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Article

Establishment of Hairy Root Transformation System for Evaluating Stress-Tolerant Gene in Jojoba

by
Bojing Li
1,
Yan Wang
1,
Wenguo Ma
1,
Jie Bing
2,
Yijun Zhou
1,3,
Yuke Gen
1 and
Fei Gao
1,3,*
1
College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China
2
College of Life Sciences, Beijing Normal University, Beijing 100875, China
3
Key Laboratory of Mass Spectrometry Imaging and Metabolomics, Minzu University of China, National Ethnic Affairs Commission, Beijing 100081, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(12), 2132; https://doi.org/10.3390/agriculture14122132
Submission received: 24 October 2024 / Revised: 18 November 2024 / Accepted: 23 November 2024 / Published: 25 November 2024
(This article belongs to the Section Crop Genetics, Genomics and Breeding)
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Versions Notes

Abstract

Jojoba is an important tropical oil crop, and jojoba oil is widely used in the aerospace lubricant and cosmetic industries. Jojoba exhibits high tolerance to droughts and high temperatures. However, there is currently a lack of rapid and effective methods for identifying stress-tolerant genes in jojoba. Here, an efficient hairy root genetic transformation system of jojoba (Simmondisa chinensis) mediated by Agrobacterium rhizogenes was established and used for the functional evaluation of ScGolS1, a putative stress-tolerant gene. First, using the leaves of jojoba as explants, transgenic jojoba hairy roots carrying the RUBY gene were obtained under sterile conditions using the “soaking co-cultivation method”. Second, we optimized the four conditions affecting hairy root genetic transformations, namely, the strains of A. rhizogenes, co-cultivation under light or dark conditions, the infection time, and the OD600 value of the bacterial suspension. The following best transformation conditions were determined, A. rhizogenes K599, light during co-cultivation, an infection time of 10 min, and bacterial suspension OD600 = 0.6, under which the transformation rate could reach 27%. Third, based on the “soaking co-cultivation method”, a new method called the “wrapping co-cultivation method” was developed, which does not require tissue cultures and can induce transgenic hairy roots of jojoba in two months. Using the “wrapping co-cultivation method”, we successfully obtained transgenic hairy roots overexpressing the ScGolS1 gene, which exhibited higher tolerance to low-temperature stress. A hairy root-based genetic transformation system of jojoba will promote the functional genomics and molecular breeding of jojoba.

