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2025 Volume 4
Article Contents
Abstract
Introduction
Materials and methods
Materials
Preparation of mixed surimi samples
Rheological characteristics of mixed surimi samples
Apparent viscosity
Dynamic rheological behavior
Strength of mixed surimi gels
Textural properties of mixed surimi gels
Cooking loss of mixed surimi gels
Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels
Color difference of mixed surimi gels
Scanning electron microscopy (SEM) of mixed surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Statistical analysis
Results and discussion
Rheological properties of surimi gels
Steady-state shear properties
Dynamic elastic properties during gelation
Strength of surimi gels
Textural properties of surimi gels
Cooking loss of mixed surimi gels
Color of surimi gels
Changes in moisture distribution of surimi gels
Scanning electron microscopy of surimi gels
Thiobarbituric acid-reactive substances (TBARS) determination
Conclusions
Author contributions
Data availability
Acknowledgments
Conflict of interest
Supplementary information
Rights and permissions
References
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ARTICLE   Open Access    

A method of genetic transformation of Xizang medical plants without tissue culture

  • 1.

    Key Laboratory of Xizang Medicine Resources Conservation and Utilization of Xizang Autonomous Region, Xizang Agriculture and Animal Husbandry University, Linzhi 860000, China

  • 2.

    School of Life Sciences, Southwest University, Chongqing 400715, China

  • 3.

    Chongqing Academy of Chinese Materia Medica, Chongqing 400065, China

More Information
  • Mirabilis himalaica, known for its medicinal properties and limited distribution in the Qinghai-Xizang Plateau, is under conservation pressure due to high market demand. Traditional transformation methods are inefficient for this species, prompting the development of a non-tissue culture approach. This study utilized mature leaves as explants and employed a cut-dip-budding (CDB) system to transform fluorescence and betacyanin reporter systems in M. himalaica, successfully transforming a high-altitude Xizang medicinal plant. The study found that mature leaves exhibited robust regeneration potential, making them ideal for large-scale transformation. The transformation system achieved approximately 76% positive rooting rate and a 2% positive transgenic plant rate. This method overcomes challenges associated with Agrobacterium tumefaciens-mediated transformation and lays the foundation for future genetic research and industrial applications of Xizang medicinal plants.

  • Introduction

    Surimi gel, known as 'concentrated myofibrillar protein'[1], is a kind of gel prepared by processing fish tissue according to fixed steps, such as rinsing, dehydration, and chopping, then adding a certain number of auxiliary materials for crushing, molding, heating and cooling. Salt-soluble myofibrillar protein (mainly myosin) of surimi denatures and unfolds after heating, and then re-crosslinks and polymerizes to form large protein aggregates[2], which is the internal mechanism of forming the gel structure. To enhance the texture and taste of surimi gel products, 2%−3% salt is added to promote the formation of the protein gel network structure and enhance the solubility and functional properties of the products[3,4]. Nonetheless, numerous studies have confirmed that excessive salt intake may result in risks of disease to human health, such as hypertension, and coronary heart disease[5]. Therefore, the development of low-salt surimi products will be widely focused on in future research.

    Recently, two strategies have been innovatively proposed to improve or maintain the gel properties of low-salt surimi products: one is to use exogenous additives or sodium salt substitutes[6−8], and the other is to exploit new processing technologies[9−11]. Glutamine transaminase (TGase) is the most effective surimi quality improver to enhance the functional properties of protein, which could catalyze acyl donors in proteins (γ-Hydroxylamine group) and acyl receptors (lysine residue, primary amine compound, etc.) undergo acyl transfer reaction to form cross-linked structures[12−14]. Previous studies by Jiang et al.[15] revealed that the cross-linking effect of TGase catalysis depends on the content and spatial distribution of available substrates. In addition, lysine, as an ideal exogenous additive, has been widely used in different meat protein gel systems, which can effectively improve the gel properties of low-salt surimi gel[16] and emulsified chicken sausage[17]. Many researchers have spent a lot of time to obtain higher-quality surimi gel products through the combination of two or more exogenous additives. Many scientists devoted themselves to obtaining higher quality surimi gel products through the combination of two or more exogenous additives. For example, Cao et al.[18] demonstrated that lysine (Lys) could bring about the dissociation of actin under the condition of the presence of TGase, promoting the cooking yield and gel properties of oxidative damaged MP. Similar findings reported by Cando et al.[19] indicated that Lys could induce the changes in protein structure, which were favorable terms to heighten the cross-linking effect of TG and improve the strength of surimi gel.

    Recently, the term ε-Poly-lysine (ε-PL) was followed with interest since it's a natural amino acid polymer produced by microbial fermentation, and was speculated that it has a similar molecular structure and potential effect to Lys[20]. ε-PL, which is generally composed of 25−30 lysine residues connected by amide bonds (by ε-Amino and α-Carboxyl group), was often used as an antibacterial agent for the preservation of meat products and aquatic products[21]. Li et al.[21] explored the effect of preservative coating with ε-PL on the quality of sea bass fillets during storage. It turned out that, ε-PL treatment evidently reduced the thiobarbituric acid and volatile base nitrogen values of bass slices during storage, inhibited the growth of microorganisms, and improved the water retention, texture, and flavor characteristics of the fish. A previous study by Cai et al.[20] proved the bacteriostatic and fresh-keeping effect of the composite coating of protein and sodium alginate on Japanese sea bass, and found that the composite coating had an effect on inhibiting the proliferation of sea bass microorganisms (Escherichia coli, lactic acid bacteria, yeast, etc.), fat oxidation, protein degradation, and nucleotide decomposition. As a natural cationic polypeptide, ε-PL has the advantages of no biological toxicity and low viscosity of aqueous solution. It is noteworthy that it can produce strong electrostatic adsorption with negatively charged amino acids[22]. Its effective modification of protein helps it fully interact with gel component molecules and groups[23], which may play a role in enhancing the texture and water retention of protein gel.

    Nevertheless, few reports are based on the addition of ε-PL to explore the influence mechanism on TGase-catalyzed surimi gel properties. The current study sought to shed light on the effects of different additions of ε-PL on TGase-induced cross-linking effect and the performance of composite surimi gel, to provide a theoretical basis and reference for further research and development of 'low salt', efficient and healthy surimi products.

    Materials and methods

    Materials

    Testing materials, including marine surimi, transglutaminase (TG, enzyme activity 100 IU/g), and ε-Polylysine (ε-PL, ≥ 99% purity) were supplied by Jiangsu Yiming Biological Technology Co., Ltd (Jiangsu, China). Egg white protein was purchased from Henan Wanbang Chemical Technology Co., Ltd (Henan, China). All other chemicals were from Shanghai Yuanye Biochemical Co., Ltd (Shanghai, China) and were at least analytical grade. The ingredients of the surimi gel are shown in Table 1.

