B.-Z. ZAIDMAN, M. GHANIM AND Y.

VAKNIN

Zaidman, B.-Z., Ghanim, M. and Vaknin, Y. (2010), Seed Sci. & Technol., 38, 758-767

Effect of seed weight on seed vigour and early seedling growth of Jatropha curcas, a biodiesel plant
B.-Z. ZAIDMAN, M. GHANIM AND Y. VAKNIN* Department of Agronomy and Natural Resources, Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel (E-mail: [email protected])

(Accepted June 2010)

Summary
The effect of seed weight on germination and seedling growth was investigated in Jatropha curcas, a tree-borne oilseed species, currently developed worldwide as a feedstock for biodiesel production. Seed weight was highly variable, from 0.28 to 0.81 g. Seed germination rate was significantly affected by seed weight: 46.2, 79.3, 84.5, and 95.3% for seeds in weight ranges, 0.39, 0.40.49, 0.50.59, and 0.6 g, respectively. Seedling shootlength and dry matter yield were significantly affected by seed weight. Seedlings grown from the heaviest seeds were 51% taller and 91% heavier than those from the lightest ones. Indirect analyses of seed vigour indicated a significant linear relationship between results of tetrazolium reduction and electrical conductivity assays and seed weight: as seed weight increased, tetrazolium reduction increased, and electrical conductivity decreased. Our results suggest that improved seed and seedling quality, as associated with greater seed weight, is attributed to better membrane integrity and increased availability of energy in the endosperm. Tetrazolium reduction and electrical conductivity assays were proven to be efficient methods to estimate J. curcas seed vigour and seedling performance and thereby to promote planting of higher quality transplants in the field.

Introduction Jatropha curcas L. is a small perennial tree or large shrub Euphorbiaceae. The tree has spreading branches and stubby twigs, and grows to 7 m high under favourable conditions. It is native from tropical America, but is now cultivated widely in tropical countries worldwide (Heller, 1996; de Jongh, 2006). It is able to thrive in a number of climatic zones with annual rainfall in the range of 2503000 mm (Achten et al., 2008); it is well adapted to arid and semi-arid conditions and therefore is considered drought tolerant (Abou Kheira and Atta 2009). All parts of J. curcas can be used, and their various uses encompass a wide range of purposes, including soil improvement, charcoal production, and manufacture of soap, lubricant and cooking-oil, fertilizers, and, with suitable treatment, animal feed (Gbitz et al., 1999; Openshaw, 2000). The oil content of J. curcas seeds is highly variable; in India, for example, it was reported to range between 23 and 45% (Jongschaap et al., 2007). Nevertheless, the potential significance of J. curcas as a feedstock for biodiesel
* Author for correspondence

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production is most remarkable. Jatropha curcas is easily propagated by both generative (direct seeding or indirect by production of seedlings) and vegetative (direct planting of cuttings) methods (Openshaw, 2000). The advantages of vegetative propagation include: true to type seedlings, faster crop growth, higher survival rate, early flowering, and enhanced yield. Disadvantages include: susceptibility to drought, adventitious root system, and shorter lifetime (Kochhar et al., 2005, 2008; Noor Camellia et al., 2009). The advantages of generative propagation include a tap-root system and drought tolerance, whereas its disadvantages include genetic variability, slow growth and development, and late flowering (Jimu et al., 2009). For quick establishment of hedges and for erosion control, direct planting of cuttings is considered easier, whereas for longlived plantations for seed-oil production, plants propagated from seeds are better suited (Heller, 1996). The plant is usually propagated on the mass scale via seeds, and according to GEXSI (2008), 85% of J. curcas projects use pre-cultivated seedlings obtained from nurseries, and the rest direct seeding or cuttings. The reason for this preference is that seedling survival and performance are highly dependent on propagation method, and J. curcas established through pre-cultivated seedlings outperformed non-rooted cuttings and direct-seeded plants (Jimu et al., 2009; Samba et al., 2007). Nevertheless, J. curcas seed germination is unreliable and it varies between 10% and 95% (Feike et al., 2008; Islam et al., 2009). The fruits of J. curcas are capsule-like and about 2.54 cm in diameter. They are green and fleshy when immature, become dark brown when ripe, and split to release up to three black seeds each about 2 cm long and 1 cm wide. Freshly harvested seeds show dormancy and post-ripening drying is necessary before they can germinate (Jker and Jepsen, 2003; Verma and Gaur, 2009), but dry seeds normally germinate readily without pretreatment. Seed germination is epigean, and under conditions of 30C and 40 60% RH is complete within 10 days (Personal information). As of 2008, approximately 900,000 ha of J. curcas had been planted worldwide, although the industry is still in its early stages. More than 85% of this cultivated land is located in Asia, and Africa accounts for approximately 120,000 ha, followed by Latin America with approximately 20,000 ha. Jatropha curcas cultivation is expected to expand enormously to 13 million ha by 2015 (GEXSI, 2008). Thus, there is a rapidly growing need for high-quality planting stock in order to meet the expected demand. The aim of the present study was to investigate the effects of J. curcas seed size on emergence and seedling development; coverage of seed parameters addressed seed vigour and viability, germination percentage and value, and that of seedling parameters included shoot and root length, dry weight, shoot-collar diameter, and leaf chlorophyll content.

