BIOTROPIKA Journal of Tropical Biology
https://biotropika.ub.ac.id/
Vol. 11 | No. 3 | 2023 | DOI: 10.21776/ub.biotropika.2023.011.03.02
MORPHOGENESIS RESPONSE OF LEAF AND PETIOLE EXPLANT OF ROOTSTOCK APPLE (Malus sylvestris Mill.) TO AUXIN AND CYTOKININ
Nafika Aulia Wirentyas1), Aminatun Munawarti1)*
ABSTRACT
Apple rootstock is a plant type used in grafting propagation. Research related to the response of morphogenesis in this plant has not been widely carried out. This study aims to examine the influence of auxin and cytokinin ratios on morphogenesis in leaf and petiole explants of rootstock apples, seeking optimal response modifications. A 2-factorial, completely randomized design (CRD) was utilized in the investigation, with variations in the types of explants and combinations of plant growth regulators (PGR) in the culture medium. There were 12 treatments with five repetitions. Explants of young apple leaf (third leaf) and petiole rootstock that had been sterilized and cut with a size of 5x5 mm for the leaf and 5 mm long for the petiole were inoculated into MS medium with cytokinin, BAP (1 ppm) combined with auxin, IBA (0, 0.1, 0.2, 0.3, 0.4, 0.5 ppm). Each culture bottle contains five explants. The results showed that the average leaf explant was capable of producing 34% callus. Meanwhile, petiole explants produced 4% callus. Callus began to form on leaf and petiole explants on the 26th and 42nd days after inoculation, respectively. Most calluses have a compact texture with green, yellowish-white, and brownish-green variations. The PGR combination with the highest percentage of callus production in leaf explants was BAP 1 ppm + IBA 0.4 ppm treatment, whereas the petiole was BAP 1 ppm + IBA 0.3 ppm treatment. Therefore, the combination of PGR with the best callus response in this study can be used as a reference in the development of apple tissue culture.
Keywords: BAP, callus formation, IBA, tissue culture of apple
INTRODUCTION
The need for fruit intake is currently increasing, especially during the COVID-19 (Coronavirus Disease 2019) pandemic, which requires people to adjust their lifestyles to be healthier. Apple is a popular fruit with various nutritional contents such as antioxidants, vitamins, fiber, and other nutrients.
Because of the benefits supplied by this fruit, it is the world’s second most significant fruit crop, with a total production of 69.5 million metric tons per year after bananas, based on 2010 data from the Food and Agriculture Organization (FAO).
Massive apple cultivation has begun to be developed in various regions of Indonesia, including East Java Province. The largest apple cultivation center in East Java is located in Batu City, Malang. Despite this, apple cultivation has declined in the last ten years. One of the contributing factors is the declining number of apples of productive age [1]. Basically, apples can be propagated generatively or vegetatively, but generative propagation often encounters problems because apple seeds have a low germination ability, so most farmers choose the vegetative method through a grafting technique known as the grafting method. Along with its development, propagation through grafting is also experiencing problems due to the limited number of rootstock apples used as rootstock [2]. This is supported by
the explanation [3] that the quality of the rootstock of apple plants in Indonesia is not healthy.
Rootstock apples that are used as root material should have a strong root system and defense to support the growth of the scion properly.
Therefore, rootstock apple cultivation needs to be done. Plant propagation in vitro by tissue culture can be employed as an alternative to providing rootstock seeds. Some of the advantages of tissue culture techniques are being able to obtain plant material in large and uniform quantities only from tissue or even from plant cells, the seeds produced are free of plant destructive organisms, and the growth of seeds that are not affected by environmental conditions such as seasons [4].
The composition of the culture media has a major influence on the growth and development of plants propagated via tissue culture. A growth regulator is a component of culture media that plays an important function in cell division and elongation. Auxin and cytokinin are two types of Plant Growth Regulator (PGR) that are often combined with various concentrations to determine the morphogenesis response of explants.
