Planta (Ber].) 81, 333--350 (1968)

Investigations on the Growth and Metabolism of Cultured Explants of D a u c u s carota I. E f f e c t s o f I r o n , M o l y b d e n u m a n d M a n g a n e s e on G r o w t h K. H. I~EUMANN* and F. C. STEWARD Laboratory for Cell Physiology, Growth and Development Cornell University, Ithaca, N. Y. Received March 5, 1968

Summary. The effects of Fe, Me and Mn on the growth of explants drawn from different carrot clones were investigated. The explants were cultured aseptically on purified basal media supplemented with coconut milk. The two components of growth, i.e. cell division and cell enlargement, seem to respond differentially to the trace elements in question. Fe plays the key role and acts as a "trigger" of the action of coconut milk in stimulating cell division. Neither Me nor Mn could replace iron in this respect. Me and Mn acting separately tend to foster growth by cell enlargement to a different degree and at different concentrations. However, when Me and Mn were added together to a medium containing iron, they seem to interact and stimulate growth by cell division and cell enlargement still further. The suggestion is made that the behavior of the explants from different clones may have been responsive to differences in the functional Fe/Mn ratio which also involves endogenous levels of trace elements as well as the exogenous levels furnished in the medium. Thus, the element Fe emerges as the key trace element which interacts with the factors present in coconut milk to induce growth in the otherwise quiescent carrot tissue. Introduction I n the controlled g r o w t h of explants f r o m the secondary p h l o e m of carrot root, as practiced in this laboratory, a basal m e d i u m p a t t e r n e d on t h a t of WHITE (1954) has been s u p p l e m e n t e d with coconut milk to secure v e r y rapid proliferative g r o w t h of the carrot tissue. The effectiveness of coconut milk has been a t t r i b u t e d to the so-called a c t i v e fraction * Present address: Justus Liebig-Universit/it, Institut ftir Pflanzenern~hrung, Braugasse 7, Gicssen, Germany. This investigation was made possible by a German-Cornell Exchange Scholarship tenable at Cornell University and awarded by the Deutsche Akadcmische Austauschdienst, Bad Godesberg. After this scholarshi p expired work continued under arrangements made possible by a grant to one of us (F. C. S.) from the National Institutes of Health, Bethesda, Md. The aseptic cultures carried out at Cornell were under the supervision of Mrs. M. O. MarEs and, at Giessen, one of us (K. H. N.) acknowledges the help of Mrs. M. MEINEL. The authors acknowledge the help of K.V.N. RAo in the preparation of the manuscript for publication.

334

K. H. NEVMA~Nand F. C. STEWARD:

with which the neutral fraction is synergistic and their combined effects being accentuated b y casein hydrolysate (SHA•TZ and STeWArD, 1952, 1964 ; POLLAI~Det al. 1961). Although several constituents of the "active" fraction as it occurs in coconut milk, or the equivalent materials from Aesculus and Zea, are known (SH~TZ and STEWARD, 1964), the full composition of the active fraction still remains to be disclosed. I t is, however, known t h a t some active constituents interact with indoleacetic acid, while others interact with myo-inositol and the effectiveness of the whole system is increased b y casein hydrolysate (SKANTZ and STEWARD, 1967; SHA~Z et al., 1967). For this reason whole coconut milk, i.e. the liquid endosperm of Cocos nuci/era, which is the nutrient for an immature embryo, is still a desirable supplement to a medium in which either proliferative or organized growth (STEwAgD, 1958; STEWAUD, MA~'ES et al., 1958) is to be achieved. The growth induced in carrot root explants by coconut milk arises from stimuli to cell division, but as cells grow they also enlarge, especially in the later stages of the typical sigmoid growth curves of the explanted tissue. Thus, early in the time-growth curve of an explant the average cell size tends to decrease and the cells remain small, later it may increase (STEWARD et al., 1952). Also, it is now known that the growth induction stimulus affects virtually all of the cytoplasmic organelles (IsxaAEL and ST~WAaD, 1966). A main. feature of the coconut-milk-induced growth is a stimulus to protein synthesis and turnover (STEWARD, BIDWELL et al., 1958; ]~IDWELL et al., 1964). Since Mo and Mn play such notable roles in nitrogen metabolism (NICHOLAS, 1961), it was important to know their bearing, if any, on the coconut milk effect. The element Fe was also investigated. The reason for this was as follows. Prior work had shown (STEwArD et al., 1961) that the tissue subjected to the coconut milk stimulus was very sensitive to cyanide and carbon monoxide at the stage of growth induction when the constituents of the coconut milk initiate the cell division. Subsequently the effect of these respiratory inhibitors was different in both ldnd and degree. Thus the growth induction stimulus which causes quiescent ceils to divide must work at sites at which protein synthesis is stimulated, respiration is affected, nucleic acids are rendered effective in protein synthesis, and at sites which are also vulnerable to cyanide and carbon monoxide (which act upon iron-containing groups) and also to gamma-irradiation (S~EWA~D et al., 1961). For these reasons, particular attention to the relationships of the coconut milk stimulus to Fe, as well as to Mo and Mn, became necessary. Although these papers relate primarily to the role of the micronutrients (Fe, Mo, and Mn) on the stimulus to growth induction of carrot ex-

Growth and Metabolism of Explants of Daucus carota. I. Growth

335

plants, the effects of other inorganic n u t r i e n t s , especially K a n d Ca, have been e x t e n s i v e l y investigated. This paper, therefore, records experiments to d e m o n s t r a t e the effectiveness, or otherwise, of the active agents i n the coconut milk when tested i n media l i m i t i n g i n Fe, Mo, and Mn, a n d also i n media lacking these trace elements. Also, the three trace elements have been added, singly a n d i n c o m b i n a t i o n , to such media so t h a t their respective effects on growth i n t e r m s of fresh weight, cell n u m b e r a n d cell size could be determined. Of the other k n o w n trace elements boron a n d zinc were consciously supplied i n the m e d i u m . I n the few experiments i n which copper was rendered limiting, its effects were small. Therefore, effects on growth due to iron, m o l y b d e n u m a n d m a n g a n e s e are first described; a later paper deals with their effects on m e t a b o h s m .

