control and kinetics of branch root formation in segments … · plant physiol. (1972) 50, 35-42...

Plant Physiol. (1972) 50, 35-42

Control and Kinetics of Branch Root Formation in Cultured RootSegments of Haplopappus ravenii

Received for publication October 22, 1971

L. M. BLAKELY, SHIRLEY J. RODAWAY, LAUREN B. HOLLEN, AND SUSAN G. CROKERDepartment of Biological Sciences, California State Polytechnic College,Kellogg- Voorhis, Pomona, California 91768

ABSTRACT

Branch root formation required only the presence of min-erals, sucrose as a carbon source, and an auxin. The numberof primordia formed was a function of auxin concentration.With naphthaleneacetic acid at 0.1 mg/I, up to 60 or morebranches were formed per centimeter of Haplopappus raveniiroot segment. Under our conditions, pea root segments formedonly five or six branches per centimeter, but tomato and radish,like H. ravenii, formed large numbers of branches. Cytokinininhibited branch formation, while gibberellic acid was withouteffect. Vitamins were not required for branch formation, al-though they enhanced elongation. Up to 5 days were requiredfor the maximum number of stable branch primordia to formunder the influence of naphthaleneacetic acid. If naphthalene-acetic acid was withdrawn earlier, fewer branch primordia de-veloped. The requirement for a lengthy exposure to naphtha-leneacetic acid, the kinetics of the response, and the ease withwhich naphthaleneacetic acid could be rinsed out of the tissuewith consequent cessation of branch root formation, were simi-lar to other hormone-regulated developmental systems. Ana-tomical and cytological studies were made of segments exposedfor various times to auxin. The segments were mostly diarch,and branches formed obliquely to protoxylem poles. Whileprimarily only pericycle-endodermis cells divided, both theseand cortex cells responded in the first 24 hours exposure tonaphthaleneacetic acid with enlarged nuclei and nucleoli, anda few cortical cells divided. Maximum nucleus and nucleolussize was reached approximately 9 hours after exposure to naph-thaleneacetic acid. Branches rarely elongated more than 5 cmbefore their meristems died. The H. ravenii culture is main-tained only by the frequent formation of new naphthalene-acetic acid-induced branches.

The initiation of branch roots in cultured root segments ap-pears to be a valuable system for studying organogenesis.Branch root primordia develop from mature cells of the peri-cycle and/or endodermis tissue adjacent to the central vascularcylinder (9). Initiation of primordia is stimulated by auxin inthe appropriate chemical and physical environment: the num-ber of branches that form in a given length of root segmentcan be used as a quantitative index of the effectiveness ofchemicals or other variables in inducing organ formation (19,22). We have recently found, with at least some species, thatcultured root segments are capable of a massive response toauxin, forming up to 60 or more branches per centimeter of

35

segment (2). The kinetics of the response to auxin has beeninvestigated and is comparable to the kinetics of response ofother developmental systems to other hormones. In this paperwe present details of our studies on branch root formation incultured root segments of Haplopappus ravenii.

MATERIALS AND METHODS

Origin and Brief History of the Root Cultures. Seeds ofHaplopappus ravenii Jackson were collected in the ProvidenceMountains of California on June 10, 1964. Identification wasbased on the number and structure of the chromosomes (13)as seen in aceto-orcein squashes of seedling radicles, and later,in cultured tissues. (Studies of the chromosomes of H. raveniicultures will be the subject of a separate communication.) OnApril 4, 1967, internode sections of 3-month-old floweringgreenhouse-grown plants were placed on agar Murashige andSkoog (14) medium, containing 10% coconut milk and 0.5mg/l NAA.1 Abundant formation of adventitious roots on thecallus tissue pieces occurred during the first few months inculture. After 5 months, a subline was initiated by subcultur-ing root tips and short root segments in liquid medium. Bythat time, all roots examined were tetraploid. This subline wasthe one used in the investigations reported herein.

