Organs formation on Jabon trees (Anthocephalus cadamba (Roxb) Miq) by in-vitro: treatment of various iba-kinetin combination

The objective of this study aimed to determine the concentration of the combination Indolebutiric acid (IBA) - Kinetin suitable for the formation of roots of the Jabon plant (Anthocephalus cadamba (Roxb) Miq) in vitro. This research is a follow-up study of the initiation and propagation of in vitro shoots of Anthocephalus macrophyllus (Roxb) Havil). The study was conducted at the Forestry Biotechnology Laboratory, Department of Forestry, Faculty of Forestry, University of Tadulako from February 2018 to August 2018. The study was arranged in a completely randomized design with the addition of IBA and Kinetin to Murashige and Skoog basic medium in various concentrations as treatments. Five concentration levels of combinations of IBA + Kinetin were studied, namely: H1(0.1 ppm IBA + 3 ppm Kinetin), H2 (0.5 ppm IBA + 1 ppm Kinetin), H3 (1.0 ppm IBA + 0.5 ppm Kinetin), H4 (1.5 ppm IBA + 0.3 ppm Kinetin), H5 (2 ppm IBA + 0.1 ppm Kinetin). The experimental results showed that the combination of 1 ppm IBA + 0.5 ppm Kinetin concentration could promote the formation of Anthocephalus cadamba roots the best compared to other treatments. Treatment of 0.1 ppm IBA + 3 ppm kinetin and treatment of 0.5 ppm IBA + 1 ppm kinetin did not respond to root formation, but encouraged the formation of the best shoots of Anthocephalus cadamba.


Introduction
The forestry industry has an important role in the Indonesian economy, doesn't it not only as a provider of the main raw materials for the downstream industry, especially logs, but also to increase economic growth through added investment value, increased export performance, state revenues through taxes and non-taxes, as well as creating business opportunities and employment.In the Production Forest Development Road Map for 2019-2045, currently the forestry industry is in a sunset industry condition, namely an industry that is difficult to develop, even though its potential is enormous.This is caused by various problems such as low business certainty, land conflicts, low productivity of land, high production costs, and low competitiveness of the wood processing industry [1].
At present, wood originating from natural forests cannot be relied upon to support demand in local, domestic and international markets.Initially, wood obtained from natural forests was able to produce millions of cubic meters, but currently the need for wood supplies is difficult to meet if you only rely on stands from natural forests.Natural forest productivity has decreased from year to year due to illegal logging, forest fires and reduced forest area due to conversion of forest land into residential, plantation and agricultural areas [2].The demand for forest product products such as wood is believed to continue to increase both domestically and abroad.However, the challenges for the timber industry from time to time are also getting tougher.Production of timber forest products also continues to decline due to a lack of supply of raw materials.Many wood processing companies closed their businesses because they did not get supplies of raw materials, and some industries even imported wood raw materials from other countries.If this condition continues, the supply of logs for the timber industry in the future may be threatened, environmental damage such as deforestation and forest degradation will worsen, and international market confidence in Indonesian wood products will decrease [3].
The availability of sufficient quantities of superior quality planting material or seedlings is a prerequisite for the success of various trees expansion programs.Availability of sufficient quantity of planting materials of superior quality is a pre-requisite for any extensive tree planting program to be successful.
There is a gap between supply and demand for wood or wood products that continues to widen, so it is a very important need to improve the quality and quality of trees.Most of the trees grown from seeds obtained from open crosses and therefore are heterozygous.As the gap between the supply and demand of wood and wood products is widening, there is an urgent need to improve the quality and quantity of trees.Most forest trees are grown from seeds obtained as a product of open pollination and are therefore heterozygous.Moreover, during the production of seeds, there is reshuffling of the genes which generates more variability among progeny.Thus, the sexually reproduced progeny of the selected superior trees need not be superior like the mother trees and also, they will not be uniform among themselves.Under such circumstances, vegetative propagation of the selected plus trees will be ideal to pass on all the favorable attributes to its progeny.However, conventional methods of vegetative propagation like grafting or budding has a limitation that the number of plantlets that can be produced may not be sufficient to meet the requirement of hundreds and thousands of planting materials.Here comes the importance of micropropagation is attaining considerable attention for obtaining large number of genetically uniform quality planting stock using in vitro methods.
One of the woody plants that has prospects for development in order to meet the needs of the wood processing industry in Indonesia is the Jabon plant (Anthocephalus cadamba (Roxb).Miq).This plant is a type of tropical tree originating from South Asia and Southeast Asia [4].
This plant is very suitable to be developed as an industrial plantation forest due to it is a fast-growing species with high production.Annual growth averages 2.2 m in height and 3.65 cm in diameter [5,6].The trunk is cylindrical and straight, the wood is yellowish white without visible fiber, the production period for Jabon is short, 5-6 years, it does not require pruning because during the growth period the branches will fall off on their own (self-pruning) so it is very good for making plywood or sawn timber.Furthermore, the increasing global demand for wood and the decreasing population of woody plants and the reduced forest area are opportunities to develop Jabon plantations as industrial forest plantations.
One of the silvicultural measures that plays an important role in the success of industrial plantation forest development is the availability of quality seeds in the required quantities.So far, Jabon plant propagation has been done by seed, where (which) is this method has drawbacks, namely it requires a longer time to produce a lot of seeds and the seeds produced cannot guarantee the same characteristics as their parents.The tissue culture technique seems to be an alternative for Jabon plant propagation which can overcome the problem of seed availability to meet the needs of various Jabon plant cultivation programs.This technology has advantages over other plant propagations, including being able to produce a large number of superior seeds in a short time, using a small amount of plant material, producing sterile or disease-free cultures (mother stock) which can be used for further propagation, and not requiring large areas.for the production of seedlings or seedlings and not depending on the season, the seeds produced have the same genetic characteristics as their parents [7][8][9].Seedlings of superior quality is one of the requirements for the success of various tree planting expansion programs.There is a gap between supply and demand for wood or wood products that continues to widen, so it is a very important need to improve the quality and quality of trees.Most of the trees grown from seeds obtained from open crosses and therefore are heterozygous.Propagation through tissue culture either by organogenesis or through embryogenesis will produce seedlings whose genetic characteristics are exactly the same as their parents.Therefore, tissue culture technology will assist breeding activities in multiplying and maintaining the superior traits of selected trees as one of the breeding activities.same as its parent [10].
In general, there are two methods of propagation through tissue culture, namely organogenesis and embryogenesis methods.The organogenesis method usually goes through several stages, namely the stages of initiation, multiplication, rooting, acclimatization and post-acclimatization enlargement of seedlings (Dodds, 1995).The use of growth regulators in plant tissue culture is very important, namely to control organogenesis and morphogenesis in the formation and development of shoots and roots and the formation of callus.There are two groups of growth regulators that are often used in tissue culture, namely the auxin group and the cytokinin group.The use of growth regulators in tissue culture depends on the desired direction of tissue growth.For the formation of shoots, cytokinin's are generally used, while for the formation of roots or callus, auxin is usually used.However, both are often needed depending on the ratio or ratio of cytokinin's to auxins or vice versa [8].The presence of one particular growth regulator can increase the activity of other growth regulators.The types and concentrations of growth regulators that are appropriate for each plant are not the same because it depends on the genotype and the physiological conditions of the plant tissue used as a source of explants [11].
Research on Jabon plant tissue culture has not been widely reported.Marzuki S et al 2016 reported success in initiating and multiplying Jabon plants at various concentrations of indole Butyric Aid (IBA) and kinetin, but had not succeeded in forming roots [12].Rooting is an important stage in tissue culture prior to acclimatization, because plantlets (plants resulting from tissue culture) that are formed without roots will find it difficult to grow and develop into perfect seedlings when acclimatized and transferred to the field.
Based on the above background, research on the formation of Jabon plant roots in vitro was carried out by adding various concentrations of the combination of Indole Butyric Acid (IBA) + Kinetin on the basic medium for plant propagation of Murashige and Skoog (MS).

