Rhizogenesis in TL-DNA-Transformed Brassica napus

Plant Physiol. (1992) 100, 1277-12820032-0889/92/100/1 277/06/$01 .00/0

Received for publication March 2, 1992Accepted July 6, 1992

Enhancement of Naphthaleneacetic Acid-inducedRhizogenesis in TL-DNA-Transformed Brassica napuswithout Significant Modification of Auxin Levels and

Auxin Sensitivity

Jacques Julliard*, Bruno Sotta, Georges Pelletier, and Emile MiginiacLaboratoire de Physiologie du Developpement des Plantes, Centre National de la Recherche Scientifique Unite de

Recherche Associee 1180, Universite Pierre et Marie Curie, Bo?te 156, 4 place Jussieu, Tour 53, Sme etage,75252 Paris Cedex 05, France (1.1., B.S., E.M.); and Laboratoire de Biologie Cellulaire, Institut National de la

Recherche Agronomique, 78000 Versailles, Frances (G.P.)

ABSTRACT

Determination of the abscisic acid and indoleacetic acid (IAA)contents of floral stem segments of nontransformed and pRi A4 TL-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor)using a high performance liquid chromatography-enzyme-linkedimmunosorbent assay procedure and mass spectrometry controlsshowed that IAA levels were not modified. The regeneration abil-ities of the in vitro cultured explants were compared on mediasupplemented with several plant growth regulator combinations.No regeneration occurred on hormone-free media, and shoot pro-duction was similar in both genotypes when supplemented withbenzyladenine. In the presence of naphthaleneacetic acid (NAA),transformed explants were characterized by faster root regenera-tion and reduced shoot organogenesis. The optimum for rootformation was the same in nontransformed and transformed plants,but the sensitivity threshold was slightly lower in the latter. TheNAA inductive period was shorter (14 versus 22 h) with trans-formed tissue. Root neoformation occurred about 72 h earlier ontransformed explants. Our results suggest mainly that there is anacceleration of the auxinic signal transduction and/or that theevents preliminary to the formation of roots occur faster in thetransformed tissues than in the normal ones.

Hairy root disease results from the infection of the plantby a soil bacterium, Agrobacterium rhizogenes. The disease isexpressed by adventitious root proliferation and is due to thetransfer, the integration, and the expression in the plant cellgenome of a DNA fragment (T-DNA, transferred DNA) fromthe Ri (root-inducing) plasmid of the bacteria (for review, see

ref. 23). The T-DNA of A. rhizogenes strain A4 consists of twodistinct regions, the TL- and the TR-DNA (9). The presenceof the TR-DNA, which is homologous to the T-DNA of theTi plasmid of A. tumefaciens coding for hormone biosynthesis(tmsl and tms2 genes), is not a prerequisite for the hairy rootsyndrome (10). Thus, tmsl and tms2 could not account foran overproduction of auxin leading to root formation in plantsinfected with TL-DNA from A. rhizogenes. Cardarelli et al. (3)have shown that even if auxin (endogenous or exogenous)plays a role in the induction of hairy root tumors, its presence

is not necessary either for T-DNA transfer or for the growthof haiiy roots. This suggests that TL rol genes (root locus)function by sensitizing the plant cell to auxin. Support forthe hypothesis of increased sensitivity of transformed cells toauxin has been provided by Shen et al. (18), who measuredroot tip elongation, proton excretion, and the transmembraneelectrical potential of protoplasts derived from nontrans-formed or transformed roots of Lotus corniculatus.However, recent data (5) have shown that the protein

encoded by the rolB oncogene is able to hydrolyze indoleglucosides. Moreover, Estruch et al. (4) have reported thatthe roiC is responsible for the release of cytokinins fromglucoside conjugates. Thus, according to these findings, onewould expect to observe an increase in IAA and cytokininsin tissues containing the TL-DNA. Until now, this has notbeen convincingly demonstrated (19, 21). Therefore, we de-cided to check these two hypotheses by comparing the abilityof transformed and untransformed tissues to regenerate budsand roots because it is well known that organogenesis iscontrolled by numerous interactions among which genotypicand hormonal factors are essential (8, 15). Thus, we haveundertaken a study using rape (Brassica napus L. var oleifera,cv Brutor) floral stem explants of nontransformed and pRi A4TL-DNA-transformed genotypes, comparing their responsesto several plant growth regulators. In addition, the levels ofendogenous auxin and ABA in the explants were measured.Here we show no differences in endogenous hormone levels,but we give evidence that differences in speed of root for-mation and number of regenerated buds were triggered byNAA'. These results are discussed with respect to the hy-pothesis of an increased sensitivity to auxin and/or a moreefficient transduction of auxin signal.

