JATROPHA CURCAS: SEED GERMINATION AND PROPAGATION METHODS Samba Arona Ndiaye Samba1, Bassirou Diallo2, Moustapha Diop3,

Marone Diatta4, Abdou Sacor Sarr5, Massaer Nguer6, Macoumba Diouf7 Institut Sngalais de Recherches Agricoles (ISRA) - Laboratoire National des Recherches sur les Productions Vgtales (LNRPV), Route des Hydrocarbures, Bel-Air, BP 3120 Dakar, Tl. (221) 832 62 98 / (221) 859 17 19/20, Fax : (221) 832 24 27, Senegal, E - mail: dgisra@isra.sn / bathie_samba@yahoo.fr / diouf_macoumba@yahoo.fr

1, 2, 5, 6

Laboratoire National des recherches sur le Productions Vgtales, Route des

Hydrocarbures, Bel Air, Tl. (221) 832 6298, BP 3120 Dakar, Sngal
3, 4

Centre de Recherche Agronomique de Saint - Louis

Direction Gnrale ISRA ABSTRACT

Studies on seed germination, growth rate, propagation by seeds and cuttings and micropropagation of Jatropha curcas have been conducted in Senegal in greenhouse conditions and in the fields to contribute to the knowledge of Jatropha curcas cultivation. Percentage germination under greenhouse conditions amounted 86 %. In vitro germination was difficult to achieve due to seed infections. However using coatless seeds allowed 20% of germination. Three month old seedlings reached a mean height growth of 24.3 cm. Three months after planting mean height of these seedlings reached 53.5 cm. Overall survival of seedlings in the station of Ndiol was 99.4% for planted seedlings and 60% for cuttings four weeks after planting. Seedling growth was faster on clayey soils than in sandy soils. Sprout number produced by seedlings followed the same trend. Percentages of dead cuttings seemed to decrease with increasing cutting length in greenhouse conditions. Disinfected of explants with HgCl2 1 alone or combined with NaClO 4 did not prevent Jatropha explants from

infections, for soaking durations varying between 15 and 35 min. 95 % of the explants placed in culture in MS0 liquid medium with added elements showed a reactivation of their axillary bud. Jatropha curcas is becoming a new perennial cash crop in Senegal, therefore special attention should be given to the way Jatropha plantations will be carried out. Key words: Jatropha curcas, Propagation methods, cuttings, seedlings, greenhouse, Micropropagation, Senegal I- INTRODUCTION Jatropha curcas L. is a tropic species from the family Euphobiaceae. Special interest has been shown in the cultivation of the species for oil extraction, especially since it is drought resistant and can be cultivated on marginal land, without competing with food production (Heller 1996; Grimm 1996; RF 1998). Today, Jatropha curcas has gained much attention (Grimm 1996; Heller 1996; Henning 2000a; Pratt et al. 2002) for both non oil producers and oil producers countries around the world. African countries are no exception and countries such as Mali and Tanzania are extracting Jatropha oil for years. The species has a high yield in oil, which can be used as fuel for diesel engines as well as for medical and insecticidal purposes (FACT Foundation, 2006). Jatropha grows on well-drained soils with good aeration and is well adapted to marginal soils with low nutrient content (FACT Foundation, 2006). It is not sensitive to day length (ICRAF, 2003). In many African countries, it is grown as a live fence and can be used to reclaim eroded areas (Heller 1996; Joker and Jepsen 2003, FACT Foundation, 2006). Jatropha can reach 6 meters or more (Heller 1996; Makkar et al., 2001). Its leaves and stems are toxic to animals (FACT Foundation, 2006), but after treatment, the seeds or seed cake can be used as an animal feed (Makkar et al. 2001). Its nitrogen - rich seed cake is a good soil fertilizer (RF, 1998; Makkar et al. 2001, FACT Foundation, 2006). Traditionally the seeds are used for medical treatments and soap production (Duke 1983; Henning 2002).

