J. AMER. SOC. HORT. SCI. 121(1):101–104. 1996.

Effects of Abscisic Acid on ex vitro Acclimatization

of Aronia arbutifolia (L.) Pers.
Wilfredo Colón-Guasp1, Terril A. Nell2, Michael E. Kane3, and James E. Barrett2
Department of Environmental Horticulture, University of Florida, Gainesville, FL 32611
Additional index words. leaf carbon assimilation, micropropagation, nonstructural carbohydrates, woody plant
Abstract. The use of abscisic acid (ABA) as an in vitro prehardening treatment to enhance ex vitro acclimatization of Stage
III Aronia arbutifolia plantlets was explored. Effects of ABA (0-4 mg·liter-1) pretreatment on ex vitro shoot growth, leaf
carbon assimilation (LCA) and nonstructural carbohydrate content were evaluated during plantlet acclimatization under
two photosynthetic photon flux (PPF) levels (450 and 650 µmol·m-2· s-1). Stage III plantlets rooted in the presence of ABA
exhibited both shoot growth inhibition and transient negative LCA rates at time of transfer ex vitro. Regardless of
treatment, maximum LCA rates were achieved by day 20 post-transplant. Pretreatment with ABA had no effect on stem
or leaf starch content at time of transplant, however, leaf and stem soluble sugar content was higher in ABA treated
plantlets than controls. Further suppression of shoot growth and alteration in the pattern of stem starch utilization
occurred at the higher irradiance level. These results indicate that ABA pretreatments provide no physiological advantage
that would facilitate ex vitro acclimatization of Aronia plantlets.

The ultimate success of shoot culture (in vitro propagation) plantlets of the woody shrub Aronia arbutifolia (Rosaceae), produce
depends on the ability to transfer and reestablish vigorously growing leaves with morphological and anatomical features similar to green-
plants from in vitro to ex vitro conditions. This involves acclimatizing house-grown plants when cultured in vitro in the presence of ABA.
or hardening-off plantlets to conditions of lower relative humidity Conceivably, medium supplementation with ABA during
and higher light levels. During the acclimatization process, tissue microcutting rooting (Stage III) could serve as an in vitro pre-
cultured plantlets undergo changes in both leaf anatomy (Capellades hardening treatment to prematurely induce developmental and physi-
et al., 1990) and physiology (Grout and Millam, 1985), which confer ological changes, which decrease water loss, increase photosynthetic
the plants with a greater potential for survival ex vitro. Even when capacity and thus increase the ability of the plantlet to survive ex vitro.
acclimatization procedures are followed, transplant survival can be The consequence of ABA pretreatment in vitro on subsequent
low due to the inability of plantlets to maintain adequate water transpiration, nonstructural carbohydrate content, and leaf carbon
relations or fully transition from a mixotrophic to a photoautotrophic assimilation rates (LCA) of rooted microcuttings during ex vitro
mode of nutrition (Grout and Aston; 1978, Wardle et al., 1983). acclimatization is unknown. Transient reductions in transpiration
A composite of anatomical, morphological, and physiological and photosynthesis have been reported for plants following ABA
features, characteristic of plants cultured in vitro under low light application in vivo (Arteca et al., 1985). In some cases, greater
intensity and high relative humidity, contribute to the decreased reductions in transpiration than net photosynthetic rate have been
survival of plantlets often observed immediately following trans- observed in ABA-treated plants (Blake et al., 1990). Where exam-
planting. These features include reduction in leaf epicuticular wax ined, sucrose/starch synthesis ratios are not significantly affected by
deposition, abnormal stomate function, poorly developed stem to ABA treatment in vivo (Sharkey et al., 1985). The objective of this
root vascularization, and limited photosynthetic competence (Brainerd study was to characterize the effects of ABA pretreatment in vitro on
and Fuchigami, 1982; Preece and Sutter, 1991). The modifications in subsequent changes in shoot growth, LCA, and nonstructural carbo-
leaf development and photosynthetic competence that typically hydrate content of A. arbutifolia plantlets during acclimatization.
occur during ex vitro acclimatization appear to be inducible. In vitro
culture of plantlets under conditions of higher light levels and lower Materials and Methods
relative humidity conditions induce anatomical modifications of
foliar epicuticular wax, stomata, and epidermal cells similar to those Stage 1/11: Culture establishment and shoot multiplication. Stems
produced on acclimatized greenhouse-grown plants (Capellades et with lateral buds of A. arbutifolia were cut from actively growing and
al., 1990). However, the physiological mechanisms regulating accli- sexually mature plants, divided into 15-mm lengths with two to three
matization remain obscure. lateral buds and rinsed in tap water for 1 h. Lateral buds were surface
Induction of similar changes in leaf developmental patterns sterilized by repeated immersion in 50% (v/v) ethanol for 1 min and
following abscisic acid treatment in vivo (Zeevaart and Creelman, then in 1.05% (v/v) sodium hypochlorite for 12 rein, followed by
1988) and in vitro (Jarret and Gawel, 1991; Kane and Albert, 1989) three 5-min rinses in sterile deionized water (Kane et al., 1991). The
suggest a possible role of endogenous ABA in the acclimatization sterilized nodal explants were transferred to 25 × 150-mm culture
process. In a previous study (Colon et al., 1990) we reported that tubes containing 15 ml of medium consisting of woody plant medium
(WPM), salts and vitamins (Lloyd and McCown, 1980), 3% (w/v)
sucrose, 2 mg·liter-1 N 6-benzylaminopttrine (BA) and solidified with
Received for publication 7 Apr. 1995. Accepted for publication 19 Sept. 1995.
Florida Agricultural Experiment Station Journal Series no. R-004463. The cost of 1.0% (w/v) TC agar (JRH Biosciences, Lenexa, Kan.) The medium
publishing this paper was defrayed in part by the payment of page charges. Under pH was adjusted to 5.5 with 0.1 N KOH before autoclaving at 1.2
postal regulations, this paper therefore must be hereby marked advertisement solely kg·cm -2 pressure for 20 min at 121 C. All cultures were placed under
to indicate this fact. a 16-h photoperiod provided by cool-white fluorescent lamps at 45
1
Current address: Escuela Agrícola Panamericana, P.O. Box 93, Tegucigalpa,
µmol·m -2·s-1. Air temperature was maintained at 25 ± 2C.
Honduras.
2
Professor. Stock cultures were maintained by subdividing shoots (15 mm
3
Associate professor. To whom reprint requests should be addressed. long bearing three axillary buds with attached subtending leaves)

