Application of Cytokinin and Mycorrhiza to Increase Production and Quality of Pineapple Seedlings from Crown Leaf Bud Cuttings

The pineapple vegetative propagation method using crown leaf bud cuttings has potential to produce seedlings of greater uniformity and in larger quantities than other methods. This study, aimed to explore the potential eff ects of concentration and frequency of Benzylaminopurine (BAP) application for increasing the production and quality of pineapple seedlings from crown leaf bud cutting propagation. The experiment used a randomized complete block design (RCBD) with two factors. The fi rst factor was the concentration of BAP (0, 200, 400, 600, 800 ppm) and the second factor was the frequency of BAP application (1, 2, 3 times). Furthermore, this study investigated the eff ects of applying mycorrhizae to accelerate seedling growth and improve seedling quality during production from crown leaf bud cutting propagation. The experiment used a RCBD with one factor (dose of mycorrhiza: 0, 50, 100, 150, 200 spores). The results indicated that the eff ects of concentration and frequency of BAP application and their interaction increased seed production as determined by sprout cuttings at 5 weeks after planting by applying 600 ppm 2 times. Generally, with increasing concentration and frequency of BAP application, leaf chlorophyll content was reduced. However, the application mycorrhiza positively infl uenced the production of seedlings from pineapple crown bud cuttings and improved seedling quality as measured by root dry weight, chlorophyll content, root infection, and phosphatase enzyme activity.


Introduction
Pineapple seedlings (Ananas comosus L.) are important for the expansion of cultivated areas and for the development of new varieties. The availability of seedlings is currently limited, yet pineapple production, on a commercial scale, requires planting material of ≥40,000 seedlings per hectare (Naibaho, 2012).
Vegetative propagation of pineapple can be done using crowns, slips, suckers, and ratoons (Hadiati and Indriyani, 2008). However, seedling production is limited and hence, cannot provide enough material for the expansion of planting areas.
The vegetative propagation method using crown leaf bud cuttings has the potential to produce seedlings uniformly and in great quantity. This method utilizes the bud meristem tissue on each leaf of the crown. Each crown leaf has dormant axillary buds and attached to each axillary of the crown. These dormant shoots have the potential to produce buds and become potential seedlings (Py et al., 1984;Hepton, 2003). Tassew (2014) reported that one pineapple crown can produce 23-32 seedlings depending on crown leaf bud size.
The propagation of pineapple using crown leaf bud cuttings is disadvantageous because it negatively aff ects the growth of roots and shoots (Chairunnisak et al., 2015). This problem can be solved by applying growth regulators (GR) to the cuttings. Eprilian (2019) stated that the application of 250 ppm IBA via soaking treatment can increase the cutting and root growth percentage as well as root dry weight.
Dirgahani Putri, M. Rahmad Suhartanto, Eny Widajati Cytokinin functions in the process of division and increasing the number of cells in plant organs forming new shoots and breaking dormancy (Gardner et al., 1991). Exogenous cytokinin applications have been reported to stimulate shoot growth (Yaish et al., 2010). One type of exogenus cytokinin that is widely used is Benzylaminopurine (BAP). Several studies related to the eff ect of BAP on the success of sprout cuttings already done. Hadiati (2011) reported that BAP application at concentrations of 200-600 ppm increase plant height and shoot numbers in a hybrid variety of pineapple stem cuttings and accelerated the time of bud rupture by 6 days compared to the control treatment. When applying growth regulators to plants both the concentration and the application time must be carefully considered. The response to growth regulators can be optimized by administering ideal concentrations during a favorable plant growth phase (Wicaksono et al., 2017).
The use of crown leaf bud cuttings as a planting material has limitations because seedling growth requires a long period of approximately 22-24 months after planting (Elfi ani and Aryati, 2012). It is possible to enhance the vegetative growth rate of seedlings by manipulating the root zone (rhizosphere) and by applying benefi cial microbes to the growth media i.e. inoculation (Lakitan, 2008). Mycorrhiza display a mutualistic symbiotic relationship between fungi and plant root systems, which plays a role in the absorption of plant nutrients, especially phosphate, increases growth rate, and positively aff ects yield potential (Suharno and Sancayaningsih, 2013).
This study aimed to investigate the eff ects of concentration and frequency of cytokinin application, as well as explore appropriate mycorrhiza applications to increase growth rate, production and quality of seedlings from pineapple crown bud cuttings. Therefore, this research is an important step towards developing pineapple propagation technology with crown leaf bud cuttings to obtain seedlings of good quality.

