Characteristics of storage reserves of triploid watermelon seeds ...

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Fatty acid profiles, starch content and germination of watermelon seeds differing in seed vigour were determined for 4 triploid and 2 diploid lines. There were ...
T. WANG, L.A. SISTRUNK, D.I. LESKOVAR AND B.G. COBB

Wang, T., Sistrunk, L.A., Leskovar, D.I. and Cobb, B.G. (2011), Seed Sci. & Technol., 39, 318-326

Characteristics of storage reserves of triploid watermelon seeds: association of starch and mean germination time T. WANG1, L.A. SISTRUNK2, D.I. LESKOVAR3 AND B.G. COBB4,5* Department of Horticultural Sciences1, 2, 4, Texas Agrilife Research3, 4, Faculty of Molecular and Environmental Plant Sciences5, Vegetable and Fruit Improvement Center3, Texas A&M University, College Station1, 2, 4 and Uvalde3, TX 77801 USA (E-mail: [email protected])

(Accepted February 2011)

Summary Fatty acid profiles, starch content and germination of watermelon seeds differing in seed vigour were determined for 4 triploid and 2 diploid lines. There were major differences in the fatty acid profiles between diploids and triploids. A major increase in the relative amounts of the unsaturated linoleic acid (C18:2) was found in triploids. Diploids had significantly higher starch levels than triploids. There was a high correlation between starch content and mean germination time. Conversions of seed storage reserves during early germination events may differentially affect seed vigour in triploid and diploid watermelon seeds.

Introduction Watermelon has long been one of the most popular vegetables in the world (Gusmini and Wehner, 2008). In Texas, watermelon was grown in 8,900 ha with a farm gate value of $53 million (USDA, NASS 2008). In the last decade, seedless watermelons produced by crossing diploid and tetraploid parents to produce sterile triploid seeds have become popular and now account for the majority of watermelon production in the country. However, even with improvements through breeding (Kapiel et al., 2005) production costs are still high since triploids are field transplanted due to the high seed cost and erratic seed germination. Seeds from interploidy crosses are atypical due to altered endosperm ploidy, which can result in seed abortion or abnormal seed development (Haig and Westoby, 1991; Scott et al., 1998). Deficiencies in cucumber triploids include inability of embryos to germinate, absence of roots, albinism and mixoploidy (Malepszy, 1998) and lower levels of ABA (Gawronska et al., 2000). For triploid watermelon the seed coats are thicker than in diploids which leads to oxygen deficit during germination (Edelstein et al., 1995; Grange et al., 2003; Ramakrishna and Amritphale, 2005). Seed treatments that increase oxygen availability, either by removing or splitting the seed coat/testa, do enhance germination (Duval and NeSmith, 1999, 2000; Grange et al., 2000; Pesis and Ng, 1986) but vigour is still lower than in diploids, which suggests additional factors contribute to low vigour. * Author for correspondence

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TRIPLOID WATERMELON STORAGE RESERVES

Triploid watermelon seeds are generally smaller than diploid seeds (Grange et al., 2000) and also have an airspace surrounding the embryo, which may contribute to water sensitivity (Grange et al., 2003). Analysis by transmission electron microscopy showed that lipid bodies and starch grains are smaller in triploids than in diploid watermelon seeds (Wang et al., 2003). This suggests that there exists a multitude of structural and biochemical characters that contribute to the low seed vigour of triploids. Seeds contain substantial quantities of at least two major food reserves in the form of complex polymers such as carbohydrates and lipids as well as proteins and phosphoruscontaining compounds. These food reserves must be either hydrolyzed or degraded to their simpler monomers which are eventually catabolized enzymatically for the production of energy (ATP) and other essential metabolites for seed development and plant growth (Desai et al., 1997) In oil storing seeds such as cucurbits lipid metabolism during germination provides energy and the carbon backbones that are essential for germination and growth (Penfield et al., 2005). Plant lipid metabolism is a complex, highly regulated and multi-compartmented series of reactions for synthesis and degradation of fatty acids as well as other lipids, such as complex acyl lipids and terpenoids (Kennedy, 1961). Starch is the primary carbohydrate that is produced during seed development and the conversion of starch into soluble carbohydrates during germination is also necessary for normal seed germination (Beck and Ziegler, 1989). Reduction in seed starch leads to reduced seed germination and seed vigour (Cao et al., 2008) This study was undertaken to determine the fatty acid composition and starch content of mature diploid and triploid watermelon seeds because of their importance for providing energy sources in seed germination and their contribution to seed vigour.

