An ecient in vitro regeneration system via callus from hypocotyl and root of Iris laevigata

is an ornamental plant with strong cold resistance. However, its low reproductive capacity limits its landscape applications. The I. laevigata wild genetic resources also need to be protected. In order to develop an effective regeneration system, the optimum agar concentration for the induction medium was determined. Two explants (hypocotyl and root) were then cultured on medium containing different concentrations of plant growth regulator (PGR). In addition, three antibiotics were evaluated for controlling endophyte contamination. 1 2,4-D g L 1 agar 7 g L − callus 6-BA 0.5 mg L − 1 + 2,4-D mg L − 1 + NAA 0.4 mg L 1 sucrose g L 1 agar 7 g L optimum callus 1 2,4-D , hypocotyl g L root differentiation rate 6-BA L g L 1 agar g L − most for I. laevigata callus penicillin G 100 mg L − results advantageous for the micropropagation and commercial horticultural application Each treatment was repeated three independent times. All data represents the mean value and standard error of three replicates. Data analyses were performed using SPSS 22.0 software. One-way analysis of variance (ANOVA) was used to determine the signicance of the various effects studied. Duncan’s Multiple Range Test was used for pair-wise comparison of the data.


Conclusions
The research determined the optimum PGR concentrations for inducting and multiplying I. laevigata calli from hypocotyl and root explants and a satisfactory means for control endophyte contamination. This research will result in the e cient and reliable reproduction of I. laevigata for landscape applications, genetic development of new Iris varieties, and preservation of the wild genetic material. Background I. laevigata is a perennial herbaceous owering plant of Iridaceae, which has attracted increasing attention in recent years. In the wild, it is primarily found along rivers, in marshes and wetlands in China, Japan, South Korea, and Russia [1,2]. Due to its gorgeous blue owers, which are larger than many Iridaceae species, it is regarded as an aquatic plant with high ornamental value [3]. It has a long history of cultivation, and a common ornamental plant in northeastern China, and many renowned botanical gardens around the world [4]. Because it has outstanding adaptability, it can be used in both land and water landscaping. In addition to its landscape applications, I. laevigata is also the parent of many popular horticultural varieties, and, therefore, considered a signi cant genetic resource for iris breeding.
Because of its native ecological origins, it has a strong tolerance for cold and waterlogged conditions. These physiological characteristics make it a valuable germplasm resource for developing new ornamental iris varieties, which will be adapted to waterlogged and cold conditions in landscaping areas [5][6][7][8].
Under natural conditions, Iris species reproduce by two methods: asexually by splitting bulbs or rhizomes, and sexually by seeds. However, neither of these two methods can achieve large-scale and e cient reproduction. Ramets not only have a low reproduction coe cient, but also cannot guarantee uniform seedling quality. In addition, researchers [9,10] continue to encounter consistent challenges to reproduce I. laevigata plants via seed germination. Furthermore, the increasing loss and damage of I. laevigata habitats pose a critical challenge to the survival of native wild populations. It has been de ned as one of the second grade endangered species in South Korea [11].
Micropropagation is a method to regenerate new plants that can be used rapidly for ornamental horticulture applications. It is also a viable strategy for increasing the number of individuals ex-situ in order to save endangered species [12]. Moreover, the establishment of a callus regeneration system is also one of the basic conditions for plant genetic transformation and further development of plant genetic resources. Reliable callus regeneration systems have been developed for several iris species with high horticultural value or in danger of extinction [13][14][15][16][17], however, there is not a reliable callus regeneration system for I. laevigata. Therefore, we are committed to developing a callus regeneration system for I. laevigata that will help promote its horticultural applications and natural resource protection.
Endophytes often affect the growth of explants and calli in plant tissue cultures and their impact varies depending on the plant species [18][19][20]. At the beginning of the experiment, we discovered that I. laevigata calli were often contaminated by transparent and sticky endophytes in the multiplication medium. If the endophytes were left untreated, the calli would gradually become mushy or turn brown and die, which obviously seriously reduced the callus regeneration e ciency.
In this research, two explants, hypocotyl and root, were used to develop a suitable medium for callus induction, proliferation, and differentiation. The survival rate of regenerated plants was used to evaluate the experimental treatments. In addition, three broad-spectrum antibiotics were tested to develop an effective endophyte inhibition method for I. laevigata micropropagation. As a result of the research, an e cient and reliable callus regeneration system for I. laevigata was successfully developed.

