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Plant Cell, Tissue and Organ Culture 72: 153156, 2003. 153 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Development of suitable protocol to overcome hyperhydricity in
carnation during micropropagation
1 2, 1*Manoj K. Yadav , A.K. Gaur & G.K. Garg1 2
Department of Molecular Biology&
Genetic Engineering;
Department of Biochemistry,
College of BasicScience and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, (Uttaranchal) *263 145, India ( requests for offprints)
Received 19 March 2001; accepted in revised form 29 July 2002
Key words: agar, hyperhydricity, metal-ions, MS medium, plant growth regulators, proliferation, shoot regenera-
tion
Abstract
Hyperhydricity during micropropagation of carnation (Dianthus caryophyllus L.) was reduced by media modi-fications. Three commercial varieties (White sim, Exquisite and Scania) tested, varied in optimal growth without
hyperhydricity. Increased concentration of iron and/or magnesium reduced hyperhydricity with 0.70.8% agar.
At some concentrations, hyperhydricity was reduced to 0% and shoot multiplication was increased. All non-
hyperhydrified micropropagated plantlets survived in a glasshouse during acclimatization.
Introduction proaches to overcome hyperhydricity include con-
tainers with good gaseous exchange, different con-
centrations of agar (Kevers and Gasper, 1986; MillerCarnation (Dianthus caryophyllus L.) is one of the
et al., 1991), the growth regulators BA, ABA (Kim etmost important commercial flowers in the world
al., 1988), IAA (Li et al., 1997) and GA (Jain et al.,3(Staby et al., 1978) being excellently suitable, in alia,
1997), the ratio of nitrate to ammonium ions (Tsay etfor cut flowers, bedding pots, borders, edging and al., 1998) and changing levels of calcium chloride,rock gardens. Gill and Arora (1988) have tested the
ammonium nitrate, potassium nitrate (Choudhary etperformance of Sim carnations for various characters.
al., 1993). In this paper, an efficient and reproducibleHyperhydricity is a serious problem during in vitro
protocol for shoot proliferation without hyperhydrici-culture of carnation, which directly affects the pro-
ty for micropropagation of caranation is reported.duction at commercial level. Thus, in vitro cultured
plantlets do not survive when transferred to soil due to
yellowing, swelling, glassiness and leaf curling of
plantlets ( Wetzstein and Sommer, 1982; Donelly and Materials and methods
Vidaver, 1984). These morphological changes have
been related to the low photosynthetic capacity of the Plant material
leaves (Kevers et al., 1984; Paek et al., 1991). In vitro
grown carnation plantlets have shown difficulties Three varieties of carnation, viz., White Sim, Exquis-during acclimatization in glasshouse due to hy- ite and Scania were obtained from Pallavika nursery,
perhydricity leading change in anatomical and mor- Rudarpur. Nodal parts were cut (about 0.5 cm),
phological characteristics (Mujib and Pal, 1995; washed with running tap water for 30 min, then
Olmos and Hallin, 1998). dipped in 0.2% Teepol for 3 5 min and rinsed three
Accumulation of gases such as ethylene and CO times in sterile distilled water. Surface disinfection2
have also been found to be responsible for hy- was achieved with 20% solution (v/ v) of sodium
perhydricity (De Proft et al., 1985). Various ap- hypochlorite for 810 min followed by four rinses in
ICPC icpcxps (TICU 3815) - product element 5100869 DISK - Sun Sep 15 14:14:09 2002
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sterile distilled water. Single nodal explants were then shoots, shoots hyperhydrified and shoots height. Re-
placed into 50 ml sterile culture medium in the wide- sults were subjected to analysis of variance and
mouthed plastic-capped glass tissue culture bottles significant differences in values were calculated,
(approximately 500 ml capacity). Different concen- where effects or interactions were statistically signifi-
trations and combinations of plant growth regulators, cant. Percent hyperhydricity was calculated from 21-
viz., NAA and kinetin in MS media were tested and day-old culture observations using an index obtained
optimized. after dividing the number of hyperhydrified shoots bythe total number of regenerated shoots, multiplied by
Culture media and conditions hundred.