    Table 1.  Surimi samples with different treatments.
    GroupsSurimi
    (g)    		TGase
    (w/w, %)    		Egg white
    Protein
    (w/w, %)    		ε-PL
    (w/w, %)    		NaCl
    (w/w, %)
    CK300−−−0.5
    TE3000.47.0−0.5
    TE + P13000.47.00.0050.5
    TE + P23000.47.00.010.5
    TE + P33000.47.00.020.5
    TE + P43000.47.00.040.5
    TE + P53000.47.00.060.5
     | Show Table
    DownLoad: CSV

    Preparation of mixed surimi samples

    The prepared block surimi was firstly put into the chopping machine (or tissue masher) and chopped for 2 min, then different mass fractions of ingredients were added, while the chopping time was extended to 5 min (the temperature should be controlled below 10 °C during this process). The exhausted surimi paste was poured into the special mold (50 mm × 20 mm), which was heated in two stages to form the heat-induced surimi gel (40 °C water bath for 40 min; 90 °C water bath for 20 min). Above prepared surimi gel underwent an ice water bath for 30 min and finally stored overnight at 4 °C for further use.

    Rheological characteristics of mixed surimi samples

    Mixed surimi samples with different treatments were determined under a Haake Mars 60 Rheometer (Thermo Fisher Scientific, Germany) with 35 mm stainless steel parallel plates referring to the method described by Cao et al.[18], and each determination was repeated three times. After centrifugation (1,000× g, 3 min) and 4 °C, the degassed surimi sol (~2 g) was equilibrated at 4 °C for 3 min before measurement.

    Apparent viscosity

    The shear stress of the mixed surimi sol was measured at shear rates between 0−100 s−1.

    Dynamic rheological behavior

    The rheological properties of the mixed surimi sols were measured in an oscillatory mode of CD-Auto Strain at 0.02% and 0.1 Hz frequency, respectively. The heating temperature range is set to 20~90 °C while the heating rate is 1 °C·min−1.

    Strength of mixed surimi gels

    The TA-XT Plus physical property analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) was used to analyze the mixed surimi gel strength of each group of samples (n ≥ 3) at room temperature, and the cube-shaped surimi gel samples are measured through the P/0.5 probe. Referring to the method previously described by Fang et al.[24], the test parameters are as follows, pre-test and test rate (1 mm·s−1); rate after measurement (5 mm·s−1); depressing degree (30%); trigger force (5 g); data acquisition rate (400 p/s).

    Textural properties of mixed surimi gels

    Concerning the method of Jirawat et al.[25], texture profile analysis (TPA) was employed to record the hardness, elasticity, cohesion, chewiness, and resilience of mixed surimi gels. It is well known that TPA can explore the textural properties of food through the texture analyzer (TA-XT Plus, Stable Micro Systems Ltd, Surrey, UK) equipped with a P/75 probe to simulate human oral chewing action and obtain the texture characteristic values related to human sensory evaluation. The physical property parameter settings were as follows: downforce (5 g), compression degree (50%), pre-test speed, test speed, and post-test speed (1.0 mm·s−1).

    Cooking loss of mixed surimi gels

    The cooking loss of surimi gel under different treatments was determined referring to the protocol of Dong et al.[26]. The cooked surimi gel sample was instantly absorbed dry and weighed (W5). The cooking loss (CL) is calculated as follows:

    CL(g/100g)=(M1−M2)/M1×100 (1)

    Where M1, weight of the sample before cooking; M2, weight of the sample after cooking.

    Low-field nuclear magnetic resonance (LF-NMR) of mixed surimi gels

    The LF-NMR of the mixed surimi gel was detected by a PQ001-20-025V NMR analyzer (Niumag Analytical Instruments Co., Ltd, Suzhou, China) referring to the previous method conducted by Li et al.[27]. Relevant characteristic parameters were set as follows: sampling frequency (200 KHZ), echo time (0.3 ms), and cumulative number (8). Keeping the surimi gel sample (approximately 2 g) at room temperature for 30 min, they were carefully put into a cylindrical nuclear magnetic tube (15 mm in diameter), and finally, the relaxation time (T2) was recorded using the Carr Purcell Meiboom Gill (CPMG) pulse sequence.

    Color difference of mixed surimi gels

    CM-5 colorimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used for surimi gel with different treatments following to the procedure of Wang et al.[28]. Several 1 cm thick slices were cut from surimi samples (n = 3) selected from different treatment groups, and the gel whiteness was calculated according to the following formula:

    Whiteness=100−√(100−L∗)2+a∗2+b∗2 (2)

    Scanning electron microscopy (SEM) of mixed surimi gels

    According to the previously described procedure by Gao et al.[29], the square-shaped surimi gels (4 mm × 4 mm × 4 mm) were immersed in 0.1 mol·L−1 phosphate buffer (pH 7.2) containing 2.5% (v/v) glutaraldehyde for 24 h. The above samples were washed using phosphate buffer (0.1 mol·L−1, pH 7.2) three times and then subsequently dehydrated in a series of alcohol solutions. The microstructures of the mixed surimi gels were imaged using an FEI Verios 460 SEM (FEI Inc., Hillsboro, OR, USA).

    Thiobarbituric acid-reactive substances (TBARS) determination

    The TBARS value was analyzed by the method described by Hu et al.[30] with slight modification. Thiobarbituric acid solution (1.5 mL) and 8.5 mL of trichloroacetic acid solution were added to the sample in turn, the mixture was bathed in water at 100 °C for 30 min. The supernatant was extracted and centrifuged twice (3,000× g, 5 min): (1) The original supernatant (5 mL) was taken and the same amount of chloroform for mixed centrifugation; (2) The second supernatant (3 mL) and petroleum ether (1.5 mL) were taken for mixed centrifugation. Finally, a small amount of lower liquid was taken to determine the absorbance (A532 nm). The final result was expressed in malondialdehyde equivalent (mg·kg−1).

    Statistical analysis

    All statistical analyses of data were investigated by statistical product and service solutions IBM SPSS Statistics version 23.0 (IBM SPSS Inc., Chicago, USA). The LSD all-pairwise multiple comparison method was used for the least significance analysis, and p < 0.05 was considered to indicate significance. The experimental data are expressed as mean ± standard deviation (SD) and plotted using Origin 2019 (Origin Lab, Northampton, MA, USA) software.

    Results and discussion

    Rheological properties of surimi gels

    Steady-state shear properties

    The steady-state shear flow curve can be used to characterize the interaction between proteins. The steady shear flow changes of apparent viscosity (Pa·s) of composite surimi with shear rate (s−1) under different treatments are shown in Fig. 1a. The apparent viscosity of all samples decreased significantly (p < 0.05) with increasing shear rate, exhibiting shear-thinning behavior[31]. In the range of shear rates from 0.1 to 100 s−1, the samples with TGase addition were always higher than the control, indicating that the induction of TGase enhanced the cross-linking of surimi proteins and formed a more stable structure. The surimi samples added with a lower proportion of ε-PL (0.005% and 0.01%) were regarded as lower viscosity fluid (Fig. 1a). The possible reason was that the low concentration of ε-PL provided a weak effect on the pH value of the surimi system. As a consequence, the content of the net charge provided was relatively small and the electrostatic interaction between the surimi proteins was weakened[20].