Culture Methods. Stock cultures of roots were maintainedby subculturing root segments at intervals of 3 weeks. Themedium used since shortly after starting the root subline, andin all experiments reported herein except as noted otherwise,contained the minerals formulation of Eriksson (8) with thezinc concentration reduced by a factor of 10, 3% w/v sucrose,0.1 mg/l of NAA, 100 mg/I of myoinositol, 0.2 mg/l of gly-cine, 0.5 mg/l of nicotinamide, 0.5 mg/l of pyridoxine, 0.5mg/l of thiamine, and 0.1 mg/l of p-aminobenzoic acid. Cul-tures were grown in 250-ml Erlenmeyer flasks with 50 ml ofmedium, or in 125-ml Erlenmeyer flasks with 20 ml, on a re-ciprocating shaker (108 strokes/min, stroke amplitude 2 cm)in dim (5 ft-c) light at 25 to 28 C. During the 3-week subcul-ture period, branches formed on inoculated segments, and thebranches extended 2 to 4 cm in length. These branches did notform secondary branches within the 3-week subculture period.Segments (1-cm long) cut from the branches in a 3-week-oldculture were used as inocula, either for subculturing or, afterrinsing with distilled water, in experiments. Three to five seg-ments were used per flask.

Assessment of Results. In most of the experiments reportedhere, the number of branches initiated in a given time intervalwas the response measured. Segments were generally harvested

1 Abbreviations: NAA: ca-naphthalene acetic acid; BA: benzyl-adenine.

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BLAKELY, RODAWAY, HOLLEN, AND CROKER

60-

50i

X 30

co 2 0 -D /Z 10/

O I 1,ix 4 3 -2 -I 0LOG NAA (PPM)

FIG. 1. Effect of NAA concentration on number of branchroot primordia formed by 1-cm cultured root segments of H.ravenii in 5 days. Each point represents the mean, + the standarderror, of nine segments.

and fixed-cleared in formalin-acetic acid-alcohol after 5 days,when all branch primordia had been formed and when theycould be readily counted. Using a stereo dissecting microscope,the length of the segments was measured, and the number ofbranches was determined. There was virtually no change inlength of the root segments during the culture period. Usuallynine to fifteen segments (three to five 125-ml flasks) were usedper treatment, and results were expressed as average numberof branches per centimeter. Standard errors were nearly alwaysconsiderably less than 10% of their associated mean values. AFortran computer program was developed for statistical analy-sis, which provided values for means, SE, F, LSD, and HSD (18).The HSD at the 0.01 % level provided a highly conservative, andthe LSD at 0.05% a more liberal, index by which to assess thesignificance of differences between treatment means.

RESULTS AND DISCUSSION

Response to Auxin. The effect of various concentrations ofNAA on branch root initiation is shown in Figure 1. Thenumber of branch primordia formed per centimeter was pro-portional to the log of the NAA concentration over the rangeof roughly 1 to 100 ,ug of NAA/liter. At a concentration ofapproximately 100 ,ug/l, up to 60 or more branches wereinitiated per centimeter of root section. With the exception ofrare malformed roots (including fasciated roots), all were capa-ble of elongating normally, producing tangled mats that eventu-ally filled the medium. The formation of so many branches percentimeter seemed surprising, since this was an order of mag-nitude greater than that reported by others. When we com-pared the response of other species (Table I), we found com-parable responses by seedling subapical segments of tomatoand radish, but not pea. Much of the work that has been doneon branch initiation in cultured segments (12, 15, 16, 21-23)has been done with pea, which apparently is capable of form-ing only relatively few primordia per centimeter. Most studieson effects of hormones on tomato roots (e.g., 6) have employedroots with intact meristems, used low concentrations of hor-mones, and/or have recorded only "emergent" laterals, leavingunrecorded the number of any primordia that fail to emerge(Pecket, (16) discussed this point some time ago). The resultssummarized in Table I suggested that H. ravenii is not unusualin the number of laterals that can be formed, under the condi-tions used here. In one of the experiments with radish roots itwas noted that primordia were well formed and easily countedat only 2 days after inoculation. However, in contrast to H.

ravenii roots, branches on radish roots failed to elongate ap-preciably when cultured more than 5 days; short (1-2 mm),stubby branches formed, which branched in turn. Elongationof H. ravenii branch roots is apparently less affected by thehigh auxin concentration, although they elongate more rapidlyif transferred to auxin-free medium after 5 days. Several stud-ies (e.g., 15, 22) have shown that more laterals are induced byauxin when the tip is removed. We have investigated this pointwith H. ravenii roots and find that, at least when the concentra-tion of auxin is around 100 ,ug/l, there is no significant effectof the tip. This may be due to the fact that the roots are veryslender (<200 [km, 15-20 cells in diameter), and the amount ofinhibitor from the tip is so small it is simply swamped; or to thefact that when segments are taken from a 3-week-old culture,the meristems have senesced (see later) and are no longer pro-ducing inhibitor. This point, however, deserves further investi-gation.We have done little work with IAA in this system. IAA

elicits responses similar to NAA when used at concentrations10 times higher than NAA. NAA was chosen for its greatereffectiveness.

