European Journal of Medicinal 4(12): 1534-1542, 2014 ISSN: 2231-0894

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Anatomical Changes during Rooting of - Avicennia officinalis and agallocha

T. Govindan1* and K. Kathiresan2

1PG and Research Department of Botany, Government Arts College, C. Mutlur-608 102, , . 2Faculty of Marine Sciences, CAS in Marine Biology, Annamalai University, Parangipettai- 608 502, Tamil Nadu, India.

Authors’ contributions

This whole work was carried out in collaboration between both authors. Both authors read and approved the final manuscript.

Article Information

DOI: 10.9734/EJMP/2014/11014 Editor(s): (1) Marcello Iriti, Department of Agricultural and Environmental Sciences, Milan State University, Italy. Reviewers: (1) Zhi-Wei Fan, Environment and Protection Institute, Chinese Academy of Tropical Agricultural Sciences, China. (2) Anonymous, The University of Douala, Cameroon. (3) Anonymous, University of KwaZulu-Natal, South Africa. Peer review History: http://www.sciencedomain.org/review-history.php?iid=618&id=13&aid=5903

Received 22nd April 2014 th Original Research Article Accepted 25 June 2014 Published 25th August 2014

ABSTRACT

Mangrove forests continue to disappear all over the world due to a number of reasons. This is the study made to screen the anatomical and biochemical changes during the rooting process. Two important have been examined based on their salt relationship natures: Avicennia officinalis subsp. australasica (salt excreting plant) and Excoecaria agallocha L (salt accumulating plant). The plants were treated with different root promoting hormones like IBA, IAA and NAA at different concentrations for three minutes each. After 45 days of the growth period root growth and anatomical

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*Corresponding author: Email: [email protected]; European Journal of Medicinal Plants, 4(12): 1534-1542, 2014

changes were observed. The roots originated from the deeper zone of the secondary xylem after several anatomical variations in the hormone treated plants. This is the first trial which may help to determine the mass propagation of these commercial and medicinally important mangroves.

Keywords: Mangroves; plant hormones; endomorphic changes; anatomical variations; subsp; australasica.

1. INTRODUCTION

It is becoming increasingly evident that the optimization of root architecture for resource capture is vital enabling the next green revolution [1]. Mangroves are woody plants that exist at the interface between land and sea in tropical and sub-tropical latitudes. These plants grow at the intertidal zones of sheltered shores, estuaries, tidal creeks, backwaters, lagoons, marshes and mud flats [2]. Mangrove forests are among the world's most productive ecosystems. However, they exist in the conditions of high salinity, extreme tides, heavy winds, high temperatures and muddy anaerobic soils [3]. There may be no other group of plants with such highly developed adaptations to extreme conditions [2]. The mangroves are of great potential value for fisheries. They serve as nursery, feeding and breeding grounds for many species of fish upon which many coastal communities depend for their livelihoods [4]. More than 80% of marine catches are directly or indirectly dependent on mangroves and other coastal ecosystems worldwide [5]. Also it showed a number of medicinal properties including anticancer, anti-diabetic, antimicrobial and antifungal activities [6].

Though it showed more beneficial impacts, mangrove forests continue to disappear all over the world. They are destroyed by man-made pressures and degraded by environmental stress factors. Destruction through human encroachment for their habitat is the primary cause of mangrove loss. Diversion of freshwater for irrigation and land reclamation has destroyed extensive mangrove forests. In the past several decades, numerous tracts of mangroves have been converted for aquaculture, fundamentally altering the nature of the habitat. Measurements reveal alarming levels of mangrove destruction. Some estimates put global loss rates at one million hectares per year with mangroves in some regions in danger of complete collapse [2]. There has been growing interest in the intensive afforestation of mangroves. It is not possible to do afforestation of the mangroves in a large tract of coastal areas for the reason that there is no adequate availability of planting stocks, with the shrinkage of mangrove habitats. It is an urgent need to develop fast and economically viable techniques to produce superior stocks for mangrove plantation. Hence, the present study revealed endomorphic variations during the root developing process of mangroves.

2. MATERIALS AND METHODS

2.1 Specimen Collection and Samples Preparation

Samples used were control and hormone-treated stems after 45 days from 3 species of mangroves: Avicennia officinalis (salt excreting plant) and Excoecaria agallocha (salt accumulating plant).

The samples were cut from the plant and fixed in FAA (Formalin - 5ml + 70% Ethyl alcohol - 90 ml). After 24 h of fixation, the specimens were dehydrated in a graded series of tertiary

1535 European Journal of Medicinal Plants, 4(12): 1534-1542, 2014 butyl alcohol as per the schedule given by [7]. Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60ºC) until tertiary butyl alcohol (TBA) solution attained super-saturation. The specimens were cast into paraffin blocks.