Several studies related to apple tissue culture with various variations of PGR concentrations have been carried out, including studies by [2], [4], and [5]. Based on this research, it is known that 6- benzylaminopurine (BAP) and indole-3-butyric acid (IBA) are hormones that can provide a good response to explant growth. Therefore, in this
Submitted : August, 4 2023 Accepted : January, 9 2024
Authors affiliation:
1) Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya
Correspondence email:
How to cite:
Wirentyas, NA, Munawarti A. 2024.
Morphogenesis response of leaf and petiole explant of rootstock apple (Malus sylvestris Mill.) to auxin and cytokinin.
Biotropika: Journal of Tropical Biology 11 (3): 133-139.
study, BAP will be combined with auxin in the form of IBA to determine the effect of varying the PGR ratio given on callus induction in explants.
The use of explant types also influences the success of tissue culture techniques. The selection of leaves as a source of explants has previously been carried out in a study [4]. Whereas culture with petiole explants has never been done before.
Leaves and petioles are alternative explants because there are many of them, and they are easy to obtain without damaging the plant. On the other hand, the use of petioles as explants on rootstock apple plants has never been done before, so in this study, both parts are used as explants. As a result, this research aims to investigate the impact of variations in the ratio of auxin and cytokinin and the ratio with the best response on the morphogenesis of leaf explants and rootstock apple petioles.
METHODS
Culture media preparation. Materials for making MS medium in the form of macro and micronutrient stock solutions, vitamins, and myoinositol were taken using a micropipette with a predetermined volume. Subsequently, 30 g/L sucrose was added, and the solution was adjusted to a volume of 600 mL. These ingredients are then poured into six Erlenmeyer bottles of 100 mL each.
Next, to each solution in the Erlenmeyer was added PGR in the form of BAP 1 ppm and IBA, which varied in concentration (0, 0.1, 0.2, 0.3, 0.4, and 0.5 ppm). After PGR was added, a pH meter was used to regulate the pH. Solutions with a pH of less than 5.8 are added with NaOH, while solutions with a pH greater than 5.8 are added with HCl. The media was cooked on a hot plate with 10 g/L of agar powder. After boiling, each solution in the Erlenmeyer was poured into ten culture bottles until a volume of 10 mL was reached in each bottle.
After that, the media was sterilized in an autoclave for 15 minutes at 121°C and 1.5 atm pressure. After sterilization, the media was stored at room temperature until it solidified.
Explants sterilization. Young leaves (the third leaf of the shoot) and petioles of healthy apple rootstock (rootstock) were selected as explants.
After that, the explants were washed with detergent and rinsed under running water for 30 minutes. The explants were then sterilized in the Laminar Air Flow (LAF) for 15 minutes by immersing them in the fungicide solution. This is done to prevent the appearance of fungal contaminants in the field during explant collection, which can inhibit the explant growth process. Next, the explants were taken and immersed in a sterile solution (30%
ProclinTM with the active ingredient NaClO 5.25%), that had been treated for 10 minutes with
two drops of tween 20. The explants were cleaned three times, each for five minutes, in the final stage with sterile distilled water [6].
Explant inoculation. Inoculated leaf explants (5x5 mm) with the abaxial section in contact with the media. While the petiole (5 mm) is positioned horizontally to the media MS supplemented in the form of cytokinin BAP 1 ppm and auxin IBA, which varies in concentration (0, 0.1, 0.2, 0.3, 0.4, and 0.5 ppm). The abaxial side of the leaf has more stomata so that the process of nutrient absorption from the medium to the leaf tissue can be optimized. Meanwhile, petiole explants are positioned horizontally to expand the area that is exposed to the culture medium. Culture maintenance is carried out by storing culture bottles in a culture room with a temperature of 18–
24 °C in the dark [2].