MATERIALS AND METHODS

Plant Material and Culture Conditions

Seeds of rape (Brassica napus L. var oleifera, cv Brutor), anAgrobacterium rhizogenes pRi A4 TL-DNA-transformed hemi-zygous (TL/-) line (gift from Dr. Primard-Brisset) were sown

'Abbreviation: NAA, naphthaleneacetic acid.1277

Plant Physiol. Vol. 100, 1992

in a greenhouse. These plants were derived from a singletransformed plant regenerated from hairy roots (C. Primard-Brisset, unpublished results) by successive back-crosses withthe Brutor parent. The comparison is made between sisterplants of the same generation: normal plants on one side andhemizygous TL plants on the other side. We can assume thatany difference between the two genotypes is a dominanteffect of the TL-DNA insertion. Plantlets were grown in 12-cm pots containing tray compost (Sterckx, Roselare, Belgium)before being planted in the ground (brown peat:clay:sand,30:50:20). Because flowering of the transformed plant isdelayed, in vitro culture experiments were carried out onplants from different sowings or from the same sowing forwhich plant flowering was synchronized by culturing youngnormal plants at 40C before they were put in soil. Bothpossibilities could be used to obtain inflorescences at thesame time, because, for a given genotype, we have neverobserved any difference in the regeneration abilities betweenplants from different sowings or between 4°C-treated anduntreated plants. Plants were drip watered with a nutrientsolution (11) containing 5 mm KNO3, 3 mm Ca(NO3)2, 0.65mM (NH4)2HP04, 0.90 mm NH4NO3, 0.75 mM MgSO4, andmicronutrients (0.3 M Mo, 24NM Bo, 9 MM Mn, 1 MM Cu, 3.5MM Zn, and 11 MM Fe provided as Fe-EDTA) at pH 5.8.

Sampling and Culture of Explants

At the stage of early flowering, whole inflorescences wereremoved and the surface sterilized for 15 min in distilledwater supplemented with Bayrochlor (Bayrol, Reichstett,France) and a few drops of Tween 80. Inflorescences werethen rinsed three times in sterile distilled water and stemsegments of 1 to 2 mm in length were removed from theirupper part using a scalpel. Segments were randomly distrib-uted in 100 x 15 mm Petri dishes containing 20 mL of RCCmedium (6) supplemented with different plant growth regu-lator combinations. BA and NAA (optima 4.4 and 5.4 MM)were added before autoclaving, but GA3 (0.06 AM) was filtersterilized and added after autoclaving. Ten explants (five ofeach genotype) per dish were cultured under continuous coolwhite fluorescent light (Mazdafluor Blanc industrie TFRS 40/BI, 16 h photoperiod, 80 ME.m-2s-') at 280C. In all theexperiments, 10 Petri dishes per treatment (i.e. 50 explantsof each genotype) were used for root and/or shoot percentageregeneration calculations. For the induction experiments, 10batches of 11 Petri dishes (1 hormone free and 10 containing5.4 Mm NAA) were prepared. The NAA-containing Petridishes were then divided into 10 equivalent batches. Every 2h, from the 1 0th to the 26th h after the start of the experiment,the explants of one batch were transferred to hormone-freemedium.

Measurements of Endogenous Hormone Levels

Sampling

During in vitro culture experiments, stem segments chosenat random were cut and immediately frozen in liquid N2. Thesamples, corresponding exactly to the in vitro-cultured stemsegments at the time they were placed on growth media,

were lyophilized and ground into powder. During sampling,fresh and dry weights were recorded.

