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Micropropagation of the Halophyte Sarcocornia fruticosa (L.) A. J. Scott.

Byline: Maria Filomena de Jesus Raposo and Rui Manuel Santos Costa de Morais

Abstract: Details of investigation to evaluate the effects of the number of nodes (one two or three) of Sarcocornia fruticosa explants on growth and multiplication rate of plantlets are presented in this paper. The responses of the 3-node explants to some supplementary sources of different aminoacids and growth regulators indol-3-acetic acid 6- benzylaminopurine and gibberellins A3 were also analysed. Plantlets from 3-node explants showed a marked increase in growth and number of lateral shoots indicating that Sarcocornia does not respond well when explants are very small. The addition of 100 mg l-1 casein hydrolysate plus 150 mg l-1 glutamine and 100 mg l-1 casein hydrolysate plus vitamins showed to be good growth promoters in micropropagating Sarcocornia giving longer plantlets and higher multiplication rates.

Keywords: Explants halophyte in vitro propagation marine biology Sarcocornia.

INTRODUCTION

Currently with the increase of soil salinity either because of natural causes or due to agricultural techniques used finding adequate agricultural fields and water to irrigate them has become a challenge to be overcome. Hence salt-tolerant and halophytic plants could be one solution. Besides the potential of old abandoned salt-marshes could be used to advantage the tides accounting for the irrigation. Sarcocornia fruticosa (Chenopodiaceae) is a perennial succulent halophyte plant with articulate stems and leaves reduced to a scale which could be used for this cultivation purpose.

Salicornia is another genus whose morphology and biochemical composition are closely related to Sarcocornia despite the annual character of Salicornia. Both genera are commonly known as salicornias.

Salicornias in general are very rich in iodine phosphorus calcium silicon zinc manganese and vitamins A C and D [1] and also in diuretic depurative and resolutive (curative) substances. Seeds are rich in edible oils (26-30% of the total lipids) highly unsaturated linoleic acid contributing with 67% for the total fatty acid content followed by oleic (17.5%) and linolenic (1.4%) acids. Salicornias like other vegetables could be a very good source of +-6 (linoleic acid) and +-3 (oleic acid) fatty acids that are essential for humans since they cannot synthesize some of the fatty acids [2]. Besides linoleic and linolenic acids after suffering desaturation and elongation by some desaturases and elongases can be converted into arachidonic acid (ARA) and eicosapentaenoic (EPA) and docosahexahenoic (DHA) acids respectively. Moreover ARA and EPA are natural precursors of prostaglandins leukotrienes and hydroxy fatty acids among other important compounds. ARA for example has been associated with prevention/treatment of some cardiovascular diseases because of its pro-aggregative and vasoconstritive actions on the platelet and antiaggregative and vasodilatative actions on the endothelium. On the other hand EPA exhibits some effects as anti-thrombotic anti-arrhythmic and anti- inflammatory agent and DHA facilitates normal growth development and function of the normal nervous system. Besides both EPA and DHA lower lipid content [3] and reduce cholesterol and triglycerides in plasma [4].

Salicornia and Sarcocornia contain L-ascorbic acid that can act as an oxygen reducing agent or regenerating primary antioxidants principally tocopherols and also as a free radical scavenger [5].

In addition major (calcium zinc) or minor minerals (iodine) are essential for the building up of and maintenance of a good/healthy skeleton (bones and cartilage formation) immune and nervous systems and to maintain the electrolyte imbalance of the body.

But growth of salicornias in the field depends on the seasons of the year and also on the production and germination of seeds. In vitro propagation can overcome these problems and can also provide a large number of plants with the same phenotypic and genetic characteristics free of pathogens throughout the year.

Nevertheless information on the in vitro propagation of salicornias is scarce with most of the experiments and observations relating to field growth and ecology. Onaindia and Omezaga [6] for example had already referred that Salicornia ramosissima (a related species of Sarcocornia) prefers locations rich in organic matter and Prehn et al. [7] and Ahmad and Anis [8] verified that casein hydrolysate and glutamine provided good results in the micropropagation of Quillaja and Cucumis.

