Establishment of tissue culture regeneration system of Ficus tikoua

Ficus tikoua Bur. is an ecologically functional perennial vine that belongs to the 20 genus Ficus in the family Moraceae. Vitro cultures are considered crucial for F. tikoua 21 embryo's rapid propagation. The present study aimed to evaluate in vitro F. tikoua 22 organogenesis using stem explants. By comparing different hormones and their ratios, the 23 medium formula suitable for different stages of differentiation was screened, and the tissue 24 culture rapid propagation system was established. The results indicated that the survival rate 25 of explants could reach 82.22% after treating them with 0.1% HgCl 2 (plus TWEEN-20) 26 surface disinfection for 14 to 16 minutes. Explants were cultured on MS medium, 27 supplemented with different concentrations of Thidiazoron (TDZ) with 6-Benzylaminopurine 28 (6-BA) and Naphthaleneacetic acid (NAA) to study their efficacy in vitro shoot organogenesis. 29


Introduction
Ficus tikoua, belonging to the family Moraceae, is a perennial creeping evergreen woody vine (Fu et al. 2000) and is a multi-purpose plant that is used for food, medicine, afforest, and ornamental purposes.Due to its many branches, fast growth, and strong climbing ability, it is suitable as an excellent ground cover plant in landscaping (Yu et al. 2018).F. tikoua can grow extensively on the surface of bare mine wasteland, such as it has been found wildly growing in active and abandoned Sb mining areas (Li et al. 2017).Zhou et al. (2022) deduced that F. tikoua is a promising plant for the care of tailings wasteland revegetation.Owing to its strong adaptability to the environment, F. tikoua is considered an excellent ground cover plant for soil and water conservation (Ji et al. 2019).Furthermore, the whole plant of wild sweet potato can be used as medicine, and it has great value in development and utilization (Yang et al. 2020).However, most of the F. tikoua is obtained by digging directly from the field, which damages the wild resources seriously.Therefore, the artificial propagation of F. tikoua is of great significance (Huang et al. 2018).
Most ficus plants are propagated by cuttings.Although the cutting survival rate is high, this propagation method takes root slowly, rooting days are often more than 60 days.The reproduction coefficient is low, and a large number of rapidly reproducing seedlings cannot be obtained (Jiang et al. 2013).Micropropagation can greatly increase the reproduction coefficient, reduce incubation time, and mostly leads to the production of innumerable true-to-type plants (Mostafa and Sarmast 2018).F. tikoua has been less reported, but the genetic transformation via a series of callus induction has been reported in other species of Moraceae, such as F. altissima, F. pandurata, and F. carica (Lin et al. 2011;Liu 2014;Liu et al. 2010).Tissue culture techniques have been widely used for the rapid large-scale propagation of plants in a sterile and controlled environment (Muthali et al. 2022).Plant hormones are a crucial part of tissue culture, such as cytokinin and growth hormone, which are both essential for shoot formation (Velcheva et al. 2005).In tissue culture, proper cytokinin and growth hormone balance is key to controlling the root-to-crown ratio (Su et al. 2011).The main plant hormones used were 6-Benzylaminopurine (6-BA), 2,4-Dichlorophenoxyacetic acid(2,4-D), and Thidiazoron (TDZ), three cytokinins, and two growth hormones, Indole-3-butyric acid (IBA) and Naphthaleneacetic acid (NAA).
The most suitable medium formulations for each stage of F. tikoua growth were selected and the tissue culture system was established.It is of great significance to accelerate the breeding speed and promote its application in the future.In this experiment, the stem section of the F. tikoua was used as an explant, and the callus induction, bud differentiation, and rooting test was carried out by adding different exogenous hormones, thereby establishing a tissue culture rapid propagation system.

Plant material
Wild F. tikoua was introduced from Hubei province as experimental material and now is planted in the greenhouse of the nursery of Northeast Forestry University.The stem segments with good characters and no disease (with bud stem ) were selected as test materials.

Surface sterilization
The stems were thoroughly washed with detergent and then rinsed with running water for 30 minutes and flushed with sterile water 3~5 times.The explants were disinfected with 75% alcohol for the 30s and rinsed in sterilized water once.Transferred the material to 0.1% HgCl2 for 8, 10, 12, 14, and 16 minutes respectively, and rinsed in sterilized water 5~6 times.The stems were dissected to retain one node on each explant, each 2 cm in length.After 2 weeks, the pollution rate, death rate, and survival rate of different disinfection times were counted.