Effects of Cytokinin and Gibberellin. The initiation ofbranch meristems induced by auxin is readily reversed in H.ravenii root segments by cytokinin (Fig. 2), as is well known inother systems (e.g., 23). BA, either alone or in combinationwith auxin, induced considerable swelling of root segments (to2-3 mm in diameter in 3 weeks) apparently due largely togrowth of cortical cells. In the presence of auxin alone thecortex undergoes little growth and is frequently sloughed off.A recent study (24) with pea root segments revealed a similarresponse. GA had no apparent effect on root initiation, eitherin the presence or absence of NAA (Table II). This is in con-trast to results reported by Brian et al. (5) for root initiation in

Table I. Branlchinig Response of 1-cm Cuiltured Root Segmenlts ofPea, Tomato anid Radish, Compared with H. ravenii

Results (experiment means) of two experiments with subapicalsegments of seedling roots, compared with range of typical ex-perimental means for H. raveiiii under the same conditions. NAAwas used at 0.1 mg,/l.

Species -No. of Branches per Centimeter

Pea 5 to 6Tomato 41 to 48Radish 23 to 57H. raveniii 35 to 60

--

x 80-U

X 60-a.crLa 40-m

Dz 20-

-3 -2 -ILOG BA (PPM)

FIG. 2. Effect of BA concentration on number of branch rootprimordia formed by 2-cm cultured root segments of H. ravenii in7 days. The medium contained 0.2 mg/l of NAA. Each point repre-sents the mean, + the standard error, of 15 segments.

36 Plant Physiol. Vol. 50, 1972



BRANCH ROOT FORMATION

stem cuttings; they concluded that GA inhibited formation ofmeristems in mature stem tissue. Butcher and Street (6), on theother hand, found that GA increased the number of emergentlaterals in tomato root cultures. It would seem, however, thatapplied GA has no effect on the process of branch root initia-tion in H. ravenii root segments.

Response to Vitamins and Other Factors. Vitamins are gen-erally required for the continuous culture of isolated roots (1).Torrey (22) has reported the beneficial effects of vitamins onbranch formation in pea root segments. H. ravenii root seg-ments, however, appear to have no requirement for exogenousvitamins insofar as the formation of branches is concerned (Ta-ble III). It even appears that they branch more abundantlywhen starved of vitamins (Table III). Three sublines of H.ravenii root cultures have now been grown for over a year inmedium lacking any vitamins or other organic constituentsother than sucrose, NAA and EDTA (iron is supplied as FeEDTA). While H. ravenii roots have no exogenous vitamin re-quirements for branch initiation or for prolonged cultivation,

Table II. Effect of Gibberellic Acid on Branch Root Formationt byCultured H. ravenii Root Segments

The gibberellic acid was filter sterilized. Means of 15 segments;LSD (0.05): 5.6.

Mean No. of Branches per Centimeter, (4 SE)GA Conc

No NAA 0.1 mg/l NAA

ng/li0 2.3 i 1.6 32.9 2.80.001 0.7 + 0.2 34.6 i 2.40.01 1.2 + 0.3 35.4 + 2.50.1 0.7 + 0.2 34.6 4 2.51.0 0.6 + 0.3 31.6 i 2.1

10.0 4.2 ± 2.6 28.0 ± 2.6

Table III. Effect of Vitaminis, Glycinie, anld Iniositol oni Branch RootFormationt by Cultured H. raveniii Root Segmenits

Roots Grown Previously In

Alediuml MMedium lackingComplete medium vitamins and glycine,

for three passages

mean no. of branches/cm, i SE4

Experiment 12Complete-autoclaved 39.7 + 2.3 52.1 ± 2.6Complete-vitamins and 36.9 4 2.3 50.2 ± 2.6

glycine filter sterilizedMinus vitamins and gly- 40.1 + 3.4 49.1 i 3.1

cineExperiment 23Complete 39.6 i 2.2 52.9 + 2.3Minus vitamins and gly- 40.2 4 2.2 50.4 ± 3.4

cineMinus inositol 35.9 + 1.6 52.9 + 2.7Minus vitamins, glycine, 39.5 + 3.5 54.5 4- 3.4and inositol

Concentrations of the vitamins (nicotinamide, pyridoxine,thiamine, paraaminobenzoic acid), glycine, and inositol are givenin "Materials and Methods.'"

