The Effects of Sevin (1-Naphthyl N-Methylcarbamate) on Morphology and Root Anatomy of Glycine max (L.) Merrill

Thi s study i nvest i gated the effects Sevi n (1-naphthyl N-methylcarbamate) had on the morphology .and root anatomy of soybeans (Glyc i ne m (L) Merr.). Morphological observations revealed that as the concentrati on of Sevin i ncreased, the cotyledons, hypocotyl and epicotyl decreased in s i ze. Absence of a pi gment (presumably anthocyanin) normally present i n the petioles and hypocotyl, was noticed at all applications of Sevin. Emergence of primary and trif oliate leaves was delayed as the concentration of Sevi n increased. Increased Sevi n concentrations produced thi n, white roots wi th numerous laterals. With i ncreased concentrat i ons, purple pigment appeared on both taproots and lateral roots. Growth rates of roots, when analyzed by Duncan's Modified Multiple Range Test, were not sign ificant between 2, 9 and 19 ppm of Sevi n, but were signifi cant at t he 1% level for 21, 25 and 55 ppm of Sevi n. Roots of treated soybeans exhibited development of lateral r oo t pr irnord i a close to the root apex . Thi s suggests t hat t he cells f ailed to e l ongate i n advance of differentiat i on. There appeared to be no change i n the quiescent center for all Sevi n concentrations. Noticeable vacuolizat i on of cort i cal, stelar , epidermal and root cap cells occurred at 21, 25 and 55 ppm of Sevi n. At i ncr ased Sevin concentrat i ons, t he root cap was usual ly sloughed off


AC KNOWLEDGE!Y£ENTS •••••••••••••••••••••••• ,. ••••••••••••••• i i i
W ithi n the last few decades, increasing de pendence upon the use of pest i c i des which are part i cularly hazardous to animal and human life, has led to laws that re gulate their production and distribution. Biologists are justly concerned with the e f fects of pestic i des on ani mals and the i r long term effects on future generations, but t h ere is less concern when an insectic i de i s a ppl i ed to a crop , unless i t i s hi ghly phy totoxic.
Research has been conducted on translocation, absorpti on, metabolism and retenti on of i nsectic i des i n plants, but few stud i es have addressed the problem of t he eff ects of insect i cides on the anatomy of t he plants. There i s no question that t he heavy use of i nsecticides has resulted in serious res i due problems i n many a gr i c ul tural areas, so i t is hi ghly des i rable to determine what levels of res i dues affect plant growth, and h ow t hi s e f fect i s expressed anatomically .
Sevi n i s t he most wi dely used carbamate i nsecti c i de i n the Uni ted States today (von Ruraker et al., 1974 ). I t i s a broad s pectrum i nsectic i de t hat controls many economically important insects, some of whi ch are the Painted Lady Butterfly larvae (Vanessa cardui), Green Cloverworm ( Plathypena scalira) and Mexican Bean Beetle (Epilachna var i vestis).
It is unique in having a low mammalian toxicity (560 mg/ kg in rats) as well as low persistanc·e in the envi ronment ( 8-135 days, dep ending on t he substrate) (Caro et al., 1974).
Soybeans are the most imp ortant cash crop i n t he United States and are an imp ortant food product for human and livestock consumpt ion (Howell, 1975). A review of the literature has revealed no s p ecifi c stud i es on the effects of Sev i n on soybeans (while applications of Sevin at 2 l b s/ acre of act i ve ingredient have been observed to be phytotoxic under field conditions). Therefore, thi s thes i s is concerned wi th the morpholo gi cal and anatom i cal effects of vari ous concentrations of Sevin on t h e soybean.
The pertinent l i terature on t he effects of i nsecti cides on p lant anatomy i s concerned wi t h the V isomer of hex achlorocyclohexane, comm only known by the trade name Li ndane.
Increased numbers of rnult i nucleate cells in the root a p ex were observed i n the roots of monocoty ledons, d i cotyledons and gymnosperms.
Lichtenste i n et al. (1962) rep orted that when corn and peas were sub j ected to 30 ppm Sev i n, t h e roots were shorter and t hick er t han the control roots, and f or most species, there was an overall reduct i on i n p l ant s i ze. Lateral roots formed wi t hi n 0.5 cm of t he root a pex a nd were shorter t han those i n the plants grown i n untreated sand. A dark purplish color appeared on t he lower p ort i ons of t h e roots.
The pr imary metaboli te of Sevi n, 1-naphthol, produced no effects on t he length of corn and pea roots, although t hey did exhibit t h e purp le color on i ts lower surfaces.
Li chtenste i n cate gor i zed t he anatomical changes as a 11 growth i nhi b i t i on" . The observat i on of d ifferent i ated P~otoxylem , metaxylem, protophloem and endoder.mis close to the root a pex, suggested t hat development of cel l s close to the root apex was 11 precoc i ous 11 • Sevi n induced growth i n-3. h ib it ion wi th not i ceable trac heary elements d ifferent i ated close t o i:he r oot ap ex i n a unique wedge-shap ed pa ttern opposite t h e proxylem poles.
Lichtenste i n also ob served proli fi c develo pm ent of lateral roots in both peas and corn treated wi t h Sevin .
Anatomi cal c hanges i n corn were d ifferent i at i on of tracheary elements close t o t he root a pex and s ignificant cell wall change i llustrated b y retention of saf r a n i n stai n in a peripheral band of cells i n t h e va scular c yl i nder t hat i n- The anatomy of soybeans has been stud i ed by ~Ji i k sche ( 1961), W eaver ( 1960), and Mitchell and Russell ( 1971).
It i s an upri ght plant, and depend i n g on the vari ety, has    Notice lack of anthocyanin in hypocotyl and epicotyl. There is a de.lay of foliar expansion and prolif'eration of .lateral roots at 21 ppm. At 2 ppm Sevin, there appears to be little effect on the soybean plant growth.

