Improved inorganic media constituents for in vitro shoot multiplication of Vitis

ScientiaHorticulturae, 32 (1987) 85-95

85

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Improved Inorganic Media Constituents for In Vitro Shoot Multiplication of Vitis R. CHI~E and R.M. POOL

Department of Pomology and Viticulture, New York State Agricultural Experiment Station, CorneU University, Geneva, N Y 14456 (U.S.A.) (Accepted for publication 16 January 1987)

ABSTRACT Ch~e, R. and Pool, R.M., 1987. Improved inorganic media constituents for in vitro shoot multiplication of Vitis. Scientia Hortic., 32: 85-95. The effects of inorganic media constituents on shoot multiplication in vitro were investigated with the Vitis hybrid 'Remaily Seedless'. The explants were 1.5-cm sub-cultured shoot tips with 3-4 nodes. An improved medium for shoot multiplication was designed. It differsfrom others in itslack of added chloride and potassium iodide, and in a lower manganese sulfate concentration. The effects of KI on shoot production were also produced by MnS04. Iodine, a non-essential element in growth and development of higher plants, can be withheld from culture media in our conditions. The effectsof manganese and KI on shoot production are discussed in relation to their involvement in auxin metabolism and transport.

Keywords: chlorine; culture medium; grapevine; iodine; manganese; organ culture. Abbreviations: A=Anderson (1978); L=Lepoivre (Quoirin et al., 1977); MS=Murashige and Skoog (1962); CD=Campbell and Durzan (1975); N N = N i t s c h and Nitsch (1969); DMRT = Duncan's multiple range test; BAP = benzylaminopurine; IAA = indoleacetic acid.

INTRODUCTION

In vitro vegetative propagation of grapevines from shoot apices (0.5-1 mm) has been reported by Chde and Pool (1982), Harris and Stevenson (1979) and Skene and Barlass (1980). In vitro culture techniques offer unparalleled potential for rapid vegetative propagation. An adequate formulation of organic nutrients for shoot multiplication has been defined for the Vitis hybrid 'Remaily Seedless' by Chde (1982) and Chde and Pool (1985). Media utilized by researchers for the culture of Vitis shoot apices vary widely 0304-4238/87/$03.50

© 1987 Elsevier Science Publishers B.V.

86 in composition (Gifford and Hewitt, 1961; Hoeffer and Gifford, 1964; Galzy, 1972; Jona and Webb, 1978; Chde and Pool, 1982 ). A comparison of inorganic nutrients has shown differential results for the establishment in culture of 'Remaily Seedless' shoot tips (Chde, 1982). Therefore, we expect that some inorganic media constituents will also prove better than others in the shoot multiplication phase. We are particularly interested in the effects of iodine, manganese and chlorine for the reasons stated below. Although iodine is not an essential element for growth and development of higher plants (Rains, 1976), KI is still a component of most tissue culture media ( Murashige and Skoog, 1962; Campbell and Durzan, 1975; Lepoivre, in Quoirin et al., 1977). Potassium iodide probably affects auxin metabolism and/or transport (Briggs, 1963; Schmidt et al., 1977; Leong and Briggs, 1982). This would explain its retention in tissue culture media when other non-essential elements have been eliminated. Manganese has been found to affect IAA oxidase systems directly as a cofactot or via its effects on natural cofactors or inhibitors ( Schneider and Wightman, 1974). Our concern for chloride comes from the well-known inhibitory effect of sodium chloride on grapevine growth and development. Chloride is the principal ion accumulated in leaves of grapevines treated with NaC1 (Downton, 1977). Most chloride salts also contain iodine as a contaminant. This report concerns the effect on shoot multiplication of some inorganic constituents in the basal medium, investigated with the goal of designing an improved medium for grapevine. MATERIALSAND METHODS The V i t i s hybrid 'Remaily Seedless' was established in culture and shoots were produced as described elsewhere ( Chde, 1982). The explants used in these experiments were 3-4-node shoots (1.5 cm) sub-cultured from media containing MS salts (except for Experiment I). The shoots were cut 2-4 mm below the oldest node, placed horizontally on the medium and lightly pressed in with the cut end submerged. For Experiment I the shoots were subcultured onto the same salts as those on which they originated and the blades of the expanded leaves ( leaves separated from the apical bud) were removed, retaining the severed petioles. The sub-cultures were incubated in pre-sterilized 25 × 100-mm plastic Petri dishes containing 30 ml of medium.