Extraction

Forty-milligram samples were stirred for 60 h at 40C in 5mL of methanol:distilled water (80:20, v/v) containing 40mg-L` butyl-hydroxytoluene as an antioxidant. To allowpurification recovery calculation, the 37. 102 Bq of each triti-ated standard was added ([3H](±)ABA, 1.85 TBq.mmol-',Amersham, UK and [3H]IAA, 1.11 TBq.mmol-', CEA, Gif-sur-Yvette, France).

Purification

The extracts were successively filtered through celluloseand 0.2-,um pore size Millipore filters, prepurified through aC18 Sep-Pack cartridge (Waters, Milford, MA), then reducedto a small volume of aqueous phase and acidified prior toHPLC purification. The extracts were injected into a RP18HPLC column (Lichrospher, Merck, Darmstadt, FRG) andeluted with a 0.2% (v/v) formic acid:methanol gradient (flowrate 0.8 mL.min-'). Fractions were collected every min for40 min, reduced to dryness, methylated with etheral diazo-methane, and taken up in distilled water prior to the ELISAtest and to scintillation counting for recovery measurement.

ELISA Measurements

The ELISA procedure with bound antigen and indirectenzyme labeling performed in the laboratory (12) has beenslightly modified. Briefly, for each purified extract, the im-munoreactivity was measured in all HPLC-purified fractions.The competition step was performed at 40C for 2 h usingeither polyclonal anti-IAA rabbit antiserum or a monoclonalanti-ABA mouse antibody (Phytoscience, Angers, France).After washings, antihormone antibodies bound to the wellsof the microtitration plates were labeled with a goat anti-rabbit (polyclonal antibodies) or a sheep anti-mouse (mono-clonal anti-ABA antibody) immunoglobulin peroxidase re-agent (Boehringer, Mannheim, FRG). Then, 0.5 mg m L` of2,2' -azino-bis(3-ethylbenzthiazoline-)-6-sulfonic acid(Sigma) was added as enzyme substrate in a perborate buffer(pH 4.6), and the result of the test was measured spectropho-tometrically at 405 nm. For the immunoreactive fractionscorresponding to ABA and IAA (based on their coelutionwith standards), hormonal content was determined fivetimes. Calculations were made by reference to a calibrationcurve established on each microtitration plate by a curvilinearregression of magnitude 4 obtained from the average of fourstandard curves.

MS Validation

For IAA and ABA, the specificity of the HPLC-ELISA testfor rape extracts was confirmed by MS. HPLC fractionscontaining IAA or ABA were submitted to extra HPLC puri-fication steps and methylated. After each step of the proce-dure, immunoreactivity was measured to ascertain that thepurified hormone was the only immunoreactive form in thefraction measured by ELISA. The identity of the purified

1278 JULLIARD ET AL.

NAPHTHALENEACETIC ACID-INDUCED RHIZOGENESIS IN T[-TRANSFORMED RAPE

IAA-methyl ester and ABA-methyl ester was obtained byMS. Full mass spectra, obtained by desorption chemical ion-ization using ammonia gas as reactant, were identical to thoseof standards. Thus, we can assert the accuracy of ABA andIAA measurements and that the possibility of overestimationdue to cross-reactions was ruled out.

RESULTSDifference in Organogenesis between Normal andTransformed Genotypes Is Expressed Only inthe Presence of NAA

Several media, hormone-free or supplemented with com-binations of NAA, BA, and GA3, have been tested. The mostsignificant results are reported in Table I.No regeneration occurred on hormone-free medium. On

NAA-free media, no root formation was observed, regardlessof the genotype. Moreover, no difference in shoot regenera-tion between normal and transformed plants was recorded.The absence of difference in shoot regeneration ability onNAA-free media between the two genotypes was clearlyattested by the results of five experiments (not shown, 50explants of each genotype per experiment). The percentagesof explants producing shoots were 91.2 ± 7.0 and 91.2 ± 8.7for normal and transformed genotypes, respectively.On media containing NAA, all stem segments produced

roots in both genotypes (Table I). However, the regenerationwas faster with transformed explants (see below). It wasworth noting that typical transformed roots did not growfaster than normal ones (Fig. 1, A and B). However, when incontact with the medium, transformed roots were still grow-ing, whereas normal ones were not (Fig. 1C). There was alsoan important difference in shoot regeneration ability; thetransformed genotype regenerated very few buds (Table I).

