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MICRO-PROPAGATION: A USEFUL TOOL FOR RAPID MULTIPLICATION OF OIL PALM (ELAEIS GUINEENSIS) HYBRIDS IN KENYA

L.N. Mimano1, C.W. Macharia1 and L.A. Wasilwa2 1 KARI-Biotechnology Centre, P.O. Box 57811-00200, Nairobi, Kenya 2 KARI Headquarters, P.O. Box 57811-00200, Nairobi, Kenya Abstract Oil Palm (Elaeis guineensis) produces palm oil used in food products, non-food derivatives, detergents and pharmaceutical industries. Kenya imports 225,000 tons of vegetable oil annually at a cost of 11 billion KES of the estimated current national annual demand of 380,000 tons of which 90% is oil palm based. Introduction of cold tolerant oil palm germplasm has enabled a crop originally associated with low land cultivation, to be planted at higher altitudes. Oil palm is the highest yielding oil crop in the world. Since the introduction of oil palm in Kenya, in 1990, only 27,000 trees are presently cultivated by smallholder farmers in six districts in western Kenya. The major limitation has been the availability of planting materials. Pre-germinated seeds are imported from Costa Rica at the cost of one dollar per seedling. Despite this high cost, the farmers demand has not been met. The objective of the experiment was to develop reliably repetitive methods for rapid micropropagation of oil palm using leaf and embryo explants. Seeds of hybrid Dura Ghana were obtained from Costa Rica. Pre-germinated seeds were planted in nurseries and maintained in a greenhouse to obtain leaf explants. Embryos excised from seed and leaf tissue were inoculated on to amended Murashige and Skoog (MS) media. Plantlets were regenerated on MS media supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and gibberellic acid (GA3). Oil palm seeds that were soaked in sterile distilled water for 3 weeks before excising the embryo gave significantly higher numbers of micro-propagated plantlets. This method produced one plantlet per embryo which was used as the mother source for obtaining leaf explants to propagate plantlets on a mass scale using callus induction. Though this method successfully produced calli from leaf explants, contamination of the cultures was a problem. The control of contamination is therefore a major issue to allow the mass production of oil palm seedlings using this method, which is currently underway. Introduction Oil palm (Elaeis guineensis), native to West Africa, is the highest yielding oil crop in the world. It is as an evergreen perennial that may be used to conserve the environment. Palm oil accounts for 21% and 47% of the global oil and fats production and trade, respectively. Malaysia and Indonesia are the world's largest producers and exporters of palm oil with 84% and 90% share of world palm oil production and export, respectively (Datuk 2003a). Compared with the major oilseed crops (soyabean, rapeseed and sunflower), oil palm is considered the most environmentally friendly with respect to land use efficiency and productivity, energy efficiency and inputs of fertilizers and pesticides and pollution potential. It contributes positively to climatic change through effective carbon sequestration and it is not a genetically modified plant (Datuk, 2003b). Palm oil is an important food and a major source of lipids. As the world population continues to increase, thus creating increasing demand, as such, oil palm will continue to be cultivated worldwide. Fruits of oil palm produce red palm oil that is rich in vitamins A and E, precursors, the deficiency of which is rampant in western Kenya and eastern Uganda (Steele and Griffee, 2001; Odenya et al., 2003 and Gitobu et al., 2004). In East Africa cold tolerant oil palm (CTOPs) are mainly grown by smallholders (Anon, 2002 and Rachier et al., 2004). The crop thrives well in rainy humid tropical climate and low-lying alluvial soils (Chinchilla, 2004). Optimum temperature requirement is 24 27OC with mean annual temperature of 20-21OC. Areas with 15001700 mm of rainfall without prolonged dry periods are most desirable for production. Oil palm requires acidic soils with a pH of 5.55.7. Cultivated oil palm begins bearing fruits at the 3rd and 4th years after planting, and attains peak fruit bearing after 12-15 years. It continues bearing fruit for 40-50 years, yielding 35-50 kg oil plant-1 year-1 (4-5 t ha-1 of oil yearly) (Wahid et al., 2004). Oil palm is the major source of edible vegetable oil for cooking fats, manufacture of margarine and ice creams. Lower grades of the oil are used in the confectionery industry and in manufacture of soaps and candles. The oil cake is used in the manufacture of animal feeds and other solid products used as fuel (Ng et al., 2002) and the sap fermented to make palm wine. Rachis and leaves are used for thatching houses (Godin and Spensley, 1997). Other uses of oil palm tree include fencing material, firewood; husk fibre for fuel or filling mattresses, pillows and cushions and for matting and trunks may be used for building. Kenya produces only 34% of the annual national requirement of edible oil estimated at 380,000 t, and imports about 225,000 t worthy about KES 11 billion (Anon, 1994; Steele and Griffee, 2001 and Chinchilla, 2004). Over 90% of the imported oil is oil palm (Oil World, 1999). To save the foreign exchange used to import palm oil and create employment, the Food and Agriculture Organisation funded a project to promote cultivation of oil palm in 2002 in Kenya. Tenera types of hybrids from Costa Rica were introduced to western Kenya in 1999