Plant material. -

a n d m e d i a c o m p o s i t i o n . - We investigated the effects on shoot multiplication of 4 macronutrient sets in combination with 2 micronutrient sets (Experiment I), the effects of 8 sets of inorganic media constituents

Experiments

87 TABLE

I

Inorganic constituents of media used I

Macronutrients (mM) NH4N03 KN03 MgSO4*7H20 KH2P04 CaCI2o2H20 NaH2PO4°H20 Ca(NO3)2°4H20 KC1 FeSO4°7H20 Na2.EDTA

A

CD 5.0 4.7 1.5 0.0 3.0 2.75 0.0 0.0 0.2 0.2

10.0 3.4 1.5 1.25 0.0 0.0 4.15 0.9 0.1 0.1

L 5.0 17.8 1.5 2.0 0.0 0.0 5.1 0.0 0.1 0.1

MS 20.6 18.8 1.5 1.25 3.0 0.0 0.0 0.0 0.1 0.1

NN 9.0 9.4 0.75 0.5 1.5 0.0 0.0 0.0 0.1 0.1

Micronutrients (#M) KI MnSO4°4H20 H3B03 ZnSO4°7H20 Na2MoO4o2H20 CuSO4°5H20 CoC12o6H20

C1

C2

C3

10.0 14.8 1.0 1.5 1.5 0.0 0.0 0.0 0.1 0.1

20.6 18.8 1.5 1.25 0.0 0.0 3.0 0.0 0.1 0.1

20.6 12.8 1.5 1.25 0.0 0.0 3.0 0.0 0.1 0.1

m 1.8 100.0 100.0 30.0 1.0 0.1 0.1

5.0 0.5 5.0 0.0 100.0 4.5 100.0 112.0 100.0 100.0 100.0 162.0 30.0 30.0 30.0 34.8 1.0 1.0 1.0 1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.0

b

c

d

5.0 0.0 5.0 0.0 100.0 100.0 5.0 5.0 100.0 100.0 100.0 100.0 30.0 30.0 30.0 30.0 1.0 1.0 1.0 1.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

1C1, C2, C3= different sets of macronutrients that were used in combinations with m,b,c,d ( = different sets of micronutrients). ( E x p e r i m e n t I I ) , a n d t h e effects o f m a n g a n e s e sulfate a n d p o t a s s i u m iodide ( E x p e r i m e n t I I I ) . T h e inorganic m e d i a c o n s t i t u e n t s are listed in T a b l e I. E x p e r i m e n t I c o m p a r e d c o m b i n a t i o n s o f t h e m a c r o n u t r i e n t s A, L, M S a n d C1 w i t h t h e m i c r o n u t r i e n t s A a n d MS. T h e m i c r o n u t r i e n t s differ only b y a lower c o n c e n t r a t i o n o f K I in A. We were t h e r e f o r e s t u d y i n g t h e effects o f K I concentration. E x p e r i m e n t II c o m p a r e d t h e salts CD, C2m, C3m, L ( w i t h m i c r o n u t r i e n t s at h a l f - s t r e n g t h ) , M S (as c o n t r o l ) , M S w i t h o u t KI, h a l f - s t r e n g t h M S , a n d N N . In E x p e r i m e n t III t h e effects of M n S 0 4 c o n c e n t r a t i o n a n d K I were investigated. T h e o t h e r m i c r o n u t r i e n t s were o f MS. T h e m a c r o n u t r i e n t s were C2. All m e d i a h a d t h e following organic s u b s t a n c e s in c o m m o n : 3 # M thiam i n e . H C 1 ; 5 5 . 5 / t M myo-inositol; 8/~M nicotinic acid; 5/~M pyridoxine.HC1; 5/~M B A P . N o a u x i n was used. T h e s e levels were f o u n d to be o p t i m a l for s h o o t m u l t i p l i c a t i o n in p r e v i o u s e x p e r i m e n t s ( Chde a n d Pool, 1985 ). All m e d i a cont a i n e d 3% sucrose a n d 0.8% " B a c t o - A g a r " ( D I F C O L a b o r a t o r i e s ) . B e f o r e autoclaving, p H was a d j u s t e d to 5.7-5.8.

