Selection of Salt-Tolerant Jojoba (Simmondisa chinensis L.) Cultivars via In Vitro Culture

Abstract

Jojoba (Simmondsia chinensis L.) is a perennial shrub adapted to dry and hot climates. It produces high-quality seed oil that is widely used in the industrial, cosmetic, and pharmaceutical sectors. Soil salinity is often a constraint to plants grown under dry and hot desert conditions. Thus, in the present study, we aim to select and establish salt tolerant-jojoba cultivars using in vitro culture. Jojoba seeds were sown on germination media that were supplemented with various amounts of seawater to achieve the final solute concentrations of 2000-, 3000-, and 5000 ppm. Shoot tips from seedlings selected on germination media with different solute levels were subsequently cultured on multiple-shoot induction media, followed by root induction media at the same solute levels as the germination media from which the explants were selected. We germinated, multiplied, rooted, and acclimatized cultivars that could sustain solute levels up to 3000 ppm. Among all cultivars selected at different salinity levels, those selected at 2000 ppm were found to have the highest growth and multiplication parameters during the multiple-shoot induction and rooting stages. In addition, these cultivars also contained high levels of chlorophyll a, b, and carotenoid contents.

1. Introduction

Jojoba, Simmondsia chinensis L. of the Simmondsiaceae family, is a dioecious, long-lived, perennial, woody shrub widely grown in semi-arid regions. It is also known as oat nut, wild hazel, coffee berry, and deer nut. Jojoba is native to the hills of Arizona, southern California, and northwestern Mexico [1]. For more than 30 years, jojoba has been cultivated in many countries around the world, such as Mexico, Chile, Argentina, Tunisia, Saudi Arabia, and Egypt [2]. The total area covered by jojoba plantations is about 18,500 hectares throughout the world. The jojoba seeds contain approximately 65% oil, which is characterized by its light, gold color as a liquid [3]. The waxy seed oil has anti-inflammatory, antimicrobial, and antifungal properties and has been used in relieving headaches, throat inflammation and in treating wounds. Besides its medical properties, jojoba seed oil is also widely used in the cosmetic, bioenergy, and pharmaceutical industries. The chemical structure of jojoba oil consists of chains of fatty acids and higher alcohols, which distinguish it from any other oil produced by plants. In addition, jojoba leaves also contain antioxidant flavonoid compounds that can treat asthma, inflammation, and cancer [4].

Salinization of water and soil is a worldwide environmental problem, especially in arid and semi-arid areas, where this problem is intensified by excessive irrigation [5]. Physiologically, salinity affects germination, photosynthesis, growth, oxidative stress, and water imbalance, eventually leading to plant death or decreased crop yield, biomass, or harvest index. This problem will likely increase due to global climate change and current irrigation practices. Various strategies have been used to mitigate the adverse effects of salinity on plants. These include selecting or generating salt-tolerant plant varieties and implementing appropriate agriculture management practices [6].

Tissue culture or other in vitro techniques are cost-effective for selecting stress-tolerant plants [7]. This technique can be performed in controlled environments with limited space and time. The in vitro culture of plant cells, tissues, or organs on a medium containing selective agents offers the opportunity to select and regenerate plants with desirable characteristics. During the selection process, plant cells or tissues will be exposed to specific abiotic (salinity, drought, ultraviolet, chill/frost) or biotic (Fusarium sp. Collectrichum sp., Helminsporium sp.) stresses in order to generate “mutant” or “variant” cultivars that can overcome the corresponding stress [8]. Examples of stress-tolerant plants developed using in vitro selection include Brassica sp., barley, citrus sp., carrot, flax, groundnut, potato, sugarcane, sweet potato, tomato [7,8], and rice [9]. To impose the selection pressure for screening of salt/salinity tolerant plants, sodium chloride (NaCl), potassium chloride (KCl), sodium sulfate (Na₂SO₄), or magnesium sulfate (MgSO₄), natural or synthetic seawater have been incorporated into the culture media [7].

Woody trees are important components of ecosystems as well as resources for bioenergy. Increased salinity in arable lands has led to numerous studies on salt responses and screening for salt-tolerant woody trees [10,11,12,13,14]. Jojoba is classified as a salt-tolerant woody plant; however, its responses to salinity are genotype-dependent. Therefore, selecting salt-tolerant jojoba varieties is essential for adopting jojoba for large-scale cultivation in semi-arid regions. Being a dioecious plant, propagation of jojoba by sexual methods is cost-inefficient due to its genetic heterogeneity and sex bias in jojoba seed populations. Large-scale vegetative propagation by rooting of semi-hardwood cuttings is hindered by the plant’s size and limited times of the year when it is suitable. Thus, in vitro propagation has emerged as an efficient way to propagate elite varieties of jojoba [15,16].

In this study, we aimed to select and propagate salt-tolerant jojoba cultivars via in vitro selection and micropropagation using culture media with different salinity levels. This is achieved by incorporating different amounts of seawater in the culture media to achieve designated solute levels. We selected, propagated, and acclimatized jojoba cultivars that could propagate in media containing as high as 3000 ppm solute level. Among all the cultivars selected at different solute levels (2000-, 3000- and 5000 ppm), those selected at 2000 ppm solute level have the highest growth, multiplication, and photosynthetic pigments content.