Materials and Methods The aseptic procedures for the culture of 2-rag explants of carrot root were those previously used (STEweD et al., 1952). To render the basal medium limiting in Fe, Mo and Mn, it was first prepared in the usual manner (WroTE, 1954) from salts (analytical reagents) in which the content of these elements was small, but the salts of the elements in question were omitted from the stock solution. One liter of inorganic basal stock solution, so prepared, was then freed from heavy metals using a method in which they were removed by co-precipitation with copper sulphide, following a general procedure described by HEWTTT(1952). AS the work progressed, experience of the removal of traces of heavy metals from the constituents of the basal medium was gained. Ix the outcome much work was done with the basal medium in a form in which the trace elements were only present in growth-limiting amounts (this basal medium is here designated B*). Later work produced basal media from which heavy metals were even more rigorously removed (designated B**). The procedures adopted for the most rigorous purification of the basal medium were as follows: 1. The basal medium, prepared without Fe and Mn, was adjusted to pI-[ 2.8; 5 ml of 20% CuSO4 was added to one liter of medium; this was shaken and then brought to boil. 2. tt~S, freshly generated in a Kipp's apparatus, was passed through the solution for 30 min. The solution was allowed to stand overnight after which it was filtered until clear through two layers of filter paper (Sehleicher and Sehiill, WeiBbandfilter). The trace elements are in this procedure co-precipitated with copper sulphide. The whole procedure was then repeated, and finally hydrogen sulphide was passed through the solution until no more precipitation of CuS was observed. 3. The purified solution was evaporated by boiling to half its volume, so that all H~S was removed. The solution was then made to its original volume and stored for use in the refrigerator; this highly purified basal stock solution is designated (B**). All glassware and other containers used in the purification procedure and subsequently in the experiments were cleaned with 0.5 % boiling EDTA-Na and subsequently washed several times (4 or 5) with glass-distilled water. The above procedure was the most rigorous used. Earlier methods used shorter time periods of contact between the precipitate and the solution; these procedures were only performed once to obtain the less purified basal medium (B*). It 23

Planta(Berl.), Bd. 81

336

K. H. N E U M A ~

and F. C. STEWARD :

will, therefore, be appreciated t h a t in this paper the convention is to refer to a "trace-element-limited basal m e d i u m " as B* a n d to a "trace-element free basal m e d i u m " as B**. Since t h e inorganic stock solution was used in combination with sucrose certain vitamins to comprise t h e basal medium, the stock sucrose a n d v i t a m i n solutions were also s u b m i t t e d to the purification procedure. (Parenthetically one m a y say t h a t the pharmaceutically pure forms of sucrose a n d vitamins, as used in these experiments, did not contain trace elements in biologically significant amounts). The water distilled from glass after a first distillation was also tested for h e a v y metals b y the dithiozone reagent. Since the growth was to be induced b y coconut milk, representative batches were analyzed for their inorganic nutrients. Typical analyses for the macron-utrients b y the methods of SCEPTER a n d MENGEL (1960) and SCm~REl~ and DELOC~ (1960), for Mo b y SCEAR~ER a n d HSFNEE (1959), a n d other trace elements b y the methods of SCEAv~LSFFEL (1960) are given in Table 1. W h e n the coconut milk was diluted (to one t e n t h b y volume) in the preparation of the final medium its contributions to the concentrations of Fe, Mn, Zn, Cu, Mo a n d B was very small; it was in fact of the order of one t e n t h of the a m o u n t normally contributed b y the basal medium. Therefore, it was not normally necessary to submit the coconut milk to h e a v y metal purification to obtain n u t r i e n t media which were deficient in these elements. Table 1. The inorganic nutrients in coconut-milk (CM), in the basal medium and in

the basal medium ( B) containing 10 % coconut milk Nutrient

Coconut milk

Basal m e d i u m B -~ CM (mg/900 ml) (rag/l)

Sample A Sample B Mean (rag/100 ml) (rag/100 ml) K Ca Mg N P S Fe Mn Zn Cu Mo Co

240.0 33.0 65.0 108.2 37.0 28.0 0.180 0.05 0.007 0.040 0.007 0.002

249.0 20.6 70.0 98.0 43.0 n.d. a 0.26 0.18 0.050 0.030 0.004 0.004

244.5 26.8 67.5 103.2 40.0 28.0 0.22 0.11 0.028 0.035 0.005 0.003

67.8 34.0 35.0 34.8 3.8 93.0 3.00 1.30 0.36 0.00 0.00 0.00

312.3 60.8 102.5 138.0 43.8 121.0 3.22 1.41 0.39 0.04 0.005 0.003

a Not determined. The methods for assessing t h e effects of the t r e a t m e n t s tried on growth were as follows. The initial a n d final fresh weight of the surface-dried explants was determined. The m e a n fresh weight so recorded referred to 9 explants per t r e a t m e n t in 3 tubes. Also, all t r e a t m e n t s were completely duplicated in populations of carrot explants from a t least two carrot roots. S~ANTZ and STEWED (1959) showed t h a t cells of tissue in the basal medium, especially when this is supplemented with casein hydrolysate, gain in fresh weight predominantly b y cell enlargement; whereas the gain in fresh weight of the tissue in the medium supplemented b y coconut milk is

Growth and metabolism of Explants of Daucus carota. I. Growth

337

largely made up of many small cells arising by division. To distinguish between these two responses, the number of cells in an average explant was determined, and then their average cell size could be calculated. The fresh tissue was macerated in a 1:1. mixture of chromic acid (10 % ) and hydrochloric acid (10 % ) for approximately 3 to 4 hours, or until the tissue could be disintegrated by shaking and by shearing it in a syringe. This procedure followed that of Bgowze and RreKLnss (1949) and the earlier anatomical methods of PRIESTLEY and SCOTT.The volume of macerating fluid to tissue was so adjusted that the density of cells in the macerate was approximately constant, and the suitable aliquots were placed on a haemocytometer slide. Usually ten replicate fields were counted and the preferred technique was to photograph the whole haemocytometer field at low magnification for permanent record. Counting could then be done at leisure on a projected image of the negative, often with the aid of an electronic counter. By appropriate standardization and sufficient replicates, the number of cells per explant was reliable to 10%. The general order of magnitude is given as follows for a 2.0 mg original carrot explant contained approximately 20,000 cells which, on the average, weighed approximately 0.1 ~zgm. After about 6 to 8 days of growth, when the tissue is multiplying most rapidly by cell division, there would usually be many cells much smaller than the original ones, and the average cell size consequently falls; subsequently many of these cells enlarge, and the average cell size might increase again until, after 21 days in a complete medium with coconut milk, the cells might even be somewhat larger than the original ones. Whenever it became necessary to assess the content of inorganic elements in the tissue, this was done spectrographically by access to the analytical services in the l~ew York State College of Agriculture (K]~WORT~u 1960).