J. A MER . SOC. HORT. SCI. 121(1): 101–104. 1996. 101

and transferring them onto fresh WPM every 5 to 7 weeks. added and the samples were placed for 20 minutes in a water bath
Stage III: Microcutting rooting and treatments. R o o t e d at 25C. Standard solutions of glucose ranging in concentrations
microcuttings were prepared by cutting 5-week-old shoots into 10- from 10 to 70 µg·ml -1 were prepared and treated in the same
mm stem segments consisting of two or three nodes. Fifteen manner. The amount of soluble sugars present in the plant extracts
microcuttings were transferred into 473-ml clear polypropylene was determined spectrophotometrically (Lambda 3A; Perkin-
culture vessels (Better Plastics, Kissimmee, Fla.) containing 100 Elmer, Norwalk, Corm.) at 490 nm.
ml of agar-solidified WPM supplemented with 2 mg·liter -1 o f Chlorophyll determinations were made following the proce-
indole-3-butyric acid (IBA) but without BA. Synthetic ABA (90% dure outlined by Bruinsma ( 1963). After 30 days of in vitro growth,
mixed isomers, Sigma Chemical, St. Louis) was prepared as a fully expanded leaves were excised, weighed, dried in a freeze
concentrated aqueous stock solution and sterilized by Millipore drier, pulverized, and added to 10 ml of 80% acetone. Solutions
filtration (pore size: 0.22 µm) before being added to molten (40C) were placed in a refrigerator in the dark at 4C for 24 h, after which
sterile medium. All cultures were maintained under the aforemen- optical densities were measured at 663, 645, and 652 nm for
tioned conditions. chlorophyll a, b, and total, respectively. Carotenoid content was
Stage IV: Ex vitro establishment. After 30 days, ABA treated and determined spectrophotometrically at a wavelength of 480 nm.
control Stage III rooted microcuttings were transplanted into plug Data were analyzed by analysis of variance by SAS General
trays (50 cells per tray) containing sterile Rootcubes (Smithers- Linear Model procedure. First (linear) or second (quadratic) order
Oasis, Kent, Ohio). Rooted microcuttings were fertilized weekly polynomials were fitted to the data by regression analysis.
with a 20N–8.8P–16.6K soluble fertilizer (200 mg·liter-1 N). Trays
were placed in a walk-in growth room at 24 ± 2C in a 16-h Results and Discussion
photoperiod provided by metal arc High Intensity Discharge (HID)
lamps (GTE, Manchester, N.H.). Rooted micro-cuttings were placed There was no significant PPF × ABA interaction on shoot growth
under two PPF treatments (450 µmol·m-2·s-1 and 650 µmol·m-2·s-1). or nonstructural carbohydrate content of planted during ex vitro
PPF attenuation was provided by polypropylene shade cloths. At acclimatization (Tables 1 and 2). Plantlets grown in vitro for 30 days
transplanting, humidity domes were placed over the trays and pro- on ABA-supplemented medium exhibited reduced shoot growth ex
gressively opened until fully removed at 10 days. Plants were watered vitro compared to control plants (Table 1). Shoot growth was further
as needed to maintain substrate moisture. inhibited at the higher irradiance level. Root growth was also reduced
Abscisic acid levels of 0, 2, or 4 mg·liter-1 were applied in vitro in the presence of ABA (data not shown). Plantlets cultured on ABA-
in Stage 111 culture in a randomized complete block design with free medium exhibited positive but very low LCA at the time of
four replications. Ten plants were chosen as subsamples for transfer to ex vitro conditions (Fig. 1 A and B). This indicates a
determination of LCA. LCA rates were determined at 0, 10,20, and capacity for rapid conversion to the photoautotrophic state upon
27 days after transfer with a Clark-type oxygen electrode transfer since Aronia plantlets exhibit negative LCA during in vitro
(Hanastech, Norfolk, England). Immediately before measure- culture (Colon et al., 1990). Leaf carbon assimilation rates were