2. Materials and Methods

2.1. Seed Disinfection and In Vitro Germination of Jojoba Seeds

Jojoba (Simmondsia chinensis L.) seeds were obtained from the National Research Center, Dokki, Egypt. The tissue culture experiment was performed at the tissue culture lab, Department of Biological Sciences, College of Science, King Faisal University, from 2019 to 2020. Jojoba seeds were first soaked in tap water for 10 min, followed by soaking in 0.5 g⋅L⁻¹ Carbomar fungicide (Anmar for Development Agriculture Trading, Riyadh, Saudi Arabia) for 15 min. Subsequently, seeds were surface disinfected with 70% aqueous ethanol for 30 s, followed by soaking in 0.1% (w/v) aqueous mercuric chloride (HgCl₂) (Sigma Aldrich, Saint Louis, MO, USA) for 5 min and a final wash in 10% (v/v) sodium hypochlorite (Clorox) solution for 5 min. The seeds were then rinsed three times with sterile tap water to remove all the traces of sodium hypochlorite. All the transfer steps from one solution to another were performed in a laminar air-flow hood.

The basic (control) germination media consisted of half-strength (½X) Murashige and Skoog (MS) [17] basic salts and vitamins (Caison Labs, Smithfield, UT, USA), supplemented with 3 g⋅L⁻¹ sucrose (Sigma Aldrich, Saint Louis, MO, USA), and 7 g⋅L⁻¹ agar (Caison Lab, Smithfield, UT, USA). Germination media with various salinities (2000-, 3000-, and 5000 ppm final solute levels) were prepared by supplementing the control germination media with appropriate volumes of seawater obtained from the Arabian Gulf (Uqair, 26.0786 N, 50.0393 E) to achieve the desired amount of solute concentrations. The solute level in the saline germination media was measured using the YSI EcoSense EC 300 electric conductivity meter (Cole-Parmer, Shanghai, China). The pH of all media was adjusted to pH 5.7 using 1 N NaOH. All media were disinfected by autoclaving at 121 °C and 15 psi pressure for 20 min.

A total of 50 seeds were aseptically cultured in each type of germination media (control, 2000-, 3000-, and 5000 ppm), with a single seed in each 55 mL tissue culture tube. Culture tubes with seeds were incubated in a growth chamber under controlled environmental conditions of 22 °C, 70% humidity, and 16/8 h of light/dark cycle. The photosynthetic photon flux density (PPFD) of 53.97 μmol/s/m² was provided by Phillips TLM 40 W/33 RS fluorescent lamps (Phillips, Memphis, TN, USA). After 4 weeks of incubation, the germination percentage (%), the number of leaves and roots (n), the length of shoot and root (cm), and the fresh seedling weight (g) were recorded.

2.2. Multiple Shoots Induction

The basic (control) multiple-shoot induction media consisted of full-strength (1X) MS basic salts and vitamins supplemented with 3 g⋅L⁻¹ sucrose, 6 g⋅L⁻¹ agar, and 2 mg⋅L⁻¹ 6-benzylaminopurine (BAP) (Caison Labs, Smithfield, UT, USA). Multiple-shoot induction media with different salinity levels (2000-, 3000-, 5000 ppm final solute levels) were prepared by incorporating an appropriate amount of seawater into the control multiple-shoot induction media. All media were adjusted to pH 5.7 and disinfected, as mentioned in Section 2.1.

Stem cuttings of approximately 1 cm length were excised from the in vitro germinated jojoba seedlings grown on various germination media and subsequently transferred to multiple-shoot induction media at the same salinity level as the germination medium, from which the stem cutting was obtained. About 10 stem cuttings were cultured in each 200 mL size culture vessel containing 50 mL of multiple-shoot induction media. All vessels were incubated in a growth chamber under controlled environmental conditions, as mentioned in Section 2.1.

For sub-culturing, stem-cutting sections from plantlets were transferred into fresh multiple-shoot induction media with the same salinity level as those from which the stem-cutting sections originated. The sub-culturing was performed at an interval of 4 weeks for 4 times. The number of shoots and leaves (n), plants height (cm), and shoot fresh weight (g) were documented at week 4 (subculture 1), week 8 (subculture 2), and week 16 (subculture 4).

2.3. In Vitro Rooting

The basic (control) root induction media consisted of half-strength (½X) MS medium supplemented with 3 g⋅L⁻¹ sucrose, 6 g⋅L⁻¹ agar and 1 mg⋅L⁻¹ of indole-3-butyric acid (IBA) (Caison Labs, Smithfield, UT, USA). Appropriate volumes of seawater were added into the basic root induction medium to obtain rooting medium with a final solute concentration of 2000-, 3000-, and 5000 ppm. All media were adjusted to pH 5.7 and disinfected, as mentioned in Section 2.1.

For root induction, 2 cm long healthy shoots were excised from a subculture of 4 multiple shoots and placed on rooting media with the corresponding salinity level as the multiple-shoot induction media. All shoots containing vessels were incubated in a growth chamber under controlled environmental conditions, as mentioned in Section 2.1. Rooting parameters, for example, the number of roots and leaves (n), length of the longest root and tallest shoot (cm), and the plantlet’s fresh and dry weight (g) were noted after 8 weeks of cultivation.

