somatic cells. Somatic embryogenesis includes initiation, proliferation, maturation, and seedling formation. Various studies show that somatic embryogenesis depends on various factors such as the type of plant, the type of explant, the type of culture medium, and the type and concentration of plant growth regulator. In general, somatic embryogenesis is done in two pathways, direct and indirect. The difference between direct and indirect somatic embryogenesis is the existence of the callus generation stage in the indirect pathway (Ghosh et al. 2018). Somatic embryogenesis has stages of induction, expression, multiplication, development, maturation, germination, and conversion into seedlings. These stages are characterized by physiological, morphological, and molecular events (Garcia et al. 2019). In the first stage of somatic embryogenesis, cultured explant-competent cells respond to induction signals and the dedifferentiation stage is induced. In the next step, the dedifferentiated cells are canalized into embryogenic developmental pathways and culminate in the generation of the embryo (Elhiti and Stasolla 2022). Plant growth regulators are considered one of the induction signals of dedifferentiation (Nic-Can and Loyola-Vargas 2016). Plant tissue culture researchers believe that auxin is required to induce somatic embryogenesis (Karami et al. 2021). Studies show that during the early stages of somatic embryogenesis, the level of endogenous auxin increases, which is associated with the activation of stress signals and changes in chromatin status. The use of auxin in the early stages of somatic embryogenesis causes profound changes in DNA methylation and the formation of embryogenic tissue. The later stages of somatic embryogenesis mainly occur in an auxin-free culture medium (Elhiti and Stasolla 2022). Studies show that among the auxins, the 2,4-D is the most suitable for inducing indirect somatic embryogenesis and embryo multiplication, but it is necessary to remove the at the subsequent expression, development, and maturation steps of somatic embryogenesis because it prevents the growth of the embryo and its conversion into a seedling (Garcia et al. 2019). Cytokinins also involved in plant growth and induce bud formation and delay leaf senescence and together with auxins stimulate cell division (Méndez-Hernández et al. 2019). It has been found that these two plant growth regulators act synergistically or antagonistically in somatic embryogenesis (Liao et al. 2015).

Somatic embryogenesis is the appropriate method in gene transfer studies. The preparation of somatic embryogenesis protocol plays an important role in genetic manipulation and plant regeneration. Using appropriate types and concentrations of plant growth regulators and explants can improve somatic embryogenesis (Hesami et al. 2018). Therefore, optimization of the somatic embryogenesis protocol is the first and most important step in gene transformation studies, but it is a laborious, time-consuming, and complex process (Hesami and Jones 2020, Hesami et al. 2020). In in vitro cultures, somatic embryogenesis processes such as dedifferentiation, re-differentiation, or differentiation are caused by genetic and environmental factors (Gürel et al. 2019, Joung et al. 2020). Therefore, it will be difficult to predict various cases, including the rate of callus formation, and the rate of embryogenesis, as well as the optimization of the factors involved in them through conventional mathematical methods (Hesami and Jones 2020, Salehi et al. 2020). Also, biological processes such as somatic embryogenesis cannot be described by a simple step-by-step algorithm (Niazian et al. 2018, Sheikhi et al. 2020). For this purpose, machine learning algorithms can be used to predict the data set (Hesami et al. 2019). The response surface method is one of the statistical and mathematical tools that can be used to predict a set of data. In this method, the relationship between different variables that affect the results can be determined. In the experimental phase, this method is used to optimize complex processes and save time and money (Farjaminezhad and Garoosi 2021, Farjaminezhad and Garoosi 2021). Box–Behnken and central composite design designs are experimental designs for response surface methodology. Box-Behnken design is more proficient and most powerful than central composite design, despite its poor coverage of the corner of nonlinear design space (Karmoker et al. 2019). In this study the effect of naphthalene acetic acid (NAA), thidiazuron (TDZ), 6-benzylaminopurine (BAP), and kinetin on callus induction, somatic embryogenesis and regeneration H. niger from leaf and petiole explants were investigated. Also, the Box–Behnken design of response surface methodology was used to optimize and predict the appropriate concentrations of plant growth regulators for callus formation, somatic embryogenesis, and regeneration in this plant.

Plant materials

H. niger seeds used in this study were obtained from the Imam Khomeini International University, Iran.

Dormancy Breaking

For breaking of seed dormancy, seeds were soaked in 200 ppm gibberellic acid and maintained at 4°C for 48 h.

Culture Media

All the experimental cultures were conducted using MS basal medium. Sucrose (30 g/L) was used as a carbon source and agar as the gelling agent at a rate of 7.0 g/L in all media. Autoclavable plant growth regulators were added before autoclaving of the medium and not autoclavable plant growth regulators were sterilized with a 0.22 µM syringe filter and added to media after autoclaving and reducing the temperature of the culture medium. The pH of the media was adjusted to 5.7–5.8 by 1N NaOH or 1N HCl. The culture media were sterilized under 1.2 bar pressure and 121°C for 20 min in an autoclave.

Seed Culture And Explants Preparation

The seeds were washed in tap water for 20 min and disinfected using 70% (v/v) ethanol for 45s and 2.5% (w/v) hypochlorite sodium with 1 drop Tween 80 for 20 min and washed in distilled water three to four times. The disinfected seeds were cultured on Petri dishes containing MS basal medium and incubated in a 16/8 h photoperiod at 25 ± 2°C in a growth chamber. The seeds germinated after 5 days of culture. After three weeks, the germinated seed with four leaves was transferred to culture jars and maintained for three weeks. The leaf (1 cm²) and petiole (1 cm) explants were obtained from these seedlings using a sharp scalpel blade.

Callus Induction, Somatic Embryogenesis, And Shoot Regeneration

The leaf and petiole explants at the prescribed dimensions were cultured on MS medium supplemented with NAA and TDZ (0, 0.25, 0.5, and 0.75 mg/L) and BAP and kinetin (0, 1, 2, and 3 mg/L) for callus induction and maintained in dark at 25 ± 2°C for 3 weeks. After three weeks, the percentage of callusing and fresh weight of callus were recorded. Then, the cultures were sub-cultured for 6 weeks in the same media at 3 weeks intervals and incubated in a 16/8 h photoperiod (2000 lux) at 25 ± 2°C in a growth chamber. After three weeks, the percentage of somatic embryogenesis and after 6 weeks the percentage of shooting, number of the shoots, the percentage of rooting, and number of roots were recorded.

Rooting And Acclimatization

The regenerated shoots (without roots) from leaf and petiole explants derived calli were moved to MS medium containing 1.63 mg/L IBA for 6 weeks and incubated in 16/8 h photoperiod (9200 lux) at 25 ± 2°C in a growth chamber and sub-cultured in the same media at 3 weeks interval. The cultures showing root initiation were shifted to hormone-free MS media and maintained for up to 6 weeks in a 16/8 h photoperiod (9200 lux) at 25 ± 2°C in a growth chamber.

After 6 weeks, the rooted shoots were uprooted and washed with sterile distilled water to remove the media and transferred to plastic pots filled with sterilized peat moss and pearlite mixture for acclimatization. The plastic pots were then covered with clear plastic, and holes were made to facilitate the exchange of gases. The regenerated plants were maintained in a 16/8 h photoperiod at 25 ± 2°C in the germinator for two weeks and the clear plastic was removed after the seedling was established.

