Growth response of sweet kaffir lime (Citrus hystrix) seeds due to kinetin and coconut water application using tissue culture technique

Sweet kaffir lime is one of Aceh’s local fruit crops, which is rarely found anymore. The problem of sweet kaffir lime cultivation is the difficulty of generative and vegetative plant propagation. The general objective of this research is to preserve the Acehnese sweet kaffir lime from extinction. One way that can be done is through plant propagation in tissue culture. The experiment was conducted in the Plant Tissue Culture laboratory of the Faculty of Agriculture, Universitas Malikussaleh. This research used a two-factor, completely randomized design, and ten replications. Two factors were tried: the concentration of kinetin (0, 1, and 2 mg/L) and the concentration of young coconut water (0, 15, 30 %). The results showed that increasing kinetin concentration could reduce shoot length, number of leaves, root length and slow the root emergence time of sweet kaffir lime in vitro. The best treatment was the 0 mg/L kinetin (without kinetin). Increasing coconut water concentration could increase shoot length, number of leaves, and root length of sweet kaffir lime in vitro. The best treatment was 30% coconut water concentration.


Introduction
Sweet kaffir lime (Citrus hystrix) in Aceh is known as "Boh Kruet Mameh" is one of Aceh's local fruit plants which is currently very difficult to obtain.Sweet kaffir limes are larger than regular kaffir limes, but the taste is sweet and sour, with a nice fragrant and fresh aroma so that they can be consumed as a fruit dish.
Sweet kaffir lime is a valuable asset in Indonesia's biodiversity.However, the cultivation of sweet kaffir lime is almost no longer practiced because many people have started to switch planting on higher economic value plants, or a land conversion has occurred.Therefore, the sweet kaffir lime is very difficult to find, and threatened with extinction.
The problem in cultivating sweet kaffir lime plants is how they are propagated vegetatively and generatively.Conventional vegetative plant propagation on a large scale will endanger the existence of the parent plant because it requires a large source of scion.Meanwhile, generatively, the number of seeds is minimal, and it is challenging to germinate conventionally.
Efforts to preserve the sweet kaffir lime plant as a genetic resource from Aceh must be carried out in various ways.A critical stage in keeping plants is to carry out plant propagation.Based on existing IOP Publishing doi:10.1088/1755-1315/1297/1/012015 2 problems, sweet kaffir lime seeds must be reproduced using a modern method, which is plant tissue culture.Plant tissue culture is one way to propagate plants artificially vegetatively by isolating plant parts in organs, tissues, cells, or protoplasts and then growing them in sterile conditions to obtain complete plants.Propagation of plant tissue culture techniques can be a solution for plants that are difficult to propagate using seeds conventionally.
Plant growth in tissue culture often requires the addition of growth regulators.Cytokinins are the most commonly added growth regulators in tissue culture media.Sources of cytokinin can be synthetic or natural, such as kinetin and young coconut water.Kinetin 1.5 mg/l produced the highest number of shoots on the growth of epicotyl explants of kasturi (Citrus microcarpa) [1].The growth medium enriched with 15% coconut water gave the best result on inducing curcuma (Curcuma longa) shoots in vitro [2].
The fruit plant research team at the Faculty of Agriculture, Malikussaleh University, has experience in research to save genetic resources from the danger of extinction.This has been done starting from the morphological and molecular characterization of plants, for example durian [3], [4], and avocado [5], [6], to plant tissue culture propagation systems [7], [8], [9] [10], [11], [12], [13], [14], [15], [16].The general aim of this research is to preserve the Aceh's sweet kaffir lime plant from extinction.The increasing amount of genetic resources support plant breeding efforts so plant development and conservation can be carried out.In addition, the success of this research is expected to reveal superior potential and serve as a basis for protecting Aceh's fruit plants from extinction or theft of genetic resources by other countries.

Place and Times
This research was conducted at the Plant Tissue Culture Laboratory, Faculty of Agriculture, Malikussaleh University, North Aceh Regency.This research was conducted from April to June 2023

Experimental design
This research used a two-factor Completely Randomized Design (CRD) method.The first factor was the application of Kinetin concentration, which consisted of three levels: 0, 1, and 2 mg/L.The second factor was the concentration of young coconut water, which consisted of three levels: 0, 10, and 30%.In this research, nine treatment combinations with ten repetitions resulted in 90 experimental units.