(Odenya et al., 2003). About 27,000 trees are currently cultivated by smallholder farmers in Bungoma, Teso, Butere/Mumias, Busia, Siaya and Kakamega Districts (Rachier et al., 2004 and Diemer et al., 2005). Since 1999 the acreage has increased by 30 to 50% annually. At least one million trees are required to produce enough fruit to warranty industrialisation. Elaeis guineensis is allogamous and propagated via seeds. Micro-propagation is an efficient tool for multiplication of allogamous material obtained by plant breeders. The potential of "in vitro" propagation of oil palm needs to be exploited in Kenya in order to expedite multiplication of planting material for commercialisation. In vitro propagation techniques provide a means of propagating oil palms introduced into Kenya in 1990 that are no longer multiplied by breeders in their country of origin. In particular somatic embryogenesis, especially with cell suspensions, offers a powerful tool for rapid multiplication of oil palm. These approaches will save foreign exchange, promote smallholder palm oil production and processing with the long term aim of crop diversification, food security and poverty alleviation. The objective of this study is to develop a rapid micro-propagation protocol for oil palm. Materials and methods Surface sterilisation. Oil palm seeds of hybrid Delix Ghana were obtained from ASD Costa Rica. The seeds were surface sterilised before excising the embryo using the following procedure. Oil palm seeds were immersed in a mild solution of water and Tween-20 and swirled for one minute. The clean seeds were then rinsed in running water for five minutes and soaked for 20 minutes in a 50% solution of Sodium hypochlorite (Jik). Thereafter the seed were immersed in 80% ethanol for 10 minutes and washed in sterile distilled water (3 times) and placed on sterile blotting paper. Seed was soaked for 7 to 21 days before the embryos were excised. Pre-germinated seeds were planted in a pre- and main nurseries at KARI Alupe. A few seedlings were transferred from KARI Alupe and maintained in a greenhouse at KARI Kabete to obtain leaf explants. Leaf explants were sterilised using combinations of ethanol, bleach and sterile water. Embryo rescue. The Murashige and Skoog (MS) (1962) salts, supplemented with benzyladenine (BA), naphthalene acetic acid (NAA) and gibberellic acid (GA3) were used for embryo culture. Macronutrients [Na NO3 (0.6g/l); CaCl2.2H2O (0.075g/l); Mg SO4.7H2O (0.25g/l); KCl (0.75g/l) and NaH2PO4.H2O (0.125g/l)], micronutrients [MnSO4.4H2O (0.1mg/l); ZnSO4.7H2O (1mg/l); H3BO3 (1mg/l); KI (0.01mg/l); CuSO4.5H2O (0.03mg/l); AlCl3 (0.03mg/l); NiCl2.6H2O (0.03mg/l) and FeCl3.6H20 (1mg/l)] and sucrose (30g) were added to 1 L of de-ionised water and calibrated to pH 5.6. Bits of cotton wool were placed in test tubes and liquid media dispensed to submerge them. This media was autoclaved at 120 OC for 20 minutes at 15 psi. The kernels were cracked with a sterile nut cracker to expose the embryo. The embryo was excised using a scalpel and forceps ensuring minimal damage. The embryos were inoculated onto liquid media with cotton wool to anchor the embryo. These cultures were incubated in the growth room at room temperature with 16 hours of fluorescent light for two months. When root and shoot primordial developed, the explants were inoculated onto organ regeneration media to initiate shoot and root formation. Leaf micro-propagation. Young leaves were obtained from oil palm seedlings maintained in a greenhouse. Leaves produced In vitro using embryo culture were also used as explants for callus induction. Different levels of 2, 4D were used to determine the best level for callus induction. Regeneration medium was essentially the same as the establishment medium but contained MS (4.43g/l), NAA (0.5mg/l, BAP (2mg/l), agar (8g/l), sucrose (30g/l) in 1 L of water at pH 4.5. The cultures were incubated in a growth room at room temperature and illuminated for 16 hours. The experimental design was a completely randomised block design with 10 replicates per treatment with two explants per replicate. Data was taken on the number of fully developed embryos, embryos with only shoots, amount of callus on leaf explants per treatment, the number of dead and contaminated treatments. Data collected was subjected to analysis of variance (ANOVA) using General Linear Model (GLM) statistical package. The means of each treatment were compared using the least significant difference (LSD) test. Results and discussions Embryo and leaf micro-propagation. Regeneration of oil palm using embryo rescue method was achieved (Plate 1). Oil palm seeds that were soaked in sterile distilled water for 3 weeks before excising the embryo gave significantly higher numbers of regenerated plantlets than those excised after 7 days. A single embryo produced a single plant indicating that embryo rescue is not the best method for mass propagation of oil palm. Although several media with leaf explants produced large white calli within 8 weeks (Table 1), embryoid differentiation was not achieved because all leaf cultures subsequently became contaminated.