Basal medium used for initial set of experiment for
shoot proliferation consisted of MS (Murashige and21 21
Skoog, 1962) salts, vitamins, 30 g l sucrose, 7 g l Results and discussion
agar (Qualigens, India). In the preliminary study, the
medium was supplemented with NAA in the range of Effect of NAA and kinetin21
0.51.3 mM corresponding to 0.100.25 mg l and
kinetin 2.314.0 mM corresponding to 0.53.0 mg Good growth and proliferation of nodal explants were21
l in different combinations. The best concentrations found in the range of 0.51.3 mM NAA and 2.314.0
of NAA and kinetin for individual varieties were mM kinetin. Incidence of hyperhydricity was high in12 12
selected to test the effect of Fe and Mg on all combinations of growth regulators in all the three
hyperhydricity of shoots. The basal concentrations of varieties. Variety White Sim gave best proliferation
iron as FeSO ?7H O along with Fe-EDTA (MS- and tall shoots at NAA (1.3 mM) and kinetin (2.34 2
Stock) and magnesium salt as MgSO ?7H O in MS mM) but with this combination there was a high4 2
medium were in the range of 100200 mM and 1.5 percentage of hyperhydricity. Variety Exquisite gave
3.0 mM, respectively. Different (0.60.8%) agar best shoot proliferation and growth with NAA (0.8
concentrations at optimal level of plant growth reg- mM) and kinetin (4.6 mM). With Scania carnation,
ulators and metal ions were examined for shoot best shoot proliferation was achieved at NAA (0.8
proliferation and hyperhydricity. mM) and kinetin (2.32 mM). These results indicated
The pH of the medium was adjusted to 5.660.1 that as the concentration of kinetin increased so did
prior to autoclaving (15 min at 120 8C). The cultures the incidence of hyperhydricity. NAA and kinetin
were grown at 2462 8C with a relative humidity gave sharp response on shoot proliferation and height
6070% under fluorescent light intensity of 24 mmol of shoots. By increasing the concentration of NAA22 21
m s , with 16-h photoperiod. from 0.5 to 1.3 mM, proliferation and height of shoots
were found to increase. However, the incidence of
Rooting, acclimatization and field transfer hyperhydricity increased in all these three varieties.
Since factors other than plant growth regulators might
Proliferated and nonhyperhydrified shoots were trans- influence hyperhydricity without affecting the shoot
ferred to half MS solidified media (0.7% agar) sup- proliferation and growth, different concentrations of
plemented with 5.4 mM NAA to develop roots in vitro metal ions (iron and magnesium) as well as agar were
for 15 days. Regenerated and unvitrified shoots with examined while plant growth regulators regime kept
well-developed roots were transferred to soil mixture constant.
(1:1 w / w sand and compost) in small plastic cups at
maintained humidity (|70%) through misting device
inside glasshouse. After hardening, plants were trans- Effect of iron and magnesiumplanted to earthen pots or on soil bed.
In the present investigation, different combinations of
Experimental design, data collection and analysis iron and magnesium were used. The concentrations of
iron and magnesium in the control were 0.10 and 1.5
Experiments were set up in completely randomized mM, respectively. White Sim carnation showed best
design with five replicates per treatment and were proliferation and no vitrification at 0.15 mM iron and
conducted thrice. Data were collected on number of 2.25 mM magnesium when NAA and kinetin con-
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Table 1. Effect of iron and magnesium on shoot proliferation and hyperhydricity on 21-day-old cultures of carnation varieties
Fe1Mg White Sim Exquisite Scania
(mM) (mM) NAA (1.34 mM) NAA (0.80 mM) NAA (0. 80 mM)
1 1 1
kinetin (2.32 mM) kinetin (4.60 mM) kinetin (2.32 mM)
NPS* %H NPS* %H NPS* %H
0.1011.5 4.760.2 55.6 2.560.7 35.7 4.060.1 40.00.1012.25 2.460.9 33.3 5.461.1 12.3 5.160.1 36.8
0.1013.0 3.360.4 0.0 2.860.8 0.0 3.960.6 0.0
0.1511.5 3.660.2 11.1 2.060.6 18.2 2.660.5 15.4
0.1512.25 4.860.2 0.0 2.060.5 0.0 4.460.9 0.0
0.1513.0 2.060.1 0.0 3.660.6 0.0 3.261.0 0.0
0.2011.5 1.960.4 15.6 2.360.7 8.3 3.660.4 35.0
0.2012.25 1.960.4 0.0 3.060.5 0.0 4.260.7 0.0
0.2013.0 1.160.3 0.0 2.860.6 0.0 6.760.1 0.0
*Significant differences among pairs for various combinations of iron and magnesium concentrations at 0.05 level of probability.
