The Laticifers and Latex of <Emphasis Type="Italic">Euphorbia Tirucalli
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THE LATICIFERS AND LATEX OF EUPHORBIA TIRUCALLI LINN. BY A. R. RAO, VIMALA K. MENON (MISS) AND MANJU MALAVIYA (MIss) (Department of Botany, University of Lucknow, Lucknow) Received June 24, 1967 I'Communicated by Prof. L. Narayana Rao, F.A.SC.(Lond.)] ABSTRACT The non-articulate, simple, feebly branched laticifers of Euphorbia tirucalli, occurring in the stem and leaf, have been investigated. The young laticifers are multinucleate, thin-walled, and full of contents, while the older ones develop a secondary wall traversed by numerous, obliquely running pit-canals. A qualitative analysis of the latex shows presence of some carbohydrates, proteolytic enzymes, amylase, etc. INTRODUCTION AND TECItNrQUES THE articulated laticifers of the rubber-yielding plants, and the biochemical assay of their latices have already been studied (Calvert, 1887; Spencer, 1939 a; Krishna Murthy and his collaborators, 1946-63). But a study of the origin and development of the non-articulate laticifers of the wild plants, and their latices, is of recent origin (Mahlberg, 1961, 1963; Rao and Malaviya, 1964, 1966 a, 1966 b, etc.). In the family Euphorbiaceae, some genera show articulate and other genera non-articulate laticifers. A number of species of the genus Euphorbia have been thoroughly investigated and illustrated (Cameron, 1936; Spencer, 1939 b; Esau, 1962). The prcscnt paper however deals with the hitherto uninvesfigated and fairly common Indian species of Euphorbia, i.e., E. tirucalli Linn. The material was fixed in formalin-propiono-alcohol (Johansen, 1940) and Foster's techniques (1946 and 1949) of clearing were used. Staining with alcoholic safranin, or with 76~o alcoholic eosin proved very useful. The procedure followed for the biochemical analysis of the latex will be mentioned later on. DISTRIBUTION AND STRUCTURE OF LATICIFERS The laticifers are totally absent in the roots. They occur in the stem and leaf profusely, penetrating into the entire plant system, The floral 61 62 A.R. RAO AND OTHERS parts could not be examined, as they were not available. The laticifers occur throughout the young and old stems, even at the extreme tips, in the cortex and pith of the stem (Text-Fig. 1 and Plate III, Figs. 1 and 2) and are particularly numerous in the nodal regions. The laticifers are present in the deciduous leaves in the spongy mesophyll (Text-Fig. 2) near the vascular elements and a few occur in the palisade region also. The mature laticifers of Euphorbia tirucalli can be classed under the category of non-articulate or simple laticifers of Esau (1962). In the primary stem, they are long, thin-walled, feebly branched, multinucleate, and full of contents (Text-Fig. 3). The primary wall of these latex tubes takes up the cellulose stains, is sinuous at places and is mostly unpitted (Text-Fig. 3). The older laticifers develop a thick, non-lignified; very closely lamellated secon- dary wall (as shown by the stains phloroglucin-conc. HC1, Foster 1949), and traversed by numerous, deep, obliquely running, pit-canals (Text-Fig. 4 and Plate Ili, Fig. 3). The lumen is very much reduced. These laticifers have very little contents and the flow of the latex from old stems is very little. The wall thickness varies from 1.5 to 3V and the entire breadth from 15 to 21/z, in primary young laticifers. In the older ones these measurements range from 9to 12# and I5 to 24/~. In the leaf, the laticifers are thin- wailed, rarely branched and full of contents. The colour of the fresh latex is white and it contains numerous crystals. These when stained with iodine and dil. sulphuric acid, become blue. According to Metcalfe and Chalk (1950), and Solereder (1908) these are starch grains. Our observations also confirm this. Apart from these crystals, large quantity of solid rubber component is also present in the latex as becomes evident after centrifugation. ONTOUEN~Y OF THE LATICIFERS The non-articulated laticifers, in genelal, originate at the embryonic stage of the plant and develop along with the growth of the other tissues (Foster, 1949, Esau, 1962). In the genus Euphorbia their development has been traced in different species from the embryonic stages. In E. tirucalli as the stages of seed and embryo development were not available the deve- lopment of the embryonic laticifers could not be traced. But the very young apical meristems and young plants raised by cuttings have been examined periodically. The laticifers seen in them must have originated from the ground meristem of the embryo, The Laticifers and Latex of Euphorbia tirucalli Linn. 63 The youngest laticifers are full of granular protoplasm and a single distinct nucleus with a nucleolus (Text-Fig. 5). They grow by "intrusive" and "symplastic" growth, as the stem matures. The nucleus generally divides (Text-Fig. 6) and as the