BIOTECHNOLOGICAL STUDIES ON Jatropha curcas L., AN IMPORTANT BIOFUEL PLANT

BIOTECHNOLOGICAL STUDIES ON Jatropha curcas L., AN IMPORTANT BIOFUEL PLANT

 M. A.Bekhit*; Solieman M.S.*; Abd Elkawi M.**; Ibrahim A. I.*and  Elhalafawi K.***

^* Plant Biotechnology Dept. Genetic Engineering and Biotechnology Research Institute (GEBRI), Minufiya University, Branch of Sadat, Sadat City, Egypt.

^**   Faculty of pharmacy, Cairo University, Egypt

^*** Molecular Biology Dept., Genetic Engineering and Biotechnology Research Institute (GEBRI), Minufiya University, Branch of Sadat, Sadat City, Egypt.

ABSTRACT

Techniques for the induction of callus from petiole and leaf explants of Jatropha curcas (L) have been developed. Callus induction from petiole and leaf explants was evaluated on a range of N⁶ benzyl adenine (BA) and α-naphthalene acetic acid (NAA) concentrations.

 Both explants were capable to form100% callus, in different levels. Callus formation and growth were significantly affected by source of explants and growth regulators. The highest amount of callus was induced from leaf segments on MS solid medium supplemented with 0.5 mg/l of BA plus 0.5 mg/l of NAA. Callus-derived shoots were strongly affected by the residual effect of growth regulators levels. Callus derived from third leaf showed the highest shoot numbers and percentage of shoot formation (90%) when cultured on MS solid medium contained 0.1 mg/l of BA. The produced callus on 0.1 mg/l BA plus 1.0 mg/l indole butyric acid (IBA) was dedifferentiated when transferred into MS medium contained 0.1 mg/l of BA. Different extracts of Jatropha curcas (L) derived from in vivo and in vitro were investigated.

        Key words: Jatropha curcas, Ephorbaceae, growth regulators,  explant type, in vitro, callus, anti-tumor activities.

INTRODUCTION

Higher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods,but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals.Although plants are renewable resources, some species are becomingmore difficult to obtain in sufficient amounts to meet increasingdemands. Destruction of natural habitats and technical difficultiesin cultivation also are driving the drastic reductions in plantavailability. (Fay, 1992)

Plant cell and organ cultureoffer promising alternatives for the production of chemicals becausetotipotency enables plant cells and organs to produce useful secondarymetabolites in vitro. Cell culture is also advantageousin that useful metabolites are obtained under a controlled environment,independent of climatic changes and soil conditions. In addition,the products are free of microbe and insect contamination.

Jatropha curcas L. (Family, Ephorbaceae) is a drought-resistant perennial, growing well in marginal-poor soil. It is adapted to arid and semi-arid conditions.  Jatropha curcas L. is one potential source of non-edible biofuel-producing energy crop (Fevereiro et al., 1986). Its importance also lies in its medicinal properties (Timir et al., (2004).  Medically it is used for diseases like cancer, piles and snakebite. The species is primarily propagated through heterozygous seeds, and thus the oil content of seed varies from 4 to 40%. The oil can be combusted as fuel without being refined. It burns with clear smoke-free flam, tested successfully as fuel for simple diesel engine. The by-products are press cake a good organic fertilizer. Oil contains also insecticides. Moreover, due to its perennial nature, seed setting requires 2 to 3 years time (Timir et al., (2004).

The aim of the present work is the optimization of callus culture of Jatropha curcas L. (Ephorbaceae), for biomass production as a potential source of biochemical. Since, the seed viability and rate of germination are low, and quality seed screening is another laborious task; seed propagation alone cannot provide quality planting material for sustainable use; thus a brief study focusing on shoot regeneration from callus cultures for faster and quality plant production. In addition, chemical analysis was investigated of some Jatropha extracts derived from in vitro and in vivo (Fevereiro et al., 1986).

MATERIALS AND METHODS

This study was conducted in the lab of plant tissue culture, Plant Biotechnology Dept., Genetic Engineering & Biotechnology Research Institute (GEBRI), Minufiya University, Branch of Sadat, Egypt, on Jatropha curcas (L). plant during the period from 2005 to 2008.

Plant material

Leaf segments and petiole of Jatropha curcas L. were used as explants that were collected from Research farm of Genetic Engineering & Biotechnology Research Institute, Minufiya University, Branch of Sadat, Egypt.

