Tissue culture of Jatropha curcas

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A report on the project which aimed to develop tissue culture protocol for the mass propagation of high oil yielding varieties of Jatropha adaptable to the Philippines.


<p>Tissue Culture of Jatropha curcas10-month Project Report (Aug. 2007 Jan. 2008, May 1, 2008 to August 31, 2008 )Portia Gamboa LapitanProfessor 7 Department of Forest Biological Sciences College of Forestry and Natural Resources</p> <p>A. Project ObjectivesTo develop tissue culture protocol for the mass propagation of high oilyielding varieties of Jatropha adaptable to the Philippines. sterilization scheme for tissues for culture best/most responsive tissue/explant for culture</p> <p> appropriate medium for callus formation, shoot induction and rooting incubation or culture conditions (light, temperature, photoperiod) for growth and development of Jatropha in culture best time for plantlets to be outplanted from the culture vessels</p> <p>B. Methodology Selection of sources of tissues for culture</p> <p>Jatropha PGL 07</p> <p>Selected and graded seeds of Jatropha used as source of explants Jatropha PGL 07</p> <p>Sources of explants were also selected from plants in the nursery and fieldJatropha PGL 07</p> <p>Mature plants of Jatropha for explants collection were also selected</p> <p>Selected plants are sectioned and cultured in different culture media for organogenesis/plantlet developmentJatropha PGL 07</p> <p>Activated charcoal in the culture medium facilitates germination of Jatropha seeds</p> <p>Jatropha Tissue Culture</p> <p>C. Accomplishments and Major Findings1. Sterilization scheme for Jatropha</p> <p>Table 1. Efficacy of sterilization schemes used for Jatropha tissue cultures Total No. of Ave. No. of Cultures Ave. PercentSterilization Scheme CulturesFungal</p> <p>ContaminatedBacterial Fungal</p> <p>ContaminationBacterial</p> <p>5% calcium hypochlorite for 10 minutes Seeds 20/trial of 10 trials 2/trial 1/trial 10% 5%</p> <p>2% Manzate for 30 min 5% calcium hypochlorite for 20 minutes Leaf Tissues Nodal sections 10/trial of 10 trials 5/trial of 10 trials 5-6/trial 3/trial 1/trial 50-60% 60% 10%</p> <p>2% Manzate for 20 min 5% calcium hypochlorite for 10 minutesYoung leaf tissues Shoot tip Young nodal sections 10/trial of 10 trials 5/trial of 10 trials 5/trial of 10 trials 6-7/trial 2/trial 3/trial 1/trial 1/trial 60-70% 40% 60% 10% 20%</p> <p>Sterilizing Jatropha seeds entailed immersion of seeds in 5% calcium hypochlorite for 10 min. For tissues from existing stocks a combination of sterilants 2% Manzate for 30 min and 5% calcium hypochlorite for 20 min was used. Younger leaf tissues were sterilized for shorter duration, 20 min., than stem sections (30 min).</p> <p>C. Accomplishments and Major Findings2. Identification of the best/most responsive tissue/explant for cultureAll tissues from seedlings were responsive to tissue culture and more responsive compared to tissues collected from adult or mature plants.</p> <p>Callus readily formed in all types of tissues</p> <p>Tissues from adult plants</p> <p>Cultures from mature/adult plants formed shoots 2 months after inoculation, tissues from seedlings 1 month after.</p> <p>J2</p> <p>Tissues from young plants/seedlings PGL 07 Jatropha</p> <p>J133</p> <p>J101 1 J10</p> <p>leaf tissuesJ02</p> <p>J13S2</p> <p>stem tissues stem tissuesT2J13S1</p> <p>root tissues</p> <p>Different tissues of Jatropha can initiate shoots07 Jatropha PGL</p> <p>J29S2</p> <p>T2J13S1</p> <p>stem tissues Different tissues of Jatropha initiating shoots#14 J13S2</p> <p>root tissuesNov 06 07Jatropha PGL 07</p> <p>The most responsive explant to shoot formation is the leaf tissue followed by stem explants (Table 2).</p> <p>Table 2. Number of explants developing shoots in the different culture media tested.Culture Medium M8 M8s M18 M18ac M20 M20ac M22 M22ac TOTAL Leaf explnt 8 1 17 1 6 2 1 2 Stem explnt 1 1 3 2 3 2 1 13 Shoot tip 1 1 2 1 5 Root explnt1</p> <p>TOTAL11</p> <p>38</p> <p>3 3 1 8</p> <p>3 25 3 12 4 4 2 64</p> <p> Two types of shoot formation, direct shoot development and the development of embryonic shoot were observed The leaf cultures had the most number of direct shoot development and embryonic shoot formation compared to stem, shoot and root cultures (Table 3).</p> <p> The direct shoot development appeared to be the more common route to shoot formation than the embryonic shoot.