Results
Effect of agar content in medium on hypocotyl callus induction Hypocotyls were harvested from 20 d old sterile I. laevigata seedlings and used as explants to induce calli in media with different agar concentrations. The callus induction rate, endogenous bacterial contamination rate, and callus appearance were recorded after 30 d ( Table 1). The medium callus induction rate with 6 and 7 g L − 1 agar was signi cantly greater than that of other treatments (4 and 10 g L − 1 ). The calli induced on the 4 g L − 1 agar medium were severely contaminated with endophytes (66.67 ± 8.82%), while the contamination rate for other medium formulations were signi cantly less and not signi cantly different from each other.
The agar concentration also affected the appearance and moisture levels of the calli (Table 1). As the agar content increased from 4 to 10 g L − 1 , the calli transformed from soggy/mushy to extremely dry.
When the agar content was low (4 g L − 1 ), the calli were soggy and became mushy. At 6 g L − 1 , the calli contained less moisture, but remained soggy. The calli appeared ideal, dry and granular, at 7 g L − 1 , which also produced a high, 56.67%, induction rate and lower (20%) contamination rate. In contrast, the 10 g L − 1 agar produced calli that were extremely dry with a low induction rate (23.33%). As a result, it was determined that adding 7 g L − 1 agar to the medium produced the most suitable callus formations for I. laevigata hypocotyl explants.  Fig. 1A). Calli were formed at the incision at the top and bottom of the hypocotyl as well as at the inevitable incisions on the hypocotyl during the experimental operation (Fig. 1B). Treatment 2's callus induction rate was 75%, and the induced calli were in good condition (Table 2). Therefore, the optimal medium for I. laevigata  As the 6-BA concentration increased from 0.5 mg L − 1 to 2.0 mg L − 1 , the mean induction rate decreased (Table 2; Fig. 2). As the 2,4-D concentration increased from 0.5 mg L − 1 to 1.5 mg L − 1 the mean value of the induction rate rst signi cantly increased and then decreased. The impact of NAA concentration followed the same general trend as 2,4-D, which initially increased and then decreased as NAA concentration increased, although the differences were not signi cant (Fig. 2). Therefore, it was determined that the most suitable phytohormone combination was 6-BA 0.5 mg L − 1 + 2,4-D 1.0 mg L − 1 + NAA 0.4 mg L − 1 , which produced a 75% callus induction rate (Table 2). Furthermore, according to the range analysis (Table 2), the in uence of the PGRs on hypocotyl callus induction are ranked as 6-BA > 2,4-D > NAA. The analysis of variance (Table 3) indicated that 6-BAP was the most signi cant factor involved in the hypocotyl induction rate (P < 0.01), while 2,4-D affected the induction rate to a lesser extent (P < 0.05), and NAA had relatively little in uence (P > 0.05). To determine the impact of 3 phytohormones on I. laevigata root callus induction, 2 types of I. laevigata root explants were investigated. One set of root explants had no brous roots, while the second root explants had the brous roots removed. Root explant segments without brous roots did not produce any calli, independent of the phytohormone concentration in the growth medium. Therefore, this manuscript will only discuss the callus induction rate from root explants which the brous roots were originally attached but removed ( Fig. 3; Table 4).
Explant root segments which originally had brous roots were collected from sterile I. laevigata seedlings (50 d old) and placed in media containing 3 different phytohormones (6-BA, 2,4-D, and NAA). Table 4 shows the callus induction rate and callus appearances resulting from the various PGR concentrations. When the 6-BA and NAA concentrations were held constant, the root callus induction rate increased as the 2,4-D concentration increased, resulting in a single peak curve (Table 4; Fig. 3). A single peak curve was also produced when the 6-BA and 2,4-D concentrations were held constant and the NAA concentration increased (Table 4; Fig. 3). The greatest callus induction rates for root explants were produced when the media contained 2,4-D at 0.5 mg L − 1 and NAA at 0.4 mg L − 1 with either 0.5 or 2.0 mg L − 1 6-BA (Table 4; Fig. 3). When the media did not contain 6-BA (treatment 1, 0 mg L − 1 ) there was no (0%) callus induction for the root explants (Table 4). When either 2,4-D or NAA were absent from the media (treatment 5 and 8), the callus induction rates were very low, 6.67% and 3.33%, respectively. As a result, it is clear that although 6-BA had the most signi cant impact on root callus induction rates, the presence of low rates of 2,4-D (0.5 mg L − 1 ) and NAA (0.4 mg L − 1 ) were essential for maximizing callus induction (Table 4). Therefore, to produce maximum callus induction rates with I. laevigata root explants, the medium must contain the 3 PGRs (6-BA, 2,4-D, and NAA) rather than just 2 PGRs. Although callus induction rates for treatment 2 (6-BA 0.5 mg L − 1 + 2,4-D 0.5 mg L − 1 + NAA 0.4 mg L − 1 ) and treatment 3 (6-BA 2.0 mg L − 1 + 2,4-D 0.5 mg L − 1 + NAA 0.4 mg L − 1 ) were not signi cantly different, 73.33% and 66.67%, respectively, treatment 2 required less 6-BA and produced numerically greater callus inductions at 45 d ( Table 4). As a result, the author's recommend the following medium formulation for I. laevigata root callus induction: 6-BA 0.5 mg L − 1 + 2,4-D 0.5 mg L − 1 + NAA 0.4 mg L − 1 + MS + sucrose 30 g L − 1 + agar 7 g L − 1 .

