HortS cience 19(6):837—839. 1984. dard curve of glucose for quantitation.

Val­
ues from samples without invertase represent
Influence of Paclobutrazol on Selected reducing sugars, and the differences in read­
ings of samples with and without invertase
represent nonreducing sugars.
Growth and Chemical Characteristics The residue remaining after ethanol ex­
traction was digested with glucoamylase ac­
of Young Pecan Seedlings cording to the method of Thievend et al. (11)
for the determination of starch. Twenty ml
Bruce W. Wood1 distilled water was added to the sugar-free
ARS, U.S. Department of Agriculture, Southeastern Fruit and Tree Nut residue and heated at 80°C for 1 hr. After
cooling, 2.5 ml of 2 m acetate buffer (pH
Research Laboratory, P.O. Box 87, Byron, GA 31008 4.8) and 25 ml of distilled water were added.
Additional index words, growth inhibitor, growth regulator, Carya illinoinensis, PP 333 Glucoamylase (250 mg) (Sigma, St. Louis,
Mo.) dissolved in 5 ml of acetate buffer then
Abstract. Soil drench applications of paclobutrazol (0, 0.5, 1, 2, 4, 8, 16, and 32 mg was added and heated at 60° with continuous
a.i.-pot'1) to greenhouse grown pecan seedlings reduced plant height, plant dry weight, shaking for one hr. The solution then was
organ dry weight, in ter node length, leaf thickness, leaf area, and chlorophyll content. filtered, diluted, and an aliquot analyzed for
Carbohydrate levels (mg-g dry weight'1 and mg-g plant'1) in treated plants increased. glucose-equivalents using the colorimetric
Total plant carbohydrate levels were unchanged at levels < 2 m g a.i.-pot'1, but plants procedure described for reducing sugars.
of reduced size showed increased levels of carbohydrates per mg of tissue. Seedlings
treated with high levels of paclobutrazol had a slight tendency for increased net pho­
tosynthesis.