Culture medium

MS (Murashige and Skoog, 1962) basal salts medium including Vitamins and 3% of sucrose was used in this study and all ingredients of medium were purchased from Sigma Company, The pH value was adjusted to 5.8 and solidified by 0.2 % of phytagel (Merck) prior to autoclaving (under pressure of 20 psi and 121^oC for 20 min.).

Surface sterilization and preparation of the explants

Leaves with petiole were washed thoroughly with running water for about 30 minutes, and then immersed in detergent (Clorox 20%, v/v) contained three drops of tween 20 and shaking for 15 minutes. All cultures were transferred into laminar-air flow cabinet. Petioles were separated from leaves and rinsed in distilled sterile water for 5 minutes and subjected to cultures. Leaves were cut into sections (0.5 cm²) which include the midrib portion. They were inoculated with their adaxial surface in contact with the solid induction medium in culture jars (25 ml). The petioles 1.0 cm long was cultured vertically in contact with the solid callus induction medium. 

Callus initiation and growth

Effect of BA and NAA on callus formation

Leaf section and petiole were cultured in MS solid medium comprised BA in rate of 0.1, 0.5 and 1.0 mg/l combined with 0.5, 1.0 and 2.0 mg/l of NAA, control was included. All cultures were incubated and maintained at 26 ± 2ºC and 16 h. light/day with 1500 lux from white florescent lamp. Data were recorded every week for four weeks as survival and percentage of callus formation, callus fresh weight, as well as, callus index.                                    

Effect of BA and NAA on callus growth

            Calli approximately 50 mg from each explant were transferred aseptically into jars (ca. 370 ml.) containing 25 ml of solidified MS basal medium and supplemented with the same levels of plant growth regulators mentioned before and 3% (w/v) of sucrose. These cultures were grown for four weeks under the culture conditions stated above. Callus growth was observed after that period and the callus relative growth (RG %) as well as growth rate was evaluated every week during incubation.

Establishment of shoots

Shoot initiation from callus

This experiment was carried out to study the effect of residual growth regulator effect on shoot formation from callus.

Third and fourth leaf and petiole-derived calli were subjected to produce callus on MS solid medium contained 0.5, 1.0 and 1.5 mg/l of BA combined with 1.0, 2.0 and 3.0 mg/l IBA for 4 weeks. Pieces of leaf and petiole-derived calli from each treatment were cultured on MS solid medium with 0.1 mg/l of BA singly or combined with 0.1 mg/l of IBA. The positive data only were recorded after 4weeks as a lot of negative responses were observed.

Measurements of Callus           

Measurement and determination:

                  The following data were measured as follow:

1. Fresh weight of callus was determined by weighing calli immediately after washing with distilled water (to remove the adhering).

2. Dry weight of callus, was determined by weighing samples dried in an oven at 60ºC until the weight became constant, according to Balbaa, et al. (1974).

3. Callus index was calculated by using the following formula: Callus index = (n x G/N) x 100 (Wakhlu and Barna 1989) where, n = total number of callused explants, G = average weight of callus rating on explant, N = Total numbers of cultured explant.

4. Growth rate (Gr) of callus cultures was calculated a (gm./day) by using the formula of Dung et al., (1981) using the following equation:

      Gr = Pt – Po / No of day (7),where Pt is the mass of callus fresh weight (mg) at the end of every 7 days during the 28 days of cultivation, Po is fresh weight of callus cultures (mg) at the beginning of every 7 day during 28 days of cultivation.

5. Relative growth-percentage (GR %) was calculated as follows:

    RG % = [(W_n - W_o)/W_o] x 100 [Djoko and Thornburg, 1992) where, W_n   is     the weight of the weight of the callus at the end of incubation and W_o is the weight of the callus at the start of the experiment.

Chemical analysis

Quantitative analysis of all samples was carried out in Principal Central Lab., Faculty of Agriculture, Cairo University, Egypt.

Chemical analysis was performed on callus dry sample derived from all selective treatments to determine the main active constituents of Jatropha curcas L. as follow:

-Total glycosides were determined According to Dubois et al., (1956).

-Total alkaloids (Sabri et al., 1973)

-Total flavonoids (Zhuang et al., 2001)

-Total lipids (Balbaa, 1986):

-Total terpenoids ( Ebrahimzadeh and Niknam, 1998)

-Total protein was determined according the method described by Abouzaid (1984).