    Figure 1.  Effect of different treatments on rheological properties of surimi.
    DownLoad: Full-Size Img PowerPoint
    Dynamic elastic properties during gelation

    The elastic modulus (G′) mainly depends on the interaction between protein molecules, which can reflect the change of elasticity in the heat-induced surimi gel. As shown in Fig. 1b, the gel process of surimi in the control group was a thermodynamic process consisting of two typical stages. In the first stage, the value of G' showed a gradual downward trend in the range of 20~50 °C. This is mainly because, (1) the activity of endogenous protease in surimi is increased; (2) the myosin light chain subunits of surimi are dissociated under the action of protease, forming myosin and actin[24]; (3) heat-induced hydrogen bonds break between protein molecules, thus enhancing the fluidity of the gel system[31]. The second stage: From 50 to 73 °C, the G' value increases rapidly. As the temperature continues to rise to 90 °C, the G 'value keeps rising steadily. At this time, the gel network structure gradually became stable and irreversible.

    Compared with several curves in Fig. 1b, TGase treatment could significantly increase the G' value. The change trend of G' value of the samples added with TGase and ε-PL were similar to that of the control group, but the former increased faster and the maximum value of G' was also significantly higher. The temperature corresponding to the first peak of G' gradually decreased (PL1≈PL2 < PL4≈TE < PL3≈PL5 < CK), indicating that the process of protein denaturation and aggregation was advanced. This result showed that the proper amount of ε-PL (0.04%) in combination with TGase can reduce the thermal denaturation temperature of protein-forming gel and improve the forming ability of composite gel, which was unanimous with the changes in texture and properties of surimi gel (Table 2). Furthermore, the addition of a small amount (0.005%) or an excessive amount (0.06%) of ε-PL made the mixed surimi protein system more unstable, affecting the ability of TG-induced gel formation as the last resort[32].

    Table 2.  Effect of different treatments on the textural properties of surimi gels.
    GroupsHardness (g)SpringinessCohesivenessChewiness (g)Resilience
    CK811.40 ± 45.28de0.81 ± 0.01a0.53 ± 0.02b498.36 ± 21.58c0.21 ± 0.01e
    TE1105.80 ± 61.83c0.83 ± 0.01a0.56 ± 0.01b510.40 ± 26.04c0.24 ± 0.00bcd
    TE + P1736.01 ± 18.49e0.80 ± 0.03a0.61 ± 0.00a348.78 ± 18.36d0.23 ± 0.01cde
    TE + P2777.45 ± 39.61de0.80 ± 0.01a0.60 ± 0.02a307.03 ± 12.97e0.22 ± 0.00de
    TE + P3869.66 ± 45.82d0.83 ± 0.02a0.63 ± 0.01a485.06 ± 22.73c0.28 ± 0.00a
    TE + P41492.80 ± 77.13a0.83 ± 0.01a0.60 ± 0.01a721.74 ± 31.55a0.26 ± 0.01ab
    TE + P51314.60 ± 84.10b0.81 ± 0.00a0.60 ± 0.02a652.36 ± 30.24b0.25 ± 0.01bc
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Strength of surimi gels

    Gel strength is one of the vital indicators to test the quality of surimi products, which directly affects the texture characteristics and sensory acceptance of the products. The strength of mixed surimi gel with TGase was significantly enhanced by 23.97% (p < 0.05) compared with the control group after heat-inducing. This enhancement is probably caused by the crosslinking promotion of TGase (Fig. 2a). A deeper explanation is that under the catalysis of TGase, the ε-amino group on lysine and the γ-amide group on glutamic acid residues undergo acylation reaction inside or between proteins, forming ε-(γ-Glutamyl)-lysine covalent cross-linking bond which promotes the production of the protein gel network[26].

    Figure 2.  (a) Catalytic reaction of glutamine transaminase and (b) effect of different treatments on gel strength and cooking loss of surimi gel. a−d/A−D: values with different lowercase letters indicate significant difference (p < 0.05).
    DownLoad: Full-Size Img PowerPoint

    On the basis of adding TGase, the strength of the mixed gel with ε-PL concentration ranging from 0.005% to 0.03% was lower than that of TE (Fig. 2b). It was speculated that the surimi sample with low concentration ε-PL was a fluid with lower viscosity, and the interaction between protein and water is strengthened, while the electrostatic interaction between proteins is further weakened[33]. This was also consistent with the change in rheological properties in Fig. 1a. Nevertheless, with the increase of ε-PL content to 0.04%, the gel strength of surimi gel significantly increased and reached the highest value (781.63 g·cm), which was about 21.03% higher than that of the TE group (p < 0.05) (Fig. 2b). The increase of gel strength in this process was obtained due to the following three possible reasons, (1) ε-PL is a cationic amino acid with positive charge, which can improve the pH value of protein or meat product system; (2) The interaction between the ε-amino group of ε-PL and the aromatic residue of protein, namely the cation-π interaction, can change the structure of meat protein[34]; (3) Protein (mixed surimi system) with high ε-amino group content, TGase has strong gel improving ability[35].

    Such synergy (ε-PL = 0.04%), as a comprehensive result of different impactors, is that after the surimi was mixed under the optimal ratio conditions, the synthesis of TGase enzyme catalysis, pH value shift, changes in protein and amino acid composition, etc., increased the strength of various forces, thus forming a denser stereoscopic network structure. Note that when the content of ε-PL reached 0.06%, the gel strength of the corresponding surimi gel decreased by 10% compared with 0.04%. Similarly, others reported the result that alkaline amino acids led to the reduction of the strength of the myosin gel of bighead carp[36].

    Textural properties of surimi gels

    TPA, also known as whole texture, is a comprehensive parameter that determines the sensory quality of surimi gel, including hardness, elasticity, cohesion, chewiness, and resilience[37]. As shown in Table 2, the addition of TGase significantly improved the texture properties of surimi gel (p < 0.05), which is related to TGase's ability to induce surimi protein to form more ε-(γ-Glu)-Lys covalent bond during heating[38] (Fig. 2a). Compared with the cross-linking induced by TGase alone, the gel hardness of the mixed surimi gel treated with ε-PL showed a similar change to the gel strength (Fig. 2b). As the concentration of ε-PL increased from 0.005% to 0.04%, the hardness of surimi gel gradually increased until it reached the maximum (1,492.80 g). At the same time, we also observed that the elasticity, cohesion, and chewiness of the composite gel reached the highest values with the 0.04% ε-PL, and these were higher than those of the TGase-only group. Similar to the findings of Ali et al.[39], the author found that the combination of ε-PL and beetroot extract can effectively replace nitrite, which produced a marked effect in maintaining the color of Frankfurt sausage and improving the texture and performance. However, the data we collated above (Table 2) also showed a clear phenomenon, that is, the addition of ε-PL with lower concentration (0.005%−0.02%) or highest concentration (0.06%) was not conducive to the combination with TGase to improve the texture characteristics of mixed surimi gel.

    Cooking loss of mixed surimi gels

    Cooking loss indicates the water holding capacity of heated surimi, which usually represents the stability of the three-dimensional network structure of surimi gel[40]. The 0.4% TGase significantly reduced the cooking loss of surimi samples (p < 0.05), which was 4.91% lower than that of the control group (Fig. 2b). When the addition of PL (combined with TGase) gradually increased from 0.005% to 0.04%, the cooking loss of surimi showed a decreasing trend and reached the minimum at 0.04%. The ε-PL-added surimi gel samples had higher cooking loss compared with that of only with TGase. Most notably, this may be due to ε-PL is able to further exert the cross-linking effect of TGase. A similar finding was reported in the study by Ma et al.[22], adding ε-PL is able to improve the solubility of myofibrillar proteins. Proteins with high solubility are more easily induced by TGase during heating, which aggravates the cross-linking between ε-PL-protein or protein-protein and forms a disordered gel network structure with relatively weaker water-holding capacity.

