Obtaining callus and seedlings of Ulmus laevis – studies of their morphogenetic capacity and in vitro rooting of seedlings
Article Category: Original Article
Data publikacji: 15 cze 2024
Zakres stron: 129 - 143
Otrzymano: 14 kwi 2023
Przyjęty: 21 mar 2024
DOI: https://doi.org/10.2478/ffp-2024-0011
Słowa kluczowe
© 2024 Natalia Gumulak-Wołoszyn et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Trees provide a habitat for a variety of organisms, ranging from microscopic fungi to insects and animals. However, the most relevant aspect from the perspective of urban forest ecosystem conservation and biotechnology is the prevention of infection by various fungal pathogens. The primary method is the development of repellents or plant varieties that are resistant to phytopathogenic fungi (Peng et al. 2021). Fungal pathogens exert high pressure on host plants, causing them to activate a number of defence mechanisms. In response, fungi attempt to combat these barriers (Priyashantha et al. 2023).
Research on fungal pathogens utilises methods derived from biotechnology. Pathogenicity can be assessed using dual cultures, in which two types of tissues are used. The first is the plant tissue (such as callus, a part of the plant or the whole plant) and the second, in this case, the fungal component. This method allows to isolate the resistant plant genotypes and assess the risk of infection under the tested conditions. Plant tissues selected for resistance can be propagated and used as a material for further regeneration by direct organogenesis (Bull and Michelmore 2022). Through breeding in this way, plants can be ensured to be free of infectious diseases and with potentially increased resistance in the future.
The European white elm (
To date, no callus has been obtained from embryos derived from immature seeds. Most of the tissues obtained were derived from buds or leaves. Tissues developing on buds mainly differentiated by indirect organogenesis to form callus at the base of the seedling (Aoun et al. 2009; Shukla et al. 2012; Välimäki et al. 2021). On leaves, the development of embryogenic callus producing by somatic embryogenesis was predominant (Conde et al. 2004).
The aim of this study is to develop reliable methods for obtaining callus and seedling tissues of
This study is based on similar elements as in the work published by K. Nawrot-Chorabik (Nawrot-Chorabik et al. 2024) due to the long-standing collaboration and the subject matter of the PhD thesis of N. Gumulak-Wołoszyn under the supervision of K. Nawrot-Chorabik.
For micropropagation, zygotic immature seed embryos from three approximately 40-year-old
Species and origin of the plant material
| No. | Species | Location and date of seed collection | Forest district | Geographical coordinates |
|---|---|---|---|---|
| 1 | Ulmus laevis | Kraków (Poland) 05.2022 | Krzeszowice |
N 50°3′19.375″ E 19°55′29.708″ |
| 2 | Bochnia (Poland) 05.2022 | Brzesko |
N 49°57′48.93″ E 20°25′37.721″ |
|
| 3 | Wisła (Poland) 06.2022 | Wisła |
N 49°39′11.057″ E 18°52′20.993″ |
Development of seed preparation and disinfection of European white elm seeds with isolation of zygotic embryos on unmodified medium with addition of cytokinin
The disinfection was based on micropropagation studies of elm species by Malá et al. (2007) and Välimäki et al. (2021) and ash by Nawrot-Chorabik et al. (2022b, 2024). At the beginning, 2400 live seeds were stripped of their seed coats and sterilised in 70% ethanol for 30 seconds. After this, they were rinsed with distilled water containing a drop of Tween 80 and placed in a beaker with a 5% polyvinyl pyrrolidone (PVP) solution for 24 hours at about 4°C. After this time, the seeds were shaken in 70% ethanol (0.5 min) and rinsed again three times in deionised water.
The seed disinfestation experiment was conducted in 16 different combinations in triplicate on one drawn origin from the three origins held, so as not to suggest later results. Each combination was represented by 50 zygotic embryos. It was tested whether the action of ultrasound prior to disinfection of the seeds proper would improve the disinfection effect, whether it would enhance tissue proliferation or not affect subsequent development. Two compounds commonly used to disinfect the plant material were also tested: sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2) at a concentration of 8% or 12% and an action time of 7 or 15 minutes to show differences.