Experimental Results and Their Interpretation The Puri/ied Basal Medium: E//ects on Growth o/Di//erent Levels o/ Trace Elements. T h e n u t r i t i o n a l t r e a t m e n t s d e s c r i b e d in T a b l e 2 w e r e a p p l i e d d u r i n g 21 d a y s of g r o w t h , a n d t h e r e s u l t a n t d a t a are also c o n t a i n e d in t h e t a b l e . A b a s a l m e d i u m (B**) f r e e d f r o m h e a v y m e t a l s (i. e. l~e, Table 2. The e]]ect o/the puri/ication o/the basal medium (B**) on the growth o]

cultured carrot explants in the presence and absence o/ coconut milk Data in mgm fresh weight per explant and cells in thousands per explant. B** denotes the Basal Medium prepared without added Fe and Mn and Mso purified from traces of heavy metals; B denotes the Basal Medium here without added Fe and Mn but not purified from traces of heavy metals. No.

1 2 3 4 5

Experimental treatment

Original Tissue Basal Medium (B**) B** -[- C~-~ B (omitting Fe, Mn) + CM B** (-kFe, Mn, Mo)a-~CM

Root A

Root B

Fresh weight

Cell number

Fresh weight

Cell number

2.0 8.0 25.0 54.0 150.0

13.3 18.6 50.0 237.0 898.0

2 5 10 30 120

14.4 17.3 22.4 291.2 918.4

a Denotes Fe at 3 ppm; ~ n at 3.6 ppm; Mo at 0.25 ppm. 23*

338

K. H. N E U M A N N and F. C. STEWAI~D"

Mo a n d Mn) gave less growth in the presence of coconut milk t h a n the same m e d i u m with •e, Mo and Mn omitted b u t without special purification; this can be seen b y comparing t r e a t m e n t s 3 and 4 of Table 2. The difference between t r e a t m e n t s 3 and 4 was due to the purification procedure; the difference between t r e a t m e n t 3 and t r e a t m e n t 2 was due to the coconut milk; the difference between 3 and 5 was due to the addiLion of the three trace elements to the purified basal solution in which these elements h a d been rendered limiting for growth. The d a t a clearly show the responses due on the one h a n d to the cell division factors of the coconut milk (cf. Nos. 2 and 3) and on the other to their interactions with different levels of trace elements (cf. Nos. 3, 4 and 5). Thus the purification procedure h a d depleted trace elements w i t h o u t which the cell division stimulus of the coconut milk could n o t act to the f u l l Over and above the points mentioned, the d a t a of Table 2 show another recurring phenomenon, n a m e l y t h a t the carrot clone labelled A seemed to be more responsive to coconut milk in the absence of added iron t h a n was the clone B (cf. t r e a t m e n t No. 3). On the other hand, when all trace elements were present the overall responses were v e r y similar (ef. t r e a t m e n t No. 5). This suggests t h a t there are different aspects of the overall growth induction system which m a y be limiting to different degrees in the different tissue stocks. This is consistent with current knowledge t h a t there are b o t h 3-indoleacetic-acid (IAA) and inositolmediated g r o w t h responses, (SHA~TZ et al. 1967), and t h a t in given carrot roots these two systems m a y be limiting to different degrees. I n other words, the trace elements m a y be involved in these two parts of the system to different degrees (see also discussion on page 346/47). Following the above experiment, the optimal ranges of Fe, Mo and Mn were investigated. The purified basal medium lacking trace elements (i.e. B**) supplemented with coconut milk was then supplemented with trace elements as follows. Each element (Fe, Mo, Mn) was added at varying concentrations to the basal medium B** which otherwise lacked these elements. This tested the specific ability of each trace element to interact with the coconut milk. These trace elements were then tested as additives to the purified medium B** in combinations taken two at a time (i.e. Fe and No; Fe and Mn; Mn and No). In doing this, one element was present in fixed amount, while the other varied in concentration. This was done for all the possible combinations. When all three trace elements were present, two of the elements being tested (e.g. Fe and No) were added in fixed amount, while the third element (in this case Mn) was varied over a wide range. This was also done for all the possible combinations. The entire b o d y of d a t a involving m a n y experiments which were repeated using cxplants from different carrot roots and with different cultures in different seasons and different years cannot be given in full. Nevertheless, out of this work there emerged the following concentration

Growth and Metabolism of Explants of Daucus carota. I. Growth

339

ranges for the trace elements in question although, within these limits, explants from individual carrot roots varied in their requirements somewhat. Additional to the purified medium (B**) plus coconut milk: Iron should be added at 3.0 to 30.0ppm (either as tartrate or EDTA-complex). Molybdenum should be added at 0.025 to 0.25 ppm 1 (as Na2MoOd). Manganese should be added at 3.6 to 36ppm (either as sulfate or EDTA-complex). As will be shown later, the optimum concentration of Fe for growth of the carrot explants, as stimulated by coconut milk, could be determined in the relative absence of Mo and Mn, though the total response caused by Fe was increased due to the addition of Mo and Mn. On the other hand, Mo and Mn gave no appreciable response when added to the purified medium (B* or B**) if iron was not also added. The key element, therefore, in all these coconut milk induced growth responses was Fe, although this also interacted with Mn and Mo. I t is interesting to note, however, that the basal medium (B) as commonly prepared with its stated content of Fe and Mn and its content of Mo as an impurity is quite adequate to elicit the full effect of the coconut milk on the growth of carrot tissue. Even if one increased the level of nutrients in the basal medium still further, by adding even more of the inorganic nutrients present in coconut milk, no appreciable further increase in growth was observed. Therefore, the inorganic content of the conventional basal medium (B) adequately supported all the growth that the growth factors of coconut milk could induce (see also STEWARDet al., 1964). Interactions o / I r o n , Molybdenum and Manganese i n the Coconut M i l k Induced Growth o/Carrot Explants. The data are to be found in Tables 3, Table 3. The interactions o] Fe (at 3.0 ppm as EDTA-Fe), Mn (at 3.6 ppm as MnSOa) and Mo (at 0.25 ppm as Na 2 MoOn) on the responses o/carrot explants to a puriiied basal medium (B**) supplemented by coconut milk (CM) All data in mg per explant after 21 days growth for each of 3 sets of carrot explants from roots A, B and C. No. Supplement None to basal medium A B (B**) Treatment 1