Parameters observation. The parameters observed in this study consisted of:
1. Percentage of surviving explants (%) Observation of live explants was carried out at the age of 4 and 8 weeks after inoculation (WAI) for each treatment with formula (1):
% 𝑠𝑢𝑟𝑣𝑖𝑣𝑖𝑛𝑔 𝑒𝑥𝑝𝑙𝑎𝑛𝑡𝑠 = ∑𝑙𝑖𝑣𝑒 𝑒𝑥𝑝𝑙𝑎𝑛𝑡𝑠
∑𝑒𝑥𝑝𝑙𝑎𝑛𝑡𝑠 𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑑 𝑥 100%(1) 2. Percentage of callus formed (%)
Observations were made from when the first callus appeared until age 8 WAI. The percentage is calculated by formula (2):
% 𝑐𝑎𝑙𝑙𝑢𝑠 𝑓𝑜𝑟𝑚𝑒𝑑 = ∑ 𝑐𝑎𝑙𝑙𝑢𝑠 𝑓𝑜𝑟𝑚𝑒𝑑
∑𝑒𝑥𝑝𝑙𝑎𝑛𝑡𝑠 𝑐𝑢𝑙𝑡𝑢𝑟𝑒𝑑𝑥 100%...(2)
3. Callus initiation times (day after inoculation/DAI)
The formation of callus that appeared was observed on apple leaf explants and petioles every day by counting the days since the inoculation was carried out.
4. Callus texture
Measurements were done at the end of the research (8 WAI) by observing the callus texture, including the friable or compact type.
5. Callus color
Observations were done by observing and determining the callus category into brownish green, yellowish white, and green when the explants were 4 and 8 WAI.
Research design. The study used a completely randomized design (CRD) with two factors. There were six treatments consisting of media with the addition of PGR in the form of BAP and IBA. Each treatment was repeated five times for all media treatments, for 60 culture bottles (Table 1).
Table 1. Research of morphogenesis response of leaf and petiole explant of rootstock apple (Malus sylvestris Mill.) to auxin and cytokinin design
PGR Combination
(ppm) Explant
BAP IBA (IB) Leaf (L) Petiole (P)
1
0.0 LIB0 PIB0
0.1 LIB1 PIB1
0.2 LIB2 PIB2
0.3 LIB3 PIB3
0.4 LIB4 PIB4
0.5 LIB5 PIB5
Data analysis. Descriptive data is explained and compared with related literature, while quantitative data is analyzed by calculating percentages presented in graphical form and carrying out normality, homogeneity, Kruskall- Wallis, and ANOVA tests. If there is a significant difference, then proceed with the 5% Mann- Whitney and Tukey tests.
RESULTS AND DISCUSSION
Explant live percentage. The calculation of the percentage of living explants aims to identify the type of treatment medium that can support the survival of the explants. Explants are said to be alive if they are not contaminated and do not experience browning. Based on the calculation results, the survival percentage of leaf and petiole explants decreased at 8 WAI, with the lowest percentage in the BAP 1 + IBA 0 ppm treatment (Table 2). According to Mayura [7], the addition of growth regulators to the medium improved the ability of the explants to survive. In addition, factors that influence the survival of explants are the type and composition of the medium and the condition of the explants themselves [8].
Table 2. Percentage of both explants aged 4 and 8 WAI
PGR Combination (ppm)
Live Explants (%) 4 WAI 8 WAI
L P L P
BAP 1 + IBA 0.0 40 0 12 0 BAP 1 + IBA 0.1 76 12 56 8 BAP 1 + IBA 0.2 88 0 56 0 BAP 1 + IBA 0.3 76 28 64 12 BAP 1 + IBA 0.4 76 12 72 4 BAP 1 + IBA 0.5 92 20 80 4
The decrease in the percentage of live explants at the age of 8 WAI could occur due to the browning of the explants. Browning is caused by the buildup of phenolic chemicals produced by explants in response to tissue injury. This is further strengthened because the apple is a tropical plant
with high phenol levels [2]. According to the findings of the study, the addition of 1 ppm BAP in all treatments was able to maintain the explant’s ability to live. In a study conducted by Alqamari et al. [9], 1 ppm NAA + 1 ppm BAP produced the best percentage of live explants. BAP is a cytokinin hormone that stimulates cytokinesis or cell division; hence its presence is important for the process of explants growth and development [10].