    Color of surimi gels

    As shown in Table 3, the high lightness (L*), low yellowness (b*), and high whiteness of surimi in the control group were relatively low, with values of 72.49, 10.42, and 70.56 respectively. After TGase was added to induce cross-linking, these values increased significantly (p < 0.05), which may be related to the photochromic effect of water molecules released from gel matrix under TGase-induced surimi protein cross-linking reported in the previous study[41]. Furthermore, on the basis of adding TGase, it was interesting to see that the L* and whiteness values of surimi gel first increased and then gradually decreased with the increase in ε-PL concentration. The influence of ε-PL combined with TGase on the whiteness of gel can be attributed to three reasons: (1) The addition of ε-PL can effectively increase the substrate content of TGase, and promote the cross-linking between proteins, so as to obtain a much more compact surimi gel structure, which affects the refractive index of light; (2) The water retention performance of the high surimi gel was significantly improved with the addition of ε-PL[42] (Fig. 2b), at this time, the L* value (positively related to the whiteness) value showed a decreasing trend with the decrease of the surface free water content of the gel sample; (3) With the increase of ε-PL concentration, some colored substances, formed due to the accelerated Maillard reaction rate during the preparation of gel, may have an adverse effect on the improvement of gel whiteness[43]. After adding TGase to induce cross-linking, although the whiteness value of composite surimi was significantly enhanced, ε-PL was not enough to further improve the whiteness value of final mixed surimi gel, and high concentration so far as to have a negative impact on the whiteness value.

    Table 3.  Effect of different treatments on the color of surimi gels.
    GroupsL*a*b*Whiteness
    CK72.49 ± 0.82e−0.92 ± 0.01e10.42 ± 0.33d70.56 ± 0.65d
    TE77.82 ± 0.34ab−0.23 ± 0.02c12.29 ± 0.29bc74.64 ± 0.26ab
    TE + P178.39 ± 0.40a−0.14 ± 0.01a12.46 ± 0.12ab75.05 ± 0.41a
    TE + P278.42 ± 0.43a−0.17 ± 0.0b12.46 ± 0.15ab75.07 ± 0.32a
    TE + P377.48 ± 0.16b−0.18 ± 0.02b12.37 ± 0.09abc74.31 ± 0.11b
    TE + P476.34 ± 0.05c−0.22 ± 0.01c12.73 ± 0.02a73.13 ± 0.04c
    TE + P575.48 ± 0.18d−0.26 ± 0.01d12.01 ± 0.14c72.70 ± 0.19c
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Changes in moisture distribution of surimi gels

    The water distribution in surimi gel products was measured by LF-NMR[44], and the results were inverted to obtain the transverse relaxation time (T2), which reflects the strength of water fluidity[45]. There are four fitted wave peaks which are assigned to four different water distribution states and wave peak ranges (Fig. 3, Table 4): strong bound water T21 (0.1−1 ms), weak bound water T22 (1−10 ms), non-flowing water T23 (10−100 ms) and free water T24 (100−1,000 ms)[46]. At the same time, similar characteristics (the main peak is centered on T23) are possessed by the water distribution of all surimi gel samples. The addition of TGase increased the proportion of non-flowing water, accompanying the significant decrease in the content of free water (p < 0.05), compared with the control group. It proved that the addition of TGase brought this phenomenon, namely the bound water in surimi gel was partially transferred to the non-flowing, which also indicated the reduction of cooking loss of surimi samples induced by TGase (Fig. 2). On the basis of adding TGase, the low level of ε-PL (0.005%, 0.01% and 0.02%) inhibited the transition of free water in surimi gel to non-flowing water, in which the proportion of T23 decreased by 2.05%, 2.74%, and 1.42% respectively compared with the surimi gel only with TGase, while T24 increased by 15.76%, 14.17%, and 4.43% respectively. The proportion of P23 in the surimi gel sample (ε-PL = 0.04%) was equivalent to that of the TE group, implying that the addition of appropriate ε-PL was not directly connected with the changes in water distribution in surimi after TGase-induced cross-linking, and slight addition would have a negative impact, which was unanimous with the above research results of cooking loss (Fig. 2a).

    Figure 3.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    DownLoad: Full-Size Img PowerPoint
    Table 4.  Effect of different treatments on the transverse relaxation time T2 of surimi gel.
    GroupsP21 (%)P22 (%)P23 (%)P24 (%)
    CK2.175 ± 0.186c1.198 ± 0.012e82.863 ± 0.691bc13.764 ± 0.091a
    TE1.938 ± 0.095d2.166 ± 0.043a85.144 ± 1.336a10.752 ± 0.033e
    TE + P12.705 ± 0.067b1.414 ± 0.009d83.435 ± 1.616b12.446 ± 0.105c
    TE + P23.005 ± 0.003a1.845 ± 0.056b82.874 ± 1.739bc12.276 ± 0.108c
    TE + P33.107 ± 0.063a1.715 ± 0.033c83.950 ± 1.587ab11.228 ± 0.086d
    TE + P41.770 ± 0.018d2.099 ± 0.059a85.021 ± 1.756a11.110 ± 0.144d
    TE + P53.121 ± 0.007a1.758 ± 0.107bc82.136 ± 1.091c12.985 ± 0.018b
    Different lowercase letters in the same column indicated significant differences (p < 0.05).
     | Show Table
    DownLoad: CSV

    Scanning electron microscopy of surimi gels

    The effect of different treatment methods on the microstructure of surimi gel was observed by SEM. As shown in Fig. 4, the microstructure of the control surimi gel (Fig. 4a) was observed to be enormously loose, uneven, and irregular, with large pores, which explained the reason why the control surimi gel had low strength and large cooking loss (Fig. 2b). In addition, the morphological properties of another group of surimi gel (TGase-induced) are appreciably different from the scanning electron micrograph mentioned above (Fig. 4b). Numerous compact and evenly distributed small pores were observed, instead of the loose structure in the control sample, which accounted for the fact that TGase could interact with proteins to form a more uniform and orderly three-dimensional network structure in the process of gel formation[47]. Moreover, similar findings were also reported in the previous studies of Dong et al.[26]. When treated with TGase and ε-PL (from 0.05% to 0.04%), the surface of the surimi gel sample gradually became flat through observation (Fig. 4f). We found that some smaller pores were formed and some larger pores were slightly supplemented visually. Remarkably, when the addition of ε-PL increased to 0.06% (Fig. 4g), the structure of surimi gel was not as uniform as before, and some large and irregular holes appeared obviously. This phenomenon might be related to moderate ε-PL, especially 0.02%, which can promote the cross-linking of TGase with protein and the aggregation/denaturation of different components, which agreed with the change of coagulation strength of surimi samples.