Adventitious buds multiplication
Adventitious buds multiplication was performed based on MS medium (with 0.5 mg/L TDZ) adding 6-BA (0.5, 1.0, 2.0 mg/L) and NAA(0.5, 1.0, 2.0 mg/L) to form an orthogonal test.The bud multiplication number was calculated after 15 days and observe the growth of seedlings.

Rooting and acclimatization
The advanceable buds (with 1~2 pair of new leaves) with a height of 2 ~3 cm were transferred to the rooting medium.And different concentrations of NAA (0.1, 0.5, 1.0 mg/L) and IBA (0.1, 0.5, 1.0 mg/L) were added to the basic medium of 1/2MS (with 1g/L activated carbon), to conduct orthogonal experiments.The rooting rate, average rooting number, and average root length were calculated after inoculation for 20 days.

Culture conditions
The light intensity was 2300 lx and the photoperiod was 16h light/8h darkness.Cultures were incubated at 25±2 ℃ and 50%~60% air humidity.All basic media were added 25 g/L sucrose and 7.8 g/LAGAR, and adjusted to pH 5.8 ~6.0.Each treatment consisted of 20 explants in triplicate.

Statistical analysis
Data processing was performed using Excel, DPS, and SPSS software for significance analysis and variance analysis.
3 Results and analysis

Effect of Mercury-disinfecting on the disinfection effect of stems at different time
The stem section of F. tikoua was disinfected with 0.1% HgCl2 (plus TWEEN-20).The highest survival rate was 82.22% when the sterilization time was 14min and 16min.The lowest mortality and contamination rates were 10% and 3.33%, respectively, at 14min and 16min of sterilization (Table 3).Compared with the sterilization time of 14min and 16min, the sterilization time of 10min had no significant difference, while the sterilization time of 8min had a significant difference.Therefore, the sterilization time can be 14 minutes or 16 minutes.

Effect of 6-BA, TDZ and NAA concentrations on callus induction of stem
The results of hormone induction are shown in Table 4 that both 6-BA and NAA had the greatest effect on stem-induced callus at level 1.The induction effect of TDZ was most obvious at level 3, and there was no significant difference between levels 2 and 3.The callus induction rate increased with the decrease of 6-BA and NAA concentration and the increase of TDZ concentration.
It can be seen from treatment group No.2 that when the concentration of 6-BA is 0.5 mg/L, the concentration of NAA is 1.0 mg/L, and the concentration of TDZ is 1.0 mg/L, the induction rate is up to 91.1% (Table 4).There was no significant difference compared with treatments No. 8 and 9, and there was a significant difference compared with the other groups (P < 0.05).
Further analysis of variance (Table 5), the Sig-value of the three hormones were all less than 0.01, indicating that all of them had a very significant effect on the callus induction of the stem node.And F-value from high to low was 6-BA >NAA>TDZ.6-BA played a major role in inducing callus in the stem segment.Therefore, among various combinations, the best callus formation was achieved in MS medium, supplemented with a combination of 0.5 mg/L 6-BA, 1 mg/L NAA, and 1 mg/L TDZ (Fig 1a) .

Effect of 6-BA, TDZ and 2,4-D concentrations on adventitious bud differentiation
As shown in Table 6, with the increase of the concentration of TDZ and 6-BA, the callus differentiation rate showed a trend of increasing first and then decreasing, and the effect on callus differentiation was greatest at the concentration of level 2. The callus differentiation rate is also affected by the concentration of 2,4-D.The greater the concentration of 2,4-D, the smaller the effect on callus differentiation.
The results of the orthogonal experiment were shown in Table 6.When TDZ concentration was constant, the callus differentiation rate increased with the increase of 6-BA concentration.The differentiation rate of callus in medium No.6 was the highest (61.11%), but not significantly different from that in medium No. 4. Therefore, further analysis of variance was conducted.
From the variance analysis (Table 7), we could see that the effect of TDZ and 6-BA on callus differentiation bud were significantly different (P< 0.05), but the difference of TDZ was more significant (P<0.01).While 2, 4-D had little effect on callus differentiation bud, with no significant difference (P >0.05).The F-value from high to low was TDZ>6-BA>2,4-D, the same conclusion can be drawn by comparing F-values.In this experiment, the best adventitious bud differentiation medium of Dioscorea was achieved in MS medium, supplemented with a combination of 2 mg/L 6-BA and 0.5 mg/L TDZ(Fig 1b).As can be seen from Table 8, when the concentration of 6-BA was 0.5 mg/L and 2.0 mg/L, the multiplication ratio increased with the increase of the NAA concentration.When the concentration of 6-BA was 1.0 mg/L, the proliferation factor first increased and then decreased with the increase of NAA concentration.In media No.5, the multiplication number of buds reached the maximum, which was 121 numbers.The newly proliferating buds grew well at this concentration.There was no significant difference between No.9 and 5 (117 numbers).In comparison, No.9 and 5 culture mediums have little difference.It indicates that when the concentration of 6-BA and NAA is the same, the formation of clumping buds is easy to be induced.A high concentration of NAA is not conducive to the increase of multiple cluster buds and the phenomenon of elongated leaves and vitrification appeared in the differentiated buds.Therefore, the optimal medium for shoot bud proliferation was MS medium inclouding 0.5 mg/L TDZ, 1 mg/L 6-BA and 1 mg/L NAA (Fig 1c).