2 LSD (0.05): 7.8; HSD (0.01): 13.7.3 LSD (0.05): 7.7; HSD (0.01): 14.1.4 Means of 15 segments.

6 FL

50F

0 40

a-u. 30

Z 20

IOF

"0 1 2 3 4DAYS

5 6 7

FIG. 3. Appearance of branch primordia in H. ravenii root seg-ments with time. Segments were harvested and fixed at daily inter-vals through 7 days after inoculation into medium with 0.1 mg/lof NAA. Each point represents the mean, the standard error, ofsix to nine segments (cf. Figs. SA and 6).

addition of the vitamin-glycine mixture to the culture mediumdoes enhance the rate at which laterals elongate (Fig. 9).The components of the vitamin-glycine supplement have not

been tested separately; a matter yet to be decided is whetherthese components interact to mask any possible promotion ofbranch formation by one or more of the components

Light appeared to have little effect on the number of pri-mordia formed per centimeter. The results of one experiment(means of 15 root segments) were, in number of branches percentimeter at 5 days: 38.3 in continuous fluorescent light of 200ft-c; 45.0 in continuous dim light of less than 7 ft-c; and 40.9in complete darkness; the LSD, 0.05, was 6.6. It should benoted, however, that Furuya and Torrey (12) observed thatlight inhibited auxin-induced lateral formation in isolated pearoot segments.The age of the root segments used has an effect on branch

initiation. Three weeks after inoculation of segments, thebranches that have formed are elongated sufficiently (2 to 4cm) and yield segments that develop maximum numbers ofwell formed primordia. Segments taken from older cultures(more than a month after inoculation) yield segments that varymore in response, often developing only few poorly formedprimordia.

Kinetics of Branch Root Initiation. In preliminary experi-ments, it was found that primordia are well formed by 5 days,and that virtually all of them subsequently elongate. On thisbasis, quantitative data on the number of branches may betaken at 5 days. The important steps that lead to branchmeristem formation appear to be completed within the first 5

days after exposure to NAA. In this section we present more

details on the role that auxin plays in the course of develop-ment during the first 5 days.The results of an experiment in which the daily course of

branch root formation was followed in root segments continu-ously exposed to NAA are given in Figure 3. Representativeroot segments are pictured in Figure 5A. Cytological and ana-

tomical observations are treated in the following section. Dur-ing the first 24 hr after inoculation the only changes visiblewith the dissecting scope were a slight swelling and darkeningin the cortex. During the next 24 hr. localized thickenings

F

Plant Physiol. Vol. 50, 1972 37

I I

f v



BLAKELY, RODAWAY, HOLLEN, AND CROKER

60

4030

2 200

U.j 1 0o-8x6

az4

z 3

2

0 1 2 3 4 5DAYS

FIG. 4. Effect of length of time of exposure to NAA on forma-tion of branch root primordia in H. ravenii root segments. All seg-ments were harvested and fixed after 5 days of culture. Segmentswere exposed to 0.1 mg/l of NAA for the number of days indi-cated, then washed in distilled water and transferred to mediumlacking NAA for the remainder of the 5-day period. Each pointrepresents the mean, + the standard error, of 12 to 15 segments(cf. Fig. SB).

(incipient primordia) began to develop on the margin of thevascular cylinder. These were rather ill defined, and root toroot variation in their number was large. At the 3rd day, therewere more incipient primordia which tended to be more distinctbut still quite small. By day 4 the primordia were larger, on theaverage, and the total number was essentially at the maximum.The primordia were quite distinct by 5 days, although they hadnot yet extended beyond the surface of the swollen and some-what ruptured cortex. By the 6th or 7th day, most primordiabegan extending through the surface. The four points betweenday 1 and day 4 (Fig. 3) fell on a straight line when plotted onarithmetic graph paper.The linear nature of the appearance of primordia with time

possibly reflects interactions between rates of development (toa visible, countable size) of individual primordia and variationin initiation time.

