TECHNICAL BULLETIN 117 ISSN 0070-2315

PRODUCTION OF CARNATION PLANTS BY SHOOT-TIP CULTURE IN VITR O

Maria Ioannou

AGRICULTURAL RESEARCH INSTITUTE MINISTRY OF AGRICULTURE AND NATURAL RESOURCES

NICOSIA CYPRUS

JULY 1990

PRODUCTION OF CARNATION PLANTS BY SHOOT-TIP

CULTURE IN VITRO

Maria Ioannou

SUMMARY

Shoot-tip culture of carnation (Dianthus caryophyllus L. ) is mainly used for the produc­tion of pathogen-free propagative stocks and for rapid clonal propagation of this crop. For pathogen exclusion, the culturing method involves the production of a single plantlet per shoot tip cultured, whereas for clonal propagation the shoot tip is chemically stimulated to generate a multitude of plantlets. The present study utilized both methods to determine the most suitable nutrient media and culturing technique for the production of healthy carnation plants from shoot tips cultured in vitro. A modified Morel's medium, supplemented with myoinositol at 100 mg/l, and with thiamine.H'Cl and a-naphthaleneacetic acid (NAA) at 1 mg/ I each, gave the best results in experiments aiming at the production of one plantlet per cul­ture. Rooted plantlets, 1 to 2 em high, were transferred to soil in 6-8 weeks from culture. Survival rate was 100% and plants reached flowering stage in 7 to 8 months from potting. All plants were healthy, and produced high-quality flowers typical of the cultivar. This tech­nique could be useful for the local production of pathogen-free propagative stocks. A modi­fied Murashige and Skoog medium, supplemented with NAA at 0.02 mg/l and kinetin at 0.5, 1 or 2 mg/l, was used for clonal propagation. The highest multiplication rate, averaging 33 plantlets per cultured tip, was obtained with the highest (2 mg/l) kinetin concentration. All shoots rooted, and plantlet development in vitro was rapid in all stages. However, plant sur­vival upon transfer to soil was rather poor.

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INTRODUCTION

Shoot-tip culture (known also as shoot-apex, meristem or meristem-tip culture) has found major application both for recovery of pathogen-free stocks and for rapid clonal propagation of many crops. When exclusion of pathogens is the goal, the culturing technique is directed towards the produc­tion of a single rooted plantlet from each shoot tip. A simple nutrient medium supplemented with a rooting hormone usually satisfies the needs of a shoot apex intended for development into a single rooted plantlet. In contrast, for clonal multiplication the shoot-tip explant is chemically stimulated to generate multiple shoots. Hence, a medium contain­ing high concentrations of inorganic salts and sup­plemented with cytokinin is usually used.

Several modificatio ns of the nutrient medium de­veloped by Morel (1964) have been employed in producing single, seedling-like carn ation plants (Di­anthus caryophyllus L. ). Baker and Phillips (1962), Stone (1963) and Phillips (1968) used a similar basal medium to investigate vario us factors affecting the growth of carnation plants from shoot apices. Stone (1968) used also the shoot-apex culture technique for the elimination of certain carnation viruses .

To obtain clonal multiplication of carnation , Hackett and Anderson (1967) used White 's medium with 5 times the inorganic salt concentration and a modified Murashige and Skoog (MS) medium. Earle and Langhans (1975) obt ained a high multiplication rate by using a gently-agitated liquid MS medium, while Davis et aI. (1977) found that a combination of stationary and gently-agitated liquid MS medium

gave the best results.

In Cyprus, carn ation ranks first in cut-flower production intended for both the local and foreign markets. A major problem is to secure healthy rooted cuttings which are essential for maximum production of high-quality flowers . Since there is no programme for local production of pathogen-free stocks, the majority of rooted cuttings needed for commercial production are imported at a significant cost to the country 's economy.

The main objective of the present work was to find the most suitable nutrient media and culturing technique for the production of carn ation plants from shoot apices cultured in vitro. Emphasis was given to procedures leading to the pro duction of one plant let per shoot-tip explant, since such a technique could be utilized for the local produ ction of pathogen-free propagative stocks. At the same time, clonal propagation of carn ation was also in­vest igated since a successful clonal propagation tech­nique could be effectively incorp orated into a healthy propagative stock programme, for rapid multiplication of pathogen-free carnation plants pro­duced with the single-plantlet technique.

MATERIALS AND METHODS

Preparation of shoot-tip ex plants.

