Proceeding of 6th ICGRC
2015
Growth of generative explants of Moringa oleifera on different culture conditions Wirdhatul Muslihatin1, Hanum Laila Rizkia1 1
Department of Biology, Sepuluh Nopember Institute of Technology, Surabaya, Indonesia Corresponding author:
[email protected]
Abstract In vitro culture is more effective propagation technique because the propagation time is faster than conventional propagation so the achievement of the results is in favor of the improvement program and faster spread of Moringa oleifera. The objectives of this study were to determine the effect of carbon source and photoperiod on the growth of generative explants of M. oleifera in vitro. The method performed were inoculation of generative explans on MS Medium, observation growth of callus, and measurement of fresht weigth of callus. The generative explants used were M. oleifera seeds grown on MS medium with 3%, 5%, 7% and 9% sucrose and then they were cultured with photoperiod of 12/12, 14/10, and 16/8 hours (light/dark). The results showed that the best response of growth callus was indicated by the explants cultured on 3% sucrose medium in the photoperiod of 12/12. Keywords: Moringa oleifera; nodule; photoperiod; sucrose
1. Introduction Moringa oleifera is plant that the origin is from Indonesia. The spread of M. oleifera is very wide, but in Indonesia, these plants become very rare nowadays. The seed germination of M. oleifera is difficult, the viability or survival plant is low, and the lack of vegetative propagation method is present [1]. M. oleifera plantation is generally done vegetatively by stem cuttings technique [2]. Because of the lack of plant multiplication technique as described previously, it is necessary to have appropriate propagation techniques that can produce M. oleifera seeds in large quantities, good quality, and in a short time. One of the alternatives that can be used is the in vitro culture technique. In vitro culture is done to get a crop in large number in a relatively short time that has physiological and morphological characteristics identic to the parent plant [3]. Propagation through in vitro culture is influenced by medium composition that includes carbon source and light or photoperiod. For in vitro culture, the roles of carbon source are to maintain the supply of carbon during in vitro culture, because the conditions of in vitro culture are not sufficient to support photosynthesis of plants culture, and the carbon source keeps the osmotic potential of cells [4]. Photoperiod is long irradiation on explants cultured, the explants need a source of energy in the process of photosynthesis and photomorphogenic [5]. Tissue culture plants form a callus or nodules. Callus and tissue nodule can be fragile, easily damaged and can be contaminated when they are stored in a relatively long time. Therefore, the protection can be done using encapsulation technique which can produce synthetic seeds. Synthetic seeds technology provides advantages such as long-lasting storage, simple distribution or dissemination, as well as to protect the seed from disease [6]. This study aimed to know the effect of carbon source and photoperiod on the growth of generative explants of M. oleifera. 2. Material and Methods 2.1. Sterilization Explants The explants used were seeds of M. oleifera. Seeds were washed with running water until they were clean, then soaked in detergent solution then rinsed with running water. Seeds were soaked in a solution of 3% antifungal and clorox for 5 minute then rinsed with running water and soaked with 96% alcohol for 5 minute. 2.2. Induction of Callus Medium used to induce callus was MS medium (Murashige and Skoog) plus 0.5 mg /L NAA and 1 mg/L kinetin. For the treatment of carbon sources variety, it used sucrose at a concentration of 3%, 5%, 7% and 9%. Medium pH was adjusted at 5.8 with the addition of 1N NaOH or 1N HCl, then the medium was sterilized by autoclave at 121C and 1.2 atm for 20 minutes. Then explants were inoculated to the medium in a sterile room in a state of LAF. Cultures were kept at room temperature of 20-25C, photoperiod of 16/8, 14/10, and 12/12 hours (light/dark). The process of callus induction carried out for 4 weeks.
International Conference on Global Resource Conservation (ICGRC)
Page 8
Proceeding of 6th ICGRC
2015
2.3. Observations Observation of growth is done by weighing the fresh weight of nodul from explant at 4th weeks using analytical balance. 3. Results and Discussion In this study, M. oleifera explants (seeds) showed growing response that is longer than the studies that have been done by [3]. The response shown by explant is the formation of nodules in areas of wounding. Nodules are a group of cells in a particular place in explants that resembles the cambium, which is often also called meristemoid. This would allow actively dividing cells [9]. Swelling of the seeds indicates that the explants grew, wherein the medium absorbed as a nutrient for the growth of nodules which will be marked by phases of proliferation (cell multiplication). This response is shown almost in all treatments (Figure 1). Sucrose (%) (B)
12/12 (1)
Photoperiode (light/ dark) (A) 14/10 (2)
16/8 (3)
3% (1)
(A1B1)
(A2B1)
(A3B1)
(A1B2)
(A2B2)
(A3B2)
(A1B3) W
(A2B3)
(A3B3)
(A2B4)
(A3B4)
5% (2)
7% (3)
9% (4)
(A1B4) Figure 1. Growth response explant of M. oleifera (in cut form) to photoperiod treatment and sucrose (observed 4 week after culture). The arrow show white nodules
From 12 treatments, there are 8 explants showed a response by forming nodules or small bumps on parts that undergo wounding namely the treatment of A1B1 (12/12 of photoperiod, 3% sucrose), A2B1 (14/10 of photoperiod, 3% sucrose), A3B1 (16/8 of photoperiod, 3% sucrose), A1B2 (14/10 of photoperiod, 5% sucrose), A1B3 (12/12 of photoperiod, 7% sucrose), A2B3 (14/10 of photoperiod, 7% sucrose), A1B4 (12/12 ofphotoperiod, 9% sucrose) and A3B4 (16/8 of photoperiod, 9% sucrose) (Figure of each treatment is not shown). White nodules were formed in all explants, while irregular shape was not same between treatments. However, for the treatment of A2B2 (14/10 of photoperiod, 5% sucrose), A3B2 (16/8 photoperiod, 5% sucrose), A3B3 (16/8 of photoperiod, 7% sucrose) and A2B4 (14/10 of photoperiod, 9% sucrose), explants showed no such response to other treatments, it does not indicate the presence of nodules formed and there is no swelling of the explants. Nodules formed increased weight of fresh explants. Fresh weight explants was measured using analytical balance at 4 weeks after inoculation. The increase of fresh weight explants is also as the result of cell multiplication. Based on the test results of two way ANOVA, it was obtained value of P