Materials and methods Plant material and size categorization Seeds were obtained from 3-year-old plants that had originated from native plants in Suriname, Central America. The plants were cultivated in an experimental plot at the Volcani Research Station, central Israel. Fruits were collected at the black maturity stage (ripe capsule), during November 2008, and seeds were removed from the fruits and kept

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in paper bags at room temperature (20 25C, 30 40% RH) for 12 months before use. A total of 3562 seeds were weighed on a Pioneer Analytical Scale (Ohaus Corp., Pine Brook, NJ, US), and were allocated to four weight groups: 0.39 g, 0.4-0.49 g, 0.5-0.59 g, and 0.6 g. Electrical conductivity and tetrazolium reduction assays Jatropha curcas seeds were divided into four groups according to the above seed-weight categories. Four samples per weight category of approximately 10 g seeds each, were placed in glass beakers, and were surface-sterilized by addition of 50 ml of 0.1% sodium hypochlorite. After soaking for 10 min, the seeds were rinsed thoroughly and 100 ml of double-distilled water were added. The beakers were then covered with aluminum foil and allowed to stand for 24 h at room temperature. After imbibition, the seeds were removed from the beakers for the tetrazolium assay a method proven to be highly predictive of J. curcas seed viability (Kak et al., 2009), and the EC of the soaking solution was measured with a Cyberscan Con510 conductivity meter (Eutech Instruments, Singapore). After imbibition, seed coats of 15 seeds per sample were carefully removed, and the endosperm was cut longitudinally to expose the embryo. Embryonic axes were detached and placed in a test tube containing 10 ml of 1% (w/v) aqueous solution of 2,3,5- triphenyltetrazolium chloride (Merck, Darmstadt, Germany). Tubes were incubated at 30C for 3 h in darkness. After the reaction, the embryos were washed with doubledistilled water, the water was removed and 10 ml of isopropanol acidified with 6N HCl was added to each tube. The reduced tetrazolium salt was extracted by shaking for 11.5 h. Absorption of 485-nm radiation by the extract was measured with a Model Genesys 5 spectrophotometer (Spectronic Analytical Instruments, Leeds, UK), Acidified Isopropanol served as blank. Germination test This took place in a greenhouse from April 18 to May 18, 2009, when the average temperature was 22.5C, with a minimum of 8.3C and maximum of 44.9C, and average relative humidity was 52.6%. Seeds ( 0.39 g (n = 312), 0.4 - 0.49 g (n = 624), 0.5 - 0.59 g (n = 1872), 0.6 g (n = 754)) were planted on April 18, 2009 in expanded polystyrene seedling trays (Polybid Agriculture, Mishmar Hanegev, Israel) containing a 70/30 peat/ perlite mixture; they were planted 2.5 cm below the surface of the medium. The trays were placed on tables in the greenhouse and watered by computer-controlled Modular micro-sprinklers with a flow rate of 25 l/h. A seed with a protruding hypocotyl of about 5 mm was considered germinated. Seedling emergence was recorded until seedlings ceased to emerge at 33 days. Germination value Germination Value (GV) an index that quantifies germinative energy by combining speed and completeness of germination was calculated according to Czabator (1962). Combining speed and completeness of germination (based on hypocotyl protrusion) into a composite score (GV) eliminates the need for subjective value judgment. Briefly, total germination is expressed as (final) Mean Daily Germination (MDG), calculated as the
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cumulative percentage of full seed germination at the end of the test, divided by the number of days from sowing to the end of the test. Speed of germination is expressed as Peak Value (PV), which is the mean daily germination of the most vigorous component of the seed lot, a mathematical expression of the break of the sigmoid curve representing a typical course of germination. The GV can then be calculated as: GV = MDG PV Seedling performance Seedling growth was measured in terms of shoot and root lengths, root-collar diameter, and shoot and root dry weight, by randomly selecting 20 seedlings from each weight category. Roots were carefully and thoroughly washed with tap water to remove growthmedium particles. Shoot and root length, and root collar diameter were measured with a LUTRON Model DC-515 digital calliper (Lutron Electronic Enterprise Co., Ltd. Taiwan). Leaf chlorophyll content was measured with a SPAD-502 Chlorophyll Meter (Konica Minolta, Osaka, Japan). Each value represented the mean of five measurements taken at the center of the leaf. Dry matter yield was determined by drying the plant material in an oven at 60C for 48 h, to achieve constant weight. Statistical analysis Data were subjected to one-way analysis of variance (ANOVA) with JMP software (SAS Institute Inc., Cary, NC, USA) to determine significant differences attributed to seed weight and growth medium. An arcsine square-root transformation was applied to the percentage data. Tukey-Kramer HSD (honestly significant difference) tests were performed to compare means of seed and seedling indices. Pearson correlation analysis was used to evaluate the relationship between the tetrazolium assays and EC assays of the seeds.