    Figure 4.  Effect of different treatments on the microstructure of surimi.
    DownLoad: Full-Size Img PowerPoint

    Thiobarbituric acid-reactive substances (TBARS) determination

    TBARS value, which can reflect the degree of lipid oxidation and rancidity of surimi, is an important indicator for the occurrence of oxidation[48]. Some substances in surimi, such as unsaturated fatty acids, will inevitably undergo oxidative decomposition to produce a large amount of malondialdehyde (MDA), the latter reacts easily with thiobarbituric acid (TBA) reagent to produce red compounds with maximum absorbance at 532 nm. As illustrated in Fig. 5, when TGase and a certain amount of PL (from 0.005% to 0.06%) were added, the TBARs value of surimi gels decreased to 2.03 and 1.67 mg/kg respectively, compared with the control group (2.26 mg/kg). This shows that the addition of both can significantly reduce the TBARS value of surimi gel (p < 0.05), considered to be a cue for the fat oxidation of surimi being inhibited. In addition, we also found that the antioxidant effect of PL was dose-dependent. The ε-PL can be chelated by ferrous ions along with the ability to scavenge free radicals like hydroxyl radicals, ensuring its antioxidant properties[33]. The ε-PL was prospectively considered as an antioxidant additive to prevent surimi protein from deteriorating induced by oxidation[49]. Similarly, making use of lysine or ε-PL as antioxidants in plant protein and meat protein has been widely reported[33, 50]. Fan et al.[42] confirmed that ε-PL addition has a significant effect on maintaining a high total phenolic content and vitamin C levels in fresh lettuce, which also shows that it has certain antioxidant activity.

    Figure 5.  Effect of different treatments on TBARS values of surimi gel. a−f: values with different lowercase letters indicate significant difference (p < 0.05).
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    Conclusions

    The addition of ε-PL at different concentrations had an apparent impact on the characteristics of TGase-induced mixed surimi gel, accompanied by the following situations: The 0.04% ε-PL provided a synergistic effect to promote the aggregation and crosslinking of surimi proteins induced by TGase; The rheology, LF-NMR and SEM results showed that the appropriate concentration (0.04%) of ε-PL apparently enhanced the initial apparent viscosity and elasticity of surimi samples, which was conducive to the formation of a more dense and uniform three-dimensional network structure, further limiting the flow of water in surimi and the exudation of hydrophilic substances; Simultaneously, the network strength was strengthened along with the texture properties of the mixed surimi gel. Furthermore, ε-PL had a strong ability to inhibit lipid oxidation in mixed surimi gel, showing a concentration dependence. When the content of ε-PL ran to lowest (0.005%−0.01%) or highest (0.6%), the improvement of the quality of surimi mixed gel was opposite to the above. The results show that ε-PL can be perceived as a prospective polyfunctional food additive to improve the texture properties, nutritional advantages, and product stability of surimi products.

  • The authors confirm contribution to the paper as follows: study conception and design: Cao Y, Xiong YL, Yuan F, Liang G; data collection: Liang G, Cao Y, Li Z, Liu M; analysis and interpretation of results: Li Z, Cao Y, Liang G, Liu Z, Liu M; draft manuscript preparation: Li Z, Cao Y, Liu ZL. All authors reviewed the results and approved the final version of the manuscript.

  • The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

    • Acknowledgments

    This work was financially supported by the Natural Science Basic Research Program of Shaanxi (No. 2023-JC-YB-146), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (No. BTBUKF202215), the Innovation Capability Support Plan of Shaanxi (No. 2023WGZJ-YB-27), the Agricultural Technology Research and Development Project of Xi'an Science and Technology Bureau (No. 22NYYF057), and Jiangsu Yiming Biological Technology Co., Ltd. in China.

  • The authors declare that they have no conflict of interest.

  • Supplementary Table S1 Primers used in this study.
    Supplementary Fig. S1 The standard curve between the HPLC peak area and betanin content (0, 1.5, 2, 3, 4, and 5 mg/ml). A linear regression was observed between the peak area and betanin contents at 0−5 mg (R2 = 0.9865).
    Supplementary Fig. S2 The contents of betanin in pYL1300H-CDGAeG transgenic plants and wild type M. himalaica. All data represent the mean ± SD (n = 3; *, p < 0.001; Student's t-test).
    Supplementary Fig. S3 Relative expression levels of tdTomato gene by qRT-PCR in tdTomato overexpression plants, respectively, compared with that in wild type (WT) M. himalaica. β-actin was used as an internal reference gene. All data represent the mean ± SD (n = 3; *, P < 0.001; Student's t-test).
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    Sun T, Han X, Jiang Y, Li Q, Xu Y, et al. 2025. A method of genetic transformation of Xizang medical plants without tissue culture. Medicinal Plant Biology 4: e002 doi: 10.48130/mpb-0024-0032
    Sun T, Han X, Jiang Y, Li Q, Xu Y, et al. 2025. A method of genetic transformation of Xizang medical plants without tissue culture. Medicinal Plant Biology 4: e002 doi: 10.48130/mpb-0024-0032
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A method of genetic transformation of Xizang medical plants without tissue culture

  • 1.

    Key Laboratory of Xizang Medicine Resources Conservation and Utilization of Xizang Autonomous Region, Xizang Agriculture and Animal Husbandry University, Linzhi 860000, China

  • 2.

    School of Life Sciences, Southwest University, Chongqing 400715, China

  • 3.

    Chongqing Academy of Chinese Materia Medica, Chongqing 400065, China

  • Received: 05 November 2024
  • Revised: 07 December 2024
  • Accepted: 13 December 2024
  • Published online: 20 January 2025
Medicinal Plant Biology  4,  Article number: e002  (2025)  |  Cite this article

Abstract: Mirabilis himalaica, known for its medicinal properties and limited distribution in the Qinghai-Xizang Plateau, is under conservation pressure due to high market demand. Traditional transformation methods are inefficient for this species, prompting the development of a non-tissue culture approach. This study utilized mature leaves as explants and employed a cut-dip-budding (CDB) system to transform fluorescence and betacyanin reporter systems in M. himalaica, successfully transforming a high-altitude Xizang medicinal plant. The study found that mature leaves exhibited robust regeneration potential, making them ideal for large-scale transformation. The transformation system achieved approximately 76% positive rooting rate and a 2% positive transgenic plant rate. This method overcomes challenges associated with Agrobacterium tumefaciens-mediated transformation and lays the foundation for future genetic research and industrial applications of Xizang medicinal plants.

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    Introduction

    • Mirabilis himalaica (Edgew.) Heimerl, commonly known as Ba Zhu in Xizang, belongs to the Nyctaginaceae family. It is the only Old World representative of a large New World genus[1]. The distribution of M. himalaica is confined to the dry-hot valleys of the Qinghai-Xizang Plateau, although it can thrive in areas with significant altitude variations, demonstrating remarkable altitude adaptability[2]. Native Xizang people have used M. himalaica since the Tang Dynasty to treat stomach disorders, nephritic edema, and gonorrhea[3]. Its limited distribution, combined with the growing demand for traditional medicinal plants, puts pressure on wild populations of M. himalaica. According to the IUCN Red List Categories and Criteria (version 3.1)[4] and the Guidelines for Application of IUCN Red List Criteria at Regional Levels (version 3.0)[5], M. himalaica is classified as 'Near Threatened' in China, based on field surveys[1]. The ecological niche and medicinal value of M. himalaica have garnered attention, prompting conservation efforts and research on its bioactive compounds. Artificial cultivation of M. himalaica was conducted in Xizang, although cultivated and wild populations exhibit differences in chemical composition[6]. Numerous active compounds have been identified in M. himalaica, most of which are phenolic acids, particularly rotenoid-type flavonoids[7,8]. For example, Linghu et al. isolated Mirabijalone E, which exhibited anticancer properties[8]. Other notable properties, such as myxospermy[9] and UV-B radiation adaptation[10], have also been investigated.

