Plant Physiol. ('1968) 43, 384-388

Relationship of Boron to Gibberellic Acid-InducedProliferation in Debudded Tobacco Plants'

John SkokDepartment of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115

Received October 9, 1967.

Abstract. Stem applications of gibberellic acid (GA) to debudded tobacco plants (Nicotianatabacum L., var. One Sucker) produce stem swellings that involve intense proliferation of,primarily, xylem tissue. Withholding boron from GA-treated debudded plants greatly reducesthe GA-induced proliferation. This response offers a system for directly demonstrating theeffect of boron on xylem formation and lignification that is unconfounded bv the effect ofboron on elongation growth.

It has been shown that total debudding of tobaccoplants cauises tumor ancd teratoma (tumor shoot)formation on the lower stem internodes and roots(6). Applications of either indole-3-acetic acid orgibberelbic acid do not prevent or modify tumlor orteratoma formation buit induce certain morphogenicresponses that do not appear in intact plants (7).Stem swel,lings are induiced by both materials andthose produiced by applied g.bberellic acid (GA)are particularly marked. They invo-lve an intensiveproliferation of tthe xylem and to a lesser degreethe bark (tisssues external to the xylem, primarilythe phloem and cortex).

Since boron appears to function in xylem forma-tion and lignification (5) experiments were carriedotit to determine whether this element is requiredfor or influences the formation of these GA-inducedproliferations and the restilts are reported here.

Materials and Methods

Tobacco plants (Nicotiana tabacurn L., var. OneSucker) were grown oIn a greenhouse bench in2 gallon glazed crocks filled with quartz sand andsui,pplied with ntutrient solutioin of the compositionprevious,ly given (9). The general growing con-ditions, method of debudding and the preparationand application of the lanollin mixtures have beendescribed also (6, 7).

In 2 separate experiments, plants 38 and 34 daysfrom sowing were transplanited to individual crockswith sand and suipplied with boron containing nii-trient (+B) for 30 and 34 days respectively.Those in the appro,priate treatment lots were thendecapitated and shifted to -- B n,utrient. At thistime the plants hadl attained a height of 30 to 35 cm

1 This work was in part conducted at the ArgonneNational ILaboratory iunider the auspices of the UnitedStates Atoiiiic Energy Commission.

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and produced 1 1 to 12 leaves. Lateral debuddingwas started 5 days after decapitation and continueduintil to.tal debuldding was accomplished in thoserequiring this treatment. Lanolin mixtures with orwithouit GA (100 ppm) were applied as a ringaround the tippermost who,le internode (below theuipper remaining node) ill the case of decapitatedand debudded plants and arotind the corres,pondinginternode in case of intact plaints. Two applica-tions were made. The first was applied 10 daysafter decapitation in *the first experiment and 7 daysin the second experiment. The second applicationin both cases was given 2 weeks following the first.The second ring- w\as applied (lirectlv below ain(adjoining the first ring.

Stem diameters were taken by averaging calip)ermeasuiremeints of the maximuinm and minimumdiameter of tihe stem alt the site of treatment ofeach individual Plant and 5 cm sections that in-cltlded the treatetd stem portion were sectionedI ouitand weighdcl.

Resultsand Discussion

,Ns fotund prev-iouisly (7), al)l)lications of G-Acauisecd marked stem swellings in debuidded pllantsbut not in intact lplantis. Withhollding bo,ron mark-edly redtuced the GA-indudced proliferationis (fig 1,tables I, II). A!l debtud,ded planits, regardless ofGA-treatment or whether boron xi as withheld ornot, produced ttimors and teratomata. Teratomadevelopment, howeever, w-as redulced wheni boronwas withhdld. The GA-iinduiced1 sw-ellings werespherical in shape in the LB plan,ts but somewhatflattened in the --B plants. 'T'ihe llants in the firstexperiment (table I, fig 1) were on a -B ntitrientfor 33 days and( those in the seconid (itable II) for35 dlaxvs at the timile the measurements were taken.

TIn both experiments about one-half of the plaitsin the inltact, -B grouip exhibited noticeable boron

SKOK-BORON AND GA-INDUCED PROLIFERATION

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2

4- 5 6m~~~~ iI

C.M.

I

4

FIG. 1. Whole, longitudinal, and cross section portionls of upper internodes of tohacco stems 23 days fromfirst GA application (fixed in chromic acid-alcohol-formalin fixativ-e which is responsible for the color contrastof the differenit tissues): 1) intact, +B, lanolin control, not proliferated; 2) intact, +B, GA-treated (roots were

removed when lanolin was wiped off), not proliferated; 3) debudded, +B, lanolini control, not proliferated; 4)debudded, +B, GA-treated, showing marked proliferation of xylem, slight proliferation of bark (tissues externalto xylem includinig prim.arily cortex and phioem), with no affect on pith; 5) dehbudded, -B, GA-treated, showingmluclh less GA-indluced proliferation iii comparison with thatt produced in 4.

Insert

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SKOK-BORON AND GA-INDUCED PROLIFERATION

Table I. Effect of Boroni antd GA on Stemiis ofThe data were obtained 23 days from first application.