Results and discussion Seeds from 3-year-old plants at the ARO experimental plot were sorted into four distinct weight groups 0.39, 0.4 - 0.49, 0.5 - 0.59, > 0.6 g representing the size variation within the whole seed population and the frequency distribution of seed weights resembles a positively skewed bell shape, with the four groups contributing 8.8, 17.5, 52.5, and 21.2%, respectively (figure 1). This high variation in seed weight is consistent with the findings of other researchers (Ginwal et al., 2004, 2005; Kaushik et al., 2007). This reported intra-specific variation in seed size is common in wild plant populations, and it is often associated with survival and reproduction (Westoby et al., 1992). In the present study, observed differences in electrical conductivity (EC) of seedsoaking solutions, and in radiation absorption by reduced tetrazolium salt were highly significant (p < 0.001): EC increased linearly and absorption decreased linearly with decreasing seed weight (table 1). Indirect evaluation of cell membrane integrity with the EC test has been used extensively in seed vigour testing research and in agriculture (Takos et al., 2006), but has not been adopted as a routine practice in forestry seed research (USDA, 2008). Poor cell-membrane integrity is usually related to low seed vigour, and

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60 50

Percent seeds

40 30 20 10 0

0.39

0.4 - 0.49

0.5 - 0.59

0.6

Seed weight group (g)

Figure 1. Frequency distribution of J. curcas seeds according to seed weight.

Table 1. Relationship between J. curcas seed weight groups and indirect seed vigour assays: electrical conductivity and tetrazolium assays. All data are shown as means SE. Means that are not significantly different are marked with the same letter. Seed weight group (g) Seed analyses Electrical conductivity (S cm g ) Formazan absorption (A480)
-1 -1