Research Implementation
The research implementation included sterilizing tools and planting space, making stock solutions, taking young coconut water, making MS media, sterilizing explants, initiating explants, and observing and analyzing data.The explants used are seeds from sweet kaffir limes from Bireuen.The criteria of the fruits are firm until ripe, bright green to yellowish green skin color, the rareness of wrinkles on the skin, juiciness of the fruit flesh, and healthiness of the fruit (not damaged by pests or disease).The criteria of the explant seeds are pithy, have a perfect shape, without wrinkles or hollow, and weight around 0.07-0.11g.
The sweet kaffir lime fruit was washed with running water and peeled; then, the seeds were taken and stored in a petri dish.The seeds were then soaked in a liquid soap solution for 10 minutes and rinsed with running water and sterile distilled water.The seeds were then soaked in 20% bayclin solution for about 20 minutes, rinsed using sterile distilled water three times, and transferred into a sterile bottle.Next, the sweet kaffir lime seeds were put into the LAFC.The seeds were then soaked in a 4 g/L fungicide/bactericide solution for 30 minutes and rinsed with sterile distilled water until clean.The seeds were soaked again in 20% bayclin solution for 5 minutes and rinsed with sterile distilled water.The seeds were then soaked in 70% alcohol for 3 minutes and then rinsed with sterile distilled water three times.The sterile seeds were drained on sterile paper in a petri dish, and the cup was closed tightly.The sterile sweet kaffir lime seeds were then removed from the epidermis so that it did not inhibit the growth of the explants.Next, the seeds were planted in culture bottles containing treatment media, two explants per bottle.Observations in this research were the shoot emergence time, shoot growth percentage, number of shoots, shoot height, number of leaves, root growth time, and callus growth.

Data analysis
Data were analyzed using ANOVA.The Duncan's Multiple Range Test (DMRT) was performed to determine whether there was any significant difference, with a 5% confidence level.

Results
The results of the F test showed that the interaction of kinetin concentration and coconut water only affected the growth percentage and number of shoots from in vitro sweet kaffir lime seed explants.The results of further tests on the percentage of shoot growth and number of shoots after 8 weeks of culture are presented in Table 1.The numbers in brackets are the transformed data √(x+0.5).
Table 1 showed the kinetin and coconut water concentration.The treatment of 0 mg/L kinetin concentration + 30% coconut water (K0A2) had the best the percentage of shoot growth and number of shoots.The influence of each treatment can be seen in other observations.Further tests on the variables of shoot emergence time, shoot length, and number of leaves after 8 weeks of culture are presented in Table 2.The numbers in brackets are the transformed data √(x+0.5).
Table 2 showed that kinetin concentration did not affect shoot emergence time, but its effect was visible on shoot length and number of leaves.An increase in kinetin concentration resulted in a decrease in leaf length and number.The coconut water concentration affected the shoots' length and the number of leaves.Increasing the concentration of coconut water can increase shoot length and number of leaves, as seen in Figures 1 and 2.  Figure 1 showed an increase in the number of shoots along with an increase in the concentration of coconut water.The number of shoots with 30% coconut water could grow more than others.The same response was also seen in the number of leaves, as shown in Figure 2.There was an increase in the number of leaves along with an increase in the concentration of coconut water.The number of shoot leaves in 30% coconut water was more than others.
The effect of kinetin concentration and coconut water was also seen in the root emergence time and root length variables.Further tests on the variables of root emergence time, number of roots, and length of roots are presented in Table 3.The numbers in brackets are the transformed data √(x+0.5).
Table 3 showed that kinetin could slow root emergence time and shorten root length.The best plant response was shown without applying kinetic (0 mg/L).The effect of coconut water was only seen in the root length variable.Increasing coconut water concentration caused the roots to grow longer.