The addition of IBA as an auxin is also important to maintain explant survival. This is consistent with the study’s findings, which revealed that the treatment with IBA had a higher survival rate than the treatment without IBA (LIB0). The LIB0 treatment had the lowest survival percentage because no exogenous auxin in the form of IBA was added. This is in line with the explanation [9] that combining auxin and cytokinin hormones can improve tissue survival, growth, and development in explants.
Callus percentage. Based on the results of observations that have been made for eight weeks, it can be seen that all types of explants are capable of forming calluses. The formation of a callus is characterized by swollen explants. As time goes by, callus begins to form in the cut area. The average callus initiation time on leaf explants occurred 26 days after inoculation (DAI).
Meanwhile, the petiole explants occurred at 42 DAI. According to Mastuti et al. [11], the formation of a callus is obtained in two stages, namely induction and proliferation. During the tissue induction stage, the explants undergo a dedifferentiation process that causes the cells that make up the mature leaf tissue to turn into meristematic cells. Meanwhile, the proliferative stage occurs when cells are actively dividing, as a result of which the number of cells increases.
The term callus in plant biology refers to the massive growth of cells that form in response to injury to plant tissue. The callus is irregular in shape (amorphous) and is totipotent or capable of regenerating all plant body parts [12]. The formation of calluses on explants is also supported by the administration of balanced auxin and cytokinin hormones, both endogenous and exogenous [13]. Even though the cytokinin hormone levels in the media treatment were much higher than the auxin hormone, all explants produced a response in the form of callus formation. This can occur due to the accumulation of endogenous hormone levels in the explants and exogenous hormones in balanced media, thus triggering callus growth (Figure 1). In leaf explants, the percentage of callus formation at 4 WAI continued to increase with the addition of IBA levels. Meanwhile, at the age of 8 WAI, the
highest percentage was found in the BAP 1 + IBA 0.4 ppm treatment (Figure 1).
Figure 1. Percentage of leaf explant forming calluses at 4 and 8 WAI. In the Mann-Whitney test, different letters indicate a significant difference.
In contrast to leaf explants, in petiole explants, the number of calluses that were successfully formed was only found in a few explants in one treatment. Of all PGR combination treatments, the PIB3 treatment (1 ppm BAP + 0.3 ppm IBA) had the highest overall average percentage of callus formation compared to the other treatments (Figure 2).
Figure 2. Percentage of petiole explants forming calluses at 8 WAI
Callus formation on both explants only occurred in the course of treatment with the addition of auxin. This is in line with the findings of research [14], which reveal that auxin plays a role in the callus induction process by stimulating cell elongation and division, while cytokinins play a part in division processes, growth, and development. Combining these two hormones can increase the ability of cells to divide, increase protein synthesis, and trigger the formation of calluses and secondary metabolites. This was also clarified by Nasution & Nasution [15], who stated that combining cytokinin and auxin could stimulate callus growth more than alone.
According to Lestari et al. [16], PGR has the ability to work effectively at certain concentrations. In addition, each plant has an optimal limit for the concentration of IBA, so if the concentration of IBA exceeds the optimal limit of
the tissue in the plant, growth inhibition will occur.
Nonetheless, the percentage of callus formation in all treatments except LIB0 (1 ppm BAP + 0 ppm IBA) increased at 8 WAI. This shows that the cells in the explants are still actively dividing to form a callus. According to Mastuti et al. [11], cleavage continues continuously until the formation of callus masses can occur due to the availability of appropriate exogenous and endogenous hormones.
