The Effect of Mixture of Alpinia Galanga, Eurycoma Longifolia Jack and Syzygium Aromaticum Crude Extract on the Growth of Saccharomyces Cerevisiae and Escherichia Coli

1. Introduction  responses such as fever or anemia are induced. IL-6 promotes the proliferation of B cells and thus is

RA (rheumatoid arthritis) is a chronic disease that involved in the production of the rheumatoid factor [6]. causes inflammation mainly in the synovium and Recently, RA has been observed to be associated with produces destruction and deformity of the joints. The high levels of IL-6 in the synovial membrane and etiology of RA remains unclear, but it is known to be serum [6, 7]. This led to the speculation that IL-6 associated with genetic and environmental factors [1].

plays a pathogenic role in RA.

Various proinflammatory cytokines, such as IL-6, Recently, an IL-6 receptor antagonist, tociliumab, TNF (tumor necrotic factor)- α, IL (interleukin)-1β, was developed and showed clinical efficacy in the and IFN (interferon)- γ, are increased in the synovial

treatment of RA [8-10].

tissue or synovial fluid of patients with RA [2, 3]. IL-6 is a pleiotropic cytokine with a wide range of Increased levels of proinflammatory cytokines lead to biological activities on various target cells. It regulates the proliferation of synovial tissue, and thereby cause immune responses, hematopoiesis, acute phase damage in the articular cartilage and bone destruction responses and bone metabolism [11]. Despite its in the adjacent area [4, 5]. In particular, IL-6 is a important physiological roles, dysregulated cytokine with various functions. overproduction of IL-6 is responsible for systemic When IL-6 is activated, acute inflammatory inflammatory manifestations and abnormal laboratory

Corresponding author: Dr. Abbas Sabbar Dkhil, lecturer, findings in patients with RA. In fact, elevated IL-6 research field: physiology. E-mail: abbassd992@gmail.com.

Association Between IL-6 (Interleukin-6) and Iron Status in Rheumatoid Arthritis Patients

levels have been observed in both serum and synovial patient, 1 mL for evaluation of ESR, whereas serum fluid in patients with RA, and there have been

samples were collected in glass tubes without correlations between serum IL-6 levels and clinical

anticoagulant, stored for one hour at room temperature, and laboratory indices of RA [12]. These findings

centrifuged (2,500 r.p.m. for 10 min at 4 °C) and then have led to the concept that interference with the

aliquoted in plastic tubes before being stored at -20 °C actions of IL-6 could represent a therapeutic approach

until analysis.

to RA.

2.2 Study Design

In RA, it is estimated that 30-60% of patients are anemic. One of the most frequent causes of anemia in

The patients were classified according to the RA patients is IDA (iron deficiency anemia). ACD

duration of RA disease [15]. As follows: (anemia of chronic disease) which does not usually

GI (Group I): the group of patients with disease respond to iron supplementation is another major

length of (less than one year) was considered as the cause of anemia in patients with RA [13]. However,

group with very early disease duration. Bloxham and his coworkers found that the majority of

GII (Group II): the group of patients with disease anemic patients were ACD, with rather fewer patients

length of (1-5) years was considered as the group with demonstrating iron deficient [14].

early disease duration.

In the current study, the authors examined whether GIII (Group III): the group of patients with disease serum levels of IL-6 are increased in patients with RA

length of (6-15) years was considered as the group and whether the increased levels are significantly

with median disease duration.

correlated with iron status. The authors compared the Group IV (GIV): the group of patients with disease serum concentrations of IL-6 in patients with RA and

length of (16-25) years was considered as the group those in normal controls and then investigated the

with long disease duration.

correlation between serum levels of IL-6 and the iron GV (Group V): the group of patients with disease status alterations of RA.

length of (more than 25) years was considered as the

2. Materials and Methods group with very long disease duration.

2.1 Subjects and clinical assessment

2.3 Methods

The study was conducted in 50 patients who had

2.3.1 Rapid test

visited Al-Sadder Medical Cityat Najaf Governorate RF (rheumatoid factor) and C-reactive protein were (Iraq) between February and May 2013, and who

performed by a rapid latex agglutination test kit for fulfilled the ACR (American College of the presumptive detection of the RF and C-reactive Rheumatology) 2010 revised criteria for the diagnosis

protein in serum.

of RA [15]. Twenty age and sex-matched healthy

2.3.2 ESR (Erythrocyte sedimentation rate) adults without any evidence of chronic inflammatory

The ICSH (International Committee on disease served as the controls. The patients underwent

Standardization in Hematology) recommends the use thorough clinical and laboratory evaluation, including

of the Westergren method [16].

complete medical history, seropositivity test for RA

2.3.3 Serum iron and TIBC (total iron binding (Rheumatoid Arthritis), CRP (C-reactive protein), and

capacity)

estimation of ESR (erythrocyte sedimentation rate). At Serum levels of iron and TIBC (total iron binding the time of clinical assessment for disease, 6 mL of

capacity) were measured spectrophotometrically in blood samples were collected intravenously from each

Biochemistry Laboratory Al-Diwanyia Teaching

Association Between IL-6 (Interleukin-6) and Iron Status in Rheumatoid Arthritis Patients

Hospital. The Colorimetric test method was used via The correlation coefficients (r) were 0.993. RANDOX reagents (RANDOX kit, U.K.) [17].