ment, leaves were cut and transferred to a leaf cuvette. Oxygen initially negative in plantlets pre-cultured with ABA (Fig. 1 A and B).
evolution was recorded using an oxygen electrode mounted in a However, ABA-induced suppression of LCA was transient. With
cooling jacket (LD2; Hansatech) connected to a temperature respect to LCA, maximum acclimatization was achieved by day 20
controlling bath and light was supplied by a red diode light source regardless of treatment. Survival of Aronia plantlets ex vitro was
(600 rim). Carbon dioxide was maintained at a constant level by 100% regardless of initial LCA at time of transplant.
adding 1 ml 1 M Na 2HCO 3 to the mat under the leaf disc (Delieu and The physiological basis for the greater initial suppression of LCA
Walker, 1983). The leaf material was illuminated for 4 min at a PPF in ABA-treated plantlets is not apparent. Low photosynthetic capac-
of 450 µmol·m -2· s-1 and oxygen evolution was measured when ity of plantlets in vitro is attributed to several factors including low
steady state was reached. chlorophyll content, reduced ribulose bisphosphate carboxylase ac-
In another experiment 2 mg·liter-1 IBA and ABA at 0, 1, 2, or 3 tivity and non-stomatal inhibition of photosynthesis following starch
mg·liter -1 were incorporated into the medium in Stage III culture in accumulation in chloroplasts (Capellades et al., 1991; Grout and
a randomized complete block design with five replications. At the Aston, 1978). No significant differences in chlorophyll or carotenoid
end of 30 days of in vitro growth and at 5, 10, 20, and 30 days post content were observed between control and ABA-treated plantlets
transplant, rooted microcuttings were assayed for nonstructural (data not shown).
carbohydrate content (leaf and stem soluble sugars and starch) and Depression of LCA in ABA treated plantlets may be the conse-
shoot growth. quence of increased stomatal resistance resulting from ABA-induced
Soluble sugars and starch content of leaf and stem tissue were stomatal closure. Stomata in leaves produced in vitro are often
extracted and analyzed with the phenol-sulfuric technique (Dubois nonfunctional but become responsive following 4 to 5 days acclima-
et al., 1956). Whole plants were collected at 1500 h and placed in tization to reduced relative humidity (Brainerd and Fuchigami,
an oven at 60C. Dried and ground leaf and stem material (0.01 g) 1981, 1982; Shackel et al., 1990). Leaves produced in vitro on Aronia
was extracted with 8 ml of 80% (v/v) ethanol at 95C for 20 min. plantlets cultured in the presence of ABA have epidermal features,
After cooling, the samples were filtered through glass microfibre including stomata, that are structurally similar to those produced on
filters (Whatman, Maidstone, England). The dried pellet recov- greenhouse-grown plants (Colon et al., 1990). Conceivably, these
ered from the filtrate was hydrated with 2 ml of 0.1 M acetate buffer stomata are functional but initially closed during acclimatization.
(pH 5.6) in a water bath at 85C for about 1 h. After cooling to 37C, However, a direct affect of ABA on carbon fixation can not be ruled
3 ml of enzyme solution (31 units alpha amylase/ml with 25.5 units out (Zeevaart and Creelman, 1988).
of amyloglucosidase/-ml, and 0.44 mg CaCl 2/ml) was added and Abscisic acid treatment had a significant main effect on
incubated in a shaking water bath for 24 h at 37C. Samples were plantlet leaf and stem soluble sugar content and leaf starch content
diluted to obtain a soluble sugar concentration between 15 and 25 over the acclimatization period (Table 2). There was also a
µg·ml -1. An aliquot of 1 ml was taken and 1 ml of 5% phenol was significant main effect of PPF level on leaf and stem soluble sugar
added and agitated. Finally, 5 ml of concentrated sulfuric acid was content and on stem starch content (Table 2). After 30 days in vitro