Terminal cut-tings, 15 to 20 em long, were ob­tained from greenhouse-gro wn carn ation plants, cv. William Sim. Each stem was shortened to 2 em and all leaves up to 0.5 em long were removed. Surface steri lization of shoot tips was not necessary (Stone, 1963). Expanding and emerging leaves were re ­moved from the stem with a borradaile needle. The terminal 0.1 to 0.3 mm of the shoot tip , which in­cluded the meristem dome plus 2 to 4 leaf primor­dia, was excised using a no. 11 surgical blade and used for the production of one plantlet per culture. A larger explant, 0.3 to 0.5 mm long, was used for clonal multiplication. Using a steril e needle, each tip was then carefully transferred to the culture vial containing the nutri ent medium. A minimum of 20 explants were used for each tre atment.

Culturing procedures and nutrient media

Production of single planUets. The culturing pro­cess in this case was accomplished in three main

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stages. In stage 1, the shoot apices were placed in­dividually in culture tubes containing liquid nutrient medium supplemented with the auxin a-naph thale­neacetic acid (NAA). Each tip was placed apex -up on a filter paper bridge, which acted as a wick, sup­plying liquid to the explant while providing support and enabling its proper aeration (phillips and Da­nielson, 1961b). The culture s were placed in a bac­teriological incubator (GCA Precision Scientific, Model 818) under continuous illumination (Phillips and Danielson, 1961a) at 2±20 C.

After 15 days all rooted tips, regardless of size, were transferred to fresh, hormone-free medium to promote further development of shoots. and roo ts (stage 2). The plantlets remained on this medium for 4 to 6 weeks, during which they re ached a height of 1.5 to 2 em and developed abundant ro ots.

In stage 3, healthy rooted plantlets, 1.5 to 2 em high, were transplanted in small pots containing a sterile soil mixture (l :2 v!v peat moss and forest soil). Potted plantlets were placed in a growth room under artificial illumination (l6h photoperiod) at 20±2CC and were watered with half-strength Hoag ­land solution. Inverted glass beakers were used to protect young plants from dehydration during their establishment in soil. The plants were transferred to greenhouse conditions 10 to 15 days from potting.

Four media formulations (Tables 1 and 2) were tested for the production of one carnation plantlet per culture. Inorganic salts were provided by either the Murashige and Skoog (1962) salt base or by the Knop (major elements) and Barthelot (trace ele­ments) solutions; TiS04 and BeS04 were omitted from Berthelot solution since their absence does not affect shoot-tip development (Stone, 1963). Iron was used in the form of Ferric Sodium EDTA in separ­ate stock solution to avoid precipitation. The pH of the medium was adjusted to 5.5 before autoclaving and the solution was dispensed in culture tubes (16x125 mm ), 4 ml of medium per tube . A strip of Whatman n042 ashless filter paper was folded to make a bridge with its ends immersed in the liquid medium. Tubes were capped with aluminum foil and sterilized by autoclaving in a pressure cooker for 10 minutes.

Table 1. Composition of nutrient media used for carn ation shoot-tip culture (based on Mor­el's medium l)

Ingredient Formula and amount used per litre of me dium

Phillips Baker Neergaard's and Phillips

Knop solution/ 500 ml 500ml lO00 ml

Berthelot solution? 0.5 ml 0.5 ml O.5 ml

glucose 40 g 40 g 40 g

NAA 1 mg 1 mg 1 mg

myoinositol 100 mg

thiamine.HCI 1 mg 1 mg I mg

adenine .so4 8 mg 8 mg

indole 1 mg

1 Morel's medium (amount per litre of medium):

Knop solution, 500 ml; Berthelot solution, 0.5 8ml; glucose, 30-50 g; NAA, 10-6_10- g; agar,

12 g; cysteine, 10-5 g; thiamine, 10-6 g; Ca pan­tothenate, 10-6_10-8 g; inositol , 10-6_10-8 g; bio ­tin, 10-6_10-8 g.

Z Knop solution:

Ca(N03hAHzO, Ig: KN03, 0.25 g; MgS04.7Hp, 0.025 g; KHzP04 , 0.25 g; dis­

tilled water, to 1000 ml.

3 Barthelot solution:

Fez(SOzh , 50 g; MnS04.7Hp, 2 g; H3B03 '

0.05 g; KI, 0.5 g; NiClz.6HzO, 0.05 g;

CoClz.6HzO, 0.05 g; ZnS04.7HzO, 0.1 g;

0.05 0.05 g; Cu4S04.5Hz0, g; TiS04.5HzO,

BeS04AHp* , 0.05 g; HZS04 (cone. ), 1ml; dis­

tilled water, to 1000 ml.

* omitted in the present work.

Production of multiple plantlets (rapid clonal propagation). The culturing procedure for clonal multiplication involved four stages. In stage 1, the

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excised shoot apices (0.3 to 0.5 nun long) were cul­tured on agar-gelled medi a in 100 m1 jars. A high cytokinin to auxin ratio was used in order to re­press apical dominance and induce production of axillary shoots (Murashige, 1979). Three levels of kinetin (0.5, 1 and 2 mg/l) were tested (Tables 2 and 5).