Time and place
This research was conducted in February until the end of August 2018 at the Forest Biotechnology Laboratory, Faculty of Forestry, Tadulako University, Palu.

Tool and material
The tools used in this study consisted of: autoclave, analytical balance, pH meter, hot plate, magnetic stirrer, water distillation apparatus, refrigerator, laminar air flow cabinet, culture rack, oven, beakers, measuring cups of various sizes, petri dishes, scalpel, knife, stir bar, test tube, funnel, pH meter, culture bottle, aluminum foil, camera and stationery.
Materials used include: chemical composition of MS basic media, casein hydrolyzate, pyridoxine-HCl, calcium pantothenate, riboflavin, ascorbic acid, Jabon plant plantlets from previous in vitro propagation as a source of explants, alcohol, spirit, tween 80, HCl 0 .1 N, 0.1 N NaOH, IBA and Kinetin growth regulators, fungicide, bactericidal, bay clean, label paper, agar-agar, sugar, distilled water and rubber bands.
This study used a completely randomized design using one factor with five (5) treatment levels of the IBA + Kinetin combination, namely: H1 = 0.1 ppm IBA + 3 ppm kinetin H2 = 0.5 ppm IBA + 1 ppm kinetin H3 = 1.0 ppm IBA + 0.5 ppm kinetin H4 = 1.5 ppm IBA + 0.3 ppm kinetin H5 = 2 ppm IBA + 0.1 ppm kinetin Each treatment was repeated six (6) times so that there were 30 experimental units.Whether there is a callus that is formed, by observing the formation of callus during the study.2. Total of roots formed were observed during the research, with a minimum root length of 0.5 cm. 3. Total of leaves formed during the study.4. Total of shoots formed during the study, both axillary and adventitious shoots.
To determine the effect of the concentration treatment of the combination of IBA + Kinetin growth regulators on the observed parameters, an analysis of variance was carried out to determine which treatment had a significant effect.While the follow-up test is a real difference to be honest or BNJ.The level of 5%, carried out for between treatments that are significantly different.