1. Introduction

Jojoba is the sole species of the Simmondsiaceae, a dioecious perennial shrub that thrives in arid and semi-arid regions [1]. Jojoba seeds contain approximately 50% oil, rendering it the most productive oil crop among perennial shrubs. The oil extracted from jojoba seeds possesses unique characteristics and thus is widely used in the aerospace lubricant and cosmetic industries. Distinguishing it from typical vegetable oils, jojoba oil is a wax ester, consisting of a mixture of straight-chain fatty acids in the ratios of 18:1, 20:1, 22:1, and 24:1 [2]. Jojoba oil exhibits high temperature resistance and oxidation resistance, coupled with strong stability and insulation properties, rendering it an excellent lubricant and insulation material. Jojoba oil has good affinity for human skin and is widely used in cosmetic products. Additionally, jojoba oil can also be used to treat diseases such as sunburn, skin abrasions, hair loss, headaches, and a sore throat [3]. Jojoba was first discovered in the Sonoran Desert, and it can survive well in desert areas with low rainfall and thus holds significant ecological value, serving as a plant that can be utilized to prevent and control desertification. Jojoba possesses strong tolerance to various environmental stresses, including droughts, high temperatures, salt, and infertility [1]. The leaves of jojoba were coated with a thick waxy layer, and thus jojoba was found to withstand extreme heat of up to 58.3 °C [4], maintaining its integrity and normal physiological functions in hot climates. Additionally, this shrub boasts a robust root system that allows it to thrive in arid regions with scant rainfall. The high stress tolerance of jojoba is related to the stress tolerance genes in its genome. Studies have shown that an overexpression of the MnSOD gene from Jojoba significantly improves the drought tolerance of the transgenic Arabidopsis [5]. With the intensification of global warming, crops will increasingly be negatively affected by environmental stresses such as high temperatures and drought stress. Thus, the stress-resistant genes identified from jojoba hold great potential for breeding stress tolerance in other crops.
Research on jojoba has primarily focused on in vitro rapid propagation [6,7], cultivation techniques [8,9], abiotic stress tolerance [10,11], and medical uses [12]. Following the completion of its genome sequencing in recent years [13,14], research on jojoba has ushered in the era of functional genomics. Currently, functional studies on jojoba genes are primarily conducted through the genetic transformation of model plants like Arabidopsis and tobacco. For instance, overexpressing the jojoba low-temperature induction gene ScTLP in Arabidopsis enhanced the plant’s cold tolerance [15]. Despite a few reported studies on the tissue culture of jojoba [16,17], effective genetic transformation technology for jojoba is still lacking.
Common methods for plant genetic transformations include electroporation, the polyethylene glycol method, particle bombardment, the pollen tube pathway method, and the Agrobacterium-mediated method. Among these, the Agrobacterium-mediated method is widely adopted due to its short cultivation period and strong genetic stability. The Ri plasmid transformation system contained in Agrobacterium rhizogenes is deemed more ideal than the Ti plasmid in A. tumefaciens, leading to its widespread use [18]. Based on the different types of crown gall alkaloids synthesized by A. rhizogenes, Ri plasmids can be categorized into four types: the agropine type, cucumopine type, agropinic acid type, and mikimopine type [19]. The hairy roots induced by A. rhizogenes exhibit characteristics such as hormone autotrophy, genetic stability, a rapid growth rate, and a high content of secondary metabolites [20]. The primary factors influencing the genetic transformation of hairy roots mediated by A. rhizogenes include the strain type, bacterial solution concentration, infection duration, co-culture time, concentration of acetyl eugenol, and type of explant. Currently, the genetic transformation of hairy roots mediated by A. rhizogenes has been successfully applied to various plants such as soybeans [21], citruses [22], watermelons [23], apples [24], lychees [25], and Salvia miltiorrhiza [26]; yet, there are no reports on its application in jojoba.
Galactinol synthase (GolS) is a key rate-limiting enzyme for the synthesis of raffinose family oligosaccharides (RFOs). It catalyzes the formation of galactinol from UDP-galactoside and inositol, providing a galactosyl group for the synthesis of RFOs [27]. RFOs encompass raffinose, stachyose, and verbascose. They accumulate under the conditions of low-temperature and osmotic stresses, playing a pivotal role in enhancing plant tolerance to these stresses. The CsGolS4 gene in cucumbers exhibits responsiveness to low-temperature and drought stresses [28]. An overexpression of CsGolS4 in cucumbers enhances the plant’s low-temperature tolerance. Similarly, an overexpression of ScGolS1 in potatoes significantly enhances the frost resistance of transgenic plants [29].
In the genetic transformation of hairy roots, it is necessary to use reporter genes such as GFP for screening transgenic hairy roots. In recent years, a novel visual reporter gene, RUBY, has been successfully applied in the screening of transgenic hairy roots. The RUBY reporter gene produces betaine, which causes the plant tissue to appear red. Betaine is a natural plant product synthesized from tyrosine as a substrate, catalyzed by three enzymes: P450 oxygenase CYP76AD1, 4,5-DOPA dioxygenase (DODA), and glycosyltransferase (GT). Researchers coupled CYP76AD1, DODA, and GT into an open reading frame and named it RUBY, which allows for the expression status of specific genes to be inferred from the produced red color [30]. The RUBY reporter gene has been used in the genetic transformation of both cannabis [31] and soybeans [32].
The RUBY reporting system developed by He et al. [30] has significant advantages over GFP and GUS reporter genes. RUBY does not require specialized equipment or expensive consumables and can be clearly observed with the naked eye [33]. It is suitable for transformation systems that are difficult to screen with antibiotics or herbicides, as well as for large-scale plant studies. Moreover, it allows for in situ observations without the need for sampling or reagent treatments, thus avoiding the risk of contamination of sterile materials and preventing external factors from interfering with gene expression patterns during sampling. In summary, the RUBY system is naturally harmless, convenient to observe, cost-effective, and is an excellent alternative to existing reporting systems [34].
To establish an effective genetic transformation system for jojoba, firstly, a sterile hairy root genetic transformation system, termed the “soaking co-culture method”, was constructed. Subsequently, four parameters influencing hairy root transformations were optimized. Based on the optimization of the “soaking co-culture method”, a “wrapping co-culture method” was established under non-sterile conditions, eliminating the need for tissue cultures. Ultimately, the stress tolerance function of the ScGolS1 gene was evaluated using the “wrapping co-culture method”. This study establishes a foundation for the functional genomics and genetic improvement of jojoba.

2. Materials and Methods

2.1. Materials

The mature seeds of jojoba are collected in Kehe Town, Huidong County, Sichuan Province. The A. rhizogenes strains used for hairy root induction include Ar.1193, K599, and C58C1. The RUBY vector, kindly gifted by Professor Zengfu Xu from Guangxi University, possesses two CaMV 35S promoters that respectively initiate the expression of resistance genes and the RUBY expression cassette. The media used in this study were indicated in Table S1.