Materials and methods Seed materials ‘Sunsweet’ and ‘Black Diamond’ seeds were the diploids used in these experiments. Triploid ‘Tri-X 313’ lot #1620(H) and lot #1610(L) and seeds of the triploid ‘Tri-X Sunrise’ lot #1901(H) and lot #1805(L) were provided by Syngenta Agribusiness. The designations of high (H) and low (L) vigour of the seed lots was based on previous germination studies of radicle emergence to 2mm and were assigned by Syngenta. Germination All seeds for each cultivar and lot used in this study were surface-sterilized by immersion in a 1 L beaker with 1% (w/v) aqueous Captan (cis-N-trichloro-methyl-thio4-cyclohexene-1,2-dicarboximide) for 5 min with constant stirring and then thoroughly rinsed with distilled water. Twenty-five seeds per replication were placed, embryo side down, into 9-cm petri dishes on two pieces of Whatman No.1 filter paper saturated with 3 ml of distilled water. The petri dishes were incubated in darkness at 30°C. Water was added to each dish as needed to replenish water loss by evaporation and imbibition. Radicle emergence to 2 mm or more was scored as germination and was recorded at 24-h intervals for 7 days. From these data, time to 50% germination (T50) and final germination 319

T. WANG, L.A. SISTRUNK, D.I. LESKOVAR AND B.G. COBB

percentage (FGP) were calculated. T50, measured as an indication of seed vigour, was determined on the basis of daily counts according to the method for estimating the mean time of plant seedling emergence (Ellis and Roberts, 1980; Orchard, 1977): Mean of N = ∑ N·D / ∑ N, where N = number of radicles emerged on day D, and D = number of days of incubation. Means of T50 were tested by Duncan’s multiple range at 5% level (SAS Institute, 1990). Lipid extraction Total lipids were extracted by the ether-ethanol method. Ground samples equivalent to 5 g of dry solid were ground with 50 ml of ethanol with a homogenizer. The mixture was filtered through Whatman No.1 filter paper in a funnel under reduced pressure. The sample was extracted into a suction flask and placed in a drying oven overnight at 95°C. The dried sample was then dissolved in 1.0 ml of methyl ether. Next, 0.2 ml of 20% tetramethylammonium hydroxide (TMAH) was added, the tube was capped and then shaken for 1 min. Finally, 2.0 ml of distilled water was added and shaken for an additional 1 min. Two hours later, the turbid upper ether layer containing the methyl esters was taken for analysis. The fatty acid methyl esters (FAMEs) were then analyzed by gas chromatograph. Gas chromatography of FAMEs The fatty acid composition was quantitatively determined by gas chromatography of the methyl esters in a Varian model 940 gas chromatograph equipped with a flame ionization detector. The operating conditions were as follows: injection port temperature 230°C, detector temperature 250°C, column temperature 160°C, carrier gas flow rate 25 ml He/min, injection volume 3.0 μl (FAME, the upper ether phase), and range setting of detector attenuation 10-11 amp/mV. Peak areas were determined by electronic integration and expressed as a percentage of total fatty acids. Each sample was done in duplicate and average results were reported. Identification of the peaks was based on comparison retention times of samples and standards (Alltech Co.). Mean separation of fatty acid contents was tested by Duncan’s multiple range at 5% level (SAS Institute, 1990). Starch determinations The procedure of starch assay was based on the protocol described by Cobb and Hannah (Cobb and Hannah, 1986) with modifications as described below. Dry seeds were ground to a powder in a coffee grinder. They were then further ground with a mortar and pestle. Ground seed (0.5 g) was placed into a 15 ml plastic test tube, 5.0 ml of 80% ethanol was added and then fully ground with a homogenizer (Brinkman Instruments, Inc., Switzerland). The suspension was filtered through a filter paper and washed 4 times with ethanol. Aluminum foil was then used to slightly wrap the filter paper containing the starch and dried overnight in an oven at 70°C. After drying, 25 mg of each sample powder was weighed into a 20 ml glass tube containing 5 ml of distilled water. In every set of assays starch standards were subjected to all procedures to account for run-to-run variation in the hydrolysis steps and colorimetric assay. Samples and standards were capped with marbles, placed in an autoclave for 320