Statistical Analysis And Box-behnken Design

The selection of explant, TDZ, NAA, BAP, and kinetin concentration was performed in a factorial experiment based on a completely randomized design in three replicate. The statistical analysis was carried out using IBM SPSS Ver.26 software. Duncan’s multiple range test was used to compare significance differences among treatment means at a 5% probability level. The box-behnken design of response surface methodology was used to assess and optimization of plant growth regulators concentration for callusing, somatic embryogenesis, and regeneration. The applied model for prediction and response analysis was:

$$y= {\beta }_{0}+\sum _{i=1}^{k}{\beta }_{i}{x}_{i}+\sum _{i=1}^{k}{\beta }_{ii}{x}_{i}^{2}+\sum _{i=1}^{k}\sum _{j=i+1}^{k-1}{\beta }_{ij}{x}_{i}{x}_{j}$$

In this model y, x_i, x_j, β₀, β_i, β_ii, β_ij, and k were the response variable, the independent variables that affect the y responses, intercepted, as linear regression coefficients for i^th factor, quadric, cross-product term, and the number of tested variables, respectively. The Box-Behnken design analysis was performed using the response surface methodology software Design-Expert, v.11 (Stat-Ease, Minneapolis, USA).

3.1. Effect of TDZ, BAP, and kinetin

3.1.1. Callus induction, callus fresh weight, and percentage of somatic embryogenesis

The leaf and petiole explants were cultured on MS basal medium supplied with TDZ, BAP, and kinetin for callus induction. The emergence of callus from the explants was observed after a week of incubation (Fig. 1). The statistical analysis indicated the significant effect of explant, TDZ, BAP, and kinetin and their interactions on the percentage of callusing and callus fresh weight (Table 1). The callus forming and growth response of both the explants were different. The highest percentage of callusing was observed in the petiole (66.67%) and the highest callus fresh weight was obtained in the leaf explant (307.72 mg/explant) (Fig. 2a). The maximum frequency of callusing (90.95%) and callus fresh weight (467.42 mg/explant) was on MS medium containing 0.25 mg/L TDZ and MS medium containing 0.5 mg/L TDZ (respectively). With increasing the concentrations of TDZ from 0.25 mg/L to 0.5 mg/L, the callus fresh weight increased from 287.12 mg/explant to 467.42 mg/explant and then decreased (Fig. 3a). Between different types and concentrations of cytokinins, the maximum callus formation (70.00%) and fresh weight of callus (363.03 mg/explant) were produced by the using of 3 mg/L BAP and 1 mg/L kinetin, respectively (Fig. 4a). After 3 weeks, the maximum frequency of callus formation (100.00%) and callus fresh weight (1207.75 mg/explant) was recorded from petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP and leaf explants on MS medium containing 0.5 mg/L TDZ and 1 mg/L kinetin, respectively (Table S1). The variance analysis indicated the significant effect of TDZ and its interactions with explant on the percentage of somatic embryogenesis (Table 1). The maximum percentage of somatic embryogenesis (92.66%) was observed on MS medium containing 0.5 mg/L TDZ (Fig. 3b). Also, the maximum percentage of somatic embryogenesis (95.71%) was recorded from petiole explants on an MS medium containing 0.5 mg/L TDZ (Fig. 5).

Table 1

ANOVA analysis of the effect of explant, TDZ, cytokinins and their interaction on *in vitro* culture of *H. niger*.
S.O.V.	df	Mean of square
S.O.V.	df	Percentage of callusing	Callus fresh weight	Percentage of somatic embryogenesis	Percentage of shooting	Number of the shoot
Explant (E)	1	1050.00 ^**	437890.26 ^**	192.86 ^ns	12.96 ^**	1.42 ^**
TDZ (T)	3	78237.30 ^**	1560490.41 ^**	87612.74 ^**	35509.99 ^**	95.50 ^**
Cytokinin (C)	6	600.00 ^**	71925.66 ^**	212.03 ^ns	2143.19 ^**	5.95 ^**
E × T	3	294.44 ^ns	164854.26 ^**	635.05 ^**	1114.18 ^**	0.49 ^*
E × C	6	655.56 ^**	119359.61 ^**	45.79 ^ns	408.81 ^**	1.94 ^**
T × C	18	624.34 ^**	69094.97 ^**	161.07 ^ns	885.12 ^**	4.50 ^**
E × T × C	18	588.89 ^**	90177.11 ^**	98.27 ^ns	960.55 ^**	4.45 ^**
Error	112	145.24	978.11	131.65	86.42	0.14
CV (%)		18.78	12.18	16.75	21.91	19.36
^** Significant at 1% probability, ^ns not significant.

3.1.1.1. Fitting the model for predicting the percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis

The petiole explant was selected for the percentage of callusing and the leaf explant was selected for callus fresh weight prediction. The interactions of the different concentrations of TDZ and BAP, and TDZ and kinetin were investigated by Box-Behnken design and shown in Table 2. The percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis were in the range from 20.00–100.00%, from 70.08 mg/explant to 1287.08 mg/explant, and from 40.00–100%, respectively. Among the experiments, experiment 4 (0.25 mg/L TDZ and 1 mg/L BAP), experiment 5 (0.25 mg/L TDZ and 3 mg/L BAP) and experiment 7 (0.5 mg/L TDZ and 3 mg/L BAP) had the highest percentage of callusing (100%) and experiment 1 (0.5 mg/L TDZ and 2 mg/L BAP) and experiment 9 (0.5 mg/L TDZ and 2 mg/L BAP) had the lowest percentage of callusing (20.00%). Also, experiment 8 (0.5 mg/L TDZ and 1 mg/L kinetin) had the highest callus fresh weight (1287.08 mg/explant), and experiment 13 (0.75 mg/L TDZ and 2 mg/L kinetin) had the lowest callus fresh weight (70.08 mg/explant). Experiment 1 (0.5 mg/L TDZ and 2 mg/L BAP), experiment 2 (0.5 mg/L TDZ and 2 mg/L BAP), experiment 5 (0.25 mg/L TDZ and 3 mg/L BAP), experiment 6 (0.75 mg/L TDZ and 1 mg/L BAP), experiment 7 (0.5 mg/L TDZ and 3 mg/L BAP), experiment 8 (0.5 mg/L TDZ and 1 mg/L BAP), experiment 9 (0.5 mg/L TDZ and 2 mg/L BAP), experiment 11 (0.5 mg/L TDZ and 2 mg/L BAP) and experiment 13 (0.75 mg/L TDZ and 2 mg/L BAP) had the highest percentage of somatic embryogenesis (100%) and experiment 4 (0.25 mg/L TDZ and 1 mg/L BAP) had the lowest percentage of somatic embryogenesis (40.00%). According to the ANOVA analysis the model was significant for the percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis (Table 3). The value of the R² were 86.80%, 74.47%, and 79.20% for the percentage of callusing, callus fresh weight, and percentage of embryogenesis, respectively; and the model was suitable for the predicting percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis within the investigated ranges. In the model for the percentage of callusing, the parameters A and B² were significant at the level of p < 0.05, but the parameters B, AB, and A² were not significant. For the callus fresh weight, the parameters A, B, and AB were not significant, but A² and B² were significant. For the percentage of somatic embryogenesis, the parameters A and AB were significant and A² and B² were not significant (Table 3). Therefore, the final predictive equation for describing the percentage of callusing, fresh weight of callus, and percentage of somatic embryogenesis was adjusted following:

Table 2

Box-Behnken design and the response and predicated values for percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis
Exp.	TDZ	BAP	Percentage of callusing (%)		Percentage of somatic embryogenesis (%)		Exp.	TDZ	Kinetin	Callus fresh weight (mg/explant)
Exp.	TDZ	BAP	Actual value	Predicted value	Actual value	Predicted value	Exp.	TDZ	Kinetin	Actual value	Predicted value
1	0.5 (0)	2 (0)	20.00	33.10	100.00	94.83	1	0.5 (0)	2 (0)	475.58	491.23
2	0.5 (0)	2 (0)	40.00	33.10	100.00	94.83	2	0.5 (0)	2 (0)	224.41	491.23
3	0.25 (-1)	2 (0)	80.00	63.91	60.00	64.60	3	0.25 (-1)	2 (0)	336.75	85.34
4	0.25 (-1)	1 (-1)	100.00	100.00	40.00	41.03	4	0.25 (-1)	1 (-1)	240.08	438.17
5	0.25 (-1)	3 (1)	100.00	100.00	100.00	94.37	5	0.25 (-1)	3 (1)	424.58	477.90
6	0.75 (1)	1 (-1)	80.00	79.71	100.00	100.00	6	0.75 (1)	1 (-1)	498.33	610.17
7	0.5 (0)	3 (1)	100.00	77.24	100.00	100.00	7	0.5 (0)	3 (1)	771.08	750.69
8	0.5 (0)	1 (-1)	80.00	77.24	100.00	91.26	8	0.5 (0)	1 (-1)	1287.08	977.15
9	0.5 (0)	2 (0)	20.00	33.10	100.00	94.83	9	0.5 (0)	2 (0)	505.08	491.23
10	0.5 (0)	2 (0)	30.00	33.10	70.00	94.83	10	0.5 (0)	2 (0)	451.50	491.23
11	0.5 (0)	2 (0)	30.00	33.10	100.00	94.83	11	0.5 (0)	2 (0)	466.25	491.23
12	0.75 (1)	3 (1)	60.00	69.71	80.00	81.03	12	0.75 (1)	3 (1)	150.45	117.52
13	0.75 (1)	2 (0)	40.00	30.57	100.00	91.26	13	0.75 (1)	2 (0)	70.08	8.84

Table 3

ANOVA for the response surface quadratic model for optimization of percentage of callusing, callus fresh weight and percentage of somatic embryogenesis
Explant: petiole				Explant: leaf
Source	df	Mean of square				Mean of square
Source	df	Percentage of callusing	Percentage of somatic embryogenesis	Source	df	Callus fresh weight
Model	5	2048.51 ^s	717.65 ^s	Model	5	1.71 × 10^{5 s}
A- TDZ	1	1666.67 ^s	1066.67 ^s	A- TDZ	1	13305.47 ^ns
B- BAP	1	0.00001 ^ns	266.67 ^ns	B- Kinetin	1	76926.20 ^ns
AB	1	100.00 ^ns	1600.00 ^s	AB	1	70857.12 ^ns
A²	1	552.05 ^ns	650.33 ^ns	A²	1	5.67 × 10 ^{5 s}
B²	1	5380.62 ^s	59.85 ^ns	B²	1	3.84 × 10^{5 s}
Residual	7	222.50	134.65	Residual	7	41974.11
Lack of fit	3	425.82 ^ns	107.51 ^ns	Lack of fit	3	81134.57 ^ns
Pure error	4	70.00	155.00	Pure error	4	12603.76
R² (%)		86.80	79.20	R² (%)		74.47
R²_adj (%)		77.37	64.34	R²_adj (%)		56.24
Adeq precision		8.14	8.46	Adeq precision		7.08
^s significant and ^ns non-significant

Percentage of callusing = 33.10–16.67 TDZ – 6.11 × 10^− 15 BAP – 5.00 TDZ BAP + 14.14 TDZ² + 44.14 BAP²

Callus fresh weight = 491.23–47.09 TDZ – 113.23 kinetin – 133.09 TDZ kinetin – 452.98 TDZ² + 372.69 kinetin²

Percentage of somatic embryogenesis = 92.24 + 13.33 TDZ + 6.67 BAP – 20.00 TDZ BAP − 15.34 TDZ² + 4.65 BAP²

3.1.1.2. Response surface analysis of the percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis

The results showed that TDZ and BAP concentrations are the important factors and with increasing their concentration, the percentage of callusing first decreases and then increased. According to Fig. 6, increasing the concentration of TDZ along with BAP increases the percentage of callusing. The best TDZ and BAP concentrations for the maximum percentage of callusing were lower than 0.5 mg/L and 2 mg/L, respectively. The highest percentage of callusing was obtained with the application of 0.25 mg/L TDZ and 1 mg/L BAP. However, the best conditions for maximizing the percentage of callusing (100.00%) were predicted by culturing of petiole explant on the MS medium containing 0.11 mg/L TDZ and 1.253 mg/L BAP. The results also indicated that the callus fresh weight depends on TDZ and kinetin concentration. According to Fig. 5b and e, increasing the TDZ concentration from 0.25 to 0.5 mg/L with increasing the kinetin concentration from 1 to 2 mg/L, increases the callus fresh weight, but then with increasing the TDZ and kinetin concentration the callus fresh weight decreases. The highest callus fresh weight is achieved by using 0.25–0.75 mg/L of TDZ and increases the callus fresh weight by decreasing kinetin concentration. In the present study, the highest callus fresh weight was achieved by adding 0.25 mg/L TDZ and 1 mg/L kinetin, but it is predicted that the highest callus fresh weight with 3273.64 mg/explant is obtained by culturing the leaf explant on MS medium containing 0.52 mg/L TDZ. By using the different concentrations of TDZ and BAP, the highest percentage of somatic embryogenesis was observed in their highest concentrations. According to Fig. 6, the highest percentage of somatic embryogenesis was obtained by applying up to 0.5 mg/L of TDZ, which is reduced by exposing the most time of cell suspension culture to this range. It is predicted that the highest percentage of embryogenesis (100%) was obtained by culture of the petiole explant on MS medium supplemented with 0.533 mg/L TDZ and 1 mg/L BAP or MS medium containing 1.159 mg/L TDZ and 3 mg/L BAP.

3.1.2. Percentage Of Shooting And Number Of The Shoot

The leaf explant has a higher frequency of shooting (42.70%) and the number of the shoot was highest in petiole explants than in the leaf explants (2.02 per callus) (Fig. 2b). The addition of TDZ to MS basal medium induced shoot formation, but it is observed that the higher concentrations of that had a negative effect on shoot formation and regeneration. The 0.25 mg/L produced the highest percentage of shooting (64.44%) and the number of the shoot (3.66 per callus) (Fig. 3c). In this study, we used two types of cytokinins including BAP and kinetin and observed that BAP is better than kinetin for shoot formation. Also, between different concentrations of BAP 1 mg/L was suitable and showed the highest percentage of shooting (56.18%) and the number of the shoot (7.24 per callus) (Fig. 3b). Therefore, the maximum frequency of shooting (93.30%) and the number of the shoot (7.75 per callus) was recorded from leaf explants on MS medium containing 0.50 mg/L TDZ and 1 mg/L kinetin and petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, respectively (Table S1).