2.4 Statistical analysis

2.3.4 Transferrin saturation percentage (%) TS% (transferrin saturation percentage) was

Data analyses were performed with SAS version calculated mathematically [18].

9.1 (SAS Institute Inc., Cary, NC, USA). All of the

2.3.5 Transferrin concentration descriptive variables were expressed as the mean ±SE

Transferrin concentration was calculated (standard error). The correlations between the mathematically [19].

concentrations of IL-6 and iron status were tested

2.3.6 Measurement of serum IL-6and ferritin using Pearson’s correlation test. The group analyses The serum concentration of IL-6 was measured

were performed using one-way ANOVA and Tukey’s using an AssayMax ELISA (enzyme-linked post-hoc analyses. For all tests, a p value less than immunosorbent assay) kit (Assaypro, USA) at

0.05 was considered statistically significant. Virology Laboratory of AL-Sadder Medical City in

3. Results

Najaf Governorate. Fifty µL each of serum sample and assay diluent were placed in each well of a

3.1. Serum concentrations of iron

96-well plate coated with a monoclonal mouse IgG TIBC (total iron binding capacity), TS% against IL-6. This mixture was incubated for two h at

(transferrin saturation percentage) and transferrin room temperature, and each well was aspirated and

concentration were significantly decreased (P < washed five times with wash buffer. Subsequently, 50

0.0001) in patients with RA compared to those of μL of Biotinylated IL-6 Antibody was added to each

healthy controls, while serum concentration of ferritin well and incubated for two h. Again, each well was

was significantly elevated (P < 0.0001) in patients washed five times with wash buffer. Following this, 50

with RA compared to those of healthy controls (Table μL of Streptavidin-Peroxidase Conjugate was added per

well and incubated for 30 min and each well was aspirated and washed five times with wash buffer.

3.2. Serum concentration of IL-6

Subsequently, 50 μL of substrate solution, which was Serum level of IL-6 of RA patients showed

prepared with equal amounts of stabilized hydrogen significant increase (P < 0.0001) in the average values peroxide (H 2 O 2 ) and tetramethylbenzidine, was added

comparatively to the healthy group. In the very early for a 20 min reaction under dark conditions. The

duration of the disease was much higher (Fig. 1). reaction was quenched by the addition of 50 μL stop

solution (0.5 N of HCl). Within 30 min, the optical

3.3. Relationship of IL-6 levels to iron status density was measured at a wavelength of 450 nm

Serum concentration of IL-6 showed inverse using the bioelisa reader ELX 800 (Molecular Device

correlations with serum iron, and serum TIBC, Co., biokit, CA, USA). The serum concentration of

respectively (Table 2).

IL-6 was determined based on a standard

3.4. Relationship of IL-6 levels to serum ferritin and concentration curve. The correlation coefficient (r) of

ESR

the standard concentration curve was 0.990. The serum ferritin was also purchased from

Serum concentration of IL-6 correlated positively and Assaypro, USA, and determined using similar significantly with serum ferritin (R = 0.9480, P < methods. The serum concentration of ferritin was

0.0001) the highest coefficient correlation being with determined based on a standard concentration curve.

ESR (R = 0.9776, P < 0.0001), respectively (Table 3).

Association Between IL-6 (Interleukin-6) and Iron Status in Rheumatoid Arthritis Patients

Table 1 Iron status in healthy control group and in the groups of patients suffering from RA.

Iron status Healthy Control RA patients (n = 50)

GV S. Iron (µmol/L)

a 10.66±1.07 a S. TIBC (µmol/L) 54.15±1.00

a 7.81±1.49 a 7.88±1.49 a 10.12±1.79

a 34.29±1.70 a 34.5±1.05 *** a 35.98±1.93 a 32.28±1.48 a S. Tf (g/L)

2.33±0.22 0.43±0.16 a *** 0.34±0.09 a *** 0.36±0.13 a *** 0.42±0.15 a *** 0.42±0.08 a *** TS (%)

39.57±2.30 13.27±3.60 a *** 11.11±2.08 a *** 11.12±2.19 a *** 12.77±2.37 a *** 14.63±1.51 a *** Ferritin (ng/mL)

131.2±5.96 220.4±23.73 ns 233.2±28.37 ** 218.2±28.47 * 198.5±27.38 ns 217.5±21.26 ns a a a a a Data are expressed as means ±SE (standard error).

The asterisks indicate significant difference based on Tukey’s multiple comparison tests. The same letters indicate no significant difference between groups based on Tukey’s multiple comparison tests. *** indicate extremely significant (P < 0.001). * indicate significant (P value 0.01 to 0.05). Ns: not significant.