Statistical analysis:

All experiments were conducted under controlled conditions and followed complete randomized design (CRD) with three replications and 10 jar for each replicate. Duncan's multiple Range Test (Sndecor and Cochran, 1982)was used with the help of MSTATE software.

RESULTS AND DISCUSSION

Callus initiation and growth:

Effect of BA and NAA on callus formation

            Callus initiation invariably occurred from the cut portions of the explants within a week of culturing. Callus initiation and growth on medium containing different levels of BA and NAA is shown in Table 1. All tested treatments formed a similar compact callus on wound surfaces of leaf and petiole explants. The best callus formation achieved with most treatments. Leaf explants kept alive till forming callus in the most of treatments, while 80% of petiole explants only formed callus in low rates of BA and NAA.

From the results, it’s clear that, Explant survival and callus formation % achieved one hundred percent with 0.5 mg/l BA + 2.0 mg/l NAA and 1.0 mg/l BA + 1.0 mg/l NAA. As for callus fresh weight, Data show that, the highest amount of callus was achieved with leaf explants grown on MS medium supplemented with 0.5 mg/l BA and 0.5 mg/l NAA (3.90 g/explant). The highest values of callus index (Fig. 1 a&b) was observed on the medium contained 0.5 and 1.0 mg/l BA plus 0.5 mg/l NAA (390 and 370), respectively.

Table 1: Effect of BA and NAA on callus formation and growth from petiole and leaf explants of Jatropha curcas

Different letters within columns indicate significant difference ( P ≤  0.05 ) according to Duncan's multiple range test.

Figure 1: Effect of BA and NAA on callus index derived from (a) petiole and (b) leaves of Jatropha curcas L.

Effect of BA and NAA on callus growth

Data in Table 2 show that, fresh amount of leaf-derived callus was significantly affected by NAA levels regardless BA concentrations. Increasing NAA levels led to decrease fresh amount of callus. NAA at 0.5 mg/l showed the highest amount of callus, while levels of BA didn’t have any effect. So, the highest fresh amount of callus was observed with 0.5 mg/l NAA combined with 0.1, 0.5 and 1.0 mg/l of BA (12.68, 10.29 and 12.12 g/jar) respectively, since no significant was observed.

Concerning fresh amount of petiole-derived callus, results differed from leaf –derived callus, whereas NAA in rate of 0.5 mg/l plus 0.5 mg/l BA recorded individually the highest value (11.14 g/jar). As for dry amount of callus, BA in rate of 0.5 mg/l and 0.5 NAA showed the best result among all treatments.

Concerning the percentage of callus relative growth, Figure  2 show that, percentage callus relative growth was significantly affected by BA and NAA levels. Leaf explants were grown on MS medium supplemented with 0.5 mg/l of both BA and NAA. Data fluctuated between 351.02% and 167.30% for petiole explants, while leaf explants was 337.24% and 142.63%. Increasing BA level from 0.1 to 1.0 mg/l differently affected the percentage of callus relative growth. Low concentration of BA (0.1 mg/l) and NAA (0.5 mg/l) showed the highest value. Increasing BA levels decreased the callus growth of both explants.

               Callus relative growth % was differently affected by explant types as well as BA and NAA concentrations, the highest value of callus relative growth % was achieved when both explants (leaf segment and petiole) were grown on MS medium contained 0.1 mg/l BA and NAA in rate of o.5 mg/l. NAA in rate of 0.5 mg/l showed the best results regardless BA levels.

Table 2: Effect of BA and NAA on callus fresh & dry weight (g/jar) and relative growth percentage derived from leaves and petiole of Jatropha curcas L.

Different letters within columns indicate significant difference ( P ≤  0.05 ) .according to Duncan's multiple range test

• RG % is relative growth % = [(W_n - W_o)/W_o] x 100 [Djoko and Thornburg, 1992).

Figure 2: Callus relative growth % as affected by BA and NAA in Jatropha curcas leaf and petiole explants.

               Where :T1= (0.1 BA+0.5 NAA), T2 =(0.1 BA+1.0 NAA), T3 =(0.1 BA+2.0 NAA), T4= (0.5 BA+0.5 NAA), T5= (0.5 BA+1.0 NAA), T6= (0.5 BA+2.0 NAA),  T7= (1.0 BA+0.5 NAA), T8= (1.0 BA+1.0 NAA), T9= (1.0 BA+2.0 NAA).