Tree size is a major problem confronting and planted 3 cm deep in 160 cm3 of 1 soil
the pecan [Carya illinoinensis (Wangenh.) : 1 vermiculite (v/v) in 15 cm clay pots. Pa­
K. Koch] industry. Trees grow rapidly and clobutrazol (50% a.i.-WP) was mixed with
commonly exceed 25 m in height, thus mak­ 200 ml of water and applied, at the time of
ing pest management, harvesting, and other plumule emergence from the soil surface, as
cultural methods both difficult and expen­ a soil drench at concentrations of 0, 0.5, 1,
sive. During spring shoot growth, large 2, 4, 8, 16, and 32 mg a.i. per pot. Plumule
amounts of energy reserves are partitioned emergence was uniform, with the range of
into vegetative growth, thus potentially de­ emergence being 3 days. Experimental de­
pleting reserves available for nut production. sign was a randomized complete block with
This characteristic likely contributes to ir­ 10 replicates. Seedlings were greenhouse
regular bearing, a major production problem grown without supplemental lighting under
(9, 16, 17). Redirecting energy reserves to a natural 16 hr photoperiod until the check
reproductive growth, and reducing tree size ceased vertical growth (about 11 weeks). Plant
may increase tree productivity and reduce height then was determined by measuring
irregular bearing. Paclobutrazol (ICI-PP333; from the root collar to the shoot apex. Leaf
1- (4-chlorophenyl) -4,4-dimethyl-2- (1,2,4- nodes were counted and total plant leaf area
triazol-l-yl)pentan-3-ol) is a new plant growth determined by a LI-COR 3100 leaf area me­
inhibitor that has exhibited retardant effects ter. The soil mix was washed from the root
in apple (3, 5, 7, 12, 13) and various orna­ system, and seedlings were separated into
mental species (2, 8). Paclobutrazol report­ shoot, leaf, tap root, and lateral root com­
edly reduces shoot growth and increases root ponents. Plant components then were lyo-
to leaf ratio and tolerance to water stress (10). phylized, weighed, ground, and assayed for
Similar influences on pecan would be desir­ starch and reducing and nonreducing sugars.
able; however, its influence and potential on Sugars were determined colorimetrically
pecan are currently unknown. How paclo­ using Nelson’s modification of Somogyi’s
butrazol influences net photosynthesis and method (6). Briefly, ground plant material
carbohydrate reserves also is unknown. This (100-500 mg) was extracted in triplicate with
study reports the influence of paclobutrazol 70% ethanol at 70°C for 4 hrs. The super­
on basic growth characteristics, net photo­ natant was collected and the extraction pro­
synthesis, and reserve energy levels of pe­ cedure twice repeated. The residue was
can. retained for starch analysis. Aliquots were
Seeds from ‘Curtis’ pecan were stratified placed in test tubes and dried at 70° in a
water bath. To one set of tubes was added 1
ml of 50 mM sodium acetate buffer (pH 4.7)
R eceiv ed fo r p u b licatio n 3 A pr. 1984. M en tio n o f
a gro w th re g u lato r in this p ap er d o es not co n stitu te to the dried sample. To another set of tubes,
a re c o m m en d atio n fo r u se b y the U S D A n o r does containing aliquots of the same sample, was
it im p ly reg istratio n u n d e r F IF R A . M en tio n o f a added 100 units of invertase (Sigma, St.
trad em ark o r p ro p rietary p ro d u ct d o es not c o n sti­ Louis, Mo.) dissolved in one ml of the above
tute a g u aran tee o r w arra n ty o f the p ro d u ct by the buffer. Tubes then were incubated at 37° for
U S D A an d does not im p ly its ap p ro v al to the e x ­ 1 hr at which time one ml of copper reagent Fig. 1. Influence o f paclobutrazol, applied as 50%
c lu sio n o f o th er p ro d u cts that m ay also be su it­ (6) was added and the tubes were heated to a .i.-W P soil d rench in 15.2 cm d iam eter p ots,
able. T he cost o f publishing this p aper w as defrayed on c a rb o h y d rate levels in g re en h o u se -g ro w n p e ­
80° for 15 min and cooled for 30 min. Sam­
in p art b y the p ay m en t o f pag e ch arg es. U n d er can seedlings. (A) P aclobutrazol effects on starch,
ples then were mixed with 2 ml of arsen- (B) red u cin g sug a rs, (C ) no n red u cin g sugars,
postal re g u latio n s, this p ap er th erefo re m ust be
hereby m arked a d v e r tis e m e n t solely to indicate this
omolybdate reagent and diluted with water. and (D) total c a rb o h y d rates in m ajo r tissues o f
fact. Sugar levels were determined colorimetric­ p ecan (ex pressed as glu co se equivalents) are
ally by measuring absorbance at 500 nm. show n. V ertical bars represent the s e o f the m ean
R e s e a r c h H o rticu ltu rist. Optical density was compared with a stan­ for 10 rep licatio n s.

HortS cience, V ol . 19(6), D ecember 1984 837

 T able 1. In flu en ce o f p aclo b u trazo l on g ro w th characteristics o f pecan seedlings. P aclo b u trazo l w as app lied as a soil d re n c h , to
p lan ts g ro w in g in p o ts co n tain in g 160 c m 3 o f 1 soil : 1 v erm iculite (v/v), w hen the p lum ule broke the soil su rface. M easu rem en ts
w ere tak en w h en p lan ts in the ch eck treatm en t ceased height grow th (11 w eeks after plum ule em erg en ce).

P aclobutrazol (m g a .i.- p o t'1)

