
This research was conducted from August to November 2021 in the Laboratory of Plant Physiology and Biotechno-logy Faculty of Agriculture, Sebelas Maret University. The study began with the sterile germination of
Sterilization was done for the tools and seeds. For the tools sterilization, tweezers, culture knife (scalpel), and petri dish are among the dissection equipment that is cleaned. The instruments were then disinfected in an autoclave for 30 minutes at 121℃ and 1 atm pressure before being stored in the oven to retain their sterility. The seeds were sterilized twice.
The medium was produced by adding varying concen-trations of BAP and 2,4-D to Murashige & Skoog (MS) based medium. Medium making starts with 30 g of sugar, 50 mL of MS macronutrients, 10 mL of MS micro-nutrients, 50 mL of Fe-EDTA, 50 mL of vitamins, 6.25 mL of BAP (0 ppm (B0); 0.5 ppm (B1); 1 ppm (B2); 1.5 ppm (B3); 2 ppm (B4)) and 6.5 mL of 2,4-D (0 ppm (D0); 0.5 ppm (D1); 1 ppm (D2); 1.5 ppm (D3); 2 ppm (D4)), followed by 1,000 mL of distilled water and pH control up until 6.2. As a controller, NaOH was used to lower pH while HCl was used to raise it. Furthermore, 8 grams of agar powder was added, heated with a magnetic stirrer until it boiled, then transferred to a culture bottle and autoclaved. The best treatment for callus induction and organogenesis was determined using a controlled media without the addition of plant growth regulators.
Explants were made by growing
The color and texture of calluses data are described and the rest parameters were analyzed by ANOVA (Analysis of Variance) 5% then continued by DMRT (Duncan Multiple Range Test) 5% if the data showed a significant result. The design used in this research was a Complete Ran-domized Design in 2 factors: Benzyl Amino Purine and 2,4-Dichlorophenoxiacetic acid with 5 levels for each concentration: 0; 0.5; 1; 1.5; and 2 ppm. 25 combinations were obtained and repeated 3 times producing 75 experi-ment units.
The percentage of inducted calluses demonstrates that BAP and 2,4-D had a considerable impact on
Table 1 . The average percentage of
2,4-D (ppm) | BAP (ppm) | 2,4-D Average | ||||
---|---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | ||
0 | 33.3 | 33.3 | 100 | 100 | 100 | 73.33a |
0.5 | 66.6 | 100 | 100 | 100 | 100 | 93.33b |
1 | 100 | 100 | 100 | 100 | 100 | 100b |
1.5 | 100 | 100 | 100 | 100 | 100 | 100b |
2 | 100 | 100 | 100 | 100 | 100 | 100b |
BAP average | 80a | 80.67ab | 100b | 100b | 100b | - |
In Table 1 , a, b are the significant concentration levels, with arepresents the least and brepresents the highest of callus induction percentage. Numbers followed by the same letter in the same rows or column are not significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
Callus developments are marked by the appearance of a bulge or swelling at the base of the explant. However, the result of the ANOVA analysis of callus appearance time showed an insignificant number (
Table 2 displayed the complete data of callus texture that is divided into 2 groups: friable and compact calluses. Compact calluses predominated among inducted calluses. When a callus was exposed to lignification, the callus consolidated, forming compact calluses. The cytokine that operates as nutrition transporters has an impact on this occurrence. Castro
Table 2 .
2,4-D (ppm) | BAP (ppm) | ||||
---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | |
0 | - | Compact | Compact | Compact | Compact |
0.5 | Friable | Compact | Friable | Compact | Compact |
1 | Friable | Friable | Friable | Compact | Compact |
1.5 | Friable | Compact | Friable | Compact | Compact |
2 | Friable | Compact | Compact | Compact | Compact |
ppm: part per million (mg/L), WAP: week after plantation.