4. Discussion

The current study showed that serum concentrations

of IL-6, were significantly elevated in patients with

L g/m

RA compared to those in healthy controls (Fig. 1). As

6 (p 20

seen in previous reports, this finding supports the

IL-

hypothesis that IL-6 is involved in the pathogenesis of

RA [20-23]. In addition to these previous observations, the significant increase in the IL-6 cytokine observed

G I II G in the current study indicates that this cytokines might G III IV G G V

rol

C ont

play a role in inducing inflammatory responses or

RA patients groups

mediating anti-inflammatory responses in the

Fig 1 Serum levels of IL-6 in healthy group and in the five

pathogenesis of RA [24].

groups of patients suffering from rheumatoid arthritis.

The present study also indicates significant decrease Data are expressed as means ±SE (standard error).

*** indicate significant difference based on Tukey’s multiple in serum iron, serum transferrin in all groups of comparison tests.

patients with RA (Table 1). As seen in previous P < 0.0001

reports, this finding supports the hypothesis that the

Table 2 Correlation between IL-6 and iron status in RA

anemia is the most frequent extra-articular

manifestation of the disease [25, 26]. S. Iron

patients.

In RA, it is estimated that 30-60% of patients are Cytokine

S. TIBC

(µmol/L)

(µmol/L)

S. Tf (g/L)

(pg/mL)

anemic. One of the most frequent causes of anemia in RA patients is IDA (iron deficiency anemia). ACD

rP * rP * r P *

IL-6 -0.1358 0.2624 -0.01167 0.9236 0.1459 0.228 (anemia of chronic disease) which does not usually

Table 3 Correlation between IL-6 and serum ferritin and

respond to iron supplementation is another major

ESR in RA patients.

cause of anemia in patients with RA [27]. Cytokine

The induction of iron sequestration in macrophages (pg/mL)

S. Ferritin (ng/mL)

ESR (mm/hr.)

rP * rP * and the decrease in iron absorption in the small IL-6 0.9480 <0.0001 *** 0.9776 <0.0001 *** intestine were shown in infections and inflammatory

*Pearson’s correlation analysis was performed. diseases. This results in the development of anaemia,

Association Between IL-6 (Interleukin-6) and Iron Status in Rheumatoid Arthritis Patients

termed ‘AI’ (anaemia of inflammation), formerly pathogenesis of RA including ACD. The inverse known as ‘ACD’ (anaemia of chronic disease). The AI

correlation between IL-6 level and serum iron and iron is a most common condition noticeable in patients

indices demonstrated that this cytokine-mediated bone suffering from inflammatory disorders (e.g. RA). It is

marrow suppression is the main mechanism for characterized by low to normal serum iron levels (i.e.

development of anemia of chronic disease in hypoferremia), low serum iron binding capacity, and

rheumatoid arthritis.

normal to elevated ferritin concentrations [28].

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May 2014, Vol. 8, No. 5, pp. 410-417

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using Random Amplified Polymorphic DNA in Indonesia

1 Nandariyah Sukaya 2 and Bambang Pujiasmanto

1. Plant Breeding Laboratory, Agrotechnology Program Study, Agricultural Faculty, University of Sebelas Maret, Surakarta 57125, Indonesia

2. Physiology and Biotechnology Laboratory, Agrotechnology Program Study, Agricultural Faculty, University of Sebelas Maret, Surakarta 57125, Indonesia

Received: January 07, 2014 / Accepted: April 30, 2014 / Published: May 30, 2014.

Abstract: Polymorphism in two types of Cereus peruvianus, short and long spines clones, and its F 1 hybrids cultivated in Indonesia were detected by RAPD (Random Amplified Polymorphic DNA) markers. High amount of mucilage (gelling polysaccharides) present in C. peruvianus was a major obstacle in isolating good quality genomic DNA. To obtain good quality DNA, the CTAB (Cetyl Trimethyl Ammonium Bromide) methode was modified. Out of 17 primers were used, and two primers OPN-05 and OPM-10 have specific loci, OPN-05 550 , OPN-05 800 and OPM-10 650 , linked with spines types in parents clones. Those can be used as molecular marker for spines type. Seventeen primers were used generated 113 loci, of which 65 loci were polymorphic in parental clones and 132

loci, of which 93 loci were polymorphic in F 1 plants. Dendrogram generated by Jaccard coefficient showed that parents’ clones had lower genetic diversity than F 1 plants. At 72% similarity, all of long or short spine parent clones grouped in one cluster according to its size of spines. At that time F 1 plants were separately grouped. None of F 1 hybrid plants grouped with its common female parents.

Key words: Genetic diversity, apple cactus, Cereus peruvianus, RAPD, hybrid.