      Genetic transformation is a critical tool for investigating the biosynthesis pathways of species-specific bioactive compounds, unique structural adaptations for plateau conditions, and genetic improvement. However, genetic transformation is limited for M. himalaica. Lan et al. established hairy-root transformation of M. himalaica[3]. Currently, there have been no reports on methods for obtaining transgenic regenerated M. himalaica through genetic transformation. Agrobacterium tumefaciens-mediated transformation is the most widely used method for genetic transformation; however, it is often inefficient, requiring the cumbersome process of tissue culture and is species- and genotype-dependent[11]. Thus, developing non-tissue culture transformation and regeneration systems is critical in plant science research, especially for non-model plants. Agrobacterium rhizogenes-mediated non-tissue culture transformation is an attractive strategy[12]. A. rhizogenes-mediated transformation has higher efficiency and better plant species adaptability compared with A. tumefaciens-mediated transformation[13]. The limitation of A. rhizogenes-mediated transformation is that it is generally used to generate transgenic hairy roots, which are difficult to convert into shoots. Recently, Cao et al. established a simple cut-dip-budding (CDB) delivery system, which enabled efficient transformation under non-sterile conditions without the need for tissue culture[11]. This method has subsequently been applied to Chinese cabbage[14], Idesia polycarpa[15], succulent plants[16], trees[17], as well as medicinal plants[18].

      Due to the special ecological niche and medicinal value of M. himalaica, we established a simple and efficient A. rhizogenes-mediated transformation system. Using leaves as explants, we successfully transformed a fluorescence and betacyanin reporter system into M. himalaica, obtaining transgenic plants. This is the first report of the transformation of a Xizang folk medicinal plant that thrives at high altitudes (over 3,000 m). Our work expands the application of the cut-dip-budding (CDB) system for plant transformation and will promote investigations into the biosynthetic pathways of bioactive compounds and genetic improvement in Xizang medicinal plants.

    Materials and methods

      Plant material and growth conditions

    • The seeds of M. himalaica were collected in Linzhi, Xizang Autonomous Region, China. These seeds were germinated in Petri dishes on wet filter paper for 2 d and then grown in an artificial climate chamber (24 °C, 16 h light/22 °C, 8 h dark). Leaves from 1-month-old M. himalaica were used for transformation.

      Preparation of Agrobacterium rhizogenes

    • The reporter constructs, pYL1300H-CDGAeG[19] and Cotton 2.0-tdTomato[20], used in this study were kindly provided by Prof. Qinglong Zhu (South China Agricultural University, Guangzhou, China) and Prof. Lu Long (Henan University, Kaifeng, China), respectively. In pYL1300H-CDGAeG, four betacyanin biosynthetic genes, including BvADH, BvCYP76AD1S, BvDODA1S, and cDOPA5GT, as well as an eGFP gene, are expressed under the control of 35S promoters. In Cotton 2.0-tdTomato, a red fluorescence gene, tdTomato, which is significantly brighter than eGFP, is expressed under the control of the enhanced 35S promoter.

      The plasmids were introduced into A. rhizogenes K599 cells via heat-shock transformation, as described by Cao et al.[11]. The transformed Agrobacteria were cultured (28 °C, 220 rpm) to reach an OD600 of 0.8–1 in TY medium (5 g/L tryptone and 3 g/L yeast extract) containing 50 mg/L streptomycin, 50 mg/L kanamycin, and 10 mM CaCl2. Three hundred microlitres of this culture were spread and cultured for 2~3 d on TY solid agar medium (5 g/L tryptone, 3 g/L yeast extract, and 15 g/L agar) until the agar medium was covered with a uniform layer of bacteria. The bacterial suspension in liquid TY medium and the slush bacterial layer from the solid agar medium were then prepared for plant infection.

      Plant transformation

    • In this study, we modified the cut-dip-budding (CDB) method reported by Cao et al.[11]. Briefly, mature leaves from 1-month-old M. himalaica were cut from the petiole and used as explants. The cut petiole ends were vacuum-infiltrated for 5 min with an A. rhizogenes K599 suspension prepared in an infection solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone (pH 6.0). Subsequently, the cut ends of the petioles were coated with bacterial layers from the TY solid agar medium. These inoculated leaves were then planted in pots containing vermiculite and covered with plastic wrap to maintain moisture. The inoculated explants were cultured under plastic wrap at 26 °C with a 16-h light/8-h dark cycle until buds developed from the leaf cuttings (approximately 1 month).

      Verification of the transgenic M. himalaica

    • The transgenic buds were first examined for purple color (indicative of the betacyanin reporter system) or red fluorescence (indicative of the tdTomato reporter system) under the 538 nm wavelength (LUYOR-3410GR). To further verify the presence of transgenic plants, genomic DNA was isolated from young leaves of M. himalaica using the CTAB method and used as templates for PCR. The primers used in genomic PCR are listed in Supplementary Table S1.

      Expression analysis of exogenous genes

    • To detect the expression levels of exogenous genes, we extracted total RNA from the wild-type and transformed regenerated M. himalaica, and reverse transcribed it into cDNA. Then it was used as a template for q-RTPCR. Beta-actin was used as an housekeeping gene in M. himalaica. The qRT-PCR primers are shown in Supplementary Table S1.

      Determination of betanin content

    • To extract betanin from M. himalaica, 1.0 g of fresh leaf tissue was ground into a fine powder using liquid nitrogen. The powdered tissue was then transferred to an Eppendorf tube containing 10 mL of distilled water. The tube was placed in an ultrasonic cleaner and maintained at 40 °C for 60 min. Afterward, the mixture was centrifuged at 8,000 rpm for 10 min, and the supernatant was collected for the comparison of betanin contents.

      To detect betanin, a Shimadzu LC-20A HPLC system with a photo-diode array detector was used (Shimadzu, Kyoto, Japan). The separation was carried out on a Kromasil-C18 column (4.6 mm × 250 mm, i.d., 5 μm) with methanol (A)-formic acid aqueous solution (0.2%, B) as the mobile phase. The isocratic elution was performed at a flow rate of 0.8 ml/min for 10 min (A:B = 15:85 v/v). The detection wavelength was 535 nm, the column temperature was 40 °C, and the injection volume was 10 μL. The authentic betanin was purchased from Shanghai yuanye (Shanghai yuanye Bio-Technology Co., Ltd, Shanghai, China). The standard curve and linear regression equations are provided in Supplementary Fig. S1.