2.2 Sectioning

The paraffin embedded specimens were sectioned with the help of a Rotary Microtome. The thickness of the sections was 10-12 µm. Dewaxing of the sections was done by following the customary procedure [8]. The sections were stained with Toluidine blue as per the method published by author [9].

Being a metachromatic stain, the toluidine blue gave remarkably good results and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage and blue to the protein bodies. Wherever necessary, sections were also stained with safranin, Fast-green and IKI (for Starch).

2.3 Photomicrographs

Microscopic descriptions of tissues are supplemented with photomicrographs wherever necessary. Images at different magnifications were captured using the Nikon Labphot-2 Microscopic Unit. For normal observation, bright field microscope was used. Since these structures have birefringent properties, under polarized light they appear bright against a dark background. Magnifications of the figures are indicated by the scale-bars.

Descriptive terms of the anatomical features given were followed from the standard anatomy book [10].

3. RESULTS

3.1 Avicennia officinalis

3.1.1 Anatomy of the normal stem

The stem has a broad well-developed periderm with 10-12 layers of phellem and about 5 layers of phelloderm (Fig. 1. 1a, 1h, 1i). The phellogen was in an active state. The cortex is broad and aerenchymatous. There are small air-chambers formed by the reticulation of partition filaments. Calcium oxalate crystals are fairly abundant in the cortex. The crystals are in the form of druses and styloids slytoids (Fig. 1.1e, 1f).

The secondary xylem of the control plant is a solid cylinder and exhibits what is known as anomalous secondary growth. There are circular bands of phloem included within the secondary xylem. These cylinders included phloem or interxylary phloem (Fig. 1.1c, 1j). The secondary xylem consists of radial rows of fibres and radial multiples of vessels (Fig. 1.1j). The pith wide comprises of circular parenchyma cells. There are circular sclereids with thin lignified walls scattered in the pith (Fig. 1.1c, 1l). Calcium oxalate crystals are abundant in the pith parenchyma (Fig. 1.1f). Branchy sclereids or stone cells occur in thin bands in the secondary phloem zone (Fig. 1.1j).

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Fig. 1. Anatomical changes during root induction on hormone treated Avicennia officinalis. 1a. T.S. of Section of control stem, 1b. T.S. of treated stem (Xylem elements oriented in horizontal planne), 1c. Control stem showing included phloem, normally oriented vessels and fibres, 1d. Treated stem showing included phloem, distorted xylem element and gum filled velles. 1e.Druses in the cortical cells of control stem, 1f. Styloids in the pith cells of control plant, 1g. Sparse crystals in the cortical cells of treated plant, 1h. Periderm, 1i. Cortex, 1j. Control stem showing breaking down of the included phloem, 1k. treated stem with intact included phloem, 1l.control stem showing secondary xylem outside of the pith, 1m. Horizontally oriented xylem elements of the treated stem, 1n. Inner xylem of treated stem showing vessels filled with dark substances

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3.1.2 Anatomy of the treated stem

Initially, the treated stem has a periderm similar to that of the control one (Fig. 12). Later, the phellogen of the treated stem becomes more active and produces a broad zone of phellem and several layers of phelloderm (Fig. 1.1b). Due to more active functioning of the phellogen, the periderm develops several irregular shallow fissures (Fig. 1.1b). The cortex also increased in thickness, double the width of the normal stem. The calcium oxalate crystals are few and sporadically seen (Fig. 1.1g). There more number of (interxylary) secondary phloem bands was observed in the treated plants (Fig. 1.1d, 1k). The included phloem elements are intact and more conspicuous (Fig. 1.1k).

More conspicuous changes are seen in the secondary xylem. In the first formed (old) secondary xylem the vessels are filled with a dark, amorphous substance. Such deposition occurred in the fibres also (Fig. 1.1d, 1n). There is also a general tendency for the disorganization of the xylem elements. In the later formed secondary xylem, lignification is highly reduced, leading to parenchymatisation of the xylem tissue (Fig. 1.2d, 1m). The vessels were fewer in frequency. The vertical orientation of the xylem elements is shifted to horizontal alignment, as seen in a cross-sectional view (Fig. 1.1d, 1m).

3.2 Excoecaria agallocha

3.2.1 Anatomy of normal and treated stem

The normal stem has a narrow, 4 or 5 layered periderm and a wide parenchymatous cortex. Tannin is abundant in the periderm and protein bodies are also seen in the cortical cells (Fig. 2.2a, 2b). There was no change in the periderm of the treated stem. However, the cortex becomes much broader, densely tanniniferous and develop many large periderm- tubes (Fig. 2.2c, 2i).

