BIOLOGIA PLANTARUM (PRAHA) 8 (6) : 427--430, 1966

Position and Extent of the Elongation Zone in the Tip of the Broad Bean L.

ALENA ADA_MKOVX and KAREL BENE~ Institute of Experimental Botany, Czechoslovak Academy of Sciences, Praha*

Received November 9, 1965

Abstract. It was found by measuring the length of the cortex cells of the root tips of the broad bean Vieia faba L. that the beginning of the elongation zone lies at about 1--2 ram from the initials and its end at about 7--8 mm fl'om the initials. Shrinkage of the object during micro- technical treatment was negligible. The autonomy of the individual tissues of the root tip was taken into account. Introduetion Biochemical investigation of anatomically strictly defined parts of the root tip has become a common method for studying cell growth and tissue differ- antiation (BRow~r and BROADBENT 1951, I~OBINSO~r and BROW~r 1952). The results of the present work should serve as an anatomical basis for a series of papers dealing with the biochemical investigation of growth and differenti- ation processes in the bean root tip. The beginning and end of the elongation zone should be established here.

Material and Methods Seeds of the broad bean Vicia faba L. cultivar Chlumeck~ were sterilized for 4 min with 70 % ethanol, washed for 8 h in running tap water and left to swell for 12 h in distilled water at 26 ~ C. The seeds were then placed onto wet paper laid on PVC plates with openings so that they lay above the opening. The plates with seeds were placed in jars with distilled water at the bottom. Thus, the were grown in the dark for 3 days at 26 ~ C. The tips of selected 3 cm roots were fixed with N~mec's fixative (chromic - potassium bichrom- ate-; N~EC et al. 1962). The water-washed objects were de- hydrated in a series with tertiary butanol, embedded in paraffin and sectioned lon- gitudinally in 8--10 B sections. The sections were treated in one of the following two ways: (1) Inverse staining with gentian violet (N~EC 1906, PRAT 1945)*; (2) PAS reaction (periodic acid- Schiff) according to McManus, as described by PEARSE (1960), Schiff's reagnet was prepared according to RAFALKO (1946)**.

* Address: Praha 6 -- Dcjviee, Flemingovo 2.

427 428 ALENA AD/~MKOV/~, K. BENE~

Material was studied from 4 different sewings (i.e. independent runs). One median section was selected from each of 20 different roots. Measurement was carried out with a Meopta (objective 20 • movable measuring eyepiece 15 • ). In the present arrangement, 1 division of the eyepiece micro- meter i.e. one revolution of the eyepiece microscrew corresponded to 36 ~, the diameter of the field to 450 ~. The zero point for measurement was the position of the initials, i.e. the boundary between the central part of the cap and the root body proper, characterized, among other things, by a change in the direction of the longitudinal cell rows (the "knee", yon GUTENBERG et al. 1955). The length of cells in the central part of the cortex was measured.*** We used the procedure of OeATRN~-FI~EROV), (1964). The sufficient number of measured cells for one field was estimated for a randomly selected root in the 8th field of vision (where, on the basis of preliminary observation, the length of cells showed greatest variability and which appears to lie in the middle of the ascend- ing part of the curve; see Fig. 1) by examining the dependence of the standard error on the number of cells measured. It was shown that it is sufficient to measure 15 cells in the given field of vision. In this manner we calculated the average length of cells in a given (the 8th) field of vision for all 20 roots. By analogy, following the dependence of the standard error on the number of roots measured, it was found that for calculating the average length of cells in a given field of vision 15 roots are sufficient to take into consideration. The average length of the whole central part of the cap was calculated from values found in the 20 roots measured. Results The results of our measurements are shown in Fig. 1. The elongation zone begins about 1--2 mm from the initials, and ends about 7--8 mm from the initials. The average length of the central part of the cap is 581 ~ 3.30 F. 50

CELL 40. .

LENGTH ./,//~'. . . !