Materials and Methods
The study was conducted in November 2018 to June 2019. The study was conducted at the Cikabayan experimental green house, Bogor Agricultural University. The leaf content analysis of nitrogen, phosphate, and potassium was done in the Department of Agronomy and Horticulture laboratory, Bogor Agricultural University. The analysis of root infections was undertaken at the Research Center for Bioresources and Biotechnology, Bogor Agricultural University. The phosphatase analysis was performed at the Indonesian Research Institute for Biotechnology and Bioindustry, Bogor.

Evaluation of Cytokinin Application to Increase the Production and Quality of Pineapple Seedlings
The fi rst experiment used a factorial randomized complete block design with two factors. The fi rst factor was cytokinin and had fi ve levels (concentrations), specifi cally: 0, 200, 400, 600, and 800 ppm. The second factor was the frequency of Benzylaminopurine (BAP) application and consisted of three levels, namely 1 times, 2 times, and 3 times. The treatments were grouped based on planting time (7, 8, and 9 days after crown wilting). Each treatment was repeated three times, so there were 45 experimental units. Each experimental unit was a seedling box of 40 cm x 30 cm and consisted of 30 pineapple crown leaf bud cuttings with a total of 1350 cuttings.
The experimental procedure was performed by spraying BAP 1 times at 2 weeks after planting (WAP), 2 times at 2 and 3 WAP, and 3 times at 2, 3, and 4 WAP using a hand sprayer according to the level of treatment on the buds. Each cutting received a BAP dose of 1 ml. Maintenance of the seedlings was done intensively during the experiment.
The measurements were taken from all seedling in each experimental unit from 5 WAP onwards. The observed variables were growth percentage, sprout percentage, seedling height, chlorophyll content, rooting percentage, root number, root length, shoot dry weight, and root dry weight. The data were analyzed using analysis of variance (ANOVA) at the level of 5%. If signifi cant diff erences were detected, a Duncan's Multiple Range Test (DMRT) was performed at the 5% level of signifi cance (Gomez and Gomez, 1984) using SAS 9.0 software.

The Potential Uses of Mycorrhiza to Increase the Production and Quality of Pineapple Seedlings
The second experiment used a RCBD with mycorrhizal doses as the treatment. The doses of Glomus sp. consisted of fi ve levels: 0 spores, 50 spores, 100 spores, 150 spores, and 200 spores. The seeds were grouped according to their size, i.e. small (0.5-3 cm), medium (> 3-5.5 cm), and large (> 5.5 cm). Each treatment was replicated three times, resulting in fi fteen experimental units. Each experimental unit consisted of 25 seedlings derived from pineapple crown bud cuttings; hence, 375 seedlings were used in total.
The procedure for the second experiment was similar to the fi rst experiment. The main diff erence was the use of growth regulator. In the second experiment only IBA was used as a growth regulator with a concentration of 250 ppm (Eprilian, 2019). Pineapple seedlings 10 WAP were transferred to nurseries based on budding size (small, medium and large). The planting media was a mixture of rice husk, compost, cocopeat, and soil with a ratio of 1 : 3 : 3 : 3 with the weight of 500 g per polybag (Kurniawan, 2014). Mycorrhiza application was performed when the seedlings were transplanted by applying mycorrhiza around the roots according to the treatment categories.
Measurements were taken from all seedlings in each experimental unit from 1 week after transplanted (WAT) onwards. The observed variables were growing percentage, seedling height, leaf number, root length, shoot dry weight, root dry weight, chlorophyll content, root infection percentage, growth of seedlings, and phosphatase enzyme activity. The data were analyzed using analysis of variance (ANOVA) at the level of 5%. If signifi cant diff erences were detected, a Duncan's Multiple Range Test (DMRT) was performed at the 5% level of signifi cance (Gomez and Gomez, 1984) using SAS 9.0 software.

Evaluation of Cytokinin Application to Increase the Production and Quality of Pineapple Seedlings
Through the use of ANOVA, signifi cant positive eff ects of the concentration and frequency of cytokinin application and their interaction were shown on the production of pineapple crown bud cuttings as measured by the cutting bud variables at 5 WAP. Improvement of the quality of crown bud cuttings signifi cantly aff ected leaf chlorophyll content which was only infl uenced by BAP concentration at 20 WAP (Table 1).

Seedling production
Towards the beginning of the experiment (5 WAP), an interaction of concentration and frequency of BAP application was detected. Table 2 shows that the cuttings sprouted at a concentration of 0 ppm BAP showed no signifi cant eff ect. When the BAP concentration was increased to 200 ppm, the sprouting of cuttings was also increased after 3 times BAP application. The sprouting of cuttings at concentrations of 400-800 ppm was signifi cantly increased after only 2 times BAP application. It was suspected that the cuttings were still active in terms of cell division, so the concentration and BAP applications were able to eff ectively increase sprouting of cuttings. According to Nuraini et al. (2016), the application of cytokinin aff ected the early growth of buds. Furthermore, it was observed that the single BAP application did not  (2019) reported that 1 BAP application by spraying did not have a signifi cant eff ect on sprouting of pineapple cuttings. After 10 WAP, the concentration and frequency of BAP application did not display any eff ect, because the cuttings already developed roots and leaves. According to Octaviani (2009) the presence of roots caused the nutrient absorption to become optimal so shoots development can be maximized.