Callus color is a parameter that can be an indicator of the compounds contained in the callus. The color of the callus ranged from green to whitish green, white, yellowish green, green, and brownish green (Fig. 3). According to Rasud and Bustaman (2018), callus color change is an indicator of the growth of cells in callus tissue. Based on the data in Table 3, it can be seen that the most common callus color found was green. Furthermore, white callus was found in B3D0 and B4D0 treatments. The white color of the callus indicates the lack of pigment in the callus cells. Yellowish green callus found in the 1.5 ppm + 2,4-D 2 ppm (B3D4) and 2 ppm + 2,4-D 0.5 ppm (B4D1) treatments were suspected as a potential embryogenic callus. Wu
Table 3 .
2,4-D (ppm) | BAP (ppm) | ||||
---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | |
0 | - | Brownish green | Whitish Green | White | White |
0.5 | Green | Green | Green | Green | Yellowish green |
1 | Green | Green | Brownish green | Brownish green | Brownish green |
1.5 | Green | Brownish green | Brownish green | Brownish green | Brownish green |
2 | Green | Green | Green | Yellowish green | Brownish green |
ppm: part per million (mg/L), WAP: week after plantation.
The weight of fresh calluses was observed on a month-old callus. Fig. 4 shows that the highest average of callus fresh weight was obtained on treatment B1D4 (0.5 ppm BAP and 2 ppm 2,4-D) with an average weight of 3,610 g. This demonstrated that this combination had the best balance of auxin (2,4D) and cytokinin (BAP), resulting in the production of optimal callus masses in
The weight of dry calluses was calculated on some samples (Fig. 5). The selected treatments are chosen as representatives for the combinations of high and low auxin and cytokinin. The harvested callus was dried in the oven at 60℃ temperature. The highest callus dry weight average was observed on treatment 0.5 ppm + 2,4-D 2 ppm (B1D4) with 0.111 g. Treatment BAP 0.5 ppm + 2,4-D 0 ppm (B1D0) has the lowest callus dry weight average of 0.027 g. However, the result of the ANOVA analysis of callus dry weight showed an insignificant number (
When a shoot reaches a length of 0.5 cm, the criteria of shoots are calculated. The ANOVA revealed that every single treatment of BAP and 2,4-D had a significant effect on the percentage of developed shoots on
Table 4 . Average percentage of
2,4-D (ppm) | BAP (ppm) | 2,4-D Average | ||||
---|---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | ||
0 | 100 | 100 | 100 | 100 | 100 | 100.00c |
0.5 | 0 | 0 | 33.3 | 66.6 | 100 | 40.00b |
1 | 0 | 0 | 33.3 | 33.3 | 66.6 | 26.67ab |
1.5 | 33.3 | 0 | 0 | 0 | 66.6 | 20.00ab |
2 | 33.3 | 0 | 0 | 0 | 0 | 6.67a |
BAP average | 33.33a | 20.00a | 33.33a | 40.00a | 66.67b | - |
In Table 4 , a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the highest of shoot forming percentage. Numbers followed by the same letter in the same rows or column are not significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
Fig. 6 displayed the complete data of the percentage of
The shoot appearance time is observed when a shoot reaches a length of 0.5 cm. The ANOVA analysis results revealed that each BAP and 2,4-D treatment has no significant effect on the shoot appearance time, as does the interaction of BAP and 2,4-D, which has no significant effect on the shoot appearance time of
The average
The ANOVA analysis result for the 2,4-D effect on
Table 5 . Average height of
2,4-D (ppm) | BAP (ppm) | 2,4-D Average | ||||
---|---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | ||
0 | 2.00 | 2.17 | 3.33 | 2.50 | 2.83 | 2.67c |
0.5 | 0.00 | 0.00 | 0.67 | 1.17 | 2.00 | 0.77b |
1 | 0.00 | 0.00 | 0.33 | 0.33 | 1.17 | 0.37ab |
1.5 | 0.33 | 0.00 | 0.00 | 0.00 | 1.00 | 0.27a |
2 | 0.33 | 0.00 | 0.00 | 0.00 | 0.00 | 0.07a |
BAP average | 0.53a | 0.43a | 0.87a | 0.80a | 1.40b | - |
In Table 5 , a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the highest of average height. Numbers followed by the same letter in the same rows or column are not significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
