Plant Physiol. (1974) 54, 931-935

Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum)Ovules Grown in Vitro'

Received for publication April 5, 1974 and in revised form June 24, 1974

ELLIOTT H. BIRNBAUM, CHARLES A. BEASLEY, AND W. MACK DUGGERDepartment of Biology, University of California, Riverside, California 92502

ABSTRACT

Boron deficiency and phytohormone interactions have beenstudied in unfertilized cotton (Gossypium hirsutum) ovulesgrown in vitro. Such ovules required exogenous indoleaceticacid and/or gibberellic acid for fiber elongation. Boron alsowas required for maintenance of fiber elongation and normalmorphogenesis throughout 14 days of culture. The amount ofexogenous boron necessary for maximum fiber elongationvaried among experiments, presumably in relation to endog-enous boron levels at anthesis. Some ovular epidermal cellsdistant from the liquid medium could be induced to elongateinto fiber even after 6 days in boron-deficient medium in re-sponse to the later addition of boron.

Boron deficiency, in the presence of exogenous indoleaceticacid, was characterized by lack of fiber development on the in-undated ovular surface and reduced fiber growth on the ovularsurface exposed to air. In the presence of gibberellic acid,boron deficiency was characterized by complete absence offiber and callusing of the entire ovular surface. When bothindoleacetic acid and gibberellic acid were added, the lack ofboron resulted in proliferation of callus laterally and upwardfrom the inundated epidermis, accumulation of brown pig-ments (presumably phenolic compounds) in the callus, and re-striction of fiber to a small area of the upper ovular surface.

If fertilization is permitted (flowers left intact until the secondday postanthesis) and ovules are aseptically transferred toculture, fibers continue to elongate in response to a completelydefined, liquid, basal medium. This continued elongation ofcotton fibers is markedly stimulated by exogenous gibberellicacid (GA3) but only slightly, if at all, by indoleacetic acid.It was concluded that IAA is the phytohormone of principalconsequence synthesized in response to the processes of fer-tilization (2). If fertilization is prevented and ovules are trans-ferred to culture, fibers fail to elongate in the presence of thesame basal medium. However, fiber elongation does proceedwhen IAA and/or GA3 are included in the basal medium. IAAalone provides for moderate fiber elongation of unfertilizedovules, GA3 provides for less fiber elongation but more callusformation from the ovular surface, and when IAA and GA3are furnished in combination, the fiber elongation response isapproximately additive (2, 3). Thus, a situation could be cre-ated where cells are completely dependent upon exogenousphytohormones for elongation and boron deficiency could beimposed at any point in the phytohormone-dependent elonga-tion phase. This report deals with various effects of boron de-ficiency and phytohormone combinations on the growth anddevelopment of unfertilized cotton ovules in vitro.

MATERIAILS AND METHODS

Boron is required for normal growth and development ofall higher plants, although to date no single specific physio-logical role has been assigned to it. During the past 2.5 decadesboron nutrition has been implicated in several general areasof plant metabolism: organic translocation, enzymatic regu-lation, plant growth regulator responses, cell division, cellmaturation, nucleic acid metabolism, phenolic acid biosyn-thesis, and cell wall metabolism (7).A system for in vitro culture of unfertilized cotton ovules in

completely defined medium (1, 3, 4) has recently been de-veloped in these labs. The system makes it possible to exposeovules to various combinations of nutrients, metabolites, andphytohormones, and thus was adopted for studies on the roleof boron in plant growth and development.

Fiber initials (epidermal cells of the ovule destined to be-come mature fibers) initiate elongation on the day of anthesis.Once elongation has been initiated the fiber cells do not divide.

'This work was partially supported by the Cooperative State Re-search Service of the United States Department of Agriculture.

In vitro culture of cotton (Gossypium hirsutum) ovules andcomposition of the basal medium have been previously de-scribed (1-4). Flowers were collected on the morning ofanthesis, and ovules were aseptically removed from each ovaryand floated on the surface of 50 ml of sterile culture mediumin a 125-ml culture flask. Ovules floated on their horizontalaxes, the upper surface remained dry, and only the lower, sub-merged surface was in direct contact with the medium. Ovuleswere cultured at 34 C in the dark. Duration of culture was 14days or as otherwise noted. To ensure that media and cul-tures were boron-free or contained only the levels desired,glass-distilled water was filtered through a column of AmberliteXE-243 (11) and used in all stock solutions and final media.Stock solutions were stored in polyethylene bottles, and cul-ture was carried out in clear polycarbonate Erlenmeyer flasks.A treatment consisted of 3 to 6 flasks, and each flask con-

tained 28 to 32 ovules. Upon termination of an experiment,twenty ovules typical of the total in each flask were selected forfiber determination. Fiber growth was estimated by the stain-destain method previously reported (5) whereby fibers werestained with toluidine blue 0, destained in acidified alcohol,and the absorbance of the destaining solution, referred to astotal fiber units, was measured. Dry weight was determinedafter pooling all sets of 20 ovules utilized in any treatment.Callus, which is produced under certain conditions of boron

