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: LUVANEE PARKIANATHAN
MATRIC NO : 860120145424 LECTURER : PROF. NASIR
SUPERVISOR : DR. YAP LIP VUN
IN VITRO REGENERATION OF CIKU (MANILKARA ZAPOTA) 1.0 Introduction Manilkara Zapota or more commonly known as ciku by the locals are well known for its luscious and sweet fruit. The Manilkara Zapota, a member of Sapotaceae family is said to have originated from southern parts of Mexico, northern of Belize and Northeastern of Guatemala and possibly believed to be the native to Yucatan Peninsula (Primack, 1997). But now, the species has widely spread across the pacific and it has various names at each specific place; baramasi (Bengal and Bihar, India); buah ciku (Malaysia); korob (Costa Rica); naseberry (Jamaica; British West Indies) and more. The Manilkara Zapota tree is slow growing, strong, wind resistant, grows up to 60 ft high in open land and races up to 100 ft high in crowded forest. It leaves are dark green, glossy, eclipse, pointed at the tip and are about 7 to 12 cm long and 2 to 4 cm wide. The flowers of Manilkara Zapota are bell-like tiny and are borne on the stalks of the leaf bases. The fruit is normally round or oval in shape, with sandy-brown scurf and the size ranges between 5 to 10 cm in diameter. When the fruit is immature, the texture is hard and tastes astringent. However, the fruit becomes soft, juicy and very sweet when it is ripe. The colour of ripe fruit is normally dark brown to reddish brown. The texture of the flesh is somewhat coarse and grainy. A Manilkara Zapota fruit carries 3 to 12 seeds which can be easily removed from the middle of fruit. The seed is hard coated, black, glossy, and has a white margin.
its germinating part would be conserved as well. Since the seeds of Manilkara Zapota are recalcitrant. 2. To determine the best concentration of BAP used for multiple shoot formation. Delimitation: Only Manilkara Zapota seeds are used in this research.The seeds are very important. To compare the effective growth of Manilkara zapota seeds with and without the seed coat. 1. 1. An analytical discussion on the limitation and delimitation are discussed below: Limitation: Access to journals online is hard.1 Rationale The seeds of Manilkara Zapota are recalcitrant and have low germination in the natural environment. and in gene manipulation. The hard-coated. In a prolonged period.2 Objectives 1. we will be able to uphold. a suitable in vitro regeneration should be developed to create the basic platform for future cryopreservation. the conventional method of seed storage is not possible. the germplasm of this species may face endangerment. An in vitro regeneration medium development for Manilkara Zapota will be studied in this research. Furthermore. Through these methodology. save and maintain the viability of this species for future generations. And only tissue culture is done on the seeds to establish its regeneration medium. To have a prolonged existence of a particular species. only few researches have been made on Manilkara Zapota. The seeds obtained must be cultured in two days time to prevent the death of seed as the seeds are recalcitrant and at the same time to avoid contamination. Therefore. . black seeds of Sapodilla are the main focus of my research.
Also in Eng’s work. The seed obtained is sterilised and sterilisation of zygotic embryo is avoided as the viability of the sterilised embryo is at stake. 1997). This may cause the embryo to be dead as it is very delicate and small in size (Eng.3. 1987). MS medium contains all the macro and micronutrients required by an explant (Dayana. For example. 2. the flesh can be made into syrupy juice concentrate.5 mg/L NAA in MS medium proved to elongate the excised Manilkara Zapota zygotic embryos and the embryo stayed green but did not show any signs of root or shoot formation. the bark which is rich in tannin content can be used as tint for fishing lines.Usage of different strength of MS medium shows different rate of viability (Saleha. this medium was focused on callus formation. 2006). it was found that mixture of 5 mg/L BAP and 0. Also the tree serves as timber resource. Also. 1999) Murashige and Skoog (1962) medium or more known as MS medium is said to be suitable the most suitable medium for plant regeneration at various temperatures. To determine the best concentration of NAA used for root formation. 2007). and also for medicinal purposes (Morton. B5 medium serves and works well as a basal medium for whole plant regeneration when the actual intentions were. 4. WPM is used to counteract the salt sensitivity in some woody species (Trigiano et al.0 Literature Review The Manilkara Zapota has many other uses than just consuming its bountiful fruits. 2007). But the most valuable product that can be extracted from Manilkara Zapota tree would be chicle or gummy latex which can emerge as second most highly produced and exported in Petén (Primack. To acclimatize the plantlets grown in vitro into normal environment. .
The whole procedure will be carried out in the laminar flow hood to maintain aseptic condition in order to eliminate contamination. 2001) The overall procedure of medium preparation. The seeds are then surface sterilised in ethanol (100%) for 5 minutes. sterilisation of seeds and culturing should be conducted in the laminar airflow hood to prevent contamination and maintain the asepticity of the whole laboratory work (Letchmana. the seeds will be excised and cultured within two days from the day the seed was obtained.0 Materials And Method Manilkara Zapota seeds variety will be used in this study. indoleacetic acid (IAA) and thiourea on Manilkara Zapota seeds showed seed germination improvement (Doijode. 3.Application of gibberellic acid . The ethanol is then discarded and 20% of sodium hypochlorite of commercial bleaching agent Clorox® is added and surface sterilised again for 20 minutes. 3. Therefore. .1 Preparation of Seeds The mature seeds will be soaked for 24 hours in water before sterilising agent is added. 2007). and also from Mr Loo Boon Kiat of Ijok. The seeds will be obtained from the Taman Pertanian of University Putra Malaysia. The Clorox® will then be discarded and the seeds will be rinsed 3 to 5 times with sterile distilled water. This variety is temperature and moisture sensitive. 3. Kuala Selangor.3 EXPERIMENT 1: Effects of Growth of Manilkara Zapota Seeds With and Without The Seed Coat.