Other growth promoters have been used with several species. For example Stefaniak et al. [9] used6-benzylaminopurine (BAP) and indol-3-acetic acid (IAA) to obtain a quicker growth of Salsola. Mei et al. [10] also used the same growth regulators to break dormancy of Atriplex. Other groups [11-13] used other growth regulators like indolbutyric acid (IBA) 24- dichlorophenoxyacetic acid (24-D) or kinetin (kin) with different plants such as Avicennia Triticum Vigna sinensis and Zea mays.

The aims of this work were to test the hypothesis that casein hydrolysate and glutamine are good growth promoters for Sarcocornia fruticosa and to evaluate how the number of nodes of the explants affects the micropropagation and multiplication rates.

MATERIALS AND METHODS

Biological Material and First Culture Conditions

Seeds and explants of Sarcocornia fruticosa were used to carry out this piece of work. Seeds were collected in November 2005 in the wetland and salt marshes of Algarve the southern part of Portugal and were stored in a dark place under room temperature (20-25 C) until the end of the project (during one year).Two sets of experiments were conducted to analyse the best conditions of plant growth and micropropagation.

Culture medium for germination of seeds was HandA [14] supplemented with 2 % NaCl (w/v) and 1 % agar (w/v). Medium pH was corrected to 7.15-7.20 as referred to by Davy et al. [15].

For culturing and in vitro propagation culture medium was initially the same but as the experiments progressed concentrations of nutrients especially nitrogen and phosphorus proved to be insufficient for growth and development of plantlets. Since then the medium was used in a double strength factor (2HandA).

Each experiment consisted of six replicates and was carried out carried out in a walk-in chamber with constant 24 hour-period light (29 E s-1 m-2) andtemperature (25 C) in order to accelerate the growth. Each replicate consisted of one explant or plantlet.

Practical work began with the disinfection of seeds that were put to germinate. Then as soon as plantlets were high enough and presented already several shoots explants with one two and three nodes were obtained and subjected to different treatments as shown below (Number of nodes and enrichment of the growth medium). Explants with three nodes were used thereafter in other experiments.

Seed Disinfection and Germination

Seeds were surface sterilised following these steps: ethyl alcohol 70 % (v/v) 5 min; sterile deionised water 1 time; sodium hypochlorite 2.5 % (v/v) + 0.5 % tween 20 (v/v) 10 min; sterile deionised water 3 times; and Benomil 1 % (w/v) 2 min.

Seed germination was carried out in Petri dishes with 30 ml solidified HandA medium.

Number of Nodes and Enrichment of the GrowthMedium

Since using too small explants (with less than 5 mm) did not grow and tissues died explants of the articulate stems with 1 2 and 3 nodes were used in order to observe the influence of the explant length and were subjected to different conditions: vitamins -1 (Vit) + casein hydrolysate (CNH) 100 mg .

Vitamins were those from the B5 medium [16]. Eight weeks later plantlets were removed to simple 2HandA medium. Plantlets without roots were prior sowed in a 98 M IBA solution to induce rooting before being transferred to the 2HandA simple medium.

Influence of BAP and IAA (Growth Regulators) and Enrichment of the Growth Medium

To induce growth of new shoots BAP (0.5 and 1.0 mg L-1) was used according to Prehn et al. [7] and Ahmad and Anis [8] but other substances were added to enrich the medium as given below:Equation

To evaluate the difference gibberellic acid (GA3) induces on the growth of the explants and on the development of new branches/shoots we used this gibberellin (5 mg L-1) with and without glutamine (gln 150 mg L-1) and/or casein hydrolysate (CNH 100 mg L-1) as given below:Equation

Statistical Analysis

Statistical analysis was performed using Statistica 6.0 (StatSoft Inc.). Differences between means (One- way Anova) were assessed by the post-hoc Tukey's Honest Significant Difference (HSD) test. Values were considered significantly different at pless than 0.05.

