Plant Cell, Tissue and Organ Culture 32: 145-151, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Photomorphogenic effects on in vitro rooting of Prunus roostock GF 655-2

Federica Rossi ~, Rita Baraldi 1, Osvaldo Facini I & Bartolomeo Lercari 2 ICentro di Studio per la Tecnica Frutticola, Consiglio Nazionale delle Ricerche, via Filippo Re 6, 40126 Bologna, Italy; 2Dipartimento di Biologia delle Piante Agrarie, viale delle Piagge 26, 56100 Pisa, Italy

Received 15 July 1991; accepted in revised form 22 June 1992

Key words: blue light photoreceptors, light quality, micropropagation, NAA, phytochrome, rhizogenesis

Abstract

The morphogenic effect of different light wavelengths on in vitro rooting of Prunus insititia GF655-2 in relation to the presence of napthaleneacetic acid (NAA) in the culture medium was investigated. Results of experiments in which plantlets were rooted in NAA enriched medium showed that the presence of auxin induced rooting even in the dark after an initial lag period. Illumination of the cultures with Red light was as effective in promoting rooting as treatment with 0.5/~M NAA; Red was more active in stimulating rooting in the short term than was NAA. The pattern of root formation resulting from the addition of NAA appeared to dominate development under White, Blue and Far Red treatments. Although it was possible to correlate the rooting response to the phytochrome photoequilibrium induced by the light treatments used, there arises a possible interference of specific Blue absorbing photoreceptors.

Abbreviations: B - Blue, FR - Far Red, HIR - High Irradiance Response, V f r - active (far-red absorb- ing) form of phytochrome, P t o t - total phytochrome, R - Red, W - White, NAA - a-naphtaleneacetic acid, B A - benzyladenine, I A A - indole 3-acetic acid

Introduction

The effects of light quality on regeneration pro- cesses and shoot differentiation have been re- ported (Hughes 1981; Lercari et al. 1986, Baraldi et al. 1988), but little attention has been paid to the interaction between plant growth regulators and light on in vitro rooting processes. Rhizogenesis is the last step in whole plant re- generation by in vitro methods, and is promoted by high auxin-cytokinin ratios. The action of the auxins in adventitious root formation can be modified by other factors (Murashige 1964, James 1983). Studies by Muir & Zhu (1983) and Ueda & Torikaba (1972) indicated the involve-

ment of phytochrome in the initiation of root apical meristems. Fuernkranz et al. (1990) have presented evidence that shows B light affects root formation in in vitro cultures of Prunus serotina. It is difficult to interpret these results in terms of the action of known photoreceptors since the level of rooting of the dark control (assumed to be low as in this study) is not reported. The authors seek to explain their re- sults in terms of oxidative decarboxylation of the auxin by light, as demonstrated by Song (1987), but their results do not preclude the photo- morphogenic action of a specific B absorbing pigment.

The work presented here is an attempt to

146

understand the effect of light and auxin on the control of rooting of in vitro grown shoots of the rootstock Prunus insititia Schneider GF655-2. Naphthaleneacetic acid (NAA), rather than I A A , was used in these experiments because it is not significantly destroyed by light (Pierik 1987).

M a t e r i a l s a n d m e t h o d s

Plant material, culture conditions and measurements

Shoot cultures of Prunus insititia Schneider GF655-2 were grown initially on a proliferation medium containing MS salt mixture (Murashige & Skoog 1962), 3 /zM thiamine, 2.7 /xM BA, 2% sucrose (w/v) and 0.65% commercial agar. The pH of the medium was adjusted to 5.25 with 0.1M KOH before autoclaving for 20 min at 120 °C. Cultures were incubated in growth cham- bers at 22 - 2 °C under 16 h light/8 h dark cycles. The light was provided by cool white fluorescent lamps at a photon flux of 60/zmol m 2 S- I at plant height. Single shoots, 15 mm long, were transferred to glass jars 10 cm in diameter and 8 cm in height, glass capped, containing a half- strength MS salts, supplemented with 3/xM thiamine, 2% sucrose, 0.65% commercial agar and, in some jars, with 0.5 tzM NAA.