It was of interest to us to determine the minimum length oftime it would be necessary to expose the root segments to NAAin order to elicit branch root formation. Accordingly, an ex-periment was set up at the same time as the one describedabove, using segments from the same batch of roots. In thisparallel experiment, roots were exposed to NAA for varyinglengths of time (zero, 1, 2, 3, 4, and 5 days) and then trans-ferred after thorough washing in distilled water (except thezero- and 5-day roots) to medium lacking NAA. All wereharvested and fixed at the end of 5 days of culture. Primordiaand branches were counted; the data are given in Figure 4,and representative segments are pictured in Figure SB. Therewas no stimulation of branching as a result of 24 hr exposureto NAA, followed by 4 days in the basal medium. The forma-tion of 1 to 3 primordia or branches (many of which grew outbeyond the cortical surface) per centimeter, and the swellingevident in the photographs, was similar to the behavior of seg-ments cultured for 5 days on the basal medium without NAAtreatment. Two days of exposure to NAA, followed by 3 daysin basal, resulted in a doubling of the number of primordia.Three days in NAA, followed by 2 days in basal, produced an

approximately 10-fold increase in the number of branches. Fourdays in NAA, followed by 1 day in basal, elicited a nearlyoptimal formation of well developed primordia, althoughsomewhat less (statistically significant at the 97.5% level) innumber per centimeter compared to those formed after 5 daysin NAA. The points for days 1 through 4 fell on a straight linewhen plotted on semilogarithmic paper (Fig. 4); i.e., the num-ber per centimeter increased exponentially rather than arithmet-ically with time of exposure to NAA.We interpret these results to indicate that the hormone must

be present not only during the earliest phases of primordiainitiation, but also up to the time that the primordia are suffi-ciently well developed to be self sustaining or stabilized. Ifthe hormone is removed at some earlier time, the incipientprimordia seem incapable of further organized development.The exponential nature of the response is what one wouldexpect of a population of developing primordia randomly vary-ing in the time, about some mean, at which stabilization isachieved. A bell-shaped curve is obtained if one plots the dailyincrement in number of branches per centimeter versius daily

IIs.i

I

Ie

F1

.4

.%.

".

I'l

i

rFIG. 5. Photographs of cultured root segments. A: Roots har-

vested and fixed at daily intervals; left to right, 0 through 7 days.These are representative segments from the experiment summarizedin Figure 3. B: Roots harvested and fixed after 5 days of culture;left to right, 0 to 5 days exposure to NAA followed by, respectively,5 to 0 days in NAA-free medium. These are representative seg-ments from the experiment summarized in Figure 4. The bar repre-sents 1 mm.

I


1

IL




interval, with the peak occurring during the 3- to 4-day inter-val.To test the possibility that some substance essential for

branch development other than NAA might have been lostduring the rinsing process, a similar experiment was performed,but this time, after rinsing, some roots were transferred tofresh NAA medium, while others were transferred to basalmedium as before. The results (Table IV) clearly implicatedNAA as the essential substance, and again showed the expo-nential kinetics.

Thus, in this developmental system, the formation of branchroots requires the presence of auxin for up to 5 days for maxi-mum response. Even though visible signs (cf. Figs. 5 and 6) ofbranch formation became evident after 2 days, organizedgrowth failed to continue or was much reduced if the hormonewas removed. Incipient primordia that failed to develop whenNAA was removed appeared to be lost in a more uniformswelling of the vascular cylinder region.

Earlier studies with branch root systems tended to indicatethat at least 2 to 3 days (21, 22) up to an undetermined numberbut more than 6 days (3) exposure to auxin was required formaximum response. In those studies relatively thick roots were

used, and they were apparently not washed when transferredfrom auxin to auxin-free medium, increasing the possibility ofauxin carryover. In one of those studies (3), interpretation was

complicated by the concomittant formation of buds, whichmay have affected branch root formation. Our findings confirma conclusion that can be drawn from those earlier studies (thata relatively prolonged exposure to auxin seemed necessary),and pinpoint the time requirements perhaps more accurately(at least for H. ravenii roots). They further extend the degreeof analysis to include a description of the kinetics of the re-

sponse, and also indicate that organ initiation could begin butcome to a halt if initiation had not gone far enough in thepresence of the hormone.

Very similar results, with a different developmental systemand a different hormone, were recently presented by Brandesand Kende (4). Formation of stable buds on moss protonemataby a cytokinin hormone required the hormone's presence for 3days. Although buds began to form before 3 days, many ofthem dedifferentiated if the hormone was removed early. Thenumber of buds that became stabilized progressed exponentiallywith time (as did H. ravenii branch root formation). As theauthors pointed out, the cytokinin's mode of action in thatsystem involves more than simply acting as a "trigger." Fromour results, we may also say that the mode of action of theauxin-type hormone NAA, acting in the branch root system, islikewise not only that of a trigger. It may be acting to turn ona "master switch" which needs to be held in the "on" position(i.e., a switch that returns to the "off" position when released).Obviously, in branch root formation, auxin must be continu-ously present until meristems have reached some critical stageof development. The fact that the hormone can be readilyrinsed out of the root tissues indicates that it is neither tightlybound to its site of action nor confined within a highly im-permeable membrane.

