The Effects of Benzil Amino Purin (BAP) and Gibberellin with in Vitro Seedling Growth of Pulesari (Alyxia Reinwardtii Bl)

Background: Alyxia reinwardtii Bl. in natural habitat becomes rare and reaches vulnerable status. The high price and high market demand made the over harvested beyond regeneration capacity of the plant while the conservation efforts are terribly limited. Presentage of seed germination is not only very low but also time consuming, therefore we conducting preliminary study on in vitro propagation using seed Alyxia reinwardtii Bl. Methods: The research was conducted in laboratory tissue culture of Medicinal Plant and Traditional Medicine Research and Development Center Tawangmangu for three months. Seeds of A. reinwardtii Bl. obtained from Pringgondani Forest, Tawangmangu Central Java. Seeds were peeled and air dryed for five days, washed using steril aquadest, soaked on 0.5% Agrept (5 minutes) then on 2.125% sodium hypochloride (5 minutes) and rinsed using sterile distilled water. This study uses a Murashige and Skoog (MS) with a completely randomized design (CRD) factorial design.. The first factor is BAP at a concentration of 3, 4, 5 mg.L-1 and the second factor is giberelin concentration of 1, 2, 3 mg.L-1. MS media with addition of BAP the concentration of 3, 4, 5 mg.L-1 and gibberellin the concentration of 1, 2, 3 mg.L-1 obtained 9 treatment combination. Results: The results showed the combination treatment of growth regulators BAP (4 mg.L-1) and giberelin (2 mg.L-1) is formed shoots and roots. The shoots were formed 30 days after planting with a height of 2 cm and the roots appear at 45 days after planting. Conclusion: Pulasari (Alyxia reinwardtii Bl.) can be propagated through tissue culture using seeds as explants.

Pulesari (Alyxia reinwardtii Bl.) is one of medicinal plants which often used as main ingredient of Jamu, an Indonesia traditional medicine. The utilization of A. reinwardtii Bl. is usually accompanied by 'adas' (fennel, Foeniculum vulgare Miller), hence the name 'adas-pulasari '. At least about 18 sorts of PDQXIDFWXUHG µMDPX ¶ IURP &HQWUDO -DYD ,QGRQHVLD FRQWDLQ µDGDV SXODVDUL ¶ DV D SULQFLSOH LQJUHGLHQW These are used as an antispasmodic and as treating RI VWRPDFK DFKH ÀDWXOHQFH FROLF IHYHU G\VHQWHU\ as a carminative and sprue. 1 The scarce status of A. rewardtii Bl. is because of its high market demand followed by its high price of the simplicia. These situations lead to the increasing of plant exploitation from the natural habitat, whereas neither any efforts on conservation nor cultivation. 2 Up to present time, propagation of A. rewardtii Bl. relies on the seeds obtained from the wild. Unfortunately, pulesari seeds germination are very low and time consuming to yield mass seedling SURGXFWLRQ %HVLGHV KDYLQJ XQFHUWDLQ ÀRZHULQJ behavior, its low growing rate play D VLJQL¿FDQW UROH on decreasing of the existance of the plant. 3 Plant tissue culture technology is a mainstay technology in the biodiversity utilization in Indonesia. The technique generates high multiplication and provides rapid production of many genetically identical plants with relatively small amounts of space, supplies and time. These methods eliminate genetic retardation due to unintentional mistakes during conventional seeding production.
The success of tissue culture of plant propagation is LQÀXHQFHG E\ SODQOHW DQG FXOWXUH PHGLXP. It also depends on the addition of plant hormones and growth regulators into the medium. Plant hormones are group of unrelated chemical substances that affect plant morphogenesis. Five major plant hormones are traditionally described: auxins, cytokinins, gibberellins, ethylene, and abscisic acid. 6,7 Auxin and cytokinin play fairly important roles in many aspects of plant growth and development. The interaction between auxin and cytokinin is particularly important to control a few development processes, such as the formation and the maintenance of meristems that are essential to establish the whole plant body. 8 Cytokinin serves to stimulate cell division and proliferation of shoots. Cytokinin effects also include on breaking of dormancy, promotion of seed germination, stimulation and nutrient mobilization, HQKDQFLQJ DQWKRF\DQLQ DQG ÀDYDQRLG V\QWKHVLV increasing resistance to disease, and stimulation of the opening of stomata. 9 &\WRNLQLQ LQÀXHQFH RQ shoot initiation &\WRNLQLQV LQÀXHQFH WKH LQLWLDWLRQ of long shoots and buds. Cytokinins can also trigger the formation of side shoots, leaves widening and stimulate the formation of shoots. 10 Giberelins in plant play an important role on stimulating WKH IRUPDWLRQ RI ÀRZHUV SROOHQV DQG VHHGV HQODUJLQJ the size of fruits, and terminating seed dormation. At low concentration gibberellin will not stimulate the root formation, otherwise at high concentration it will do. Gibberellin added for differentiation or multiplication cell function, especially the formation of callus. The use of gibberellin in the plant will affect plant height, root length size and leaf area. The most obvious effect of gibberellin is modifying plant growth, namely cell division that led to the increase in the number of cells and cell enlargement, so that root growth is faster and longer many. 11, 12 Research concerning propagation and cultivation of A. reinwardtii Bl. in Indonesia still few, therefore in vitro propagation study of A. reinwardtii Bl. is needed. The research aimed to obtain breeding methods in tissue culture pulesari (Alyxia reinwardtii Bl.).