The shelled seeds were disinfected in 70% ethanol (0.5 min) and then rinsed under running water (10 min). After this, they were transferred to a beaker with a 5% PVP solution for 24 hours. Once the time had elapsed, the seeds were transferred to a clean beaker and rinsed in ethanol (0.5 min). Depending on the combination, ultrasound (42 kHz) was used at this stage using an ultrasound generator (CHEMLAND, Stargard Szczeciński, Poland), which treated the seeds for 10 minutes, after which time they were transferred to a beaker. The seeds were washed several times with sterile water and transferred to a sterile beaker. Depending on the combination, the beaker was supplemented with the appropriate concentration of H2O2 or NaOCl and the seeds were disinfected for 7 or 15 minutes, respectively (Table 2). The seeds were then washed several times with sterile water to rinse off the disinfectant and transferred to a sterile filter paper. After isolation, embryos were lined onto petri dishes containing WPM culture medium (Lloyd and McCown 1980; Välimäki et al. 2021) without any growth regulators. Assumed combinations are presented in Table 2.
Methods used to decontaminate
| Combination | The use of ultrasounds [45kHz] | Disinfectant used | Disinfectant concentration [%] | Disinfection time [min.] |
|---|---|---|---|---|
| 1 | + | NaOCl | 8 | 7 |
| 2 | 15 | |||
| 3 | 12 | 7 | ||
| 4 | 15 | |||
| 5 | H2O2 | 8 | 7 | |
| 6 | 15 | |||
| 7 | 12 | 7 | ||
| 8 | 15 | |||
| 9 | − | NaOCl | 8 | 7 |
| 10 | 15 | |||
| 11 | 12 | 7 | ||
| 12 | 15 | |||
| 13 | H2O2 | 8 | 7 | |
| 14 | 15 | |||
| 15 | 12 | 7 | ||
| 16 | 15 |
Adaptation of a suitable culture medium for Ulmus laevis explants
Four types of media were used in the study: DKW (Driver and Kuniyuki 1984), modified MS (Murashige and Skoog 1962; Patent No: P.433288), LV (Litvay et al. 1985) and WPM. The MS, DKW and LV media selected for this experiment are commonly used for culture in our laboratory. The medium WPM was considered based on the publication of Välimäki et al. (2021). Each medium was supplemented with the same growth regulator 6-benzylaminopurine (BA) at 4.440 μM/l. Growth regulators were prepared at a concentration of 1 mg substance per 1 ml solvent. A total of 600 seeds were disinfected, and embryos were isolated onto the specified culture media at 100 per combination. Three replicates were performed. Cultures were stored in a phytotron at 25°C, 40% humidity and a photoperiod of 16 hours light and 8 hours dark with a warm white light intensity of 60 μmol m−2 s−1 (Nawrot-Chorabik 2024). The culture was carried out for 21 days. During this time, tissues were passaged onto a suitable sterile medium as required.
The experiment used 20 embryos per combination in three replicates (2880 seeds in total) and selected: 6-benzylaminopurine (BA), kinetin (KIN), thidiazuron (TDZ) and gibberellin A (GA3) in various combinations. Growth hormones were tested in the following combinations: BA and KIN, BA and TDZ, BA and GA3, at well-defined concentrations (Result section) followed by determination of which combination formed callus and which combination formed differentiated tissues containing root or cotyledon. The culture medium was supplemented with 20 g/l sucrose and 7 g/l agar. The plant material was cleaned and subjected to a two-step dis-infection process under sterile conditions. During this process, a half-potency culture medium was prepared. In the laminar chamber, each bottle of sterilised nutrient solution was supplemented with a different combination of growth regulators. BA and KIN, BA and TDZ, and BA and GA3 were added through sterile 0.22 μm PES syringe filters at constant concentrations. After 10 days of culture in the dark, the tissues were transferred to the light of the phytotron for 11 days. After 21 days of culture on Petri dishes, the tissues had fully differentiated, and the combination with the most proliferation of tissues with shoot or leaf elements in good health was selected. Once the tissues had reached a minimum length of 1.5 cm, they were cut in half, removing any callus tissue buds if necessary, and then passaged onto the next medium for seedling micropropagation.
