Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LII, s. II a. Biologie vegetală, 2006 PARTIAL RESEARCH ON THE MORPHOLOGY, PHYSIOLOGY AND PHYTO-CHEMICAL COMPOSITION OF THE SEEDS OF PALIURUS SPINA-CHRISTI MILLER (RHAMNACEAE) UNDER VARIOUS BEFORE GERMINATING TREATMENTS Ş. G. BURDA, PĂDURE IOANA MARCELA, D. E. MIHĂIESCU, ANA FELICIA ILIESCU Abstract: In this paper we are following the fundamental morphological, structural and physiological study that would explain the advantages of certain methods for before germinating treatments of the seeds of Paliurus spina-christi Miller. A first stage included a morphological research for completing the description of the fruit of the given species provided in specialized literature. The structural study of the Paliurus seeds was conducted through transversal sections followed by quantitative microscopic analysis (including by stereomicroscope). The seeds stored in layers under various conditions were studied by analyzing the physiological changes that occur as a result of a variation in external factors. For the study of the phytochemical composition of the seeds we took into account the abscisic acid revealed by way of extracting the selective solvents, followed by GC-MS, derived fat acids, and triglycerides, followed by GC-MS as well as the glucides, using also derivational reactions and GC-MS analysis. Key Words: Paliurus spina-christi, germinating treatment, morpho-structural, biochemical, abscisic acid Introduction Paliurus spina-christi Miller is a shrub (syn. P. australis Gaertn., P. aculeatus Lam., Rhamnus paliurus L.) is one of the few woody species from the Dobrogea dry plane. A xerophyte shrub or tree, growing spontaneously in our country, 3 up to 6 meters in height, erect, with diffuse growth and kneed shoots, with hard thorns, alternately leafed, smooth, dark green leaves with a gleaming upper surface. It has yellow-green quinquepetalous flowers, growing on axial raceme; they smell like vineyard flowers. It blooms in April August. Dry fruit of 2-3 cm in diameter, with a woody seed, having a circular radial nerved winglet around it. The fructification period is in October December [4], [8], [9]. University of Agronomic Sciences and Veterinary Medicine Bucharest, (U.S.A.M.V.B.), Dept. of Arboriculture and Landscape Horticulture, 59 Marasti Blvd., 011464 Bucharest, Romania, E-mail:stefanburda@yahoo.com Dept. of Botany and Plant Physiology, U.S.A.M.V.B., E-mail: nepeta@k.ro Dept. of Botany and Plant Physiology, U.S.A.M.V.B., E-mail: danmih@usamv.ro Dept. of Arboriculture and Landscape Horticulture, U.S.A.M.V.B., E-mail: afiliescu@gmail.com 43
The species presents ornamental value for landscaping in Southern of Romania. It is remarkable for the color of its leaves, but mostly for the shape of its fruits. Due to its many yet very sharp thorns it is suitable for hedges [8]. Moreover, P. spina-christi is a very rare plant of the Romanian flora, [4] raising the issue of its preservation in situ as well as ex situ. It is also a plant with medicinal uses in Mediterranean regions [1], [2]. The paper presents a parallel multiple analyses of the fruits and seeds of P. spinachristi. Morpho-structural analyses of fruits (including seed) to fo llow their structure and its consequences for the germination process. Biochemical analyses to follow the development of the phytohormones in the seeds of P. spina-christi that has been differently prepared before germination. Thus we aim at bringing explanations for choosing a certain multiplication technology [5], [6] for this species relying on physiological and biochemical data [3]. Materials and methods Morpho-structural analyses The fruits of Paliurus were collected from mature plants growing in the collection of D. Brandza Botanical Garden, Bucharest. They have been harvested in November from the plants to create the various pregermination treatments and in February from the ground for morpho-structural studies. During the experiment the seed were not extracted from the fruits, following a lower cost technology to propagate it [5], [6]. The pregermination treatments and sowing process have been realized in U.S.A.M.V. Bucharest. While