2.2. Establishment of Hairy Root Transformation System Using “Soaking Co-Culture Method” Under Sterile Conditions

The RUBY vector was transferred into K599 competent cells, according to the product manual of K599 chemically competent cells (Shanghai Weidi Biotech, Shanghai, China). For the preparation of A. rhizogenes suspension, monoclonal cells were inoculated into 3 mL of a TY liquid medium containing 50 mg·L−1 of streptomycin and 50 mg·L−1 of kanamycin at 28 °C with shaking at 200 rpm. For the preparation of an infection solution, 50 μL of a bacterial suspension was inoculated in 50 mL of a TY liquid medium containing kanamycin (50 mg L−1) and streptomycin (50 mg L−1) and cultured by shaking at 28 °C for 48 h. The cells were collected by centrifugation at 5000 rpm for 10 min and then suspended in an SSM liquid medium (OD600 = 0.8) (Table S1).
For the hairy root induction of jojoba, tender green leaves were surface-disinfected with 75% ethanol for 30 s, dipped in a sodium hypochlorite solution (4%) for 10 min, then rinsed 5 times with sterile water. Then, the sterilized leaves of jojoba were cut into pieces that were about 1 cm2, soaked in the infection solution containing K599 for 20 min. The leaves were then inoculated in a CM medium (Table S1) for 4 d under darkness. The leaves were washed with a WM liquid medium (Table S1) to remove the A. rhizogenes on the surface and then inoculated in an RIM medium (Table S1) to induce hairy roots.

2.3. Optimization of Hairy Root Transformation System Using “Soaking Co-Culture Method”

The four parameters affecting hairy root genetic transformations were optimized, including the A. rhizogenes strains, co-cultivation under light or dark conditions, the infection time, and the OD600 value of the bacterial suspension used for infection. The strains used were Ar.1193, K599, and C58C1 (Shanghai Weidi Biotech, Shanghai, China). The infection time was set to 5 min/10 min/20 min/30 min. Co-cultures were conducted under dark or light conditions. The OD600 values of the bacterial suspension were set to 0.4, 0.6, 0.8, and 1.0.
The hairy root induction rate and hairy root transformation rate were used to evaluate the effects of different parameters on the induction efficiency and transformation efficiency of hairy roots in jojoba, respectively. The hairy root induction rate (%) = the number of hairy root explants produced/total number of explants × 100%. The hairy root transformation rate (%) = the number of positive hairy roots/total number of hairy roots × 100%.

2.4. Induction of Hairy Roots in Jojoba Using “Wrapping Co-Culture Method” Under Non-Sterile Conditions

For the preparation of jojoba seedlings, the seeds were disinfected in a potassium permanganate solution for 2–3 min, rinsed with clean water 4–5 times, soaked in ultrapure water overnight, and germinated on a damp cotton cloth. After germination, the jojoba seedlings were moved to a 28 °C greenhouse with a photoperiod of 16 h/8 h (light/dark) for cultivation. One month-old seedlings were used for the induction of hairy roots using the “wrapping co-culture method”.
For the preparation of the bacterial suspension and plates used for the infection of the jojoba root, a single colony of A. rhizogenes K599 harboring the RUBY vector was picked and placed into 3 mL of a TY liquid medium containing the corresponding antibiotics and incubated with shaking at 28 °C and 200 rpm overnight. Up to 100 μL of the obtained bacterial suspension was inoculated into 200 mL of a TY liquid medium containing antibiotics and incubated with shaking at 28 °C and 200 rpm for 24 h to obtain a large volume of the bacterial suspension. A total of 200 μL of the bacterial suspension was evenly spread on a TY solid medium containing the corresponding antibiotics and incubated at 28 °C for 48 h to obtain bacterial plates.
For the induction of the hairy roots in the jojoba, the lateral roots and most of the leaves of jojoba seedlings were cut off, and the cutting surface was wrapped with A. rhizogenes cells from the bacterial plates. Then, the seedlings were planted into a pot containing moist vermiculite, and then, 5 mL of the A. rhizogenes suspension was watered at the root of each seedling. The inoculated jojoba seedlings were cultured in a greenhouse with a photoperiod of 16 h/18 h (light/dark) until the formation of hairy roots.

2.5. Genomic DNA PCR Analysis of rolB and GT Genes

Genomic DNA was extracted from transgenic and wild-type jojoba tissues using the FastPure Plant DNA Isolation Mini Kit-BOX 2 (Novozymes, Nanjing, China) and was used as a template for a PCR analysis with corresponding primers (Table S2). The amplification conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, and primer extension at 72 °C for 1 min. After the final amplification cycle, a final extension was performed at 72 °C for 5 min.