2.4. Acclimatization of Plantlets

After 8 weeks of cultivation in respective root induction medium, the rooted shoots were carefully removed from the culture jars and then washed with running tap water to remove residual agar around the roots. Subsequently, the roots were washed with 1 g⋅L⁻¹ Carbomar fungicide (Anmar for Development Agriculture Trading, Riyadh, Saudi Arabia) to reduce the risk of contamination during the acclimatization. The plantlets were then transferred to the greenhouse at the Agriculture and Veterinary Research and Training Center, King Faisal University, for acclimatization in a plastic tray (200 cells) containing a moist mixture of (1:1:1:1) sand, vermiculite, perlite, and peat moss. The rooted plantlets were sprayed with a fine mist of water for 3 weeks.

2.5. Determination of Chlorophyll a, b, and Carotenoid Contents

Three randomly selected plantlets at the multiple-shoot induction and rooting stages were used for the determination of chlorophyll a, b, and carotenoid content. A 0.1 g fresh leaf sample from an individual plantlet was removed and placed into 10 mL of 80% acetone. The samples were kept in the dark until the color of the plant tissues was bleached (3–4 days). The absorbance of the supernatant at 444 nm, 644 nm, and 662 nm wavelengths was measured against the solvent (80% acetone) as a blank using a G1369 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The chlorophyll a, b and carotenoid contents were calculated according to [18].

2.6. Determination of Sodium (Na), Potassium (K), Magnesium (Mg), and Calcium (Ca) Compositions

Three randomly selected 8-week-old plantlets cultivated in respective root induction media were excised from their media and used for the determination of Na, K, Mg, and Ca contents. The whole plantlets were cleaned with distilled water to remove the residual agar and dried in an oven at 50 °C. A 0.5-g powdered-dried sample was digested in a 50-mL volumetric flask with 2.5 mL of concentrated (95–97%) sulfuric acid (H₂SO₄) (Scharlab, Barcelona, Spain). The digestion was performed on a hotplate at approximately 270 °C. Hydrogen peroxide (H₂O₂) (Sigma-Aldrich, Saint Louis, MO, USA) solution was added bit-by-bit until the digestion was completed (a clear solution was obtained). After digestion, deionized water was added to achieve a final volume of 50 mL in the volumetric flask. The resultant clear solution was then used for Na, K, Mg, and Ca composition determination using an Atomic Absorption Flame Photometer (Shimadzu-AA7000, Tokyo, Japan) according to the method of [19].

2.7. Peroxidase (POD) Enzyme Assay

2.7.1. Preparation of Plant Extract

The plant extract was prepared from jojoba leaves that were collected from plantlets grown in multiple-shoot induction media with various salinity levels and subsequently kept in a −80 °C freezer until used. The thawed leaf samples were ground in 0.1 M potassium phosphate pH 5.8 buffer (1 g leaf/10 mL buffer) by using an ice-cold mortar and pestle. The leaf debris from the mashed leaf samples was removed by centrifugation at a relative centrifugation force of 8000× g at 4 °C. The clear supernatants were stored in a −80 °C freezer until used [20].

2.7.2. Composition and Condition of Enzymatic Reaction

Peroxidase activity was measured using guaiacol (Fisher Scientific, Waltham, MA, USA) as the substrate. The product was quantified using a G1369C spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) at a wavelength of 420 nm. The substrate-free blank assay consisted of 2.6 mL of 0.1 M potassium phosphate pH 5.8 buffer, 0.1 mL of distilled water, 0.2 mL plant extract, and 0.2 mL of 0.2 M hydrogen peroxide (H₂O₂) (Sigma-Aldrich, Saint Louis, MO, USA). Each enzyme assay consisted of 2.5 mL of 0.1 M potassium phosphate pH 5.8 buffer, 0.1 mL of 20 mM guaiacol, 0.2 mL plant extract, and 0.2 mL of 0.2 M hydrogen peroxide. The absorbance at 420 nm for each enzyme assay was recorded at 30 s intervals for a duration of 5 min [20]. Two in vitro plantlets were used for plant extract preparation for each treatment, and three enzyme assays were performed for each plant extract sample.

2.8. Statistical Analysis

The experiment was conducted with a completely randomized design with 10 replicates. The data were statistically analyzed using Statistica 6 program ANOVA/MANOVA [21]. The significance of differences among means was detected using the Least Significant Test (L.S.D) at p < 0.05.

3. Results

3.1. The Effect of Salinity on Jojoba Seed Germination

The solute concentration of the basic (control) germination media (i.e., the media without seawater) was 800 ppm due to the presence of the Murashige and Skoog (MS) basic salts in the culture media. Seawater taken from the Arabian Gulf was added to the basic media to obtain germination media with the final solute levels of 2000-, 3000- and 5000 ppm. A total of 50 sterile Simmondsia chinensis L. seeds were cultured in each of the treatments (control, 2000-, 3000- and 5000 ppm germination media). The numbers of germinated seeds were recorded in the 4th week after sowing the seeds on respective culture media. The average germination rate for each treatment was computed from three independent germination experiments. An average of 33% of seeds germinated in the control media. In the 2000 ppm germination media, a 3% reduction in the germination rate was seen as compared with that of the control media. A further 3% reduction was observed when comparing the germination rate in the 3000 ppm germination media with that of the 2000 ppm. The germination rate of seeds in the 5000 ppm germination media was 15%, almost half of that in the control media.