Embryo on cotton wool soaked in media

Shoot development

Oil palm plantlet

Stages of oil palm development in vitro using embryo rescue

sequence of events

Plate 1Oil palm embryo culture

Table 1 Response of oil palm leaf explants to 2, 4D. Data obtained from leaf explants obtained from embryo rescue 2, 4D mg/L 0.5 1.0 No callus formed 1.5 2.0 2.5 Leaf explant covered with callus 3.0 No callus formed Remarks on callu No callus induction formed Some callus Moderate on leaf edges callus formation

Contamination in cultures from leaf explants was either from poor sterilisation protocol or poor incubation condition. Embryo culture will be useful for the production of sterile explants. Calli induced from leaves of plants grown aseptically by embryo rescue were inoculated onto media to mass produce the required plants of the genetically improved oil palm hybrids. Leaves, inflorescences and roots can be used as explants but young leaf spears are often preferred. Leaf explants can be easily surface sterilised and give higher clonability rates (Rajanaidu et al., 1997). From the explants, callus is initiated, followed by embryogenesis, shoot and root regeneration, hardening of ramets for the nursery and finally field evaluation. The earliest reports of successful vegetative propagation of oil palm by tissue culture were in the mid 1970s (Jones, 1974). Now, about 20 oil palm laboratories are in operation throughout the world with capacity ranging from 10,000 200,000 plantlets per year. As compared to seed production, tissue culture of oil palm offers numerous advantages (Sogeke, 1998). It allows rapid multiplication of uniform planting materials with desired characteristics. This enables improvement of planting materials using existing individuals which have all or

