STUDIES ON LATICIFER DEVELOPMENT IN CALOTROPIS PROCERA AN IMPORTANT PLANT YIELDING HYDROCARBON AND IMPROVEMENT OF ITS GROWTH POTENTIAL Ashwini Kumar and Neetu Vijay Bio-Technology Lab, Department of Botany University of Rajasthan, Jaipur - 302 004, India. Energy Plantation Demonstration project and Biotechnology Center E-mail: msku31@yahoo.com ABSTRACT: The non articulated laticifer cell present in C.procera (Asclepiadaceae) are distributed in cortex, phloem region, pith and among parenchyma cells present in the stem. The transverse and longitudinal sections of the plants treated with plant growth regulators gave varying distribution of non articulated cells. Treatment with all growth regulators i.e. Indole acetic acid, Indole butyric acid, alpha Naphthalene acetic acid, Gibberalic acid and Zinc Sulphate had a positive effect on plant height, number of branches, per cent dry matter and per cent heptane extractables. The maximum increase in dry matter, plant height and per cent heptane extractables was recorded in GA3 treatment among all the growth regulators. ZnSO4 also promoted dry matter, plant height and per cent heptane extractables. 176 1. INTRODUCTION Calotropis procera (Asclepiadaceae) is one of the most abundantly available plant in the semi arid and arid conditions of India. In the present time biomass is considered one of the most promising renewable energy sources. Currently attention is focused mainly on biomass improvement and laticifer development for this purpose plantation of fast growing and high regeneration capacity shrub C.proera. Hydrocarbon provides an alternative to petroleum[1,2,3,4]. Source of hydrocarbon is white milky latex which is present in specialized laticifer cells. Laticifers are found in 12,500 species belonging to 900 genera some 22 families mostly of dicotyledons. Mostly laticiferous plnts belong to the dicot families Apocynaceae, Asclepiadaceae, Asteraceae, Euphorbiaceae, Papavaraceae and Sapotaceae. Mahlberg et al (1987)(5) reported fossil laticifers in some brown coal deposits. Laticifers are classified as non-articulated and articulated types[6]. The Calotropis procera has non-articulated laticifer (NAL) originate from single cells develop with the plant and finally become multinucleate. Laticifer, the specialized cells or tubes containing latex constitute an organized storage or excretion system. Laticiferous cells grow by intrusive as well as symplastic growth growing tips follow the course of middle lamella. Pectinase may be present in latex: this may be facilitate intrusive growth and also loosen wall material of laticifer itself to simplify extension growth[7]. The basic nutritional requirement of the laticifer are obtained from adjacent cells, possibly phloem cells to which they are often associated [8,9]. Apocynaceae has non-articulated laticifers while Euphorbiaceae contains both articulated and nonarticulated laticifers whereas Euphorbia bears nonarticulated ones. Calotropis procera is known for bearing non articulated branched laticifer [10,11]. The branched NAL develop only in primary tissues, in leaves they follow the vascular bundles these laticifers commonly associated with phloem may send out branches between palisade cells or downward between spongy mesophyll cells. [12], studied development of NAL in Euphorbia marignata. The NAL in C.gigantea originate from single cells and develop into long tube like structure that may branch but usually do not anastomose [11,13]. They exist either singly or in the groups parallel elongated cells. These cells undergo elongation with free nuclear division. During present investigations also laticifers were confined with the primary tissues and are generally found in closely associated groups of parallel elongated cells. The NAL are branched coenocytes with actively growing tips, its dense cytoplasm contains numerous vesicles. Generally they contain large central vacuole and peripheral cytoplasm. A large number of vesicles produced in them probably from the endoplasmic reticulum and dictyosomes. The starch reserve in the amyloplast have been reported in Euphorbia species [14]. The role of starch grains in differentiation of mature laticifer needs to be further illustrated. The plastid in NAL have few lamellae possess phytoferritin and accumulate small amount of starch [15] as the laticifer plastids do not become photo synthetically green .The laticifer nuclei are highly lobed and possess dilated perinuclear spaces. The outer nuclear membrane of adjacent nuclei are connected by segments of rough ER. The role of perinuclear dilation in the latex production has not been determined. In Calotropis procera NAL occur in vascular tissues particularly in phloem, phellem and parenchyma. These may enter leaves and develop branches, extending into the mesophyll, sometimes reaching the hypodermis or epidermis. The laticifer initial grow more rapidly than the neighboring cells, their nuclei enlarge and divide without subsequent wall formation. The cells elongate mostly by apical growth, often at both ends, so that the elongating cells tips intrude between the immediately adjoining cells. The