B**q-CM

2

B**q-CM q-Fe

5

Mn C

8

A

7

120 103 118

3/[o B

6

17

C

13

165 112 148

A

--

Mn q- Mo B

22

C

10

186 121 129

A

B

8

C

8

8

205 111 130

1 The convention here adopted is parts per million (ppm), which is equivalent to milligrams per liter (rag/l).

340

K. H. N E V M A ~ a n d F. C. STEWARD :

4 a n d 5 a n d i n F i g s . 1 a n d 2. T a b l e 3 c o n t a i n s d a t a f r o m t h r e e e x p e r i m e n t s i n w h i c h t h e i n f l u e n c e s of F e , M o a n d M n w e r e t e s t e d . T h e e l e m e n t s w e r e a d d e d a t t h e f i x e d l e v e l s s t a t e d i n t h e c a p t i o n of t h e t a b l e . I n all t h r e e e x p e r i m e n t s (A, B a n d C) a v e r y s t r o n g i n f l u e n c e of i r o n a d d e d t o t h e p u r i f i e d t r a c e e l e m e n t - f l e e m e d i u m ( B * * ) o n t h e e f f e c t i v e n e s s of t h e Table 4. The growth o/carrot explants milk in a purl]led basal medium (B**) Mo (at 0.25 ppm as Na2Mo04) I n Expt. 4 a all data in rag fresh cells • 1000 per explant. E x p t . No.

after 21 days in culture as a]/ected by coconut and with added Fe (at 3.0 ppm as E D T A - F e ) , and M n (at 0.36 to 36 as ppm MnS04) weight per explant; in Expt. 4 b all data in

Treatraent

Supplement to basal medium (B**) None

4a

4b

1

B**

2

3 4 5

B** ~ CM B** ~ CM + 0.36 ppra Mn B** ~- CM ~ 3.6 p p m Mn B * * - k C M + 3 6 ppra Mn

8

1

B**

2 3 4 5

B** B** B** B**

-k CM -k CM + 0.36 p p m Mn -k CM + 3.6 ppra Mn q- CM + 36 p p m l~n

Fe

Mo

Fe ~ Mo --

--

--

25 -18 --

94 131 150 223

20 -24 --

18.6

--

--

--

50.0 -78.6 --

650.0 585.6 742.0 900.0

68.6 -68.3 --

580.8 672.0 951.2 1029.6

129 142 175 190

Table 5. The growth o] carrot explants as affected by coconut milk in a purified basal medium (B**) and with added Fe (at 3.0 ppm as E D T A - F e ) , M n (at 3.6 ppm as MnSOa) and Mo (at 0.025 to 2.5 ppm as Na~Mo04) I n Expt. 5 a all data in mg fl'esh weight per explant; in Expt. 5 b all data in cells • 1000 per explant. Culture period 21 days. Expt. No.

5a

5b

Treatment

1 2 3 4 5

B** B** B** B** B**

1

B**

2 3 4 5

B** B** B** B**

+ + + +

CM CM + 0.025 p p m Mo C ~ -~ 0.25 p p m Mo C1~ + 2.5 ppra Mo

+ C1~ + CM + 0.025 p p m Mo + C1~ + 0.25 p p m Mo q - C M + 2 . 5 p p m Mo

Suppleraent to basal medium (B**) None

Fe

M_n

Fe + Mn

8 25 -20 --

94 93 152 76

18 -24 --

146 128 130 150

18.6 50.0 -68.6 --

784.0 400.0 486.0 294.4

78.6 -68.3 --

598.0 675.0 873.6 898.0

Growth and Metabolism of Explants of Daucus carota. I. Growth

341

coconut milk was detected. I n fact the tissues in the media without added Fe were virtually unable to respond to the cell-division-inducing effect of the coconut milk. Neither Mn nor Mo could substitute for Fe in this respect. I n other words, the action of coconut milk in growth induction b y cell division seems to be specifically dependent on a sufficient exogenous supply of iron. The influences of Mn and Mo on the growth as induced b y an Fe coconut milk interaction varied from experiment to experiment and with the stock from which the carrot explants were drawn. For example, the carrot clone "C" responded well to Fe, additionally to Fe plus Mn and to Fe plus Mo, but it did not show any additional response to Fe plus Mo plus Mn. B y contrast, the carrot clone " B " , which also responded clearly to Fe, showed further but small responses to Mn, Mo, and M~ plus Mo. (Later, reference is made to the sources and significance of variations of this type, see p. 346. Based on previous experiences (see above) it was suspected t h a t the variations in the responses of the tissue from the different carrots (e.g.A, B and C of Table 3) to the same treatment m a y have been due to their sensitivity to Mn and Mo. To test this possibility in the experiments of Tables 4 and 5 and Figs. 1 and 2, various concentrations of Mn and Mo were used at a fixed concentration level of Fe. One should distinguish here between the influences of Fe, Mn and Mo on cell division (cf. Table 4b and 5b) and on cell enlargement (el. Figs. 1 and 2); to do this the growth was recorded in terms of fresh weight per explant; cells per explant, and the average size of cells (~gm/cell). The conclusions from Tables 4 and 5 and Figs. 1 and 2 are as follows : 1. The essentiality of Fe for the action of coconut milk as a cell division inducer (cf. Table 3) is confirmed (cf. Tables 4 and 5). 2. Adding Mn to an Fe containing but Mo-free medium tended to cause cell growth (el.Fig. 1), but at the higher concentrations of manganese (36 ppm) cell division was especially stimulated. 3. At constant Fe concentration (3.0 ppm) and various Mn concentrations (0.36 to 36.0 ppm) the further responses to 0.25 p p m Mo, though small, were consistent. The tendency of the further effect due to Mo was to increase cell number and to decrease average cell size at the higher Mn concentrations (Fig. 1). 4. Adding Mo to the Mn-free, Fe-containing medium tended to increase cell enlargement at the expense of cell multiplication (Fig. 2a). The extent of these responses was not dependent upon the Mo concentration above the minimum used. 5. At constant Fe concentrations (3.0 ppm) and various Mo concentrations (0.025 to 2.5 ppm) the further responses due to added Mn (3.6 ppm) were appreciable (Fig. 2). Mn tended to increase cell number