</p> <p>Callus from leaf of mature plant Direct shoot developing forming embryonic shoot from callus of leaf tissue Embryonic shoot formed in Jatropha culture (left)Jatropha PGL 07</p> <p>Table 3. Type of shoots formed in different culture media by different explantsEmbryonic shoot Direct shoot development TOTAL</p> <p>Culture leaf stem shoot root leaf stem shoot root Mdium M8 M8s 1 8 1 1 2 2 12 3</p> <p>M18M18ac M20 M20ac M22 M22acTOTAL</p> <p>41 1 1</p> <p>21 1 1 5</p> <p>1</p> <p>1</p> <p>222 4 3 1 2 2 1</p> <p>2</p> <p>22</p> <p>343 9 6 4 2</p> <p>1 6 5 4</p> <p>7</p> <p>1</p> <p>2</p> <p>43</p> <p>73</p> <p>C. Accomplishments and Major Findings3.1 Appropriate medium for callus formation</p> <p>Callus formed in all culture media tested.</p> <p>Growth and development of callus varied depending upon J7 the culture media1-week old</p> <p>M15</p> <p>M18</p> <p>M202-week old</p> <p>Callus morphology differed</p> <p>M15</p> <p>M18</p> <p>M20Jatropha PGL 07</p> <p>White cottony callus formed in M28ac</p> <p>Callus in M22 produced shoots. Embryonic shoots were produced compared to direct shoot development in the other media</p> <p>Nodular calli (left) differentiated to shoots weeks after (right)Jatropha PGL 07</p> <p>Different tissues of Jatropha initiating shoots from callus</p> <p>Jatropha PGL 07</p> <p>C. Accomplishments and Major Findings3.2 Appropriate medium for shoot induction and growthDifferent Modified Murashige and Skoog media (Lapitan 1988) with varying concentrations and combinations of IAA, IBA, NAA, Kinetin, BAP and gibberellinswere effective for different developmental changes in Jatropha tissues cultured.</p> <p>T2J40 Cultures are more responsive to media without</p> <p>than with activated charcoal (AC)</p> <p>M22 w/ AC</p> <p>M22 w/o ACJ29S2</p> <p>M18 w/ ACT2J32</p> <p>M18 w/o AC</p> <p>Change in auxin induced the development of new axillary shoot in less than one week Jatropha PGL 07</p> <p>#14</p> <p>Oct 31 07</p> <p>Jatropha PGL 07</p> <p>The culture media M8, M18 and M20 induced shoot formation better than the rest of the media tested, with M18 registering the highest number of cultures forming shoots followed by M8 and M20 (Table 2).</p> <p>Table 2. Number of explants developing shoots in the different culture media tested.Culture Medium Leaf explnt8 1</p> <p>Stem explnt1 1</p> <p>Shoot tip1 1</p> <p>Root explnt1 -</p> <p>TOTAL11 3</p> <p>M8 M8s M18 M18ac M20 M20ac M22 M22acTOTAL</p> <p>171</p> <p>32</p> <p>2-</p> <p>3-</p> <p>253</p> <p>62 1 2 38</p> <p>32 1 13</p> <p>1 5</p> <p>31 8</p> <p>124 4 2 64</p> <p>J6</p> <p>J102</p> <p>J13S22</p> <p>J2</p> <p>Different tissues of Jatropha initiating shoots in M18Jatropha PGL 07</p> <p>Shoot tips of seedlings growing faster in M8 (left) than in M18</p> <p>Increasing sucrose of culture medium induced even more shoots to form in the cultures.</p> <p>Gibberellins (medium M7) enhanced and improved shoot growth. Shoots big enough for rooting just after 2 weeks.</p> <p>Protocol for shoot proliferation of mature tissues has also been established. Leaf and nodal tissues were induced to develop multishoots in media M8 and M18.</p> <p>C. Accomplishments and Major Findings3.3 Appropriate medium for rooting</p> <p>Rooting is enhanced in media with activated charcoal</p> <p>T2J13S1</p> <p>A rooted leaf</p> <p>protocol for rooting still has to be improved. Outplanting trial result was not that encouraging. Survival was only 30%.</p> <p>C. Accomplishments and Major Findings4. Incubation or culture conditions (light, temperature, photoperiod) for growth and development of Jatropha in culture</p> <p>absence of light can cause the browning of cultures. Shoot elongation appeared not enhanced by short-term exposure to dark treatment.</p> <p>AcknowledgementUniversity of the Philippines Los Banos (Basic Research/Trust Fund) ICRISAT, India; UPLB-CHED Jatropha Research Project; homegrown plantations in Los Banos and Batangas. Chancellor Luis Rey I. Velasco who encouraged the researcher to conduct this kind of study in support of the Biofuel Act of the Philippines and UPLB; Vice Chancellor Enrico P. Supangco who looked for funds for the project to push through; Dr. Arturo S.A. Castillo who opened his Jatropha collections as source of materials for the project; Ms. Melecia C. Gibe the laboratory technician of the project.</p>