Transformed Explants Regenerate Roots Faster thanUntransformed Ones after NAA InductionEffect of NAA Concentration

In an attempt to determine if the faster root regenerationof the transformed genotype compared with the normal one

Table I. Effects of Plant Growth Regulators (PGRs) onOrganogenesis from Floral Stem Segments of Normal andTL-DNA-Transformed B. napus cv Brutor

Percentage of regeneration was determined on the basis of thenumber of explants forming shoots and/or roots as a percentage ofthe total number of explants (50 explants per treatment). For shootregeneration, two classes of explants were considered according tothe number of regenerated buds per explant. Data were collectedafter 32 d of culture. Data from one of two sets of experimentsgiving similar results are reported.

Normal TransformedPG Rs

Shoots ShootsRoots Roots

NAA BA GA3 21 -5 21 -5

Am % %

0 0 0 0 2 04.4 0.06 0 88 60 0 100 52

5.4 100 14 0 100 0 05.4 4.4 100 76 32 100 4 0

was due to a difference in sensitivity to NAA, the effect ofNAA concentration, from 5.4 nm up to 540 AM, was tested.With 540 Mm NAA in the medium, no root formation occurredand most of the explants assumed a typical appearance, withalmost complete detachment of the epidermis. This phenom-enon, more marked in the transformed genotype, was alreadyobservable with 5.4 Mm NAA in the medium. With NAAconcentrations lower than 0.54 ,AM, no organogenesis oc-curred. Thus, root regeneration was possible on media sup-plemented with NAA from 0.54 up to 54 ,uM. The optimumfor root organogenesis, assessed by testing several NAAconcentrations within the range 0.54 to 54 AM, was similarly5.4 gM for both genotypes. Moreover, whatever the NAAconcentration used, transformed explants always regeneratedfaster than normal ones. Using 5.4 ,uM NAA, root formationwas faster (on average 3 d earlier) with the transformedgenotype. Within 8 d, both genotypes showed 100% rootregeneration (Fig. 2). The difference in the speed of rootregeneration at 5.4 AM NAA became clear from the observa-tions from seven other independent experiments reported inTable II. When NAA concentration was increased up to 54JAM, root organogenesis was delayed (4 d for transformedgenotype and 6 d for the normal one) and commenced slowly(Fig. 2). Thus, the difference between the two genotypes wasincreased. On the other hand, on the medium supplementedwith 0.54 Mm NAA, the delay in root formation was greater,and the percentage of explants forming roots was limited forthe transformed genotype and close to zero for the normalBrutor genotype (Fig. 2). At this concentration of NAA, onlysome transformed stem segments were able to form strongroots (one or two in general), which grew very fast (comparedwith growth in 5.4 ,M NAA).

Effect of Pretreatment with NAA

To distinguish between the effects of NAA on root induc-tion and root growth, responses to exposure to NAA forvarious lengths of time were recorded. Figure 3 shows that 1d of exposure to induction medium was sufficient to inducefurther root organogenesis when explants were transferredto hormone-free medium. Nevertheless, the root-inducedstate was acquired about 8 h earlier for the transformedgenotype than for the normal one.

Transformed and Normal Explants Have the SameHormonal Content at the Time of Removal

For IAA, there was clearly no difference between the twogenotypes (Table III). The only difference in hormonal levelsconcerned ABA; transformed plants showed slightly but sig-nificantly lower ABA contents (Table III).

DISCUSSION

The first observation from the in vitro culture study wasthe absence of root regeneration from TL-transformed rapeexplants cultured on hormone-free medium. Thus, trans-formed cells containing the full-length TL-DNA, known fortheir root-formation capacity (2, 19), were not able to expressthis ability under these conditions. Cardarelli et al. (3) have

1279


Figure 1. Root organogenesis from floral stemsegments of normal and TL-DNA-transformedB. napus var oleifera, cv Brutor on mediumsupplemented with 5.4 gM NAA. Normal (A)and transformed (B) explant after 8 d of culture.C, Normal (left) and transformed (right) explantsafter 21 d of culture. Bars = 2 mm (A, B) and10 mm (C).