               * RG % is relative growth % = [(W_n - W_o)/W_o] x 100 [Djoko and Thornburg, 1992)

Increasing NAA level more than 0.5 inhabited the growth, for both explants. petiole explant that was grown on MS medium contained 0.1% mg/l BA and 0.5 mg/l NAA

As for callus growth rate (Gr),Figure 3 (a & b) illustrate the variation in callus growth rate and the effect of the different combinations of BA and NAA. These results clearly indicate that, callus growth rate was significantly affected by BA and NAA balance. Values of treatments reached its maximum after three weeks of incubation. These results clearly observed that, 0.5 mg/l of both BA and NAA showed to be the most suitable auxin/cytokinin balance level in order to induce optimal amount of callus (0.33 g/day) after 21 days of incubation comparing with the other treatments. It was clear that, callus growth rate depended on auxin/cytokinin balance and incubation period.

Figure 3: Callus growth rate (g/day) as affected by BA and NAA in Jaqtropha  carcus L. (a) petiole (b) leaf explant.

                 Where :  T1= (0.1 BA+0.5 NAA),T2= (0.1 BA+1.0 NAA), T3=(0.1 BA+2.0 NAA),  T4= (0.5 BA+0.5 NAA),T5= (0.5 BA+1.0 NAA), T6= (0.5 BA+2.0 NAA), T7= (1.0 BA+0.5 NAA), T8= (1.0 BA+1.0 NAA), T9= (1.0 BA+2.0 NAA).

* Growth rate (Gr) was calculated according to Dung et al.,(1981) using the following equation;  (Gr) = Pt – Po / No. of day (7), where Pt is the mass of callus fresh weight (g) at the end of every 7 during the 28 days of cultivation, Po is fresh weight of callus cultures (g) at the beginning of every 7 day during 28 days of incubation.

Establishment of shoots

Shoot regeneration from callus

As for callus formation, In vitro leaf segments and petiole explants of Jatropha curcas were cultured on MS medium with indole-3-butyric acid (IBA) and 6-benzyladenine (BA) in different levels (Table 3). Calli were differently affected by source of explant and BA and IBA concentrations. Calli were induced from third leaf explants showed the highest values on MS medium contained 0.5,1.0 and1.5 mg / l BA combined with 1.0 mg / l  IBA (90, 80,90%, respectively). On the contrary, the  fourth  leaf  explants recorded the lowest values, while petiole explants achieved a positive result on MS medium contained 1.0 mg/l BA plus 2.0 mg/l IBA (90%), in addition 1.5 mg/l BA plus 3.0 mg/l IBA (80%), no significant was appeared between them. From the results, it was evident that, callus formation could be induced from third leaf or petiole depending on growth regulator concentrations and its residual effect on shoot regeneration later.

Data in Table 4 show that, In vitro leaf and petiole explants of Jatropha curcas were cultured on MS medium with indole-3-butyric acid (IBA) and 6-benzyladenine (BA). Adventitious shoots were induced from the callus grown on MS medium contained combinations of 1.0 mg/l IBA and 1.0 mg/l BA. However, 0.1 mg / l BA induced the highest regeneration frequency.

Table 3: Effect of BA and IBA on callus formation percentage derived from leaves and petiole of Jatropha curcas L.

Different letters within columns indicate significant difference (P ≤ 0.05), according to Duncan's multiple range test.

Table 4: Effect of BA and IBA on shoot formation percentage and shoot number from leaves and petiole of Jatropha curcas L.

Different letters within columns indicate significant difference ( P≤  0.05 ) according to Duncan's multiple range test

Callus derived from third leaf showed the highest shoot formation (90%) compared to the fourth leaf. On the other hand, petiole failed to form any shoots on this concentration, while on medium contained 0.1 mg/l BA plus 0.1 mg/l IBA recorded 60% shoot formation and 9 shoots. Shoot numbers from third leaf-derived callus and grew on 0.1 mg /l BA recorded the highest value (23.5 shoots),

However, Wei  et al. (2004), induced adventitious buds directly from the surface of epicotyl explants from Jatropha curcas under the condition with the combinations of 0.1 mg/L IBA and 0.2-0.7 mg/l BA. Also, Mjlpuri et al. (2000) found that, shoot regeneration frequency of Jatropha curcas varied between 19.6 and 100% depending on explant types and BA and IBA balance. This result may be almost agree with the finding Sujatha and Mukta, (1996), who deeloped techniques to obtain shoots from third leaf and petiole of Jatropha curcas.