3.1.2.1. Fitting The Model For Predicting The Percentage Of Shooting And Number Of The Shoot

The leaf explant was selected for the percentage of shooting and the petiole explant was selected for the number of the shoot prediction. The interactions of the different concentrations of TDZ and BAP, and TDZ and kinetin were investigated by Box-Behnken design and shown in Table 4. The percentage of shooting and number of shoots were in the range of 50.00–100.00% and from 1.00 to 8.50 per callus, respectively. Among the experiments, experiment 4 (0.25 mg/L TDZ and 1 mg/L BAP) had the highest percentage of shooting (100%) and experiment 2 (0.50 mg/L TDZ and 2 mg/L BAP), and experiment 13 (0.75 mg/L TDZ and 2 mg/L BAP) had the lowest percentage of shooting (50.00%). Also, experiment 4 (0.25 mg/L TDZ and 1 mg/L BAP) had the highest number of the shoot (8.50 per callus) and experiment 7 (0.50 mg/L TDZ and 3 mg/L BAP) and experiment 9 (0.50 mg/L TDZ and 2 mg/L BAP) had the lowest number of the shoot (1.00 per callus). The results showed that the model was significant for the percentage of shooting and number of the shoot (Table 5). The value of the R² was 78.40% and 97.01% for the percentage of shooting and number of the shoot, respectively. So, the model was suitable for the predicting percentage of shooting and number of the shoot within the tested ranges. In the model for the percentage of shooting, the parameters A and B were significant, but AB, A² and B² were not significant. Also, for the number of shoots, the parameters A, B, A² and B² were significant, but AB was not significant. Therefore, the equation for the percentage of shooting and number of the shoot was following:

Table 4

Box-Behnken design and the response and predicated values for percentage of shooting and number of the shoot.
Percentage of shooting (%)					Number of the shoot
Experiment	TDZ	BAP	Actual value	Predicted value	Experiment	TDZ	BAP	Actual value	Predicted value
1	0.5 (0)	2 (0)	60.00	63.56	1	0.5 (0)	2 (0)	1.50	1.49
2	0.5 (0)	2 (0)	50.00	63.56	2	0.5 (0)	2 (0)	2.00	1.49
3	0.25 (-1)	2 (0)	80.00	78.31	3	0.25 (-1)	2 (0)	5.50	6.08
4	0.25 (-1)	1 (-1)	100.00	100.00	4	0.25 (-1)	1 (-1)	8.50	8.43
5	0.25 (-1)	3 (1)	70.00	70.57	5	0.25 (-1)	3 (1)	6.50	6.00
6	0.75 (1)	1 (-1)	66.67	65.01	6	0.75 (1)	1 (-1)	3.00	3.22
7	0.5 (0)	3 (1)	60.00	61.65	7	0.5 (0)	3 (1)	1.00	1.72
8	0.5 (0)	1 (-1)	80.00	80.54	8	0.5 (0)	1 (-1)	3.67	3.52
9	0.5 (0)	2 (0)	75.00	63.56	9	0.5 (0)	2 (0)	1.00	1.49
10	0.5 (0)	2 (0)	60.00	63.56	10	0.5 (0)	2 (0)	2.00	1.49
11	0.5 (0)	2 (0)	75.00	63.56	11	0.5 (0)	2 (0)	1.50	1.49
12	0.75 (1)	3 (1)	60.00	57.79	12	0.75 (1)	3 (1)	2.25	2.04
13	0.75 (1)	2 (0)	50.00	53.87	13	0.75 (1)	2 (0)	1.50	1.49

Table 5

ANOVA for the response surface quadratic model for optimization of percentage of shooting and number of the shoot
Explant: leaf			Explant: petiole
Source	df	Mean of square of percentage of shooting	Source	df	Mean of square of number of the shoot
Model	5	363.64 ^s	Model	5	12.89 ^s
A- TDZ	1	896.21 ^s	A- TDZ	1	31.51 ^s
B- BAP	1	535.25 ^s	B- BAP	1	4.90 ^s
AB	1	136.07 ^ns	AB	1	0.39 ^ns
A²	1	17.67 ^ns	A²	1	14.60 ^s
B²	1	156.57 ^ns	B²	1	3.55 ^s
Residual	7	71.57	Residual	7	0.28
Lack of fit	3	10.33 ^ns	Lack of fit	3	0.43 ^ns
Pure error	4	117.50	Pure error	4	0.17
R² (%)		78.40	R²		97.01
R²_adj (%)		62.97	R²_adj		94.88
Adeq Precision		8.22	Adeq Precision		19.18

Percentage of shooting = 63.56–12.22 TDZ − 9.44 BAP + 5.83 TDZ BAP + 2.53 TDZ² + 7.53 BAP²

Number of the shoot = 1.48–2.29 TDZ − 0.90 BAP + 0.31 TDZ BAP + 2.30 TDZ² + 1.13 BAP²

3.1.2.2. Response Surface Analysis Of Percentage Of Shooting And Number Of The Shoot

The results showed that by increasing the TDZ and BAP concentration, the percentage of shooting decreased. According to Fig. 7, increasing the concentration of TDZ along with BAP decreases the percentage of shooting. The best TDZ and BAP concentrations for the maximum percentage of the shooting were lower than 0.5 mg/L and 2 mg/L, respectively. The highest percentage of shooting was obtained with the application of 0.25 mg/L TDZ and 1 mg/L BAP. However, the best conditions for maximizing the percentage of shooting (100.00%) were predicted by culturing of leaf explant on MS medium with 0.18 mg/L TDZ and 2.89 mg/L BAP. The results also indicated that the number of shoots depends on TDZ and kinetin concentration. According to Fig. 7, increasing the TDZ concentration from 0.25 to 0.75 mg/L with increasing the BAP concentration from 1 to 3 mg/L, decreases the number of the shoot. In the present study, the highest number of the shoot was achieved by adding 0.25 mg/L TDZ and 1 mg/L BAP, but it is predicted that the highest number of the shoot obtained by culturing the petiole explant on MS medium with 0.1 mg/L TDZ and 3 mg/L BAP (8.61 shoots per callus).

