The acclimatization of banana plantlets cv. Barangan Merah resulting from in vitro culture using organic medium

Barangan Merah is a potential type of banana to be developed under in vitro condition. Acclimatization is the final stage in the propagation by in vitro culture. The use of acclimatization medium from organic materials in the surrounding environment can be an alternative in acclimatization process. This study aims to obtain the optimal organic medium in acclimatization process for the growth of Barangan Merah banana seedlings resulting from in vitro culture. The planting material used plantlets from in vitro regeneration of banana cv. Barangan Merah from Tissue Culture Laboratory, Agricultural Faculty, Universitas Syiah Kuala. The experiment used the individual pot technique with the application of organic medium types, namely husk charcoal, sand, leaf litter compost, and oil palm fruit bunches compost. The results of this research showed that the highest number of seedlings that were still survive until the end of the acclimatization process was found in the husk charcoal medium (71.43%). The best seedling growth according to the increasing of leaves number, leaf size, and plant height in the acclimatization process was found in leaf litter compost medium.


Introduction
Banana cv.Barangan Merah is a type of banana that has high economic value because of the nutritional content of vitamin C and beta-carotene [1,2], sweet taste and delicious aroma [3].This banana variety has limitations in its propagation because it does not produce seeds.Conventional propagation of banana plants used saplings took longer and caused these plants to be susceptible to soil-borne diseases such as Fusarium wilt.Therefore, non-conventional tissue culture techniques were employed to produce healthy banana cv.Barangan Merah seedlings quickly.This technique is a way to grow banana parts in the form of weevil (sucker) pieces into plantlets under aseptic conditions in vitro [4,5].
In vitro propagation of Barangan Merah banana plants needs to be accompanied by good plantlet acclimatization before being transplanted into the field.Acclimatization of micropropagation plants by in vitro to the field is very important because of the significant differences between the micropropagation and field environments.This is because plantlets in the in vitro environment are in conditions of minimal pressure and an optimal environment for their growth [6].Plantlet development during the acclimatization process is greatly influenced by the use of planting medium.The planting medium 1297 (2024) 012057 IOP Publishing doi:10.1088/1755-1315/1297/1/012057 2 needed for plantlet acclimatization was medium that did not decompose easily, porous, able to hold water well, had high nutrient elements and was available in large quantities according to the needs of the seedlings.This medium include compost, husk charcoal, and sand [7].Suitable acclimatization medium has an effect on root development and nutrient absorption for plant growth.The use of organic materials as an acclimatization medium can improve the physical and chemical properties of the soil [8].Organic material as a planting medium produces the best plantlet growth during the acclimatization process [9].
The use of husk charcoal as acclimatization medium produced the best average plant height and average number of leaves for acclimatization of Barangan Merah bananas [10].The use of organic materials alone as an acclimatization medium has not been found in research on acclimatization of Barangan Merah bananas.Therefore, this research aims to obtain the optimal organic medium in acclimatization process for the growth of Barangan Merah banana seeds resulting from in vitro culture.

Materials
The planting material used plantlets from in vitro regeneration of banana cv.Barangan Merah.That plantlets have grown to the cap of the culture bottle and has roots.The equipment and materials that used were plastic pots with a diameter of 10 cm, polybags with a diameter of 20 cm, transparent plastic bags with a diameter of 20 cm, husk charcoal, sand, leaf litter compost, oil palm fruit bunches compost, and mixture of soil + compost + husk charcoal with a ratio of 1:1:1.Other supporting tools were camera and writing tools.

Methods 2.2.1 Experimental design.
The research was conducted at the Tissue Culture Laboratory of Agricultural Faculty, Universitas Syiah Kuala from May to July 2023.The number of plantlets that were acclimatized was 19 plantlets.The plantlets were obtained from the Tissue Culture Laboratory, Faculty of Agriculture, Universitas Syiah Kuala.The research employed four treatments of organic medium, namely husk charcoal, sand, leaf litter compost, and oil palm fruit bunches compost, using the individual pot technique in the experiment.After one month, the seedlings that survive until the end of the acclimatization period were transplanted into soil + compost + burnt husk (1:1:1).