Ef feet on Root
With increased concentrations of Sevin, both tap roots and lateral roots exhibited marked difference in size and number rather than difference in the day of emergence.
The roots were white and thin in comparison with the control roots (Figs. 2, 3). Many of the lateral roots emerged only a short distance from the root apex, and this distance decreased with increased concentrations of Sevin.
At lower concentrations of Sevin, the lateral roots were longer and more prolific than at higher concentrations.
After 10 days growth, the tap roots and lateral roots con-

RADIOASSAY OBSERVATIONS
The symplast consists of the interconnected living elements of plants (e.g., protoplasm) while the apoplast is the nonliving portion of plants (e.g., intercellular spaces, xylem vessels). "Specific Activity"(corrected counts per minute-:-weight of sample) and "Total Activity " (specific activity X sample weight) were computed and used to derive percent distribution values (individual plant part total activity.;.~ of total activity for the entire plant) of 1 4c-sevin applied to various plant organs (Fig, 6,     since very little 1 4c-sevin was translocated to the meristematic areas of the soybean (i.e., shoot and root apices and pods) (arrows in Fig. 7). There appeared to be some 14 concentration of the C-Sevin in the symplast, but true symplastic movement is doubtful.
The primary leaf treatment of 1 .6 ~Ci 1 4c-Sevin per plant showed that 49% of the radioactivity remained on the treated leaf with the nex t higher percentage in the other leaves (31 %). The roots, stems and pods accounted for the remaining 20% of the 14c-sevin uptake (Figs. 6, 8, Table 4).
The faint outline of the stem (arrows) could possibly be due to aphids which had fed on the area of the leaf treated with 1 4c-sevin and transmitted this to other plant parts at subsequent feedings.
As with the primary leaf treatment, the other leaves received 13%, while the remaining 17% of the 1 4c-sevin might be attributed to insect transport (arrows). The evidence suggests that no symplastic movement had taken place since the meristematic areas (m) were deviod of labelled 14c-sevin.

ANATOMICAL ANALYSES
For purposes of discussion, the description of the tissue systems in the root tips of Glycine ~ are discussed as follows: 1) Quiescent center, 2) Root cap and epidermis, 3) Cortex and 4) Vascular cylinder.

The Effects of Sevin on the Quiescent Center
Examination of the quiescent center of the plants treated with 2, 9 and 19 ppm of Sevin revealed little or no appreciable differences from the control (Fig 10). The quiescent centers of plants treated with 21, 25 and 55 ppm did not appear appreciably different from the controls, although the hemispherical outline of the quiescent centers was somewhat less well defi ned than those of the controls (Figs. 11,13).
The Effects of Sevin ..9E. the Root Cap and Epidermis The root cap was not affected at 2, 9 and 19 ppm of Sevin. At 21 ppm, hypoplasia of the root cap and epidermal tissues, previously reported by Lichtenstein et al~ (1962), was observed (Fig . 11). In many instances, the root cap was partially sloughed off or w~s completely absent. The cells of the root cap appeared to be more compressed than those of the controls and were also rounded in appearance difference from the controls while at 21, 25 and 55 ppm, a noticeable difference in structure had occurred.
At 21 ppm, the cells of the cortex were apparently compressed. At 25 ppm the apical meristem was compressed and the cortex appeared to bulge out at approximately 300 ).ll11 from the quiescent center (Fig. 12). The cortical cells at 55 ppm were difficult to distinguish from the vascular cylinder (Fig. 13). The cells appeared shorter, more compressed and more vacuolate than in the controls.

