Apical shoot tip culture of tomato

Scientia Horticulturae, 10 (1979) 337--344 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

337

APICAL SHOOT TIP C U L T U R E OF TOMATO

F.J. NOV/(K and IRENA MASKOVA

Institute of Experimental Botany of the Czechoslovak Academy of Sciences, Sokolovskd 6, 772 O00lomouc (Czechoslovakia) (Received 7 November 1978)

ABSTRACT Nov~k, F.J. and Ma~kov~, I., 1979. Apical shoot tip culture of tomato. Scientia Hortic., 10: 337--344. We studied the effect of phytohormones on growth and development of apical shoot tip of t o m a t o (Lycopersicon esculentum Mill.). Whole plants rarely developed on basal medium containing N A A (~-naphthylacetic acid). K (kinetin) and BAP (6-benzylamino purine) stimulated shoot development within the range of 0.1--10 ~M. BAP at 5 and 10 uM induced shoot multiplication. The clonal progenies were phenotypically uniform and without chromosomal changes or meiotic irregularities. The potential uses for propagating genetically valuable t o m a t o lines under in vitro conditions are discussed.

INTRODUCTION

Plantlet production from apical meristems under in vitro conditions has been described for a number of species (Hussey, 1978). The technique can be used as an effective method of vegetative plant propagation, at the same time eliminating virus infection in the clonal progeny (Quak, 1977). De Langhe and De Bruijne (1974), and Padmanabhan et al. (1974) described regeneration in t o m a t o explants (Lycopersicon esculentum Mill.) from leaf
338 proposed by De Fossard et al. (1974). The aim of our experiments was also to determine the possibilities of clonal tomato plant propagation for genetic and selection purposes. MATERIALS AND METHODS Shoot tips were taken from 10
from 0.1 to 10 tzM was insufficient to induce plant development from shoot tips. Weak plantlets developed sporadically (10%), sometimes forming rootlets on media with low IAA level (0.1 and 1 uM) (Fig. 1A). Upon transplanting into soil, all these plants died. The NAA response was concentration-dependent (Fig. 1B). The NAA level of 0.1 pM induced sporadic (8%) development of shoots without roots. The concentration of 1 pM stimulated shoot development with a vigorous root system. In some cases the development of roots suppressed that of a stem, which gradually gave rise to a sizeable callus. An intensive formation of friable, yellowish callus occurred on media with 5 or 10 pM NAA.

339

E f f e c t o f c y t o k i n i n s . -- Apical meristems cultivated on basal medium con-

taining K (0.1--10 ~M) gave rise to dark green plants, which formed roots after 30 days. Higher K concentrations (1--10 ~M) were more favourable for shoot development (Fig. 1C). After 10 days of culture, green compact callus was formed at the base of an explant. R o o t s gradually arose from the callus. R o o t formation was most intensive at 5 pM K. R o o t e d plants were successfully transplanted into soil and grown to flowering. Shoot development was also stimulated at all BAP levels (Fig. 1D). In contrast to the K effect, there was a more intense suppression of r o o t development, which began to form only after 40 days of culture provided the concentration was 1 ~M. Callus developed at the base of all explants, and most intensively at 5 uM BAP. Higher BAP rates (5 and 10 pM) induced shoot multiplication with simultaneous callus development at their bases. The highest BAP concentrations (10/~M) induced chlorophyll disintegration; the plants were chlorotic and rooting was sparse. E f f e c t o f g i b b e r e l l i n . -- All the tested GA3 levels were suitable for shoot

development, the lower ones even for the development of roots which arose as soon as 20 days (Fig. 1E). The plants in test-tubes were rather elongated; however, their p h e n o t y p e became normal once they were transplanted into soil. A u x i n - - c y t o k i n i n interaction. - - All combinations of IAA and K stimulated

shoot development, while at 0.1 pM and 1 ~M K roots only arose in combination with IAA levels of 10 pM, or 5 and 10 ~M. Media with 5 or 10 pM K yielded complete plant development within the whole range of IAA concentrations (Fig. 2). The intensity of callus formation at the explant base % 1UC

A

B

C

+-I. + +

D

++ ~÷

U@

++ i )"

<

~

2C

I'1 I-I o ~

i

z

I&l Ili

m

anc

2¢

r~

II

u

u U

~n

i

B

bC 8C I0C

%

[]

MULTIPLE

SHOOT

CALLUS GROWTH: +LOW; 0"1

I

S

IAA

' 10

' 0"1

++MODERATE;

÷÷÷

1' '

o' 1

S

NAA

' IO

HIGH

.1 . 5 . IO .