RESULTS AND DISCUSSION

Sarcocornia did not seem to react positively to culturing of explants on media enriched with growth

Table 1: Length and Number of Nodes of the Main Shoot of Sarcocornia Plantlets Number of Lateral Shoots and

###Number of Nodes/Shoot of 1 to 3-Node Explants of Sarcocornia Under Different Growth Media (During Seven

###Weeks) Seven Weeks After Being Transferred to a Medium with no Treatment

###Growth medium###N of nodes of###Main axis/shoot###Lateral shoots

###explants

###Length (mm)###N of nodes###N shoots###N of nodes/ shoot

###2HandA###3###383 b###7.0+1.0###5.0+0.25###1

###2###343 bc###6.0###4.0+0.3###1

###1###91 e###2.7+0.6###0###0

###2HandA+CNH###3###385 b###6.7+0.6###1.0+0.3###2

###2.0+0.25###1

###2###292 c###7.0###1.0+0.25###1

###1###121 e###4.0+1.0###0###0

###2HandA+Vit###3###293 bc###4.7+0.6###2.0+0.25###1

###2###375 b###7.0+1.0###1.0###3

###2.0+1.0###1

###1###181 d###4.8+0.5###0###0

###2HandA+CNH+Vit###3###505 a###9.0###4.0+0.3###4

###4.0+0.25###1

###2###325 b###7.8+0.5###4.0###3

###2.0+0.3###1

###1###373 b###7.0+1.0###5.0+0.25###1

promoters/phytohormones as other plants. As a matter of fact all the additives seemed to cause death of the fragile small explants (results not shown). After that we started using explants with one to three nodes.

Number of Nodes and Enrichment of the Growth Medium

When comparing growth of the different Sarcocornia explants with 1 to 3 nodes after a 3-week period it was already possible to observe a better development of explants with 3 nodes under simple medium 2HandA with no supplements. Moreover more than the addition of casein hydrolysate (CNH) or vitamins (vit) the number of nodes of the explant was the main factor for the better growth of sarcocornias. One third of those 3- node explants had already showed out the principal root (results not shown).

After seven weeks in a medium enriched with 100mg L-1 CNH+Vit difference between growth of explants with different number of nodes was significant those with 3 nodes presenting the best development and having a good rooting system (results not shown).

However when vitamins were added alone explants with 2 nodes apparently showed the best growth. However both groups of 2- and 3-node explants presented one third of rooted plantlets (Table 4). On the other hand CNH alone did not induce differences on the growth of any of the explants (results not shown).

Seven weeks after being transferred to simple 2HandA medium plantlets whose explants had been under CNH+Vit grew further and already presented 4 shoots per explant each with 3 nodes (Table 1).

In general treatment of 3-node explants with CNH+Vit was the most efficient giving rise to better developed plants and higher multiplication rates with more secondary shoots and more nodes per shoot. This effect was even more evident with plants coming from 1-node explants which showed the best characteristics among similar explants under different development conditions (Table 1).

Influence of BAP and IAA (Growth Regulators) and Enrichment of the Growth Medium

Development of the 3-node explants of Sarcocornia was affected by the compounds added to enrich the growth medium. Growth increased mainly with CNH+gln presenting 75% rooted plantlets. These plantlets also showed more ramified secondary/lateral shoots (Table 2) showing therefore higher multiplication rates. However when growth medium was enriched with CNH+gln+BAP it did not induce positive differences in relation to the results obtained with CNH+gln (Table 2). These results are in agreement with the ones of the first experiment. In both experiments a significant increase in plant growth was verified when medium was enriched with casein hydrolysate (CNH) and vitamins (Table 1) or glutamine (Table 2). Similar results have already been recorded by Prehn et al. [7] with Quillaja saponaria. They also bserved an increase of the main shoot length and -1 ooting percentage when 100 mg Lr -1 CNH and 150 mg L glutamine (gln) were added to the growth medium.

This assay also showed that micropropagation of salicornias depends more on the number of nodes of the explants than on nutritional supplements. For a good development we need at least two-node explants growth always being better when 3-node explants are used.

Influence of GA3 on the Development of Explants

Gibberellins are involved in the regulation of cell elongation; they determine plant height and fructification and thus they are economically important [17].