88 Incubation conditions. - Illumination was by a 1:1 mixture of "Gro-Lux" (F40GRO) and "Cool White" (F40CW) fluorescent tubes (Lifeline series, manufactured by Sylvania). The tubes were 14 cm apart and 30 cm above the cultures. Total radiant energy measured at the explant level was 1900/~W cm -2. Daylength was 10 h. Temperatures varied from 21°C in the dark to 27°C in the light. Design of the experiments. - Experimental designs were completely random with sub-sampling. Each treatment had 6 plates each containing 3 explants. The treatment designs were structured as a 4 X 2 factorial for Experiment I and a 2 X 2 factorial for Experiment III. Statistical analysis was by the components of variance method to determine if specific factors had effects and/or interacted. T r e a t m e n t means were compared using DMRT, when appropriate, to determine which media induced best shoot multiplication. Variates recorded after 8-9 weeks were the number of shoots produced per explant (shoot/explant) and the number of nodes on those shoots. From these data we calculated, per explant, the number of shoots having at least 3 nodes and their percentage (% shoots i> 3 nodes/explant), the average number of nodes on the shoots produced (nodes/shoot) and the total number of nodes as a measure of vegetative vigor (total nodes/explant). RESULTS Effects of macronutrients and potassium iodide concentration (Experiment I). - The contributions of the macronutrients to the total sum of squares were significant for each variate ( Table II). KI contributed significantly to shoots / explant and nodes/shoot but not to % shoots >I 3 nodes/explant and total nodes/explant. Therefore all the simple effects are listed in Table III for the first 2 variates, while only the average effects for each macronutrient are given for the latter two. The means were compared with a D M R T within each variate to ascertain which medium gave the best performance. The macronutrients MS produced more shoots and macronutrients L produced larger shoots as measured in nodes (Table III). In general, media with the lower KI concentration produced either fewer or the same number of shoots. Lower KI concentration also resulted in larger shoots. Effects ofvlifferent inorganic salt formulae (Experiment II). - Analyses of variance showed significant treatment terms for all variates. The means of the 8 treatments were compared using D M R T (Table IV). More shoots were obtained with L salts. The shoots were largest on MS salts. Removing KI from the latter medium did not diminish the number of shoots produced but decreased their size. Differences in the aspect of the shoots produced were noticed. Sb.~ots that

98 7 3 1 3 91 177 (21) 275(21)

Among plates Among t r e a t m e n t s Macronutrients KI conc. Interaction Among plates w i t h i n t r e a t m e n t s Among explants w i t h i n plates Among explants

16 331 5 899 4 669 750 480 10 431 13 209 29 540

55 20 16 3 1 35 45 100 ** * NS

57 212 8 033 6 928 810 295 49 180 82 986 14 099

41 6 5 1 0 35 59 100

%SS

SS

F

SS

%SS

% Shoots/> 3 nodes

Shoots/explant

** NS NS

F 144 38 25 11 2 106 198 342

SS 42 11 7 3 1 31 58 100

%SS

Nodes/shoot

** ** NS

F 119 946 42 254 38 114 2 464 1 676 77 692 111 151 231 096

SS

52 19 17 1 1 33 48 100

%SS

Total n o d e s / e x p l a n t

NS

NS

F

1Missing values in parentheses; d f = degree of freedom; SS = sum of squares; F = test of significance; * = significant at P = 0.05; ** = significant a t P = 0.01; N S -- non-significant.

dr'

Source of variation

Analyses of variance. Effects of m a c r o n u t r i e n t s a n d p o t a s s i u m iodide c o n c e n t r a t i o n on shoot p r o d u c t i o n from 'Remaily Seedless' shoot explants of 3-4 nodes (1.5 c m ) sub-cultured in vitro for 8-9 weeks

T A B L E II

QO

11 bcd 10bcd 6 d

13 bc 7 cd 6 d

19 a

63 a 59a 62a

51 b

4.2 a 3.3 b 3.3 b

3.4 b

Low KI

Nodes/shoot

3.5 b 3.1bc 2.7 c

3.0 bc

High KI

ISeparation of means within variatesby Duncan's multiple range test (P=0.05). 2The micronutrients were of MS, two concentrations of potassium iodide (KI) being compared (1.8and 5/tM).