Effect of IBA and NAA concentrations on adventitious bud rooting
According to Table 9, when NAA was added to the rooting medium alone, the rooting rate gradually decreased with the increase of concentration, and the average number of rooting increased first and then decreased.When IBA was added separately to the medium at concentrations of 0.1mg/L and 1.0mg/L, the rooting rate and the average number of rooting were zero.When IBA and NAA acted together, higher concentrations of IBA and NAA inhibited the rooting.The highest rooting rate was 68.52% and the average number of rooting was 10.67% in medium No.13, which was higher than that in medium with single excitation.
Therefore, the best medium to promote rooting was a combination of 1/2 MS (plus 1g/L activated carbon) with 1 mg/L IBA and 0.1 mg/L NAA (Fig 1d).

Discussion
Plant development can be manipulated by the addition of plant growth regulators at particular stages of growth or maturation (Phillips et al. 2019).For genetic improvement of the species， the major prerequisite is to develop an efficient and reliable plant regeneration system without compromising the genetic stability of the species/genotypes (Aggarwal et al.

2015).
The tissue culture of woody plants is more difficult than that of herbaceous plants because propagation techniques are complex and require more time and effort.F. tikoua is a plant of the genus banyan in the mulberry family, whose stem segment contains white milk.If the disinfection test before the tissue culture test is not complete, it will lead to the phenomenon of large areas of bacteria growth in the later period.Residual toxic HgCl2 on the explants will cause damage to the plants, resulting in increased mortality after disinfection.
Therefore, the determination of the optimal time for surface disinfection is more conducive to the exploration of hormone medium with different ratios.It was concluded that 0.1% HgCl2 (plus TWEEN-20) treatment for 14 ~16min was the best way to disinfect the stem nodes of F.

tikoua.
The ratio of auxin to cytokinin controls the direction of plant morphogenesis (Raisz 1965).Different kinds and proportions of hormones have different effects on plant growth and development.In tissue culture, various combinations of hormone concentrations should be screened step by step to determine the ideal culture conditions.Low concentrations of cytokinin promote the germination of F. tikoua, while high concentrations of cytokinin inhibit the growth of F. tikoua.When callus was induced in the stem segment of F. tikoua, 6-BA, NAA, and TDZ all have significant effects on callus induction in the stem segment.But the effect of 6-BA is more obvious.When the concentrations of 6-BA, NAA, and TDZ were 0.5 mg/L, 1.0 mg/L, and 1.0 mg/L, respectively, the callus induction rate was as high as 91.1%.At higher concentrations of growth hormone, dense brown guaiac tissue appeared at the base of F.
tikoua stem segments that severely affected the nutrient uptake of the plant, resulting in a reduced budding rate and poor growth.Therefore, during the primary culture of buds, the concentration of growth hormone needs to be reduced to inhibit the production of healing wounds to avoid the formation of healing wounds on the nutrient uptake of F. tikoua stem segments.
The orthogonal test of different species and mass concentrations of plant growth

Table 1
Orthogonal experimental design of stem-induced callus

Table 2
Orthogonal experimental design of callus differentiation adventitious bud

Table 3
Effect of Mercury-disinfecting on the disinfection effect of stems at different time

Table 4
Effect of different hormone concentrations on callus induction of stem

Table 6
Effect of different hormone concentrations on adventitious bud differentiation

Table 8
Effect of different concentrations of hormones on shoot bud proliferation

Table 9
Effect of different concentrations of hormones on adventitious bud rooting

Table 10
Effect of different matrix combinations on transplantation survival rate of tissue