Responses of “Benihoppe” strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro

Abstract The study was aimed to assess the effects of exposure of the “Benihoppe” strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro. The results showed that propagation and rooting were inhibited by lower (0.2, 0.5 mg/L) and higher (40.0 mg/L) concentrations of La3+, respectively. Propagation and rooting were promoted by a moderate concentration (1.0, 10.0, and 15.0 mg/L) of La3+, and a concentration of 1.0 mg/L was found to be optimal. Under 1.0 mg/L of La3+ treatment, certain trends associated with changes in the levels of hormones and 12 nutrients, with the exception of Ca, were observed during plant growth in vitro, and a greater balance was observed in the trends associated with changes in the amounts of elements at the rooting stage, as compared to the propagation stage. Furthermore, the elements or hormones with the highest effects on the two stages of propagation were Co and K (positive), and TDZ and Fe (negative), while those that affected the two stages of rooting were S and Zn ( positive), and IAA and Fe (positive). Our findings suggest that the use of 1.0 mg/L of La3+ is cost-effective, and can help achieve strawberry propagation and rooting in vitro.

Efeito do tratamento de lantanídeos na propagação rápida in vitro e no enraizamento do morango "Cara Vermelha" Resumo: O objetivo deste estudo é avaliar o efeito do tratamento de lantanídeos na propagação rápida in vitro e no enraizamento do morango "Cara Vermelha".Os resultados mostram que concentrações menores (0,2; 0,5 mg/L) e maiores (40,0 mg/L) de La 3+ inibem a propagação e o enraizamento do morango, respectivamente.As concentrações médias (1,0; 10,0 e 15,0 mg/L) de La 3+ promovem a propagação e o enraizamento, sendo 1,0 mg/L a concentração ideal.No tratamento com 1,0 mg/L Introduction Since the 1970s, scientists have studied the effects of rare earth elements (REEs) that act as microelement fertilizers on crop yield and found that REEs could promote germination and seedling growth in rice, wheat, barley, and vegetable oil crops (Xiong et al. 1998, Hong et al. 2000, Hong et al. 2003, Liu et al. 2006).Consequently, the agricultural uses of REEs, especially La 3+ , has received considerable attention in the last few decades.Lanthanum is the most abundantly occurring REE in the soil.Owing to its chemical properties resembling those of Ca 2+ and its higher charge density, the effects of La 3+ on plant growth were observed to be mainly calcium dependent.In the presence of La 3+ , the atomic percentages of Na, Mg, Cl, K, and Ca were reduced and those of Mn and Fe were increased in cucumber seedling leaves (Zeng et al. 2000).Furthermore, the activities of enzymes belonging to the reactive oxygen species (ROS) scavenging system,, such as superoxide dismutase (SOD), catalase (CAT), ascorbate-specific peroxidase (AsA-POD), and dehydroascorbate reductase (DHAR), could be increased via the treatment of wheat leaves with La 3+ ,which indicates that La 3+ can increase plant tolerance to environmental stresses (Zhang et al. 2003, Zhang et al. 2006, Xu et al. 2007).However, the extent of application of La 3+ to plants in vitro has been limited (Song et al. 2002, Guo et al. 2012).
The strawberry plant is now cultivated in all arable regions, as it yields an important fruit that is consumed in both fresh and processed forms (Félix et al. 2022, Aung et al. 2019) .During the past decade, the strawber-ry plantation area and output have improved by 31.6%and41.4%, and 0.3847 million hectares and 8.8613 million tons, respectively.In 2020, the strawberry plantation area and output in China were 0.1272 million hectares and 3.3367 million tons, which constituted 33.06% and 36.26% of the area and output observed worldwide, respectively (FAO, 2020).Several major strawberry plantations are located in the Shandong, Jiangsu, Hebei, Liaoning, Zhejiang, Sichuan, and Anhui provinces of China.Farmers have traditionally used stolons for the vegetative propagation of strawberry plants.However, this system of propagation is slow and has resulted in the spread of plant diseases.In recent years, the micropropagation of strawberries has been considered an appropriate method for the production of numerous homogeneous virus-free plants via meristem culture (Su et al. 2022, Zhang et al. 2017, Shu et al. 2019, Wang et al. 2021).
Because of the need for a higher number of strawberry seedlings derived via tissue culture and the potential value of La 3+ treatment for plant growth, we conducted an in vitro experiment to study the effect of La 3+ treatment on strawberry propagation and rooting.More specifically, the effects of La 3+ on the bud germination rate, propagation coefficient, fresh weight, plant height, leaf area, SPAD value, rooting coefficient, root surface area and root tips, trends in changes in TDZ and IAA levels, nutrient accumulation, and Lanthanum nutrient interactions were determined, in order to further assess hormone and nutrient uptake patterns under La 3+ treatment.