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BIRNBAUM, BEASLEY, AND DUGGER

A

4

i

A A

A

100 /xM BIAA IAA IAA GAGA GAKin

A

4 - B ( - )IAA IAA IAA GAGA GAK n

FIG. 1. Phytohormone effects on growth of unfertilized cottonovules cultured in B(+) and B(-) media for 14 days. TFU valuesfor heavily callosed treatments (high dry weights; i.e., B(-) +

IAA, GA3 and Kin; B(-) + IAA and GA3; B(-) + GA3) pri-marily represent extent of callusing rather than fiber develop-ment.

deficiency, also undergoes slight staining; consequently, TFU2values for callused tissues are exaggerated. Therefore, dryweight is an important measure of the physiological conditionof callused ovules and the ratio of TFU-dry weight is moremeaningful for comparing such treatments than either TFUor dry weight individually, and is presented where appropriate.The higher the ratio the more "normal" the growth (i.e., themore fiber development and less callus).

RESULTS

PHYTOHORMONE-BORON INTERACTIONS

Boron-sufficient Culture[B(+)]. Ovules yielded an averageTFU value of about 4 following 14 days culture in basalmedium (containing the usual 100 /pM boron) supplementedwith 5 /1M IAA (Fig. 1). The ovules remained white andmimicked in vitro growth of fertilized ovules, producing fiberover their entire surfaces. If identical medium was supple-mented with 0.5 /-M GA3 only, about 1.5 TFU were recorded.The little fiber that was produced was restricted to the drysurface and some callusing was evident on ihe lateral andinundated sides of the ovules. If the basal medium containedboth IAA and GA3, the average TFU value was about 5.5,and little or no callusing occurred.

Boron-deficient Culture[B(-)]. In 14 days culture in B(-)

2 Abbreviations: TFU: total fiber units; Kin: kinetin.

medium containing IAA only, fiber length was markedly re-duced to 0.5 TFU. Fiber development was restricted to the dryupper surface; the inundated surface remaining fiberless, butuncallused. In B(-) medium containing GA3 only, fibers wereessentially absent and ovules were completely covered bycaLus. A TFU value of 1.33 was obtained for such tissue.While this value is higher than that for the IAA only treat-ment, it primarily represents stain taken up by callus and notactual fiber units. In B(-) medium containing both IAA andGA3, TFU were only about 15% of the amount produced on asimilar but B(+) treatment, and callus increased dramatically.More fiber and less callus w.s produced on such ovules thanwi.h GA3 alone, but normal growth was less than with IAAalone. Thus, it is apparent that in a B(-) medium, IAA de-creased the callus-promoting and brown pigment-inducingeffects of GA3. Figure 2 shows the typical growth responsesobserved, and Figure 1 the numerical data for the above in-teractions. Sensitivity of the cotton ovule to boron was re-flected in the fact that essentially the same amount of growth(TFU and dry weight) occurred for ovules cultured in glassflzsks without added boron as for ovuies cultured in B(+)medium in boron-free polycarbonate flasks.

BORON CONCENTRATION EFFECTS

In the presence of IAA and GA3, increases in boron con-centration through 100 uM yielded concomitant increases infiber production and decreases in dry weight (Fig. 3). Underthe s:me conditions, if IAA was the only exogenous phyto-hormone, increasing boron concentrations resulted in increasesin TFU; callusing of the inundated surface did not occur,hence there was little change in dry weight (Fig. 4). Theoptimum boron concentration for ovule development variedamong experiments, e.g., Figure 5 indicates that 10 [kM B for14 days was limiting to growth; however, in the experimentrepresented by Figure 6, 10 /tM B produced as much fiber as100 tiM, after 14 days of culture. It is thought that the level ofendogenous boron in ovules when they are collected from theparent plants varies in relation to environmental influences, andthis determines the amount of exogenous boron that is neces-sary for optimum growth.

Subsequent experiments were conducted with the phyto-hormone complement conducive to optimum growth in boron-sufficient medium (5 AM IAA, 0.5 /IM GA3, a-nd 0.05 pMKin). Kinetin was found to have a slight positive effect onfiber growth in B(+) medium supplemented with IAA and GA!(6.07 TFU with Kin; 5.55 TFU without; Fig. 1) and was there-fore included in the media used in the experiments describedbelow.