Effects of Sevin .£!! the Vascular Cylinder
The vascular cylinder was affected primarily at higher dosages of Sevin. These caused an appearance of maturing tracheary elements and a development of lateral root primordia opposite the protoxylem poles close to the quiescent center. At 2, 9 and 19 ppm there were no readily observable differences between the treated roots and the controls.
Higher dosages of 21, 25 and 55 ppm of Sevin produced short, flat, maturing tracheary elements approxiamtely 300 µm from the quiescent center (Figs. 11,12). This suggests that the root apex did not advance before dif'ferentiation had taken place or dif'ferentiation occurs more rapidly than meristematic growth. Lichtenstein et al. (1962) labelled this process as a "growth inhibition " .

DISCUSSION
One of the most noticeable differences in the morphology of soyb eans fol lowing the application of Sevin was the absence of pigment (presumably anthocyanin) which is normally present in the stem and hypocotyl.
Hydroxylation is the primary means of oxidation of Sevin (Kuhr, 1970). The hydrolysis of Sevin to its aromatic r ing components, 1-naphthol, or to its N-methyl carbamate Auxin (Indol Acetic Acid) is produced in the apical meristem of the shoot and is transported toward the root apex (Galston and :Davies, 1970 Sevin is applied to the foliage of soybeans at concentrations ranging from 1 .O to 2.5 lbs Active Ingredient/ acre or 1.0 to 1 .5 Actual Ingredient/ 100 gallons of water (Thompson, 1967). These concentrations range from 1 to 9 ppm in the field environment. Actual grower applications  2) In all treated plants a pigment, presumably anthocyanin, that is normally present in untreated plants was absent from the hypocotyl and epicotyl.
3) Emergence of primary leaves was delayed with increasing concentrations of Sevin. At higher concentrations the trifoliate leaves never fully expanded even though primordia were present.
4) The r oots were abnormally whi te and thin. As Sevi n concentration increased, numerous lateral roots app eared close to ,the root apex. A purple color on the ro ots was noticed at 21, 25 and 55 ppm of Sevin.

58.
RAD I OASSAY OBSERVATIO NS 1) When 1 4c-sevin was applied to the root 64% of the radioactivity was translocated into the primary and trifoliate leaves. The autoradiographs suggest that transport of 1 4c-sevin is primarily apoplastic and possibly concentration of 1 4c-sevin into the symplast occurs.
2) With foliar application to the primary and trifoliate leaves, 49 and 70% respectively, of the 1 4c-sevin remained at the application site. As a result of infestations with aphids, some 1 4c-sevin may have been translocated to other plant parts.

ANATOMICAL ANALYSES
1) The quiescent center for all concentrations of Sevin showed little or no difference from the controls, but at doses of Sevin of 21 ppm and above, the hemispherical outline of the quiescent center appeared less well defined than those at 2, 9 and 19 ppm of Sevin.
2) At increased concentrations, noticeable hypoplasia of the root cap and epidermal tissues occurred. At 21, 25 and 55 ppm of Sevin, the epidermis was a flattened, roughened area against the cortex.
3) At 21 ppm of Sevin, the cortex was a shortened and flattened into compressed files of cells, and at 2~ ppm, the cortex appeared to bulge at approximately JOO )J!ll from the quiescent center. At 55 ppm, the cells of the cortex were difficult to distinguish from the vascular cylinder, and appeared more compressed and vacuolate than the controls. 4) Higher concentrations of Sevin affected the vascular cylinder by possibly inhibiting the rate of elongation, therefore, causing the appearance of maturing tracheary elements close to the quiescent center.
Lateral root primordia were similarly affected at higher concentrations of Sevin.

5)
The phloem, endodermis and parenchyma of the vascular cylinder showed no change in appearance in any of the treated plants. 60.