342

K. H. NEVMA~ and F. C. STEWAI~D:

and to decrease cell size at all Me concentrations used. The evident toxicity due to 2.5 ppm Me in the absence of Mn was not evident after adding Mn to the medium. Treatments Original explan~s Grown in Basal Medlum(B**)

Cells in Thousands/expiant IOOO 600 200

20

mgm Fresh Weight/explant 60 I00 140 180

i

E

t

~

i

i

I

i

i

~ugm/cell 0.1

J

0.43

Growth in Basal Medium(B * ~ ) la. +CM + further supplements None +3.6 pprn +3.0pprn +3.0ppm +3.0ppm + 3.0ppm

--1 :Z]

Mn Fe Fe +O.36ppm Mn Fe+ 3.6ppm Mn Fe + 36. ppm Mn

I i i

0.5 0.23 O. 12 0.22 0.23 0.25

Growth in Basal Medium(B**) +CM + 0.25ppm Igo + I b. further supplements ---1

None +3.6ppm Mn +3.Oppm Fe +3.Oppm Fe + 0.36 ppm Mn +3.0ppm Fe+3.S ppm Mn +3.0 pprn Fe + 36. ppm Mn

0.30 0.35 0.22 0.2I

1 f

]

0.18 O. TT

Fig. 1. The effects on growth (mg/explant; cells/explan~ and cell size in ~g/cell) of carrot explants as affected by coconut milk in a purified basal medium (B**) and in l a with added Fe (3.0 ppm) and Mn (at 0.36 to 36.0 ppm) and, in i b, with Me (at 0.25 ppm)

Treatments

Cells in Thousands/explent lOOO 600 200

Original explents Grown in Basal Medium(B**)

i

i

i

J

mgm Fresh Weight/explant 60 [00 140 180 ~ J , , ~ , i t ,

20

i

pgm/cell 0.1 0.43

Growth in Basel Medium ( B * * ) 2(:I.+CM + further supplements None +0.025 ppm Mo +3.0 ppm Fe +5.Oppm Fe +O.025ppm Me § Fe +0.25 pprn Me +3.Oppm Fe +2.5pprn Me

0.5 0.3 0.12 0.23

I

Z i I

i

0.31 0.26

I I

Growth in Basal M e d i u m ( B * * } +CM+5.6ppm Mn+ ~b. further supplements None +0.25 ppm Me +3.0 ppm Fe +5.Opprn Fe +O.025ppm Me +3.0 pprn Fe + 0.25ppm Mo +5.Oppm Fe + 2.5 ppm Me

r-Z

i J

0.23 0.35 0.24 023 0.15 037

Fig. 2. The effects on growth (mg/explant; cells/explant and cell size in t~g/cell) of carrot explants as affected by coconut milk in a purified basal medium (B**) and in 2a with added Fe (3.0 ppm) and Me (at 0.025 to 2.5 ppm) and, in 2b, with Mn (at 3.6 ppm)

Growth and Metabolism of Explants of Daucus carota. I. Growth

343

The progressive effects due to Fe, Fe plus Mn and Ye plus Ma plus Me when these are added to a purified basal medium (B**) plus coconut milk are conveniently shown in the schematic diagram of Fig. 3 with reference to (a) growth in fresh weight (mg/explant) and (b) in cell number (thousands per exp]ant) and (c) in the size of the explants and cells per explant relative to the original.

E//ects Due to Chelating Agents. The basal medium as normally prepared and when supplemented with both coconut milk and casein hydrolysate usually supports extra growth attributable to casein hydrolysate Initial Explants Fresh Weight _ _Cells per explant (in thousands) 2.0 E~ 13.3 .~ X4 8,0 XI2 25.0"

Xl.5

18.6 X4

B* ~ X 49

94

9

B :~'~ ~'CM Fe + CM e

50.0 X 50.3

B * * + F, + M n § X75

150

X55.8 B*ak+Ft

X87.5

742

~'Mn+Mo+CM X7[.5

175 a

951.2 e

c

b

Fig. 3 a--c. Scheme to summarize the effects of Fe, Mn and Me on (a) growth in fresh weight in mg/explant, (b) cell number in thousands, (e) growth relative to initial explants after 21 days in B** supplemented with CM, Fe, Mn and Me

(SHANTZ and ST]~WAI{D, 1959). While the casein hydrolysate effect is largely due to its role as a source of reduced organic nitrogen, it nevertheless m a y also affect the availability of the metals b y chelation (MARTELL and CALV~, 1958). Another familiar chelating agent (ethylenediamine tetra-acetic acid = EDTA) has found use in the supply of various essential heavy metals to plants in the form of their complexes with t h a t substance. This being so, m a n y experiments were performed to see what effects these chelating agents might have on the growth of the tissue as it was affected b y the trace elements in question. The data will not be reported, but the following ideas emerged. When casein hydrolysate was added to media in which, b y purification, the Fe, Me and Mn levels had been rendered limiting, the growth due to the remaining traces of these elements was actually r e t a r d e d - - p r e sumably because Shcy were less available when complexed than when free. The same result could be obtained by using an excess of EDTA. Nevertheless, when the supply of the trace elements was at the optimum level normally used, the casein hydrolysate increased growth (in its role