pointed tip of the laticifer advances through the surrounding tissue, more nuclear divisions take place leading to a multi- nucleate stage (Text-Figs. 7 and 8). As in Tabernaemontana coronaria Willd (Rao and Malaviya, 1966 b) the laticifer here also joins with an adjoining vegetative cell by the dissolution of their cell walls (Plate III, Fig. 4). A small branch is thus produced (Text-Figs. 9 and 10). The nuclei and protoplasm migrate into this branch, which grows further and sometimes dichotomises (Text-Fig. 11). A few young laticifers do not elongate but bend a little (Text-Figs. 12 and 13). The old ones develop a very thick secondary wall, traversed by numerous~ obliquely running pit- canals. In such laticifers, the lumen is practically reduced to minimum. In the leaves, development of the laticifers is both by the embryonic laticifers as well as by distinct laticifer primordia, di'fferentiated from the spongy mesophyll near the vascular elements (Text-Fig. 14). They are larger in sizc than the surrounding cells and are distinctly uninueleate (Text-Fig. 14). They increase in size, elongate and develop into small laficifers (Text- Figs. 15-17). Other laticifers which are of embryonic origin and are pIesent in the leaves also, develop in the same way as in the stem (Text-Figs. 18 and 19). This type of development of laticifers by distinct laticifer primordia in the lcaf, has been mentioned by Esau (1962) in some plants and in Yatropha by Rao and Malaviya (1964). A QUALITATIVE CHEMICAL ANALYSIS OF 7I-1E LAT13gK A preliminary qualitative analysis of the freshly collected latex shows the presence of some enzymes, carbohydrates, etc. Paper chromatography, and standard biochemical tests have been done as mentioned by Rao and Malaviya (1954, 1966 a, 1966 b). In the case of amylase, viscometer test was not used. Instead, 1 gin. of water-soluble starch was dissolved in some warm water and then scdium chloride was added and the. volume was made 100 c.c. To 5 ml. of this 1% starch solution, a known amount of latex between 0.3 ml. and 0.6 ml. was added. This was incubated at 40 ° C. for 1 hour, and was cooled for about 5 mrs. at 20 ° C. Then, one drop of iodine solution was added and the enzyme activity tested. When no blue colour persists it was presumed that the starch has been acted upon by the enzyme present in the latex. The colour which is blue for a very brief period at the beginning gradually becomes violet, brown, then pink to colourless. Phy p o~ Pl~ , . ...+, .." • .+:-:.+- + ° ~.:'÷. ° • '" o 0 o •~ o~ \ .o °° :" • Q • 0 o 0 n ~ o / .~t,~ r-- i-~a:i, 4 TBx'r.Fios. 1-19 The L~ticifers attd Latex of Euphorbia tirucalli Linn. 65 This is a wcll-known slightly modified amylase tcsL mentioned in Methods in Enzymology. The results of the analysis are summarised below in Table I. TABLE I £oloar Proteolytie Milk- of Free amino-acids Carbohydrates Amylase Peroxidase Dehydro. enzyme genase clotting fresh latex enzyme Milky white : No clear spots. One bri ht Enzyme present. Present; Present; Present, but in No ¢olouring It seems that Ac- Absent spot, corres- Activity very Activity tivity fast small amounts, pigment pre- free amino-acids ponding pro- fast; Klett vigorous as it takes 15- sent me present in bably to glu- reading 167 18 n~nutes for only very small cose the of traces clotting fresh milk Proteolytic activity has been measured in Klett Summerson photoelectric colorimeter. CONCLUSIONS The non-articulate, feebly branched laticifers of Euphorbia tirucalli resemble the laticifers of the Euphorbiaceae in general and the genus Euphorbia in particular (Esau, 1962). The ontogenetic development of the laticifers of Euphorbia tiruealli, is also not different from those of the other members of Euphorbiaceae. The laticifers of Jatropha (Rao and Malaviya, 1964) in the younger stages resemble those of E. tirucalli. But they differ in the older stages in that E. tirucalli develops a very thick, non-lignified, but closely lamellated secondary wall traversed by numerous pit-canals. The branching of laticifers is more profuse in Jatropha than in Euphorbia tirucalli. The biochemical analysis of the fresh latex shows that the constituents of the fresh latex in datropha and E. tirucalli are slightly different. The fresh latex is white in E. tirucalli whereas it is coloured in Jatropha. Free amino-acids are present in large amounts in the latex of Jatropha, but they are practically negligible in E. tirucalli. The enzyme dehydrogenase is present in detectable quantities in Jatropha but is absent in E. tirucalli. Other constituents are very similar in the two species. A more detailed and quantitative assay of the enzyme present in the latex of E. tirucalli may throw up some further interesting features of bio- chemical or therapeutic interest. Since E. tirucalli is a wild species and occurs abundantly, its latex can be used for economic purposes on a larger scale than the latex of other plants.