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20 minutes, and then cooled in ice. For starch determinations 5 ml of 0.05 M citrate buffer (pH 4.5) containing 4 mg/ml amyloglucosidase (from Rhizopus mold, Sigma chemical Co., St. Louis, Montana) was added to the solubilized starch samples. All samples and standards were incubated in a water bath at 55°C for 10 hours. After incubation, 1.0 ml of samples and starch standards as well as 1.0 ml of DNS (dinitrosalicyclic acid) regent were added, and then incubated for 15 minutes in a water bath at 95°C. After removing the samples from the water bath, 0.33 ml of sodium potassium tartrate (Rochell’s salt) solution (40% in dd H2O; 4 mg/10 ml) was added and then cooled in ice to room temperature. Four replications of each sample were read in a microplate reader (Dynatech MR 700, Alexandria, Virginia) at 610 nm using 0 percent starch standards as the reference blank. Percent starch was determined from starch standards included in the assays.

Results and discussion Table 1 shows time to 50% germination (T50), and final germination percentage (FGP) of triploid (3x) and diploid (2x) watermelon seeds. Total germination percentage was significantly less for Tri-X 313 lot #1620 (L) and TRI-X SUNRISE LOT #1805 (L) than for the others. For the diploid cultivars, T50 (defined as radicle emergence to 2mm) was significantly less than any of the triploids. For triploids, T50 for both H and L lots of Tri-X 313 were lower than the H and L lots of Tri-X Sunrise (table 1). Cole (Cole, 1994) reported that seed germination varied considerably by parental genotypes; therefore, a certain degree of the germinating performance of triploids could be regarded as the influence of genetic variability. In table 2, analysis of the fatty acid composition of the total seed lipid from the dry watermelon seeds of triploids and diploids indicates that there were marked differences in the composition of the major saturated and unsaturated fatty acids. Our analysis shows that six methyl ester peaks were found in the fatty acid profile of watermelon samples: myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) acid. The seed oils of curcurbits have been reported to contain palmitic, stearic, oleic and linoleic acid as the main fatty acids (Akoh and Nwosu, 1992; Jacks et al., 1972). Bhatia (Bhatia et al., 1971) also indicated that linoleic acid was the principal fatty acid in all cucurbits (e.g., watermelon, muskmelon and cucumber) except pumpkin where oleic acid was the major fatty acid. While linoleic acid predominates the concentrations of each acid are highly variable between species (Bemis et al., 1967). The results in table 2 showed that there was a marked level (18.5-21.3%) of myristic acid (C14:0) in the 313H and diploids but was absent from the other triploid cultivars. Palmitoleic acid (C16:1) was absent in all four triploid seed lots, but was present in the diploid lots (table 2). Similar results were also reported in mutants of sunflower seeds (Cantisan et al., 1999). Palmitic acid levels ranged from about 7 to 17%, being the highest for 3x TRI-X Sunrise lot#1901 (H) and the two diploids. Among the unsaturated fatty acids, linoleic acid (C18:2) was the principal constituent of the seed lipids from the different cultivars (‘Tri-X 313’ and ‘Tri-X Sunrise’) of triploids 321

322

2.9 bx

T50z 2.8 b

76.0 b 3.2 a

90.7 a

lot #1901 (H)

3x ‘Tri-X Sunrise’

(18:1)

(18:2)

Oleic

Linoleic

18.9b

54.0 b

14.5a

4.1b

27.3b

84.9a

4.5c

3.4b

---

7.1c

---z

11.6c

63.5 b

10.2b

9.9a

---

16.9a

---

lot #1901 (H)

3x ‘Tri-X Sunrise’

15.1c

87a

2.6c

3.4b

---

6.9 c ?

---

lot #1805 (L)

3x ‘Tri-X Sunrise’

42.2a

35.0c

10.7 b

7.8 a

11.2

13.7b

21.3

lot # WV8133 (H)

2x ‘Sunsweet’

2.0 c

88.0 a

2x ‘Sunsweet’

46.3a

38.9c

10.7 b

8.2 a

9.6

11.7b

20.1

lot #18193

2x ‘Black Diamond’

2.1 c

91.3 a

2x ‘Black Diamond’

Not detected. y Means separation within rows for each variable using Duncan’s multiple range test at p < 0.05. x Starch content (W:W) was determined by quantifying glucose after hydrolysis by amyloglucosidase as described in the Material and methods.

z

Starch Contentx

(18:0)

---

Palmitoleic (16:1)