K. H. NEUMANNand F. C. STEWARD:

344

as a nitrogen supply), whereas E D T A still depressed it somewhat. Nevertheless, EDTA-Fe or ETDA-Mn (in stoichiometrically equivalent amounts) will serve as the sole source of the element in question. Under these circumstances the E D T A complex dissociated and liberated enough of the metal for it to be ionically absorbed. Since a role of molybdenum is to permit nitrate to be reduced, one might expect that casein hydrolysate could eliminate the requirement for molybdenum. Although in many experiments in presence of casein hydrolysate the tissue failed to respond by growth to added molybdenum, one cannot exclude another role of molybdenum than its effects upon nitrate reduction (cf. S T ~ w ~ ) et al., 1968). Beneficial and Toxic Levels o/ Manganese and Molybdenum: Their Relations to I r o n and to Variation between Carrot Roots. I n the growth of carrot explants, as it is induced by coconut milk added to a basal nutrient medium, variations are observed in the responses of explants from different carrot roots from the same stock submitted to the same treatments. This being so, all experiments have commonly been repeated using explants from at least two carrots concurrently. Any clue to the source of this variation between such similar carrot roots would, therefore, be valuable. The data to follow are open to the interpretation that variation in the behavior of tissue from one carrot to another may be a function of their relative sensitivities to Mn and Mo as modified by Fe.

Table 6 shows the optimum and toxic levels for Mo in explants drawn from each of two carrot roots (A and B) in the absence of Mn and using a rigorously purified basal medium (B**). In the basal medium Table 6. The influence o/ various molybdenum concentrations on the coconut milk induced growth of carrot explants from roots A and B, in a purified basal medium (B**) supplemented with Fe (3.0 ppm as EDTA-Fe) but lacking added Mn Data in mg fresh weight per explant after 21 days of growth. No.

Treatment

A

B

1

B** ~- CM

2 3 4

B** A-CM ~-0.025 ppm as Na~MoOt B** ~- CM + 0.25 ppm as Na2MoO 4 B** ~- CM ~- 2.5 ppm as Na~MoO4

72 120 81 59

94 93 152 76

alone (treatment 1) the growth due t o coconut milk was obviously limited b y lack of trace elements (Mn and Mo), but the disparity between the two sets of explants was considerable. When Mo was restored (treatments 2 to 4) both sets of carrot exp!ants responded but reached their highest growth at very different levels of Mo (carrot A at 0.025 ppm;

Growth and Metabolism of Explants of Daucus carota. I. Growth

345

carrot B at 0.25 ppm); thereafter both batches of explants showed Mo toxicity to different degrees. Table 7 shows similar evidence for Mn although for this experiment a basal medium which was less purified (B*) was employed. The content of trace elements that still remained in the medium (B*) permitted the substantial growth shown in the coconut-milk-supplemented medium. Table 7. The influence o / F e (3.0 ppm as Fe-tartrate), Mo (0.25 ppm as Na2MoO~) and various M n concentrations (4.5 to 90 ppm as MnS04) on growth o/carrot explants cultured in a puri/ied basal medium (B*), supplemented with coconut milk ( C M ) /or each o/two sets o/explants/tom roots A and B Data in terms of mg fresh weight per explant after 21 days of growth. No. Treatment

Further Supplement to B* d- CM None A

1 2 3 4

B*-4-C)~ 86 B* ~ CM-]-4.5 ppm Mn as MnSO4 91 B* ~ CM ~- 45 ppm Mn as MnSO4 70 B* ~- CM -{-90 ppm Mn as MnSO4 56

~ Mo

~- Fe d- Mo

B

A

A

B

116 97 44 34

104 118 89 93 72 40 62 20

158 140 177 182

150 148 157 145

B

The behavior of the batches of explants from the two roots (A and B) was somewhat different, as shown by the levels of added Mn that gave the greatest growth, and by the subsequent degree of Mn toxicity. Adding Mo in the absence of Mn increased the growth of explants from clone A more than those from B. Increasing the concentrations of Mn beyond 4.5 ppm seemed to be toxic and progressively decreased the growth in explants of both the clones A and B. The addition of Mo at 0.25 ppm did not counteract the toxic effects due to the higher concentrations of Mn at 45 ppm and 90 ppm. But, the best response in growth due to Fe plus Mo occurred at different levels of Mn in each of the two clones A and B (at 90 ppm of Mn for A; at 45 ppm of Mn for B). The differential response m a y have been due to the different endogenous levels of Fe and Mn in the tissue so that the most favorable Fe/Mn ratio was established by the use of different exogenous concentrations of Mn. Nevertheless the beneficial effect of Fe was again due to its triggering effect on the coconutmilk-mediated cell division. These effects are consistent with the view, stated earlier, that added Fe promotes cell division by coconut milk, while Mo and Mn tend to favor greater cell enlargement. Apparently the explants from different roots have different sensitivities to these elements; this m a y be in part due to (a) their different initial content of trace elements, (b) possible genetic differences in requirement even though

K. H. N~VHAN~ and F. C. STEWAI~D:

346

t h e c a r r o t r o o t s c a m e f r o m t h e s a m e v a r i e t y a n d source ~. Therefore, t h e r e l a t i o n s of different b a t c h e s of c a r r o t e x p l a n t s t o w a r d exogenous t r a c e e l e m e n t s m a y b e a p o t e n t f a c t o r in t h e v a r i a b i l i t y of response to the g r o w t h - i n d u c i n g s u b s t a n c e s of c o c o n u t milk, etc. to which reference has been m a d e . W h e n assessing t h e v a r i a b i h t y in t h e g r o w t h of c a r r o t e x p l a n t s from a given s t o c k of California-grown r o o t s i t was f o u n d t h a t , u n d e r s t a n d a r d conditions, t h e g r o w t h o b t a i n e d f r o m 9 r o o t s (designated A to I) v a r i e d as shown in T a b l e 8. A n a l y s i s were m a d e of t h e m i n e r a l - n u t r i e n t c o n t e n t of t h e original p h l o e m tissue f r o m each of these r o o t s a n d also of t h e c u l t u r e d e x p l a n t s g r o w n f r o m t h e m in a p e r i o d of 28 d a y s . These d a t a are shown in T a b l e 8, w i t h special reference to t h e t r a c e elements F e , Mn, Zn a n d Cu. Table 8. Variability in the growth o/ explants /rom di//erent carrot roots o/the same cultivar ]rom the same source in relation to their mineral content