The number of petiole explants that responded to form a callus was 4–12%. This amount is lower when compared to the callus produced on leaf explants. This was caused by the large number of petiole explants that were contaminated by both fungi and bacteria (Figure 3). Contamination by fungi is indicated by the appearance of mycelia, or white fibers covering the explants. These contaminants quickly spread to all explants in a short time. Meanwhile, bacterial contaminants are characterized by the appearance of mucus around the explants and can quickly cause the death of the explants [17]. The number of contaminated petiole explants can also be affected by their thicker structure than leaves, so a greater sterilant concentration is required. In addition, apple plants have petioles with more trichomes than the leaves [18]. The presence of trichomes in explant sources can be a habitat for various microbes so that they are susceptible to contaminants, both fungi and bacteria [19].
Callus texture. Callus texture is critical in determining the quality of callus formed by explants. Based on the type of texture, callus can be divided into compact and friable callus types.
Compact-type calluses have a denser texture and do not cut easily. Meanwhile, the crumb callus type has a loose texture, is easily separated between its parts, and contains more water (Figure 4). Several factors may influence callus texture, namely the composition of the nutrient medium, the growth hormone used, the culture environment conditions, and the type of plant used as a source of explants [20].
A good callus is thought to have a crumb texture because it is easy to separate into single cells for
0 10 20 30 40 50 60 70 80 90
BAP 1 + IBA 0
BAP 1 + IBA 0,1
BAP 1 + IBA 0,2
BAP 1 + IBA 0,3
BAP 1 + IBA 0,4
BAP 1 + IBA 0,5
Percentage of calluses (%)
Treatment (ppm)
a a
a a
ab
a ab
bc bc
bc
bc
0 10 20 30 40 50 60 70 80 90
BAP 1 + IBA 0
BAP 1 + IBA 0,1
BAP 1 + IBA 0,2
BAP 1 + IBA 0,3
BAP 1 + IBA 0,4
BAP 1 + IBA 0,5
Percentage of caluuses (%)
Treatment (ppm)
4 WAI 8 WAI
A B
Figure 3. Contaminated petiole explants ( ). (A) Fungal contamination on PIB5 explants aged 7 DAI; (B) Bacterial contamination on PIB3 explants aged 5 DAI
1 cm 1 cm
Figure 4. Callus texture on apple leaf and petiole explants (A) Callus crumbs of LIB2 explants aged 4 WAI; (B) Compact callus of LIB4 explants aged 8 WAI; (C) Callus crumbs of PIB3 explants aged 8 WAI; (D) Compact callus of PIB5 explants aged 8 WAI.
suspension culture. Good callus quality depends on the desired goal [15]. In this study, the callus texture formed was dominated by compact texture.
These results are in accordance with research [2]
on apple leaf culture, which also produces calluses with a compact texture. According to Afiyah et al.
[21], callus with a compact texture is caused by the hormones auxin and cytokinin, which can affect the water potential in cells. Callus with a compact texture can be formed due to differences in the ability of plant tissues to absorb nutrients and PGR [22]. Compact callus has the potential to develop into organs such as roots and shoots indirectly through the process of organogenesis, even though its growth is relatively slow [15].
Callus color. The color of calluses indicates the success rate of cell development. Calluses have various colors, such as green, yellow, white, or red,
depending on the pigment content of the cells that make up the callus [18]. Based on the results of callus studies on leaf explants and rootstock apple petioles, they showed green and yellowish-white colors (fresh green and green rice on the color chart) at the age of 4 WAI.
The bright color of the callus indicates that the condition of the callus cells is still in good condition and has not yet entered the stationary phase [20]. However, at the time of observation, reaching the age of 8 WAI, there is a callus that changes color to brownish green (olive green on the color chart) (Figure 5). The discoloration of the callus at the age of 8 WAI is an indication of a change in the growth phase and a decrease in regeneration power, along with the increasing age of the cells that make up the callus [15].