30"

20"

10"

D/STANCE FROM INITIALS (MM)

Fig. 1. Dependenceof the mean cell length in the central part of the cortex in the root tip of the broad bean Viciafaba L. on their distance from the initials. ELONGATION ZONE IN THE ROOT TIP 429

Discussion The root tip of the broad bean has been studied from a similar point of view by WAGNER (1937) and recently by GRAY and SCHOL~S (1951), HOLMES and co-workers (1955) and JENSEI~ (1955). Even if the position of the cells measured is reported, either with respect to the physical tip of the roots or with respect to the initials, the results are mutually comparable, the data differing by the length of the central part of the cap. WAONER (1937) reports the length of the rhizodermal cells alone up to 3.5 mm from the tip. Similarly, J~NSE~r (1955) examined only the first 3 mm of the root tip. The paper by HOLMES and co- -workers (1955) shows an apparently identical curve of dependence of the length of cells on their distance from the tip, just as that of GRAY and SCHOLES (1951). None of the last-mentioned papers give all the details of the measuring procedure and the processing of the results. The position of the elongation zone found in the present paper generally agrees with the data of GRAY and SCrOLlS (1951) and of HOL~ES et al. (1955). We have determined the end of the elongation zone to lie about 7--8 mm from the initials, but the length of cells is great there and it is difficult to estimate it. At about 2--3 mm from the initials, the cell length obtained in the present work does not differ significantly from the data of the above authors. However 10 mm from the tip, substantially lower values were found than are given by the above authors. The position and range of the elongation zone can be determined (1) accord- ing to Sachs, i.e. by following marks on living roots, but this prevents the estimation of the autonomy of individual tissues (see below), (2) by determining the mitotic index which, however, indicates only the end of the meristematie zone or the beginning of the elongation zone, (3) by measuring the cell length which supplies data only oll the -- sensu stricto -- longitudinal growth and disregards the fact that we are dealing here with volume growth, the growth in width preceding the growth in length, see e.g. JE~CSEN (1960). In the present paper we used only the third-named method, the growth in width not being taken into account. We have only measured cells of the central part of the cortex. It is known that the individual parts of the root tip do not begin and probably do not terminate longitudinal growth simultaneously, i.e. at the same distance from the root tip or from the boundary between the cap and the root body (JENs~N 1955). As far as is known, these differences are not such as to affect the preparation of the material for the above mentioned biochemical analyses when it is necessary to test selected one-millimeter segments. The results reported here were obtained by measuring fixed and paraffin-embedded roots. With material the microtechnieal treatment brings about considerable shrinkage of objects, whereas here the shrinkage is negligible (AD/I.MKOV.&, unpublished). Aeknowledfment We are indebted for stimulating discussions to Drs. HadaSovA, Opatrn~ and Pokorn~.

