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Obtaining callus and seedlings of Ulmus laevis – studies of their morphogenetic capacity and in vitro rooting of seedlings

Natalia Gumulak-Wołoszyn
Natalia Gumulak-Wołoszyn
, 
Małgorzata Sułkowska
Małgorzata Sułkowska
 and 
Katarzyna Nawrot-Chorabik
Katarzyna Nawrot-Chorabik
   | Jun 15, 2024
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Article Category: Original Article
Published Online: Jun 15, 2024
Page range: 129 - 143
Received: Apr 14, 2023
Accepted: Mar 21, 2024
DOI: https://doi.org/10.2478/ffp-2024-0011
Keywords
© 2024 Natalia Gumulak-Wołoszyn et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1.

Experiment to select a suitable disinfection of Ulmus laevis seeds in purpose to isolate suitable combination. Disinfection without ultrasound in 8% H2O2 for 15 minutes proved to be suitable for disinfection of European white elm seeds. A – combination with 8% H2O2 for 7 minutes without ultrasound; B – combination with 8% H2O2 for 7 minutes with ultrasound; C – combination with 8% H2O2 for 15 minutes without ultrasound; D – combination using 8% H2O2 for 15 minutes with ultrasound; E – combination using NaOCl at 8% or 12% for 7 or 15 minutes with or without ultrasound; F – combination using H2O2 at 12% for 7 or 15 minutes with or without ultrasound.
Experiment to select a suitable disinfection of Ulmus laevis seeds in purpose to isolate suitable combination. Disinfection without ultrasound in 8% H2O2 for 15 minutes proved to be suitable for disinfection of European white elm seeds. A – combination with 8% H2O2 for 7 minutes without ultrasound; B – combination with 8% H2O2 for 7 minutes with ultrasound; C – combination with 8% H2O2 for 15 minutes without ultrasound; D – combination using 8% H2O2 for 15 minutes with ultrasound; E – combination using NaOCl at 8% or 12% for 7 or 15 minutes with or without ultrasound; F – combination using H2O2 at 12% for 7 or 15 minutes with or without ultrasound.

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.
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.

Figure 3.

Culture medium adaptation experiment for a single random origin collected from seeds of Ulmus laevis embryos on culture media: DKW (A), mod. MS (B), LV (C) and WPM (D) supplemented in 4.440 µM/l BA. Photographs were taken after 28 days of culture. Disinfection of the plant material included a two-step disinfection with H2O2 disinfectant at a concentration of 8%.
Culture medium adaptation experiment for a single random origin collected from seeds of Ulmus laevis embryos on culture media: DKW (A), mod. MS (B), LV (C) and WPM (D) supplemented in 4.440 µM/l BA. Photographs were taken after 28 days of culture. Disinfection of the plant material included a two-step disinfection with H2O2 disinfectant at a concentration of 8%.

Figure 4.

Ulmus laevis explants obtained from embryos of trees from Kraków (1), Bochnia (2) and Wisła (3) on DKW, LV, mod. MS and WPM supplemented in 4.440 µM/l BA. Photographs were taken after 28 days of culture. Disinfection of the plant material included a two-step disinfection with the disinfectant H2O2 at a concentration of 8%.
Ulmus laevis explants obtained from embryos of trees from Kraków (1), Bochnia (2) and Wisła (3) on DKW, LV, mod. MS and WPM supplemented in 4.440 µM/l BA. Photographs were taken after 28 days of culture. Disinfection of the plant material included a two-step disinfection with the disinfectant H2O2 at a concentration of 8%.

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
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

Figure 6.

Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators kinetin and 6-benzylaminopurine added to 1 litre of medium. The majority of callus was obtained. Tissues grown on the WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA were the most viable among the others
Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators kinetin and 6-benzylaminopurine added to 1 litre of medium. The majority of callus was obtained. Tissues grown on the WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA were the most viable among the others

Figure 7.

Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators gibberellin A and 6-benzylaminopurine added to 1 litre of medium. The majority of calluses were obtained with an educated shoot or shoots. Tissues grown on the WPM medium supplemented with 1.444 µM/l GA3 and 4.440 µM/l BA were selected for further seedling studies
Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators gibberellin A and 6-benzylaminopurine added to 1 litre of medium. The majority of calluses were obtained with an educated shoot or shoots. Tissues grown on the WPM medium supplemented with 1.444 µM/l GA3 and 4.440 µM/l BA were selected for further seedling studies

Figure 8.

Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators thidiazuron and 6-benzylaminopurine added to a 1 litre of medium. For the most part, the tissues turned brown or died
Ulmus laevis tissues obtained from seed-derived embryos. Combinations of concentrations of two growth regulators thidiazuron and 6-benzylaminopurine added to a 1 litre of medium. For the most part, the tissues turned brown or died

Figure 9.

Ulmus laevis seedlings developing on rooting WPM media. A single main shoot developed on the IBA-supplemented medium (A) irrespective of concentration, with callus growing at its base. The leaves were small but developed along the entire shoot. On the medium supplemented with NAA (B), a callus grew independently of concentration from which a minimum of two shoots developed. The leaves remained viable from the middle of the shoot, allowing them to reach a larger size at the top
Ulmus laevis seedlings developing on rooting WPM media. A single main shoot developed on the IBA-supplemented medium (A) irrespective of concentration, with callus growing at its base. The leaves were small but developed along the entire shoot. On the medium supplemented with NAA (B), a callus grew independently of concentration from which a minimum of two shoots developed. The leaves remained viable from the middle of the shoot, allowing them to reach a larger size at the top

Figure 10.

Ulmus laevis seedlings developing on rooting WPM media. On medium supplemented with 4.920 µM/l IBA (A), callus was cream-coloured and the main root was browning. The tissues died at a rapid rate. On medium supplemented with 9.840 µM/l IBA (B), the seedlings produced a cream-coloured callus at the base. Their roots died most rapidly, resulting in the death of whole seedlings. On medium supplemented with 5.370 µM/l NAA (C), the callus tissue was orange in colour, and large numbers of root hairs developed from it. The seedlings had long, vigorous shoots with green leaves. On medium supplemented with 10.740 µM/l NAA (D), the seedlings achieved the required parameters. The callus in the roots was orange in colour with a large number of trichome roots growing out of it. In some cases, the beginnings of a primary root could be observed.
Ulmus laevis seedlings developing on rooting WPM media. On medium supplemented with 4.920 µM/l IBA (A), callus was cream-coloured and the main root was browning. The tissues died at a rapid rate. On medium supplemented with 9.840 µM/l IBA (B), the seedlings produced a cream-coloured callus at the base. Their roots died most rapidly, resulting in the death of whole seedlings. On medium supplemented with 5.370 µM/l NAA (C), the callus tissue was orange in colour, and large numbers of root hairs developed from it. The seedlings had long, vigorous shoots with green leaves. On medium supplemented with 10.740 µM/l NAA (D), the seedlings achieved the required parameters. The callus in the roots was orange in colour with a large number of trichome roots growing out of it. In some cases, the beginnings of a primary root could be observed.

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.
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.

Figure 12.

Ulmus laevis seedlings in passage from WPM medium with the addition of 10.740 µM/l NAA (A) or 5.370 µM/l NAA (B) to soil mixed with perlite at a ratio of 3:1 (C)
Ulmus laevis seedlings in passage from WPM medium with the addition of 10.740 µM/l NAA (A) or 5.370 µM/l NAA (B) to soil mixed with perlite at a ratio of 3:1 (C)

Figure 13.

Comparison of a pair of Ulmus laevis callus grown on WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA. The callus was divided into two equal parts by weight and one was transferred to the dark (A) and the other to the light (B) in the phytotron
Comparison of a pair of Ulmus laevis callus grown on WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA. The callus was divided into two equal parts by weight and one was transferred to the dark (A) and the other to the light (B) in the phytotron

Figure 14.

Comparison of the mass of 45 calluses of U. laevis. Each callus divided into two parts came from a single zygotic embryo. One part of the callus was cultured in the light of the phytotron and another in the dark of the phytotron. The experiment lasted 21 days on WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA. Paired t-test: t = 9.4479, df = 44, p-value = 3.737e-12
Comparison of the mass of 45 calluses of U. laevis. Each callus divided into two parts came from a single zygotic embryo. One part of the callus was cultured in the light of the phytotron and another in the dark of the phytotron. The experiment lasted 21 days on WPM medium supplemented with 4.646 µM/l KIN and 4.440 µM/l BA. Paired t-test: t = 9.4479, df = 44, p-value = 3.737e-12

P-values of mean tissue sizes at combinations of specific concentrations of growth regulators using Pearson’s correlation coefficient. At a significance level of p = 0.05

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

Methods used to decontaminate Ulmus laevis seeds

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

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″

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
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