Discussion
The test results showed that kinetin and coconut water concentration only affected the growth percentage and number of shoots; however, the effect was also seen individually on other variables (Table 1-3).Regarding the interaction, the best value was shown at 30% coconut water concentration without adding kinetin.Likewise, with other variables, the influence of a single factor showed that giving kinetin could reduce sweet kaffir lime explant growth.The opposite could be seen in the coconut water treatment, which was that applying coconut water could increase sweet kaffir lime explant growth.
Kinetin concentration could reduce the value of shoot length, number of leaves, and root length and slow the emergence of shoots.The best treatment was seen at 0 mg/L kinetin (without kinetin).This might be caused by the type of explant used: the whole seeds without splitting.Seeds that already have endogenous hormones stimulate growth and organ formation.Some plants do not need exogenous hormones because they can be toxic and inhibit the growth of the explant.Seeds can still grow even without the addition of exogenous hormones.Endogenous hormones in the seeds cause this but in small concentrations.The addition of exogenous hormones must be to the needs of seed organogenesis [17].Media treatment without cytokinin triggered the growth of musk orange seed explants [18].
The results of this research are different from tissue culture propagation of halved sweet kaffir lime seeds, obtained from previous research.This research showed that adding the cytokinin hormone Benzyl Amino Purine (BAP) could increase the percentage of shoot growth and number of leaves and speed up the growth time of shoots.The condition of the zygote contained in intact seeds is not damaged, and the endogenous hormones in the seeds are not divided, so they have high growth ability [16].The whole seeds have high apical dominance, so the emergence of buds on intact seeds is higher than on split seeds [10].Therefore, the seed division factor is important for the growth of sweet kaffir lime seed explants.
Another reason for the lack of influence of kinetin concentration on the growth of sweet kaffir lime seed explants is possibly due to differences in the type of cytokinin used.In previous research, BAP was used, while in this research, kinetin was used.BAP is an adenine-derived cytokinin that is most active in cell division and stimulates shoot growth to influence the percentage of explant survival [17].The effect of BAP application is also more consistent than kinetin [20].
The concentration of coconut water can increase shoot growth percentage, number and length of shoots, number of leaves, and root length.This clearly showed the positive influence of the coconut solution on the growth of sweet kaffir lime seed explants (Figures 1 and 2).Coconut water, an organic ingredient containing compounds such as cytokinins, can also increase shoot growth.[21] stated that adding coconut water to MS media could increase the percentage of shoot growth in Cymbidium orchid subcultures in vitro.
In research by [22] and [23], young coconut water treatment of 15% and 20% produced the highest number of shoots in ginger rhizome shoot tissue culture.Likewise, research by [24] stated that giving 200 ml/l coconut water on MS media produced many shoots on sticky banana explants in vitro.This shows that the explants still need exogenous hormones from coconut water to stimulate the number of shoots.
The concentration of coconut water can also increase the growth of sweet kaffir lime roots.Research by [25] explains that giving 10% -50% coconut water can stimulate root formation; roots begin to appear 12 days after planting pepper shoot explants in vitro.This is because coconut water contains the hormone auxin, which plays a role in root induction.The auxin content in coconut water in the form of IAA is 198.55 mg/l [22].Exogenous IAA can increase the endogenous auxin activity of explants in stimulating root formation.

Conclusions
Increasing kinetin concentration could reduce shoot length, number of leaves, root length and slow the root emergence time of sweet kaffir lime in vitro.The best treatment was the 0 mg/L kinetin (without kinetin).Increasing coconut water concentration could increase shoot length, number of leaves, and root length of sweet kaffir lime in vitro.The best treatment was 30% coconut water concentration.

Figure 1 .
Figure 1.Number of sweet kaffir lime shoots in vitro 8 weeks of culture.(a) Applied with 30% coconut water; (b) Applied with 15% coconut water; (c) Applied with 0% coconut water.

Figure 2 .
Figure 2. A number of sweet kaffir lime leaves 8 weeks of culture.a) Applied with 30% coconut water; (b) Applied with 15% coconut water; (c) Applied with 0% coconut water;.

Table 1 .
Effect of the interaction of kinetin and coconut water on the growth percentage and number of shoots of sweet kaffir lime in vitro a .
b Data in the same column followed by different letters are significanly different by Duncan's Multiple Range Test at the 5% level.c

Table 2 .
Effect of kinetin and coconut water concentration on bud emergence time, shoot length, and number of leaves of sweet kaffir lime in vitro a .
a Notes are referenced using alpha superscripts b Data in the same column followed by different letters are significanly different by Duncan'sMultiple Range Test at the 5% level.c

Table 3 .
Effect of kinetin and coconut water concentration on root emergence time, number of roots, and roots length of sweet kaffir lime in vitro a .Notes are referenced using alpha superscripts b Data in the same column followed by different letters are significanly different by Duncan's Multiple Range Test at the 5% level.
a c