Plant material and experimental design:
The experiment was carried out in the plant tissue culture laboratory of the Agricultural College at Anhui Science and Technology University.The strawberry plantlet used in our experiments was of the "Benihoppe" variety and had been sub-cultured 4-5 times.LaCl 3 was obtained from Shanghai Zhanyun Chemical Co., Ltd.The basic media used for propagation and rooting were composed of MS + TDZ 0.1 mg/L + sucrose 30 g/L + agar 7 g/L (pH 6.0) and ¼ MS + IAA 0.1 mg/L + sucrose 20 g/L + agar 7 g/L (pH 6.0), to which LaCl 3 was added at concentrations of 0, 0.2, 0.5, 1.0, 10.0, 15.0, 20.0, and 40.0 mg/L.Five bottles were used for culturing three plantlets during every treatment process with three replicates.The average height of plantlet was about 1.5 cm, and they were cultured at a temperature of 26 ± 1 °C and light intensity of 1500-2000 lux for 12 h on a daily basis.

Sampling and measurement:
The rate of propagation and the number of buds were investigated after the plantlets were cultured for 30 and 45 d.The fresh weight was determined and the SPAD value was measured with an SPAD-502PLUS instrument at 45 d.The height and leaf area of plantlets were measured after they were cultured for 45 d using the LA-S Image Analysis System (Wanshen Detection Technology Co., Hangzhou, China).The number of roots was investigated after the plantlets were cultured for 15 d and 30 d, and root characteristics (length, surface area, root tips) were determined after the plantlets were cultured for 30 d using the LA-S Image Analysis System (Meng et al. 2021).
Chemical analysis: Analysis of La, macronutrient, and micronutrient levels: We introduced 0.1-0.2g of dry sample into the inner tank during PTFE digestion and performed digestion with 5 mL of nitric acid overnight.After covering the tank with the inner cover and screwing on the outer stainlesssteel cover tightly, the solution containing digestive enzymes was incubated with the sample at 80 °C for 2 h, 120 °C for 2 h, and 160 °C for 4 until the solution was almost completely dehydrated.The residues were washed into a 25-mL volumetric flask 3 times and fixed to scale with 1% HNO 3 .Tests were simultaneously performed using the blank solution.
Hormone levels: Fresh samples (5 ± 0.01 g) were crushed with liquid N 2 and then introduced into a 50-mL centrifuge tube with a plug.Then, 10 mL of acetonitrile and 2.5 mL of water were added to this mixture.After allowing the samples to soak in the solution for 10 min and shaking the contents using a tissue grinder for 15 min, 2.5 g of sodium chloride was added into the centrifuge tube.The contents were agitated for 10 min and centrifuged at 4000 rpm for 5 min.Then, the supernatant was filtered through a 0.22-µm filter membrane, and the solution was used for TDZ testing.
A fresh sample (0.5 ± 0.01 g) was used for IAA extraction.We added 10 times the volume of acetonitrile solution and internal standard (400 ng D-IAA) before extraction overnight at 4 °C in a 50-mL glass tube.After centrifuging the contents at 12000 g for 5 min, the supernatant was collected, and the sediment was dissolved in 5 volumes of acetonitrile solution for the second extraction process.Then, the supernatant was combined with 35 mg of C18 filler, and the mixture was shaken vigorously for 30 s, and centrifuged at 10000g for 5 min.The supernatant was blow-dried with nitrogen and re-dissolved in 400 µL methanol.Then, the liquid was filtered through a 0.22-µm filter membrane for IAA testing.The TDZ and IAA levels were quantified using a high-performance liquid chromatography system (Agilent 1260, USA) equipped with a mass selective detector (Agilent 6420A,USA).We performed the chromatographic separation of TDZ and IAA using the Acquity HSS T3 capillary column (2.1 mm × 100 mm, 1.7 µm) and the Poroshell 120 SB-C18 column (2.1 mm × 150 mm, 2.7 µm), respectively.All analyses were conducted at Nanjing Ruiyuan Bio-tech Co., Ltd.(China).