Agar used: 0.7%.
NPS: number of proliferated shoots.
%H: percent hyperhydricity (ratio of hyperhydrified shoots/total number of proliferated shoots).
centrations were optimal. Exquisite showed maxi- and RNA, these processes are known to influence
mum proliferation at 0.10 mM iron and 2.25 mM hyperhydricity (Fontes et al., 1999).
magnesium.
Scania gave no hyperhydricity at concentration Effect of agar
combinations of 0.10 and 3.0 mM, 0.15 and 2.25 mM,
0.15 and 3.0 mM, 0.20 and 2.25 mM and 0.20 and 3.0 Low agar concentration (0.6%) promoted shoot pro-
mM iron and magnesium. These results indicated that liferation in all varieties (Table 3). Agar concentration
higher concentrations of iron (0.20 mM) and mag- had no effect on hyperhydricity in variety White Sim
nesium (3.0 mM) were observed to decrease shoot and the other two varieties it was seen only using
proliferation in White Sim, while Scania showed an 0.6% agar. All the results for shoot proliferation and
increase proliferation except Equisite where shoot growth without hyperhydricity were significant (p#
proliferation was not found to be effected (Table 1). 0.05) when compared with respect to agar concen-Thus, the concentrations of iron and magnesium trations. Growth and shoot proliferation decreased
beyond 0.2 and 3.0 mM might not suit for micro- with increased agar amount throughout the range
propagation of carnation cultivars. examined. These plantlets were survived well upon
These data indicated that iron and magnesium play transfer into glasshouse after rooting. All acclimatized
an important role in controlling hyperhydricity but plants showed 100% survival.
magnesium was more potent to overcome hy-Table 2. Correlation coefficient between number of regeneratedperhydicity (Table 1). Correlation existed among rateshoots, height (cm) and percent hyperhydricity when medium was
of shoot proliferation, growth and hyperhydricity. Insupplemented with iron and magnesium ions
general, there was positive correlation between shootNumber of Percentheight and proliferation while negative correlationproliferated shoots hyperhydricity
was observed between shoot height, proliferation anda a
Height (cm) 0.4 20.5
hyperhydricity in all varieties (Table 2). Hy- b b0.8* 20.3perhydricity has been reported to induce disorder in c c
0.7* 20.1protein synthesis (Ziv, 1991). Increased magnesium
aavailability forms magnesium-ATP complexes re- No. of proliferated shoots 20.7*b
20.4quired for various enzymes during protein biosyn-c
20.3thesis. Besides, magnesium also plays important rolea b cin physiological processes including photosynthesis, White Sim, Exquisite, Scania.
respiration as well as biosynthesis of protein, DNA *Significant correlation.
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Table 3. Role of agar on shoot proliferation and hyperhydricity ethylene evolution in the culture atmosphere of magnolia cul-
during in vitro culture of carnation varieties after 21 days at tured in vitro. Physiol. Plant. 65: 375379
optimum concentrations of plant growth regulators and metal ions Donelly VA & Vidaver W (1984) Leaf anatomy of red raspberry
transferred from culture to soil. J. Am. Soc. Hort. Sci. 109:Variety Agar (%) Number of Percent
172176proliferated shoots* hyperhydricity
Fontes MA, Otoni WC, Carolino SMB, Brommonschenkel SH,
WS 0.6 6.060.2 0.0 Fontes EPB, Fari M & Louro RP (1999) Hyperhydricity in
WS 0.7 5.560.6 0.0 pepper plants regenerated in vitro: involvement of BiP (Binding
WS 0.8 2.860.4 0.0 Protein) and ultrastructural aspects. Plant. Cell. Rep. 19: 81 87
EX 0.6 5.660.5 17.0 Gill APS & Arora JS (1988 ) Performance of sim carnations under
EX 0.7 4.860.6 0.0 subtropical climatic conditions of Punjab. Indian. J. Hort. 45:
EX 0.8 3.560.6 0.0 329335
SC 0.6 8.160.5 22.0 Jain A, Husain H & Kothari SL (1997) Micro propagation of
SC 0.7 7.960.7 0.0 Dianthus caryophyllus L. control of vitrification. J. Plant.