The culture of 60 tissues randomly selected from stage “Adaptation of growth regulators to seedling proliferation or callus development” (3 replicates of 20 tissues each) having a root or stem was conducted in a plant growth chamber (Biogent, Fito, Poland) at 23 ± 1°C and 70% humidity, under white light 380–710 nm, photoperiod 12 hours, on the WPM medium with 20 g/l sucrose and 7 g/l agar with 2.887 µM/l GA3 and 2.220 µM/l BA. During the 21-day culture period, tissues were passaged onto new media with identical composition if necessary. After 28 days, tissues with a minimum of one leaf and root buds were transferred onto rooting media in a tall glass dish (80 mm × 45 mm) with a volume of 226 cm3 covered by a 90 mm diameter glass Petri dish. For rooting, WPM media with naphthaleneacetic acid (NAA) or Indole-3-butyric acid (IBA) were used at a concentration of 5.370 µM/l NAA, 4.920 µM/l IBA, 10.740 µM/l NAA or 9.840 µM/l IBA.
After 14 days, the tissues were transferred to a new medium in a tall dish, but the composition of each medium was significantly altered. The content of macro-nutrients, micronutrients, vitamins, sugar and the respective growth regulator was halved, and the amount of solidifying agent was reduced to 6 g × dm−3. After an additional 14 days, the tissues were redeposited in the high dish containing medium again halved in the chemical content, but without the addition of the growth regulator. The tall pans, covered with a Petri dish, were protected with parafilm and transferred to a germinator outside the phytotron within reach of natural light. The room temperature was maintained at 20 ± 1°C. After 14 days, the tissues were washed in sterile water to remove any residual culture medium. If necessary, callus developing at the roots was removed, yellowing leaves were removed, and roots were shortened to 1 cm in length and placed in a plant growing medium with perlite at a ratio of 3:1. Seedlings were watered with sterile water every 4 days.
A total of 45 calluses obtained from the stage “Adaptation of growth regulators to seedling proliferation or callus development” culture were used, and they were divided into three replicates of 15 tissues each. Tissues so far were cultured for 10 days in the dark and 11 days in phytotron light on the WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA. Each tissue was weighed, cut in half and repotted onto a new medium. This provided two tissues from one initial tissue that could be compared. One part, containing 45 different calluses, was transferred to the dark in the phytotron, and the other part was still kept in light. After 21 days, the experiment was terminated and the results were recorded.
For analysis, the Wilcoxon rank sum test with continuity correction was used along with the Kruskal–Wallis test to determine the performance of individual tissue culture experiments. Pearson’s correlation coefficient was used to determine average tissue sizes in the presence of growth regulators. The same coefficient along with the Kruskal–Wallis test was used to compare the concentration of growth regulators in rooting seedlings. Paired t-test was used to compare weights of callus tissues grown in the dark. Statistical analyses were performed in RStudio version 2022.07.2 + 576 (library tidyverse). A p<0.05 value was considered significant.
The diversity of decontamination methods proved essential in selecting the optimal procedure for sterilising the plant material.
Decontamination without ultrasound in 8% H2O2 for 15 minutes was selected for further study (Fig. 1, 2). Only in the case of combination No. 14, in addition to the abundance of living tissues, attention was also paid to their health status. The tissues were partially browned but reached sizes above 5 mm in diameter.
Figure 1.
Experiment to select a suitable disinfection of
Figure 2.
Pairwise comparisons using Wilcoxon rank sum test with continuity correction conducted on 16 combinations of a disinfested elm plant material. Three replicates of 50 tissues were summarised. Kruskal–Wallis chi-squared = 1648.7, df = 15, p-value < 2.2e-16.
Once the disinfection method had been developed, it was important to select a suitable culture medium for the culture. After selecting combination 14 with disinfection in 8% H2O2 for 15 min (Tab. 2, Combination 14), European white elm seed embryos were plated onto media supplemented with 4.440 µM/l BA: DKW, modified MS, LV and WPM. After 28 days of culture in the phytotron, differences were observed in the number of developed embryos and their condition on media supplemented with cytokinin BA (Fig. 3).
Figure 3.
Culture medium adaptation experiment for a single random origin collected from seeds of
Based on the results, MS with BA was selected for further experiments. On the other substrates, tissues did not show potential for differentiation. Those that developed sparingly on the DKW medium and modified MS died within a week. Tissues that reached a size of 0.7 mm on the WPM medium were considered optimal for further studies because of their growth rate. The dying process then does not occur drastically due to the constant growth or the capacity for organogenesis.