100

80

00

60

40

20

O

0 10 20 30DAYS

Figure 2. Effect of NAA concentration (0.54 lAM, squares; 5.4 gM,circles; 54 /AM, triangles) on root organogenesis from floral stemsegments of normal (closed symbols) and TL-DNA-transformed(open symbols) B. napus var oleifera, cv Brutor. The frequency ofexplants producing roots was determined by considering the num-

ber of explants forming roots as a percentage of the total numberof explants (50 stem fragments per treatment).

shown that both auxin-either endogenous or exogenous-

and TL-DNA are required for root induction in carrot cells.Indeed, Ri plasmid T-DNA confers to plant cells the capabilityto differentiate roots upon stimulation by auxin (3).However, in rape, when NAA was added in the medium

at 5.4 ,LM, both genotypes were able to produce roots as ifauxin was the only limiting factor for root induction. Never-theless, in these culture conditions, the presence of the TL-DNA was responsible for a faster root regeneration, a reduced

Table II. Frequencies of Root Organogenesis from Floral StemSegments of B. napus cv. Brutor Normal and TL-DNA-TransformedPlantsThe frequencies of root production are means ± SE of observa-

tions taken from seven independent experiments (each with 50explants) performed from several plant cultures. Explants werecultured on NAA-containing media. n, Number of experiments inwhich each treatment was included and scored.

Days Normal Transformed n

4 0.0 50.0 ± 2.8 35 3.0 ± 2.6 86.0 ± 13.9 46 16.0 ± 5.8 99.2 ± 1.8 57 56.0 100 18 79.3 ± 10.6 99.3 ± 1.6 69 87.3±13.3 100±0 3

11 100 ± 0 100 ± 0 3


NAPHTHALENEACETIC ACID-INDUCED RHIZOGENESIS IN TL-TRANSFORMED RAPE

100

80

rf3

860

40

20 -

10 14 18 22 26

HOURS OF CULTUREON INDUCTIVE MEDIUM

Figure 3. Root organogenesis in floral axis segments of normal (0)and TL-DNA transformed (0) B. napus cv Brutor, cultured on induc-tion medium (5.4 AM NAA) for various periods of time. Results are

an average value of three experiments. Bars indicate SE. Data were

collected after 21 d. In each experiment, the frequency of explantsproducing roots was determined by considering the number ofexplants forming roots as a percentage of the total number ofexplants (50 stem segments per treatment).

number of shoots formed, and a weaker response to BAsupply. These observations cannot be explained by an alter-ation of cytokinin metabolism or perhaps a modified absorp-tion of BA because shoot regeneration ability was similar forboth genotypes on media supplemented with BA (60% ofexplants producing shoots, data not shown), BA plus GA3(90%), or GA3 alone (<20%, data not shown). Thus, theweaker response to BA supply of transformed explants on

NAA-containing media seemed to be the consequence of an

inhibition of shoot formation-perhaps due to an imbalancebetween auxin and cytokinin compared with the normalexplants-rather than the consequence of an inability torespond to BA. Endogenous IAA measurements allow us toassert that the differences in the organogenesis on mediasupplemented with NAA were not the consequence of a

higher auxin content preexisting in transformed cells. Theresults of IAA measurements were in disagreement with therecent finding that the rolB oncogene codes for a 13-glucosi-dase, which is supposed to release free IAA from inactive 3-

glucosides (5). In this respect, it is worth noting that a rolB-GUS gene fusion was expressed in the vascular tissue ofinflorescences of tobacco (1). Thus, the only hypothesis inagreement with our results seemed to be a difference insensitivity to NAA, which was previously reported (3, 18).To check the possible difference in sensitivity to NAA, the