Acclimatization process and condition.
The acclimatization process was started with preacclimatization (hardening off) by placing the plantlets that were still in the bottle into the acclimatization room.Acclimatization process in this experiment was carried out in several stages.The first stage was to remove the plantlet from the bottle and carefully removed the agar medium that was still attached to the roots using water.The second stage was soaking the plantlets in the solution of fungicide 1 g L -1 then the plantlets were drained.The third stage was to plant the plantlets inside pot containing acclimatization medium according to the treatment.Plantlets were watered until they were in medium of water field capacity.Plantlets were tightly closed using transparent plastic.Acclimatized seedlings were placed in a place not exposed to direct sunlight with a temperature of 27ºC and air humidity of 43%.The seedlings that had been acclimatized for 4 weeks transplanted into polybags containing soil + husk charcoal + compost with a composition of 1:1:1.

Data observation.
Research parameters were observed for 4 weeks after planting.The observed parameters were percentage of live seedlings, plant height, number of leaves, leaf length, and leaf width.That parameters were observed at the beginning of acclimatization and the beginning of transplanting.The pH, total nitrogen (N), and organic carbon (C) content were analyzed at the Soil and Plant Research Laboratory, Faculty of Agriculture, Universitas Syiah Kuala.

Percentage of live seedlings = number of 𝑙𝑖𝑣𝑒 𝑠𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝑠 number of seedling planted in each treatment
(1)

Percentage of live seedlings
Pre-acclimatization (hardening) was carried out by placing the bottle of explant in the acclimatization room for one week (Figure 1d shown by arrow).This process served to maintain plant growth when adapting to the environment outside the bottle during acclimatization.Plantlets were planted in pots containing medium according to the treatment (husk charcoal, sand, leaf litter compost, and oil palm fruit bunches compost).Each individual seedling in the pot was covered with transparent plastic to keep the air humidity high (Figure 1d).The temperature was higher and the humidity was lower in the acclimatization room than when incubating plantlets in bottles.The acclimatization room had a temperature of 27ºC and relative humidity of 43% (Figure 1d).Covering was done so that the seedlings experience gradual environmental changes before being transplanted to an outdoor environment and exposed to direct sunlight.The in vitro growth environment is in optimal conditions with low light intensity, high humidity, aseptic conditions, and the medium contains lots of sugar and nutrients.This condition allows plantlets to grow heterotrophically and results in the plantlets formed having abnormal morphology, anatomy and physiology.Plantlets can wilt or dry out quickly and can die due to environmental changes when transferred from culture to the greenhouse or field.The intensity of sunlight is higher and the air humidity is much lower in a greenhouse or radiation field environment than in a culture bottle.The higher water potential of the substrate compared to medium water containing sucrose can cause plantlets to wilt quickly due to water loss [6].During the acclimatization process, plantlets required high air humidity for survival.Therefore, all plantlets were covered with transparent plastic to maintain humidity [9].
According to table 1, the live percentage of banana seedlings cv.Barangan Merah in medium husk charcoal was highest (71.43%).This showed that husk charcoal was highly effective for plantlets adaptation in the acclimatization process.compared to the percentage of live seedlings in palm oil fruit bunches compost medium (50%), Leaf litter compost medium (40%), and sand (0%).This is in accordance with seedlings that were acclimatized in the treated acclimatization medium were showed in Figure 2. Seedlings in charcoal husk medium (Figure 2a) had better appearance than seedlings in medium of sand (Figure 2b), palm fruit compost (Figure 2c), and leaf litter compost (Figure 2d).Husk charcoals come from incomplete burning of rice husks so that it takes the form of coarse grains, light and black in color.Husk charcoal as a planting medium has many pores for air circulation, is able to bind water, does not rot easily and is a source of potassium.These properties make it easier for the roots to penetrate the medium so that the roots develop well [11].Incomplete combustion of rice husks can increase the carbon and nutrient content of the resulting husk charcoal [12].Furthermore, the charcoal medium is a buffer so that it could neutralize if there is a mistake in administering the nutrients contained in the fertilizer and the nutrients could be absorbed by the roots [13].The sand medium tended to compact after watering until field capacity, causing the seedling leaves to become translucent and subsequently dying (Figure 2b shown by an arrow).There were no seedlings in the sand medium were survive at the end of the acclimatization process (Table 1).The same result was shown by research [14] on acclimatization of Tembesu plantlets (Fagraea fragrans Miq.) which all died 3 weeks after acclimatization.Research [15] on banana acclimatization showed that using sand alone produced the lowest growth percentage during 4 weeks of acclimatization.This may occured because sand has very poor water holding capacity, thereby affecting the relative humidity of plants.5