14 b

L C1 A

Average KI effects

Low KI

High KI

% Shoots >/3 nodes

Shoots/explant

MS

Macronutrients

43 a 29 b 19 b

50 a

Average KI effect

Total nodes/explant

Effectsof macronutrients and potassium iodide concentration on shoot production from 'Remaily Seedless'shoot explants of 3-4 nodes (1.5cm) sub-culturedin vitrofor 8-9 weeks I'2

T A B L E III

¢X)

91

TABLE IV Effectof differentinorganicsalt formulaeon shoot multiplicationfrom 'RemailySeedless'shoot explants of 3-4 nodes (1.5 cm) sub-culturedin vitro for 8-9 weeks1 Basal salts

Shoots/explant % Shoots >/3 nodes Nodes/shoot Total nodes/explant

CD C2m (see Table I) C,~m (see Table I) L, 1/2 micronutrients MS MS, n o K I MS, half-strength NN

25 26 26 49 23 29 4 5

b b b a b b c c

49ab 49 ab 44 ab 41 ab 56a 42ab 38 b 19 c

2.5 bc 3.1 ab 2.6 bc 2.4 bc 3.5a 2.5 bc 2.0 cd 1.5 d

59 b 74 b 68 b 111 a 64 b 70 b 9 c 9 c

1Separation of means within columns by Duncan's multiple range test (P = 0.05)

were greener and of a better constitution were produced with C2 m and C3m salts, followed by those on MS and CD. With L salts (half-strength micronutrients), the shoots grew in dense clusters and were of lesser quality with regard to constitution and appearance. Effects of manganese sulfate concentration and potassium iodide (Experiment III). - The effects of these two factors were tested with the salt formulations C2m, C2b, C2c and C2d, and a 2 × 2 factorial was analyzed. The manganese term made no significant contributions to the total sum of squares for any of the variates (Table V). The iodine term was significant for shoots/explant, nodes / shoot and total nodes/explant. The interaction between manganese and iodine was significant for shoots/explant and total nodes/explant. To ascertain that one of the new salt formulations gave better performances, the four treatments were also compared to MS and L salts, our previous bests. New analyses of variance were made and the means of the six treatments were compared with DMRT's for each variate (Table VI). Shoot multiplication on C2d salts was significantly greater than on any of the other media. The salts, C2c, produced larger shoots than C2d and C2b. The effect of KI, tested with C2 macronutrients, was significant with the lower manganese concentration. There was a dramatic increase in shoot multiplication with both low Mn and no KI. DISCUSSION

Effects of macronutrients and potassium iodide concentration. - The results replicate findings made when establishing shoots in culture (Ch~e, 1982 ). In particular, shoot production from leafless explants was favored by MS salts (above other formulae) and decreased by reducing KI.

1 1 1 20 47 (1) 70

Manganese Iodine Interaction Among plates within treatment Among explants within plate Among explants

553 1 490 3 634 4 389 15 493 25 557

2 6 15 18 62 102

NS * **

9 90 0 1 825 3 139 5 064

0 2 0 36 62 100

%SS

SS

F

SS

%SS

% Shoots/> 3 nodes

Shoots/explant

NS NS NS

F

0.7 2.7 0.7 9.6 15.0 28.6

SS

2 9 2 33 52 100

%SS

Nodes/shoot

NS * NS

F

6 413 7 554 22 033 30 603 87 236 153 838

SS

4 5 15 21 58 103

%SS

Total nodes/explant

NS * **

F

1Missing values in parentheses; d f = degree of freedom; SS = sum of squares; F = test of significance; * = significant at P = 0.05; ** -- significant at P = 0.01; N S = non-significant.

df 1

Source of variation

Analyses of variance. Effect of manganese sulfate concentration and potassium iodide on shoot production from 'Remaily Seedless' shoot explants of 3-4 nodes sub-cultured in vitro for 8-9 weeks