Once the results were obtained, the entire experiment was repeated on all three origins in triplicate. The isolated embryos were subjected to a two-step disinfection with 8% H2O2 for 15 minutes. Thus prepared were plated on DKW media, mod. MS, LV and WPM supplemented with 4.440 µM/l BA. The culture was maintained under sterile conditions in the phytotron and completed after 21 days. Representative dishes were selected for photography (Fig. 4).
Figure 4.
Tissues developing on the media after 28 days of culture showed significant variation in size, abundance (Fig. 5) and health condition.
Figure 5.
Pairwise comparisons using Wilcoxon rank sum test with continuity correction. The effect of culture media on tissues was investigated. Four types of culture medium were considered in three replicates. Analysis was carried out on all origins with averaging of values. One hundred embryos were used in each of three replicates. Kruskal–Wallis chi-squared = 28.673, df = 3, p-value = 2.623e-06
Explants from the Wisła Forest District (Tab. 1, Origin No. 3) developing on WPM medium supplemented with 4.440 µM/l BA were distinguished from other combinations. On the DKW substrate, the explants took on a dark beige colour. Green microshoots were also frequent, which were cut off and passaged onto the new medium. This proved to be the optimal medium for origin 2. LV medium and mod. MS did not stimulate the development of
By varying the growth regulators, it was possible to match the appropriate combination to obtain callus or seedlings
Figure 6.
Figure 7.
Figure 8.
| Growth regulator and its concentration | 6-benzylaminopurine | ||||
|---|---|---|---|---|---|
| 2.220 µM/l | 4.440 µM/l | 6.660 µM/l | 8.880 µM/l | ||
| Kinetin | 2.323 µM/l | 0.238100 | 0.213310 | 0.238100 | 0.238100 |
| 4.646 µM/l | 0.213310 | 0.238100 | 0.213310 | 0.238100 | |
| 6.969 µM/l | 0.213310 | 0.213310 | 0.238100 | 0.238100 | |
| 9.292 µM/l | 0.238100 | 0.23810 | 0.213310 | 0.238100 | |
| Gibberellin A | 1.444 µM/l | 0.213300 | 0.238080 | 0.213300 | 0.238080 |
| 2.887 µM/l | 0.213300 | 0.21330 | 0.238080 | 0.213300 | |
| 4.331 µM/l | 0.238080 | 0.21330 | 0.213300 | 0.238080 | |
| 5.774 µM/l | 0.213300 | 0.238080 | 0.213300 | 0.213300 | |
| Thidiazuron | 2.270 µM/l | 0.091578 | 0.406010 | 0.287300 | 0.406010 |
| 4.540 µM/l | 0.287300 | 0.091578 | 0.406010 | 0.406010 | |
| 6.810 µM/l | 0.287300 | 0.28730 | 0.091578 | 0.287300 | |
| 9.080 µM/l | 0.406010 | 0.28730 | 0.287300 | 0.091578 | |
European white elm explants obtained on the WPM medium supplemented with specific growth regulators showed high variation depending on the amount of hormones.
Kinetin has a potentiating effect on the cytokinesis step and thus, in combination with BA, induced the proliferation of callus in most cases. The higher the concentration of kinetin, the more compact the tissue became and, on passage, it easily broke down into smaller, rapidly dying parts. The higher the concentration of BA, which causes the formation of new shoots, the more numerous the tissue developed through the process of intermediate organogenesis. The maximum concentration of both regulators led to the formation of white, spreading callus from which single transparent shoots grew. Flexible and fully viable tissues with a pale beige colour were obtained at concentrations of 4.646 µM/l KIN and 4.440 µM/l BA, indicating that the concentrations of the regulators were correctly selected for the growth of
Gibberellin A, due to its germination-stimulating properties, showed very good results in the development of
Thidiazuron is a cytokinin used in the tissue culture for plant regeneration and organogenesis. It showed organogenetic effects on European white elm embryos. At the lowest concentration, it did not strongly affect shoot development in tissues, nor did it promote callus development. Tissues reached approximately 10 mm in length. However, the higher the concentration of TDZ was, the tissues browned considerably. The callus, from which shoots developed, died rapidly. Maximum concentrations of both of these regulators resulted in the dwarfing or dying of part or all of the explants. Each tissue completely died after a period of about 35 days. The resulting explants did not show the expected potential, and development was not directed along one of the
After 42 days of culture on varying concentrations of media with NAA and IBA and a further 14 days on medium without growth regulators, the tissues showed differences in the number of shoots developing by indirect organogenesis (Tab. 4). On the medium supplemented with IBA, a single shoot developed from each tissue in the root of which callus was observed. The shoot had at least four leaves along the entire shoot (Fig. 9A). On medium supplemented with NAA, a minimum of two shoots were differentiated from the callus developing in the root. Each shoot had a minimum of two leaves, which quickly fell away from the bottom of the shoot. This resulted in more developmental energy being transferred to the top leaves. The shoots were thicker and longer compared to shoots developing on IBA medium, and trichome roots developed in large numbers from the callus (Fig. 9B). Out of a total of 60 tissues developing on the WPM medium with lower concentrations of IBA, 15 survived and 23 with higher concentrations.