Jatropha is fast growing and produce seeds after approximately two years, depending on many factors such as rainfall conditions and either the plant is propagated from cuttings or from seeds. Research on cultivation and propagation of Jatropha curcas is very limited, especially in the sahelian region of Africa. But quick establishment of live fences is usually achieved by directly planted cuttings. Unfortunately researches have shown that plants propagated by cuttings show a lower longevity and possess a lower drought and disease resistance than plants propagated by seeds (Heller, 1996). Besides, the more suitable cutting length has not been found yet. But traditionally, cuttings of fifty to sixty centimeters long, or slightly more, are generally used by farmer to establish their live fences. Today the species is going to be planted at a large scale in Senegal for biofuel production purposes. This will require a very large amount of cuttings and seeds. The adequate length of cuttings needs then to be found in order to get more cuttings by reducing the usual length traditionally adopted. But Henning (2000c) has noticed that pre-cultivation of Jatropha seedlings in poly-ethylene bags is more appropriate and helps to accelerate the installation of a plantation by at least 3 months compared to cuttings. The aim of the present paper is to contribute to the knowledge of Jatropha curcas cultivation through the presentation of preliminary results of different studies that are being carried out by the Senegalese Agricultural Research Institute in different aspects. Seed germination, horticultural and in vitro vegetative propagation and growth rate studies of Jatropha curcas in Senegal (Dakar and Saint - Louis) are especially concerned by these results

II- MATERIAL AND METHOD 2.1. In vitro seed germination of Jatropha curcas Four in vitro germination trials have been conducted using one lot of mature J. curcas seeds harvested in Thies (Silma district, on 27/06/2006). In the first trial, 50 seeds were disinfected under a laminar flow cabinet (sterile environment) with C2H2OH 70, then with HgCl2 1 during 10 min and soaked in distilled water during 2 h. In the second test another sublot of 50 seeds were disinfected only with HgCl2 1 during 30 min. In the third test, 50 different seeds were scarified with H2SO4 96-98 % and disinfected with HgCl2 1 during 20 min. In the fourth trial, 50 other seeds of the same lot were mechanically decorticated (seed coat removed) and disinfected with HgCl2 1 during 20 min. Disinfection with HgCl2 1 % was always followed by six 5 min rinsing periods with sterile distilled water. Then, all seeds were put for germination tests in glass containers containing agar solutions, the whole being placed at 28 C room temperature. 2.2. Seeds germination under greenhouse conditions Three germination tests were conducted under greenhouse conditions using sublots containing 50 seeds which were directly sown in poly - ethylene bags after 12 h soaking in drinking water. One seed was sown by bag and the sowing was superficially made, approximately 1 cm deep. Seed lots originated from Thies (Senegal) and were collected in June 2006, oven - dried to low moisture content (5 - 7%) and stored in air-tight containers in a cold room at 4 C, during 90 days prior to sowing 2.3. Vegetative propagation of Jatropha with cuttings under greenhouse conditions Plant materials used to produce cuttings were collected on 31/01/07 in Ndiar (Thies), situated at 14 N and 17 W, and were gauged the same day to avoid drying. Cuttings were planted in polyethylene bags (7 cm x 20 cm) 5 days after, using different cutting lengths (3 cm, 5 cm, 10 cm and 15 cm). For each length, 25 cuttings were used.