0.39 191.79 0.46 a 0.45 0.02 d

0.4 - 0.49 172.22 2.35 b 0.61 0.02 c

0.5 - 0.59 155.54 2.43 c 0.79 0.01 b

0.6 140.72 1.15 d 0.99 0.01 a

can be indirectly monitored by measuring leakage of electrolytes during imbibition (Cheng et al., 2005). The tetrazolium assay, on the other hand, is widely accepted as a tool for testing seed viability and vigour (Peters, 2000). Briefly, colorless soluble tetrazolium salt is metabolically reduced by cellular NADH and NAPDH to insoluble red formazan salt that can be assayed by spectrophotometer; the absorption is proportional to cellular activity. We found that tetrazolium assays were negatively correlated with EC assays (R2 = 0.95, P < 0.0001). The present tetrazolium and EC data suggest that J. curcas seed vigour was directly related to seed weight: heavier seeds were more vigorous than lighter ones, and were characterized by lower values of EC and higher values of absorption (table 1). Germination percentages (figure 2A), germination values (figure 2B) and germination rates (figure 2C), differed significantly among seed-weight groups. Germination percentages ranged from 46.2% for seeds of the 0.39 g group up to 95.3% for those of the 0.6 g group, and the corresponding GV values ranged from 5.32 to 31.34, respectively. In general, heavier seeds germinated much faster and reached significantly higher maximal germination percentages than lighter ones. Seedling emergence started in all seed-weight groups 8 days after sowing, but germination rates varied significantly different: seedling emergence from 0.6-g seeds ended on the 15th day after sowing, and
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100

35

Percent germination

90 80 70 60 50 40 30 20 10 0

Germination value

b c

30 25 20 15 10

5 0 0.39 0.4 - 0.49 0.5 - 0.59 0.6

0.39

0.4 - 0.49

0.5 - 0.59

0.6

Seed weight group (g)

100

Seed weight group (g)

Cumulative germination (No. seeds)

90 80 70 60 50 40 30 20 10 0 6

0.39 0.4 - 0.49 0.5 - 0.59 0.6

10

12

14

16

18

20

22

24

Days after sowing

Figure 2. Relationship between J. curcas seed weight groups and germination indices. A. Germination percentage, B. Germination value, C. Cumulative germination. Seeds were categorized into 4 distinct weight groups and seeded as follows: 0.39 g (n = 312), 0.4 - 0.49 g (n = 624), 0.5 - 0.59 g (n = 1872), 0.6 g (n = 754). Germination percentage, Germination value, and Cumulative germination were calculated for each tray. All data are shown as means SE. Means that are not significantly different are marked with the same letter.

this completion time gradually increased with decreasing seed weight, up to an additional 7 days for seeds of the 0.39g group (figure 2C). Germination value (GV), which integrates total germination at the end of the experiment and germination energy, also varied significantly between seed weight groups. Germination value of 0.6 g seeds was 5.9 times as great as that of the 0.39 g seeds (figure 2B). Similar relationships between seed size, on the one hand, and germination rate and seedling growth, on the other hand, were reported by Dagar et al. (2004). Seed germination and the subsequent seedling growth require large amounts of energy and nutrition, which can be provided only from seed reserves, because the germinating seed lacks a mineral uptake system and photosynthetic apparatus (Bewley, 1997). These reserves are mainly in the form of lipids, proteins, and starch in the embryo or endosperm, and their relative amounts vary among different species. In J. curcas seeds, the major storage reserve is lipid, in the form of triacylglycerol, which is stored in oil bodies in the endosperm (Annarao et al., 2008). Yang et al. (2009) found that endosperm-stored oil content started to decrease 60 h

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after imbibition, and was almost completely depleted after 6 days; they suggested that the oil was mobilized and consumed during the germination and post-germination stages in J. curcas. Thus, in the present study, larger seeds ( 0.6 g group) probably contained more oil reserves, and therefore achieved faster and more complete germination. It was found that seed weight significantly (p < 0.001) affected seedling shoot length. Seedlings that emerged from heaviest seeds had 51.35% longer shoots than those from lightest seeds, but root length was not affected by seed weight (figure 3A). Dry matter yield was also found to be significantly (p < 0.001) affected by seed weight (figure 3B). Dry weight of seedlings gradually increased as seed weight increased, and seedlings that emerged from the lightest seeds were 91.2% lighter than those from the heaviest ones (figure 3B). Root length was not affected by seed weight (figure 3A) while root collar diameter was significantly smaller in seedlings from 0.39 g seeds than that of those from all the other weight groups (figure 3C).