The growth parameters of the germinated seedlings from all salinity treatments were assessed in the 4th week after sowing (

Table 1. Growth parameters of jojoba (Simmondsia chinensis L.) seedlings in various saline germination media.

Salinity (ppm)	Shoot Length (cm)	Leaf Numbers (n)	Root Length (cm)	Root Numbers (n)	Fresh Weight of Whole Seedling (g)
Control	4.57 ab ± 0.061	6.14 a ± 0.610	5.14 a ± 0.003	29.86 a ± 0.910	4.86 ab ± 0.008
2000	5.90 a ± 0.052	5.8 a ± 0.820	6.1 a ± 0.223	26.8 a ± 0.230	4.55 ab ± 0.021
3000	2.94 b ± 0.071	2.75 b ± 0.340	5.38 a ± 0.530	23.13 a ± 0.208	3.2 b ± 0.802
5000	4.0 ab ± 0.120	4.4 ab ± 0.240	6.4 a ± 0.210	18.8 a ± 0.304	7.0 a ± 0.504

Footnote: The growth parameters of seedlings germinated in various germination media were measured four weeks after sowing. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

, Figure 1A). The means of shoot length, leaves number, and fresh seedling weight were significantly different (p < 0.05) among seedlings germinated in media with different salinity levels, while those of the root length and root numbers were not significantly different. Seedlings germinated in the 2000 ppm germination media had the highest average shoot length, while those germinated in the 3000 ppm germination media had the lowest among all four salinity treatments. Interestingly, seedlings germinated in the 5000 ppm had a comparable average shoot length as those in the control media, despite the high salinity of 5000 ppm germination media.

The increase in media salinity reduced average leaves numbers in seedlings present in 2000, 3000, and 5000 ppm germination media, compared with those growing in the control germination media. Moreover, the increased salinity resulted in the reduction of root numbers in a salinity-level-dependent manner, even though the differences were not statistically significant. On the other hand, salinity was found to increase the root length of the seedlings, even though the differences were not statistically significant. Seedlings from both 2000 and 5000 ppm germination media had similar average root lengths, which were higher than those present in the control and 3000 ppm germination media. The seedlings from the 2000 ppm germination media have similar mean fresh weight compared to those from the control media. Among all seedlings, those grown in the 3000 ppm media had the lowest fresh weight, while seedlings from the 5000 ppm media had the highest fresh weight. In summary, seedlings from the 3000 ppm media had the lowest means in shoot length, leaves numbers, and fresh weight, while seedlings from the 5000 ppm media had the highest means in root length and fresh weight despite the short shoots and low numbers of leaves and roots.

3.2. The Effect of Salinity on Jojoba Multiple-Shoot Induction

Stem cuttings from seedlings that were germinated on each type of germination media (control, 2000-, 3000-, and 5000 ppm) were excised and transferred to multiple-shoot induction media with the same salinity level. Subculturing of stem cuttings was performed every 4 weeks. The growth and multiplication parameters (number of shoots and leaves, shoot length, and plantlet weight) of the plantlets from subcultures 1, 2, and 4 were recorded (

Table 2. Multiplication parameters of explants from plantlets grown on different saline multiple-shoot induction media.

Subculture	Salinity (ppm)	Shoot Numbers (n)	Leaf Numbers (n)	Plant Length (cm)	Fresh Weight of Plantlet (g)
(A) Main effect of salinity
	Control	3.67 b	11.0 b	3.08 b	3.18 b
	2000	30.04 a	173.05 a	17.66 a	17.91 a
	3000	14.17 ab	67.72 ab	8.22 ab	6.88 ab
	5000	7.73 b	38.93 ab	5.8 ab	6.53 ab
(B) Main effect of subculture
Sub1		5.83 b	21.75 b	5.38 b	3.13 b
Sub2		12.09 b	61.59 b	7.95 b	7.22 b
Sub4		31.90 a	184.05 a	16.98 a	20.0 a
(C) The interaction effect of salinity and subculture
Sub1	Control	3.8 c ± 0.002	9 d ± 0.010	3.8 bc ± 0.800	3.76 c ± 0.730
Sub1	2000	5.5 bc ± 0.200	27.5 c ± 1.600	6.38 b ± 0.200	2.0 d ± 0.230
Sub1	3000	5.33 bc ± 0.4	11.5 d ± 2.4	4.17 b ± 0.7	1.08 cd ± 0.52
Sub1	5000	9 b ± 0.1	37.6 c ± 0.06	6.8 b ± 0.002	6.7 c ± 0.82
Sub2	Control	3.5 c ± 0.8	11 d ± 0.5	3 bc ± 1.2	1.03 cd ± 0.90
Sub2	2000	21.29 b ± 1.1	119.57 b ± 0.03	12.86 b ± 0.03	14.04 b ± 0.34
Sub2	3000	10 b ± 0.6	40.67 c ± 0.9	7 b ± 0.5	4.08 c ± 1.03
Sub2	5000	8.6 b ± 1.2	46 c ± 1.02	6.2 b ± 0.04	4.6 c ± 0.007
Sub4	Control	3.7 c ± 0.8	14.3 d ± 0.04	2 b c ± 0.6	5.1 c ± 0.03
Sub4	2000	66.86 a ± 1.03	392.85 a ± 0.001	35.36 a ± 1.2	39.96 a ± 0.62
Sub4	3000	27.17 b ± 0.04	151 b ± 0.7	13.5 b ± 0.03	15.48 b ± 0.70
Sub4	5000	5.6 bc ± 0.001	33.2 c ± 0.03	4.4 b ± 0.9	6.46 c ± 0.003