starting the sowing for this species the following morphological features and internal structure are taken into account to choose the treatments before germination, as well as the specifications from specialized studies. The fruits and seeds of P. spina-christi have been observed macroscopically by undergoing measurements and external morphological analyses (measures, colour, weight, and specific formations on the tegument). For the internal morphological and structural analyses we have resorted to a specific working protocol. Due to the fact the seeds were very hard, a special treatment was required to soften the tissues in hot water. Thus, the dry fruits were placed in a recipient, boiled water was then poured over them and they were left to cool down to the room temperature. The same treatment was applied to the seeds, but, given their small size, they were left to cool for a shorter period of time (we did not wait for a complete cooling!). The fruits have been cut with a penknife, while the seeds have been cut with a blade. The sectioned fruits (transversal, lateral a nd longitudinal) have been studied with a magnifying glass and pictures have been taken. The microscopic studies of the seeds have been conducted using an IOR microscope, with 10x oculars and 10x and 25x objectives. For the transversal section of the seeds and in analyzing the reserve substances form the endosperm we have used the reaction to colour of the starch to potassium iodine. 44
Biochemical analyses The variants chosen for the treatments applied before germination on the seeds of P. spina-christi: Variant V1 single-layer seed harvested in February from the ground and stored at room temperature (18 20C) in hermetically confined recipients; Variant V2 multiple layers seeds stored in a cool place (5 10C) and planed in spring, in March; Variant V3 seeds harvested in November and directly planed. The analysis of the phytochemical compounds was conducted with a gas chromatograph connected to a mass spectrometer Agilent 5973 (GS MS) (picture 10). The column is of type DB5 - MS of 25m length, 0.25X film and inner diameter of 0.25mm, programmed temperature from 50 o C to 320 o C with a rising rate of 14 o C/minute to 200 o C and 3 o C /minute to 320 o C. It has split less injector, with 1 to 1 injection, 320 o C in temperature, split rate 1:60. The carrier gas flows at a rate of 0.7ml/minute. The working temperature of the qvadrupole is of 200 o C, of the transfer line 230 o C, and of the scan area of 10 700 uam. The preparation method was the following: - the seed are extracted from the fruits; - the seeds are then weighed and grinded; - methanol is added the result is the methanolic extract (abscisic acid is solvable in methanol); - acetonitrile (CH3CN) and BSTFA bis(trimethylsilyl) trifloroacetamide are added - the probes are kept at 105 o C for one hour in recipients (derivation) closed with Teflon corks extreme-temperature resistant; - the analysis under the GS MS. In the analysis of the compounds from the seeds of P. spina-christi, nine reference substances were used: 6-furylamino-purine, naphthylacetic acid, 34-butiric indolyl acid, nicotinic acid, X-amino butyric acid, indole acetic acid IAA (auxine), gibberellin, BAP, and abscisic acid. The first 8 substances are usually used in creating the culture environment for micro-multiplication, while the abscisic acid was separately highlighted in reference substance as it was the main biochemical compound analyzed. The method of analysis applied in this paper has a much simpler protocol for the preparation of the probes in point of the chemical substances and the extracting methods, mostly in the case of the abscisic acid as against the methods applied in similar experiments. The methods already in use for the preparation of the probes involve using chemical substances (biochemicals, liquefied gas etc) which are expensive and difficult to purchase for the Romanian labs [5], [7], *. 45