Number and size of leaves at the time of acclimatization and transplanting
Table 2 showed the number of leaves, leaf width, leaf length, and height of banana cv.Barangan Merah plantlets before acclimatization varied because the original plantlets also varied in height, number and size of leaves (leaf length and width).The number of leaves on seedlings treated with husk charcoal media before acclimatization was 3 leaves (Table 2), and did not increase until before transplanting (Table 3).The number of leaves on seedlings treated with Palm Fruit Bunch Compost (4 leaves) and Leaf Litter Compost (2.8 leaves) before acclimatization (Table 2) increased to 4.5 and 3.5 leaves respectively before transplanting (Table 3).This showed that the seedlings from the charcoal husk medium had not shown an increase in the number of leaves, even though they had a higher nitrogen content compared to other treatments of acclimatization medium (Table 4).2) each experienced an increase at the end of acclimatization (before transplanting) (Table 3).This showed that the seedlings in that three medium of acclimatization experienced growth.Seedling growth was related to the medium and availability of nutrients in the acclimatization medium, such as nitrogen (Table 4).
The higher plantlets were found in the sand medium (Table 2) did not survive until the end of acclimatization or before transplanting (Table 3).This showed that taller seedlings were not necessarily able to adapt well when acclimatized in sand medium.This was different from the seedlings in the Husk Charcoal, Palm Oil Fruit Bunches Compost, and Leaf Litter Compost medium which remained survive and growing.Seedlings on Leaf Litter Compost medium showed above average growth (Table 3) even though the number of leaves, leaf size (length and width), and plant height were lower before acclimatization compared with seedlings on Husk Charcoal and Palm Oil Fruit Bunches medium.Figure 3 showed a graph of the Barangan Merah banana seedlings growth on several acclimatization medium.The numbers for each seedling growth parameter were obtained from subtracting the parameter values for number of leaves, leaf width, leaf length and plant height before the transplanting process (Table 3) and before the acclimatization process (Table 2).The seedling growth graph showed that the best seedling growth was found in the leaf litter compost treatment, namely the number of leaves increased by 0.7, leaf width increased by 1.14 cm, leaf length increased by 1.7 cm, and plant height increased by 1.14 cm.This was in accordance with the results of the analysis of the highest total N content found in leaf litter compost medium i.e. 0.83% total N (Table 4).The high N content in the acclimatization medium is closely related to seedling growth during the acclimatization process.As mentioned in research [16] that the nitrogen nutrient was needed by plants for leaf formation, thus influencing the increase in the number of leaves.

Figure 1 .
Figure 1.Plantlets to be acclimatized (a and b), measurements plantlet (c), hardening (showed by arrow) and covering per individual plant on acclimatization process (d)

Figure 2 .
Figure 2. Barangan Merah banana seedlings in charcoal husk medium (a), sand (b), palm fruit compost (c), and leaf litter compost (d) at the end of the acclimatization process

Figure 3 .
Figure 3.The increasing of leaves number, leaf width (cm), leaf length (cm), and plant height of seedlings in the acclimatization process of banana seedlings cv.Barangan Merah

Table 1 .
Percentage of live seedlings in the acclimatization process of banana seedlings cv.Barangan Merah.

Table 2 .
The average number of leaves, leaf width, leaf length, and plant height before the acclimatization process of banana seedlings cv.Barangan Merah.

Table 3 .
The average number of leaves, leaf width, leaf length, and plant height before the transplanting process of banana seedlings cv.Barangan Merah.

Table 4 .
Analysis of Total N (%) and Organic C (%) in acclimatization medium of banana seedlings cv.Barangan Merah.