most of the desired qualities such as good oil yield and composition, slow vertical growth and disease resistance. Additionally, it also opens new avenues for producing novel planting materials via genetic engineering, because tissue culture is the means for regeneration of tissues transformed with genes for traits of interest. Favourable reports of plantings of liquid culture plantlets suggested that the technology can be exploited for oil palm clone production (Soh et al., 2001). The liquid culture system offers advantages in reproducibility, versatility and efficiency with high potential for scaling up propagule production. Based on field performance data from various sources, improvement of oil yield (20% to 30%) by clonally propagated materials over seedling planting materials is achievable (Soh et al., 2001). There are indications that selection for resistance to major oil palm diseases will be easier than it is currently based on differences in susceptibility shown by clones to Ganoderma, Fusarium and blast (Purand-Gasselin et al., 1999). The difficulty lies in achieving true-totype reproduction of plants selected as ortets, especially with the incidence of mantled abnormality. The percentage of abnormality in the field has generally been maintained at a tolerable level of less than 5% (Maheran et al., 1995). Prudent selection of good ramets at both in vitro and nursery stage is practised to reduce abnormality level. Maheran et al. (1995) reported that clones appearing normal at both stages gave a low level of abnormality in the field (about 2.2%). It was estimated that the initial investment on clonal materials will be covered in the sixth year after which the returns from clonal materials will be much higher than conventional planting materials due to their higher productivity. Conclusions and recommendations Production oil palm is economically viable and environmentally sustainable. The effort to narrow the gap between commercial yield and potential yield will continue to be given priority in Kenya. The low overall national oil yield averages suggest that besides good planting materials, good crop management and environment are critical for the realization of yield potential. In addition, it is important that the plantations to quickly adopt new technologies, particularly in fertilizer management. This will be a challenge for research scientists, extension agents and farmers. In Kenya, oil palm tissue culture maybe employed both as a means for producing high yielding oil palm germplasm for commercial planting and to rapidly multiply introduced hybrids (Deli Ghana, Tanzania Ekonaor, Tanzania Bermeda) or developed hybrids for commercialisation. Based on current demand for oil palm, seedlings micro-propagation technologies should be developed to provide cheap plant material. Acknowledgment The authors wish to acknowledge the Director, KARI through Kenya Agriculture Productivity Programme for provision of the funds that supported this work. We would also like to thank Mrs Mary Wabule (the Assistant Director of Horticulture and Industrial Crops); Dr. S. M. Wokabi (the Centre Director KARI-Kabete) and Dr. Simon Gichuki (Program Co-ordinator Biotechnology Centre) for their continuous support. We also wish to acknowledge the technical assistance of Ms Jostina Ngundo and Mr. Lazarus Kisuya (Officer in Charge KARI Alupe) for maintenance of plant material at the pre-and post nursery management stage. References Anon. (1994). Oil seed Crops Production Handbook. Rural Oil Protein Production and Processing Project (ROPPP) Anon. (2002). Oil palm in western Kenya. FAO Agricultural Services Bulletin AG21. Magazine spotlight. Food and Agriculture Organization of the United Nations Rome. http://www.fao.org/ag/magazine/0202sp1.htm Chinchilla C.M.I. (2004). Agronomic performance of cold-tolerant (stress tolerant) oil palm germplasm in western Kenya: Dry season evaluation. FAO Kenya Oil Palm Report (TCP/KEN/2902) Datuk Yusuf Basiron. (2003a). Palm oil: The Driving Force of the World Oils and Fats Economy. Oil palm industry economic journal - Volume 4 No 1 Datuk Yusuf Basiron. (2003b). Selling the Green Palm Oil Advantage? Oil palm industry economic journal - Volume 4 No 1 Diemer P., Chinchilla C., and Griffee P. (2005). Smallholder oil palm manual. Editors Wasilwa, L.A., Rege. R., Ayaga, M. http://www.ecoport.org/EP.exe$PassCheckStart?ID=E180 Gitobu J.K., Mitoko G., Mukunya D., Tauta C., Wasilwa L. and Kabutha C. (2004). Reducing food insecurity at the community and household level in Kenya: Promotion of integrated gender responsive nutrition and agricultural policies and programs. International Centre for Research on Women. Winrock International, Nairobi, Kenya. Godin V.J. and Spensley P.C. (1997). Crop Production Digest No.1. Oil and Oil Seeds. Tropical Products

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