Stearic

8.7cy

(16:0)

Palmitic

18.5

lot #1610 (L)

lot #1620 (H)z

(14:0)

3x ‘Tri-X 313’

3x ‘Tri-X 313’

Myristic

Fatty Acid

Table 2. Fatty acid composition (%) and starch content (%) of diploid and triploid watermelon seeds.

y

3.3 a

68.0 b

lot #1805 (L)

3x ‘Tri-X Sunrise’

Time to 50% germination. Final germination percentage. x Means separation within rows for each variable using Duncan’s multiple range test at p < 0.05.

z

88.0 a

lot #1610 (L)

lot #1620 (H)

FGPy

3x ‘Tri-X 313’

3x ‘Tri-X 313’

Table 1. Germination of diploid and triploid watermelon seeds at 30oC in the dark under laboratory conditions. Final germination was evaluated after 7 days.

T. WANG, L.A. SISTRUNK, D.I. LESKOVAR AND B.G. COBB

TRIPLOID WATERMELON STORAGE RESERVES

that markedly contributed to the total percentage of fatty acids while lower levels of other fatty acids were measured (table 2). Bhatia et al. (1971) also reported that linoleic acid was the largest class of fatty acids in cucurbits although they found lower concentrations of linoleic acid than in this study. The linoleic content found in TRI-X SUNRISE LOT #1805 (L) (87%) was extremely high and is equal to that found in safflower (Daood and Alani, 1986; Ichihara and Noda, 1980). The starch content of diploids was significantly higher than that of the triploids (table 2). The starch content of the triploid ‘Tri-X 313’ and ‘Tri-X Sunrise’ ranged from 11.6 to 27.3%. There were clear differences in starch content between the two cultivars, with a markedly higher starch content for ‘Tri-X 313’ lots than for ‘Tri-X Sunrise’ lots. Perhaps unexpectedly, triploids with low germination, lot #1610 (L) and lot #1805 (L), had higher starch content than those with high germination, (Tri-X 313 lot #1620 (H) and Tri-X 313 lot #1620 (H)), within the same cultivar (table 2). However, the starch content of watermelon seed lots analyzed in this study did not correlate with the final germination percentage (FGP). In contrast to total germination, T50 (time to 50% germination) was highly correlated with starch content (figure 1). Our previous work (Grange et al., 2003; Grange et al., 2000) and that of others (Duval and NeSmith, 1999, 2000) has shown that oxygen uptake is restricted in triploids due to the thick seed coat. In addition, the ability of seeds to germinate under oxygen deficit is dependent on the availability of carbohydrates (Huang et al., 2003; Kato-Noguchi et al., 2008; Loreti et al., 2003; Perata et al., 1997). During germination, lipids are converted into carbohydrates for use by the developing embryo by means of β−oxidation (Baker et al., 2006; Eastmond and Graham, 2001; Goepfert and Poirier, 2007; Mclaughlin and Smith, 1994) and the oxygen requiring glyoxylate cycle, first characterized by Beevers (Beevers, 1961; Canvin and Beevers, 1961). Slack (Slack et al., 1977) showed that lipid metabolism was limited by oxygen transport through the seed coat. Also, isocitrate lyase activity peaks at 3 days

3.2 3.0

T 50

2.8 2.6 2.4 2.2 2.0 15

20

25

30

35

40

Starch Content Figure 1. Correlation of starch content with Mean Germination Time of 4 Triploid and 2 Diploid Cucumber lines. Starch (W:W) was determined as described in Materials and Methods.

323

T. WANG, L.A. SISTRUNK, D.I. LESKOVAR AND B.G. COBB

of germination (Slack et al., 1977) which suggests that initial germination events may depend on carbohydrates and not lipids. Seeds with mutations deficient in any of these conversions are characterized by poor germination (Andre and Benning, 2007; Footitt et al., 2002; Zheng et al., 2008). Therefore, the correlation of starch content and T50 could be due to early metabolic events that rely more on starch and not lipids. To provide energy for the plant, both lipid and starch can be involved to break down these storage compounds and to mobilize their products during germination. The reduction of any of these conversions could lead to the alteration of fatty acid profiles observed and could lead to reduced availability of stored starch for energy use during germination and/ or lipids needed for cell integrity during development and germination. Therefore, the alterations of any of seed storage reserves could certainly result in decreased seed vigour.

Acknowledgements The author thanks Syngenta Agribusiness for providing seeds for this study.

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