Several workgroups also observed that application of gibberelic acid (GA3) could improve shoot growth of Xanthium pennsylvanium [18] Quercus robur [19] and Prunus avium [20]. Nevertheless Sarcocornia did not answer positively to the GA3 enrichment of the medium. In fact after 6 weeks of treatment with GA3 (Table 3) all explants presented a growth depression when compared with control plantlets and growth

Table 2: Length and Number of Nodes of the Main Shoot of Sarcocornia After a 4-Week Period of Different Treatment

###Conditions and Results of 4 and 6 Weeks After Transferring Plantlets to the Simple 2HandA Medium. Number of

###Lateral Shoots and Number of Nodes Per Shoot are also Indicated

###After 4 weeks of treatment###After 4 weeks 2HandA medium###2 weeks later

###Main axis/shoot###Main axis/shoot###Lateral shoots###Main axis/shoot###Lateral shoots

###Length (mm)###N of###Length (mm)###N of###N of###N of###Length (mm)###N of###N of###N of

###nodes###nodes###shoots###nodes/###nodes###shoots###nodes/

###shoot###shoot

###2HandA###8.51.7###2.8+0.5###10.02.0 e###3.30.58###0.3+0.06###1###-###-###-###-

###(control)

###CNH###17.01.7###5.3+1.15###28.05.3 ab###5.71.53###1.70.58###2###44.04.0 b###7.50.71###1###3

###1.70.58###1###31###2

###1###1

###Gln###12.01.0###3.7+0.5###12.30.6 e###4.01.0###0.30.58###2###35.05.0 b###6.32.52###0.3###3

###0.30.58###1###0.06###2

###1.7

###0.58

###CNH+gln###19.09.1###4.8+0.96###33.36.5 a###7.01.73###1.0###2###65.07.1 a###9.0###3###2

###1.80.5###1###2.7###1

###0.58

CNH+gln+0.5BAP###16.02.6###4.0+1.0###21.32.9 b###4.70.58###1.30.58###2###22.03.9 c###4.70.58###1.7###2

###1.0###1###0.58###1

###1

CNH+gln+1BAP###17.31.2###4.3+1.15###17.31.2 cd###5.01.0###1.0###2###23.03.2 c###5.01.0###1###2

###1.0###1###1.3###1

###0.96

###0.5BAP###9.31.5###3.3+0.5###17.35.5 bcde###5.71.53###0.71.15###1###25.07.2 bc###5.31.15###0.3###2

###0.58

###1BAP###12.21.1###5.0+1.0###13.61.5 de###4.0###0.50.58###1###-###-###-###-

0.5BAP+0.1IAA###11.51.7###4.0###14.51.0 de###4.50.58###0.30.58###1###17.55.0 c###5.0###0.5###1

###0.58

###-1###-1

###Lateral Shoots and Number of Nodes Per Shoot are also Indicated

###After 6 weeks of treatment###After 3 weeks 2HandA medium###6 weeks later

###Main axis/shoot###Main axis/shoot###Lateral shoots###Main axis/shoot###Lateral shoots

###Length (mm)###N of###Length (mm)###N of###N of###N of###Length###N of###N of###N of

###nodes###nodes###shoots###nodes/###(mm)###nodes###shoots###nodes/

###shoot###shoot

###C (2HandA)###31.30.82 a###4.70.52###38.51.29 b###5.51.05###4.0###2.0###77.31.50 b###10.20.75###12.51.0###3.50.55

###C+GA3###19.00.82 c###3.40.55###23.51.29 c###4.50.58###2.31.03###1.90.55###59.32.63 c###7.82.22###7.01.41###3.00.71

###C+GA3+gln###19.30.96 c###3.30.96###20.51.73 c###3.50.58###2.0###1.80.96###44.02.65 d###7.01.73###3.72.08###4.01.0

###C+GA3+CNH###15.80.96 d###3.0###20.52.08 c###3.81.26###4.31.71###2.40.48###75.74.04 b###9.31.53###7.33.51###3.20.45

C+GA3+gln+CNH###16.31.26 d###3.30.5###20.52.08 c###4.30.96###3.30.96###3.0###72.02.65 b###10.71.15###11 1.41###5.00.82

###C+gln###21.01.41 c###4.0###37.51.71 b###6.20.75###3.31.26###1.0###74.34.51 b###10.31.15###122.00###2.80.45

###C+CNH###26.51.91 b###4.30.96###37.31.89 b###6.30.5###3.31.50###2.30.50###73.32.63 b###10.70.82###9.02.45###3.20.41

###C+CNH+gln###32.81.50 a###5.0###55.02.16 a###6.40.55###6.0###1.60.55###88.51.29 a###10.80.84###11.20.84###2.80.45

###-1###-1

depression was even more evident nine weeks after transferring plantlets to the simple 2HandA medium. Plants that had been treated with GA3 alone (C+GA3) or in combination with glutamine (C+GA3+gln) were the smallest the latter ones showing half of the height of the C+CNH+gln" treated plants.