Multiple index analysis:
The multiple index (MI) is the ratio of the index (propagation coefficient, fresh weight, height, area, etc.) for strawberry tissues subjected to a certain treatment to that of the index for control tissues subjected to the same treatment.MI values < 1indicates a decrease in the index value and suggests an inhibitory effect.If MI values are equal to 1, it indicates that there are no effects, and if they are > 1, it indicates a net increase in the index value and suggests that a growth-promoting effect could be observed.

Four types of trends in changes in hormone,
La, and nutrient levels: Four trends were observed with regard to the changes in the levels of hormones, La, and nutrients during strawberry propagation and rooting in vitro after treatment with 1 mg/L of La 3+ .The "R" (rising) trend indicated that the concentration in the first stage was greater than that at the beginning and that the concentration in the second stage was greater than that in the first stage.
The "R-D" (rising to dropping) trend indicated that the concentration in the first stage was higher than that at the beginning, while the concentration in the second stage was lower than that at the first stage.The "D-R" (dropping to rising) trend indicated that the concentration in the first stage was lower than that at the beginning, and the concentration in the second stage was greater than that in the first stage.The "D" (dropping) trend indicated that the concentration in the first stage was lower than that at the beginning, and the concentration in the second stage was lower than that at the first stage.
Data analysis: DPS 7.05 statistical analysis software was used for data analysis.All results were recorded in terms of the arithmetic means with standard deviation calculated for three replicates.Differences were tested using ANOVA, followed by the Duncan test at a significance level of 0.05.Values with different letters refer to significant differences between the different treatments or different growth periods (p<0.05).
Furthermore, in order to distinguish between the levels of nutrients and hormones observed in plants treated with 1 mg/L of La 3+ and CK, important values were calculated using the following formula: (Value of indices with 1 mg/L of La 3+ treatment -Value of indices with CK)/ Value of indices with CK.