elongating apices repeatedly thus forming a branched system. Non articulated laticifers originate in primary tissue and continue intrusive growth only in living tissue that not lost their ability to divide [16]. NAL of C.procera are most common in the stem, cortex and proliferate at the nodes from where they spread into leaves via the parenchymatous tissue of the petiole and into pith through nodal leaf gaps. STUDIES ON LATICIFER DEVELOPMENT IN CALOTROPIS PROCERA AN IMPORTANT PLANT YIELDING HYDROCARBON AND IMPROVEMENT OF ITS GROWTH POTENTIAL Ashwini Kumar and Neetu Vijay Bio-Technology Lab, Department of Botany University of Rajasthan, Jaipur - 302 004, India. Energy Plantation Demonstration project and Biotechnology Center E-mail: msku31@yahoo.com ABSTRACT: The non articulated laticifer cell present in C.procera (Asclepiadaceae) are distributed in cortex, phloem region, pith and among parenchyma cells present in the stem. The transverse and longitudinal sections of the plants treated with plant growth regulators gave varying distribution of non articulated cells. Treatment with all growth regulators i.e. Indole acetic acid, Indole butyric acid, alpha Naphthalene acetic acid, Gibberalic acid and Zinc Sulphate had a positive effect on plant height, number of branches, per cent dry matter and per cent heptane extractables. The maximum increase in dry matter, plant height and per cent heptane extractables was recorded in GA3 treatment among all the growth regulators. ZnSO4 also promoted dry matter, plant height and per cent heptane extractables. 176 2nd World Conference on Biomass for Energy, Industry and Climate Protection, 10-14 May 2004, Rome, Italy Earlier workers compared laticifers with blood vessels of animals. Today, laticifers are envisaged as component of secretary tissue. Laticifer store substance that do not re enter plant metabolism; that may also have some role in water balance and transport of oxygen in the plant. Latex may play a role in wound healing and as a defense against microorganisms and herbivores. 2. METHODOLOGY A 50 ha. Bioenergy plantation demonstration centre has been established in the campus of the University of Rajasthan, Jaipur, to conduct the experiments on large scale cultivation of Calotropis procera with the objective of developing optimal conditions to increase its growth potential and number of non articulated laticifers. Seeds of C.procera were collected in the month of March 2002 to May 2002 from different localities of Jaipur. Seeds were dried in shed and stored in cool and dry place and used for raising seedlings in the polythene bags filled with sand : manure : clay in 1 : 1 : 1, in the month of June, 2002. one month old seedling transferred to pot in July, 2002. Treatment of different growth regulators and nutrient was given at an age of three month old plants. Plant growth regulators viz. Indole acetic acid (IAA) , Indole butyric acid (IBA), alpha naphthalene acetic acid (NAA), Gibberalic acid (GA3) were applied in the form of foliar spray at the 50 ppm concentration. Total twelve sprays were given at an interval of fifteen days from September, 2002 to February, 2003. Each growth regulator solution was applied in a constant amount c.a. 15 ml. Inorganic nutrient Zinc Sulphate (ZnSO4) at a concentration of 50ppm was applied in the form of foliar spray at constant amount c.a. 15 ml. Control plants were sprayed with double distill water of equal amount. Samples were taken six months after the last spray. Anatomy of each treated sample was conducted to see the effect on laticifer development just below the third node. Per cent biocrude was find out to see the effect on productivity. 3. RESULTS Application of plant growth regulators influenced the plant height, number of branches, per cent dry matter and per cent heptane extractables to carrying level. Although no attempts was made to quantify the presence of non articulated laticifers but hand cut sections in their transverse and longitudinal views represented the presence of laticifers cells in cortex, phloem, and parenchyma and pith region to varying extent as seen in different treatments. Attempts were made to cut the sections of the stem selecting the sections just below the third node of the main branch. The Indole acetic acid application has less dry matter production and accordingly heptane extractable were also low. Probably greater proportion of cells were of parenchymatous nature. The cross section gives a clear view of the presence of laticifers in subepidermal cortical, blast fiber region, secondary phloem, intraxylary phloem and pith. In the longitudinal section non articulated laticifers were found to associated to the phloem cells in abundance. This support the contentions of Fay et al, 1989 [9], that the laticifers are often associated with the phloem cells from which they drive their basic requirements. The Indole butyric acid (IBA) treatment which promoted root development to a greater extent also favored increase in plant height, number of branches, fresh weight and dry weight. The latex cells were prominently associated with the phloem , inter as well as intra xylary