Effects of different PGRs and concentrations on root callus multiplication
The callus was inserted into the multiplication medium of different PGRs concentrations for 45 d ( Table 5; Fig. 4).
Interestingly, there were no signi cant differences in the endophyte contamination rates among the experimental groups with different PGR content. All PGR combinations resulted in multiplication rates equal to or greater than 21.67%, with the greatest numerical value of 73.33%. The multiplication rates were inversely proportional to the NAA concentrations. As 6-BA and 2,4-D were held constant at 0.5 mg L − 1 and the NAA concentration increased from 0.2 to 0,.6 mg L − 1 , the multiplication rate decreased from 73.33-28.33%. At the same NAA concentration (0.4 mg L − 1 ), the multiplication rate was greater at the lowest (0.5 mg L − 1 ) 6-BA and 2,4-D concentrations. The two highest multiplication rates were at the two lowest PGR concentrations, treatment 1 and 2. As a result, it was decided that treatment 1 (6-BA 0.5 mg Page 8/25 L − 1 + 2,4-D 0.5 mg L − 1 + NAA 0.2 mg L − 1 + MS + sucrose 30 g L − 1 + agar 8 g L − 1 ), with a multiplication rate of 73.33%, was the most suitable medium formula for root callus multiplication.

Effects of different PGRs and concentrations on hypocotyl and root callus differentiation
Three media formulations were evaluated to determine their impact on hypocotyl callus differentiation ( Table 6). The greatest differentiation rate (39.72%) was achieved after 60 d with a medium containing IBA 0.5 mg L − 1 + 6-BA 1.5 mg L − 1 + NAA 1.0 mg L − 1 + MS + sucrose 30 g L − 1 + agar 8 g L − 1 (Table 6).
These results con rm that IBA is signi cantly involved in hypocotyl callus differentiation. No adventitious shoots were produced without adding IBA to the media and the resulting shoots were green and strong when 0.5 mgL − 1 IBA was added to the media.  Within a certain concentration range, the induction rate was directly proportional to the 6-BA and KT concentrations, and inversely proportional to the concentration of NAA (Fig. 5). According to the range analysis (Table 7), the in uence of each factor was ranked as 6-BA > KT > NAA. The analysis of variance indicated that both 6-BA and KT had a signi cant impact on the differentiation rate (P < 0.01), while NAA had relatively little in uence (P > 0.05) (Table 8). Therefore, future research should investigate the use only the two PGRs, 6-BA and KT, for I. laevigata root differentiation medium.