Result
Plant propagation and rooting with different concentrations of La 3+ La 3+ had significant effects on the propagation and rooting of strawberries in vitro (Figure 1, Tables 1 and 2).Compared to the control (treated with 0 mg/L of La 3+ ), the changes in the values for the propagation coefficient (30 d or 45 d), fresh weight, height, rooting coefficient (15 d or 30 d),and root tips were similar.La 3+ treatment (0.2-40 mg/L) was observed to "promote growth at low concentrations and inhibit growth at high concentrations".Under 1.0 mg/L of La 3+ treatment, the propagation coefficient (30 d or 45 d), plant height, rootingcoefficient (15 d or 30 d), and root surface area were the highest, and notable variations were observed, as compared to those observed with other treatments.There were notable variations in the fresh weight, leaf area, SPAD, root length, and root tips, compared to those of the control.When treated with 40.0 mg/L of La, no significant decrease was observed in the propagation coefficient (30 d or 45 d), fresh weight, plant height, leaf area, root length, and root tips, as compared to the parameters for the control.The MI (Figure 2) of the strawberry plant varied between 0.60 and 2.65 for the root tips (0.2 mg/L of La treatment) and root surface area (1.0 mg/L of La 3+ treatment) at all La 3+ concentrations (0.2-40 mg/L).
The MI values of 5, 3, and 7 out of 11 indices were lower than 1.0 at three concentrations of La 3+ (0.2, 0.5, and 40.0 mg/L), which demonstrated the inhibitory effects of La 3+ at these concentrations.In 4 out of 5 inhibiting indices, 3 out of 3 inhibiting indices could be observed in the bud propa-gation phase after treatment with 0.2 and 0.5 mg/L of La 3+ , while 7 out of 7 inhibiting indices could be observed both in the bud propagation and rooting phases after treatment with 40.0 mg/L of La 3+ .This indicates that treatment with low concentrations of La 3+ (0.2 or 0.5 mg/L) mainly negatively affected strawberry rooting in vitro, while treatment with high concentrations of La Hence, it can be assumed that treatment with La 3+ at a concentration of 1.0 mg/L was optimal for both strawberry bud propagation and rooting in vitro.The accumulation of La and hormones after treatment with 1 mg/L of La 3+ Notably different trends were observed in the changes in La accumulation during the propagation and rooting of strawberries in vitro after treatment with 1.0 mg/L of La 3+ (Figure 3).In general, the La concentration increased slowly at the first stage (30 d) and more rapidly at the second stage (45 d) of propagation, with the concentration at the latter stage (4.76 mg/kg) being ten times higher than that at the former stage(0.45mg/kg).The La 3+ concentration peaked (9.63 mg/kg) at the first stage of rooting (15 d), and then substantially declined to 3.48 mg/kg at the second stage of rooting (30 d).Hence, the "R" and "R-D" trends were observed during propagation and rooting, respectively.
In this experiment, TDZ and IAA were mainly used for the propagation and rooting of strawberries in vitro.TDZ is an exogenous cytokinin, and IAA is an endogenous auxin that has been synthesized by the plant or added during experiments.At two stages of propagation (30 d, 45 d) and rooting (15 d, 30 d) after treatment with 1.0 mg/L of La 3+ , the accumulation patterns for TDZ and IAA were notably different, compared to those observed for the control.The same "R-D" trend was observed for TDZ during both La treatment and for the control during propagation, though there were differences in concentration values, while the "R"trend was observed for IAA after La 3+ treatment, and the "R-D" trend was observed for IAA in the control.
The accumulation of macroelements after 1 mg/L La 3+ treatment Responses of "Benihoppe" strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro 7 the levels of S, P, K, Ca, and Mg were significantly higher than those observed with CK (30 d); the levels of S, P, and Mg were notably higher than those of CK (0 d), while the levels of K and Ca were significantly lower than those observed with CK (0 d).During the second stage of propagation (45 d), the content of Ca was notably higher than that observed with CK (45 d) and La (30 d), and substantially lower than that for CK (0 d).Interestingly, during the two stages of rooting, the same "D-R" trend was observed for the levels of five macroelements.No obvious difference was observed in the levels of Ca and Mg between CK (15 d) and La (15 d); the levels of Na were an exception.The accumulation of microelements after 1 mg/L La 3+ treatment The concentrations of microelements (B, Mn, Cu, Co, Zn, and Fe) during the propagation and rooting of strawberries in vitro have been shown in Table 4.As observed for the accumulation of macroelements during the rooting stage after either La 3+ and CK treatment, the same trends of change were observed for the levels of four microelements, i.e., the "R" trend was observed for Mn, and the same "D-R" trend was observed for B, Cu, and Co, while the "D" trend was observed only for B during propagation.When the same treatment was administered (CK or La treatment), the same types of changes in levels were observed for both Zn and Fe during both propagation or rooting, which was indicative of the synergistic effects of Zn and Fe on strawberry growth in vitro.

Importance of hormones and nutrients during the two stages of propagation and rooting
As shown in Figure 4, under 1.0 mg/L of La 3+ treatment, the levels of 10 out of 12 nutrients were increased, and TDZ accumulation was higher than that of the control at the first stage of propagation (30 d).During the second stage of propagation (45 d), only the concentrations of B, Mn, Zn, and Ca were significantly higher, while those of Na, Fe, and TDZ were significantly lower than those observed for the control.The most influential elements or hormones in the two stages of propagation were Co and K (both positive), and TDZ and Fe (both negative).During the first rooting stage (15 d), the levels of all 12 nutrients and accumulated IAA were more than those for the control, and for 7 out of 12 nutrients, IAA resulted in a more notable extent of accumulation.The ability of elements to be accumulated could be shown as follows: S>Zn>Fe>Na>B>P>IAA>Mg; the accumulation of IAA, Fe, Zn, Mn,Cu, and Mg was significantly increased, and that of K, B, S, and P was significantly decreased, as compared to the control at the second stage.Hence, the elements or hormones that mostly influenced the two stages of rooting were S and Zn (both positive), and IAA and Fe (both positive).
The above results showed that 1.0 mg/L of La 3+ treatment would result in a greater extent of accumulation of many nutrients at the first stage of propagation or rooting, as compared to the control.During the second stage, a substantial decrease in the concentrations of Fe and TDZ could probably contribute to increased bud propagation, and a notable increase in the concentrations of IAA and Fe could be beneficial for rooting (Figures 4,5).