Implementation
a. Tool sterilization is carried out using an autoclave, namely by inserting the tools to be used the autoclave.Furthermore, the autoclave is connected to electricity until the pressure reaches 15 psi and this pressure is maintained for 30 minutes.Furthermore, the tool is removed and then stored in the oven before use.b.Media preparation is carried out by weighing the chemicals according to the concentration of the composition of the Murashige and Skoog (MS) basic media and then adding amino acids and vitamins according to the needs of the MS basic media.Then five liters of basic media containing vitamins and amino acids were made and each one liter was added with a combination of IBA + Kinetin with the concentration according to the treatment then the pH was adjusted to 5.8 using HCl or NaOH.Then put it in a sterile culture bottle that has been tightly closed and labeled according to the treatment.Culture bottles containing MS basic media added with a combination of IBA + Kinetin according to treatment and labeled respectively H1 (0.1 ppm IBA + 3 ppm Kinetin), H2 (0.5 ppm IBA + 1.0 ppm Kinetin), H3(1.0 ppm IBA +0.5 ppm Kintein), H4 (1.5 ppm IBA +0.3 ppm Kinetin) and H5 (2.0 ppm IBA + 0.1 ppm Kinetin).The bottles containing the treatment were sterilized using an autoclave at a pressure of 5 psi which was maintained for 15 minutes and then removed from the autoclave to be stored in the media storage area.c.Explant sterilization is carried out by removing the explant from the culture bottle and then cutting it 3-4 cm long and having 2 buds.Next, the leaves are removed.The cut explants were put into bottles filled with sterile distilled water and placed in a laminar air flow cabinet.The explants were then transferred into a Petridis containing 2% tween 80 and left for 2-3 minutes for further planting.d.Planting and maintenance of culture.Before planting, the bottles containing the treatment were sprayed with 85% alcohol before being put into the laminar.Next, the explants are planted using a spirit lamp according to the treatment then placed in a culture room equipped with a lamp and always maintained in sterile conditions with a temperature of 18 0 C.

Results and discussion
Table 1.Observational data on the effect of various combinations of IBA + treatment Kinetin on the presence or absence of Jabon callus formation in vitro.The BNJ test results showed that the H3 combination treatment (1 ppm IBA + 0.5 ppm Kinetin) produced more leaves and was significantly different from the other treatments.This is in line with Wareing and Phillips, 1986 that growth hormone plays an important role in the formation of organs in tissue culture, especially growth regulators (hormones) of the auxin and cytokinin groups.Furthermore Gunawan, 1988 states that the addition of growth regulators at the appropriate concentration and type with endogenous hormones will provide an optimum biochemical, physiological and anatomical response as shown in the formation of leaves, shoots, roots and callus in this experiment.
Treatment H3 (1 ppm IBA + 0.5 ppm Kinetin), seems to be a balanced concentration of auxin and cytokinin to promote the formation of leaves, shoots and roots.This treatment, although the leaves and shoots were not as many as the other treatments, this treatment gave a complete organ formation response.Treatments H1 (0.1 ppm IBA + 3 ppm Kinetin) and H2 (0.5 ppm IBA +1 ppm Kinetin) did not form roots at all, so this treatment is not suitable to induce the rooting stage (Pictures 1 and 2).
The interesting thing from the results of this study is that an increase in auxin concentration (IBA) which is not balanced with the appropriate cytokinins also does not encourage good root formation, and conversely an increase in cytokinins (kinetin) also does not encourage good shoot formation if it is not balanced with auxin balance (IBA) as shown in this study (tables 1, 2 and 3).This is in accordance with Gunawan's statement, 1988, that the ratio between auxin and cytokinin will determine the direction of organ formation in tissue culture.These results are also in line with the results shown by Durzan and Bonga, 1987 in an in vitro regeneration study of conifers [14].All of the medium formulations tried were able to promote callus growth and development.Basic MS medium formulation with the addition of 2 ppm 2,4-D and 15% coconut water and MS0 medium with the addition of 2 ppm 2,4-D, 0.2 ppm BAP and 15% coconut water is a medium that is very effective in inducing callus.This is based on the speed of inducing callus, the nature of callus which is compact, white in color and the nature of the cells which continue to actively divide [15].
The results of this study indicated that callus formation on Jabon was not formed in the H1, H2, H3 treatments (Figures 1, 2 and 3).But in the H4 and H5 treatments (Figures 4 and 5) callus formation occurred.This shows that not all treatments given were able to form Jabon callus.

Conclusions
In conclusion, let's summarize.
• The addition of a combination of growth regulators H1 (0.1 ppm IBA+3 ppm Kinetin) encouraged the formation of good shoots, but did not form roots. On the other hand, the combination of growth regulators H5 (2 ppm IBA + 0.1 ppm Kinetin) was able to promote good root formation but could not promote good shoot and leaf formation.• The addition of H3 (1 ppm IBA + 0.5 ppm Kinetin) can form good roots, buds, and leaves even though the number of buds formed is not as good as in the H1 and H2 treatments.• Callus was only formed in the H4 and H5 treatments.