METHODS
The research was conducted in laboratory tissue culture of Medicinal Plant and Traditional Medicine Research and Development Center Tawangmangu for three months. Selected ripe seeds of A. reinwardtii Bl. which had dark brown husk were collected from 3ULQJJRQGDQL )RUHVW 7DZDQJPDQJX &HQWUDO -DYD 6HHGV ZHUH SHHOHG DQG DLU GU\HG IRU ¿YH GD\V washed using steril aquadest (s.a), soaked on 0.5% Agrept (w/v) for 5 minutes then on 2.125% sodium hypochloride (v/v) for 5 minutes and rinsed three times XVLQJ V D DQG SODFHG RQ /DPLQDU $LU )ORZ /$) prior to planting. Murashige and Skoog (MS) media were prepared according to directed composition. 15 The research used a completely randomized design (CRD) factorial with two factors with 3 repetitions. 7KH ¿UVW IDFWRU ZDV WKH FRQFHQWUDWLRQ RI JURZWK UHJXODWRUV %$3 DW D FRQFHQWUDWLRQ RI DQG PJ / 1 and the second factor was giberelin concentration RI DQG PJ / 06 PHGLD ZHUH PRGL¿HG ZLWK DGGLWLRQ RI %$3 ZLWK WKH FRQFHQWUDWLRQ RI DQG PJ / and gibberellin with the concentration of 1, 2, DQG PJ / with 3 repetitions.

From the observation and measurement of in vitro
Gibberellin has an effect on growth and germination of the embryo. Gibberellins will stimulate amylase synthesis, this enzym will catalize amylum found in endosperm into glucose as source of energy for growing. A stanted plant will grow normally by adding of gibberellins. Gibberellins also terminate a period of seed dormantion. From the above result if can be concluded that shoot and root initiation time of seed of Alyxia reinwardtii Bl. on MS media HQULFKHG ZLWK %$3 PJ / DQG JLEEHUHOOLQ PJ / 1 ZHUH DQG GD\V DIWHU SODQWLQJ UHVSHFWLYHO\ The best growth of shoot and root of A. reinwardtii Bl. seed could be obtained from MS media enriched ZLWK %$3 PJ / DQG JLEEHUHOOLQ PJ / .
In conclusion, pulasari (Alyxia reinwardtii Bl.) can be propagated through tissue culture by using seeds as explant.