Footnote: Explants (1 cm stem cuttings) were excised from seedlings grown in control, 2000-, 3000-, and 5000 ppm germination media. These explants were transferred to multiple-shoot induction media with the same salinity levels as the germination media from which the explants derived. The growth and multiplication parameters were measured at 4 weeks after the transplantation. Subcultures (Sub-) of stem cutting from plantlets were performed after the growth and multiplication measurements were taken. In each sub-culturing, stem cuttings were transferred to fresh multiple-shoot induction media with the same salinity level as the previous multiple-shoot induction media. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

) on the same day as stem cuttings were excised for subsequent subculturing.

The data (Table 2A) indicated that plantlets from 2000 ppm (Figure 1B), 3000 ppm (Figure 1C), and 5000 ppm (Figure 1D) multiple-shoot induction media had a higher number of shoots and leaves, as well as longer shoot lengths and higher plantlet weights as compared to the those on control media. Among all the plantlets grown on various multiple-shoot induction media, those in the 2000 ppm media had the highest number of shoots and leaves, longest shoot and highest plantlet weight. On the other hand, plantlets grown in 5000 ppm multiple-shoot induction media were found to have the lowest means in the above-mentioned parameters. As revealed in Table 2B, each subculturing increased the number of shoots and leaves, the length of the tallest shoot, and plantlet weight.

The interacting effects of subculturing and salinity are documented in Table 2C. In subculture (Sub-) 1, the highest mean values in all measured parameters were obtained in the plantlets growing in the 5000 ppm multiple-shoot induction media. Plantlets in the multiple-shoot induction media without seawater (control) had the lowest means in all measured parameters except for the plantlet weight. As for plant height, plantlets from the 2000- and 3000 ppm multiple-shoot induction media had lower means than those in the control media. In Sub-2 and -4, the control was found to have the lowest means in all parameters. In contrast to Sub-1, plantlets in the 2000 ppm multiple-shoot induction media recorded the highest values in all parameters in both Sub-2 and -4. When comparing plantlets through different subcultures, those in the control media showed no significant differences in the means of all parameters except for the plantlet weight. As for plantlets in the 2000 ppm multiple-shoot induction media, a consistently significant increase in all parameters was observed. Similarly, a consistent increase in all parameters was observed for plantlets in the 3000 ppm multiple-shoot induction media, even though the increase in shoot height was statistically insignificant. Similar to the plantlets in the control media, those in the 5000 ppm multiple-shoot induction media did not show any significant changes in all the parameters throughout Sub-1 to Sub-4. Indeed, in these plantlets, decreased trends were observed from Sub-1 to Sub-4 in the number of shoots and shoot length.

3.3. Effects of Salinity on Jojoba Rooting

Growth parameters such as the number of roots and leaves, the length of root and shoot, as well as plantlet fresh and dry weight, were documented eight weeks after transplanting the 2 cm shoots from the multiple-shoot induction media to rooting media (

Table 3. Rooting parameters of explants grown on different saline rooting media. Multiple-shoot induction media.

Salinity (ppm)	Root Number (n)	Root Length (cm)	Leaves Number (n)	Shoot Length (cm)	Plantlet Fresh Weight (g)	Plantlet Dry Weight (g)
Control	0.27 a ± 0.646	1.09 a ± 2.700	2.27 b ± 1.380	0.77 b ± 0.408	0.22 ab ± 0.869	0.063 ab ± 1.380
2000	0 b ± 0.000	0 b ± 0.000	3.92 a ± 0.428	1.31 ab ± 0.076	0.43 a ± 0.589	0.083 a ±0.301
3000	0.14 a ± 0.590	0.36 a ± 1.596	2.96 ab ± 0.135	1.52 a ± 0.038	0.32 ab ± 0.001	0.052 bc ± 1.002
5000	0 b ± 0.000	0 b ± 0.000	2.10 b ± 0.223	0.75 b ± 0.001	0.16 b ± 1.132	0.028 c ± 0.234

Footnote: Explants (2 cm shoot) were excised from Sub-4 plantlets grown in control, 2000-, 3000-, and 5000 ppm multiple-shoot induction media. The explants were transferred to rooting media with the same salinity levels as the multiple-shoot induction media where the corresponding plantlets were grown. The rooting parameters were measured 8 weeks after the transplantation. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

).