The method used in this experiment was drawn out in the Plant Physiology Lab of the Faculty of Horticulture of the U.S.A.M.V. Bucharest and is still conducting tests and pending completion. The method of analyzing the chemical compounds by gas chromatograph connected to a mass spectrometer relies on the hypothesis of identifying the chemical types and it involves a rapid preparation of the plant material with the result being a quality analysis. The results of the given biochemical analysis do not identify the compounds in a simple state, but show the presence of their mono-, bi- and trimethylsilyl esters as a result of the derivation reaction with the BSTFA. Results and discussion The morpho-structural analyses The Paliurus fruit is dry, 2-3 cm in diameter, with a woody seed, surrounded by a wavy circular winglet with radial nerves (fig. 1) [9]. The epicarp is felted and it continues with the winglet, and the seed is hard and intensely lignified with three seminal cavities. In crosssection we observed the tissue of the fruit epicarp and the way the pattern of the seminal cavities, while in longitudinal section we followed the texture of the tissue from the winglet around the fruit. The transversal and longitudinal sections have revealed the specific seed tissues, while the lateral has shown the morphology and the way the embryo is placed. In order to conduct the analyses the seeds are being extracted from the fruits. During extraction, the features of the P. spina-christi seeds should be taken into account, because they are small and not very resistant to breakage (the woody epicarp of the fruit should be broken for extraction). The external characteristics of the P. spinachristi seeds are: The shape: obovate, slightly convex; dimensions: 3 5 mm in diameter, 1 1,5 mm thick. MMB = 15,384 17,272 g; colour: shades of brown; surface of seed coat: smooth, shiny; hilum: darker-coloured, it appears slightly clear in the line of the seed; micropyle: point-shaped, of a lighter colour, placed on a small prominence; raphe: appears as a slightly visible stripe on the surface of the seed coat. Fig. 1 - The morphlogy of fruit in Paliurus spina-christi Micropile Raphe Hil e Fig. 2 - The morphology of P. spina-christi seed 46
The inner morphological characteristics of the Paliurus seeds were observed by macroscopic analyses in different sections. In longitudinal and transversal section one can easily see the tissues of the seminal tegument: the outer coat of the seed of a light brownish colour and the brown tegument. Here one can also notice the whitish endosperm, which indicates that Paliurus seeds are albuminous. The cotyledons are plane and they occupy the largest surface of the section, joining the tegument on the margin of the seed, were the endosperm is absent. In lateral section one can notice the tegument and the obovate shape of the cotyledon ( fig. 3), identical to that of the seed. The embryo is of white-yellowish colour and it is straight with a clearly shaped hypocotyl and radicle. For the structural analysis the transversal sections of the seed and cotyledon were made. The cross-section of the seed presents the outer coat of the seed represents by a brown epidermis and the palisade sclerenchyma with elongated cells. The tegument is dark brown with cells slightly shaped. The endosperm has polyhedral cells with thin walls, under the tegument. Inside the endosperm cells, sporadically starch highlighted through the colour reaction appeared (fig. 4). Cotyledon obovate Fig. 3 - Lateral section trough seed: front face of the cotyledon Seed coat Hipocotyl Radicle Epiderm Palisade sclerenchyma Testa Tegmen Endosperm Fig. 4 - Transversal section through the Paliurus seed (oc. 10x, ob. 25x) 47
Adaxial palisadic tissue Adaxial epidermis Spongy tissue with vascular bundles Abaxial palisadic tissue Abaxial epidermis Fig. 5 - Crossing section through cotyledon: bifacial structure (oc. 10x, ob. 10x) In the transversal section of the seed, the seed presents the same tissues as in the previous section. The tissues that make up the seminal tegument, the outer coat of the seed and the tegmen and the endosperm where the starch can be better seen. The transversal section of the cotyledon, under a microscope with a 10X ocular and objective, shows its bifacial structure with two palisadic tissues, one exterior and the other interior, and also the external and internal epidermis. Inside one can see the lacunose tissue crossed by vessel fascicles (fig. 5). Biochemical analyses In conducting the phytochemical analysis we have followed mainly the abscisic acid deemed to be the main compound involved in the dynamics of the seminal dormant and germination [3.5]. Consequently we have followed the growing hormone AIA to highlight the dynamics of the physiological balance in point of plant hormones, under various before germinating treatment conditions for the P. spina-christi. 48