Discussion Application of La 3+ in vitro
Lanthanum is a trivalent rare earth metal that has been used in pharmacological and electronic industries as well as in agriculture (Che et al. 2011, Chen et al. 2001, Sabine et al. 2005).The positive or negative effects of La 3+ on plants were observed mainly via the culture process or field experiments in many countries, and applications involving its use for commercial purposes were developed gradually.China was the first country to use commercial REE-based fertilizers (more than 25% of La 3+ was included in the "CHANGLE"REE fertilizer) for crop production; this resulted in an increase in yield of more than 5% (Zheng et al. 2004, Xiong et al. 1998, Xiong et al. 2000).
The findings of several studies have shown that seed germination, plant growth, yield, and many related indices were promoted by low concentrations of La 3+ and inhibited by high concentrations of La 3+ (Xie et al. 2002, Zheng et al. 1993, Liu et al. 1996).For example, in comparison to CK, the germination rate, germination index, dry weight Responses of "Benihoppe" strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro 9 per seedling, and vigor index of rice were significantly restrained upon treatment with 1500μg/mL of La 3+ , while they were improved upon treatment with low concentrations of La 3+ (100-500μg/mL).Another experiment showed that the application of La 3+ increased the total aboveground biomass of rice seedlings, and treatment with La 3+ at concentrations of 80 and 100 mg kg −1 and 12 kg ha −1 greatly increased the 2-AP (by 6.45-43.03%)and mature grain levels, compared to those of the control (Luo et al. 2021).Furthermore, La also exhibited beneficial effects under many different types of stresses.The treatment of young seedlings subjected to phosphorus deficiency stress with La 3+ at 150 mg L −1 effectively improved phosphorus-use efficiency (PUE) in the roots, stems, and leaves of Vigna angularis seedlings via the regulation of root elongation and activation of root activity and acid phosphatase (APase) activity (Lian et al. 2019).Additionally, when rice seedlings were treated with an adequate concentration of La 3+ (0.06 mmol L −1 ) under acid rain stress (pH 3.5 and 2.5), an increase in the plasma membrane H+-ATPase activity and a decrease in the relative growth rate were observed, while the application of 0.12 mmol L −1 La 3+ resulted in a synergistic interaction with acid rain (Liang et al. 2018).Under cadmium (Cd) stress, La 3+ could be used as a regulator to improve the Cd tolerance of maize by alleviating Cd-induced oxidative damage (Dai et al. 2017).Further, another experiment concluded that the La-mediated decrease in Cd accumulation in wheat was probably a consequence of both the decreased Cd uptake due to the down-regulation of TaNramp5, and reduced root-to-shoot uptake resulting from the down-regulation of TaHMA2 (Yang et al. 2019).
Such experiments were carried out in vivo, while hardly any information was available about the plant response to La 3+ in vitro, which has limited the use of this plant in the huge plant tissue culture market.The large-scale consumption of strawberries in China has necessitated the development of a large number of tissue culture seedlings that could be used by farmers.Hence, 7 concentrations of La 3+ were used in the propagation and rooting medium of strawberry plants in vitro, and their effects were assessed in this study.The results showed that a concentration of 1.0 mg/L of La 3+ was optimal for both the propagation and rooting of strawberries in vitro, based on the fact that the values of 11 out of 12 indices tested after treatment with 1.0 mg/L of La 3+ were significantly better than those of the control.With regard to the core indices, under 1.0 mg/L of La 3+ treatment, the propagation coefficients of strawberry plants increased to 13.73 (30 d) and 19 (45 d), and the values of the rooting coefficients were up to 5.93 (15 d) and 13.18 (30 d).These values were notably higher than the values observed with other treatments.La 3+ treatment at lower concentrations (0.2 and 0.5 mg/L) or higher concentrations (40.0 mg/L) have more or less negative effects, indicating that 1.0 mg/L of La 3+ was a cheap and useful exogenous additive that could facilitate propagation and rooting in the "Benihoppe" strawberry.