A relative plantation depth, taking into account the length of cuttings, was adopted. It was fixed to one third (1/3) of the cuttings length, taking into account the fact that i) the length of cuttings traditionally used by the producers is approximately 60 cm and 2) cuttings are traditionally planted at an approximate depth of 20 cm, corresponding to 1/3 of the length. So, the following depths of plantation were adopted in this study conducted in nursery conditions: 1 cm, 1.7 cm, 3.3 cm and 5 cm, respectively for cuttings of 3 cm, 5 cm, 10 cm and 15 cm long. After plantation, cuttings were placed during three weeks in a mini - plastic greenhouse conceived for that purpose. Mean temperatures under the greenhouse were 35 - 40 C in the daytime and 30 - 35 C in the evening. Cuttings were then moved and placed in another greenhouse, at ambient temperature (27 - 32 C in the daytime and 19 in 25 C at night). The following data were noted at the beginning of the trial: length and diameter of cuttings, number of buds per cutting, and depth of plantation. 2.4. Propagation from seedlings and cuttings on station Since 2006, nursery and field research works on Jatropha curcas are being conducted in the station of Ndiol, 25 km far from of Saint - Louis. 1600 Jatropha seedling have been produced in nursery and two planted plots of Jatropha implemented. The first one covers 0.76 ha and contained 500 planted seedlings raised in poly ethylene bags, with 4m x 4m spacing. The second one covers 0.03 ha and contains 80 cuttings raised also in poly ethylene bags with 2m x 2m spacing. Seedling and cutting plantations were done the same day on January 15, 2007. Three (3) rectangular subplots (P1, P2, P3) of 96 m2 each were randomly distributed in the main plot of planted seedlings (PS). Each subplot contains 12 plants. In the second main plot with cuttings (PC), three subplots (P1, P2, P3) of 12 m2 were randomly installed. Each subplot contains 6 cuttings. Subplots P1 and P2 of PS main plot are characterized by clay - sandy soils and P3 by clayey soils. Subplots of PC main plot are all located in a sandy soil.

2.5. Micropropagation of Jatropha curcas 2.5.1. Disinfection trials Thirty explants of one to two years old, stemming from young seedlings raised in greenhouse were used as plant basic materials. Explants were placed in culture in Murashige and Skoog, 1962 medium (MS) without plant growth hormones after disinfection. They were subjected to various treatments (disinfectant chemicals: NaClO, HgCl2 1,) with different times of soaking (15, 20, 25, 30, 35 min). 2.5.2. Micropropagation The same plant basis material was used. Explants were disinfected and placed in the same liquid medium to which we added the following elements: sugar 30 g/l; activated charcoal 2 g/l; adenine sulphate 25 g/l; glutamine 100 mg / l; agar 8 g/l; BAP 2 mg / l. III- RESULTS 3.1. Seed germination of Jatropha curcas Percentage germination under greenhouse conditions was high and amounted 86 % 15 days after sowing. In vitro germination was more difficult to achieve: all seeds were infected in the first, second and third trials with no germination at all. But in the fourth trail where coatless seeds were used, 20% of germination has been achieved (one seed out of five), the rest being infected too. 3.2. Growth rate of Jatropha curcas Jatropha curcas seed lots used in these trails originated from Thies (Senegal). Seeds were collected in June 2006, stored in a cold room at 4 C during 90 days prior to sowing. One thousand seeds weighted 556.9 g. Three months after sowing seeds in black polyethylene bags maintained in a greenhouse, mean height growth of seedlings varied between a minimum of 15 cm and a maximum of 33 cm. Then, the range (max - min) was large and amounted 18 cm. The overall mean of seedlings was 24.3 cm (standard error = 1.1 cm). Three months after

planting the seedlings in Bel Air, mean height of plants reached a mean height of 53.5 cm (standard error = 2.3 cm). Therefore the range between seedlings increased to 40 cm, varying from a minimum of 27 cm to a maximum of 67 cm. In the station of Ndiol (Saint - Louis), overall dead rate was 0.6% for planted seedlings and 40% for cuttings two weeks after planting. This rate remained at the same level two weeks later. Seedling growth was faster on clay - sandy (subplot1: 18.92 cm) and clayey soils (subplot2: 18.08 cm) than in sandy soils (subplot3: 11.75 cm; Figure 1). Sprout number produced by seedlings followed the same trend; with 4.3, 4.1 and 1.6 sprouts for subplot1, subplot2 and subplot3 respectively.
25 Growth (cm) / Sprout number 20 15 10 5 0 Subplot1 Subplot2 Subplot3 Growth (cm) Sprout number

Figure 1. Seedling growth and number of sprouts produced as a function of soil types (clay sandy, clayey and sandy soils) in the station of Ndiol. 3.3. Propagation of Jatropha curcas by cuttings Five weeks after plantation under greenhouse conditions, only 12% of 10 cm long cuttings (C10cm) produced open buds (Table 1). Percentages of dead cuttings seemed to decrease with increasing cutting length with 100, 60, 24 and 28%, respectively for C3cm, C5cm, C10cm and C15cm. One week later, the percentage of cuttings with open buds increased to 60% for C10cm and started for C5cm and C15cm with 8 and 16% respectively.