P aram eter 0 0 .5 1.0 2 .0 4 .0 8 .0 16.0 3 2 .0 (5% ) S ig n ifican c e3

P lan t h eig h t (cm )y 2 6 .0 17.5 5 .4 5.3 5 .2 5.2 5.1 5 .0 4 .0 L *Q **

In te m o d e length 2 0 .0 14.3 7 .2 4 .5 4.1 3.1 2 .0 2 .0 1.9 L *Q **
(m m )x
T o tal p lan t d ry w tw (g) 13.45 11.59 9 .1 4 9.11 6.71 5 .67 6 .0 8 5 .3 0 2 .1 6 L *Q **
S tem dry w t (g) 1.70 0 .7 0 0 .4 0 0 .5 7 0 .4 5 0 .3 2 0.41 0 .3 4 0.31 L *Q **
L e a f d ry w t (g) 2 .3 2 1.83 1.72 1.75 1.32 1.08 1.26 0 .9 8 0 .4 4 L **Q *
T ap ro o t d ry w t (g) 8 .35 8 .2 8 6 .1 0 6 .13 4.41 3.81 4 .0 2 3 .5 6 1.58 L *Q *
L ateral ro o t dry w t (g) 1.08 0 .7 8 0 .9 2 0 .6 6 0 .5 3 0 .4 6 0 .3 9 0.41 0 .3 4 L *Q *
T o tal p lan t le a f area 523 326 332 326 237 200 222 156 66 L *Q *
(cm 2)
L eav es p e r p lan t (n o .) 7 .4 7 .3 5 .0 4 .8 4 .6 4 .4 4 .5 4 .0 0 .3 L *Q *
R o o t:to p ra tio v 2 .3 2 3 .4 5 3.33 2 .7 8 2 .7 0 3.12 2 .5 0 2 .8 6 0 .2 9 NS
L e a f area: le a f d ry w t 225 178 193 186 180 185 176 159 25 L *Q *
ratio (cm 2- g '1) •n

L e a f ch lo ro p h y ll 3 9 .0 4 0 .5 4 2 .0 4 3 .5 4 5 .5 4 5 .5 4 6 .0 4 9 .0 2.1 L*
(|xM -cm '2)
u
N et p h o to sy n th esis 10.6 10.7 10.9 11.0 11.2 11.4 ... 1.3 NS
(m g C 0 2dm -2h -')
zL in e ar (L); Q u ad ratic (Q ); no sig n ifican ce (ns ); 5% level (*); 1% level (**).
yP lan t h eig h t tak en 11 w eek s a fter p lu m u le em erg ence. T his co rresponds to the cessation o f heig h t grow th o f the ch eck . P lant height
w as tak en fro m the ro o t c o llar to the ap ical bu d .
xIn tem o d e len g th is an av erag e o f the d istan c e from the basal le a f to the apical bud d ivided by leaf n u m ber.
wM ean s o f 10 rep licatio n s.
vD ry w eig h t b asis. R o o t is co m p rised o f b o th tap and lateral roots and top is co m prised o f b oth stem and leaves.
uD ata w ere n o t co llected .