102 J. AMER . SOC. HORT. SCI. 121(1): 101–104. 1996.

 Table 1. Effects of ABA (0, 1, 2, and 3 mg·liter-1) on shoot length (mm) of Aronia arbutifolia rooted
microcuttings grown ex vitro under 450 and 650 µmol·m-2·s-1 PPF for 5, 10, 15, 20, 25, and 30 days.

Treatment Shoot length (mm)

PPF ABA Days post-transplant
-2 -1 -1
( µ m o l · m · s ) (mg·liter ) 0 5 10 15 20 25 30

450
0 15 18 23 28 37 59 93
1 14 17 21 24 30 49 75
2 13 14 17 19 24 38 61
3 13 14 17 19 23 39 61
Linear * ** ** ** ** ** **
Quadratic NS NS NS NS NS NS NS
650
0 16 20 24 28 35 46 61
1 12 13 16 17 20 30 44
2 12 13 15 16 20 26 35
3 11 12 15 16 20 29 41
Linear ** ** ** ** ** ** **
Quadratic * ** ** ** ** ** *
PPF × ABA NS * NS NS NS NS NS
PPF (t test) NS NS * * * * *
NS,*,**
Significant or nonsignificant at P ≤ 0.05 or 0.01, respectively.

(0 days after transfer ex vitro), ABA pretreatments had no signifi- treatments were observed by 20 days after transfer. Beyond 10 days
cant effect on leaf or stem starch content (Fig. 2 A and B and Fig. post-transplant the increased shoot growth observed (Table 1) was
3 A and B) as determined at the time of transfer. Starch content perhaps due tore-mobilization of carbohydrate from leaf and stem
rapidly increased before and then declined after day five post- carbohydrate reserves to areas of active growth.
transplant in both leaf and stem tissues. The accumulation of Based on starch utilization, Stage III Aronia rooted plantlets
starch from new photosynthate is unlikely since LCA were low or remain highly dependent on carbohydrate reserves for sustained
negative during this period. The possibility of carbohydrate growth and development of photosynthetically competent leaves
translocation from the roots was not examined. Stem starch during initial acclimatization. Starch reserves became depleted
utilization was delayed in plantlets pre-cultured with ABA and within the first 20 days. Lack of sufficient energy reserves for both
maintained at the higher PPF (Fig. 3B). adventitious root formation and growth maintenance would ex-
Leaf (Fig. 2 C and D) and stem (Fig. 3 C and D) soluble sugar levels plain the poor ex vitro survival of unrooted Stage II Aronia
were greater in ABA-treated plantlets than in control plantlets at the microcuttings (Kane et al., 1991). Treatments that increase stem
time of transfer. Under the higher PPF, transient elevations in soluble starch content might facilitate ex vitro survival of more problem-
sugar levels were observed in both leaf (Fig. 2D) and stem (Fig. 3D) atic species. While in vitro application of ABA induced starch
tissues of ABA-treated plantlets. However, no differences between deposition in other plants (Smart and Trewavas, 1983), no such