3.2. Effect Of Naa, Bap And Kinetin

3.2.1. Callus induction, callus fresh weight and percentage of somatic embryogenesis

The leaf and petiole explants were cultured on MS basal medium containing NAA, BAP, and kinetin for callus induction. The statistical analysis indicated the significant effect of explant, NAA, BAP, and kinetin and their interactions on the percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis (Table 6). The callus forming and growth response of both explants were different. The highest percentage of callusing was observed in the petiole explant (59.88%), the highest callus fresh weight was obtained in the leaf explant (174.83 mg/explant), and the highest percentage of somatic embryogenesis was recorded in the petiole explant (54.38%) (Fig. 8 ab). The maximum frequency of callusing (85.71%), callus fresh weight (246.76 mg/explant), and percentage of somatic embryogenesis (72.90%) were recorded on MS medium containing 0.75, 0.50, and 0.50 mg/L NAA, respectively. With increasing the concentrations of NAA from 0.25 mg/L to 0.5 mg/L, the callus fresh weight increased from 67.57 mg/explant to 246.76 mg/explant and then decreased (Fig. 9 ab). Between different types and concentrations of cytokinins, the maximum callus formation (67.50%), callus fresh weight (269.09 mg/explant), and percentage of somatic embryogenesis (64.30%) produced by the addition of 3 mg/L BAP, 2 mg/L kinetin, and 1 mg/L BAP, respectively (Fig. 10 ab). After 3 weeks, the maximum frequency of callus formation (100.00%) was obtained from leaf explant on MS medium with 0.5 mg/L NAA and 2 mg/L BAP, MS medium with 0.5 mg/L NAA and 3 mg/L BAP, MS medium with 0.75 mg/L NAA and 1 mg/L BAP, petiole explant on MS medium with 0.5 mg/L NAA and 3 mg/L BAP, MS medium with only 0.75 mg/L NAA, MS medium with 0.75 mg/L NAA and 1 mg/L BAP, MS medium with 0.75 mg/L NAA and 2 mg/L BAP, and MS medium with 0.75 mg/L NAA and 3 mg/L BAP. Also, the maximum callus fresh weight (1182.08 mg/explant) was recorded from leaf explants on MS medium containing 0.5 mg/L NAA and 2 mg/L kinetin. The highest percentage of somatic embryogenesis (100.00%) was recorded from leaf explants on MS medium containing 0.5 mg/L NAA and 2 mg/L BAP and MS medium containing 0.5 mg/L NAA and 3 mg/L BAP and from petiole explant on MS medium containing 0.5 mg/L NAA and 1 mg/L BAP, MS medium containing 0.5 mg/L NAA and 3 mg/L BAP, MS medium containing 0.5 mg/L NAA and 2 mg/L kinetin, MS medium containing 0.75 mg/L NAA and 1 mg/L BAP, and MS medium containing 0.75 mg/L NAA and 2 mg/L BAP (Table S2).

Table 6

ANOVA analysis of the effect of explant, NAA, cytokinins and their interaction on *in vitro* culture of *H. niger*.
S.O.V.	df	Mean of square
S.O.V.	df	Percentage of callusing	Callus fresh weight	Percentage of somatic embryogenesis	Percentage of shooting	Number of the shoot	Percentage of rooting	Number of the root
Explant (E)	1	2350.02 ^**	342392.77 ^**	7555.91 ^**	928.72 ^**	0.60 ^**	6789.43 ^**	30.91 ^**
NAA (N)	3	67996.58 ^**	559559.67 ^**	46643.59 ^**	4225.94 ^**	14.07 ^**	2371.97 ^**	17.64 ^**
Cytokinin (C)	6	3234.41 ^**	121638.23 ^**	3331.28 ^**	879.12 ^**	3.04 ^**	270.30 ^**	1.93 ^**
E × N	3	635.47 ^**	128492.66 ^**	3212.80 ^**	754.12 ^**	0.35 ^**	1884.35 ^**	12.29 ^**
E × C	6	2254.65 ^**	227376.09 ^**	1495.27 ^**	459.97 ^**	2.06 ^**	249.98 ^**	3.34 ^**
N × C	18	1747.35 ^**	107253.00 ^**	2597.23 ^**	672.70 ^**	5.41 ^**	690.86 ^**	4.36 ^**
E × N × C	18	1140.25 ^**	133475.03 ^**	1860.95 ^**	430.27 ^**	2.10 ^**	520.27 ^**	2.37 ^**
Error	112	103.69	612.22	80.94	2.50	0.008	1.85	0.011
CV (%)		17.92	19.08	18.87	19.75	18.48	19.23	17.26
^** Significant at 1% probability, ^ns not significant.

3.2.1.1. Fitting the model for predicting of percentage of callusing, callus fresh weight and percentage of somatic embryogenesis

The petiole explant was selected for the percentage of callusing and somatic embryogenesis and the leaf explant was selected for callus fresh weight prediction. The interactions of the different concentrations of NAA and BAP, and NAA and kinetin were investigated by Box-Behnken design and shown in Table 7. The percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis were in the range of 40.00–100.00%, from zero to 1194.08 mg/explant, and from 40.00–100%, respectively. Among the experiments, experiment 5 (0.25 mg/L NAA and 3 mg/L BAP), experiment 6 (0.75 mg/L NAA and 1 mg/L BAP), and experiment 12 (0.75 mg/L NAA and 3 mg/L BAP) had the highest percentage of callusing (100%) and experiment 2 (0.50 mg/L NAA and 2 mg/L BAP) and experiment 10 (0.50 mg/L NAA and 2 mg/L BAP) had the lowest percentage of callusing (40.00%). Also, experiment 9 (0.50 mg/L NAA and 2 mg/L kinetin) had the highest callus fresh weight (1194.08 mg/explant) and experiment 3 (0.25 mg/L NAA and 2 mg/L kinetin) and experiment 5 (0.25 mg/L NAA and 3 mg/L kinetin) had the lowest callus fresh weight. Experiment 1 (0.50 mg/L NAA and 2 mg/L BAP), experiment 2 (0.50 mg/L NAA and 2 mg/L BAP), experiment 5 (0.25 mg/L NAA and 3 mg/L BAP), experiment 6 (0.75 mg/L NAA and 1 mg/L BAP), experiment 7 (0.50 mg/L NAA and 3 mg/L BAP), experiment 8 (0.50 mg/L NAA and 1 mg/L BAP), experiment 9 (0.5 mg/L NAA and 2 mg/L BAP), experiment 11 (0.5 mg/L NAA and 2 mg/L BAP) and experiment 13 (0.75 mg/L NAA and 2 mg/L BAP) had the highest percentage of somatic embryogenesis (100%) and experiment 4 (0.25 mg/L NAA and 1 mg/L BAP) had the lowest percentage of somatic embryogenesis (40.00%). The model was significant for the percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis (Table 8). The value of the R² were 80.38%, 79.09%, and 78.79 for the percentage of callusing, callus fresh weight, and percentage of embryogenesis, respectively. So, the model was suitable for the predicting percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis within the tested ranges. In the model for the percentage of callusing, the parameters A, A² and B² were significant at the level of p < 0.05, but the parameters B and AB were not significant. For callus fresh weight, the parameters A and B² were significant, but B, AB and A² were not significant. For the percentage of somatic embryogenesis, the parameters A and AB were significant but the parameters B, A² and B² were not significant (Table 8). The predictive equation for the percentage of callusing, fresh weight of callus, and percentage of somatic embryogenesis was following:

Table 7

Box-Behnken design and the response and predicated values for percentage of callusing, callus fresh weight, and percentage of somatic embryogenesis
Exp.	NAA	BAP	Percentage of callusing (%)		Percentage of somatic embryogenesis (%)		Exp.	NAA	Kinetin	Callus fresh weight (mg/explant)
Exp.	NAA	BAP	Actual value	Predicted value	Actual value	Predicted value	Exp.	NAA	Kinetin	Actual value	Predicted value
1	0.5 (0)	2 (0)	60.00	56.21	100.00	94.83	1	0.5 (0)	2 (0)	875.58	956.63
2	0.5 (0)	2 (0)	40.00	56.21	100.00	94.83	2	0.5 (0)	2 (0)	905.58	956.63
3	0.25 (-1)	2 (0)	80.00	72.82	60.00	64.60	3	0.25 (-1)	2 (0)	0.00	395.82
4	0.25 (-1)	1 (-1)	80.00	81.09	40.00	41.03	4	0.25 (-1)	1 (-1)	60.58	0.00
5	0.25 (-1)	3 (1)	100.00	100.00	100.00	94.37	5	0.25 (-1)	3 (1)	0.00	0.00
6	0.75 (1)	1 (-1)	100.00	100.00	100.00	100.00	6	0.75 (1)	1 (-1)	212.58	184.50
7	0.5 (0)	3 (1)	100.00	84.48	100.00	100.00	7	0.5 (0)	3 (1)	109.83	474.27
8	0.5 (0)	1 (-1)	75.00	69.48	100.00	91.26	8	0.5 (0)	1 (-1)	132.28	312.11
9	0.5 (0)	2 (0)	60.00	56.21	100.00	94.83	9	0.5 (0)	2 (0)	1194.08	956.63
10	0.5 (0)	2 (0)	40.00	56.21	70.0	94.83	10	0.5 (0)	2 (0)	1181.08	956.63
11	0.5 (0)	2 (0)	60.00	56.21	100.00	94.83	11	0.5 (0)	2 (0)	1171.08	956.63
12	0.75 (1)	3 (1)	100.00	100.00	80.00	81.03	12	0.75 (1)	3 (1)	782.08	661.70
13	0.75 (1)	2 (0)	100.00	86.15	100.00	91.26	13	0.75 (1)	2 (0)	838.08	986.54

Table 8

ANOVA for the response surface quadratic model for optimization of percentage of callusing, callus fresh weight and percentage of somatic embryogenesis
Explant: petiole				Explant: leaf
Source	df	Mean of square				Mean of square
Source	df	Percentage of callusing	Percentage of somatic embryogenesis	Source	df	Callus fresh weight
Model	5	1007.87 ^s	751.55 ^s	Model	5	4.57 × 10^{5 s}
A- NAA	1	226.67 ^ns	1066.67 ^s	A- NAA	1	5.23 × 10^{5 s}
B- BAP	1	337.50 ^ns	266.67 ^ns	B- Kinetin	1	39442.18 ^ns
AB	1	100.00 ^ns	1600.00 ^s	AB	1	99250.20 ^ns
A²	1	1496.31 ^s	788.51 ^ns	A²	1	1.95 × 10 ^{5 ns}
B²	1	1192.12 ^s	26.60 ^ns	B²	1	8.77 × 10^{5 s}
Residual	7	175.70	144.50	Residual	7	86238.53
Lack of fit	3	249.96 ^ns	97.16 ^ns	Lack of fit	3	1.67 × 10^{5 ns}
Pure error	4	120.00	180.00	Pure error	4	25670.67
R² (%)		80.38	78.79	R² (%)		79.09
R²_adj (%)		66.37	63.64	R²_adj (%)		64.16
Adeq precision		5.91	8.16	Adeq precision		6.17
^s significant and ^ns non-significant

Percentage of callusing = 56.21 + 6.67 NAA + 7.50 BAP – 5.00 (NAA × BAP) + 23.27 NAA² + 20.77 BAP²

Callus fresh weight = 956.62 + 295.36 NAA + 81.08 kinetin + 157.52 (NAA × kinetin) – 265.45 NAA² – 563.43 kinetin²

Percentage of somatic embryogenesis = 94.83 + 13.33 NAA + 6.67 BAP – 20.00 (NAA × BAP) − 16.89 TDZ² + 3.10 BAP²

3.2.1.2. Response surface analysis of percentage of callusing, callus fresh weight and percentage of somatic embryogenesis

The results showed that NAA and BAP concentrations are important factors and with increasing their concentration, the percentage of callusing first decreases and then increased. According to Fig. 11, increasing the concentration of NAA along with BAP increases the percentage of callusing. The best NAA and BAP concentrations for the maximum percentage of callusing were upper than 0.5 mg/L and 2 mg/L, respectively. The highest percentage of callusing was obtained with the application of 0.75 mg/L NAA and 1 mg/L BAP. However, the best conditions for maximizing the percentage of callusing (100.00%) were predicted by culturing of petiole explant on MS medium containing 0.15 mg/L NAA and 1.282 mg/L BAP. The results also indicated that the callus fresh weight depends on NAA and kinetin concentration. According to Fig. 11, increasing the NAA concentration from 0.50 to 0.75 mg/L with increasing the kinetin concentration, increases the callus fresh weight, but then with increasing the NAA and kinetin concentration the callus fresh weight decreases. The highest callus fresh weight is achieved by using 0.50–0.75 mg/L of NAA and increases the callus fresh weight by increasing kinetin concentration. In the present study, the highest callus fresh weight was observed by adding 0.50 mg/L NAA and 2 mg/L kinetin, but it is predicted that the highest callus fresh weight with 4518.24 mg/explant is obtained by culturing the leaf explant on MS medium with 0.60 mg/L NAA and 2.30 mg/L kinetin. By using the different concentrations of NAA and BAP, the highest percentage of somatic embryogenesis was observed in their highest concentrations. According to Fig. 11, the highest percentage of somatic embryogenesis was obtained by applying up to 0.5 mg/L of NAA and up to 2 mg/L BAP. It is predicted that the highest percentage of embryogenesis (100%) was obtained by culture of the petiole explant on MS medium supplemented with 0.60 mg/L NAA and 0.425 mg/L BAP.

3.2.2. Percentage Of Shooting, Number Of The Shoot, Percentage Of Rooting And Number Of Root

The leaf explant has a higher frequency of shooting (10.36%), a number of the shoot (0.54 per callus), a percentage of rooting (13.43%), and a number of the root (1.04 per callus) (Fig. 8 cd). The addition of NAA to MS basal medium induced shoot formation, but it is observed that the higher concentrations of that have a negative effect on shoot formation and regeneration. The 0.50 mg/L produced the highest percentage of shooting (22.26%) and the number of the shoot (1.28 per callus). Also, the highest percentage of rooting (14.24%) and the number of roots (1.19 per callus) were obtained using 0.75 and 0.50 mg/L NAA, respectively. We used two types of cytokinins including BAP and kinetin and observed that BAP is better than kinetin for the shoot and root formation (Fig. 9 cd). Among different concentrations of BAP, 3 mg/L was suitable for shoot formation and 1 mg/L was better for root formation. The highest percentage of shooting (15.21%) and the number of the shoot (1.16 per callus) were achieved by using 3 mg/L BAP and the highest percentage of rooting (12.08%) and the number of the root (0.98 per callus) obtained by adding of 1 mg/L BAP (Fig. 10 cd). Therefore, the maximum frequency of shooting (80.00%), number of shoots (6.27 per callus), percentage of rooting (76.67%), and number of roots (5.44 per callus) was recorded from leaf explants on MS medium containing 0.50 mg/L NAA and 3 mg/L BAP (Table S2).