K

01

I' '

S

BAP

. . I0

. 0"1 .

I

5

;0

pM

GA3

Fig. 1. Frequency (%) of explants differentiating shoots and roots on media with various phytohormones.

340

Ijll 1

%

÷

100 8Q

u_

O z

O m

in

O O z

6C 4O 20

< I,-

Z

lad

lid u-

U'I im

O O a¢

U

2C

't-

[]

MULTIPLE

+ + +++ + + +++ -k, + +++ +++ +++

U

40 6C

+

SHOOT

It"

u

80

CALLUS GROWTH:+ LOW; +-I- MODERATE;

100

% IAA

0i'1

K

I

i

1

5

~

0

+ + + HIGH

l

i

l

l

i

l

i

i

l

i

i

i

0"1

1

S

I0

01

1

S

10

0"1

l

5

10

0"1

1

5

10

,M

pM

Fig. 2. Frequency (%) of explants differentiating shoots and roots on media with IAA and K at various concentrations.

was directly proportional to plication occurred when 1.0 the media. There was a trend to root on the media with NAA and °

the increased K concentrations. Shoot multior 5 pM IAA and 5 ~M K were combined in formation with simultaneous callus proliferation K (Fig. 3). Nevertheless, well-developed shoots

!

+

I00

++

+

8O 6C

++ +++11++Hi

n

a

II

GC RC I0©

N A A%

rl+++llllrl+++ll

i]++

~÷++÷

uIHIuu]IIJlU o':'T ~ t'°T:";, +7 "~ ;':C°°o~"7 + ~'~o"'?, ', 0"1

I

5

10

,o . pM

Fig. 3. Frequency (%) of explants differentiating shoots and roots on media with various concentrations of NAA and K.

341 and roots occurred in some combinations with 1:0.1 pM, 0.1:5 ~M and 0.1:10 gM, NAA:K, respectively. An inhibitory effect of IAA in combination with BAP on r o o t formation was apparent (Fig. 4). R o o t e d plants developed only on the medium with 0.1 or 1.0 pM IAA + 1 pM BAP. Shoot multiplication and intensive callus formation occurred at higher BAP levels (5.0 and 10.0 pM). To ensure the rooting of shoots, it was necessary to transfer them to a medium without growth regulators or with 1.0 #M NAA. The plants obtained in this manner were successfully transferred to soil. The combination of NAA--BAP seemed to be the least suitable from the point of view of the meristem-isolated plant development (Fig. 5). R o o t e d shoots developed at a low frequency in the combinations with the lowest NAA level only (0.1 pM). All other combinations of NAA and BAP induced whitish friable callus w i t h o u t organogenesis. The plants transplanted into soil had a tendency to flower very early. Usually an inflorescence with 2--6 flowers was formed in the axil of the first true leaf. The plant development in the soil continued in a normal way. We have n o t observed any phenotypical deviations from the standard type of 'Moneymaker' in the regenerated population. The habit of the plants corresponded to the clonal progeny. s t u d i e s . - - Chromosomal analysis was carried o u t in 30 young single-stemmed plants. At least 5 root tips and 35 metaphases were counted on each plant. We found no deviations in diploid chromosome number. The anthers were taken from the buds of the first inflorescences to observe Chromosome

%

llll

•4.÷ + ÷+÷ 4..÷ ÷,÷

4-4"

100

4"

8C

0

I,-

z

0 Z

o

m

0

bc 4© 2C

D D ~ z W ell

20

O m

a

4"+

o

Qg

4~ 6C

I~l

MULTIPLE

SHOOT

8a 10~

%

IAA BAP

CALLUS GROWTH: -I-LOW;÷÷MODER4TE; .4-4-.4-4"HIGH I 0"1

i I

I 5

0"1

i 10

| 0"1

i I

I S

1

I 10

i 0"1

i I

, 5

5

J 10

i

i

*

i

0"1

1

S

10

10

pM

~M

Fig. 4. F r e q u e n c y (%) o f e x p l a n t s differentiating s h o o t s and roots on m e d i a w i t h I A A and B A P at various concentrations.