Salinity negatively impacted root formation for shoots that were transplanted to the rooting media containing the same salinity level as the multiple-shoot induction media in which the corresponding plantlets were grown. In vitro shoots from the 2000 and 5000 ppm multiple-shoot induction media failed to root. Exceptionally, some shoots from the 3000 ppm multiple-shoot induction media were able to root in rooting media with the corresponding salinity level (Figure 1E). Despite failing to root, the shoots derived from the 2000 ppm multiple-shoot induction media produced a significantly higher number of leaves, longer shoots, and higher fresh and dry weight as compared with shoots from shoots originating from control, 3000 ppm, and 5000 ppm multiple-shoot induction media, when grown in rooting media with the same salinity levels as their respective.

3.4. Acclimatization

The success of any in vitro micropropagation protocol largely depends on the survival and growth performance of the propagated plantlets ex vitro [22]. In the present study, the acclimatization procedures applied were successful using the previously mentioned experimental conditions. Almost 100% of the regenerated plants selected from control, 2000-, 3000-, and 5000 ppm salinity media survived and showed vigorous growth. All these in vitro-derived plants did not display any phenotypic variation during subsequent vegetative development.

3.5. The Effect of Salinity on the Photosynthetic Pigments Content in Jojoba Shoots

3.5.1. The Effect of Salinity on the Photosynthetic Pigment Content in Jojoba (Simmondsia chinensis L.) Shoots Growing in the Multiple-Shoot Induction Media with Various Salinity Levels

Photosynthetic pigment content present in the shoots of the plantlets growing the multiple-shoot induction media with various salinity levels was tabulated in

Table 4. Chlorophyll a, b, and carotenoid content from plantlets grown on multiple-shoot induction media with different salinity levels.

Salinity (ppm)	Chlorophyll a (mg/100 g F.W)	Chlorophyll b (mg/100 g F.W)	Carotenoid (mg/100 g F.W)
Control	18.69 ab ± 2.593	7.93 a ± 1.444	19.28 ab ± 1.593
2000	12.27 b ± 3.440	3.62 a ± 2.781	14.74 b ± 1.444
3000	30.44 a ± 3.311	5.35 a ± 1.605	33.84 a ± 0.897
5000	13.41 ab ± 2.441	3.26 a ± 3.363	15.96 ab ± 0.311

Footnote: Leaves were excised from control, 2000-, 3000-, and 5000 ppm multiple-shoot induction media plantlets. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

. Among all plantlets, the plantlets grown in 3000 ppm multiple-shoot induction media demonstrated the highest means in chlorophyll a (Chl a) and carotenoid content, which were much higher than those found in plantlets grown in all other media. On the other hand, the plantlets grown in the control multiple-shoot induction media had the highest chlorophyll b (Chl b) content, followed by those from 3000 ppm multiple-shoot induction media.

3.5.2. The Effect of Salinity on the Photosynthetic Pigment Content in Jojoba (Simmondsia chinensis L.) Shoots Growing in the Rooting Media with Various Salinity Levels

Table 5. Chlorophyll a, b, and carotenoid content of plantlets grown on rooting media with different salinity levels.

Salinity (ppm)	Chlorophyll a (mg/100 g F.W)	Chlorophyll b (mg/100 g F.W)	Carotenoid (mg/100 g F.W)
Control	17.42 a ± 0.084	15.98 a ± 0.776	21.57 a ± 1.135
2000	22.19 a ± 1.899	26.10 a ± 3.269	27.48 a ± 2.281
3000	19.16 a ± 1.583	19.56 a ± 1.399	23.41 a ± 1.429
5000	6.567 a ± 4.741	4.88 a ± 4.893	5.79 a ± 4.276

Footnote: Plantlets from multiple-shoot induction media with different salinity levels were transplanted to rooting media with the same salinity level as those of the multiple-shoot induction media which they were derived from. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

shows the photosynthetic pigments content in the jojoba shoots growing in the rooting media with various salinity levels. The plantlets in the 2000 ppm rooting media displayed the highest means in all three types of photosynthetic pigments content, followed by those in the 3000 ppm rooting media.

3.6. The Effect of Salinity on the Mineral Content in Jojoba Shoots

Calcium (Ca²⁺), potassium (K⁺), sodium (Na⁺), and magnesium (Mg²⁺) ion contents of shoots that grew in the rooting media with various salinity were tabulated in

Table 6. Mineral content from plantlets grown on rooting media with different salinity levels.