Intact anid Debuidded Tobacco Platlts

Means + S. E. mean'Stem diam Fr wt

Plant's per at site of of stem sectionTrealtment treatment treatment of treated part

Number i1iiii gIntact, + B, 5 16.0 ± 0.47 9.20 ± 0.39lanolin controlIntact, -B, 5 14.6 ± 0.41 8.01 ± 0.39lanolin controlIntact, +B, 10 18.9 ± 0.18 11.10 0.30GA-treatedDebudded, +B, 5 15.3 ± 0.54 9.08 ± 0.26lanolin controlDebudded, -B, 5 14.2 0.23 7.52 ± 0.11lanolin controlDebudded, +B. 20 33.0 ± 0.24 26.00 ± 0.40GA-treatedDebudded, -B. 20 21.5 ± 0.65 15.12 ± 0.61GA-treated

Both stem diameters and stemii weights differ significantly at the 1 % level between: debudded, lanolin control anddebudded, GA-treatecl with or without boron; debudded, +B, GA-treated and debudded, -B, GA-treated; andintact, +B, GA-treated and debu-dded, +B, GA-treated. Other differences significant at the 1 % level occurbetween intact, +B, lanolin control and intact, +B, GA-treated in case of stem diameters and between debudded,+B, lanolin control and debtidded, -B, lanolin control in case of stem weights.

Table II. Effect of Boroni on GA-Inidutced Stemn Pro-lifer-ation of Debudded Tobacco Plants

The data were obtained 28 days from first appli-cation.

Means ± S. E. mean1(15 plants per treatment)

Stem diam Fr wtBoron at site of of stem sectiontreatment treatment of treated part

111i11 g

+B 34.4 ± 0.29 26.98 ± 0.64-B 24.8 ± 0.59 18.00 ± 0.72

Stem diameters and stem weights of the 2 lots differsignificantly at the 1 % level.

deficiency sympitoms in the.ir terminail buds ancdsmal!l upper leaves by ,the tenth day on -B nutrient.In 2 to 3 more days all the plants of this groupshowed marked symptoms and in ianother day or sotheir tips were dead. The debudded plants ex-hibited boron deficiency symptom's after 20 to 21days on -B nutrient in the form of petiole-crackingin their upper leaves (these leaves are larger andolder than the upper-most leaves of the intaot plantsthat continued producing lleaves).

The GA-induced prolliferation involves mainilythe xylem and to a lesser degree the bark. Witih-holding boron markedly reduces ithe proliferationof the xylem 'but 'has ilittle or no effect in reducingthe lesser pro,liferated bark tissue (fig 1). Theboron deficient 'tissues were about one-fourth toone-fifth as proliferated as the similarly treated

boron sufficient tissues. Relaltionships betweenboron and 'lignification have been shown previotusly(1, 2, 5). Since certain phenolic compounds 'thatmay serve as lignin prec-ursors 'have been shown toaccumulate in boron deficient ti'ssues in amountsconsiderably above 'those in normail tissues (3, 4,8, 9), it is possible that boron may have a functionin the polymerization o'f these materials into lignin.

The mechanism of GA-induced xylem prolifera-tion and the relationship and function of delbuddingin its elicitation are not known. This response,however, offers a u;seful experimental model forstudying the requirement of boron in xylem forma-tion in tissue that is potentially capable of prolifer-ating. It permits measuring lignification in re-sponse to boron unconfounded 'by other growthparameters 'that are also influenced by boron. Ithas been pointed out (1, 2) that attempts to deter-mine the effect of boron on lignification on thebasis of lignin analyses are difficult and may bemisleading. Such analyses may actually show lowerpercentage 1ignin value's for + B plants than forboron deficient plants be,cause 'of a diluting effectof 'lignin concentration. This is catused by con-tinued growth, stem elongation and dry weightproduction in the boron sufficient plants and thecessation or reduction 'of cllongation growth indeficient plants. Lignin 'analyses on stem sectionsof equal lengths or weights from such groups ofplants are actualily made on relatively young, grow-ing tissues in the case of +B plants and on maturetissues that have stopped growing in the case ofdeficient plants. Use of GA-induced stem prolifer-ation for the study of the influence of boron or

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PLANT PHYSIOLOGY

other factors on lignification and xy-lem formationiperm,its making use of specific internodes of com-

parable ages and tdevelopment. Elon1gation growthis not involved and the major response that occtursin this system entails xylem proliferation.

Literature Cited

1. DUTTA, T. R. AND \W. J. 'MCILRATH. 1964. Ef f ectsof boron on growth and lignificationl in suniflowertissue and orrgan cultures. Botan. Gaz. 125:89-96.

2. MCILRATH, \\. J. AND J. SKOJE. 1964. BoroIn nu-

trition and lignification in sunflow-er and tobaccostems. Botan. Gaz. 125: 268-71.

3. PERKINS, H. J. AND S. ARANOFF. 1956. Identifi-cation of the blue fluoresceint compounds in borondeficienit planits. Arch. Bioclhemil. Biophiys. 64:506-07.

4. REED, H. S. 1947. A physiological study of borondeficiency- in plants. H-ilgardia 17: 377-411.

5. SKOK, J. 1958. The role of boron in the plantcell. Chap. 15. In: Trace Elements. C. A.Lamb, 0. G. Bentley, and J. M. Beattie, eds.Academic Press, New York. p 227-43.

6. SKOK, J. 1967. Tumor and teratoma inductionin tobacco plants (lehudl(lidng. Planit Physiol.42: 767-73.

7. SKOK, J. Morphooenic responises of (lebudded to-bacco plants to ,gibberellic acid and indole-3-aceticacid. Plant Physiol. 43: 215-23.

8. \WATANABE, R.. W%V. CIIORNEY, J. SKOK, AND S. H.\VENDER. 1964. Effect of boron deficienicy on

polyphenol production in the suniflow-er. Phvto-chemistry 3: 391-93.

9. WATANABE, R., WX J. MCILRATH, J. SKOK, N.CHORNEY, AND S. H. WVENDER. 1961. AccI11nU-lation of scopoletin glucosi-de in boron-deficienttobacco leavue. Arch. Biochemi. B,iophys. 94:241-43.

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