Leaf chlorophyll content was determined plants also had a darker green coloration,
by punching out one cm diameter leaf discs than those from untreated plants, the color
with a cork borer and extracting the chlo­ apparent within 2 weeks after treatment. Pa­
rophyll using 80% acetone. The optical den­ clobutrazol had no significant influence on
sity of the chlorophyll extract was measured net photosynthesis; however, there was a
immediately according to the method of Ar- slight trend for increased rates with increas­
non (1) at 645 and 663 nm. Amon’s equa­ ing levels of paclobutrazol (Table 1).
tions w ere tran sfo rm ed to give m olar Carbohydrate levels (mg-g dry weight"1)
concentrations (22.22) D .D .645 -f 9.057 in all plant parts were affected by paclobu­
O .D .663 = pM C hll'1) as described by Ev­ trazol (Fig. 1). Starch levels increased in tap
ans (4). ; and lateral roots and in stem tissues as treat­
Net photosynthesis was measured by the ment concentration increased. Yet there was
standard open-system differential analysis no influence on leaf starch (Fig. 1-A). Starch
method as described previously (15). Mea­ levels in seedling pecan were highest in the
surements were made on the attached 4th tap root, followed by the stem, leaf, and lat­
leaf of each seedling. Leaves were fully ex­ eral root tissues. Reducing sugars increased
panded at time of measurement. with treatment levels in leaves, lateral roots,
Paclobutrazol altered normal growth char­ and stem. There was no influence on reduc­ F ig. 2. In flu en ce o f v ario u s levels o f p a c lo b u ­
acteristics of pecan seedlings greatly such ing sugars in the tap root. (Fig. 1-B). Non­ trazol (50% a .i.-W P ), app lied as a soil dren ch ,
that plant height, intemode length, total plant reducing sugars were increased in all plant o n p e c a n s e e d lin g c a r b o h y d r a te le v e ls , e x ­
dry weight, dry weights of each plant com­ components with low levels of paclobutra­ p ressed as m g-g p lan t dry w e ig h t'1 and as
ponent, leaf number and leaf area, and leaf zol. High levels reduced such sugars in both m g -p la n t'1. V ertical b ars rep re se n t the S£ of the
dry wt ratio were reduced (Table 1). Effects the tap and lateral roots (Fig. 1-C). Total m ean fo r 10 rep licatio n s.
of paclobutrazol on inhibiting intemode, leaf soluble carbohydrates were increased in all
midrib, and root (observed) length are con­ plant parts, with the greatest increase being lings treated with 1 mg paclobutrazol than in
sistent with the reported mode of action of at the concentrations of paclobutrazol < 2 mg the check. These same treatment levels (< 2
paclobutrazol (i.e., inhibiting gibberellin a.i.-pot'1 (Fig. 1-D). The majority of energy mg a.i.-pot'1) did not affect the level of car­
biosynthesis) (3, 13). There were no signif­ reserves in seedling pecan plants was in the bohydrates when expressed as mg-plant'1 (Fig.
icant differences in the ro o t: shoot ratio (Ta­ tap root; followed by the stem, leaf and lat­ 2), even though plant dry weight and height
ble 1); how ever, the 0.5 and one mg eral roots, respectively. The general increase were reduced by 36% and 80%, respectively
treatments tended to increase the ratio. This in carbohydrates with paclobutrazol treat­ (Table 1). Available reserve energy for fu­
effect has been reported previously for low ment was inversely correlated with plant and ture growth is presumably increased. Prelim­
levels of paclobutrazol on apple (10). The plant component dry weight, and presum­ inary observations of mature pecan trees
ratio change was due to a decline in shoot ably was due to energy or carbohydrate sur­ indicated paclobutrazol increased carbohy­
weight rather than an increase in root dry pluses generated as a result of reduced demand drate levels and the percentage of kernel of
weight. for growth. bearing trees, suggesting productivity may
Paclobutrazol, in addition to causing a re­ Pecan carbohydrate reserves, based on increase with increased energy reserves (14).
duction in leaf area, reduced the leaf area:leaf mg-g p la n t1, were increased with most pa­ Paclobutrazol offers potential as a growth
dry weight ratio. The increase in leaf thick­ clobutrazol treatments but highest in plants retardant that may increase pecan productiv­
ness was accompanied by increased chloro­ treated with < 2m g a.i.-pot'1 (Fig. 2). Car­ ity, provide a means of controlling irregular
phyll content (Table 1). Leaves from treated bohydrate levels were 60% greater in seed- bearing, and facilitate crop management by