    Results

      Screening the most suitable explants for non-tissue culture transformation

    • The selection of adaptive explants is crucial for non-tissue culture transformation. To identify the most suitable explants for transformation, we focused on the regeneration capabilities of different tissue parts of Mirabilis himalaica. We conducted experiments using three types of explants: root segments, stem segments, and leaves. The results indicated that after approximately 10 d, the stem segments successfully developed adventitious roots; however, they did not produce any regeneration buds even after 40 d. Root segments failed to produce either buds or roots. In contrast, the leaves of M. himalaica demonstrated robust regenerative potential, rooting and surviving within about 10 d. After approximately 30 d, adventitious buds began to emerge at the base of the petioles, with each petiole averaging 1.3 adventitious buds (Table 1). Furthermore, experiments investigating petioles of varying ages—mature and juvenile—showed that while both types of leaves could root, the mature petioles exhibited superior performance in both rooting and budding.

      Table 1.  Statistics on leaf regeneration and buds emergence of Mirabilis himalaica.

      Experiment No. of explants No. of buds Ratio of buds/explants
      I 114 148 1.30
      II 135 177 1.31
      III 100 132 1.32

      In comparison to other tissue parts used as explants, petiole leaves offer accessibility and convenience, which is particularly significant for large-scale genetic transformation, ensuring a reliable source of explants. In conclusion, the mature leaves of M. himalaica exhibited robust regeneration and budding capacity, making them ideal explants for the non-tissue culture transformation of M. himalaica.

      Establishment of transformation system using leaf petioles as explants

    • To establish a non-tissue culture transformation system using leaf petioles as explants (Fig. 1a), we employed A. rhizogenes K599 carrying the pYLTAC380H-CDGAeG construct, in which four betacyanin biosynthetic genes and an eGFP gene were assembled using the TGSII-UNiE system[19]. The petioles of M. himalaica were immersed in the infection solution and placed in a vacuum chamber for 5 min (Fig. 1b). Subsequently, we inoculated the leaf petioles with A. rhizogenes K599 (Fig. 1c) and inserted them into moist vermiculite for cultivation (Fig. 1d & e). Hairy roots were induced after approximately 10 d, and budding emerged after 30 d (Fig. 1f & g).

      Figure 1. 

      Genetic transformation of Mirabilis himalaica using the modified CDB protocol. (a) Petiolate leaves were cut and used as explants. The site of infection by A. rhizogene was enlarged. (b) The explants soaked in A. rhizogenes K599 suspension were then subjected to vacuum pressure. (c) Coating the cut sites with A. rhizogenes K599 layers from agar media. (d)−(e) Explants inoculated with A. rhizogenes K599 were cultured in soil. (f) The root of 10 d old explants. (g) Buds emerged after about 30 d. Scale bars: 1 cm.

      The red color of betacyanin was used to monitor transformation events. Compared to the wild type, the buds and roots of positive transformants of M. himalaica accumulated higher levels of betacyanin (Fig. 2a−d). To confirm the transformation of M. himalaica, we conducted genomic PCR to examine the presence of exogenous, rolB and rolC genes. As shown in Fig. 2e, four betanin biosynthetic genes, rolB and rolC gens were amplified in the transgenic M. himalaica plants. This indicates that the A. rhizogenes-mediated transgenic process has successfully inserted the target genes into M. himalaica genome. The leaves of transgenic M. himalaica plants are redder than those of the wild type (Fig. 2f). Simple extraction of betacyanin using water also indicates that the genetically modified M. himalaica may accumulate more betacyanin (Fig. 2g). To further confirm whether the genetically modified M. himalaica accumulates more betanin, we used qRT-PCR to detect the expression levels of genes involved in betanin biosynthesis and employed HPLC to measure the content of betanin. The expression levels of four betanin biosynthetic genes were significantly increased in the transgenic plants (Fig. 2h−k). The betanin contents in transgenic M. himalaica is 5−6 folds higher compared with that in the wild type (Fig. 2l & Supplementary Fig. S2). To analyze the genetic transformation efficiency using this procedure, we repeated the genetic transformation experiments three times, using 100 explants each time. The average rooting rate was about 89%, and the rate of positive roots (exhibiting red color) was also about 78%. However, the rate of positive buds was relatively low, at only 1.7% of the total explants (Table 2).

      Figure 2. 

      Genetic transformation of M. himalaica using betacyanin as a reporter. (a) & (b) The above-ground part of M. himalaica, showing the (a) non-transgenic, and (b) transgenic. White arrow indicates red bud with high betacyanin contents. (c) & (d) Roots of M. himalaica. White arrow indicates red roots with high betacyanin contents. (e) Genomic PCR detect four exogenous genes. M: marker; NC: negative control (sterilized water was used as templates); PC: positive control (plasmid pYL1300H-CDGAeG was used as templates); WT: wild type M. himalaica. (f) The betacyanin accumulated in pYL1300H-CDGAeG transgenic M. himalaica leaf. (g) The leaf of pYL1300H-CDGAeG transgenic M. himalaica (right) have higher betacyanin contents compared with wild type M. himalaica (left). (h)−(k) Relative expression levels of betanin biosynthetic genes by qRT-PCR in transgenic M. himalaica, respectively, compared with wild-type (WT) M. himalaica. All data represent the mean ± SD (n = 3; *, p < 0.001; Student's t-test). (l) Analysis of betanin in leaves of M. himalaica by HPLC.

      Table 2.  Statistics of M. himalaica transformation efficiency using betacyanin as a reporter.

      Experiment Explants Rooting explants Transgenic roots Transgenic plants Rooting rate Positive rooting rate Positive plant rate
      I 100 84 75 2 84% 75% 2%
      II 100 90 80 1 90% 80% 1%
      III 100 92 79 2 92% 79% 2%

      Establishment of transformation system using tdTomato as a reporter

    • Using betacyanin as a reporter requires at least three genes, which complicates plasmid construction and may also reduce transformation efficiency. In addition, many plants in Caryophyllales, such as M. himalaica, also produce betacyanin which might interfere the judgement. Therefore, we employed tdTomato as a reporter, an exceptionally bright red fluorescent protein that is about six times brighter than eGFP. We used A. rhizogenes K599 carrying a construct in which tdTomato was driven by the 35S promoter to infect Mirabilis himalaica leaves following the same protocol. The transgenic plants were checked using a portable fluorescent lamp (Fig. 3a−d). The transformants were further confirmed by genomic PCR (Fig. 3e). Further qRT-PCR results showed that the expression levels of tdTomato gene was increased in the transgenic plants (Supplementary Fig. S3). We conducted three repeated experiments as well, using 100 explants each time. The positive rooting rate and the positive plant rate were similar to those obtained using betacyanin biosynthetic genes, at 73% and 2%, respectively (Fig. 3f).

      Figure 3. 

      Genetic transformation of M. himalaica using tdTomato as a reporter. (a) & (b) Screening positive plants with a portable fluorescent lamp. Two representative plants growing closely together are displayed here. The plants enclosed in solid rectangular boxes are positive plants and arrow with solid line showed the fluorescent elicited by tdTomato protein. The plants enclosed in dotted rectangular boxes were negative transgenic M. himalaica and the arrow with dotted line indicated negative fluorescent signal. (c) & (d) The fluorescent of positive tdTomato transgenic plants. The roots enclosed in dotted rectangular boxes represented negative transgenic roots and the arrows with solid line showed the fluorescent elicited by tdTomato protein. (e) Genomic PCR detect the tdTomato, rolB and rolC genes in transgenic plants. M: marker; NC: negative control (sterilized water was used as templates); PC: positive control (plasmid Cotton 2.0-tdTomato was used as templates); WT: wild type M. himalaica. (f) Statistics of M. himalaica transformation efficiency using tdTomato as a reporter. Scale bar: 1 cm.