3.1.2.1. Fitting the model for predicting of percentage of shooting, number of the shoot, percentage of rooting and number of root

According to the Box-Behnken design analysis, the model was not suitable for the percentage of rooting and number of the root. The leaf explant was selected for the percentage of shooting and the petiole explant was selected for the number of the shoot prediction. The interactions of the different concentrations of NAA and BAP were investigated by Box-Behnken design and shown in Table 9. The percentage of shooting and number of the shoot were in the range of 0.00–80.00% and from 0.00 to 2.50 per callus, respectively. Among the experiments, experiment 7 (0.50 mg/L NAA and 3 mg/L BAP) had the highest percentage of shooting (80.00%) and experiment 3 (0.25 mg/L NAA and 2 mg/L BAP), experiment 4 (0.25 mg/L NAA and 1 mg/L BAP), and experiment 13 (0.75 mg/L NAA and 2 mg/L BAP) had the lowest percentage of shooting (0.00%). Also, experiment 6 (0.75 mg/L NAA and 1 mg/L BAP) had the highest number of the shoot (2.50 per callus), and experiment 1 (0.50 mg/L NAA and 2 mg/L BAP), experiment 5 (0.50 mg/L NAA and 2 mg/L BAP), experiment 4 (0.25 mg/L NAA and 1 mg/L BAP), experiment 5 (0.25 mg/L NAA and 3 mg/L BAP), experiment 8 (0.50 mg/L NAA and 1 mg/L BAP), experiments 9, 10 and 11 (0.50 mg/L NAA and 2 mg/L BAP), experiment 12 (0.75 mg/L NAA and 3 mg/L BAP), and experiment 13 (0.75 mg/L NAA and 2 mg/L BAP) had the lowest number of the shoot (0.00 per callus). The model was significant for the percentage of shooting and non-significant for the number of the shoot (Table 10). The value of the R² was 75.55% for the percentage of shooting. So, the model was suitable for the predicting percentage of shooting within the tested ranges. In the model for the percentage of shooting, the parameters A, B and AB were not significant, but A² and B² were significant. The final equation for describing the percentage of shooting was following:

Table 9

Box-Behnken design and the response and predicated values for percentage of shooting and number of the shoot.
Percentage of shooting (%)					Number of the shoot
Experiment	NAA	BAP	Actual value	Predicted value	Experiment	NAA	BAP	Actual value	Predicted value
1	0.5 (0)	2 (0)	15.00	33.71	1	0.5 (0)	2 (0)	0.00	0.21
2	0.5 (0)	2 (0)	20.00	33.71	2	0.5 (0)	2 (0)	0.00	0.21
3	0.25 (-1)	2 (0)	0.00	0.00	3	0.25 (-1)	2 (0)	1.75	0.23
4	0.25 (-1)	1 (-1)	0.00	0.00	4	0.25 (-1)	1 (-1)	0.00	0.13
5	0.25 (-1)	3 (1)	20.00	31.30	5	0.25 (-1)	3 (1)	0.00	1.38
6	0.75 (1)	1 (-1)	35.00	27.97	6	0.75 (1)	1 (-1)	2.50	1.63
7	0.5 (0)	3 (1)	80.00	61.15	7	0.5 (0)	3 (1)	2.50	0.73
8	0.5 (0)	1 (-1)	40.00	50.32	8	0.5 (0)	1 (-1)	0.00	0.73
9	0.5 (0)	2 (0)	40.00	33.71	9	0.5 (0)	2 (0)	0.00	0.21
10	0.5 (0)	2 (0)	35.00	33.71	10	0.5 (0)	2 (0)	0.00	0.21
11	0.5 (0)	2 (0)	50.00	33.71	11	0.5 (0)	2 (0)	0.00	0.21
12	0.75 (1)	3 (1)	7.50	15.05	12	0.75 (1)	3 (1)	0.00	0.38
13	0.75 (1)	2 (0)	0.00	0.00	13	0.75 (1)	2 (0)	0.00	0.48

Table 10

ANOVA for the response surface quadratic model for optimization of percentage of shooting and number of the shoot
Explant: leaf			Explant: petiole
Source	df	Mean of square of percentage of shooting	Source	df	Mean of square of number of the shoot
Model	5	998.37 ^s	Model	5	0.56 ^ns
A- NAA	1	84.38 ^ns	A- NAA	1	0.09 ^ns
B- BAP	1	176.04 ^ns	B- BAP	1	0.00 ^ns
AB	1	564.06 ^ns	AB	1	1.56 ^ns
A²	1	3982.76 ^s	A²	1	0.06 ^ns
B²	1	1339.91 ^s	B²	1	0.76 ^ns
Residual	7	230.83	Residual	7	1.32
Lack of fit	3	261.94 ^ns	Lack of fit	3	3.08 ^ns
Pure error	4	107.50	Pure error	4	0.00
R² (%)		75.55	R²		23.37
R²_adj (%)		58.08	R²_adj		-0.31
Adeq Precision		6.70	Adeq Precision		1.92

Percentage of shooting = 33.71 + 3.75 NAA + 5.42 BAP − 11.87 (NAA × BAP) − 37.97 NAA² + 22.02 BAP²

3.2.2.2. Response Surface Analysis Of Percentage Of Shooting

The results showed that by increasing the NAA and BAP concentration together, the percentage of shooting decreased. According to Fig. 12, increasing the concentration of NAA along with BAP decreases the percentage of shooting. The best NAA and BAP concentrations alone for the maximum percentage of the shooting were upper than 0.5 mg/L and 2 mg/L, respectively. The highest percentage of shooting was obtained with the application of 0.50 mg/L NAA and 3 mg/L BAP. However, the best conditions for maximizing the percentage of shooting (100.00%) were predicted by culturing of leaf explant on MS medium containing 0.49 mg/L NAA and 1.72 mg/L BAP.

Plant growth regulators play an important role in the somatic embryogenesis of plants. Among them, auxins induce somatic embryogenesis but hinder their development, and after induction, their concentration should be reduced or removed from the culture medium. Studies show that depending on the type of plant, different types and concentrations of plant growth regulators have different effects on somatic embryogenesis. Plant regeneration through somatic embryogenesis also depends on the genotype. Different plant genotypes respond differently to plant growth regulators. Also, different explants are used for somatic embryogenesis. The type and size of the explant also have a great effect on somatic embryogenesis (Gulzar et al. 2020). For example, Kocak et al. (2014) have reported that leaves and petioles of Cyclamen persicum show a better response to callus induction than ovules and ovaries.