DURATION OF BORON REQUIREMENT

Concentration and Duration of Culture. Progression ofovule growth in the presence of 10, 50, and 100 /_M B wasfollowed by terminating treatments at 5, 8, 11, and 14 dayspostanthesis (Fig. 5). With 50 and 100 jcM B, normal growthoccurred, growth at 100 pM being better than at 50 ,uM bothin terms of TFU and dry weight. In both boron concentrations,ovules were growing actively at day 14. In 10 juM B the rate offiber production rapidly dropped between the 11th and 14thday of culture. During those last days of culture in the treat-ment containing 10 pM B, dry weight continued to increase ata slightly faster rate than in treatments containing 50 and 100[kM B, an additional sign of boron deficiency.Boron Deletion during Growth. Ovules were precultured

in 0, 1, 5. and 10 /-M B for 5, 8, 1 . or 14 davs. washed with

0

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n

(I.)

z

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0.4k

932 Plant Physiol. Vol. 54, 1974

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Plant Physiol. Vol. 54, 1974 BORON DEFICIENCY IN COTTON OVULES

FIG. 2. Ovules grown for 14 days ili (a) B(+) medium containing IAA, GA3 and Kin; (b) B(+) medium containing IAA only; (c) B(-)medium containing IAA, GA3 and Kin; (d) B(-) medium containing IAA only. Concentrations when present: IAA, 5 ,uM; GA3, 0.5,uM; Kin,0.05 AM. In treatment a fiber occurred on all surfaces of the ovule. In b less fiber was produced than in a, however, the entire ovule was covered.Treatment c resulted in callusing of the inundated surface of the ovule and reduced fiber production on the upper surface. And in treatment dfiber production on the upper ovular surface was reduced; the lower surface produced no fiber or callus. (Left to right: a, b. c, d).

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media for 14dy.AdaogteoueA utrdwt o

Boro AdditionAduring Growth. The converse of the aboveM0.6

0.6 - A4 ~

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0 5 10 25 50 100 250 500 1000

,UM H3B03

FIG. 3. Effects of boron concentration on development (TFU,

dry weight, and TFU/dry weight) of unfertilized cotton ovules

cultured in the presence of 5 gmM IAA, 0.5 mM GA3, and 0.05 mm

Kin.

B(-) medium, and subcultured in B(-) medium for the re-

mainder of the 14-day culture period. The higher the B con-

centration and the longer the B(+) preculture, the greater the

resultant TFU value (Fig. 6). Irrespective of the B concentra-

tion, ovules transferred to B(-) medium on days 5 and 8 were

callused when the experiment was terminated at day 14. Ovules

transferred to B(-) medium on day 11I appeared less deficient

(more fiber and less callus) than those transferred on days 5 or

8, yet were obviously inferior to ovules left in B-containingmedia for 14 days. And among the ovules cultured with B for

14 days, there was improvement with increasing B concentra-

tion.

Boron Addition during Growth. The converse of the above

_xperiment, preculture in B(-) medium for varying periods,followed by introduction of boron to the system, was carriedout in three ways, each giving essentially identical results. Ineach case, ovules were precultured in B(-) medium for 0, 1,2, 3, 4, 5, 6, or 14 days. In the first of the experiments, 0.5 mlof 10 mm B (100 ,uM final concentration) was asepticallyadded to the 50 ml in each culture flask on the appropriate day,and the culture was allowed to proceed through day 14 (Fig. 7).In the Eecond experiment, ovules were transferred from B(-)to B(+) medium on the appropriate day and, again, culturecontinued through day 14. The B(+) medium, prior to intro-duction of the boron-deficient ovules, had contained otherovules cultured when the experiment was initiated in order tomimic the normal changes in media under usual conditions of

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10 25 50 100

,pM H3803FIG. 4. Effects of boron concentration on development (TFU,

dry weight and TFU/dry weight) of unfertilized cotton ovulescultured in the presence of 5 MM IAA.

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BIRNBAUM, BEASLEY, AND DUGGER

en a, 7 e-

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o 50 uM H3B03

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LL

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5 8 11 14

DAYS POSTANTHESISFIG. 5. TFU and dry weight development on unfertilized cotton

ovules cultured for 5, 8, 11 or 14 days in media containing 10, 50,or 100 uM boron plus 5 yM IAA, 0.5 j4M GA3, and 0.05 uM Kin.