TABLE V

eX~ t~

93 TABLE VI Effects of manganese sulfate concentration and potassium iodide on shoot production from 'Remaily Seedless' shoot explants of 3-4 nodes (1.5 cm ) sub-cultured in vitro for 8-9 weeks ~ Medium 2 C2m C2b C2c C2d L MS

MnSO4

KI

(/zM)

(/~M)

100 100 5 5 4.5 100

5 0 5 0 0.5 5

Variation coefficient

Shoots/explant

% Shoots/> 3 nodes Nodes/shoot

Total nodes/explant

31 b 25 b 22 b 45 a 27 b 21 b

48a 46a 49a 47 a 47a 48a

82 67 65 121 75 59

0.35

0.13

3.0 ab 2.8 b 3.4a 2.8 b 3.lab 3.0ab 0.14

b b b a b b

0.32

~Separationof means within columns by Duncan's multiplerange test (P-- 0.05 ). 2See Table I.

The responses to KI seem inconsistent with its possible role in IAA transport. Phototropic responses are known to be mediated by lateral transport of auxin (Briggs, 1963), and KI inhibits phototropism (Schmidt et al., 1977). If KI increased endogenous IAA levels by opposing its transport, then lowering KI should improve IAA diffusion through the explant into the medium and thereby release axillary buds from auxin inhibition. The reverse was observed. Since shoot growth depends on a specific balance of endogenous and exogenous hormones, one could argue that such balance for maximum shoot production was established with media containing higher KI concentrations. The effects of exogenous cytokinin/auxin ratios on shoot production were demonstrated (Chde and Pool, 1982). Supra-optimal BAP additions, creating a high cytokinin/auxin ratio, inhibited shootgrowth (Chde and Pool, 1985). Such high ratios might occur if decreased KI results in lower IAA concentrations. Effects of different inorganic salt formulae. - Only effects relevant to improving shoot multiplication are discussed. Shoot multiplication was best on L salts (halfed micronutrients). However, shoots were of a size inadequate for micropropagation, of lesser quality and grew in dense clusters, which made dissection difficult. Cultures on C2m, C3m and MS salts were less dense with shoots of adequate size. C2m and C3m salts, which differ from MS salts by the substitution of calcium nitrate for calcium chloride, produced shoots of better quality. This is consistent with reported chloride sensitivity of grapevines (Woodham, 1960). Multiplication of shoots from leafed explants on MS salts was not statistically different when KI was witheld, but shoots were smaller. With leafless explants, lowering KI resulted in fewer but larger shoots (see above), and we suggested that witholding KI facilitated IAA removal from the explant, resulting in a supra-optimal cytokinin/auxin ratio. Perhaps IAA production by the

94

leaves ( Leopold and Kriedemann, 1975) maintained a hormone balance favorable to shoot multiplication. Effects of manganese sulfate concentration and potassium iodide. - As discussed above, removing KI did not decrease shoot multiplication on MS salts. However, twice as many shoots grew on L salts with halfed micronutrients. It is possible that lowering MnS04, in addition to lowering KI, improved shoot multiplication. Both nutrients may affect IAA action ( Stonier et al., 1968; Rains, 1976; Schmidt et al., 1977). Furthermore, L salts do not contain chloride to which grapevines are sensitive (Woodham, 1960) and which are a possible source of iodine contamination. In this experiment, it was verified that high Mn concentrations might mask KI effects. Media C2m and C2b with high manganese levels did not show an effect, while C2c and C2d with low manganese did. Maximum shoot multiplication occurred with no KI. This is consistent with our hypothesis that KI inhibits IAA transport (Briggs, 1963; Schmidt et al., 1977), and manganese inhibits IAA oxidation (Stonier et al., 1968). Lowering Mn or KI would decrease endogenous auxin, resulting perhaps in decreased inhibition of axillary growth. CONCLUSION

Iodine,a non-essentialelement for growth and development of higher plants, can be removed from tissue culture media under certain conditions. Its effects interact with those of M n and are masked by a high M n concentration. Substitutingcalcium nitratefor calcium chlorideimproved the qualityof grapevine shoots produced in culture. The medium C2d promoted shoot multiplication above the M S and L saltsthat gave best resultspreviously.