Figure 9.
Number of tissues isolated from individual 30 primary seedlings showing multistemming ability. From the initial 15 tissues on WPM medium supplemented with 5.370 µM/l NAA, 35 separate seedlings were obtained at the end. From the initial 15 primary tissues on WPM medium supplemented with 10.740 µM/l NAA, a total of 48 separate tissues were obtained
| Number of tissue | Amount of NAA growth regulator per litre of medium | |
|---|---|---|
| 5.370 µM/l | 10.740 µM/l | |
| 1 | 2 | 3 |
| 2 | 2 | 3 |
| 3 | 3 | 2 |
| 4 | 2 | 4 |
| 5 | 2 | 2 |
| 6 | 3 | 3 |
| 7 | 3 | 3 |
| 8 | 2 | 3 |
| 9 | 2 | 4 |
| 10 | 2 | 4 |
| 11 | 3 | 4 |
| 12 | 2 | 3 |
| 13 | 2 | 4 |
| 14 | 2 | 2 |
| 15 | 3 | 4 |
However, 15 randomly selected tissues from cultures with NAA were included in the experiment in order to make the results comparable with tissues developing on IBA media. All tissues were cultured as planned.
Seedlings developing on medium originally containing the growth regulator 4.920 µM/l IBA showed variable differences in development. Out of 15 seedlings, 8 died. The others developed slowly, the callus in the root was cream-coloured, browning in some places. On the medium containing 9.840 µM/l IBA, 10 seedlings survived and had longer shoots and creamy callus. IBA also induced the production of one trichome root at the lower concentration, and in the presence of 9.840 µM/l, 6 tissues grew a short, browning main root. Seedlings developing on medium with 5.370 µM/l NAA developed longer shoots, larger and more numerous leaves and a large number of trichome roots. Seedlings growing on a medium containing 10.740 µM/l NAA in terms of shoot length, size and number of leaves performed most favourably (Fig. 10, 11). They also developed viable main shoots and trichome roots. Calluses formed in the roots were compacted by the trichome roots, did not disintegrate and were orange-brown in colour.
Figure 10.
Figure 11.
Pairwise comparisons using Wilcoxon rank sum test with continuity correction. The effect of the concentration of specific growth regulators on the growth of pedunculate elm seedlings was investigated: 5.370 µM/l NAA (Concentration_1), 10.740 µM/l NAA (Concentration_2), 4.920 µM/l IBA (Concentration_3) and 9.840 µM/l IBA (Concentration_4). Kruskal–Wallis chi-squared = 35.405, df = 3, p-value = 1e-07.
Within 14 days of culture under sterile conditions with access to natural light, tissues that were originally grown on the WPM medium with the IBA regulator died. Tissues derived from the WPM medium with 10.740 µM/l NAA (Fig. 12A) or 5.370 µM/l NAA (Fig. 12B) remained viable, so they were all cleaned of medium and superfluous elements and transferred to soil with perlite.
Figure 12.
After 28 days, seedlings transferred to soil with perlite grown on medium with 5.370 µM/l NAA had shorter shoots and roots, and the leaves were fully developed. Seedlings grown on medium containing 10.740 µM/l NAA after transfer to soil with perlite reached an average size of 1.5–2 times larger than on medium supplemented with 5.370 µM/l. The large number of root hairs and their length showed the positive effect of the higher concentration on initial development. Leaves continued to be healthy and fully coloured (Fig. 12C).
An experiment was carried out on changes in the weight of callus obtained in step 3 (Fig. 13).