2.6. qRT-PCR Analysis of Transgenic Hairy Roots

The total RNA was extracted from the red hairy roots and the wild-type roots of jojoba [35]. The integrity of the extracted RNA was assessed using 1%-agarose gel electrophoresis. The purity and concentration of the RNA samples were measured with NanoDrop 2000 (Thermo Fisher Scientific, Cleveland, OH, USA). A reverse transcription synthesis of the cDNA was conducted, according to the instructions provided by the FastQuant cDNA First Chain Synthesis Kit (Tiangen, Beijing, China). Primers were designed using Primer 5.0 and were subsequently sent to a company for synthesis (Table S2). Quantitative reverse transcription PCR (qRT-PCR) was performed using a QuantStudio3 instrument (Thermo Fisher Scientific, Cleveland, OH, USA). The reaction system comprised 2x Fastfire qPCR PreMix, 10 μL; template, 2 μL; RNase-Free ddH2O, 6.8 μL; and upstream and downstream primers, each at 0.6 μL. The operating conditions were set as follows: 95 °C for 1 min, 95 °C for 15 s, and 60 °C for 1 min, for 40 cycles. The internal reference gene was 18S rRNA (NCBI accession number: AF094562), and gene expression levels were calculated using the 2−ΔΔCt method.

2.7. Low-Temperature Treatment of Jojoba Seedlings and qRT-PCR Analysis of ScGolS Genes

A qRT-PCR analysis was performed to identify the ScGolS gene induced by a low temperature. After germination, the jojoba seedlings were transplanted into 10 L pots in a greenhouse maintained with a 16 h/8 h light/dark cycle. After one month of growth, sixty seedlings with consistent growth were randomly selected for a 4 °C low-temperature stress treatment. At 0, 24, 48, and 72 h, four fully developed leaves from the top of the seedlings were collected and frozen immediately with liquid nitrogen and then stored at −80 °C for future use. The experimental procedure for the qRT-PCR analysis of the ScGolS genes is described in Section 2.6.

2.8. Construction of ScGolS1 Gene Expression Vector

CDs of ScGolS1 were isolated from the total RNA extracted from jojoba leaves, and the upstream and downstream primers carried the cleavage sites of Xba I and Kpn I, respectively (Table S2). The RUBY vector was digested with the same restriction enzymes and purified to recover the digested product. Finally, using a homologous recombination technique, ScGolS1 was linked to the RUBY vector to obtain the RUBY–ScGolS1 construct.

2.9. Evaluation of Low-Temperature Tolerance of Plant Tissues Using NBT Staining

NBT staining was conducted to evaluate whether an overexpression of ScGolS1 in hairy roots enhances tolerance to low-temperature stress in jojoba. A. rhizogenes K599 containing empty RUBY and RUBY–ScGolS1 plasmids were used to infect the roots of jojoba seedlings, and the obtained transgenic hairy roots were used for an evaluation of low-temperature tolerance. For low-temperature stress treatment, the hairy roots were incubated at 4 °C for 7 days. NBT staining was used to evaluate the low-temperature tolerance of the hairy roots transformed with an empty vector and the ScGolS1 gene.

2.10. Statistical Analysis

The SPSS 26 software was used to conduct all statistical analyses. To determine significance (p < 0.05) between experiments, either Student’s t-test or a one-way ANOVA was applied, followed by post-hoc Duncan’s new multiple range tests.

3. Results

3.1. Establishment of Hairy Root Transformation System (Soaking Co-Culture Method) for Jojoba

A sterile genetic transformation system for the hairy roots of jojoba, termed the “soaking co-culture method”, was established using leaves of jojoba as explants and a vector carrying the RUBY visual reporter gene (Figure 1). Using this method, hairy roots were obtained within 2 months, with 6.25% to 18.18% of the obtained hairy roots appearing red.
To confirm that these red hairy roots are transgenic positive, we performed a genomic DNA PCR amplification analysis. The rolB gene in the Ri plasmid and GT gene on the RUBY vector were detected in the red hairy roots, while neither the rolB gene nor the GT gene was detected in the wild-type roots of jojoba (Figure 2A,B). This result indicates that the T-DNA region of the A. rhizogenes Ri plasmid and RUBY plasmid in transgenic hairy roots successfully integrated into the genome of jojoba.
qRT-PCR was used to analyze the expression of the GT, CYP76AD1, and DODA genes in the RUBY expression cassette. The results reveal that there was a certain level of expression for the GT, CYP76AD1, and DODA genes in the red hairy roots, whereas no expression of these genes was detected in the wild-type roots (Figure 2C).

3.2. The Impact of Various Strains of A. rhizogenes on the Induction and Transformation Rates of Hairy Roots in Jojoba

Three A. rhizogenes strains, i.e., K599, Ar.1193, and C58C1, were used to infect jojoba leaves. After 60 days of cultivation, it was found that the leaves infected by the K599 and Ar.1193 strains developed hairy roots, while the C58C1 strain could not induce hairy roots (Figure 3A,B). The K599 strain has a relatively high hairy root induction rate of 17.55% (average), while the Ar.1193 strain has a hairy root induction rate of less than 10% (Table S3). In summary, the K599 strain has a better effect on inducing hairy roots in jojoba leaves than Ar.1193 and C58C1.

