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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

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    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

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