Chemical analysis

Preliminary phytochemical screening was performed for root, stem, leaf and seed samples of wild Jatropha curcas L. and samples of callus derived from leaves and petiole to be compared with that of the origin plant. Data in Table 5 show that, it’s evident that, callus derived from different organs contain the same main chemical groups  compared with the wild plant, whereas, all calli contained glycosides, alkaloids, flavonoids, lipids and terpenoids as well as protein. It’s cleared from screening that, the highest value of total glycosides was  determined  with leaf  callus  (0.12%), while  in

Table 5 : Phytochemical analysis of in vivo and in vitro samples

case of total alkaloids was found in stem (0.81%). As for total flavonoids, total lipids and total protein, plant seeds recorded the highest amount (10.44%, 19.45% and 35.0%) respectively. leaf of wild plant was superior for total terpenoids (14.0%). From analysis, it’s evident that, petiole-derived callus contained the same amount of protein as of plant seeds. Therefore, it could be concluded that, the undifferentiated cells contain all genetic information which present in the mother plant and required for the manufacture of secondary metabolite.

Conclusion

            From this study, it’s clear that, callus from Jatropha curcas can berapidly induced, so it could be exploited this feature for secondary metabolites production and plant regeneration. In this concern, further study should be carried out to produce intact plantlets. Also, the suggested protocol of shoot formation from callus of Jatropha curcas may be used for future transgenic research. Interestingly, callus derived from leaf and petiole explants is very important source of protein and flavonoids as well as other components.

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دراسات بیوتکنولوجیة على نبات الجاتروفا، کنبات مهم للوقود الحیوی

متولی حسن بخیت*، منال محمد سید أحمد*،  مصطفى عبد القوی**،ابراهیم عبد المقصود*، خلیل الحلفاوی***

^*   قسم البیوتکنولوجیا النباتیة– معهد الهندسة الوراثیة والتکنولوجیا الحیویة – جامعة المنوفیة – مصر

^**   کلیة الصیدلة ، جامعة القاهره، مصر

^***   قسم البیولوجیا الجزیئیة – معهد الهندسة الوراثیة والتکنولوجیا الحیویة – جامعة المنوفیة – مصر

أجریت هذه الدراسة بقسم البیوتکنولوجیا النباتیة– معهد بحوث الهندسة الوراثیة والتکنولوجیا الحیویة – جامعة المنوفیة، فرع السادات، مصر. بهدف انتاج الکالس من الأجزاء النباتیة لورقة نبات الجاتروفا بالتقنیات الحدیثة، وقد تم تقییم الکالس الناتج من نصل وعنق الورقة بأستخدام ترکیزات مختلفة من البنزایل ادینیین ونفثالین حمض الخلیک، واظهر کلا من نصل وعنق  الورقة قدرة کبیرة فی تکوین الکالس (100%)، وقد تأثر  تکوین ونمو الکالس معنویا بمصدر الجزء النباتی المنزرع ، باﻹضافة الى منظمات النمو. وحقق نصل الورقة أعلى کمیة من الکالس عندما زرع على بیئة موراشیج وسکوج الصلبة المحتویة على 0.5 ملجم/لترمن البنزایل ادینیین مع 0.5ملجم/لتر من نفثالین حمض الخلیک. کما تأثرت الأفرع الخضریة الناتجة من تکشف خلایا الکالس بالأثر المتبقی من ترکیزات منظمات النمو المستخدمة فی تکوین خلایا الکالس. وکانت أعلى نسبة للأفرع الخضریة المتکونة وعدد الأوراق للکالس النامی على بیئة موراشیج وسکوج الصلبة المحتویة على 0.1 ملجم/لتر من البنزایل ادینیین. وقد تم تقدیر المواد الفعالة الرئیسیة (الجلیکوزیدات، القلویدات، الفلافونیدات، الیبیدات، التربینات) فی کل من المستخلصات الناتجة من زراعة الأنسجة وأجزاء النبات البری، کما تساوت کمیة البروتین فی کل من الکالس الناتج من عنق الورقة وبذور النبات.