342

10C 8C Id,,

0 z 0

6(

i0 0 z

4C

+++ ++

2C

I-=

17

IB M4 M4 ..

"-

-4-+-I-

+++ ++

++

+++

+

+

[]

+

÷+

+

I1

u

2C

z

+

~C

0 0

6C OC 10C

[]

MULTIPLE

SHOOT

CALLUS GROWTH: +LOW;

Jr +4. MODERATE; + + HIGH

o~ NAA

BAP

' 0'1

' 1

I 5

0'i

I

IoI

0'1

I I

Ill001 5

1

1

I I

I I S 10

5

I

o'1

I

I

1

s

10

I

lo

FM

Fig. 5. Frequency (%) of explants differentiating shoots and roots on media with NAA and BAP at various concentrations.

meiosis. There were no irregularities in meiosis and microsporogenesis. The meiotic analysis was not made in older inflorescences, however fruit set in meristem-derived plants was the same as in control ones grown from seeds. The fruits developed viable seeds. DISCUSSION

Our results show that regeneration of intact plants of L. esculentum from isolated shoot tips can be controlled by manipulation of the cytokinin and auxin levels in the culture medium. Only IAA had a slight influence on shoot and root development. In contrast to our observations, Kartha et al. (1977) obtained a complete plant regeneration from isolated t o m a t o meristems on a medium with 10.0 t~M IAA. However, Kartha et al. (1977) added IAA to the medium after filter sterilization, while we sterilized IAA in the medium by autoclaving. Moreover, they used another cultivar ('Starfire'). NAA was especially effective for root initiation and formation and for callus proliferation, but complete plant regeneration was obtained at one level only (1 t~M). Similar morphogenetic responses were also described for callus induction on t o m a t o leaf explants (Kartha et al., 1976). Cytokinins in the culture medium were sufficient for complete plant development from isolated shoot tips. This conclusion is in agreement with the observations of Kartha et al. (1977), who tested BAP and zeatin (Z)

343 effects on the development of tomato meristems. BAP or Z alone induced plant formation from a leaf callus of L. esculentum (Kartha et al., 1976) and higher Z levels induced shoot bud formation in the stem-segmentderived callus culture of tomatoes (De Langhe and De Bruijne, 1974). The optimal cytokinin concentration was about 5.0 pM, which is in full agreement with our observations. A positive influence of cytokinins on the induction and stimulation of shoot development has been shown in a number of species (Murashige, 1977). Moreover, BAP induces shoot multiplication by means of suppressing apical dominance (Hussey, 1976), which can be used in mass plant propagation by in vitro techniques. The testing of the BAP-influence on isolated t o m a t o shoot tip developm e n t has confirmed another well-known effect of cytokinins, namely inhibition of root formation {Murashige, 1977). On the other hand, the shoot tips cultivated on media with K rooted well, even at high levels. Kartha et al. {1977) observed root formation on media with BAP and Z in cultures older than 60 days. This fact is explained by a high level of endogenous auxins in t o m a t o stem tissue (De Langhe and De Bruijne, 1974), conditioning root formation even in the presence of exogenous cytokinin. This conclusion is also in agreement with the fact that shoots transferred onto medium without growth regulators root quickly. GA3 induced shoot and root development at lower concentrations than auxins or cytokinins. A stimulating effect of GA3 on the development of isolated meristems has also been observed in potatoes (Solanum tuberosum) and carnations (Dianthus sp.) at low concentrations (up to 0.5 pM) (unpublished). We feel that these observations are in agreement with the conclusion of Murashige (1963) on the stimulating effect of gibberellins on the in vitro growth of organs. Auxin--cytokinin combinations control, to a certain extent, the organogenetic processes of isolated t o m a t o shoot tips. L. esculentum represents a good experimental object for studying p h y t o h o r m o n e interactions. Moreover, the m e t h o d of shoot tip cultures can be used for cloning valuable t o m a t o genotypes for genetic and breeding purposes. They can also be exploited for the elimination of systemic virus diseases, which represent a serious problem in tomato growing. Plant propagation from isolated meristems by in vitro culture is suitable from the point of view of maintaining the genetic identity of the material. This fact is also confirmed by the results of our cytological analysis of regenerants. The m e t h o d in question eliminates the danger of genetic changes in regenerants which often occur in plants differentiated from callus cultures, e.g. cytological chimerism of plants of L. peruvianum regenerated from a callus of anther and stem origins (Sree Ramulu et al., 1976a,b}.