838 HortScience, V ol . 19(6), D ecember 1984

 controlling tree size. Pecan physiology seems ity o f ‘D e l i c i o u s ’ a p p le s . H o r tS c ie n c e J .N . B e M iller (e d s.), M eth o d s o f carb o h y ­
to be highly sensitive to paclobutrazol, and * 17(5):474. (A bstr.) drate chem istry, vol. 6. A cadem ic P ress, N ew
there is an apparent absence of short-term 6. H o d g e, J .E . and B .T . H o freiter. 1962. D e ­ Y ork.
adverse effects at reasonable treatment lev­ term in a tio n o f red u cin g sugars and c a rb o ­ 12. T u k e y , L .D . 1983. E x p lo rin g the use o f p.
h y d ra te s, p p . 3 0 0 -3 9 4 . In: R .L . W h istler, 3 3 3 , a new gro w th re g u la to r fo r apple trees.
els. These data indicate that, under the de­
and J .L . W o lfra m (e d s.). M ethods in c a r­ P A F ru it N ew s 6 2 :6 4 -6 6 .
scribed set of experimental conditions, levels
bohydrate chem istry, vol. 1. A cadem ic Press, 13. W illia m s, M .W . and L .J . E d gerton. 1983.
over 4 mg-pot"1 were an overdose and re­ N ew Y ork. V eg etativ e gro w th c o n tro l o f apple and p ear
sulted in several undesirable effects. 7. M iller, S .S . 1982. G ro w th and b ranching o f tr e e s w ith I C I-P P 3 3 3 ( p a c lo b u tra z o l) a
ap p le se edlings as influ e n ced by p ressure- ch em ical analog o f B ay le to n . A cta H ort.
Literature Cited in je c te d p la n t g r o w th re g u la to rs . H o r t­ 1 3 7 :1 1 1 -1 1 6 .
S cience 1 7 (5 )7 7 5 -7 7 6 . 14. W o o d , B .W . 1984. In flu en ce o f p a c lo b u tra ­
1. A m o n , D .I. 1949. C o p p e r en zy m es in iso ­
lated chloroplasts: Polyphenoloxidase in B eta 8. S h anks, J .B . 1980. C hem ical d w arfing o f zol on selected g ro w th and chem ical c h a r­
v u lg a r is . P lan t P h y sio l. 2 4 :1 -1 5 . several o rnam ental green h o u se crops w ith acteristics o f p ecan . H o rtS c ien ce 19(2):204.
2. Barrett, J.E . and C .A . B artuska. 1982. PP333 pp3 33. P roc. P lant G ro w th R eg u la to r W o rk ­ (A b str.)
effe cts o n stem elo n g atio n d e p en d en t on site ing G roup 7 :4 6 -5 1 . 15. W o o d , B .W . and J .A . P ayne. 1984. In flu ­
o f ap p licatio n . H o rtS c ien ce 17(5):737—738. 9. S p arks, D . 1979. P hy sio lo g y -site, gro w th , ence o f single a p p licatio n s o f inse ctic id e s on
3. C u rry , E .A . an d M .W . W illiam s. 1983. flo w erin g , fruiting, and n u tritio n , pp. 2 1 1 - net p h o to sy n th esis o f pecan . H ortS c ien ce
P ro m a lin o r G A 3 in creases p ed icel an d fruit 239. In: R .A . Jaynes (e d .), N ut tree culture 1 9 (2 ):2 6 5 -2 6 6 .
len g th an d le a f size o f ‘D e lic io u s’ apples in N orth A m erica. N orth. N ut G row . A ssn. 16. W o rle y , R .E . 1979. P ecan y ield , q u ality ,
tr e a te d w ith p a c lo b u tr a z o l. H o r tS c ie n c e 10. S w ietlik, D . and S .S . M iller. 1983. T h e e f­ n u tlet set, and spring grow th as a resp o n se
18(2):214—215. fect o f p aclobutrazol on grow th and response to tim e o f fall d efo liatio n . J. A m er. S oc.
4. E v a n s, J .R . 1983. N itro g en an d p h o to sy n ­ to w ater stress o f apple seedlings. J. A m er. H ort. S ci. 104(2): 1 9 2 -1 9 4 .
th esis in th e flag le a f o f w h eat (T r itic u m a e s - Soc. H ort. Sci. 108(6): 1 0 7 6 -1 0 8 0 . 17. W orley, R .E . 1979. Fall defoliation date and
tiv u m L .). P lan t P h y sio l 7 2 :2 9 7 -3 0 2 . 11. T h ie v en d , P ., C . M e rcier, and A . G uilbot. se asonal c a rb o h y d rate c o n c en tratio n o f p e ­
5. G reen e, D .W . 1982. E ffect o f p p3 3 3 and its 1972. D eterm ination o f starch w ith glucoa- can w o o d tissue. J. A m er. S oc. H ort. Sci.
an alo g s o n v eg etativ e g ro w th an d fru it q u al­ m y lase , pp. 1 0 0 -1 0 5 . In: R .L . W h istler and 104(2): 1 9 5 -1 9 9 .

HortS cience 19(6):839-841. 1984.