3.3. The Effect of Co-Cultivation-Period Lighting on the Induction Rate and Transformation Rate of Hairy Roots in Jojoba

Co-culture is a critical period, wherein A. rhizogenes transfer exogenous genes into explants. To explore the effect of co-cultivation-period lighting on the genetic transformation efficiency of the hairy roots of jojoba, two conditions, co-culture with darkness or light, were used in the induction of transgenic hairy roots. The results show that the induction rate and transformation rate of hairy roots under co-culture with light were higher than those under dark conditions (Figure 3C,D, Table S3). Therefore, light during the co-culture period is conducive to inducing the leaves of jojoba to produce hairy roots. In the present study, the K599 strain and co-culture with light were used for subsequent experiments.

3.4. The Effect of the Infection Time on the Induction Rate and Transformation Rate of Hairy Roots in Jojoba

To investigate the effect of different infection times on the efficiency of the genetic transformation system of hairy roots in jojoba, four infection times were set at 5 min, 10 min, 20 min, and 30 min, with the other conditions remaining the same for each group. The results indicate that, with an increase in the infection time, the induction of hairy roots first increased and then decreased, with the highest induction rate occurring at 10 min (Figure 3E, Table S3). Under the four different infection times, there was no significant difference in the transformation rate of hairy roots, which ranged from 13.08% to 22.73% (Table S3). These results indicate that infection time has an impact on the induction rate of hairy roots in jojoba but has little effect on the transformation rate of hairy roots (Figure 3E,F, Table S3).

3.5. The Effect of the Bacterial Solution Concentration on the Induction Rate and Transformation Rate of Hairy Roots in Jojoba

The concentration of A. rhizogenes suspension is an important factor affecting genetic transformation. In this study, jojoba leaves were infected with a bacterial suspension with OD600 values of 0.4, 0.6, 0.8, and 1.0. The results indicate that all the selected concentrations of the bacterial suspension induced hairy roots (Figure 3G, Table S3). Among the five bacterial suspension concentrations tested, the highest induction rate of hairy roots was observed at an OD600 of 1. However, at this same OD600 value, the transformation rate of hairy roots was found to be the lowest (Figure 3G,H, Table S3).

3.6. Establishment of Hairy Root Transformation System of Jojoba Without Tissue Culture

Based on the optimization of the “soaking co-culture method”, the K599 strain with OD600 = 0.6 was used to infect the main roots of the jojoba seedlings, and transgenic hairy roots of jojoba were successfully obtained without a tissue culture. The specific operational steps are shown in Figure 4. After about 60 days of cultivation, one-to-more red hairy roots were formed at the root of about 25% of the infected jojoba seedlings, and these plants were chimeric with the wild-type stem and leaf and transgenic hairy roots.
To verify that the red hairy roots obtained by the “wrapping co-culture method” are transgenic hairy roots, a PCR analysis of the rolB gene and GT gene was carried out. It was found that the rolB and GT gene on the RUBY vector were detected in red hairy roots, while the rolB gene and GT gene were not detected in wild-type roots (Figure 5A,B). qRT-PCR was used to detect the expression of the GT, CYP76AD1, and DODA genes in the RUBY expression cassette. The results show that these three genes were expressed in red hairy roots, while the expression of the GT, CYP76AD1, and DODA genes could not be detected in wild-type roots (Figure 5C).

3.7. Genetic Transformation and Molecular Identification of ScGolS1 in Jojoba

Four ScGolS genes were identified in the jojoba genome. A gene expression analysis under low-temperature conditions showed that ScGolS1 was notably upregulated (Figure 6). We selected ScGolS1 to evaluate the efficacy of the established hairy root transformation system for jojoba (Figure 7A). It was found that the red hairy root DNA contained the ScGolS1 gene and the GT gene (Figure 7B,C). qRT-PCR analysis showed that the ScGolS1, GT, CYP76AD1, and DODA genes were expressed highly in red hairy roots, while ScGolS1 gene expression was relatively low in wild-type roots, and the GT, CYP76AD1, and DODA genes were not expressed in wild-type roots (Figure 7D).

3.8. Overexpression of ScGolS1 Enhanced Low-Temperature Tolerance of Jojoba

The ScGolS1 gene was ligated into the RUBY vector (Figure 7A). Jojoba with empty and overexpressed ScGolS1 hairy roots were subjected to low-temperature treatment, and then, their low-temperature tolerance was evaluated by NBT staining. The NBT staining revealed that compared with wild-type hairy roots, transgenic hairy roots stained lighter, indicating that their tissues accumulated less superoxide anions and had strong low-temperature tolerance (Figure 7E,F). These results indicate that an overexpression of ScGolS1 enhanced the low-temperature tolerance of jojoba.