Rooting and transplanting of adventitious shoots
The adventitious shoots were multiplied, then pruned to a suitable height, and placed in the rooting culture medium. After 30 d, the regenerated seedlings were cultured in water and then transplanted to potting soil. The potted seedlings were then placed in a greenhouse for 30 d and maintained a 90% survival rate.
The impact of three antibiotics on the control of endophytes in calli During the experiment, it was found that the endophytes severely inhibited the induction and multiplication of calli. Therefore, we investigated the impact of adding different combinations of penicillin G (50, 100, 200, 300 mg L − 1 ), carbenicillin disodium (50, 100, 300, 500 mg L − 1 ) and cefotaxime sodium (50, 100, 300, 500 mg L − 1 ) to media to control endophytes.Each antibiotic had a positive impact on endophytic bacteria control (Fig. 6). The penicillin G bacteriostatic rate was signi cantly greater than the other two antibiotics at all concentration levels (Fig. 6). The inhibition rate increased gradually as the penicillin G concentration increased and reached the greatest inhibition (54.29%) at the 300 mg L − 1 concentration (Fig. 6A).
Besides controlling the endophytes, antibiotics also have the potential to negatively impact plant materials and cause callus browning and death. Each of the three antibiotics resulted in a varying degrees of browning (Fig. 6B). Overall, penicillin G produced the least browning compared to other two antibiotics, carbenicillin disodium and cefotaxime sodium. The lowest two penicillin G concentrations, 50 and 100 mg L − 1 , produced the lowest browning rates. As the penicillin G concentration increased from 100 to 300 mg L − 1 , the browning rate signi cantly increased (Fig. 6B). Therefore, penicillin G was selected as the superior antibiotic to both control endophytes and limit callus browning ( Fig. 6A; 6B). The addition of penicillin G at 100 mg L − 1 provided the best balance between inhibiting endophytes and limiting browning ( Fig. 6A; 6B; 6C).

Discussion
In tissue culture, the concentration of gelling agent has an important effect on the induction rate and callus growth [21][22][23]. Differences in gel strength, mineral content, and inhibition compounds can also in uence callus growth [24]. This research evaluated the effect of agar content on the growth of explants and calli. As the agar content increased, the media strength and the degree of drying increased. In addition, as agar content increased, the induction rate increased and the callus granulation became more obvious. Interestingly, in the process, the contamination rate decreased. The greatest callus induction rate was achieved (56.67%) when the agar content reached 7 g L − 1 . However, when the agar concentration increased to 10 g L − 1 , the medium became too dry, which resulted in a decrease in the callus induction rate. Other researchers [21,25] have shown that the optimal agar concentration will change according to the species and genotype. In our research we determined that the optimum agar concentration for I. laevigata callus induction was 7 g L − 1 . Furthermore, different kinds of gelling agents, such as gellan gum [26] and phytagel [23], can also result in differences in plant regeneration.
The source of explants has a decisive in uence on the ability and e ciency for in vitro regeneration.  [32] reported that Brassica oleracea var. capitata calli were induced with brous root explants (15,20, and 25 days old). Initially, the calli appeared at the incision sites at both ends of the root segment, and then the calli were induced over the entire explant. The brous root explants produced the greatest induction rate (96.00%) at 20 d [32]. In our research, the thicker adventitious roots were selected as explants on which the thinner brous roots were excised. Unlike Li et al. [32] research, the I. laevigata root induced calli appeared at only one root end (Fig. 7A), but did produce calli at the brous root excise locations along the root (Fig. 7B). It is speculated that the roots used for root explants were too old (30 d) after aseptic seedling process, therefore, resulting in the decline callus induction. However, the brous root explants were more tender and readily resulted in calli.
The types, concentrations, and combinations of plant growth regulators play an important role in plant regeneration [33,34]. Moreover, even for the same plant species, different explants respond differently depending on the type and level of PRGs [35]. Analysis of variance for the hypocotyl induced callus results indicated that 6-BA and 2,4-D were relatively important factors affecting the induction rate, while NAA had little effect. However, when root segments were used to induce calli, the induction rate was very low if only two PGRs (6-BA, 2,4-D or NAA) were used. The combination of the three PRGs was more conducive to callus induction than any combination of two PRGs. In the callus differentiation culture, NAA had little impact on the differentiation of root induced calli among the three PRGs (6-BA, KT, and NAA). Additional research should be conducted with only 6-BA and KT, and other PGR combinations and concentrations to increase the differentiation rate for root induced calli.
Also, the hypocotyl induced calli did not differentiate when 6-BA 1.0 mg L − 1 and NAA 0.4 mg L − 1 were added to the medium, but the differentiation rate increased when IBA 0.5 mg L − 1 was added. Obviously, IBA played a key role in the differentiation of adventitious shoots. Similar results have been shown concerning auxins being crucial for adventitious shoot differentiation [36,37]. This is because auxins can rapidly regulate cytokinin synthesis [36,37]. In conclusion, the balance between culture medium PGRs and PGRs in explants and calli are consequential.
Endophytes that would not cause a disease when living in plant tissue [38], can often lead to explant browning and necrosis in calli in plant regeneration systems [19,20]. It is well established that adding antibiotics to the culture medium is an effective method to eliminate endophyte contamination in plant tissue cultures [20,39,40]. In this research, we studied the impact of three antibiotics (penicillin G, carbenicillin disodium, and cefotaxime sodium) on endophyte contamination. We determined that adding penicillin G 100 mg L − 1 to the culture medium was the best strategy to inhibit endophyte contamination Although, we did not identify the endophytes involved in our research, researchers often use targeted antimicrobial agents to control know endophyte species [39,41]. For example, Han et al. [42] determined that Staphylococcus epidermidis was responsible for the contamination of I. germanica in micropropagation. Future research should investigate which endophyte species are involved in I. laevigata contamination to better target the control of the endophytes.