344 ACKNOWLEDGEMENTS The authors wish to express their thanks to Mr. I. K l em ent for technical assistance and Miss J. P o t o m k o v ~ for the English translation of the manuscript. REFERENCES De Fossard, R.A., De Myint, A. and Lee, E.C.M., 1974. A broad spectrum tissue culture experiment with tobacco (Nicotiana tabacum) pith tissue callus.Physiol. Plant., 31: 125--130. De Langhe, E. and De Bruijne, D., 1974. Massive propagation in vitro of tomato plants with maintenance of genetic stability.3rd Int. Congr. Plant Tissue and Cell Cult. 21-27 July 1974, Univ. Leicester (U.K.),No. 42 (abstract). Gamborg, O.L., Miller, R.A. and Ojima, K., 1968. Nutrient requirements of suspension cultures of soybean root cells.Exp. Cell Res., 50: 151--158. Hussey, G., 1976. In vitro release of axillaryshoots from apical dominance in monocotyledonous plantlets. Ann. Bot., 40: 1323-1326. Hussey, G., 1978. The application of tissue culture to the vegetative propagation of plants. Sci. Progr. (Oxford), 65: 185--208. Kartha, K.K., Gamborg, O.L., Shyluk, J.P. and Constabel, F., 1976. Morphogenetic investigations on in vitro leaf culture of tomato (Lycopersicon eseulentum Mill. cv. Starfire) and high frequency plant regeneration. Z. Pflanzenphysiol., 77 : 292--301. Kartha, K.K., Champoux, S., Gamborg, O.L. and Pahl, K., 1977. In vitro propagation of tomato by shoot apical meristem culture. J. Am. Soc. Hortic. Sci.,102: 346--349. Murashige, T., 1963. The role of gibberellin in shoot differentiation in tobacco tissue culture. In: P.R. White and A.R. Grove (Editors), Plant Tissue Culture. McCutchan, Berkeley, pp. 401--409. Murashige, T., 1977. Manipulation of organ initiationin plant tissue cultures. Bot. Bull. Acad. Sin., 18: 1--24. Murashige, T. and Skoog, F., 1962. A revised m e d i u m for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant., 15: 473--497. Nov~k, F.J., 1968. Modifikace orceinovJ barv~c~ metody vhodn~ pro n~kterd druhy zelenin. Genet. a Slecht. (Praha), 4: 96--98. Padmanabhan, V., Paddock, E.F. and Sharp, W.R., 1974. Plantlet formation from Lycopersicon esculenturn leaf callus.Can. J. Bot., 52: 1429--1432. Quak, F., 1977. Meristem culture and virus-freeplants. In: J. Reinert and Y.P.S. Bajaj (Editors), Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture. Springer-Verlag, Berlin--Heidelberg--New York, pp. 598---615. Sree Ramulu, K., Devreux, M., Ancora, G. and Laneri, U., 1976a. Chimerism in Lycopersicon peruvianum plants regenerated from in vitro cultures of anthers and stern internodium. Z. Pflanzenzilchtr.,76: 299--319. Sree Ramulu, K., Devreux, M. and De Martinis, P., 1976b. Origin and genetic analysis of plants regenerated in vitro from periclinalchimeras of Lyeopersicon peruvianum. Z. Pflanzenziicht., 77 : 112--120. v