1. Introduction  In the arid Northeastern region of Brazil, the ‘mandacaru’ plants are used as an animal fodder [5]

Apple cactus Cereus peruvianus Mill., popularly and also as a medicinal plant exhibiting antimicrobial known in Brazil as mandacaru is a common activity against Streptococcus epidermidis, S. ornamental cactus species found in gardens of tropical taphylococcus aureus, Pseudomonas aeruginosa and and subtropical countries. In the northeast region of Escherichia coli [6]. The shoots are also served as Brazil ‘mandacaru’ is designated as C. jamacaru food for large and small wild and domesticated instead of C. peruvianus [1]. animals, while the flowers and fruits provide food for Apple cactus has several uses. Heteropolysaccharides insects and wild birds [1]. Since 1991, C. peruvianus (Homo -D Galacturonan) are used in the purification which was introduced in 1984 has been cultivated as a of industrial wastewater [2]. Gums extracted from the

fruit crop in Israel [7].

shoots are used in cosmetic preparations [3]. A pectic Although it is unknown that who took it and when compound (pectic acid) may be used in the it were introduced, cactus apples were also found in preparation of jams, jellies, gums, yogurts, and other Indonesia, cultivated as an ornamental plant by products [4]. community but were not collected yet by research

Corresponding author: Nandariyah Sukaya, M.Sc. , institute. A survey the authors have done shown that

research field: plant breeding. E-mail: sukoyots@yahoo.co.id.

Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using 411

Random Amplified Polymorphic DNA in Indonesia

there were two types of apple cactus grown in were added with 800 L chloroform. Debris was Indonesia, short spines clones type (spine size smaller

removed by centrifugation at 12,000 r. p. m. for 10 than 2 mm) even spineless and long spines clones type

min with a microcentrifuge at room temperature. (spines size more than 20 mm). Both types were

Supernatant (700 L) was transferred to a new micro cultivated separately and were predicted of more than

tube and was added with an equal volume of

10 years with a height of plants reached over 3 m. chloroform: isoamyl alcohol (24:1) and centrifuged at Except that, both types are flowering but usually not

15,000 r. p. m. for 10 min. Supernatant (600 L) was fructify on the rainy season in October to April. Intra

transferred to a new micro tube and was added with 25 and inter hand-pollination between two types we have

L sodium acetate and 650 L cold isopropanol. The done revealed that only pollination between different

solution was centrifuged at 15,000 r. p. m. for 10 min. types produced fruits, which means cactus apples are

The obtained pellet DNA was added with 1 ml 70% SI (self-incompatible). SI is genetically controlled

cold ethanol and was centrifuged at 15,000 r. p. m. for mechanism that prevents fertilization in fertile

5 min. The process was replicated with 96% cold hermaphroditic plants when they are selfed or crossed

ethanol. The DNA pellet was air-dried and suspended to another plant with an identical incompatibility. At

in 200 L purified sterile distillated water and stored least 28 of 98 genera of the Cactaceae family exhibit

at 4 °C. The DNA was purified with Gene Clean III SI [8]. Beside fruits from hand pollination, we also

kit (Biogene): 50 L DNA was transferred to a new obtained a few fruits from open pollination on long

micro tube and was added with 150 L NaI and 5 L spines clones. F 1 hybrids seed were planted and all of

silica, mixed gentle by inverted and incubated for 5 seedlings have long spines, indicating that long spines

min. The solution was centrifuged at 10,000 r. p. m. are dominant character.

for 30 s. The DNA pellet was washed with 375 L The aims of this research were to investigate the

new wash (kit) and centrifuged at 13,000 r. p. m. for genetic diversity of the two types of clones and its F 1 30 s. The process was repeated twice at 14,000 and hybrid plants at the DNA level used RAPD (Random

15,000 r. p. m. respectively. Quantification and purity Amplification Polymorphism DNA).

of DNA were determined with Nano View spectrophotometer (Thermo Scientific). Sample with

2. Materials and Methods

purity ratio above 2.0 extraction process was repeated. Thirty eight fresh shoot sections, 4 from each short

The isolated DNA was used as a template in the

PCR reactions. The PCR reactions were performed in hand pollination between S  L; from L  S, and from

(S) or long (L) spines type, 10 F 1 hybrid plants from

a total reaction volume of 25 L containing 10 ng of open pollination of long spines clones (Lop) template DNA, 0.2 M of each dNTP, 0.2 L primer, respectively were collected from green house of

2.5 M MgCl 2 ,1  PCR buffer and 2 unit Taq-DNA agrotechnology program study, Agricultural Faculty,

polymerase (Operon). Ten-mer primers were used in University of Sebelas Maret, Surakarta, Central Java,

the amplification reactions. Among 31 primers were Indonesia. The material was individually prepared

tested, 17 primers (OPA-02, OPA-04, OPA-12, according to CTAB method with minor modifications

OPB-01, OPB-04, OPB-07, OPD-02, OPD-03, OPD-08, [9]. For DNA extraction, 100 mg of fresh shoot

OPF-09, OPL-11, OPM-02, OPM-10, OPN-04, sample was grounded separately in mini bead beater-8

OPN-05, OPN-06 and OPN-18) were used in the with 1.5 mL CTAB buffer (NaCl were increased to 5

amplification reactions using the C. peruvianus DNA. M) for 10 min and were incubated at 65 °C for 60 min.