According to Lizawati [22], a green callus indicates that chlorophyll is present in the callus tissue. The presence of green pigment in calluses is caused by the content of cytokinins, which play a role in inhibiting cell aging. This is backed by the results of calculating the percentage of live explants and callus formed on study’s findings, which revealed that most calluses were green in all treatments with a cytokinin content of 1 ppm.
Meanwhile, the brownish color of the callus indicates signs of cell aging. This can be anticipated by subculture periodically on the callus.
Discoloration to brown (browning) in the callus can also be triggered by cell phenolic substance production [15].
According to Azzahra et al. [2], rootstock apple leaf explants are more prone to browning than other explants, such as shoots. This is because the leaf tissue is thinner, so it is susceptible to cutting, which triggers injury to the tissue and impacts the physiological function of the explants.
Figure 5. Callus color diversity in leaf (A-C) and petiole explants (D-F). (A) Green on DIB4 explants aged 8 WAI, (B) Yellowish white on DIB2 explants aged 4 WAI, (C) Brownish green on DIB5 explants aged 8 WAI, (D) Yellowish white on PIB1aged 16 WAI, (E) Green on PIB5 aged 6 WAI, (F) Brownish green on PIB5 aged 8 WAI
1 cm A 1 cm B
D
1 cm C
1 cm
B 1 cm
A 1 cm C 1 cm
D 1 cm E 1 cm F 1 cm
The apple plant is also classified as a tropical plant with high levels of phenolic compounds, so if there is injury to the tissue, it will be easily oxidized [23]. Cutting apple leaf explants will cause the polyphenol oxidase (PPO) enzyme to oxidize phenolic compounds and trigger an quinone reaction, forming a brown color [24].
Treatment with the best callus response.
Based on the results of the parameters that have been calculated and observed, it can be seen that the treatment with the best response of leaf explants was the 1 ppm BAP + 0.4 ppm IBA treatment. This is supported by the results of calculating the percentage of live explants and callus formed on leaf explants, which was higher than other treatments at the age of 8 WAI.
Meanwhile, in the LIB5 treatment (1 ppm BAP + 0.5 ppm IBA), the percentage of live explants at 4 WAI and 8 WAI was relatively stable, but these explants were not optimal in forming calluses like in the LIB4 treatment (1 ppm BAP + 0.4 ppm IBA).
Several treatments on petiole explants, such as PIB1 (1 ppm BAP + 0.1 ppm IBA), PIB3 (1 ppm BAP + 0.3 ppm IBA), PIB4 (1 ppm BAP + 0.4 ppm IBA), and PIB5 (1 ppm BAP + 0.5 ppm IBA), succeeded in forming calluses. However, petiole explants showed a lower percentage of callus formation than leaf explants. This was also influenced by the number of petiole explants that were contaminated.
CONCLUSION
Based on the study’s findings, it is possible to deduce that the addition of auxin variations in the form of IBA (0.1, 0.2, 0.3, 0.4, and 0.5 ppm) and cytokinin in the form of BAP (1 ppm) in culture medium produces callus on both leaf expanses and petioles. Most of the calluses produced have a compact texture with variations in green, yellowish-white, and brownish-green. Treatments with a ratio of 1 ppm BAP + 0.4 ppm IBA and 1 ppm BAP + 0.3 ppm were able to produce the highest percentage of callus on leaf and petiole explants, respectively, compared to other treatments. It is necessary to have an appropriate sterilization method for rootstock apple leaf petiole explants. In addition, various combinations of cytokinin concentrations also need to be added to determine the effect of cytokinins more accurately.
ACKNOWLEDGMENT
The author would like to express gratitude to the Laboratory of Physiology, Tissue Culture, and Plant Microtechnics (FKM), Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, for
providing facilities and infrastructure during the research process.
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