Uptake and distribution of La, hormones, and nutrients Some studies have focused on the effects of REEs on La and nutrient uptake in plants, including the changes in content and distribution (Wang et al. 2011, Han et al. 2005, Yuan et al. 2017, Liu et al. 2022).For example, under La 3+ treatment (0-8 mg/L) conditions, the La content increased and K content decreased in both the roots and leaves of V. faba L. seedlings.In addition, the levels of Mg, Mn, Ca, Fe, Zn, Na, and P in the roots changed in an inversely proportional manner with an increase in extraneous La 3+ levels, as compared to the levels in the leaves, indicating the imbalance and redistribution of these elements in different organs.Besides, when Ce 3+ and Tb 3+ were used as test REEs, to investigate their effects on horseradish, the distribution behaviors of mineral elements (K, Ca, Mg, Cu, Mn, and Fe) and heavy metals (Cd, Pb, and Cr) could be affected by the type and concentration of REEs and the growth of the plant (Wang et al. 2008).In this experiment, four trends were observed in the changes in the levels of La and 12 nutrients during propagation and rooting.Overall, Ca was the only element for which the "R-D" trend was observed after La 3+ and CK treatment during both propagation and rooting, along with a substantial decline in the Ca levels from 15.74 g/kg at the start of the experiment to 6.10 g/kgat the end of these processes, indicating that the manner in which Ca was utilized during propagation and rooting was the same.The "R" trend and "R-D" trend were observed during propagation and rooting, respectively, with regard to changes in the content of La (Figures 3, 5).Furthermore, during propagation, three macroelements, i.e., Mg, P, and Ca, and one microelement, i.e., B, showed the same types of changes in content after treatment with both1.0mg/L of La 3+ and CK, while a total of 9 elements out of 11, including 5 macroelements (P, K, S, Ca, and Mg) and 4 microelements (B, Cu, Co, and Mn), exhibited the same trend of changes in content when treated with 1.0 mg/L of La 3+ and CK, suggesting that a greater extent of imbalance in the trends associated with changes in levels could be observed during the propagation stage, as compared to the rooting stage (Figure 5).Responses of "Benihoppe" strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro 11 The response of plants to La 3+ involves complex physiologic and biochemical processes, including changes in the concentration and distribution of hormones.During the germination stage of the rice seed, low concentrations of La 3+ (20-900 μg/mL) resulted in an increase in the levels of IAA, GAs, and CTK, and treatment with 1200-1500 μg/mL La 3+ resulted in contrasting results.In the presence of 0.1 or 1 mM La 3+ , the ABA content increased by 32% and 87% in the intact roots of corn plants, and decreased by 50% and 60%in protoplasts; thus, contrasting effects were observed (Liu et al. 2008).The entire strawberry plant showed continuous IAA enrichment during the rooting stage under treatment with1.0 mg/L of La 3+ , which suggested a positive relationship between La 3+ and IAA accumulation.Though the same "R-D" trend was observed for TDZ, trends in the changes in levels were different upon treatment with 1.0 mg/L of La 3+ and CK during propagation.These changes indicated that the difference in the changes in levels of TDZ during propagation and continuous accumulation of IAA during rooting upon treatment with 1.0 mg/L of La 3+ would be beneficial for plant growth.