Studies show that many plants require auxin for somatic embryogenesis. Auxins play an essential role in the early stages of somatic embryo development (Nic-Can and Loyola-Vargas 2016). For example, in date palm, the amount of somatic embryogenesis increased 20 times with the application of a low concentration of 2,4-D (Abohatem et al. 2017). Cytokinins are also involved in plant growth and cause the formation of lateral buds and delay the aging of leaves. Auxins and cytokinins have synergistic activity and stimulate cell division (Singh and Sinha 2017). A high ratio of these two plant growth regulators causes branching and a low ratio causes rooting (Kotov and Kotova 2018). In the process of somatic embryogenesis, these two plant growth regulators act synergistically or antagonistically (Méndez-Hernández et al. 2019). Plant growth regulators change the genetic program of somatic cells by expressing different genes and regulating different stages of somatic embryogenesis (Loyola-Vargas and Ochoa-Alejo 2016). Most of these genes are related to transcription factors, proteins involved in the cell cycle, biosynthesis of plant growth regulators, and proteins involved in signaling (Leljak-Levanić et al. 2015).

In this study, the effect of NAA, TDZ, BAP, and Kinetin on callus formation, somatic embryogenesis, and regeneration was investigated and it was observed that the efficiency of TDZ is higher than NAA in somatic embryogenesis and regeneration of H. niger plant. The highest percentage of shooting and number of shoots was obtained by using TDZ. Also, we used two types of explants including leaf and petiole, but their response was different depending on the types and concentrations of plant growth regulators. Generally, we observed maximum regeneration and number of the shoot in the leaf explants on MS medium containing 0.50 mg/L TDZ and 1 mg/L kinetin and petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, respectively.

Although TDZ is classified as a cytokinin in the plant growth regulator classification due to its natural cytokinin response, it shows cytokinin and auxin-like responses in different plant species in vitro culture. The mode of action of TDZ has not been fully determined and it may be involved in the accumulation or synthesis of endogenous plant growth hormones (Ghosh et al. 2018). The application of TDZ induced somatic embryogenesis in grape (Dhekney et al. 2016), rubus (Fiola et al. 1990), and geranium (Visser et al. 1992). For example, Mithila et al. (2003) reported that higher concentrations of TDZ (5–10 µM) are more effective for the somatic embryogenesis of Saintpaulia ionantha. Ghosh et al. (2018) reported that 9 µM TDZ is most effective for somatic embryo formation in half-high blueberry. The leaf explants of Scaevola aemula induced somatic embryos on MS medium supplemented with 0.2 mg/L 2,4-D and 0.2–0.5 mg/L BA and seedlings produced on MS medium with 0.5 mg/L BA (Bhalla and Sweeney 1998). Liang et al. (2020) reported that the root explants of Scaevola sericea produce 7.6 somatic embryos on MS medium supplemented with 2.5 mg/L TDZ. The leaf explants of Primulina tabacum induced somatic embryos if cultured on a TDZ-containing medium, but when transferred to a medium containing BA, produced somatic embryos and adventitious shoots (Yang et al. 2012). The optimum conditions for regeneration of Gentiana triflora cv. WSP-3 from leaf, stem, and root explants were the MS medium with 5–10 mg/L TDZ and 0.1 mg/L NAA for the leaf and stem explants, and 10 mg/L TDZ and 1 mg/L NAA for the root explants (Hosokawa et al. 1996). In the Ocimum basilicum L. the shoots were induced on the MS medium supplemented with 0.1–10 µM BA or TDZ (Fraj et al. 2016).

In this study, for the first time, the Box-Behnken design of response surface methodology was used to optimize and predict the concentration of plant growth regulators for callus formation, somatic embryogenesis, and regeneration of H. niger. The results showed that the use of these plant growth regulators has a significant effect on callus formation, somatic embryogenesis, and regeneration of H. niger from leaf and petiole explants. By using different concentrations of TDZ, BAP, and kinetin, the maximum frequency of callus formation, callus fresh weight, percentage of somatic embryogenesis, frequency of shooting, and the number of the shoot were recorded from petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, leaf explants on MS medium containing 0.5 mg/L TDZ and 1 mg/L kinetin, petiole explants on an MS medium containing 0.5 mg/L TDZ, leaf explants on MS medium containing 0.50 mg/L TDZ and 1 mg/L kinetin, and petiole explants on MS medium containing 0.25 mg/L TDZ and 1 mg/L BAP, respectively. Also, the highest percentage of callusing (100.00%), callus fresh weight, percentage of embryogenesis, percentage of shooting, and the number of the shoot were predicted by culturing of petiole explant on the MS medium containing 0.11 mg/L TDZ and 1.253 mg/L BAP, leaf explant on MS medium containing 0.52 mg/L TDZ, petiole explant on MS medium with 0.533 mg/L TDZ and 1 mg/L BAP, leaf explant on MS medium with 0.18 mg/L TDZ and 2.89 mg/L BAP and petiole explant on MS medium with 0.1 mg/L TDZ and 3 mg/L BAP. By using different concentrations of NAA, BAP, and kinetin, the highest percentage of callusing and somatic embryogenesis was obtained in different treatments, but the maximum callus fresh weight was recorded from leaf explants on MS medium containing 0.5 mg/L NAA and 2 mg/L kinetin and highest frequency of shooting, the number of the shoot, percentage of rooting, and the number of the root observed from leaf explants on MS medium containing 0.50 mg/L NAA and 3 mg/L BAP. Also, it is predicted that the highest percentage of callusing, callus fresh weight, percentage of embryogenesis, and percentage of the shooting were obtained by culturing of petiole explant on MS medium containing 0.15 mg/L NAA and 1.282 mg/L BAP, leaf explant on MS medium with 0.60 mg/L NAA and 2.30 mg/L kinetin, petiole explant on MS medium with 0.60 mg/L NAA and 0.425 mg/L BAP, and leaf explant on MS medium containing 0.49 mg/L NAA and 1.72 mg/L BAP.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Roghayeh Ahmadpour. The first draft of the manuscript was written by Roghayeh Ahmadpour and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgment

The authors are grateful to the University of Zanjan and Plant tissue culture laboratory of Imam Khomeini International University for financial and technical support.

Competing Interests

The authors declare they have no financial interests.

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Prediction of the concentration of plant growth regulators for somatic embryogenesis and regeneration of Hyoscyamus niger using Box-Behnken design of response surface methodology

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Abstract

Figures

Key Message

Introduction

Materials And Methods

Plant materials

Dormancy Breaking

Culture Media

Seed Culture And Explants Preparation

Callus Induction, Somatic Embryogenesis, And Shoot Regeneration

Rooting And Acclimatization

Statistical Analysis And Box-behnken Design

Results

3.1. Effect of TDZ, BAP, and kinetin

3.1.1. Callus induction, callus fresh weight, and percentage of somatic embryogenesis

3.1.2. Percentage Of Shooting And Number Of The Shoot

3.1.2.1. Fitting The Model For Predicting The Percentage Of Shooting And Number Of The Shoot

3.1.2.2. Response Surface Analysis Of Percentage Of Shooting And Number Of The Shoot

3.2. Effect Of Naa, Bap And Kinetin

3.2.1. Callus induction, callus fresh weight and percentage of somatic embryogenesis

3.2.2. Percentage Of Shooting, Number Of The Shoot, Percentage Of Rooting And Number Of Root

3.2.2.2. Response Surface Analysis Of Percentage Of Shooting

Discussion

Conclusion

Declarations

Author Contributions

Acknowledgment

Competing Interests

References

Supplementary Files

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Journal Publication

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