Figure 13.
Comparison of a pair of
Tissues developing under the light of the phytotron continued to be viable. The average weight after 21 days of culture was 0.163±0.031 g. Explants developing in the dark showed varied responses to the lack of light. Eleven of them increased their weight while continuing to take up nutrients from the medium, while the others stopped developing and started to lose weight. The average weight after 21 days was 0.129±0.027 g. The average weight of the initial callus was 0.121±0.027 g (Fig. 14).
Figure 14.
Comparison of the mass of 45 calluses of
In this study, the
The protocol for obtaining tissues has shown that callus and seedling cultures can be used to maintain the genetic diversity of many biodiversity-enriching species. It further revealed that elm seed embryo can be successfully cultured. In the study of Fenning et al. (1993), proliferating
Teixeira da Silva et al. (2015) highlighted the importance of disinfection. Pre-disinfection time and detergent concentrations were also considered during the propagation of
The effect of light and darkness on the cultured tissues established how external conditions can influence physiological processes. The incubation process was carried out without light in the phytotron to make them autotrophic, and this was found to improve the growth of callus (Siddique and Islam 2018). Earlier Kintzios et al. (2002) reported that the induction of the callus of
The average weight of the tissues showed that there are differences between growing in light or dark. However, calluses were observed to form after 6 days in the dark condition in the presence of sucrose at 2% concentration. However, under light conditions, calluses already increase their weight after the first few days. Callus tissue was brittle regardless of light conditions and colour varied under all conditions. This corroborates Wahyuni et al. (2020): on MS medium supplemented with 2,4-D and BAP hormones and sucrose at different concentrations, differences were evident in the incubation of
After obtaining viable shoots that demonstrated the ability to produce root hairs, an attempt was made to root them. The NAA and IBA regulators used for this are commonly used for elm seedling proliferation (Mezzetti et al. 1988; Corchete et al. 1997; Lee et al. 2021; Välimäki et al. 2021). According to a study on
In the case of
However, obtaining seedlings from buds can be similar to growing them from embryos. In this study, the seed expresses multiple seedlings which were still viable after separation.
However, in the case of buds (Välimäki et al. 2021), the seedlings did not show multiple shoot, which would have contributed to achieving more seedlings. In a research study carried out by Teixeira da Silva et al. (2017) on
It was further discovered that despite genotypic differences, few of the tissues are susceptible to consistent regeneration conditions. Differences in structure and growth speed were evident between tissues. The refinement of the rooting method for
The development of micropropagation processes is a very important issue. Regeneration of adult
The callus and cuttings of white elm, therefore, make it possible to develop research in this area, as it has shown to significantly reduce the time to wait for breeding results. Pasternak and Steinmacher (2024) described that the use of organogenesis methods helps to optimise the seedling production process and the increase in the multiplication coefficient. This study showed with a high degree of certainty that the survival rate of callus depended on the chemical composition of the substrates on which they were located. However, it proved crucial to select growth regulators according to the type of tissues required for individual study. In turn, seedlings obtained under sterile conditions, according to the developed procedure, finally provide the basis on which to implement extensive pathogenicity studies.
The establishment of dual cultures involving callus or seedlings from a single genotype together with pathogen tissue can provide more information than other studies have done so far. Pathogenicity studies are time-consuming and difficult to carry out, but with a well-prepared material, problems such as infection can be avoided. Mohaddab et al. (2022) emphasise that dual cultures can be used to, among other things, produce secondary metabolites and increase physiological tasks as a response to stress.
Based on the result of the study, it can be concluded that the plant material should be taken straight from the plus tree with no signs of disease. There is every possibility that seedlings cultured in vitro and ex vitro after transfer to soil with perlite will be suitable for plantation establishment after 4 weeks. Given the rapidly progressing Dutch elm disease, protocols for obtaining a large number of seedlings in a short period of time may be essential when developing conservation methods. Also, the act of culturing callus in the laboratory should be focused more from seeds rather than from bud and cuttings. This established method of growing seedlings will increase the rate of seedling production for to meet the ever growing demand of silviculturist. As for the formation of callus on the culture medium, this is an improvement that can contribute to a lot of new research and analyses. Using the protocol from this paper to obtain calluses, they can be successfully cultured for further research in various branches of biotechnology and beyond.