The interaction of BAP and 2,4-D did not have a significant influence on the number of
Table 6 . Average number of
2,4-D (ppm) | BAP (ppm) | 2,4-D Average | ||||
---|---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | ||
0 | 2.67 | 4.00 | 6.67 | 6.00 | 8.67 | 5.60c |
0.5 | 0.00 | 0.00 | 0.67 | 3.33 | 5.33 | 1.87b |
1 | 0.00 | 0.00 | 1.33 | 0.67 | 1.33 | 0.67a |
1.5 | 0.67 | 0.00 | 0.00 | 0.00 | 2.67 | 0.67a |
2 | 0.67 | 0.00 | 0.00 | 0.00 | 0.00 | 0.13a |
BAP average | 0.80a | 0.80a | 1.73ab | 2.00b | 3.60c | - |
In Table 6 , a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the highest of leaves number. Numbers followed by the same letter in the same rows or column are not significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
In this study, the flavonoid content test was applied to 10 samples of callus and plantlets that had formed stems, leaves, and roots and 1 field sample of
Table 7 . Flavonoid compound on field and callus samples of
Sample | Flavonoid compound (% b/b) |
---|---|
Field | 3.27 |
B0D0 | 4.35 |
B0D4 | 1.61 |
B1D0 | 2.10 |
B1D4 | 1.94 |
B2D0 | 2.87 |
B2D4 | 1.79 |
B3D0 | 2.88 |
B3D4 | 1.87 |
B4D0 | 2.98 |
B4D4 | 1.97 |
B0D0: Treatment of BAP 0 ppm + 2,4-D 0 ppm, B0D4: Treatment of BAP 0 ppm + 2,4-D 2 ppm, B1D0: Treat-ment of BAP 0.5 ppm + 2,4-D 0 ppm, B1D4: Treatment of BAP 0.5 ppm + 2,4-D 2 ppm, B2D0: Treatment of BAP 1 ppm + 2,4-D 0 ppm, B2D4: Treatment of BAP 1 ppm + 2,4-D 2 ppm, B3D0: Treatment of BAP 1.5 ppm + 2,4-D 0 ppm, B3D4: Treatment of BAP 1.5 ppm + 2,4-D 2 ppm, B4D0: Treatment of BAP 2 ppm + 2,4-D 0 ppm, B4D4: Treatment of BAP 2 ppm + 2,4-D 2 ppm.
Bioactive compound analysis was conducted using GCMS (
Table 8 . List of major compounds in field samples and B1D2 plantlets using the GC-MS analysis method.
No. | Compounds | Structure | Field sample | B1D2 Plantlet sample |
---|---|---|---|---|
1 | Benzene ethyl | ![]() | + | + |
2 | Benzene, 1-3-Dimethyl | ![]() | + | + |
3 | Pentadecanoic acid | ![]() | + | + |
4 | 9-Octadecenamide | ![]() | + | + |
5 | Octadecamethyl cyclononasiloxane | ![]() | ‒ | + |
6 | Benzene, 1,2-dimethyl | ![]() | ‒ | + |
7 | Hexadecamethyl cyclooctasiloxane | ![]() | ‒ | + |
8 | Eicosamethyl-cyclodecasiloxane | ![]() | ‒ | + |
9 | Tetracosamethyl cyclododecasiloxane | ![]() | + | ‒ |
The + sign indicates the presence of chemical compounds in the test sample with GC-MS.
Based on the data in Table 8,
The explants’ meristematic tissue contains endogenous hormones that aid in the formation of calluses. When endogenous hormones were coupled with BAP and 2,4-D, cells that would be actively differentiated began to pro-liferate. This finding was consistent with Fadhilasari
According to Robles
The color and texture of the callus were observed in this study as callus morphology. The texture of a callus is used to determine its quality. Friable and compact callus were the two types of callus texture identified. According to Karimi
According to Li
A certain concentration of cytokinin combined with auxin proved to be able to help explants produce calluses. However, the interactions between BAP and 2,4-D in this research do not affect the weight of fresh callus (Table 9). Cytokinin itself has an important role to develop a good callus quality. Mahadi
Table 9 . Average of
2,4-D (ppm) | BAP (ppm) | 2,4-D Average | ||||
---|---|---|---|---|---|---|
0 | 0.5 | 1 | 1.5 | 2 | ||
0 | 0.07 | 0.37 | 0.77 | 0.70 | 2.01 | 0.78a |
0.5 | 0.09 | 0.12 | 0.66 | 0.69 | 0.70 | 0.45a |
1 | 0.13 | 0.33 | 0.98 | 0.77 | 1.63 | 0.77a |
1.5 | 0.85 | 1.61 | 2.17 | 0.97 | 3.47 | 1.81b |
2 | 0.81 | 3.61 | 2.16 | 1.96 | 2.97 | 2.30b |
BAP average | 0.40a | 1.20b | 1.35b | 1.02ab | 2.16c | - |
In Table 9 , a, b, c are the significant concentration levels, with arepresents the least, brepresents mild, and crepresents the highest of callus fresh weight. Numbers followed by the same letter in the same rows or column are not significantly different at 5% level DMRT. The (-) sign indicates there is no interaction.