Seedling quality
BAP concentration and application did not show a signifi cant eff ect on seedling height, rooted cuttings, root number, root length, shoot dry weight, root dry weight except chlorophyll content ( Table 3).
The highest chlorophyll content was detected at 0 ppm BAP and was signifi cantly higher than at BAP concentrations of 200-800 ppm. The seedlings with 0 ppm BAP had a greener color compared with 200-800 ppm BAP. This was caused by high BAP concentrations disrupting the synthesis of chlorophyll. According to Kocot et al. (2011), the application of cytokinin can aff ect the formation of chlorophyll, specifi cally via supporting the transport of Mg 2+ ions into leaf mesophyll cells. However, the application of cytokinins at high concentration results in reduced accumulation of Mg 2+ , thereby disrupting chlorophyll synthesis.

The Potential Uses of Mycorrhiza to Increase the Production and Quality of Pineapple Seedlings
The application of mycorrhizal fungi showed no signifi cant eff ect on production and quality of seedlings except for the root dry weight. Furthermore, there was no signifi cant eff ect on production. This shows that mycorrhiza can be applied safely to pineapple bud cutting seedlings, which can be seen from the percentage of growing cuttings (100%) ( Table 4). Further, mycorrhiza function as a biological barrier against root pathogen infections (Prihastuti, 2007).  The mycorrhizal application tended to increase the root dry weight and maximum root dry weight was obtained with the application of 200 spores (91 mg) and this was signifi cantly higher than the control (0 spores) 79 mg (Table 4). The infected plants were of larger volume and length; hence, increasing mycorrhizal dose resulted in corresponding increases in dry weight. These results were similar to Djazuli (2011) which stated that mycorrhizal application at a dose of 30 g per pot signifi cantly increased the root dry weight of Pimpinella pruatjan. According to Prasasti et al. (2013), mycorrhizal application increased the absorption of water and plant nutrients and increased the dry weight of plants.
The highest chlorophyll content was found in the mycorrhizal application of 200 spores but this did not diff er signifi cantly from the application of 100 spores (Table 5). Based on root infection, almost all mycorrhizae were infected in their roots, and therefore aff ected the dry weight of the roots and leaf chlorophyll. This was caused by the ability of mycorrhiza to increase the levels of nitrogen and phosphate of plant tissue which aff ect chlorophyll levels. Basela and Mahadeen (2008) stated that nitrogen was a key element in the formation of amino acids for protein synthesis which has a structural role in chlorophyll. According to Naisumu et al. (2017), phosphate also plays an important role in the formation of chlorophyll especially regarding the stability of chlorophyll molecules. Roots, which were infected with mycorrhiza, impacted the growth speed of pineapple seedlings. This can be seen from the increase in growth speed of seedlings which were treated with mycorrhiza (5 mm per week) compared to those which were not treated with mycorrhiza (4 mm per week). However, this eff ect was not statistically signifi cant.
Based on Table 5, seedlings that were not treated with mycorrhiza did not have phosphatase enzyme activity, but the seedlings that were amended with mycorrhiza showed an increase in phosphatase activity. The highest phosphatase activity was seen in mycorrhiza applications of 100 spores. The activity of the phosphatase enzyme in the application of 100 spores was correlated with the phosphate content available in the leaves (Table 6). Margalef et al. (2017) stated that the higher the activity of the enzyme phosphatase, the more phosphate would be produced.
The eff ects of mycorrhizal dose on the N, P and K content of pineapple crown leaf bud cuttings showed that the application to the pineapple crown did not diff er signifi cantly in terms of nutrient uptake (N, Note: Values followed by the same letters in the same column are not signifi cantly diff erent according to DMRT at ɑ = 0.05. 1) each value represents one sample P and K; Table 6). The results of this leaf analysis showed that mycorrhizal application can not improve the quality of seedlings from pineapple crown bud cuttings.

Conclusion
The eff ects of concentration and frequency of BAP application and their interaction increased seed production sprout cuttings at 5 weeks after planting by applying 600 ppm 2 times. Generally, with increasing concentration and frequency of BAP application, leaf chlorophyll content was reduced. However, the application mycorrhiza positively infl uenced the production of seedlings from pineapple crown bud cuttings and improved seedling quality as measured by root dry weight, chlorophyll content, root infection, and phosphatase enzyme activity.