By week number seven, the percentage of cutting with open buds reached 80% for C10cm, and 60% and 20% for C15cm and C5cm. Seven weeks later, this percentage did not varied for C10cm and C5cm but increase to 60% for C15cm. Globally, we noticed that 100% of C3cm, 80% of C5cm, 20% of C10cm and 40% of C15cm were dead. In the station of Ndiol, cuttings had the same mean growth (4.2 cm) in subplots 1 and 3 with standard errors of 1.11 cm and 1.40 cm, respectively. In subplot 2, cutting growth was very low (0.67 cm 0.42 cm). Sprout number produced by cuttings was greater in subplot 1 with a mean of 3.2 (standard error = 0.31). The number did not vary in the other subplots: 2.7 (standard error = 0.21).

Table 1. Cumulative number of cuttings (3, 5, 10 and 15 cm long) with opened buds and cumulative number of dead cuttings as a function of time (5, 6, 7 and 14 weeks after plantation) in greenhouse conditions, n = 25 for each cutting length.
Variables 5 weeks after plantation 3 cm Cuttings with open buds Rotten cuttings Total 0 25 25 5 cm 0 15 15 10 cm 3 7 10 15 cm 0 6 6 6 weeks after plantation 3 cm 0 25 25 5 cm 2 20 22 10 cm 15 9 24 15 cm 4 9 13 7 weeks after plantation 3 cm 0 25 25 5 cm 5 20 25 10 cm 20 5 25 15 cm 14 10 24 14 weeks after plantation 3 cm 0 25 25 5 cm 5 20 25 10 cm 20 5 25 15 cm 15 10 25

3.4. Micropropagation of Jatropha curcas 3.4.1. Disinfection trial All explants disinfected with HgCl2 1 alone or combined with NaClO 4 were subjected to infections for soaking durations varying between 15 and 35 min. However with NaClO 8 as disinfectant chemical and for a soaking time of 25 min, 25% seed germination was reached, but infection percentage was still very high (75%). Table 2. Germination rate (%) and percentages of infection (%) of Jatropha curcas seeds under different disinfectant chemicals and different soaking durations.
Disinfectant chemical HgCl2 1 HgCl2 1 HgCl2 1 HgCl2 1 HgCl2 1 + NaClO 4 HgCl2 1 + NaClO 4 NaClO 8 Soaking duration (min.) 15 20 25 30 25 + 5 25 + 10 25 Germination rate (%) 0 0 0 0 0 0 25 Infection (%) 100 100 100 100 100 100 75

3.4.2. Micropropagation Ninety five percent (95 %) of the explants placed in culture in MS0 liquid medium with added elements (sugar 30 g/l, activated charcoal 2 g/l, adenine sulphate 25 g/l, glutamine 100 mg / l, agar 8 g/l and BAP 2 mg / l) reacted after 10 days with a reactivation of their axillary bud which produced one to two leaves that spread well after one month in vitro culture. None of the remaining explants has shown a morphogenetic reaction, probably because of their infection.