0

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DAYS IN B(-) MEDIUM PRIOR TOB (IOO,uM H3803) ADDITION

FIG. 7. Typical growth in 14 days (TFU, dry weight andTFU/dry weight) of unfertilized cotton ovules cultured for vary-

ing periods in B(-) medium and subcultured to B(+) medium.Media contained 5 AM IAA, 0.5 uM GA3, and 0.05 AM Kin.

U)

zD

cc:LLim

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7-

6

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,AM H3B03 100 10 5DAYS OF B() 14 14 11 8 5 14 11 8 5 14 11 8 5

PRECULTURE

FIG. 6. TFU development on unfertilized cotton ovules pre-cultured in media containing 1, 5 or 10 gM boron for 5, 8, 11 or 14

days, washed with B(-) medium and subcultured in B(-) medium

for the remainder of the 14-day culture period. Media contained5 uM IAA, 0.5 ,M GA3, and 0.05 aM Kin.

full term (14-day) culture. These ovules were discarded priorto introduction of the B(-) ovules. In the third of this seriesof experiments, ovules were subtransferred on the appropriatedays to fresh B(+) medium and allowed to grow 14 days more.

For example, if transferred on day 6 they were terminated after20 days of culture (6 days in B(-) and 14 in B(+) medium).TFU and dry weight data for the three experiments indicatedthat the sooner ovules contacted boron the more fiber theyproduced; though even if ovules were without boron until day6, its introduction at that point still resulted in some fibergrowth. Progression of boron-deficiency symptoms occurred as

described earlier for ovules cultured directly in B(-) medium.Ovules supplied with boron after one day of B(-) preculturewere normal in appearance at the end of the 14 days, thoughthey had less TFU than ovules cultured immediately in B(+)medium. (This was so even if ovules were left in B(+) mediumfor 14 days after the one day of B(-) preculture.) Beyondone day, the longer ovules were cultured in B(-) medium be-fore transfer to B(+) medium, the greater was the increase in

callus, reduction in fiber development, and restriction of fiberto the center of the upper ovular surface. Similar experimentsinvolving medium supplemented with only IAA producedsimilar results, though without proliferation of callus.

DISCUSSION

Epidermal cells of the cotton ovule that will extend into lintfibers (fibers of commerical interest) begin to elongate on theday of anthesis. They continue to elongate in vitro if fertilizationhas occurred (2) or if IAA and/or GA are furnished exoge-nously (1). In B(+) medium, exogenous IAA induces fiber

elongation: GA3 brings about less fiber development and causes

9

934 Plant Physiol. Vol. 54, 1974

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BORON DEFICIENCY IN COTTON OVULES

some proliferation of callus, and combination of the two re-sults in additive fiber production and only slight callus forma-tion. In the absence of boron, if only IAA is added, elongationof fiber cells is reduced and restricted to the upper ovularsurface; if boron is deleted and only GA3 is added, elongationof fiber cells is prevented and callus formation is stimulatedover the entire ovular surface. Combination of IAA and GA3in B(-) medium resulted in ovules intermediate in quality be-tween those grown with each phytohormone individually, i.e.,callus proliferated from the inundated and lateral surfaces ofthe ovule and fiber was restricted to the uncallused center ofthe upper ovular surface. Thus, it appears that IAA, whethersynthesized endogenously in fertilized ovules or added to thegrowth medium of unfertilized ovules, may be of greater im-portance to fiber production and normal ovular developmentthan GA3.

B(-) preculture experiments indicated that normal mor-phogenesis of unfertilized ovules could be restored by additionof boron before the third day of culture. This, together withthe observation that abnormal growth in the absence of boronoccurred primarily on the ovular surface in direct contact withthe culture medium, suggests that it takes 2 to 3 days for boron,or substances through which boron functions, to be depletedfrom cells of the epidermis into the medium. Cells more dis-tant from the medium can be induced to elongate into fibereven later in the culture period by addition of boron to themedium. Thus, it seems that a lower level of boron, or inter-mediate substances, not sufficient to promote fiber elongation,can maintain an arrested metabolic condition in these cells.A constant supply of boron appears to be necessary to main-

tain fiber elongation and prevent callusing of epidermal (andsubepidermal) cells. Therefore, it would seem that boron is notrequired for cell division and, in fact, its absence promotes thedivision-inducing (callus-forming) capacity of GA3. Rather,it seems that boron is required for fiber elongation in responseto IAA.

Skok (12) reported that boron-deficient sunflower seedlingswere more resistant to X-irradiation, suggesting that reducedmetabolic activity coincided with lower boron levels. He furtherobserved that cell maturation rather than division appeared tobe most affected by boron deficiency. In our system, boron de-ficiency-induced arrest of fiber elongation tends to support theideas that boron is involved in cell maturation, and that borondeficiency results in reduced metabolic activity. However, moresevere boron deficiency (GA3-related) involving proliferationof callus, indicates high, though aberrant, metabolic activity.Perhaps low levels of boron result in reduced, yet still normalmetabolic activity (and consequently, retarded maturation)while even lower levels allow the observed aberrant growth tooccur.