0 1. From the first experiment. with 1 mg/L of NAA and 1 mg/L of BAP. 3.4 EXPERIMENT 2: Multiple Shoot Induction on Manilkara Zapota seeds.0 2.0 1.0 3.0 4.0 5. blot dry and cultured immediately on MS medium of 30% sucrose concentration.0 1.5 1.0 1. The experiment is reciprocated for 3 replications.0 1. The table below shows the different concentrations used for each treatment which is named MZC.0 GA3 1. The table below shows the different concentrations used for each treatment which is named MZA.0 1. seed with better viability rate will be taken into account.About 6 sterilised seed with the seed coat on will be taken out.0 CONCENTRATION (mg/L) NAA 1.0 1.0 1.0 3. Thus new seeds with similar morphology of the first experiment’s success will be cultured on MS medium with different concentrations of BAP. . TABLE 1: VARIOUS COMBINATION OF PLANT GROWTH REGULATORS ON MUTIPLE SHOOT FORMATION MZC BAP MZC 1 MZC 2 MZC 3 MZC 4 MZC 5 MZC 6 0.0 1. Another 6 sterilised seeds will be excised to expose the endosperm and cultured immediately on the similar concentration medium.0 1.4 EXPERIMENT 3: Root Induction on The Shoots of Manilkara Zapota Shoots formed from the second experiment will be cultured again in this experiment to induce root formation using different concentrations of NAA.
0 3.0 3.0 Data Collection From the first experiment. The second experiment will be observed for 21 days in 3 days interval for multiple shoot induction. The acclimatization of the plantlet will be carried out in the nursery annexe with time intervals of spraying water to the plant and acclimatize the plant.0 1.0 1. The whole plant which has been successfully cultured as whole plant with shoots and roots will be planted into soil containing pots.0 1. 4.TABLE 2: VARIOUS COMBINATION OF PLANT GROWTH REGULATORS ON ROOT FORMATION MZA NAA MZA 1 MZA 2 MZA 3 MZA 4 MZA 5 MZA 6 0.0 1.0 1.0 1.5 1.0 CONCENTRATION (mg/L) BAP 1.0 5.0 4.0 1.0 1. The data will be collected once in every 3 days for 15 days.0 1.5 EXPERIMENT 4: Acclimatization of successfully cultured whole plant of Manilkara Zapota. The . the parameter that will be measured will be the effects of growth with and without the seed coat. The third experiment is observed for root induction for 21 days as well in 3 days interval.0 2.0 1.0 GA3 1.
from the second experiment.0 Tentative Activity / month Literature review 2009 DEC JAN FEB MAR APR MAY JUN JULY AUG SEP OCT Total (RM) 1000. 6. Item Research materials and supplies Travel and transport Special services & Miscellaneous Stationery 8.0 Expected Results From the first experiment.00 700. we can expect that BAP with higher concentrations will give rise to multiple shoot formation.00 . and compared using the SPSS/16 software. From the third experiment.00 400. And from the fourth experiment.0 Statistical Analysis All collected data will be evaluated.fourth experiment will be considered for nearly 14 days with close care and attention given everyday. we can expect that seeds without the seed coat will be the best viable seed for germination.0 Costing / Budget A budget of approximately of RM2000 to RM2500 will be needed to conduct this experiment.00 100. The overall time frame taken for data collection will be around 2 to 3 months. we can expect that lower concentrations of NAA will give rise to root formation. Also. 7. analysed. 5. we will be able to acclimatize the plantlets when proper attention and care given.
W.S. Boussaïd.. and Marrakchi. 2001. H.. Dayana. Chawla. Using Petioles Culture. Introduction to Plant Biotechnology. S. Journal of Applied Horticulture.Sample collection Laboratory work Data collection & analysis Thesis writing Thesis Submission Viva preparation References Arous. Science Publishers. M. 2002. 538 p.). A Preliminary Study of In Vitro Establishment of Gnetum Gnemon Linn. M. University Industry Selangor. Plant Regeneration From Zygotic Embryo Hypocotyls of Tunisian Chili (Capsicum annuum L. Bachelor Thesis. . 2006. Malaysia. 3(1): 17-22.
Bachelor Thesis. 2:128-139. 2001. 2005. CRC Press. S. Trigiano. Malaysia. . Bachelor Thesis. Saleha. Introduction to Plant Tissue Culture. H. 1987. pp.K. 426 p. Malaysia. and Temples: Conservation and Development in The Maya Forest Of Belize. Timber. 26-27. Letchmana. Micropropagation of Rhizophora Apiculata Using Petioles and Leaf Explants. Sapodilla. T. Tourists. 2003. Miami. pp 96-99. Eng.B. 1999.K. Zygotic Embryo Culture. 2007.Van Royen) Following Liqiud Nitrogen Exposure. D. Bachelor Thesis. Morton. Primack. Jha. R. B. Bray. J. 2007. Florida. Seed Storage of Horticultural Crops. Effeccts of Desiccation and Sucrose Preculture on Survival of Zygotic Embryos of Ciku (Manilkara Zapota L. pp. Fruits of Warm Climate. 1997. and Galletti. D. Micropropagation of Piper Sarmentosum Leaf and Petiole Explants. Orient Blackswan. Science Publishers. Haworth Press. Island Press.N. R. K.393-398. S. Malaysia. 2007.Doijode..D. Plant Tissue Culture Concepts and Laboratory Exercises. University Industry Selangor. Plant Tissue Culture: Basic and Applied.. 2: 24-37. Razdan. and Gray. Embryo and Endosperm Culture. University Industry Selangor. M. and Ghosh. University Industry Selangor.J. L. Guatemala And Mexico.
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