Conclusions
This is the rst report on the in vitro regeneration system of calli from I. laevigata. It was determined that there was no signi cant difference in callus induction rates between the two explants (hypocotyl and root). The greatest hypocotyl callus induction rate (75%) was achieved with 6-BA 0.5 mg L − 1 + 2,4-D

Experimental method
In this research, we established an I. laevigata callus regeneration system using hypocotyls and root segments as explants. The optimal medium agar content for callus induction was determined by the assessing the callus induction rate, endophyte contamination rate, and hypocotyl callus growth. In addition, the research identi ed the formula for adding phytohormones to the medium to maximize callus induction, multiplication, and differentiation for two I. laevigata explant seedling sources (hypocotyls and roots). The differentiated adventitious shoots were then rooted and transplanted. Standard MS medium was used in the experiment with the addition of sucrose at 30 g L − 1 . All experiments were conducted in a tissue culture chamber at 25 ± 1 °C with a light intensity of 25 µmol m − 2 ·s − 1 for 14 h d − 1 .

Explant sources
Seeds of I. laevigata were collected in September 2018 from Tahe, Heilongjiang province, China, and then stored in low-temperature sand in the Northeast Forestry University experimental nursery from October to December 2018. After December 2018, the seeds were kept in a 4 °C refrigerator. The seeds were sterilized with 75% alcohol for 10 s and 2% NaOCl for 25 min, followed by washing with sterile deionized water and then inoculated into the culture dish with wet lter paper. They were then cultured for 20 d until sprouting 2-3 leaves and roots. The leaves and roots of the sterile seedlings (20 d old) were removed, leaving a hypocotyl about 1 cm long to be used as explants. In addition, sterile seedlings (20 d old) were cultured in rooting medium for 30 d, and then the roots were cut off and the brous roots were removed. These roots were then cut into 1 cm long segments and used as explants. All the operations were carried out in sterile conditions.

Medium preparation
Standard MS medium was used in all the experiments with sucrose added at 30 g L − 1 . The various PGRs at assigned concentrations were added to media, along with antibiotics according the experimental design. The media were then adjusted to pH 5.9 and sterilized at 121 °C by autoclaving after adding different agar concentrations. All Petri dishes used in the experiments were sterilized at 121 °C by autoclaving. Thirty ml of each medium was poured into separate sterilized Petri dishes, allowed to cool to coagulate, and sealed with Para lm in a sterile environment. The Petri dishes were then stored for 3 d and observed to con rm their sterility prior to being used.