The amplifications were performed using a Perkin Samples were transferred to new 2 mL micro tube and

Elmer DNA Thermal Cycler. The PCR conditions

412 Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using

Random Amplified Polymorphic DNA in Indonesia

were: denaturation for 5 min at 96 °C, 45 cycles of

spines [17].

three steps, denaturation at 94 °C for 30 s, annealing Successful amplification of RAPD bands from all at 35 °C for 1 min, and extension at 72 °C for 2 min;

parent clones and F 1 plants were obtained from 17 with a final extension for 7 min at 72 °C. Amplification

primers. Polymorphic banding patterns (Fig. 1) reflect products were separated by electrophoresis on 1.0%

genomic variability in apple cactus was grown in agarose (Sigma) gels. Gels were run for 4 h at 3.2

Indonesia. The 17 primers generated 113 loci, of V/cm in TAE buffer, stained with ethidium bromide

which 65 loci were polymorphic in parental clones (0.5 mg/mL) and photographed under UV light. 1 kb

and 132 loci, of which 93 loci were polymorphic in F 1 DNA Ladder (vivantis) was used as size marker.

plants (Table 1).

Bands were analyzed by comparing the RAPD The number of loci for each primer varied from 2 profiles of each plant in terms of presence or absence

(OPB-07) to 14 (OPN-06) with average of 7.2 loci per of each DNA locus. The similarities between plants

primer. This results were lower than the average were calculated using the Jaccard’s coefficient and

number of bands obtained from callus tissue (11.7) of UPGMA cluster analysis was performed using the

C. peruvianus maintained in different growth NTSYS-pc software [10].

regulator combinations [18], and the average number of bands obtained from F1 (11.5) descendents of C.

3. Results and Discussion

peruvianus somaclones in Brazil [19] and from

Genomic DNA extraction using CTAB has somaclonal variation (15.6) in C. peruvianus [20]. routinely been obtained from various plant tissues,

Size of the amplification products ranged from 150 to however firstly we have difficulty in extracting DNA

1200 bp. This results lower than the size of amplified from C. peruvianus. The purity ratio of the DNA was

products range (500-5000 bp) from callus [18], from obtained more than 2.3. Difficulty were solved with

F 1 descendents of C. peruvianus somaclones increased the concentration of NaCl to 5 M on CTAB

(250-7500 bp) in Brazil [19] and from somaclonal buffer and was obtained good DNA with purity ratio

variation (300-5.500 bp) in C. peruvianus [20]. among 1.7 to 2.0. A number of investigators have

Polymorphism in F 1 plants (74.3%) was higher than comment on the difficulty of extracting genomic DNA

the polymorphism detected in parental clones (57.1%). from cactus tissues because of the high This result was same with esterase-detected

polysaccharide-base mucilage content and other polymorphism in C. peruvianus regenerated in vitro interfering secondary compounds [11-15]. To reduce

(R 1 ) (55.9%) which was higher than the R 0 (29.8%) or eliminate mucilage content on shoot tissue, DNA

[19]. The high polymorphism in R 1 populations may could be extracted from other tissues like roots [16] or

have resulted from in vitro-induced mutations in

Fig. 1 RAPD profile of C. peruvianus clones and its F 1 hybrid plants obtained with primer OPF-09 (A) and OPN-06 (B). (M:

DNA ladder; line 1-4 Long spines clones (L) ; line 5-8 Short spines clones (S); lines 9-18 F 1 plants of S  L crossing; lines

20-24 F 1 plants from open pollinated on Long spines clones.

Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using 413

Random Amplified Polymorphic DNA in Indonesia Table 1 Number of Loci in Parent Clones and F 1 Hybrids of C. perunianus.

Parental Type

Hybrids

Primer Short spines (S)

Long spines (L)

LOP: Open Pollination on Long spines; NTL: Number of Total Loci; NPL: Number of polymorphic Loci.

Fig. 2 RAPD profile generated by primer OPM-10 (A) and OPN-05 (B) for Long Spines clones (L) and Short Spines clones (S). M: DNA marker. Arrow: specific locus.

constitutive gene coding esterase isozymes in soma detected in Pinus thunbergii [22]. RAPD markers clones [21].

were not associated with the morphological character Out of 17 primers were used. Two primers,

of C. peruvianus soma clone shoots [23]. RAPD have OPM-10 and OPN-05 have specific loci. OPM-10 650 ,

been used as sex-linked marker in Hippophae OPN-05 550 and OPN-05 800 were linked with spines

rhamnoides [24, 25]; H. salicifolia [26] Myristica types in clonal parents. The three loci present only in

fragrans [27].

long spines type clones (Fig. 2) that can be used as Dendrogram by Jaccard’s coefficient showed that molecular marker that may or may not link with

similarity of parent clones and F 1 plants ranged from phenotypic expression of a trait. No linked between

30% to 86% with SLH-1 and SLH-5 have highest RAPD markers and morphological variations has been

similarity (Table 2 and Fig. 3). Dendrogram showed

Table 2 Jaccard’s coefficient similarity among 38 Accesions C. peruvianus by RAPD polymorphisms using 17 primers.