Similar results, indicating a requirement for the continuouspresence of a hormone during tissue response, have recentlybeen presented for the role of ethylene in abscission promotion(10), gibberellin in amylase production (7), and auxin in cellelongation (11).

Anatomical and Cytological Studies on Branch Root Initia-tion. Sections 10 ,um thick, from roots harvested after zero, 1,2, 3, and 4 days in NAA medium, are shown in Figure 6. Rootswere typically diarch (branches usually formed in two rows),occasionally triarch.

Table IV. Effect ont Branichinig of Washinig H. raveili Cultured RootSegmentts Exposedfor Various Times to Medium Conttaininig NAAAfter 1 to 4 days in the original medium containing 0.1 mg/I

NAA, segments were washed thoroughly in distilled water andtransferred to fresh medium with or without 0.1 mg/l NAA. Allsegments were harvested after a total of 5 days. Segments culturedfor 5 days on either the original NAA medium, or on mediumlacking NAA, formed respectively, 52.7 + 2.4 and 4.0 i 0.7branches per centimeter (means of 12 segments).

W'ashed, then Transferred at IndicatedDay to Fresh Medium Containing

Day

-No >NAA 0.1 mg/l iNAA

mtean no. branches/cml

1 1.4 0.7 52.2 i 3.52 4.6 1.6 51.4 i 3.83 15.9 3.0 52.9 i 2.84 43.4 ± 4.12 50.5 + 1.8

Means of eight segments.2 Significantly different (P = 0.05) from mean for segments

grown 5 days on NAA.

After 1 day in NAA, nuclei and nucleoli of living cells hadenlarged. This was true of cells in the cortex as well as of cellsin the pericycle-endodermis region. Mitotic figures were ob-served in some cortical cells after 24 hr in NAA (Fig. 6). Corti-cal cells apparently responded initially to the influence of NAAin a fashion similar to pericycle-endodermal cells, to the extentthat their nuclei and nucleoli became swollen and at least somecell divisions ensued. Measurements were made on the size ofnuclei and nucleoli in cortical cells in squashed segmentsharvested at various intervals after inoculation (Fig. 7). Maxi-mum size was reached about 9 hours after inoculation.

After 2 days, localized areas in the pericycle-endodermisregion showed clear signs of primordia initiation (Fig. 6). Onlya few cells, in many sections studied, had what appeared to becasparian strips. Thus it was not possible to delineate the endo-dermis unambiguously, although it appeared that both pericycleand endodermis cells were involved in primordia initiation (9).Few cell divisions occurred in the cortex, which becamecrushed and ruptured as primordia developed further (Fig. 6).Primordia formed at a slight angle to protoxylem poles, com-mon in diarch roots (9).The number of cells in a 1-cm segment versus time is shown

in Figure 8. The initial number of cells in a 1-cm root segmentis about 15,000. Only a small percentage of these (primarilythe pericycle-endodermal cells that form primordia) are actu-ally progenitors of the population of cells that develops in 5days.

Elongation of Branch Roots. As mentioned earlier, mostprimordia are capable of elongation to form branch roots a fewcentimeters long. Such branches develop even in the originalmedium, but will do so more rapidly if segments are rinsed andtransferred to auxin-free medium after primordia have formed.It was also observed that roots elongated more rapidly inmedium containing the mixture of vitamins, inositol, and gly-cine which is listed in "Materials and Methods," than if thesesupplements were omitted. Data in Figure 9 illustrate this re-sponse, as well as the time course of elongation.

Branch roots typically stopped elongating after reaching 3 to5 cm in length (3 to 4 weeks after inoculation), and their tipssubsequently turned brown. Soon after meristem necrosis, how-ever, a branch formed just behind the dead tip of most elon-

39Plant Physiol. Vol. 50, 1972



40 ~~~~BLAKELY, RODAWAY, HOLLEN, AND CROKER PatPyil o.5,17

xH

.~

....

3m

A.,ow.

AL

....