Moreover in contrast with the results observed by Anand et al. [21]) and Ford et al. [20] who obtained an improvement of rooting either with Ipomoea and Prunus cuttings development of salicornias roots was severely inhibited when treated with GA3+CNH (Table 4). All the plants previously treated with GA3 showed a root growth depression as observed by other authors on different kinds of plant cuttings [19 22-24]. One of the explanations for rooting inhibition could be that explants were exposed to light as it happened with Carvalho et al. [22] and Carvalho [25].

Table 4: Effects of Gibberellic Acid (GA3) Glutamine###

###(gln) and Casein Hydrolysate (CNH) on the###

###Rooting of Sarcocornia###

Enrichment of the growth medium###Number of rooted plants

###(%)

###Control (2HandA simple medium)###100

###C+GA3###50

###C+GA3+gln###67

###C+GA3+CNH###33

###C+GA3+gln+CNH###67

###C+gln###100

###C+CNH###100

###C+gln+CNH###100

During this assay best growth results were obtained when explants were treated with a medium enriched with casein hydrolysate (CNH) plus glutamine (gln) (Table 3). Nine weeks after being treated and transferred to simple medium these plants showed a growth significantly higher than the plants from all the other conditions. Moreover by the end of treatment (first 6-week period) all of them were already rooted (Table 4).

In addition CNH+gln" induced a decrease in the differences between plants of the same group plants obtained being more homogeneous and regular despite the reduced number in nodes/shoot of the lateral shoots. Actually this combination promoted lateral shooting when GA3 was also added to the medium (Table 3). Data obtained from these experiments indicate that supplementary sources of nitrogen in the form of aminoacids (glutamine) and oligopeptides (casein hydrolysate) appear to be necessary for micropropagation of salicornias along with explants with at least three nodes.

ACKNOWLEDGEMENTS

This research was supported by the project ProducAPound o Experimental da Planta Halofila Salicornia AGRO" Medida 8 Desenvolvimento Tecnologico e DemonstracAPound o AccAPound o 8.1 Desenvolvimento Experimental e DemonstracAPound o.

REFERENCES

[1] A la dACopyrightcouverte des plantes Plantes recensACopyrightes: Salicorne [page in the internet cited 2009 July]. Available from: http://plantes.sauvages.free.fr/pages_plantes/plante_salicorn e.htm[2] Lauritzen D. Food enrichment with marine omega-3 fatty acids. Int Food Ingred 1994; 1/2: 41-4.

[3] Gordon PT Ratliff V. The implications of omega-3 fatty acids in human health. In: Flick Jr GJ Martin RE editors. Advances in Seafood Biochemistry Composition and Quality. Lancaster USA: Technomic Publishing 1992.[4] Simopoulos AP. The importance of the ratio of omega- 6/omega-3 essential fatty acids. Biomed Pharmacother 2002; 56: 365-79. http://dx.doi.org/10.1016/S0753-3322(02)00253-6

[5] Cuvelier ME. Antioxidants. In: Morais RM editor. Functional Foods: an introductory course. Porto: Escola Superior de Biotecnologia/UCP 2001; p. 95-105.[6] Onaindia M Omezaga I. Natural regeneration in salt marshes of northern Spain. Ann Bot Fen 1999; 36: 59-66.

[7] Prehn D Serrano C. Berrios CG Arce-Johnson P. Propagation of Quillaja saponaria Mol. starting from seed. Bosque 2003; 24: 3-12.

[8] Ahmad N Anis M. In vitro mass propagation of Cucumis sativus L from nodal nodes. Turk J Bot 2005; 29: 237-40.