4. Discussion

Jojoba is an important oil crop [4]. At present, the lack of an effective genetic transformation system hinders the gene function research and molecular breeding of jojoba. In the present study, two hairy root genetic transformation methods of jojoba were established, and one of them does not require tissue culture techniques. These genetic transformation methods contribute to advancing further molecular biology research on jojoba. Although a jojoba transformation method based on A. tumefaciens has been reported [36], it requires a long time, and our laboratory has been unable to reproduce this method. The entire process of the genetic transformation method based on A. rhizogenes established in this study only takes two months, and that our gene transformation method does not require a tissue culture is more convenient.
A. rhizogenes play a crucial role in the transformation process, because they are accountable for the efficiency of genetic transformations [37]. A. rhizogenes have a wide range of host plant cells, and different strains have different induction abilities for plant hairy roots [38]. The type of the crown gall alkaloid of A. rhizogenes plays a decisive role in the range of hosts they can infect [39]. There are many types of A. rhizogenes used for hairy root induction research, but K599, Ar.1193, and C58C1 are the three A. rhizogenes with a high frequency of use and high efficiency in hairy root inductions [40,41,42]. Therefore, we chose them for the induction of jojoba hairy roots. Although the three strains of A. rhizogenes selected all belong to the agropine type, they exhibit differences in their ability to induce hairy roots in jojoba. Specifically, the K599 strain demonstrates a stronger transformation capability, with both the hairy root induction rate and transformation rate exceeding those of the Ar.1193 and C58C1 strains. In cucumbers, the K599 strain exhibited a better induction ability than other strains [43], which is consistent with our results. But the hairy root transformation rate of strain A4GUS is higher than that of strains R1000, R1601, and ATCC15834 in Artemisia vulgaris [44]. This may be because different A. rhizogenes strains have different infecting abilities on plants.
Light irradiation affects the transformation frequency of hairy roots. In this study, both the induction rate and transformation rate of hairy roots in jojoba under co-cultivation with light were higher than those under co-cultivation in darkness, and the leaves of jojoba were more prone to death during co-cultivation in darkness. The higher induction rates of the hairy roots of the leaves of jojoba under co-cultivation in light may be due to the speculation that light can regulate the plant’s biological clock and gene expression [45]. As one of the most crucial environmental factors, light can influence the growth and development of roots via the auxin signaling pathway [46]. Thus, it is speculated that some genes related to the growth and development of hairy roots may be activated during co-cultivation under light, thereby promoting the induction and transformation of hairy roots.
The infection time affects the efficiency of Agrobacterium infection and adsorption on explants. If the infection time is too short, the Ri plasmid in A. rhizogenes cannot be effectively integrated into the plant genome. If the infection time is too long, the Agrobacterium strains will continue to propagate, the incision will turn brown, and the ex-plants may also turn black and rot, inhibiting the production of hairy roots [47]. In this study, the induction rate of hairy roots in the leaves of jojoba after 10 min of infection was higher than other infection times. Similarly, the induction rate of hairy roots in kiwifruit was the best under 10–13 min of infection [48]. We speculated that about 10 min of infection might be beneficial to the formation of hairy roots. The concentration of the bacterial solution has a great influence on the induction of plant hairy roots [49]. In this study, the transformation rate of hairy roots reached a peak when the concentration of the bacterial suspension OD600 was 0.6. Similarly, the transformation rate was the highest when OD600 = 0.6 in citruses [50], and the highest when OD600 = 0.7 in strawberries [40].
GolS genes play a crucial role in plant responses to abiotic stress. To date, identifications of the GolS family have been conducted in many plants. In Arabidopsis, seven GolS members have been identified, with AtGolS1 and AtGolS2 being induced by droughts, salt, and heat stress, while only AtGolS3 is induced by cold stress [51]. In jojoba, four ScGolS genes were identified, and the number of members in the GolS family is comparable to that of cucumbers [52] and tomatoes [53]. It was observed that only the expression of ScGolS1 is significantly upregulated under cold stress, and jojoba hairy roots overexpressing ScGolS1 exhibited higher tolerance to a low temperature. Interestingly, although an overexpression of AtGolS1, the homologous gene of ScGolS1, in Arabidopsis enhanced the cold tolerance of transgenic plants, AtGolS1 is not induced by a low temperature.
In this study, the transgenic hairy roots of jojoba were successfully generated under sterile and non-sterile conditions. Compared with classical genetic transformation methods [54] that require sterile conditions, the “wrapping co-culture method” developed in the present study has multiple advantages. First, this method eliminates the need for complex instruments such as a clean bench, thereby reducing the investment of experimental equipment and enabling the genetic transformation to be conducted in a field with rudimentary conditions. Secondly, the simple operation process reduces the labor input and time cost during the plant genetic transformation. Lastly, this method does not require a tissue culture, which allows plants that are difficult to sterilize, such as pistachios [55], to be transformed genetically.
A hairy root genetic transformation system was successfully used to study gene functions [50], to produce a large number of secondary metabolites [44], and to obtain regenerated plants [56]. Jojoba has strong stress tolerance to environmental stresses, but the identification of stress tolerance genes mainly relies on Arabidopsis transgenic technology [15]. The hairy root system established in this study can complete the genetic transformation of specific genes and stress tolerance evaluations in 2–3 months. Therefore, our study will promote the mining of the stress-tolerant gene resources of jojoba. Moreover, the hairy root system established in this study can be used to investigate the function of protein-coding genes, including the regulation of transcription factors on downstream genes [25] and protein interactions [57], and noncoding genes including microRNAs (miRNAs) [58] and long non-coding RNAs (lncRNAs) [59].