effect of NAA concentration, from 5.4 nm to 540 lM, was

tested in an attempt to establish a difference in dose-responsebetween the two genotypes. A wide range of concentrationwas chosen because a 100- or 1000-fold increase in sensitivityto NAA was reported between normal and hairy root proto-plasts in several species (17). The results shown in Figure 2indicate that the optimum for root formation was similar in

both plants (5.4 AM) but that the sensitivity threshold was

lower in transformed plants. However, the difference never

exceeded a 10-fold decrease. The induction of roots on 0.54AM NAA, possible only from transformed material, is a goodargument supporting a difference in sensitivity to NAA or a

modified endogenous IAA metabolism. However, this con-

clusion is not definitely convincing because a high NAAconcentration (54 ylM, Fig. 2) does not inhibit the formationof roots more in transformed tissue than in normal tissue, as

we would expect considering the classic effect-dose curves

(18). In the same manner, transformed root elongation was

less inhibited on 5.4 jLM NAA (Fig. 1C).The most evident difference between the two kinds of

tissue is their reactivity as a function of time. First, theinductive period with NAA was shorter with transformedtissues (14 h) than for normal ones (22 h) (Fig. 3). Second,there was also a 72-h delay before the appearance of roots,again in favor of the transformed tissues (Table II). Theseresults suggest that if transformed cells competent for rhizo-genesis are not more, or are only slightly more sensitive toauxin, they react faster than the homologous and normalcells. Thus, the TL genes could act on biochemical changesstarting from the transduction chain of events following theperception of the auxinic signal and ending with the forma-tion of root primordia. Of course, during this time, numerousand different metabolic and cellular changes occur, but, as

far as we know, morphological observations have not re-

vealed differences in the growth of the roots between thetwo genotypes (Fig. 1, A and B). Nevertheless, it remains tobe explained how TL genes are involved specifically in an

acceleration of the rhizogenic process. It was suggested byMaurel et al. (13) that only some cellular responses to auxincould be selectively altered by rol genes. Furthermore, TLgenes responsible for the rhizogenic response could be trig-gered by NAA. For instance, it was demonstrated that auxinplays a central role in the regulation of the root-inducing rolBgene in tobacco (14). Our results do not allow us to answer

precisely this very complex question because the transductionchain following the perception of auxin is far from beingunderstood (7). However, our results suggest that the trans-duction chain of events is accelerated. This explanation aboutthe mechanism of action of TL genes is at least complemen-tary, if not different, from the two hypotheses proposed, i.e.higher sensitivity (17, 18) or increase in auxin levels (5).

It is surprising that the only difference in endogenous plantgrowth regulators we found between normal and trans-formed stem segments concerned ABA. This result is inagreement with those obtained for several organs, i.e. termi-

Table MII. Auxin and ABA Contents of Normal and TL-DNA-Transformed Floral Stem Segments of B. napus cv Brutor

Each value, in fmol- [mg dry weight]-' ± SE, iS an average of fivereplicates. Results of two independent experiments are given.

Normal (N) Transformed (TL) N/TIIAA 3291.7 + 878.2 3071.0 ± 414.2 1.07

1349.4 ± 75.3 1246.1 ±43.9 1.08ABA 1568.0 ± 126.5 1158.0 ± 98.9 1.35

980.4 ± 72.6 754.9 ± 45.8 1.30

1281


nal bud, young and mature leaves, and root system, of 45-d-old aeroponically grown plants and has already been noticedin tobacco plants cultured in vitro (F. Pelese, J. Julliard,unpublished results). It is well known that the hairy rootsyndrome is correlated with the expression of rolA, B, and Cgenes of the TL-DNA of A. rhizogenes (16, 20, 22). Thus, thelowered ABA content may be due to an interaction phenom-enon or to the expression of another TL-DNA gene. We are

now investigating ABA content in other transformed plants.

ACKNOWLEDGMENTS

The authors wish to acknowledge the assistance of Drs. J. Einhomand L. Kerhoas (Laboratoire de Spectrometrie de Masse, INRA deVersailles) for MS analyses and A. Martin-Canadell for the plantmaintenance in the greenhouse.

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Rhizogenesis in TL-DNA-Transformed Brassica napus