Inducing Precocious Germination in

Asexual Embryos of Cacao
Yi-Chang Wang and Jules Janick
Department of Horticulture, Purdue University, West Lafayette, IN 47907 F ig. 1. (Left) A sex u al em b ry o o f caca o at the
“ sp a d e ’’ shape e q u iv alen t to 100-day-old z y g ­
Additional index words, cacao, radicle, inhibitor, Theobroma cacao otic em b ry o s; (Center) em bryos w ere co n sid ­
ered g e rm in ated w ith a distin ct ex tru sio n o f
Abstract. Renewing liquid medium induced radicle development in up to 60% of radicle fro m the hypoco ty l; and (Right) g e r­
asexual embryos of cacao (Theobroma cacao L.). In combination with medium renewal, m inating m atu re, 180 day zygotic em bryo.
radicle development was promoted further by eliminating casein hydrolysate and by
reducing the concentration of Murashige and Skoog salts in liquid medium.
opaque embryos to culture tubes containing
Precocious germination is a common fea­ ganic salts according to Murashige and Skoog liquid basal medium (10 ml medium/tube)
ture of cultured embryos (2). Asexual em­ (9); i-inositol, 100 mg; nicotinic acid, 0.5 with different sucrose concentrations (0%,
bryos of cacao proliferated in agar-gelled basal mg; pyridoxine-HCl, 0.5 mg; thiamine-HCl, 1.5%, 3%, 9%, w/v) and placed on the Rol-
medium (without growth regulators) rarely 0.1 mg; glycine, 2.0 mg; casein hydrolysate, lodrum apparatus (TC-6, New Brunswick
germinate. Asexual embryogenesis could 1 g, and sucrose, 15 g (1.5%) with pH ad­ Scientific Co.) rotating at 15 rpms. Media
provide a rapid method for asexual propa­ justed to 5.7 before autoclaving. “ Spade­ were kept constant or renewed twice (day 10
gation of cacao if embryogenesis could be shaped” translucent embryos, about 7 mm and day 20), or 4 times (day 5, 10, 15, 20).
induced in somatic tissue and germination in length, equivalent to about 100- to 120- There were 12 treatments (4 sucrose concen­
could be achieved. Asexual embryogenesis day-old zygotic embryos developing in vivo trations x 3 medium transfer states) with 10
also could be exploited as a method for in (Fig. 1) were transferred to basal medium embryos/treatment; the experiment was ter­
vitro production of cocoa butter and cocoa with 3% sucrose for 1 week where they be­ minated after 25 days. Germination ranged
solids (5) if asexual embryos would develop came opaque. The culture room was main­ from 0% to 60% (Table 1). The optimum
to maturity and if precocious germination tained at a constant 26°C. Light was provided renewal frequency was at the 10-day interval
could be prevented. This study was initiated from cool-white fluorescent lamps (45 |xmol with germination highest at 3% or 9% su­
to investigate factors influencing precocious s -1m -2 PAR) for 16 hr daily. crose.
germination in asexual embryos of cacao. In a series of preliminary experiments in The interaction of medium renewal and
Asexual embryos of cacao, initiated from agar-gelled medium, precocious germination salt concentration on precocious germination
immature zygotic embryos (10, 11), were was not induced by adding gibberellic acid was tested in a 2nd experiment. Embryos
proliferated on an agar-gelled basal medium (GA3) at 0.1, 1, 5, or 10 mg/liter, by na- were transferred into liquid basal medium
consisting of the following per liter: inor- phthaleneacetic acid (NAA) at 0.5, 1, or 5 with 9% sucrose and 2 strengths of MS salts
mg/liter, by light treatments, 0, 45, or 180 (4.33 g/liter = lx, or 2.16 g/liter = l/2x).
(xmol s -1m ~2 PAR, or by injury to the hy- Medium was renewed on day 10. After 21
R e ceiv ed fo r p u b licatio n 16 Jan . 1984. Journal
pocotyl. Leaching asexual embryos in dis­ days, radicle development was 25% (3/12)
P ap er N o . 9 7 6 9 o f th e P u rd u e U n iv . A g ricu ltu ral
E x p erim en t S ta tio n .T h e co st o f pu b lish in g this p a ­
tilled water or renewing liquid medium in with full-strength MS salts and 43% (10/23)
p e r w as d efra y ed in p art b y the p ay m en t o f pag e 3% sucrose induced radicle extrusion and with half-strength MS salts, whereas epico-
c h a r g e s . U n d e r p o s ta l r e g u la tio n s , th is p a p e r elongation (Fig. 1). tyl elongation was 8% (1/12) in full-strength
therefo re m u st be h e reb y m ark ed a d v e r tis e m e n t The effect of medium renewal on preco­ MS salts and 26% (6/23) in half-strength MS
solely to in d icate th is fact. cious germination was tested by transferring salts.

HortScience, V ol . 19(6), D ecember 1984 839