Various workers have suggested that there is a direct re-lationship between boron nutrition and IAA metabolism; how-ever, they disagree concerning the nature of the relationship.Jaweed and Scott (10) found greater amounts of IAA in boron-deficient sunflower plants. Coke and Whittington (6) reportedthat both low boron and high IAA concentrations resulted in

reduced elongation of bean roots and that roots grown in excessIAA recovered more rapidly in media containing high con-centrations of boron. Extracts of boron-deficient root apicesappeared to contain higher than normal concentrations ofIAA-like substances. Thus, it was suggested that boron de-ficiency was related to IAA excess either because IAA accumu-lated due to impairment of growth processes in which it wasinvolved or because IAA oxidase was inactivated by phenolicinhibitors. Eaton (9), on the other hand, observed similaritiesbetween boron deficiency and IAA deficiency, and Dyar andWebb (8) found that meristems of boron-sufficient bean plantsdid not respond to naphthaleneacetic acid, whereas boron-deficient meristems did, suggesting that the concentration ofendogenous auxin was suboptimal in the boron-deficient plants.Brown pigments (probably phenolic compounds) accumulatedin cotton ovules grown in B(-) medium, and the coincident re-duced fiber elongation might be considered a sign of IAA de-ficiency. Furthermore, callusing in response to GA3 in boron-deficient ovules suggests an imbalance in the relationship ofIAA and GA3. These observations tend to support the latterhypothesis that boron deficiency and IAA deficiency might berelated. However, reduced fiber elongation can also be causedby IAA toxicity (1), and this too might be the case duringboron deficiency.

Further work in this area is necessary to determine the exactnature of these relationships and whether they are directlyrelated to boron metabolism or are merely secondary effects.

LITERATURE CITED

1. BEASLEY, C. A. 1973. Hormonal regulation of growtli in unfertilized cottonovxles. Science 179: 1003-1005.

2. BEASLEY, C. A. AND I. P. TING. 1973. The effects of plant gr-owth substanceson iin itro fiber development from fertilized cotton ovules. Amer. J. Bot.60: 130-139.

3. BEASLEY, C. A. AN-D I. P. TING. 1974. Effects of plant growth substances onitl vitro fiber dlevelolpment from tinfertilize(d cotton ovules. Anmer. J. Bot. 61:188-194.

4. BEASLEY. C. A. AND I. P. TiNG. 1974. Plvyteliormone effects on i'n lvitro cottonseed (le-elop)ment. 8th International Conference on Plant Growth Substances.Tokyo, Japan. In piess.

5. BEASLEY, C. A., E. H. BIRNBAUM, W. M. DUGGER, AND I. P. TING. 1974. Aquantitative procedure for estimation of growth of cotton fibers. Stain Tech-nology. 49: 85-92.

6. COKE, L. AND W. J. WHITTINGTO-N. 1968. The role of boron in plant growth.IV. Interrelationships between boron and Indol-3yl-acetic acid in the me-tabolism of bean radicles. J. Exp. Bot. 19: 295-308.

7. DU-GGER, W. A1. 1973. Functional aspects of boron in plants. In: E. L. Kothny,ed., Trace elements in the environment. Advances in Chemistry Series 123.American Chemical Society, Washington, D. C. pp. 112-129.

8. DYAR, J. J. AND K. L. WE-BB. 1961. A relationship between boron and auxin in14C translocation in bean plants. Plant Phvysol. 36: 672-676.

9. EATON, F. 'M. 1940. Interrelations in the effects of boron and indoleacetic acidin plant growth. Bot. Gaz. 101: 700-705.

10. JAWEED, 'I. 'I. AND E. G. SCOTT. 1967. Effect of boron on ribonucleic acid andindoleacetic acid metabolism in the apical meristems of sunflower plants.Proc. WVest V'a. Acad. Sci. 39: 180-193.

11. KuNiN, R. 1973. A macroreticular boron-specific ion-exchange resin. Traceelements in the en-ironment. In: E. L. Kothny, ed., Advances in ChemistrySeries 123. American Chemical Society, Washington, D.C. pp. 139-143.

12. SKOK, J. 1957. Relationship of boron nt3trition to radiosensitivity of sunflowerplants. Plant Physiol. 32: 648-658.

Plant Physiol. Vol. 54, 1974 935

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