REFERENCES Anderson, W.C., 1978. Tissue culture propagation of Rhodendrons. In Vitro, 14:177 (abstract). Briggs, W.R., 1963. The phototropic responses of :higherplants. Annu. Rev. Plant Physiol., 14: 311-352. Campbell, R.A. and Durzan, D.J., 1975. Induction of multiple buds and needles in tissue culture of Piceaglauca. Can. J. Bot., 53: 1652-1657. Chde, R., 1982, In vitro micropropagation of Vitis.Ph.D. Thesis, Cornell University, Ithaca, pp. 1-155. Ch~e, R. and Pool, R.M., 1982. The effectsof growth substances and photoperiod on the development of shoot apices of Vitiscultured in vitro.Scientia Hortic., 16: 17-27. Ch~e, R. and Pool, R.M., 1985. In vitro propagation of Vitis:the effectsof organic substances on shoot multiplication.Vitis,24: 106-118. Downton, W.J.S., 1977. Photosynthesis in salt-stressedgrapevines. Aust. J. Plant Physiol., 4: 183-192. Galzy, R., 1972. La culture in vitrodes apex de Vitisrupestris.C.R. Acad. Sci.,Paris,274: 210-213.

95 Gifford,E.M., Jr. and Hewitt, W.M.B., 1961. The use of heat therapy and in vitroshoot tip culture to eliminate Fanleaf virus from the grapevine. Am. J. Enology Vitic.,12: 129-135. Harris, R.E. and Stevenson, J.H., 1979. Virus elimination and rapid propagation of grapes in vitro. Comb. Proc. Int. Plant Propag. Soc., 29: 95-106. Hoeffer, L.L. and Gifford, E.M., Jr., 1964. Growth in vitro of excised stem tip of Vitis vinifera. Am. J. Bot., 51:677 (abstract). Jona, R. and Webb, K.J., 1978. Callus and axillary-budcultureof Vit~svinifera'Sylvaner Riesling'. Scientia Hortic., 9: 55-60. Leong, T.Y. and Briggs, W.R., 1982. Evidence from studies with acifluoffenfor participation of a flavin-cytochrome complex in blue lightphotoreception for phototropism of oat coleoptiles. Plant Physiol., 70: 875-881. Leopold, A.C. and Kriedemann, P.E., 1975. Plant Growth and Development. McGraw-Hill, New York, pp. 1-545. Murashige, T. and Skoog, F., 1962. A revisedmedium for rapid growth and bioassays with tobacco tissue cultures.Physiol. Plant., 15: 473-497. Nitsch, J.P. and Nitsch, C., 1969. Haploid plants from pollen grains. Science, 163: 85-87. Quoirin, M., Lepoivre, P. and Boxus, P., 1977. Un premier bilan de 10 ann~es de recherches sur les cultures de m~rist~mes et la multiplication in vitro de fruitierligneux. Compte Rendu des Recherches, Station des Cultures Fruitibreset Maralch~res de Gembloux, Belgium, pp. 93-117. Rains, D.W., 1976. Mineral metabolism. In: J. Bonner and J.L. Varner (Editors), Plant Biochemistry.Academic Press, N e w York, pp. 1-907. Schmidt, W., Hart, J.,Filner,P. and Poff, K.L., 1977. Specificinhibitionof phototropism in corn seedlings.Plant Physiol.,60: 736-740. Schneider, E.A. and Wightman, F., 1974. Metabolism of auxin in higher plants. Annu. Rev. Plant Physiol., 25: 487-513. Skene, K.G.M. and Barlass, M., 1980. Micropropagation of grapevine. Comb. Proc. Int. Plant. Propag. Soc.,30: 564-570. Stonier, T., Rodriguez-Tormes, F. and Yoneda, Y., 1968. Study of auxin protector.IV. The effect of manganese on auxin protector-I of the Japanese Morning Glory. Plant Physiol.,43: 69-72. Woodham, R.C., 1960. The chloride status of the irrigatedSultana vine and its relation to vine health. Aust. J. Agric. Res., 7: 414-427.