Salinity (ppm)	Ca2+ (g/kg)	K+ (g/kg)	Na+ (g/kg)	Mg2+ (g/kg)
Control	1.63 a ± 0.751	17.74 a ± 2.708	7.71 b ± 1.229	0.08 b ± 0.268
2000	1.61 a ± 0.623	12.28 a ± 1.727	26.99 ab ± 4.632	1.67 b ± 0.212
3000	3.08 a ± 1.829	17.05 a ± 2.851	46.78 a ± 1.771	2.14 ab ± 0.079
5000	2.43 a ± 1.032	8.82 a ± 1.455	72.87 a ± 1.378	4.75 a ± 0.973

Footnote: Shoots were excised from plantlets of the control, 2000-, 3000-, and 5000 ppm multiple-shoot induction media and transplanted into rooting media with the same salinity levels as their corresponding multiple-shoot induction media. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

. The highest Na⁺ and Mg²⁺ ion contents were recorded in the plantlets grown in the 5000 ppm multiple-shoot induction media. On the other hand, the highest Ca²⁺ and K⁺ ion contents were recorded in the plantlets that were grown in the 3000 ppm multiple-shoot induction media. The Na⁺ content in plantlets grown in rooting media with different salinity levels varied considerably as compared with those of Ca²⁺, K⁺, and Mg²⁺. The Na⁺ content in the plantlets was highly correlated with the salinity of the media in which the plantlets were grown.

3.7. The Effect of Salinity on the Peroxidase (POD) Activities in Jojoba Shoots

As shown in

Table 7. Peroxidase activity of different salinity treatments.

Salinity (ppm)	Enzyme Activity (unit/min/g)
Control	0.147 a ± 0.0370
2000	0.054 b ± 0.0395
3000	0.052 b ± 0.0206
5000	0.06 b ± 0.0081

Footnote: Plantlets grown in their respective multiple-shoot induction media were used for the preparation of plant extracts. Mean values succeeded by the same letter in a column are not significantly different at p = 0.05 as measured by the Least Significant Test (L.S.D).

, the highest mean peroxidase activity was observed in the plantlets grown in control multiple-shoot induction media, while plantlets grown in multiple-shoot induction media at 2000-, 3000-, and 5000 ppm solute levels displayed comparably lower peroxidase activity. The peroxidase activities among plantlets grown in media supplemented with seawater to achieve the 2000-, 3000-, and 5000 ppm solute levels were not statistically different.

4. Discussion

The germination results indicated that in vitro germination of jojoba seeds in the basic germination media, containing MS basic salts, vitamins, and 3% sucrose, was low (33%). High salinity (5000 ppm) reduced the germination rate to half of that in the basic (control) germination media. The germination result implies that the jojoba seeds can tolerate moderate salinity (2000 and 3000 ppm salinity) as there were only small differences in the germination rate (approximately 3%) among the basic, 2000, and 3000 ppm germination media.

Even though seedlings in the 5000 ppm germination media had the lowest average number of roots, moderate shoot length, and the number of leaves, they had the longest average root length and fresh seedling weight among those grown on germination media with various salinity. Seedlings in the 3000 ppm germination media were found to have the lowest or second lowest values for each growth parameter (Table 1). Germination of jojoba seeds was reduced as salinity levels in the media increased (Table 1). Jojoba plants are known as salt-tolerant plants; however, the responses of jojoba plants to salinity are highly genotype-dependent. In addition, being a dioecious plant, the seeds produced by even a single plant can be highly heterogeneous [15,23]. Therefore, seedlings germinating in the germination media with the same salinity level might also be highly heterogeneous; for instance, those germinated in the control media would consist of seedlings that can germinate at 800 ppm (the solute level of the control media) or above. Similarly, seedlings germinating in the 2000 ppm media might withstand a 2000 ppm solute level or higher.

High multiplication, i.e., the excessive number of shoots and leaves, was observed in explants grown in moderately saline conditions (2000 and 3000 ppm salinity), in accordance with Taha [24]. Fayek et al. [25] have reported that jojoba plantlets grown at 2000 ppm salinity have the highest average shoot length. Similarly, Mohasseb et al. [26] also documented that moderate salinity indeed enhances the shoot length of jojoba plantlets. In fact, in the present study, plantlets that were cultured onto 2000 ppm multiple shoot induction media produced the longest shoots among all salinity treatments (Table 2A). In addition, the highest number of shoots and leaves and plantlet fresh weight was also noted in plantlets grown in the 2000 ppm multiple-shoot induction media. These observations were consistent with numerous previous reports [24,25,26,27]. Repeated subculturing in the fresh multiple-shoot induction media with the same salinity was expected to improve all multiplication and growth parameters, as shown by the data presented in Table 2B. This was due to the acclimatization of the plantlets to the salinity of the respective culture media. The acclimatization effects were most obvious for plantlets subculturing on multiple-shoot induction media with 2000 ppm salinity. Drastic increases in the number of shoots and leaves were observed compared with those in control, 3000-, and 5000 ppm media (Table 2C).

Roussos et al. [27] have reported no significant differences in the rooting rate and root length of jojoba plantlets grown at different salinities. The present study also indicated similar results for seedlings grown in germination media with various salinities (Table 1), in which seedlings germinated and grown in germination media with different salinities showed no significance in the number of roots and length of roots. However, when explants selected from different salinities were excised and transferred to rooting media with salinity levels corresponding to those of the multiple-shoot induction media that they were derived from, shoots from 2000 and 5000 ppm salinity levels failed to produce any roots, while explants produced from 3000 ppm salinity levels produced fewer and shorter roots as compared to those grown in control rooting media (Table 3). It was surprising to note that explants originating from the 2000 ppm salinity treatment, which showed the highest growth and multiplication in the multiple-shoot induction media as compared to plantlets derived from media with different salinity, failed to root in the 2000 ppm rooting media. Nevertheless, these rootless plantlets produced the highest number of leaves and fresh and dry weight when cultured in the 2000 ppm rooting media (Table 3).