Treatment with 2 ppm concentration of BAP was signi-ficantly different from any other treatment and can be concluded as the best treatment for increasing the fresh weight of callus. Acid secretion causes cell membrane relaxation by activating the enzyme at a certain pH. This enzyme is responsible for breaking the links between cellulose molecules in the cell wall. Turgor pressure occurs when cells absorb water molecules in response to an increase in soluble material concentration in the vacuole, allowing them to expand. Table 9 shows that the existence of 2,4-D alone has a significant effect on the weight of fresh calluses. Treatments with 0; 0.5; and 1 ppm were observed to have a similar effect to the fresh weight of calluses. However, the effect of those treatments was significantly different from treatment 1.5 ppm and 2 ppm concentration of 2,4-D.
The result also showed that higher concentrations of 2,4-D were able to produce higher callus dry weight. This indicates the significant effect of 2,4-D to callus weight. Another research by Abdelmaksood (2017) showed that auxin concentration plays a big role in callus quality, a combination of auxin and cytokinin was also a better treatment for the high frequency of callus. The drying process of calluses will reduce 60% to 90% its weight as in the research conducted by Osman
Those number was calculated based on the results of three replications of the calculation per treatment, and the MS base media with growth regulator concentration provided the best response. This statement is in accordance with the research of Paramartha
The long circumstances of each explant’s shoots vary. This can happen because the absorption of nutrients for regeneration varies depending on the explant. Shoots explant with no new shoot growth and a length of buds are conceivable because there is still sufficient nutritional content from the previous culture on the body of the explant. The appropriateness of the regulatory compounds administered during multiplication can also influence the length of the shoots. According to Buko and Trine (2020), the number of internodes indicates a potential mass increase because each node usually has at least one axillary shoot.
The number of shoots was calculated after 4 weeks of initiation. The result revealed that the presence of 2,4-D alone had a substantial effect on the number of shoots generated. However, the single treatment of BAP and the interaction of BAP and 2,4-D gave no significant effect on the number of shoots. Table 10 displays the result of the DMRT test on a single treatment of 2,4-D concentration. The treatment with 0.5; 1; 1.5; and 2 ppm showed no significantly different effect on the number of shoots formed. However, these treatments gave significantly different effects with treatment 2,4-D 0 ppm.
Table 10 . Average number of
2,4-D concentration (ppm) | Number of shoots |
---|---|
0 | 2.00b |
0.5 | 0.53a |
1 | 0.20a |
1.5 | 0.27a |
2 | 0.07a |
In Table 10 , a, b are the significant concentration levels, with arepresents the least and brepresents the highest of number of shoots formed. Numbers followed by the same letter in the same column are not significantly different at 5% level DMRT.
The parameter of the number of leaves can be observed right after observing the parameter of the number of shoots. Khan
Flavonoids have been found several times in various parts of
The results of the analysis indicated that in vitro cultivation was able to produce more types of active compounds than cultivation in the field. This is thought to be caused by differences in the organs used as samples and the presence of a self-protection system formed in the plantlets after explant injury. The GCMS method has pre-viously been applied to
Another compound found in both types of samples is
According to Falowo
Callus and shoots of
The authors would like to thank Sebelas Maret University for funding the Domestic Universities Colla-borative Research.
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