    Discussion

    • In this study, we developed a simple non-tissue culture transformation system for the Xizang folk medicinal plant Mirabilis himalaica using Agrobacterium rhizogenes-mediated genetic transformation technology. This method has been successfully applied to medicinal plants, as it overcomes many challenges associated with the Agrobacterium tumefaciens-mediated transformation method, including long transformation periods, rigorous sterile conditions, and limitations regarding species and genotype[15,18]. Due to the unique growth habits of plateau plants, a stable genetic transformation system has not yet been established for many Xizang medicinal plants[3]. In our study, leaves were first selected as explants. Subsequently, we successfully obtained transgenic Mirabilis himalaica via A. rhizogenes-mediated genetic transformation. This procedure is simple and does not require tissue culture. Furthermore, using leaves as explants is advantageous because they are the most abundant tissues in plants and are relatively easy to obtain. The establishment of this transformation procedure for Mirabilis himalaica lays a solid foundation for future gene function identification through gene overexpressing or editing[21,22]. Furthermore, the roots of M. himalaica can also serve as a chassis for synthetic biology, similar to the endosperm of rice[23].

      The reporter is crucial for monitoring genetic transformation, especially in non-tissue culture procedures. Unlike A. tumefaciens mediated tissue culture transformation, antibiotics are not the preferred selection markers for non-tissue culture transformation[12]. Pigments, such as betacyanin, and fluorescent proteins are the most commonly used reporters for the visualization of transformation[15,24]. However, plants belonging to the Caryophyllales order produce betacyanins pigments themselves. The endogenous betacyanin may interfere with the identification of transformants when betacyanin is used as a reporter. Thus, betacyanin is not a preferred reporter for Caryophyllales plants. In this study, we used tdTomato as an alternative reporter, which is smaller than the four betacyanin biosynthetic genes or the RUBY reporter. This simplification aids in plasmid construction and is suitable for large-scale genetic transformation. Nevertheless, the fluorescent signal was relatively weak in the leaves of transgenic M. himalaica in our study (Fig. 3b). The presence of pigments, such as chlorophyll and betacyanin, might hinder the fluorescent signal. Notably, betaxanthins, the intermediates of betacyanin, emit visible green fluorescence under blue light excitation[25]. More suitable fluorescent proteins should be screened to improve the visualization of transformations in Caryophyllales plants. An alternative reporter could be herbicide resistance genes, such as the BAR or EPSPS gene. For example, phosphinothricin (Basta) has been used for bulk selection of transgenic sweet potato[24].

      Our study demonstrated that the transgenic adventitious roots of Mirabilis himalaica could produce shoots. The method we used is similar to the recently reported cut-dip-budding (CDB) method[11]. Using this approach, transformants have been successfully obtained from several other plants, including two herbaceous plants (Taraxacum kok-saghyz and Coronilla varia), a tuberous root plant (sweet potato), and three woody plant species (Ailanthus altissima, Aralia elata, and Clerodendrum chinense)[11]. More recently, transformation systems for some medicinal plants, including Pugongying (Taraxacum mongolicum), Dihuang (Rehmannia glutinosa), Danshen (Salvia miltiorrhiza), and Yuanzhi (Polygala tenuifolia), have been established using this method[18]. Compared to the results reported by Cao et al.[11], the rate of positive plants in our study was relatively low (Fig. 3f, Table 2), averaging 2%. In addition to potential species differences, several factors should be considered in future studies to increase the conversion rate from roots to shoots, such as environmental conditions and plant hormones. Nonetheless, the establishment of a genetic transformation system for Mirabilis himalaica is beneficial and will facilitate the development of genetic transformation systems for other Xizang medicinal plants.

      Acknowledgments

      • This work was financially supported by the National Natural Science Foundation of China (Grant No. U20A20401). Thanks to Prof. Qinglong Zhu (South China Agricultural University, Guangzhou, China) and Prof. Lu Long (Henan University, Kaifeng, China) who kindly provided pYL1300H-CDGAeG and Cotton 2.0-tdTomato constructs.

      Author contributions

      • The authors confirm contribution to the paper as follows: study conception and design: Zhang F, Lan X; data collection: Sun T, Han X, Jiang Y, Li Q, Xu Y; analysis and interpretation of results: Zhang F, Han X; draft manuscript preparation: Lan X, Sun T. All authors reviewed the results and approved the final version of the manuscript.

      Data availability

      • All the data generated or analyzed during this study are included in this published article and its supplementary information files.

      Conflict of interest

      • The authors declare that they have no conflict of interest. Dr. Fangyuan Zhang is the Editorial Board member of Medicinal Plant Biology who was blinded from reviewing or making decisions on the manuscript. The article was subject to the journal's standard procedures, with peer-review handled independently of this Editorial Board member and the research groups.

      Supplementary information
      • Supplementary Table S1 Primers used in this study.
      • Supplementary Fig. S1 The standard curve between the HPLC peak area and betanin content (0, 1.5, 2, 3, 4, and 5 mg/ml). A linear regression was observed between the peak area and betanin contents at 0−5 mg (R2 = 0.9865).
      • Supplementary Fig. S2 The contents of betanin in pYL1300H-CDGAeG transgenic plants and wild type M. himalaica. All data represent the mean ± SD (n = 3; *, p < 0.001; Student's t-test).
      • Supplementary Fig. S3 Relative expression levels of tdTomato gene by qRT-PCR in tdTomato overexpression plants, respectively, compared with that in wild type (WT) M. himalaica. β-actin was used as an internal reference gene. All data represent the mean ± SD (n = 3; *, P < 0.001; Student's t-test).
      Rights and permissions
      • Copyright: © 2025 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (3)  Table (2) References (25)
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    Cite this article
    Sun T, Han X, Jiang Y, Li Q, Xu Y, et al. 2025. A method of genetic transformation of Xizang medical plants without tissue culture. Medicinal Plant Biology 4: e002 doi: 10.48130/mpb-0024-0032
    Sun T, Han X, Jiang Y, Li Q, Xu Y, et al. 2025. A method of genetic transformation of Xizang medical plants without tissue culture. Medicinal Plant Biology 4: e002 doi: 10.48130/mpb-0024-0032
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  • Introduction
  • Materials and methods

  • Plant material and growth conditions

  • Preparation of Agrobacterium rhizogenes

  • Plant transformation

  • Verification of the transgenic M. himalaica

  • Expression analysis of exogenous genes

  • Determination of betanin content

  • Results

  • Screening the most suitable explants for non-tissue culture transformation

  • Establishment of transformation system using leaf petioles as explants

  • Establishment of transformation system using tdTomato as a reporter

  • Discussion
  • Acknowledgments
  • Author contributions
  • Data availability
  • Conflict of interest
  • Supplementary information
  • Rights and permissions
  • About this article
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