Ad blhl., A

FIG. 6. Photomicrographs of 10 /Am sections of roots shown in Figure 5A. Paraffin-embedded, safranin-fast green stain. Numbers in lowerright of each photograph indicate number of days after inoculation in medium with 0.1 mg/i of NAA. Light lines indicate protoxylem poles.Arrow in upper middle day 1 photograph indicates a mitotic figure in cortex, seen also in several other sections. Essentially, all cortex nucleiand nucleoli enlarged in response to NAA, although only few divided. In upper right day 1 photograph, note enlarged nuclei and nucleoli oftwo pericycle cells, presumably primordium initials. The heavy line in the day 0 photograph represents 50 Mmr.

gated laterals, which was capable of elongating another fewcentimeters, before its tip in turn died. The secondary branchesin turn often formed branches. It is this pattern of developmentthat leads to dense mats of intertwining roots in older cultures(over a 2- to 3-month period) before medium depletion andaccumulation of staling products (the medium eventually turnsdark brown) cause cessation of growth.

It was of interest that branches formed just behind the ne-crotic tips of elongated roots. Perhaps an auxin or otherbranch-inducing substance moves from the older portions ofthe root toward the tip. It might accumulate at the tip. be inac-tivated if the tip is functioning, or be counteracted by sub-stances (cytokinin?, see 17) produced by an active tip. It mightlead to tip necrosis. Street and associates (summarized, 20)have studied meristem longevity in tomato and other rootcultures. They present evidence for a feedback mechanism be-

tween the meristem and older parts of the root. It appeared thatolder parts formed an auxin-like substance which was trans-located toward the tip and which could lead to tip senescence;the amount produced was thought to be directly related to thegrowth rate of the tip. This hypothesis was offered to accountfor cyclic variations in growth rate. Adding IAA to the mediumhad little effect on meristem longevity, but low concentrations(0.1 [Lgll) of NAA markedly decreased meristem longevity.Certain antiauxins increased longevity and also counteractedthe effect of added NAA. Sucrose levels above 1I% also de-creased longevity. Part of the explanation for the relativelyslow growth rate and early meristem necrosis in our H. raven iiroots may lie in our use of NAA and relatively high (3%) su-crose in the medium. It has not been possible, however, tomaintain these roots in the absence of NAA, in spite of re-peated attempts. Even in the absence of NAA, meristems cease





growing after only a few centimeters of growth; one or twobranches may form behind the dead tip, but while these may inturn grow a short while, further branching and growth fail tooccur. Added auxin is required for the indefinite growth of aculture, which requires the frequent formation of newmeristems.We have yet to investigate the fate of NAA in the medium.

It is curious that elongating branches do not in turn branch

I

_j 30-0

c. x,,L ! s . ,^0

-I 10 1

z

hi

_J ~~~~NAA

0>~~~~~~~ 0

FIG. 7. Changes with time in nuclear and nucleolar volume ofcells in cultured H. ravenii root segments. The segments werestained with cyanine R, then squashed for microscopic examinationand measurement. Most nuclei were from the cortex. Points repre-sent means of 60 measurements (10 each on 6 root segments), ±the standard error. O: Cultured in NAA-free medium; 0: culturedin medium with 0.1 mg/l of NAA. (In calculating volumes, nucleiand nucleoli were assumed to be prolate spheroids.)

6

2 4/x NAA

_J 3 z

w

*<)I 2 3 4 5DAYS

FIG. 8. Number of cells in 1 cm H. ravenii root segments atvarious times after inoculation. Q: Cultured in NAA-free medium;0: cultured in medium with 0.1 mg/l of NAA. Cells were sepa-rated by the chromic acid method, and counted in a planktoncounting chamber. The increase in cell number apparently com-mences at about 24 hr after inoculation into medium with NAA,and progresses exponentially. Points represent means of threecounts, based on 30 one cm segments, + the standard error.

_5I

5 e

_I

Z' 2

DAYS

FIG. 9. Elongation of branch roots formed on 1-cm H. raveniiroot segments. Each point represents the mean length of 17 to 89branch roots in cultures harvested at the days (after inoculation)indicated. To facilitate measurements, a suboptimal concentrationof NAA (0.01 mg/l) was used, so that fewer branches would form.*: Roots cultured in complete medium listed in Materials andMethods; segments averaged 8.2 branches per cm. 0: Vitamins,glycine, and inositol omitted; segments averaged 7.5 branches percm.

profusely when left in the original medium (they usually onlybranch once, and then just behind the necrotic tip as notedabove), although they will branch profusely if excised andplaced in fresh medium with NAA. Is the NAA inactivatedsomehow, or do the roots produce a substance that counteractsNAA? If something is produced in the older parts of theroots which translocates toward the tip and is responsible forthe branch that develops there (and possibly responsible alsofor tip necrosis), what is its nature? These and many other un-answered questions must be the subject of future studies.