phloem and in the parenchyma cells found in the cortex and the pith region. It also increased per cent heptane extractables. Application of alpha naphthalene acetic acid (NAA) adversely affected the plant height but it promoted number of branches , number of leaves, dry matter and per cent heptane extractables. Laticifers cells are well distinguished in the cortical and secondary phloem region. They were also prominently distributed in the pith region. The association of laticifer cells ith the phloem cells is more clearly seen in the longitudinal sections where the laticifer cells are in continuous chains. Increase in plant height was maximum in GA3 treated plants. GA3 also increased number of branches, dry matter and per cent heptane extractables. The localization of laticifer cells in the cortical region is distinct and as seen in the longitudinal section (LS) , these cells are probably greater in number and more elongated. In the same ratio the heptane extractable were recorded in the GA3 treated plants which were maximum among all the growth regulator treatments. The biosynthesis of growth regulator IAA is supported by ZnSO4 which helps in tryptophan biosynthesis a precursor of IAA. The application of ZnSO4 had almost similar effect as the IAA in the plant height, number of branches and leaves were slightly reduced, but the size and thickness of the leaves was much more in ZnSO4 treated plants. The heptane extractable were maximum in plants treated with ZnSO4. the size of the laticifers was greater and cells were closely ad pressed forming thick channels. 177 2nd World Conference on Biomass for Energy, Industry and Climate Protection, 10-14 May 2004, Rome, Italy Effect of Different Treatments on Growth and Productivity of C.Procera Treatment Plant Height (cm) Number of Branches % dry matter % Heptane extractables Control 109 6 9.88% 3.78% IAA (50ppm) 135 9 10.11 % 3.82% IBA (50ppm) 143 10 16.2% 4.03% NAA(50pp m) 120 14 29.6% 5.13% GA3 (50ppm) 180 15 30.8% 5.245% ZnSO4 (50ppm) 135 7 26% 5.64% 4. DISCUSSION Non articulated vessels are essentially branched coenocytes with actively growing tips continuous with the remote part of the cells, where the protoplasm is almost senescent. When the laticifer cut its protoplast tends to become damaged because of the sudden loss of turgor pressure [17]. The development of a large central vacuole and formation of dense globules or particles subsequently released into the vacuole are common ultra structural feature of laticifer differentiation . Latex particles may develop in the cytoplasm [18,19] in small vacuoles [20] in association with rough ER [21] or in golgi vesicles[22]. These particles originated de novo in the cytoplasmic matrix, where they remain at maturity, or may be shifted to the vacuole at some later stage, as in Calotropis gigantea [13]. Alternatively latex particles may arise and remain with in the vacuole. The NAL generally contain latex in to distinct vesicles which are single membrane bound. The limiting membrane of the vesicles seems to be derived from tonoplast. The electron dense latex particles may be derive from vacuolar sap may accumulate on the vesicles. The measurement of cyclo artinol, the laticifer specific terpinol in E.pulcherimma showed that young leaf contain the highest latex and laticifer starch contents on the basis of dry weight and leaf area. The laticifer growth and starch accumulation stops upon cessation of leaf expansion [23]. The high latex content in young leaves may provide protection against insect and animal predation. The starch concentration in laticifers differs from plant organs and related to the primary photosynthate site. The latex contains fuel oils, carbohydrates, organic acids, alkaloids, glycosides etc. in solution and also various suspended particles, including terpenes, resins and rubber [16,24]. C28–C30 triterpenes and their esters and various polycyclic diterpenes and cyclic irritants are found in latex. In addition to this latex also contains cardiac glycosides [10]. Alkaloids in Calotropis, cartenolides in latex and osteoid starch grains present both in vivo and in vitro [10,11]. Triperpenols and phyto sterols are abundant in Euphorbia latex. Triterpenoids and their esters comprise 40% of the latex dry weight in E.pulcherrima [23]. Significantly, the biosynthesis of several compounds affects development of laticifers in culture. Attempts to grow laticifers in vitro have had only limited success [16]. Callus cultures from ovary and shoot tip explant were used for laticifer differentiation in C. gigantea. MS medium with auxins such as IAA, IBA, NAA, Cytokinin Such as Kinetin, BAP and other growth regulators including adenin sulphate (ads) alone or in combination (at a range of concentration) was used for callus cultures and development of laticifers [10,11]. The largest number of the NAL of Calotropis was obtained after 160 d of culture on media containing 1 mg/L IAA. Observations with Calotropis callus show a close relationship between the phytohormone-induced initiation of laticifers and the age of the culture [11]. 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