1-4: LSP (long spines parents clones); 5-8: SSP (short spines parent clones); 9-18: F 1 from crossing SSP  LSP; 19-28: F 1 from open pollination of L (LOP); 29-38: F 1 from crossing LSP  SSP.

Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using 415

Random Amplified Polymorphic DNA in Indonesia

Fig. 3 Dendrogram among parent clones and its F 1 hybrid plants of C. peruvianus based on UPGMA cluster analysis of the RAPD profiles derived from 17 primers, by Jaccard’s similarity coefficient.

that parental clones have higher similarity (low level There was no grouping of R 1 plants according to the

soma clones parental (R 0 ) of Cereus peruvianus [19]. vegetatively propagated with stem cutting and had

of genetic diversity) than F 1 plants. Apple cacti were

4. Conclusions

limited genetic variability. Low level of genetic diversity of C. peruvianus has been reported as result

RAPD markers can be used to detect genetic from vegetative propagation [28]. Hybridization diversity of Apple cactus (Cereus peruvianus) clones increased the genetic variability of germplasm. At cut

and its F 1 hybrids grown in Indonesia. Out of 17 point of 72% similarity, all of long spine parent clones

primers were used, and two primers OPN-05 and (LSP 1-4) and most of shot spine parent clones (SSP 1,

OPM-10 having specific loci, OPN-05 550 , OPN-05 800

2 and 4) grouped in one cluster according to its size of and OPM-10 650 , linked with spines types in parents spines. At that time F 1 plants were separately grouped.

clones can be used as molecular marker for spines Dendrogram showed that hybrids from the same

type. The seventeen primers generated 113 loci, of parents’ type (SLHn, LSHn, and LOPn) tend to

which 65 loci were polymorphic in parental clones

and 132 loci, of which 93 loci were polymorphic in F 1 plants grouped with its common female parent. This

grouped according to its hybrids. None of F 1 hybrid

plants. Dendrogram generated by Jaccard coefficient result was same with grouping of 18 sugarcane

showed that parents clones had lower genetic diversity cultivars based on RAPD markers, whose cultivars

than F 1 plants. At 72% similarity, all of long or short have common female parent, in different groups [29].

spine parent clones grouped in one cluster according

416 Genetic Diversity of Apple Cactus, Cereus peruvianus Mill. Clones (Cactaceae) and Its F 1 Hybrids Using

Random Amplified Polymorphic DNA in Indonesia

to its size of spines. At that time F 1 plants were

[13] E.J. Edward, R. Nyffeler, M.J. Donoghue, Basal cactus phylogeny: Implications of Pereskia (Cactaceae)

separately grouped. None of F 1 hybrid plants grouped

paraphyly for the transition to the cactus life form, with its common female parents.

American Naturalist 92 (2005) 1177-1188. [14] .N. Korotkova, T. Borsch, N. Quanndt, K. Taylor, P.

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May 2014, Vol. 8, No. 5, pp. 418-424

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture, Example of Two Tunisian Cultivars

1 2 Sana Gammoudi 1 , René Lecoustre and Mohamed Ben Salah 1. Arid and Oases Cropping Laboratory, Arid Lands Institute, Medenine 4119, Tunisia

2. UMR AMAP CIRAD-BIOS, Boulevard de la Lironde TA A51/PS2 - 34398 Montpellier Cedex 5, France

Received: February 01, 2014 / Accepted: May 07, 2014 / Published: May 30, 2014.

Abstract: The present study was carried out to verify the statistical relationships between the characteristic parameters in terms of vegetative aerial architecture of the date palms for simulating realistic 3D models. The vegetal material was composed of two Tunisian varieties of Phoenix dactylifera L., “Barhi” and “Rochdi”. The observations are taken place in Gabes and on one pair of palms per main stem and offshoot for each cultivar. The analysis of the characteristic dimensions of the pinnae and rachis allowed the determination of a minimum sample. The geometrical analysis confirmed the existence of a strong correlation between rotation angles and radial angles. The architectural analysis of the two Tunisian cultivars revealed that the distribution of characteristic parameter values of pinnae was the outcome of a regionalized variable along the rachis. This statistical study of relationships between the characteristic parameters in terms of vegetative aerial architecture of the two varieties allows executing a new measurement protocol for computing and simulating realistic 3D models.

Key words: Phoenix dactylifera L., modeling, architecture, minimum sample, correlation.