Carrot root C J E B G tt D F A

Growth (mg/fresh wt. per explant)

ppm initial tissue

final tissue

Fe

Mn

Zn

Cu

Fe

Mn

Zn

Cu

107 109 137 144 172 188 205 212 233

2.4 3.1 5.0 6.5 5.4 4.1 4.0 6.3 5.0

0.9 0.8 1.6 2.4 0.9 0.6 0.8 2.5 0.9

1.3 1.3 1.9 2.0 2.2 1.2 2.2 3.5 1.6

0.4 0.3 0.5 0.5 0.5 0.4 0.6 1.0 0.4

19.7 15.2 13.8 17.2 13.4 13.3 10.7 11.3 13.4

30.3 16.4 27.6 20.5 15.1 9.5 11.3 15.0 15.7

15.0 10.9 8.7 11.0 8.0 6.8 5.5 6.8 6.7

trace trace 0.48 0.47 0.19 0.31 trace trace 0.17

F e , )/In, Zn a n d Cu were p r e s e n t in t h e original tissue a t low levels - v e r y m u c h lower t h a n in t h e c u l t u r e d tissue. F u r t h e r m o r e t h e r a n g e of r e l a t i v e v a r i a t i o n was g r e a t e r f r o m r o o t t o r o o t in t h e initial tissue, especially for F e , t h a n in t h e tissue as cultured. I n o t h e r words, t h e e v i d e n t v a r i a b i l i t y in g r o w t h is m o r e l i k e l y to b e r e l a t e d to t h e initial t r a c e e l e m e n t c o n t e n t of t h e e x p l a n t in which i t was i n d u c e d t h a n t o t h e final c o n t e n t which, in fact, reflects t h e result of t h e g r o w t h which h a d occurred. H o w e v e r , t h e c o n t e n t of P, K , a n d Ca in t h e i n i t i a l e x p l a n t s was f o u n d to be m u c h m o r e u n i f o r m a n d less s u b j e c t to change as t h e tissue grew. W h e r e a s t h e c o n t e n t of t h e t r a c e elements in t h e tissue c h a n g e d 2 In this laboratory clones of carrot explants from a given root are now regarded as being responsive to what is termed growth-inducing System I (mediated by IAA) or System I I (mediated by inositol). It is, therefore, highly suggestive that these differential sensitivities to trace elements may go hand in hand with the predominance of one or the other growth promoting system for the carrot root in question.

Growth and metabolism of Explants of Daucus carota. I. Growth

347

manyfold as a result of the growth induction, the corresponding changes in macronutrients were much smaller. This again suggests that there is a more intimate involvement of Fe and Mn in the growth stimulus mediated b y coconut milk. In general terms, however, the greatest growth occurred in the initial tissue with the highest Fe/Mn ratio (e. g. 5.5 for carrot tissue A of Table 8) and the lowest growth with tissue (C of Table 8) with the lowest Fe/Mn ratio (e.g. 2.6). Between these extremes, however, the data are somewhat scattered, though growth was broadly correlated with the Fe/Mn ratio of the initial explants. (Later reference is to be made to Fe/Mn interactions in Paper I I which is to follow). The basal medium as commonly prepared permits the full effect of the coconut milk stimulus and also allows the tissue as it grows to absorb its full complement of trace elements; the main source of variability in growth due to mineral elements is their content in the initial tissue. The best way at present to allow for this is to adopt the technique of this laboratory by which all tests and growth experiments are routinely carried out on populations of explants derived from one carrot root, although they are habitually replicated by the use of explants from two or more roots. Discussion

Based on the evidence here reported, the following interpretations can now be made. In the building up of fresh weight during growth in the carrot-coconut milk bioassay used, cell enlargement tends to compete with cell division.. The respective contributions of cell division and cell enlargement to the weight of the carrot explants can be influenced by the elements Fe, Mo and Mn, by their individual concentrations, and by their interactions. Fe interacts with factors in the coconut milk and so stimulates cell division that it serves as a "trigger" of the coconut-milk action. Neither Mn nor Mo can act as a substitute for Fe in this respect. Mn and Mo acting separately, however, tend to foster cell growth; but, whereas Mo in the absence of 5In suppresses cell division, Mn in the absence of Mo tends to stimulate it at higher concentrations (36 ppm). These effects, however, occur only alter cell division has first been initiated by the Fe-eoconut milk interaction. The role of Fe in growth induction by coconut milk is strongly reminiscent of the action of cyanide and carbon monoxide which, at the outset, eliminate the "triggering action" of coconut milk on cell division. All these effects (STwwA~D, SHANTZ et al., 1961) therefore must be mediated at sites at which Fe-containing compounds are important.

348

K. H. NEu~A~x and F. C. STEWAt~D."

Even though the effect of Mn and Mo added separately to an Fecoconut milk-containing basal medium (B**) m a y foster cell enlargement, when added together they interact to stimulate cell division somewhat and to an extent which depends upon the concentrations used. Since Fe interacts with t h a t part of the coconut milk system which stimulates cell division and thereafter Mn and Mo separately and in combination have supplementary effects, there will obviously be concentrations of these three nutrient elements which together keep the two aspects of growth b y cell division and cell enlargement in the best balance. By selecting the best concentration of each element in the presence of the other two (cf. Figs. 1 and 2 and Tables 4 and 5), one can arrive at the following minimum levels at which good growth without toxicity is achieved: Iron at 3.0 p p m Manganese at 3.6 p p m Molybdenum at 0.25 p p m or less. Depending somewhat upon the carrot stock, even higher levels of manganese and molybdenum can, however, be tolerated (see above). The variation of explants from different carrot roots in their res. ponse to the same treatment seems to be more connected with the endogenous properties, including the Fe/Mn ratio, of the initial explants than with the micro-nutrient content of cultures at the end of the experiment. Now t h a t carrot cells can be kept in suspension culture, there are obvious ways to overcome the inherent variability of the freshly cut explants from carrot roots. These involve the possible use of clones of cells maintained in, or adjusted to, culture media of strictly regulated composition with respect to the trace elements. This paper, therefore, establishes the broad requirements of cultured carrot tissue for the principal trace elements and it anticipates the need to understand how the elements in question affect the metabolism of the tissue even as t h e y are here shown to affect the growth induced b y coconut milk. Inasmuch as different clones of explants from different roots react differently to coconut milk, one m a y also anticipate t h a t further work m a y be needed to ascertain whether each and all of the trace elements are equally, or indiscriminately, involved in the contribution to the whole coconut milk stimulus which is made up b y its different growth regulating moieties. These are tasks for the future. Refereilces BIDW~LL, R. G. S., R. A. B ~ , and F. C. STeWarD: Protein synthesis and turnover in cultured plant tissue : Sources of carbon for synthesis and the fate of the

protein breakdown products. Nature (Loud.) 208, 367--873 (1964).