Table 2. F values from GLM of ABA (0, 1, 2, 3 mg·liter -1 ) and PPF (450 and 650 µmol·m -2 ·s -1 ) two factor
experiment on leaf and stem soluble sugar and starch content (mg·g-1 dry weight) after transfer from an in vitro
to an ex vitro environment.

Sugar Starch
Plant Model F Model F
part R2 Source df value R2 Source df value
Leaf 0.67** ABA 3 11** 0.77** ABA 3 7.53**
PPF 1 16** PPF z 1 0.28 NS
D A Ty 4 30** D A Ty 4 82**
PPF × ABA 3 0.55 NS PPF × ABA 3 0.58 NS
DAT × PPF 4 2.68* DAT × PPF 4 0.82 NS
DAT × ABA 12 3.02** DAT × ABA 12 1.34 NS
Stem 0.53** ABA 3 5** 0.76** ABA 3 1.05 NS
PPF 1 29** PPF 1 6*
DAT 4 6** DAT 4 74**
PPF × ABA 3 0.67 NS PPF × ABA 3 0.49 NS
DAT × PPF 4 8** DAT × PPF 4 4**
DAT × ABA 12 1.56 N S DAT × ABA 12 1 . 0 0N S
z
Photosynthetic photon flux.
y
Days after transfer.
NS,*,**
Significant or nonsignificant at P ≤ 0.05, or 0.01, respectively.

J. A MER. SOC. HORT. SCI. 121(1): 101–104. 1996. 103

Fig. 1. Effect of ABA(0–4 mg·liter-1)pre-treatment on subsequent leaf carbon
assimilation (LCA) of rooted A. arbutifolia plantlets during acclimatization
under 450 (A) and 650 µmol·m-2· s-1 (B) PPF for 27 days. Values are mean ± SE.

response was observed in Aronia.

Although development of cutinized greenhouse-type leaves are
induced in vitro in the presence of ABA, the suppression of shoot
growth and LCA that occur provide no physiological benefit that
Fig. 2. Leaf soluble sugar and starch content of A. arbutifolia rooted plantlets pre-
would facilitate ex vitro acclimatization of Aronia plantlets. How-
treated in vitro with ABA (0, 1, 2, and 3 mg·liter -1) and grown under 450 (A and
ever, studies of the ABA effects on acclimatization of more C) and 650 µmol·m-2·s-1 (B and D) PPF during a 30 day growing period after
problematic species could prove useful. transfer from in vitro. Values are mean ± SE.