1. Introduction  inflorescence are produced laterally to the palm leaves.

In the context of the present study, we focus on the Plant architecture is defined like the whole of the architectures and the geometry of the palms of this structural forms which the plant presents through its

species.

existence; topology is the way in which its organs are Several studies have dealt with architecture in laid out the ones linked to the others, while the Arecaceae. The first measurement of date palm geometry describes the size and arrangement in the architecture has been done in 1989 [4] and later in space of these organs [1]. Date palm, Phoenix dactylifera 2002 [5]. MOCAF Phoenix network, which is a L. (Arecaceae), is a dioecious monocotyledon; its Euromediterranean project, carries out studies on date vegetative propagation through shoot cuttings is palm but always remaining bounded to the other palm widely practiced. Belonging to the Phoeniceae tribe [2]

trees researches.

classified in the model of Corner or the mode of Tomlinson for the trees carrying of the rejections

2. Materials and Methods

according to the botanist [3], the date palm is built The vegetal material was composed of two Tunisian with one vegetative axis with apical continues growth cultivars: “Barhi” and “Rochdi”. The measured palms and a massive crown of leaves with thorny base; the were taken in the palm groves of Gabes in Tunisia.

Observations were conducted on one pair of palms per Corresponding author: Sana Gammoudi, Ph.D. candidate, research field: plant biology. E-mail: main stem and offshoot for each variety. sanagammoudi85@gmail.com.

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture,

Example of Two Tunisian Cultivars

Modeling a palm tree needs the measurements of The variance of r (r 2 ) may be the estimated by: various metric and geometrical parameters which are

N h C LDa h CDb h necessary to feed the model able to simulate the

fronds of the date palm. / n Mean Da Mean Db

Metric measurement related to the rachis where n is the number of paired measurement and N characteristics including the rachis length from the

(h) is the number of couple of measurements for given insertion on the stem to the extremity, widths and

h distance on the non oriented axe. height taken every 10 cm and length of the spiny and

The minimum useful sample size is defined as to be pinnate parts measured with one tape meter.

m=1– r 2 .

Metric characteristics of the pinnae throughout the If the two variables are positively auto-correlated, rachis counting pinnae length, pinnae opening at the

then m < n.

first and second third of their length, pinnae width at

If they are not, m = n.

the base, the first and the second third of their length, If they are negatively auto-correlated, it can be they were measured by means of digital caliper.

expected that m > n.

Geometrical characteristics of the pinnae, these

3. Results

parameters are three main insertion angles of pinnae relative to the rachis directions: axial angle is the

3.1 Width and Height of Rachis Sections angle between the main directions of the pinnae and

The position on the rachis is normalized relatively the rachis; radial angle is the orthogonal projection of to the total length of rachis. Width and height are the angle between the main direction of the pinnae and normalized relatively to their maximum value. For the line joining right and left insertion points on the each palm tree separately (Figs. 1 and 2) shows the rachis; rotation angle is the angle between the dot groups for the measurements taken every 10 cm insertion scare and a perpendicular to the main on the rachis of the fronds. The width and height data direction of the rachis. noted for the two cultivars show that the width and The sample size of the metric characteristics of the height of the rachis are strongly dependent of their rachis and the pinnae will be estimated using

position on the rachis.

techniques issued from the regionalized variables The semi-variograms (Figs. 3 and 4) for the two theory. Considering Da and Db are the random cultivars are of exponential nature showing the variables recorded on frond A and frond B of the significant deviation of the rachis dimension variables studied palm tree. along the rachis. The ratio data Height/Width of rachis According to recent studies [6], respecting the sections notes for the two cultivars (Fig. 5) varied hypothesis of spatially homogeneous variances and between 0.5 and 2.5 along the rachis. As the variable covariances [7], the co-variance between Da and Db width of rachis is strongly regionalized, the may be estimated using the following classical regionalized variables theory can be used for estimators: estimating a minimum useful sample (Table 1). It

1 CDa h

gives a sample size of 3 sections along the rachis for Nh

Dai Mean Da

the two cultivars.

Daj Mean Da

1 3.2 Length of Pinnae CDb h

Dbi Mean Db

Nh

The position on the rachis is normalized relatively Dbj Mean Dd

to the total length of rachis and the pinnae length is

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture,

Example of Two Tunisian Cultivars

Barhi1

Rochdi 1, width

Barhi 2

ight

Rochdi 2, width

he

Rochdi 1, height

and

ianc

Rochdi 2, height

idth d w

mi-var

Se

aliz m

Nor

Relative position on the rachis

Relative distance on the rachis

Fig. 1 Width and heigh－Rochdi. Fig. 4 Semi-variogram of rachis width－Barhi.

Rochdi 1

Barhi 1, width

Rochdi 2

ight

Barhi 2, width

Barhi 1

Barhi 1, height

idth

Barhi 2

and he idth

Barhi 2, height

d e w aliz

Ratio height/w

m Nor

Relative position on the rachis

Relative position on the rachis

Fig. 2 Width and height－Barhi. Fig. 5 Ratio height/width of rachis sections－All palms.

Rochdi 1-A

Table 1 Estimation of utile minimum sample size for rachis section width estimations.

Rochdi 1-B

Rochdi Barhi Variance of r (x, y)

Useful min sample

ianc

located on one or the other sides of each frond. It

mi-var

seems that the length of the pinnae is strongly related

Se

to its position along the rachis. The two figures show an acceleration of the size difference between the pinnae at the time of transition between spines and

Relative distance on the rachis

leaflets. This phenomenon should not be regarded as discontinuity but as acceleration. The semi-variograms

Fig. 3 Semi-variogram of rachis width－Rochdi.