While identifying the fat acids the following have been discovered: miristic acid, behenic acid, arahic acid, palmitic acid, oleic acid, linoleic acid and stearic acid (fig. 6). The amino acids from the Paliurus seeds the following have been identified: alanine, glycine, isoleucine, leucine, valine, proline, aspartic acid, tyrosine, and tryptophan (fig. 7). In identifying the indolyl acetic acid IAA (auxine) one faces the problem of visualizing the characteristic pic (fig. 8) in full-scan or TIC (total ion count), that is why it is recommended to set the apparatus in SIM (single ion monitoring) mode. On the TIC chromatogram one can see the pics of all substances existing inside the probe which makes it difficult to single out one certain compound. In order to avoid this large crowd of pics on the graphic one resort to setting the apparatus according to the characteristic ion of the analyzed substance (the mass specter of the reference substance is used as reference point). By using the SIM system for the IAA one can remove the unpleasant likelihood of its pic being obscured by the pic of the glucitol, as it appears in the TIC. In the IAA analysis one can see a maximum (expressed in surface percentages) for variant V3, for seeds planted directly on the ground in autumn, and a minimum for variant V1 for the single-layered seeds stored in a dry place at room temperature. In highlighting the abscisic acid in TIC one can clearly see the pic of the reference substance with the largest area, but the probes have very small pics. This led to the necessity of setting the apparatus to show a more detailed graphic, working in SIM mode. This clearly shows the ABA pics for the probes, while one can notice a reversely proportional variation of this phytohormone as against the IAA. Thus, the V1 has the highest concentration of ABA, while the V1 has the lowest. This is very well highlighted by scanning the probe according to 262, 393 and 408 ions, were the point of the pics is placed on an approximately common axis (fig. 9). Conclusions New data have been brought referring to the morphology and structure of the fruits and seeds of P. spina-christi and they also confirm the major role the structure plays in choosing the before germinating treatments. As a result of this research one can see that the issue of the hormonal balance for the seeds of P. spina-christi is consistent to other dendrological species which have been studied in specialized literature [3]. Thus, for the variant of seeds planted in autumn on the ground, which is the closest to the natural germination conditions, the quantity of abscisic acid is the lowest, while of the IAA is the highest, showing the most favourable hormonal balance for the germination. 49
In choosing the technology for seminal propagation for the Paliurus, one should however take into account besides the physiological, also the anatomical [6.8}and mostly the environmental (the biotic / non-biotic) hazard. If a technology of autumn planting is chosen, one is facing variations in humidity which reduce the softening effect of the woody tissue from the fruit exocarp, but mostly the risks posed by rodents and birds (the seeds are planted not very deep in the ground). From the above mentioned follows that it is recommended to select a technology with a low environmental risk and which ensures a hormonal balance favourable for germination. After ruling out other variants, the V2 variant is advisable, with multiple layered seed in recipients, in cooling rooms or cellars, which provide a constant level of humidity for the softening of the seeds and an above low temperature to launch the synthesis of the