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349

BROW~, R., and P. I~ICKLESS: A new method for the study of cell division and cell extension with some preliminary observations on the effect of temperature and of nutrients. Proc. roy. Soc. B 186, 110--125 (1949). HEWITT, E. J. : Sand and water culture methods used in the study of plant nutrition. Tech. Comm. No. 22, Commonw. Bur. Hort. and Plantation. Reading: Bradley & Son. 1952. ISREAEL, H. W., and F. C. STEWARD: The fine structure of quiescent and growing carrot cells: Its relation to growth induction. Ann. Bot., N. S. 30, 63--79 (1966). KE~NWO~THY, A. L. : Photoelectric spectrometer analysis of plant materials. Proc. Counc. on Fertilizer Application. 1960, p. 39--50 (1960). MA~TELL, A. E., u. M. CALVIN: Die Chemic der Metallchelatverbindungen. Transl. by H. SPECKEm Weinheim: Verlag Chemic GmbH 1958. NICHOLAS, D. J. D. : Minor mineral nutrients. Ann. Rev. Plant Physiol. 12, 63--90 (1961). POLLARD, J . K . , E . M . S~NTZ, and F. C. STEWARD: Hexitels in coconut milk: Their role in nurture of dividing cells. Plant Physiol. 36, 492--501 (1961). SCH~RE~, K., u. I-I. W. D]~LOC~: Zur Bestimmung kleinster Schwefelmengen in biochemischcn Substanzen. Z. Tierphysiol., Tierern~hr. Fnttermittelk. 15, 67--76 (1960). - - , u. W. H S ~ E R : Kationenabtrennung durch Ionenaustauscher bei der photometrischen Molybd~nbestimmung mit Dithiol. Z. Pflanzenern~hr. Diing. Bodenk. 86, 49--56 (1959). - - , u. K. MENGEL: Die Bestimmung yon K, Na, Ca, Mg und P in physiologischen Fliissigkeiten. Z. Tierphysiol., Tiererni~hr. Futtermittelk. 15, 1--17 (1960). SCm~U~L6FFEL, E. : ~ b e r die colorimetrische Bestimmung der Mikron~hrstoffe Kupfer, Zink, Kobalt, Mangan, Eisen und Molybdi~n aus einer Asehenl6sung dutch fraktionierte Extraktion. Landwirtsch. Forsch. 13, 278--286 (1960). SHANTZ, E. M., and F. C. STEWED : The growth-promoting substances in coconut milk. J. Amer. chem. Soc. 74, 6 1 3 3 ~ 1 3 5 (1952). - - - - Investigations on growth and metabolism of plant cells. VII. Sources of nitrogen for tissues cultured under optimal conditions for their growth. Ann. Bot. (Lond.) 23, 371--390 (1959). - - - - Growth-promoting substances from the environment of the embryo. II. The growth-stimulating complexes of coconut milk, corn and Aesculus. In: l~6gulateurs Naturels de la Croissance v6g6tale (J. P. NITSCH ed.), p. 59--75. Paris: Centre National de la Recherche Scientifique 1964. - - A growth substance from the vesicular embryo sac of Aesculus. Paper read at VI Internat. Congr. on Plant Growth Substances, held at Carleton University, Ottawa, 1967. - - M. SUGII, and F. C. STY.WARD: The interaction of cell division factors with myoinositol and their effect on cultured carrot tissue. Ann. N. Aead. Sci. 144, 335--356 (1967). STEWED, F. C. : Growth and organized development of cultured cells. III. Interpretation of the growth from free cell to carrot plant. Amer. J. Bot. 45, 709-713 (1958). -R. G. S. B I D W E L L , and E. W. YEMM: Nitrogen metabolism, respiration and growth of cultured plant tissue. J. exp. Bot. 9, 11--51 (1958). - - S. M. C~VLIN, and F. K. MILLAI~: Investigation on growth and metabolism of plant cells. I. New techniques for the investigation of metabolism, nutrition and growth in undifferentiated cells. Ann. Bot., N. S. 16, 57--77 (1952).

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STEWARD F. C., M. O. MAPES, and K. MEARs : Growth and organized development of cultured cells. II. Organization in cultures grown from ferley suspended cells. Amer. J. Bot. 45, 705--708 (1958). - - - - and J. SMIT~ : Growth and organized development of cultured cells. I. Growth and division of freely suspended cells. Amer. J. Bot. 45, 693--703 (1958). - - K . H. NEUMAWN, and K. V. N. RAo: Investigations on the growth and metabolism of cultured explants of Daucus carota. II. Effects of iron, molybdenum and manganese on metabolism. Planta (Berl.) 81, 351--371 (1968). - - and E. M. S~ANTZ: The growth of carrot tissue explants and its relation to the growth factors in coconut milk. II. The growth-promoting properties of coconut milk for plant tissue cultures. Ann6e biol. 30, 399--410 (1959). - - , with E. M. S~A~TZ, M. O. M~ES, A. E. KE~T, and R. D. HOLSTEN: The growthpromoting substances from the environment of the embryo. I. The criteria and measurement of growth-promoting activity and the responses induced. In: R~gulateurs Naturels de la Croissance v~g~tale (J. P. NITSCH, ed), p. 45--58. Paris: CNRS. 1964. - - - - J. K. POLLARD, M. O. MAPES, and J. MITRA: Growth induction in explanted cells and tissues: Metabolic and morphogenetic manifestations. In: Molecular and cellular synthesis (D. RUDNICK, ed), p. 193--246. New York: Ronald 1961. W~IITE, P. R. : The cultivation of animal and plant cells. New York: Ronald 1954. Prof. F. C. STEWARD Laboratory of Cell Physiology Growth and Development, Cornell University, 252 Clark Hall, Ithaca, N.Y. 14850, U.S.A.