5. Conclusions

Here, an efficient hairy root transformation system for jojoba, an important dry-land oil crop, was established. Through optimization, we determined that the suitable parameters for the established “soaking co-culture method” are to infect the explants with A. rhizogenes K599 for 10 min under OD600 = 0.6 and to co-culture with light for 4 d. Furthermore, a “wrapping co-culture method” that does not require a tissue culture was developed, which can induce the transgenic hairy roots of jojoba in two months. Overexpression of ScGolS1 through the “soaking co-culture method” enhances the low-temperature tolerance of jojoba, indicating the effectiveness of our established genetic transformation method for evaluating stress-tolerant genes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14122132/s1, Table S1. Composition of the media used in the present study. Table S2. Sequences of the primers used in the present study. Table S3. The effect of different parameters on the induction rate and transformation rate of jojoba hairy root.

Author Contributions

Conceptualization, B.L., Y.Z. and F.G.; formal analysis, J.B. and Y.G.; funding acquisition, Y.G. and F.G.; investigation, B.L., Y.W., W.M., Y.G. and F.G.; supervision, Y.Z.; writing—original draft, B.L.; writing—review and editing, F.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Natural Science Foundation of China (grant nos. 32472209 and 31670335).

Data Availability Statement

Data are contained within the article.

Acknowledgments

We are very grateful to Zengfu Xu, College of Forestry, Guangxi University, China, for kindly providing the RUBY plasmid.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Workflow of hairy root transformation by “soaking co-culture method” in jojoba.
Figure 1. Workflow of hairy root transformation by “soaking co-culture method” in jojoba.
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Figure 2. Molecular validation of transgenic hairy roots. (A) rolB gene fragment detection; M, DL2000 DNA Marker; 1, K599 DNA amplification product (positive control); 2, wild-type roots (negative control); 3–5, red hairy roots; (B) GT gene fragment detection (M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2, wild-type roots (negative control); 3–5, red hairy roots); (C) expression levels of GT, CYP76AD1, and DODA genes in wild-type roots and red hairy roots.
Figure 2. Molecular validation of transgenic hairy roots. (A) rolB gene fragment detection; M, DL2000 DNA Marker; 1, K599 DNA amplification product (positive control); 2, wild-type roots (negative control); 3–5, red hairy roots; (B) GT gene fragment detection (M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2, wild-type roots (negative control); 3–5, red hairy roots); (C) expression levels of GT, CYP76AD1, and DODA genes in wild-type roots and red hairy roots.
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Figure 3. The effects of A. rhizogenes strains, co-culture with light, infection time, and OD600 values on the induction and transformation rates of hairy roots in jojoba. (A) The induction rates with different strains; (B) the transformation rates with different strains; (C) the induction rates with light or darkness during co-culture; (D) the transformation rates with light or darkness during co-culture; (E) the induction rates with infection time; (F) the transformation rates with infection time; (G) the induction rates with OD600 values; (H) the transformation rates with OD600 values. Different lowercase letters indicate significant differences (p < 0.05).
Figure 3. The effects of A. rhizogenes strains, co-culture with light, infection time, and OD600 values on the induction and transformation rates of hairy roots in jojoba. (A) The induction rates with different strains; (B) the transformation rates with different strains; (C) the induction rates with light or darkness during co-culture; (D) the transformation rates with light or darkness during co-culture; (E) the induction rates with infection time; (F) the transformation rates with infection time; (G) the induction rates with OD600 values; (H) the transformation rates with OD600 values. Different lowercase letters indicate significant differences (p < 0.05).
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Figure 4. Workflow of hairy root transformation without tissue culture in jojoba. Firstly, some roots and most leaves from the jojoba plant were removed, then the cut root was wrapped with A. rhizogenes carrying the gene to be expressed, cultured on a solid medium. Next, the seedling was planted in a pot and watered with A. rhizogenes suspension carrying the gene to be expressed. Finally, the jojoba seedling was grown in a greenhouse for about one month until transgenic hairy roots were obtained.