Literature Cited
Arteca, R.N., D. Tsai, and C. Schlagnhaufer. 1985. Abscisic acid effects on photosyn-
thesis and transpiration in geranium cuttings. HortScience 20:370–372.
Blake, T.J., W. Tan, and S.R. Abrams, 1990. Antitranspirant action of abscisic acid
and ten synthetic analogs in black spruce. Physiol. Plant 80:365–370.
Brainerd, K.E. and L.H. Fuchigami. 1981. Acclimatization of aseptically cultured
apple plants to low relative humidity. J. Amer. Soc. Hort. Sci. 106:515–518.
Brainerd, K.E. and L.H. Fuchigami. 1982. Stomatal functioning of in vitro and greenhouse
apple leaves in darkness, mannitol, abscisic acid, and CO2.J. Expt. Bot. 33:388–392.
Bruinsma, J. 1963. The quantitative analysis of chlorophylls a and b in plant extracts.
Photochem. Photobiol. (Chlor. Metabol. Symp.) 2:241–249.
Capellades, M., R. Fontarnau, C. Carulla, and P. Debergh. 1990. Environment
influences anatomy of stomata and epidermal cells in tissue-cultured Rosa multi-
flora. J. Amer. Soc. Hort. Sci. 115:141–145.
Capellades, M., R. Lemeur, and P. Debergh. 1991. Effects of sucrose on starch
accumulation and rate of photosynthesis in Rosa cultured in vitro. Plant Cell Tissue
Organ Cult. 25:21-26.
Colon, W., M. E. Kane, and D. L. Ingram. 1990. Effects of abscisic acid on
photosynthesis, growth and development of Stage III Aronia arbutifolia (Ro-
saceae). Proc. Fla. State Hort. Soc. 103: 178–182.
Delieu, T.J. and D.A. Walker. 1983. Simultaneous measurement of oxygen evolution
and chlorophyll fluorescence from leaf pieces. Plant Physiol. 73:534–541. Fig. 3. Stem soluble sugar and starch content of A. arbutifolia rooted plantlets pre-
Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956. Colorimet- treated in vitro with ABA (0, 1, 2, and 3 mg·liter -1) and grown under 450 (A and
ric methods for determination of sugars and related substances. Analytical Chem. C) and650~molm -’s-’ (B and D) PPF during a 30 day growing period after
28:350-356. transfer from in vitro. Values are mean ± SE.
Grout, B.W.W. and M.J. Aston. 1978. Transplanting of cauliflower plants regener-
ated from meristem culture. II. Carbon dioxide fixation and the development of greenhouse and field, p. 71-93. In: P.C. Debergh and R. H. Zimmerman (eds.).
photosynthetic ability. Hort. Res. 17:65–71. Micropropagation Technology and Application. Kluwer Acad. Publ., Boston.
Grout, B.W.W. and S. Millam. 1985. Photosynthetic development of micropropa- Shackel, K.A., V. Novello, and E.G. Sutter. 1990. Stomatal function and cuticular
gated strawberry plantlets following transplanting. Annu. Bot. 55:129–131. conductance in whole tissue-cultured apple shoots. 1990. J. Amer. Soc. Hort. Sci.
Jarret, R.L. and N. Gawel, 1991. Abscisic acid-induced growth inhibition of sweet potato 115:468–472.
(Ipomea batatas L.) in vitro. Plant Cell, Tissue, and Organ Culture. 24:13-18. Sharkey, T.D., J.A. Berry, and K. Raschke. 1985. Starch and sucrose synthesis in
Kane, M.E. and L.S. Albert. 1989. Abscisic acid induction of aerial leaf development Phaseolus vulgaris as affectedly light, CO2, and abscisic acid. Plant Physiol.
in Myriophyllum and Proserpinaca species cultured in vitro. J. Aquatic Plant Mgt. 77:617–620.
27:102-111. Smart, C.C. and A.J. Trewavas. 1983. Abscisic-acid-induced turion formation in
Kane, M. E., B. Dehgan, and T.J. Sheehan. 1991. In vitro propagation of Florida native Spirodela polyrrhiza L. 1. production and development of the turion. Plant Cell
plants: Aronia arbutifolia. Proc. Fla. State Hort. Soc. 104:287-290. Environ. 6:507–514.
Lloyd, G. and B. McCown. 1980. Commercially-feasible micropropagation of Wardle, K., E.B. Dobbs, and K.C. Short. 1983. In vitro acclimatization of aseptically
mountain laurel Kalmia latifolia, by use of shoot-tip culture. Proc. Intl. Plant Prop. cultured plantlets to humidity. J. Amer. Soc. Hort. Sci. 108:386–389.
Soc. 30:421-427. Zeevaart, J.A.D. and R.A. Creelman. 1988. Metabolism and physiology of abscisic
Preece, J.E. and E.G. Sutter. 1991. Acclimatization of micropropagated plants to acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:439473.

104 J. AMER . SOC. HORT . SCI. 121(1):101–104. 1996.