auxine and reduce the quantity of the ABA. Abundance Ion 117.00 (116.70 to 117.70): paliurus test 3 23-05-2006.D\ data.ms 300000 280000 260000 linoleic oleic 240000 220000 200000 180000 palmit ic stearic 160000 140000 120000 100000 80000 miristic arahic behenic 60000 40000 20000 Time--> 0 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 Fig. 6 - The revelation of the trimetilsilil esters of the fat acids derivated by BSTFA by tracing the chromatogram inside the characteristic ion M = 117 Abundance 9000000 TIC: PALIURUS TEST 1 20-05-2006.D\ data.ms TIC: paliurus test 2 23-05-2006.D\ data.ms (*) TIC: paliurus test 3 23-05-2006.D\ data.ms (*) 8500000 8000000 7500000 7000000 6500000 6000000 isoleucine 50 5500000 5000000 4500000 alanine valine leucine 4000000 3500000 3000000 2500000 glycine 2000000 1500000 1000000 500000 Time--> 0 8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00 Fig. 7 - The revelation of the trimetilsilil esters of the amino acids derivated by BSTFA
Abundance 3200 Ion 202.00 (201.70 to 202.70): PALIURUS TEST 1 20-05-2006.D\ data.ms Ion 202.00 (201.70 to 202.70): paliurus test 2 23-05-2006.D\ data.ms (*) Ion 202.00 (201.70 to 202.70): paliurus test 3 23-05-2006.D\ data.ms (*) 3000 Abundance 2800 2600 IAA ion 202 2000 1800 1600 1400 1200 1000 800 600 400 200 Ion 202.00 (201.70 to 202.70): PALIURUS TEST 1 20-05-2006.D\ data.ms Ion 202.00 (201.70 to 202.70): paliurus test 2 23-05-2006.D\ data.ms (*) Ion 202.00 (201.70 to 202.70): paliurus test 3 23-05-2006.D\ data.ms (*) V3 V 2 V 1 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Time--> 20.45 20.50 20.55 20.60 20.65 20.70 20.75 20.80 0 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 22.50 Time--> Fig. 8 - The revelation of the IAA by visualising the chromatogram in the ion M = 202 Abundance 3500 3000 Ion 408.00 (407.70 to 408.70): paliurus V1 SIM 24-05-2006.D\ data.ms Ion 408.00 (407.70 to 408.70): paliurus V2 SIM 24-05-2006.D\ data.ms (*) Ion 408.00 (407.70 to 408.70): paliurus V3 SIM 24-05-2006.D\ data.ms (*) V1 2500 2000 V2 1500 V3 1000 500 Time--> 0 23.00 23.50 24.00 24.50 25.00 25.50 26.00 26.50 27.00 27.50 28.00 Fig. 9 - The revelation of the ABA by reanalyzing the probes in SIM-MS for the 262, 393, 408 ions 51
As to the method of analysis, its novelty, speed, precision and effectiveness are noticeable, as well as its potential for further development for other species. Nevertheless the results do not depend only on the complexity of the analysis protocol, but also on the users getting more familiarized with the performances of the computer programs adjacent to the chromatographs. This can be noticed in the wide range of methods to highlight the picks of the IAA and ABA so as just single them out or to present them as against several analyzed probes. As a final conclusion, for the propagation of the P. spina-christi, it is better for the seeds to be extracted from fruits, and for avoiding biotic risks (insects, rodents, and birds) it is advisable to layer them until spring. These technologies removes the necessity for expensive protected spaces used in the technologies presented in field research studies. REFERENCES 1. BRANTNER A., MALES Z., PEPELJNJAK S. & ANTOLIC A., 1996. Antimicrobial activity of Paliurus spina-christi Mill. (Christ s thorn), J. of Ehnopharmacol. 52: 119-122. 2. BRANTNER A. & MALES Z., 1999. Quality assessment of Paliurus spina-christi extracts, J. of Ethnopharmacol. 66: 175 179. 3. BURZO I. DOBRESCU AURELIA, 2005. Fiziologia plantelor, vol. VII, Ed. Elisavaros, Bucharest. 4. CIOCÂRLAN V., 2000. Paliurus in Flora ilustrată a României, Ed. Ceres, Bucharest. 5. DAVIES P. J. 2004 Plant hormones, Kluwer Acadamic Publishers, Dordrecht. 6. KRUSSMANN G., 1981, La pepiniere, La maison rustique, Paris. 7. TAYLOR B.I., BURBRIGE A., THOMPSON A. J., 2000, Control of abscisic acid synthesis, J. of experim. Botany 51: 350. 8. TĂTĂRANU D. 1960, Arbori şi arbuşti forestieri şi ornamentali, cultivaţi în R.P.R. Ed. Agrosilvică, Bucharest. 9. TUTIN T. G., HEYWOOD V. H., BURGES N. A., MOORE D. M., VALENTINE D. H., WALTERS S. M., WEBB D. A., 1980, Flora europaea, vol. 5, Cambridge University Press, Cambridge. *** www.armyacademy.ro 52