Three weeks from culture initiation clusters of shoots were !: a.Ilsferred to fresh medium for further multiplication (stage 2). The same basal medium as in stage 1, but supplemented with NAA and kinetin at 0.02 and 2 mg/l, respectively, was used. The clus­ters remained on this medium for 3 weeks.

In stage 3, individual shoots 0.5 to 1 em long, were detached and transferred to a fresh medium for further shoot development and rooting. This medium was similar to that in stages I and 2 except that it was hormone free.

Cultures of all stages were incubated under artifi­cial illumination at 20±2°C as described for cultures aiming at the production of single plantlets.

After 3 weeks on rooting medium, rooted plant ­lets (2 to 3 em high) were transplanted to soil (stage 4). Upon removal from the medium, roots were washed thoroughly to remove agar and plant­lets were potted in sterile soil mixture (1:2 v/v peat moss and forest soil). Plantlet handling and weaning procedures were identical to those already described for the production of single plantlets.

A modified MS medium (Table 2) solidified with Difco "Noble" agar (highly purified) was used throughout the culture. The pH of the medium was adjusted to 5.7 before autoclaving and prior to add­ing agar. The solution was autoclaved for 3 min to dissolve agar and then dispensed in glass jars (40 m1 per jar). The vials were capped with aluminum foil and autoclaved in a pressure cooker for 10 minutes.

Table 2. Modifications of Murashige and Skoog (MS) medium used for carnation shoot-tip culture (mg/l)

Ingredient Single plantlet Multiple plantlet production production

MSsalt base1

4303.5 4303.5

glucose 40000 40000

myoinositol 100 100

thiamine.HCI 0.4 0.4

NAA 1.0 0.02

kinetin 0.0-0.5 0.5-2.0

IMS salt base (mg/l):

1650; KN03, 1900; CaCI2.2H20 , 493.8; NH4N03,

MgS04.7H20, 370.6; KH2P02, 170; FeNaEDTA, 170; 6.2; MnS04.4HP, 8.6; KI, 0.83; H3B03,

N~Mo04.2H20 , 0.25; CuS04.5HP, 0.025; CoCI2·6Hp,0.025.

RESULTS AND DIS CUSSION

Production of single plantlets. Shoot tips cultured on modified MS high-salt me­

dium (Table 2) rooted and grew rapidly regardless of the level of Kinetin added (Table 3). All plant ­lets, however, had very turgid shoots with translu ­cent leaves and lacked the blue-green color charac­terizing normal carnation plants. Some improvement in plantlet appearance was noticed after develop ­ment for 2 weeks on hormone-free medium, during stage 2. However, their survival upon transfer to soil was rather poor, ranging from 15 to 77%. Best results were obtained when the kinetin concentra­tion in the initial medium was 0.01 mg/l (Table 3).

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Table 3. Effect of kineti n concentration on rooting and urvival of carn ation shoo t tips cul­tured on Murashige and Skoog medium for production of single plantlets

Kinetin No. of tips No. of rooted No . of plants concent­ cultured platle ts surviving to ration produced maturity (mg/l)

o 60 56 15

0.01 60 58 45

0.5 60 59 9

The percentage of rooted explants was much lower on Phillips (37%) than on Baker and Phillips medium (85%), apparently because the presence of indole in the former medium had an inhibitory ef­fect on shoot-tip rooting (fables 1 and 4) . These resul ts contradict those of Phillips (1968) who found that, in the presence of NAA, indole accelerated growth but did not influence rooting of carnation shoot tips. Despite higher percentage in rooting, rooted plantlets produced on the Baker and Phillips medium were rather slen der and thei r surviral in soil was rather poor. Best results in both ro ot and shoot development were obtained with Neergaard's mediu m. Although growth on this medium was not as rapid as on MS medium, all roo ted plantlets had the normal blue-green color and all survived and grew to flowering in 7 to 8 months from potting (Table 4). Evidently, Knop and Berthelot solutions

fully sat isfy the needs of carnation shoot apices in inorgan ic salts whenever one plantlet per tip is de­sired.

Table 4. Rooting and survival of carn ation shoot tips cultured on various nutrient media for production of single plantlets

Nutrient No. of tips No. of ro ote d No . of plants medium cultured plantlets surviving to

produced maturity

Phillips 60 22 17

Baker & Phillip s 60 51 40 Neergaard's 60 58 58

Production of multiple plantlets (rapid clonal propagation).

A multitude of shoots was obtained on modified MS medium with a high cytokinin to auxin ratio (Table 2). The average multiplication rate (number of rooted plantlets produced/number of shoot t ips cultured) increased from 8 to 33 as the kinetin con­centration in the medium increased from 0.5 to 2 mg/l (Table 5). All shoots separated and transferred to hormone-free medium grew rapidly into rooted plantlets. However, such plantlets had very turgid shoots and their leaves were thick and translucent , with pale-green color. Their survival upo n transfer to soil was poor , irrespective of the kinetin concen­tration in the culture medium (fable 5).