(Figs. 8 and 9) relating to the pinnae lengths may be normalized relatively to its maximum value measured

considered as fitting to a periodic model.

on left or right side of the frond. For each studied As the variable length of pinnae is strongly palm tree (Figs. 6 and 7) shows the evolution of the

regionalized, the regionalized variables theory can be dot groups for the length measurements of all pinnae

used for estimating a minimum useful sample (Table

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture,

Example of Two Tunisian Cultivars

2). It gives a sample size ranging between 5 and 6 for the two cultivars.

Rochdi1 left side

Rochdi 1 right side

ngths of pinnae

Demi-variance

d e le aliz

Rochdi2 left side

Rochdi 2 right side

Left side

Right side

Nor

Distance

Fig. 9 Semi-variogram of pinnae length Barhi. Relative position on the rachis

Table 2 Details of the estimation of utile minimum sample Fig. 6 Length of pinnae－Rochdi.

size for pinnae length estimation.

Rochdi Barhi

Barhi 1 left side

Variance of r (x, y)

Useful min sample

Barhi 1 right side

3.3 Width of Pinnae at the One Third of Its Length

The position on the rachises is normalized relatively

ngths of pinnae

Barhi 2 left side

d le

to the total length of rachis and the pinnae width is

e aliz

Barhi 2 right side

normalized relatively to its maximum value measured

on left or right side of each measured frond of the two

Nor

cultivars (Figs. 10 and 11) showing that the width of the pinnae is strongly dependent of its position on the

Relative position on the rachis

rachis. As the variable width of pinnae is strongly

Fig. 7 Length of pinnae－Barhi.

regionalized, the regionalized variables theory can be

used for estimating a minimum useful sample (Table 3). It gives a sample size ranging between 4 and 7 for the two studied cultivars.

3.4 Relation between Rotation Angle and Radial Angle of Pinnae

Demi-variance For the two cultivars (Figs. 12 and 13) showed the

dot groups and the linear regression including its equation and signification coefficient. “Barhi” y =

Left side

1.9711x - 37.816 with R² = 0.37 and “Rochdi” y =

1.8329x - 42.352 with R² = 0.39. This result confirms Distance

Right side

the strong correlation between rotation angle and

Fig. 8 Semi-variogram of pinnae length Rochdi.

radial angle of pinnae.

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture,

Example of Two Tunisian Cultivars

y = 1.9711x - 37.816 R² = 0.3682

Rochdi 1 left side

Rochdi 1 right side

ngth

Rochdi 2 left side

idth at 1/3 of le ed w

Rochdi 2 right side

pinnae

Rotation angle

Normaliz

Barhi

Relative position on the rachis

Radial angle

Fig. 10 Width of pinnae at 1/3 of their lengths－Rochdi. Fig. 13 Radial angle f (Rotation angle) Barhi.

Barhi 1 left side

Barhi 1 right side

Barhi 2 left side

ngth idth at 1/3 of

le

Barhi 2 right side

Relative position on the rachis

Fig. 11 Width of pinnae at 1/3 of their lengths－Barhi.

Table 3 Details of the estimation of utile minimum sample size for pinnae width estimation.

(b)

Barhi 1 Barhi 2 Rochdi 1 Rochdi 2

Fig. 14 Simulation of fronds. (a) “Rochdi” fronds. (b)

Variance of r (x,y) 0.31

0.32 0.3 0.17 “Barhi”.

Useful min sample 4.2

3.5 Simulation of Fronds

y = 0.2123x + 19.895

R² = 0.3892

All these geometrical and morphometric parameters allow the fronds simulation of the two observed cultivars “Barhi” and “Rochdi” (Fig. 14).

4. Discussion

Rotation angle The authors’ results confirm the results obtained in other studies [8], and it shows that the width and the height of rachis section are strongly dependent on their

position on the rachis. The ratio height on width for Radial angle

Rochdi

rachis sections throughout the rachis is regular

Fig. 12 Radial angle f (Rotation angle) Rochdi.

according to previous studies [9]. It appears adjustable

Modeling of Date Palm (Phoenix dactylifera L.) Vegetative Aerial Architecture,

Example of Two Tunisian Cultivars

to a statistical function curve whose type remains to be All these characteristics are used as a taxonomical discovered according to knowledge in biomechanics.

index to differentiate between the two Tunisian The modeling of the relationship between width and

varieties. This study allows the frond simulations of height of the rachis indexed on the position through

the two observed Tunisian cultivars “Barhi” and this rachis may allow us to reduce the observation to

“Rochdi”.

only one of the two metric parameters and shorten the Complementary studies will be undertaken in order observation time. Recent studies have shown that

to determine the architecture and geometry of the date width variation throughout the rachis is adjustable to

palm inflorescence.

an exponential function, knowing that the coefficient