The secondary xylem of the control plant has regular radial files of thin walled fibres and fairly wide, angular, thick walled, radial multiples of vessels (Fig 2.2c, 2i, 2j). Gelatinous fibres are abundant and occur in broad tangential bands (Fig. 2.2e, 2f). Starch grains were abundant in the xylem fibres (Fig. 2.2h). Secondary phloem had randomly oriented elements. Protein bodies are common in the phloem parenchyma (Fig. 2.2d).

In the treated stem, the fibres are thin walled and less lignified. The vessels are grouped into clusters with fewer radial multiples (Fig. 2.2k, 2l). The xylem rays have a dense tannin content (Fig. 2.2k, 2l, 2g). Another conspicuous feature in the secondary xylem of the treated plant is the presence of tangential bands of tanniniferous, parenchymatous cells (Fig. 2.2l). Crystals and starch grains are also frequent in the parenchyma bands (Fig. 2.2g). The secondary phloem is quite broad and regularly organized into radial files (Fig. 2.2k). Phloem elements are distinctive. Phloem rays are also tanniniferous. Both cortical cells and vascular tissues have greater concentrations of tannin than the control plant. Protein bodies are abundant in the phloem parenchyma. While tannin is found in phloem rays, protein is formed in the phloem parenchyma cells (Fig. 2.2k).

3.3 Origin of Roots in the Treated Stem

The adventitious root, after treatment, originates from deeper zone of the secondary xylem. The root develops from the tanniniferous tangential band of parenchyma found in the middle part of the secondary xylem (Fig. 2.2m, 2n). The parenchyma cells as well as the xylem rays become meristematic and give rise to radially directed derivatives which develop into the

1538 European Journal of Medicinal Plants, 4(12): 1534-1542, 2014 lateral root. The lateral root emerges out by piercing the rough outer secondary xylem and bark tissues.

Fig. 2. Anatomical changes during root induction on hormone treated Excoecaria agalloclha. 2a T.S. of cortex of the control plant, 2b. Periderm tissue of the control plant, 2c. Periderm tube in the treated stem, 2d. Secondary phloem, 2e. T.S. of secondary xylem showing gelatinous fibers, 2f. T.S. of inner secondary xylem, 2g. T.S. of secondary xylem of treated stem, 2h.T.S. of secondary xylem of normal stem, 2i&j. Secondary xylem of control stem, 2k.Outer portion of the stem showing pith, secondary phloem and secondary xylem, 2l inner portion of the secondary xylem, 2m. Origin of the root parenchyma from secondary xylem tissue, 2n. Basal portion of the lateral root originating from the parenchymatous tissue

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4. DISCUSSION

The rooting through vegetative methods is a complex phenomenon which involves very different events. Several authors strongly proved that [11-16] the process of rooting can be divided into two phases: (i) formation of root primordia which occurs inside the materials, and (ii) growth which mostly occurs outside of the plant materials.

Anatomical studies on the structure of young stems of two plants before and after treatment for air-layering showed that the plants selected have built self-defensive mechanisms against injuries caused during the experiment. Deposition of abundant tannin and calcium oxalate crystals is one of the defense systems noticed. The periderm which is the external protective barrier of plants is more expressed in the treated plants.

The origin of the adventitious roots is facilitated by the development of parenchymatous bands in the secondary xylem. This band is developed by the wound stimulus coupled with hormone application. The parenchymatous bands exhibit distinct accumulation of tannins. Thus, air-layering of the lateral branches seems to be a preferable technique for the propagation of the selected mangroves. In this investigation the visible adventitious roots were formed from the species, Excoecaria agallocha. The other species examined did not show the visible root but we have observed the root promotion from the anatomical studies. Similar findings reported in cutting from 10 genotypes of E. cretica did not root while the samples from 11 other genotypes rooted only with the application of 0.5g of IBA. A higher concentration of IBA decreased rooting percentage [17]. High rooting percentages, about 97% and 100%, respectively, were achieved in IBA treated in vitro rooting. This is the greatest response achieved from the growth regulator IBA [18]. The auxin requirements in each phase have been a matter of controversy in all the processes including the biochemical deposition [19-23].

5. CONCLUSION

This is the study majorly concentrated on to analyse the anatomical and biochemical changes during root development of control and hormone treated mangroves. This trial may help to mass propagation of mangroves and this study will be an important step to conserve the commercially and medicinally important mangroves.

CONSENT

All authors declare that ‘written informed consent was obtained from the patient (or other approved parties) for publication of this case report and accompanying images.

ETHICAL APPROVAL

All authors hereby declare that all experiments have been examined and approved by the appropriate ethics committee and have therefore been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=618&id=13&aid=5903

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