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IV- DISCUSSION Jatropha curcas seed lots used in our trails originated from Thies (Senegal). The seed weight per 1000 seeds was 556.9 g which means that the average weight of a seed was 0.5569 g which is lower than the average of 0,750 g found by FACT Foundation (2006). But these two results fit within the 0.53 to 0.74 g interval found by Makkar et al. (1998). Seeds were harvested in June 2006, stored in a cold room at 4 C during 90 days prior to sowing. Seed germination percentage under greenhouse conditions reached a cumulative rate of 86 % within 15 days after sowing. However, this good performance remained lower than the one obtained in South Africa with a mean rate above 93 % and a completed germination within 9 days only. According to FACT Foundation (2006), with good moisture conditions the germination of Jatropha curcas seeds takes 10 days. Jatropha seeds are oily and do not store for long (Kobilke, 1989). Research on viability of Jatropha seeds has shown a decrease due to term of storage: seeds older than 15 months show viability below 50 % (Kobilke, 1989). However many other factors can influence the establishment by direct seeding of Jatropha curcas: seeding depth, date and quality of seed, soil moisture content, soil preparation quality (Heller, 1996). In vitro germination of Jatropha curcas seeds was hard to achieve, since almost all seeds were infected in all our trials with no germination except the last one, with coatless seeds, but where rate of germination did not exceed 25%. In greenhouse conditions, the results have shown in one hand that cuttings shorter than 10 cm have a very low survival rate and are not suitable for Jatropha curcas propagation. In the other hand, percentages of dead cuttings seemed to decrease with increasing cutting length. But besides this variable, many others can influence the establishment of Jatropha curcas by direct planting of cuttings: cutting material (length, diameter of branch, age, location of the cutting in the tree), cutting time, storage of cuttings, fungicide treatment, planting time,

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preparation of planting hole, depth of planting, soil moisture content, weed clearance (Heller, 1996). Micropropagation of Jatropha curcas is still a problem. All explants disinfected with HgCl2 1 alone or combined with NaClO 4 were subjected to infections for soaking durations varying between 15 and 35 min. However with NaClO 8 as disinfectant chemical and for a soaking time of 25 min, 25% seed germination was reached, but infection percentage was still very high (75%). Ninety five percent (95 %) of the explants placed in culture in a MS0 liquid medium with added elements (sugar 30 g/l, activated charcoal 2 g/l, adenine sulphate 25 g/l, glutamine 100 mg / l, agar 8 g/l and BAP 2 mg / l) reacted after 10 days with a reactivation of their axillary bud which produced one to two leaves. According to many unpublished papers and testimonies, Jatropha curcas is one of the most difficult plants to deal with for in vitro propagation. Basic difficulty usually encountered is obtaining rooting. A brief note on Jatropha tissue culture works based on experiments conducted has been presented by Sanket (2004) and is as follows: Jatropha axillary buds of fresh shoots are ideal for culture initiation; a combination of BAP (1 mg/l), adenine sulphate (10 mg/l) and citric acid (30 mg/l) has been proven to be suitable on MS medium for shoot multiplication, elongation and shoot buds induction; necrosis of leaves and entire tissue mass is a serious problem and requires anti - oxidants and more growth regulators; elongated shoots resulted into rooting on MS medium supplemented with IAA and IBA (rooting success is still limited); rates of shoot multiplication and elongation are slow as compared to other species, therefore more efforts needed to accelerate the growth.

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Jatropha curcas growth rate has been variable. Three months after sowing, mean height growth of seedlings produced in greenhouse conditions ranged between 15 cm and 33 cm and showed variability among seeds, since soil medium in poly - ethylene bags and water supply were constant. Three months after transplanting the seedlings, the range between seedlings height varied from 27 cm and 67 cm, showing probably a cumulative variability, among seeds and among micro sites (soil characteristics, ), which influenced the seedling growth rate. Growth rate differences were noticed in the station of Ndiol between seedlings (16.25 cm, error standard = 0.90) and cuttings (3 cm; error standard = 0.7 cm). Seedlings have also produced more sprouts (3.3) than cuttings (2.8). CONCLUSION Jatropha curcas is becoming a new perennial cash crop for many regions of the world. This makes that special attention should be given to the way Jatropha plantations will be carried out. Jatropha curcas culture can be done in marginal lands or intercropped with food producing species (millet, sorghum, maize, ) even though some authors claim that the root system of cuttings, not having a tap root, but more lateral roots, is less suitable than that of the seeded plants with tap roots in intercropping systems (the side roots make the plants use more space rather than depth). Studies must be undertaken on this feature. Anyway, if well managed, Jatropha curcas culture should not affect food-producing species yield and can become a very interesting development tool for no oil producers countries. However researches must be performed in many aspects such as: Productivity studies of different provenances of Jatropha curcas in different ecological zones and selection of the best provenances on the basis of seeds and oil productivities; Optimal nutritional and physiological requirements studies of Jatropha curcas according to ecological zones;