[9] Stefaniak B Wozny A Li V. Plant micropropagation and callus induction of some annual Salsola species. Biol Plant 2003; 46: 305-8. http://dx.doi.org/10.1023/A:1022879400747

[10] Mei B No EG McWilliams E Gould JH Newton RJ. In vitro regeneration of fourwing salt bush Atriplex canescens (Pursh) Nutt. J Ran Manag 1997; 50: 413-8. http://dx.doi.org/10.2307/4003309[11] Al-Bahany AM Al-Khayri JM. Micropropagation of grey mangrove Avicennia marina. Plant Cell Tissue Org Cult 2003; 72: 87-93. http://dx.doi.org/10.1023/A:1021205731719[12] Sakhabutdinova AR Fatkhutdinova DR Bezrukova MV Shakirova FM. Salicylic acid prevents the damaging action of stress factors of wheat plants. Bulg J Plant Physiol 2003: 314-9.[13] Younis ME El-Shahaby OA Nemat-Alla MM El-Bastawisy ZM. Kinetin alleviates the influence of water logging and salinity on growth and affects the production of plant growth regulators in Vigna sinensis and Zea mays. Agronomie 2003; 23: 277-85. http://dx.doi.org/10.1051/agro:2003010

[14] Hoagland DR Arnon DI. The water-culture method for growing plants without soil. Calif Agric Exp Stat 1950; 347: 1- 32.[15] Davy AJ Bishop GF Costa CSB. Salicornia L. (Salicornia pusilla J. Woods S. ramosissima J. Woods S. europaea L. S. obscura P.W. Ball and Tutin S. nitens P.W. Ball and Tutin S. fragilis P.W. Ball and Tutin and S. dolichostachya Moss). J Ecol 2001; 89: 681-707. http://dx.doi.org/10.1046/j.0022-0477.2001.00607.x

[16] Dixon RA. Plant Cell Culture a practical approach chp 1. Oxford: IRL Press 1987.

[17] Slater A Scott NW Fowler MR. Plant Biotechnology: the genetic manipulation of plants. In: Plant Tissue Culture [book in the internet]. Oxford University Press; 2003 [cited 2003 April 14]. Available from: http://www.oup.com/uk/booksites/ content/0199254680[18] Orkwiszewski JAJ Maksymowych R Maksymowych AB. Regulatory role of indole-3-acetic acid and gibberellic acid in vegetative development of Xanthium pennsylvanicum. Am J Bot 1979; 66: 532-7. http://dx.doi.org/10.2307/2442502

[19] Smith DJ Schwabe WW. Acceleration of early growth of seedlings and rooted cuttings of Quercus robur L. Forestry 1984; 57: 143-57. http://dx.doi.org/10.1093/forestry/57.2.143 [20] Ford Y-Y Taylor JM Blake PS Marks TR. Gibberellin A3 stimulates adventitious rooting of cuttings from cherry (Prunus avium). Plant Growth Reg 2002; 37: 127-33. http://dx.doi.org/10.1023/A:1020584627919

[21] Anand VK Chibbar RN Nanda KK. Effect of GA3 and IBA on rooting and on the sprouting of buds on stem cuttings of Ipomoea fistulosa. Plant Cell Physiol 1972; 13: 917-21.

[22] Carvalho MAM Monteiro WR Dietrich MC. Histological aspects of root formation in petioles of detached leaves of Pereskia grandifolia (Cactaceae): natural conditions and effects of GA3 and dark. Ann Bot 1989; 63: 505-14.

[23] Kaway Y. Effects of exogenous BAP GA3 and ABA on endogenous auxin and rooting of grapevine hardwood cuttings. J Jap Soc Hort Sci 1997; 66: 93-8. http://dx.doi.org/10.2503/jjshs.66.93[24] Igwilo N. Presence of axillary bud and application of plant growth hormones on rooting and tuberization of Yam (Dioscorea sp) vine cuttings. Glob J Agric Sci 2003; 2: 128- 30.[25] Carvalho MA. AlteracAes histologicas e bioquimicas na formacAPound o de raizes em folhas destacadas de Pereskia grandifolia Haw. [MSc Thesis in the internet). Campinas Brasil: Unicamp Digital Library; 1983 [cited 2009 Sept 28]: Available from: http://libdigi.unicamp.br
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