* When the method was tested in more detail, it was confirmed that is is necessary to mordan t with but that mordanting with tartrate can be omitted. A critical step for the selectivity of staining cell walls is the differentiation in 50% methanol during dehydration. The basis of the method apparently lies in the binding of tannin to a component of the cell wall and the affinity of gentian violet (a mixture of related basic dyes) for tannin. Thus, the inversion staining according to Nfimoc is identical with other methods based on the affinity of tannin for the cell wall, such as Foster's method (see JOHANSE~r 1940) where the bound tanning is revealed by the action of ferric 430 ALENA ADfi_MKOVfi_, K. BENE~ chloride. Bound tannin can be also demonstrated by azo-coupling (DIxoN 1959; it is not chemi- cally clear why DIXON recommends oxidation with periodic acid after mordanting with tannin). The chemical aspects of the binding of tannin are taken up in the paper by HARMS (1959). The question of the affinity of basic dyes for tannin can be considered in connection with the vital staining of "full" vacuoles. ** The PAS reaction was used for morphological studies of plant tissues by yon STosc~ (1955). To apply the PAS reaction to this work was recommended by van DuIJN. *** The individual parts of the meristem and the elongation zone here bear the same names as the corresponding parts of the mature root. When speaking of the root cap we have in mind the central part of the cap (including the columella), as measured from the initials along the longitud- inal axis of the root. References BROWN, R., BROA-DBENT, D.: The development of ceils in the growing zones of the root. --- J. exper. Bet. 1 : 249--263, 1951. DIxoN, K.: Oxidized tannin-azo method for protein in tissues. -- Am. J. Path. 35 : 199--211, 1959. GRAY, L. H., SCHOLES, M. E.: The effect of ionizing radiations on the brood bean root. VIII. Growth rate and histological analyses. -- Brit. J. Radiol. 24 : 82--92, 176--180, 228--236, 285--291, 348--352, 1951. voN GUTTENBERG, H. etal.: Studien tiber die Entwicklung des Wurzelvegetationspunktes der Dikotyledonen II. -- Planta 46 : 179--222, 1955. HARMS, It.: Welchen histotopochemischen Aussagewert besitzt die Tannin -- Eisenreaktion nach Salazar. -- Acta histochem. 8 : 130--I57, 1959. HOLr~ES, B. E. et al.: The nucleic acid content of cells in the meristcmatic, elongation and fully elongated segments of roots of Viciafaba. --- Exp. Cell'Res. 8 : 101--113, I955. JENSEN, W. A.: A morphological and biochemical analysis of the early phases of cellular growth in the root tip of Viciafaba. -- Exp. Cell Res. 8 : 506~522, 1955. JE~SEN, W. A.: Relation of primary cell wall formation to cell development in plants. -- In: RUDNICK, D. (ed.): Synthesis of Molecular and Cellular Structure. Ronald, New York, 1960. JOHANSEN, D. A.: Plant Microtechnique. -- McGraw Hill, New York, 1940. N~MEC, B.: Uber inverse Tinktion. -- Ber. dtsch, bet. Ges. 24 : 528--531, 1906. N~MEC, B. et al.: Botanickd~ mikrotechnika. [Botanical Microtechnique.] N(~SAV, Praha, 1962. OPATRN~-FI~EROVJ~, Z.: Dor Einfluss yon K -- Humat auf Teilung und Litngenwachstum der Wurzelspitzen von Allium cepa L. -- Biol. Plant. 6 : 122--131, 1964. PEARSE, A. G. E.: Histochemistry Theoretical and Applied. -- Churchill, London, 1960. PR.~T, S.: Rostlina pod drobnohledem. [The plant under the microscope.] -- Cs. graf. Unie, Praha, 1945. RAFALKO, J. S. : A modified Feulgen technique for small and diffuse chromatin elements. --- Stain Tochnol. 21 : 91--93, 1946. ROBINSON, E., BROWN, R.: The development of the enzyme complement in growing root cells. -- J. oxper. Bet. 3 : 356--374, 1952. yon STOSC~, H. A.: Zur Darstellung pflanzlichor Meristeme. -- Ztschr. wiss. Mikr. 62 : 305--310, 1955. WAGNER, :N.: Waehstum und Teilung der Meristemzellen in Wurzelspitzen. -- Planta 27: 550--583, 1937.

A. AD.~MKOVJ~, I(. RENEW,~stav experimentAlni botaniky ~SAV, Praha: Poloha a rozsah pro, dlutovaei z6ny v ko~enov~ ~pi~ee koflskdho bobu Vicia faba L. -- Biol. Plant. 8 : 427--430, 1966. M6~enim d6lky bun~k primgtrni kflry ko~enov)%h ~piSek koflsk6ho bobu Viola faba L. bylo zji~t6no, ~o poSg~tek prodlu~ovacf zony levi v oblasti 1 a~ 2 mm od iniciA1 a jeji konec ve vzdAle- nosti 7 a~ 8 mmod inicigd. Smrsknuti objektu p~i mikrotechnick6m zpracovAnl bylo nopatrnd. Byla uvA~ena autonomic jednotliv~ch pletiv ko~enovd ~pi6ky.

A. A)IAMHOBA, H. ]~EIIEIH, HHCT14TyT 31