Figure 4. Workflow of hairy root transformation without tissue culture in jojoba. Firstly, some roots and most leaves from the jojoba plant were removed, then the cut root was wrapped with A. rhizogenes carrying the gene to be expressed, cultured on a solid medium. Next, the seedling was planted in a pot and watered with A. rhizogenes suspension carrying the gene to be expressed. Finally, the jojoba seedling was grown in a greenhouse for about one month until transgenic hairy roots were obtained.
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Figure 5. Molecular validation of transgenic hairy roots. (A) rolB gene fragment detection, M, DL2000 DNA Marker; 1, bacterial solution (positive control); 2, wild-type roots (negative control); 3–5: red hairy roots; (B) GT gene fragment detection; M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2: wild-type roots (negative control); 3–5: red hairy roots; (C) expression levels of GT, CYP76AD1, and DODA genes in wild-type roots and red hairy roots.
Figure 5. Molecular validation of transgenic hairy roots. (A) rolB gene fragment detection, M, DL2000 DNA Marker; 1, bacterial solution (positive control); 2, wild-type roots (negative control); 3–5: red hairy roots; (B) GT gene fragment detection; M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2: wild-type roots (negative control); 3–5: red hairy roots; (C) expression levels of GT, CYP76AD1, and DODA genes in wild-type roots and red hairy roots.
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Figure 6. qRT-PCR analysis of four ScGolS genes under low-temperature stress. The expression levels of ScGolS1 (A); ScGolS2 (B); ScGolS3 (C); and ScGolS4 (D). ** represents a significant difference compared to a 0 h treatment, p < 0.01, * represents a significant difference compared to a 0 h treatment, p < 0.05.
Figure 6. qRT-PCR analysis of four ScGolS genes under low-temperature stress. The expression levels of ScGolS1 (A); ScGolS2 (B); ScGolS3 (C); and ScGolS4 (D). ** represents a significant difference compared to a 0 h treatment, p < 0.01, * represents a significant difference compared to a 0 h treatment, p < 0.05.
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Figure 7. Schematic diagram of vector construction, molecular identification, and histochemical staining. (A) Schematic diagram of RUBY–ScGolS1 vector construction; (B) ScGolS1 gene fragment detection, M, DL2000 DNA Marker; 1, RUBY–ScGolS1 plasmid (positive control); 2, H2O (negative control; 3–5, red hairy roots; (C) GT gene fragment detection; M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2: wild-type roots (negative control); 3–5: red hairy roots; (D) qRT-PCR analysis of wild-type roots and hairy roots overexpressing ScGolS1; (E) Histochemical staining of untreated hairy roots expressing empty vector control (left) and hairy roots overexpressing ScGolS1 (right); (F) Histochemical staining of low-temperature treated hairy roots expressing empty vector control (left) and hairy roots overexpressing ScGolS1 (right).
Figure 7. Schematic diagram of vector construction, molecular identification, and histochemical staining. (A) Schematic diagram of RUBY–ScGolS1 vector construction; (B) ScGolS1 gene fragment detection, M, DL2000 DNA Marker; 1, RUBY–ScGolS1 plasmid (positive control); 2, H2O (negative control; 3–5, red hairy roots; (C) GT gene fragment detection; M, DL2000 DNA Marker; 1, RUBY plasmid (positive control); 2: wild-type roots (negative control); 3–5: red hairy roots; (D) qRT-PCR analysis of wild-type roots and hairy roots overexpressing ScGolS1; (E) Histochemical staining of untreated hairy roots expressing empty vector control (left) and hairy roots overexpressing ScGolS1 (right); (F) Histochemical staining of low-temperature treated hairy roots expressing empty vector control (left) and hairy roots overexpressing ScGolS1 (right).
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Li, B.; Wang, Y.; Ma, W.; Bing, J.; Zhou, Y.; Gen, Y.; Gao, F. Establishment of Hairy Root Transformation System for Evaluating Stress-Tolerant Gene in Jojoba. Agriculture 2024, 14, 2132. https://doi.org/10.3390/agriculture14122132

AMA Style

Li B, Wang Y, Ma W, Bing J, Zhou Y, Gen Y, Gao F. Establishment of Hairy Root Transformation System for Evaluating Stress-Tolerant Gene in Jojoba. Agriculture. 2024; 14(12):2132. https://doi.org/10.3390/agriculture14122132

Chicago/Turabian Style

Li, Bojing, Yan Wang, Wenguo Ma, Jie Bing, Yijun Zhou, Yuke Gen, and Fei Gao. 2024. "Establishment of Hairy Root Transformation System for Evaluating Stress-Tolerant Gene in Jojoba" Agriculture 14, no. 12: 2132. https://doi.org/10.3390/agriculture14122132

APA Style

Li, B., Wang, Y., Ma, W., Bing, J., Zhou, Y., Gen, Y., & Gao, F. (2024). Establishment of Hairy Root Transformation System for Evaluating Stress-Tolerant Gene in Jojoba. Agriculture, 14(12), 2132. https://doi.org/10.3390/agriculture14122132

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