Determination of agar content in callus induction medium
Hypocotyl explants were inserted into media with 4, 6, 7, and 10 g L − 1 of agar to induce callus formation.
The induction rate, endophyte contamination rate, and callus appearance were recorded after 30 d (Table 1). Each treatment included 10 explants and three replications.

Callus induction from two explants
The explants, hypocotyls and root segments, were placed into the medium horizontally and assure that the explants were in full contact with the medium. The induction rate and callus appearance were evaluated after 45 d ( Table 2; Table 4). Each treatment included 20 explants and three replications.

Callus multiplication
Healthy calli of almost uniform size were selected and inoculated on the multiplication media with the different experimental formulations ( Table 5). The optimal multiplication medium was determined by comparing the multiplication rate, the contamination rate, and the callus appearance after 45 d. Each treatment included 20 explants and three replications.

Callus differentiation
Healthy calli of almost uniform size were selected and inoculated on the differentiation medium with different experimental formulas (Table 6; Table 7). After being cultured for 60 d, the differentiation rate and adventitious shoot appearance were recorded. Each treatment included 20 explants and three replications.

Multiplication, rooting, and transplanting of adventitious shoots
The adventitious shoots were transferred to MS medium with IBA 0.5 mg L − 1 + 6-BA 1.5 mg L − 1 + NAA 1.0 mg L − 1 + sucrose 30 g L − 1 + agar 9 g L − 1 for further subculture. Subsequently, the clumps were separated into 4 adventitious shoots and their leaves were clipped to 2 cm. The treated shoots were inoculated on the MS medium containing NAA 0.2 mg L − 1 for rooting. After 30 d of rooting, the culture bottle caps were removed and added to sterile deionized water to acclimate the in vitro regenerated plantlets. Three days later, the plantlets were removed from the bottles and washed carefully to remove any residual medium from the roots. The plantlets were grown hydroponically for 20 d after cleaning the roots and leaves. The seedlings were then transplanted into pots lled with orchard soil and vermiculite (3:1). The survival rate of transplanted plants was calculated after 30 d.

Elimination of Endophytes by antibiotics
The calli with endophytic bacteria were placed into multiplication media, which included different combinations of penicillin G (50, 100, 200, 300 mg/L), carbenicillin disodium (50, 100, 300, 500 mg/L) and cefotaxime sodium (50, 100, 300, 500 mg/L). The inhibition and browning rates of calli were monitored to determine the most suitable antimicrobial agents for I. laevitaga calli. Each treatment included 20 explants and three replications.

Statistical analysis
Induction rate (%) = the number of induced explants/the number of total inserted explants × 100% Contamination rate (%) = the number of explants contaminated by endophytes/the number of total inserted explants × 100% Multiplication rate (%) = the number of multiplicated calli/the number of total inserted calli × 100% Differentiation rate (%) = the number of differentiated calli/the number of total inserted calli × 100% Browning rate (%) = the number of browning explants/the number of total inserted explants × 100% Survival rate (%) = the number of plantlets survived after transplanting/the number of total transplanted plantlets × 100% Each treatment was repeated three independent times. All data represents the mean value and standard error of three replicates. Data analyses were performed using SPSS 22.0 software. One-way analysis of variance (ANOVA) was used to determine the signi cance of the various effects studied. Duncan's Multiple Range Test was used for pair-wise comparison of the data. Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by the National Natural Science Foundation of China (No.31670344) and Fundamental Research Funds for the Central Universities (No. ) .

Figure 2
Single-factor analysis of the impact of PGRs on the hypocotyl callus induction rate. Each value represents the mean ± SE of three independent experiments.   Single-factor analysis of the effect of PGRs on the differentiation rate of root induced calli. Each value represents the mean ± SE of three independent experiments.

Figure 6
Impact of antibiotic concentrations on endophyte inhibition and browning rates for calli. (A) Effect of three antibiotics on endophyte inhibition rates for calli; (B) effect of three antibiotics on the browning rate of calli; (C) effect of penicillin G concentrations on endophyte inhibition and browning rates.