Relationship between La and the metabolism of mineral nutrients and hormones
It has been speculated in many studies that La was analogous to Ca due to the similarities in their characteristics.Because La had the same binding sites as Ca, and it could inhibit the efflux of extra-cellular Ca 2+ , partially inhibit the generation of intracellular Ca 2+ , and replace Ca in many enzymatic reactions observed during the treatment process or in certain organs (Brown et al. 1990, Li et al. 2011, Hu et al. 2004, Wang et al. 1998).We conducted an experiment in which a low concentration of La 3+ (1 mg/L) could substantially promote Ca accumulation in strawberry plants, compared to that observed in the control during the propagation stage, while the Ca accumulation resulting from La 3+ treatment during rooting occurred to a greater extent, but no significant differences were observed, as compared to the control.
La influences the ionic fluxes in organs or cells in different ways.The conflicting results shown above indicate that it was challenging to understand the metabolism of mineral nutrients and hormones in plants due to the plants being of different types, including C3 or C4, monocotyledons or dicotyledons, and annual or perennial plants, and because of the differences in the treatment durations and analytical methods used during experiments.

Conclusion
Our findings revealed that the use of La 3+ at a concentration of 1.0 mg/L was optimal for "Benihoppe" strawberry growth in vitro, as it resulted in the highest bud and root induction coefficients during different stages of propagation and rooting, while lower (0.2, 0.5 mg/L) and higher (40.0 mg/L) concentrations of La resulted in higher or lower inhibitory effects, respectively.Under 1.0 mg/L of La treatment, the concentrations of La, hormones, and 12 nutrients were found to be varied during plant growth in vitro, with the exception of Ca, and a greater balance was observed in the levels of elements in the rooting stage than in the propagation stage.We concluded from the findings that the regulation of growth of the "Benihoppe" strawberry in vitro with 1 mg/L of La 3+ treatment was complicated and was affected by four trends associated with changes in the levels of La, hormones, and nutrients, of which the first two influential elements or hormones in different stages of propagation were Co and K (both positive), and TDZ and Fe (both negative) and those in the two stages of rooting were S and Zn (both positive), and IAA and Fe (both positive).

Figure 1 -
Figure 1-Variations in the propagation(left) and rooting(right) of strawberry plants treated in vitro with different concentrations of La 3+ .
3+ (40 mg/L) negatively affected both strawberry bud propagation and rooting in vitro.With these exceptions, the MI values of all Responses of "Benihoppe" strawberry (Fragaria×ananassa Duch.) to La3+ treatment during propagation and rooting in vitro Zhang et al. (2023) 11 indices were higher after treatment with three concentrations of La 3+ (1.0, 10.0, and 15.0 mg/L), as compared to those of the control, indicating that La 3+ treatment resulted in growth-promoting effects at these concentrations.The MI values of 10 out of 11 indices (with the exception of the leaf area) were larger than those observed at two other La 3+ concentrations (10.0 and 15.0 mg/L) under 1.0 mg/L of La 3+ treatment.

Figure 2 -
Figure 2-Multiple index (MI) of strawberry plants in vitro after treatment with different concentrations of La 3+ .

Figure 4 .
Figure 4. Important values of nutrients and hormones in strawberry plants treated with 1 mg/L of La 3+ and CK.The higher the value, the greater the degree of importance (positive or negative) of hormones and nutrients

Figure 5 -
Figure 5-The hypothesis of growth regulation in strawberry plants in vitro upon treatment with 1 mg/L of La 3+ based on four types of trends associated with changes in the levels of hormones and nutrients.Italic letters indicate the same trends of changes in the propagation and rooting stages with the same treatment, while underlined letters indicate the same trends associated with changes after treatment with La 3+ and CK during propagation or rooting; arrows pointing up or down are indicative of values that are higher or lower than the value of CK.
La3+ treatment during propagation and rooting in vitro

Table 1 .
Effects of treatment with different concentrations of La 3+ on the propagation of strawberry in vitro.

Table 2 .
Effects of treatment with different concentrations of La 3+ on the rooting of strawberry in vitro.

Table 3 -
Effects of 1 mg/L of La 3+ treatment on macroelements in strawberry plants in vitro.

Table 4 -
Effects of treatment with 1 mg/L of La 3+ on microelements in strawberry plants in vitro.
During the second stage of rooting, the levels of Mn, Zn, and Fe were 13.55, 132.39, and 311.82 mg/kg, respectively.These levels were double of those observed with CK (0 d), which suggested that increased amounts of Mn, Zn, and Fe were required during the rooting process.