It was astonishing that the plantlets that were grown in the 2000 ppm multiple-shoot induction media displayed the lowest value in average chlorophyll a (Chl. a) and carotenoid content, even though these plantlets have the highest multiplication and growth rate among all the salinity treatments. Nonetheless, the plantlets from the 3000 ppm salinity treatments, which has the second highest multiplication and growth rate, were found to have corresponding high Chl. a and carotenoid content (Table 2 and Table 4). The content of Chl. a, b, and carotenoids in plantlets grown in rooting media with various salinity levels were consistent with the measured growth parameters (the number of leaves, shoot length, and plantlet fresh and dried weight). This was implicated by the observation that high Chl. a, b, carotenoid content, and high growth were recorded concurrently in the plantlets grown at the 2000 and 3000 ppm salinity levels (Table 2 and Table 5).

Previous studies from Fayek et al. [25] and Roussos et al. [27] have reported that increased salinity (level of Na⁺) in the culture media increased the Na⁺ content while lowering the K⁺ and Ca²⁺ content in the plantlets. In accordance with the reported previous investigations, significantly high levels of Na⁺ have been observed in plantlets grown in rooting media supplemented with seawater (2000-, 3000-, and 5000 ppm). However, no significant difference was observed in the K⁺ content among the control, 2000-, and 3000 ppm plantlets. Only the 5000 ppm salinity treatment displayed a decrease in the K⁺ level as compared with the control. Interestingly, in contrast to the previous reports, Ca²⁺ content in the 3000 and 5000 ppm salinity treatment has increased as compared with the control and 2000 ppm. These observations may imply that the jojoba cultivars that were selected from the in vitro culture in the present study could tolerate up to 3000 ppm salinity; therefore, plantlets grown at moderate salinity (2000 and 3000 ppm) did not show any reduction in K⁺ or Ca²⁺ uptake despite the increase in Na⁺ uptake. This hypothesis was also supported by the growth and multiplication data obtained in the present study. The uptake of Mg²⁺ was correlated with the media’s salinity, in which higher Mg²⁺ content was observed in plantlets grown in media with increasing salinity (Table 6).

Increased activities of antioxidant enzymes have been associated with plant salinity tolerance [28]. In Broussonetia papyrifera, a salt-resistant woody plant, the peroxidase (POD) activity was differentially affected by salinity in different parts of the plant. At 50 mM NaCl, the plants demonstrated decreased POD activities in the stems but not in the leaves. On the other hand, the POD activities decreased remarkably in the leaves but not in the stems when the plants were grown with 100 and 150 mM NaCl [29]. In the present study, we have observed that plantlets grown in multiple-shoot induction media with 2000-, 3000-, and 5000 ppm salinity have lower POD enzyme activities as compared with the control. Due to the small size of the plantlet, the whole plantlets have been used for the enzyme assays; therefore, we could not determine whether the POD activities were differentially affected in the leaves and stem, but the overall decrease in POD activities in jojoba plantlets exposed to salinity was in accordance with that reported in B. papyrifera [29]. Using RNA sequencing and various physiological and chemical analyses, Alghamdi et al. [30,31] have attempted to elucidate the physiological and molecular mechanisms for salt tolerance in jojoba. The authors proposed that the upregulation of genes encoding fructose-bisphosphate aldolase and transketolase in jojoba plants enhanced photosynthesis. In addition, the reactive oxygen species scavenger, such as ascorbate peroxidase (APX), also increased its activity and encoding gene transcript during salt stress, suggesting its involvement in reducing reactive oxygen species under salt stress.

5. Conclusions

In this study, jojoba plants that could withstand 5000 ppm salinity were successfully selected. However, the multiplication and growth of plantlets derived from the 5000 ppm salinity level were lower than those selected under 2000 ppm and 3000 ppm salinity. The seedlings selected at the 2000 and 3000 ppm grew well in the multiple-shoot induction and rooting media. Even though the plantlets in 2000 ppm have higher values in all growth and multiplication parameters in the multiple-shoot induction stage, the chlorophyll and carotenoid content in the plantlets were not in concordance with the observed high growth and multiplication. On the other hand, plantlets from 3000 ppm salinity, which performed slightly lower than those from 2000 ppm salinity in the multiple-shoot induction stage, have shown high concordance levels of chlorophyll a and carotenoid content. Only plantlets selected at the control and 3000 ppm salinity managed to root in the rooting media with salinity corresponding to their respective original selection levels. Despite failing to root, plantlets originating from the 2000 ppm have shown higher growth and yield parameters as compared to plantlets selected at different salinity levels. The mineral analyses have implied that the increased uptake of Na⁺ did not affect the uptake of K⁺ and Ca²⁺ in plantlets grown in 2000 and 3000 ppm salinity media. The peroxidase enzyme activities in all salinity treatments (2000, 3000, and 5000 ppm) were similar among each other but less than half of that in the control treatment.