180

1.0

a b b

Seedling dry weight (g)

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Shoot Root c d

a b

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.39 0.4 - 0.49 0.5 - 0.59 0.6
c

Length (mm)

140 120 100 80 60 40 20 0 0.39

a

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0.5 - 0.59

0.6

Seed weight group (g) Root collar diameter (mm)

8 7 6 5 4 3 2 1 0 0.39 0.4 - 0.49 0.5 - 0.59
b a a

Seed weight group (g)

0.6

Seed weight group (g)

Figure 3. Relationship between J. curcas seed weight groups and the following seedling traits: A. shoot and root length, B. shoot and root dry weight, C. root collar diameter. All data are shown as means SE. The Tukey-Kramer HSD test was applied to separate means. Means that are not significantly different are marked with the same letter.

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These results suggest that after completing germination, seedlings allocate much of their resources to shoot and to a lesser extent to root development, so that heavier seeds exhibit better seedling growth than lighter ones. It is our experience that as long as the soil is properly irrigated and aerated, pot grown J. curcas seedlings exhibit rapid vegetative growth above ground while the roots remain relatively underdeveloped. This disproportionate resource allocation to the shoot could be explained by the fact that water as well as oxygen are amply available and thus root elongation is less promoted. A support for this hypothesis comes from a recent study by Achten et al. (2010) who found that under extreme drought, pot-grown J. curcas seedlings stopped growing while the biomass allocation to the roots was significantly enhanced. Our results are consistent with those obtained by Ginwal et al. (2004), who found that seed weight was significantly and positively correlated with seed kernel weight, seed oil content, kernel oil content, and seedling growth traits, i.e., height and root collar diameter. Leaf chlorophyll content was fairly similar among seedlings produced from seeds of all weight groups (table 2). Leaf chlorophyll content is a key indicator of the physiological status of plants (Gitelson et al., 2003), because chlorophylls (a and b) are essential pigments for the conversion of light energy to stored chemical energy. Factors affecting chlorophyll synthesis are genotype, availability of nutrients (especially nitrogen, since 75% of leaf nitrogen is incorporated in chlorophyll), light availability, water stress, presence of insects, senescence, and herbicide use (Steele et al., 2008). Thus, in a given genotype, leaf chlorophyll content can serve as an integrator of all the environmental and nutritional variables that ultimately affect plant quality (Richardson et al., 2002; Wang et al., 2005). In the present study, seed size had little effect on seedling leaf chlorophyll content suggesting that the differences we showed in seed vigour among the weight groups were not related with the photosynthetic capacity of the growing seedling. At this point
Table 2. Relationship between J. curcas seed weight groups and seedling leaf chlorophyll content (SPAD Minolta Company-defined values that are proportional to the amount of chlorophyll present in the leaf). Means that are not significantly different are marked with the same letter. Seed weight group (g) Seedling analysis Chlorophyll content (SPAD value) 0.39 31.09 0.87 a 0.4 - 0.49 31.97 0.75 a 0.5 - 0.59 32.47 0.72 a 0.6 30.94 0.76 a

we have no reliable explanation for this outcome. Expanding the study to other genetic lines or germinating the seeds under less than optimal conditions might reveal significant differences in leaf chlorophyll content. In conclusion, it was found the high level of intra-specific variation in seed size to significantly influence both seed germination and seedling performance. In general, the larger the seeds within the range studied here, the better they germinated and the better they performed as young seedlings. The fact that the common practice of tetrazolium assays was negatively correlated with EC assays, suggests that use of the EC assay in conjunction with seed weight measurements could provide better estimations of seed lot

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performance, initially in germination, and then in young seedling growth. At this point we recommend a minimum seed weight of 0.4 g to produce high vigour seedlings. Extension of these simple measurements to other genetic lines offers the possibility to increase our ability to predict potential performance of future seed lots, and to provide the growers with valuable information prior to the common practice of mass producing seedlings in future J. curcas-based biodiesel projects. Acknowledgements This research was partially funded by a grant from the ARO director. References
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