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Management of Jatropha curcas plantations according to ecological zones to optimize grain yield per unit area;

Studies on attacks and diseases which can affect seeds and oil content productivity of Jatropha curcas plantations;

In vitro propagation using various methods (from axillary buds, tissue culture, ); Constitution of Jatropha gene banks (cold room, laboratory, greenhouse, fields); Gene viability tests and renewal of stocks. Disinfection of basic plant material REFERENCES

Duke, J. A. 1983. Handbook of Energy Crops. Purdue University. Center for New Crops and Plants Products. Unpublished FACT Foundation: Handbook on Jatropha Curcas First draft March 2006 - www. factfuels.org Grimm, C. (1996). The Jatropha project in Nicaragua. Bagani Tulu (Mali) 1: 10-14. Heller, J. 1996. Physic nut. Jatropha curcas L. In: International Plant Genetic Resources Institute (IPGRI), Promoting the conservation and use of underutilized and neglected crops. (Prom Underused Crops) 1:166. Henning, R. 2000a. The Jatropha Booklet. A Guide to the Jatropha System and its Dissemination in Zambia. GTZ-ASIP Support Project Southern Province. Bagani GbR. Henning, R. 2000b. The Jatropha Manual.A guide to the Integrated Exploitation of the Jatropha Plant in Zambia. Henning, R. 2000c. Use of Jatropha curcas oil as raw material and fuel: an integrated approach to create income and supply energy for rural development. Experiences of the Jatropha Project in Mali, West Africa. Presentation at the International Meeting Renewable

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Energy - A Vehicle for Local Development - II. Folkecenter for Renewable Energy, Denmark, August 2000. Henning, R. (2002). Using the Indigenous Knowledge of Jatropha The use of Jatropha curcas oil as raw material and fuel. IK Notes. No.47. August. World Bank. ICRAF, 2003. Jatropha curcas. Agroforestree

Database.http://www.worldagroforestrycentre.org/Sites/TreeDBS/AFT/SpeciesInfo.cfm?SpID =1013. Jker, D and J. Jepsen (2003). Jatropha curcas L. Seed leaflet No. 83 August 2003. Danida Forest Seed Centre. Denmark. Kobilke, H. (1989). Untersuchungen zur Bestandesbegrndung von Purgiernu (Jatropha curcas L.). Diploma thesis. University Hohenheim, Stuttgart Makkar, H.P.S, Becker, K and B. Schmook (2001). Edible provenances of Jatropha curcas from Quintna Roo state of Mexico and effect of roasting on antinutrient and toxic factors in seeds. Institute for Animal Production in the Tropics and Subtropics (480), University of Hohenheim, D-70593 Stuttgart, Germany. Pratt, J.H.; Henry, E.M.T.; Mbeza, H.F.; Mlaka, E. and L.B. Satali (2002). Malawi Agroforestry Extension Project Marketing & Enterprise Program Main Report. Publication No. 47. Malawi Agroforestry. 2002 RF (1998). The Potential of Jatropha curcas in Rural Development and Environment Protection An Exploration. Concept paper. Rockerfeller Foundation and Scientific & Industrial Research & Development Centre, Harare, Zimbabwe 1998. Sanket T. 2004. BioForum > Biology Methods Forums > Botany and Plant Biology http://www.protocol-online.org/forums/index.php?s=1597595bec3dace7b0ae14ee14a40ae2 &showtopic=3575&st=0&p=6761&#entry6761

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