Acknowledgments-We thank Dr. J. Wu and 'Mrs. Ruth Blakely for manyhelpful stuggestions and discussions.

LITERATURE CITED

1. ABERG, B. 1961. Vitamins as growth factors in higher plants. Handb. Pflan-zenphysiol. 14: 418-449.

2. BLAKELY, L. M., S. J. RODAWAY, AND L. B. HOLLEN. 1969. Branch root ini-tiation in cultured root segments. Abstracts, XI International BotanicalCongress, Seattle, Wash., p. 16.

3. BONNETT, H. T., JR., AN-,D J. G. TORREY. 1965. Chiemical control of organ for-mation in root segments of Convolvulus cultured in vitro. Plant Physiol. 40:1228-1236.

4. BRANDES, H. AND H. KENDE. 1968. Studies on cytokinin-controlled bud forma-tion in moss protonemata. Plant Physiol. 43: 827-837.

5. BRIAN, P. W., H. G. HEMMING, AND D. LOVE. 1960. Inhibition of rooting ofcuttings by gibberellic acid. Ann. Bot. 24: 407-419.

6. BUTCHER, D. N., AND H. E. STREET. 1960. The effects of gibberellins on thegrowth of excised tomato roots. J. Exp. Bot. 11: 206-216.

7. CHRISPEELS, M. J., AND J. E. VARNER. 1967. Gibberellic acid-enhanced synthe-sis and release of a-amylase and ribonuclease by isolated barley aleuronelayers. Plant Physiol. 42: 398-406.

8. ERIKssoN, T. 1965. Studies on the growth requirements and growth measure-ments of cell cultures of Haplopappus gracilis. Plant Physiol. 18: 976-993.

9. ESAU, K. 1965. Plant Anatomy. Wiley, New York. pp. 509-513.10. DELA FUENTE, R. K. AND A. C. LEOPOLD. 1969. Kinetics of abscission in the

bean petiole explant. Plant Physiol. 44: 251-254.11. DELA FUENTE, R. K. .AND A. C. LEOPOLD. 1970. Time course of auxin stimula-

tions of growth. Plant Physiol. 46: 186-189.12. FURUYA, M. AND J. G. TORREY. 1964. The reversible inhibition by red and

far-red light of auxin-induced lateral root initiation in isolated pea roots.Plant Physiol. 39: 987-991.

13. JACKSON, R. C. 1962. Interspecific hybridization in Haplopappus and its bear-ing on chromosome evolution in the Blepharodon section. Amer. J. Bot. 49:119-132.

14. IMURASHIGE, T. AND F. SKOOG. 1962. A revised medium for rapid growth andbioassays with tobacco tissue cultures. Plant Physiol. 15: 473-497.

15. PECKET, R. C. 1957. The initiation and development of lateral meristems in

41Plant Physiol. Vol. 50, 1972



42 BLAKELY, RODAWAY, HOLLEN, AND CROKER Plant Physiol. Vol. 50, 1972

the pea root. I. The effect of young and of mature tissue. J. Exp. Bot. 8: 20. STREET, H. E. 1967. The aging of root meristems. Soc. Exp. Biol. Symp. 21:172-180. 517-542.

16. PECKET, R. C. 1957. The initiation and development of lateral meristems in 21. TORREY, J. G. 1950. The induction of lateral roots by indoleacetic acid andthe pea root. II. The effect of indole-3-acetic acid. J. Exp. Bot. 8: 181-194. root decapitation. Amer. J. Bot. 37: 257-263.

17. SHORT, K. C. AN-D J. G. TORREY. 1970. Natural occurrence of cytokinins in 22. TORREY, J. G. 1956. Chemical factors limiting lateral root formation in isolatedroots of pea seedlings. Plant Physiol. 46: S9. pea roots. Physiol. Plant. 9: 370-388.

18. STEEL, R. G. D. AND J. H. TORRIE. 1960. Principles and Procedures of Statis- 23. TORREY, J. G. 1962. Auxin and purine interactions in lateral root initiation intics. McGraw-Hill, New York. isolated pea root segments. Physiol. Plant. 15: 177-185.

19. STREET, H. E. 1966. The physiology of root growth. Annu. Rev. Plant Physiol. 24. TORREY, J. G. AND D. E. FOSKET. 1970. Cell division in relation to cytodiffer-17: 315-344. entiation in cultured pea root segments. Amer. J. Bot. 57: 1072-1080.



control and kinetics of branch root formation in segments … · plant physiol. (1972) 50, 35-42...

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