Table 5. Effect of kinetin concentration on proliferation ofcarn atio n shoot tips cultured on Murash ige and Skoog medium for rapid clonal propagation

Kinetin No.of tips Ave rage multi­ No .of plant­ No . of plants con cent­ culture d plication lets potted surviving to ration rate* maturity (rng/l )

0.5 40 8 20 5

1.0 40 2 1 20 4

2.0 40 33 20 5

* Number of rooted plantlets produced/number of tips cultured.

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Rapid shoot development , due to high concentra­tion of inorganic salts in the medium, probably was the main cause of translucency in the produced plantlets and of poor survival in soil. As noted by Constantine (1986), vitrification (hyperhydration or translucency) is most frequently observed in very rapidly gro wing cultures , and translucent plantlets do not establish successfully in compost. Davis et al. (1977) encountered the same problem when the re ­volving flask technique (liquid culture) was used in the multiplication stage; they attributed the problem to excessive uptake of water, nutrients and, espe­cially, growth regulators through the surface of leaves and stems. Earl e and Langhans (1975) ob­tained a high multiplication rate using slowly rotat ­ing liquid medium during the shoot multiplication stage. However, some plants showed fasciation, while survival rate of potted plants was low (45­75%). In the present study, leaves became exces ­sively broad and thick when the kinetin concentra­tion in the medium was increased to 2 mg/l. Thus, lowering the kinetin concentration in the multiplica­tion medium and/or the major elements conce ntra­tion in the rooting medium might be helpful in im­proving the quality of the plants.

In all experiments, contamination was rather low, ranging between 2 and 6%. No surface sterilization of the shoots was used, because previous work (Stone, 1963) showed that it is not necessary.

Prospects for carnation shoot-tip culture in Cy­prus

The successful propagation of carnation by shoot ­tip culture in vitro is affected by many factors . Few of them were elucidated in the present study, while many others still require furthe r investigation. How ­ever, the nutrient media and culturing techniques used in the present study could be utilized in the production of carnation plants from shoot tips cul­tured in vitro, especially for single plantlet produc­tion. This method could be utilized for the produc­tion of healthy propagative stocks, which are presently imported at a significant cost. It must be emphasized, however , that for the successful imple ­mentation of a carnation clean stock programme, shoot-tip culture should be supported by a strong Virus-indexing programme in order to confirm pa­thogen elimination from propagative stocks.

REFERENCES Bake r, R. and DJ. Phillips. 1962. Obtaining patho­

gen-free stock by shoot-tip culture . Phytopa­thology 52:1242-1244.

Constantine, D.R. 1986. Micropropagation in the commercial environment. Plant Tissue Culture and its Agricultural Applications (Withers, L.A., and P.G. Alderso n, eds), pp. 175-186. University Press, Cambridge.

Davis, M.J., R. Baker and U. Hanan. 1977. Clonal multiplication of carnation by micropropaga­tion. Journal of Horticultural Science 102:48­53.

Earle, E.D. and R.W. Langhans. 1975. Carn ation propagation from shoot tip s cultured on liquid medium. HortScience 10: 608-610.

Hakcett, W.P. and l A. Anderson. 1967. Aseptic multiplication and maintenance of differentiat ­ed carnation shoot tissue derived from shoot apices . Proceedings of the American Society for Horticultural Science 90:365-369 .

Morel, G.M. 1964. Tissue culture, a new mean s of clonal propagation in orchids. American Or­chid Society Bu1Jetin 33:473.

Murashige , T. and F.Skoog . 1962. A revised medi ­um for rapid growth and bioassays with tobac­co tissue cultures . Physiologia Plantarum 15:473-497.

Murashige, T. 1979. Plant tissue culture and its im ­portance to agriculture. In Practical Tissue Culture Applications (Maramorosch, K., and H. Hirubi, eds), pp . 22-24. Academic Press, New York.

Phillips, DJ. and L. Danielson. 1961a. Effects of constant fluorescent light on the growth of carnation tips. Colorado Association Bu1Jetin 132.4p.

Phillips , DJ. and L. Danie lson . 1961b. Filter paper wicks for support in growing carnation shoot tips. Colorado Association Bulletin 133. 4p.

Phillips, DJ . 1968. Carn atio n shoot-tip culture. Col­orado State University Experiment Station Technical Bu1Jetin 102. 22p.

Stone, a .M. 1963. Factors affecting the growth of carnation plants from shoot apices . Annals of Applied Biology 52: 199-209.

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Stone, a.M. 1968. The elimination of four viruses from carnation and sweet william by rneris­tern-tip culture. Annals of Applied Biology 62:119-122.

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