SC 0.8 2.960.6 0.0 Biochem. Biotechnol. 6: 3537
Kevers C, Coumans M, Coumans-gilles MF & Gasper T (1984)*Significant differences among pairs in different varieties at various Physiological and biochemical events leading to vitrification ofagar concentrations at 0.05 level of probability. plants cultured in vitro. Physiol. Plant. 61: 6974WS: White Sim (NAA1kinetin, 1.3412.32 mM) and (iron1 Kevers C & Gasper T (1986) Vitrification of carnation in vitro:magnesium, 0.1512.25 mM). change in water contents, extracellular space, air volume and ionEX: Exquisite (NAA1kinetin, 0.8014.6 mM) and (iron1 levels. Physiol. Veg. 24: 647653magnesium, 0.1012.25 mM). Kim KW, Byun MS & Kang MS (1988) Effect of ABA and agar inSC: Scania (NAA1kinetin, 0.8012.32 mM ) and ( iron1
preventing vitrification of carnation plantlets cultured in vitro. J.magnesium, 0.2013.0 mM). Kor. Soc. Hort. Sci. 29: 208215
Li Y, Wang L, Ye M, Shen D, Li Y, Wang LH, Ye MM & Shen DL
(1997) The factors influencing vitrification of tissue-culturedConclusioncarnation plantlets. Plant. Physiol. Commun. 33: 256258
Miller RM, Kaul V, Hutchinson SF & Richards D (1991 ) Adventiti-In the present investigation, we have showed that
ous shoot regeneration in carnation (Dianthus caryophyllus) frompronounced effect of iron and magnesium on hy- axillary bud explants. Ann. Bot. 67: 3542perhydricity in carnation during in vitro shoot prolif- Mujib A & Pal AK (1995) Inter varietal variation in response to in
vitro cloning of carnation. Crop. Res. Hisar. 10: 190194eration. Thus, modified concentrations of iron andMurashige T & Skoog F (1962) A revised medium for rapid growthmagnesium may be exploited to overcome hy-
and bioassays with tobacco tissue culture. Physiol. Plant. 15:perhydricity. Each variety required different regimes
473497of plant growth regulators and metal ions, it showed Olmos E & Hallin E (1998) Ultrastructural differences of hy-that response is genotype specific. perhydric and normal leaves from regenerated carnation plants.
Sci. Hort. 75: 91101Paek KY, Han BH & Choi SL (1991) Physiological, biochemical
and morphological characteristics of vitrified shoots regeneratedAcknowledgements
in vitro. Kor. J. Plant. Tiss. Cult. 3: 151162
Staby GI, Robertson JL, Kiplinger DC & Conover CA (1978)M.K. Yadav wishes to thank to Dr S.J. Amdekar for Chain of Life. Ohio Florists Assoc, Ohio State University,
Columbushelping in statistical analysis and Department ofTsay H, Tsay HS & Drew RA (1998) Effect of medium com-Biotechnology, Govt. of India for providing a scholar-
positions at different recultures on vitrification of carnationship.
(Dianthus caryophyllus) in vitro shoot proliferation. Acta. Hort.
461: 243249
Wetzstein HY & Sommer HE (1982) Leaf anatomy of tissue
cultured Liquidambar stryraciflua during acclimatization. Am. J.ReferencesBot. 69: 15791586
Ziv M (1991) Vitrification: Morphological and physiological dis-Choudhary ML, Prakash ML & Prakash P (1993) Effect of different
orders ofin vitro plants. In: Debergh PC & Zimmerman RH (eds)levels of agar and MS macro salts on the production of hardenedMicropropagation: Technology and Application (pp. 4569).carnation in vitro. Adv. Hort. Forest. 3: 165169Kluwer Academic Press, Dordrecht, The NetherlandsDe Proft M, Maene J & Debergh P (1985) Carbon dioxide and