Thirty shoots were used per each treatment and the treatments were run in duplicate. The experiment was conducted twice. Analysis of variance was used to evaluate the effect of light treatments on percent rooting and the mean number of roots per shoot.

Rooting percentages were determined under a dim green safe light for all treatments. Root extension (number × length of roots) was de- termined at the end of the experimental period.

Light treatments and sources

For experimental purposes the explants were maintained under continuous illumination ac- cording to the schedule shown in Fig. 1. White light was provided by Philips 40W 33RS Cool White fluorescent tubes. Blue light was obtained by filtering the same light source through a Lee 141 cinemoid filter (Lee Filters Ltd, Hants, UK),

8

b [

c

d

e I I

I I 0 14 21 28

Fig. 1. Experimental protocol. The experimental cultures were treated as follows: a) 28 days of darkness; b) 7 days of light followed by 21 days of darkness; c) 14 days of light followed by 14 days of darkness; d) 21 days of light followed by 7 days of darkness; e) 28 days of light. The light treat- ments used were W, R, B and FR as specified in Materials and methods.

while Red light was produced via a Lee 106 filter. Far Red was produced by using Linestra incandescent tubes filtered through plexiglass PG 501/3 (Impla, s.p.a. Italy) and KG 3/2 mm glass filters (Schoot & Geinz, Mainz, Germany). The spectral emission curves (Fig. 2) and total photon fluence rates of these light sources were measured with a calibrated LiCor 1800 Spec- troradiometer. The photon fluence rates were 36/zmol m - 2 s - 1 for W, R and B and 12/zmol m-2 s ~ for FR. The phytochrome photoequilib- rium (Pfr/Ptot) values were obtained multiplying the emission spectrum by the absorption spectra for Pr and Pfr from purified rye phytochrome and correcting for 86% maximal photoconver- sion to Pfr (Lercari & Deitzer 1987).

Regression analysis was performed to examine linear relationships between rooting percentage,

1"1 I 'E o E ~ o.65

0 400

B /

R

L

6 0 0 600 700 800 900

Wavelength (nm)

Fig. 2. Emission spectra of W, B, R and FR sources as described in Materials and methods.

147

root extension and phytochrome photo- equilibrium.

Results

Figure 3 shows the result of experiments in which the appearance of roots on shoots growing under various light regimes in the presence or absence of NAA was monitored weekly up to 28 days. A very low rooting percentage occurred for the dark control without NAA. This increased slightly for shoots exposed to FR light but to a much greater extent for shoots exposed to B, R and W light.

After 7 days of R light without auxin, about 80% of shoots rooted, while under W and B the rooting percentages were about one third. Root- ing in R light was always higher during the entire experimental period, reaching the maximum at 28 days. Under W and B light the final rooting percentage without auxin supply was approxi- mately 80% and 70% respectively.

After an initial lag period of at least 7 days, NAA dramatically enhanced root production of dark grown shoots. FR light appeared to elimi- nate the initial lag period, although the response reached saturation more slowly and never reached the 100% attained by the dark grown shoots.

The interaction between NAA, W, R and B appears to be more complex. Under these re- gimes, the presence of NAA resulted in initially lower root appearance and, in R and B light treatments, root appearance did not differ sig- nificantly from the respective control (-NAA) by day 28. Under W light condition, NAA en- hanced root emergence by day 28.

Figure 4 shows rooting percentage found on day 28, when the shoots were grown under W, R, B, FR, with and without NAA, using the various time regimes outlined in Fig. 1. A com- parison of these data with those in Fig. 3 shows that shoots exposed to W, R, B light for 7 days continued to develop additional roots during the ensuing dark period. NAA effects on rooting in W, B and R and in the dark are similar to those seen in Fig. 3.

Root number per plant (Fig. 5) was also de- termined on day 28 for the same shoots as in Fig. 4. NAA increased the number of roots per shoot in the dark, as well as in W, R and FR. Although there was quite a lot of variation, there did not appear to be any significant effect of B on the number of roots developed per shoot. It is evi- dent from these data that R is the most effective light regime for the production of roots and this effect is enhanced further by the presence of NAA.

Although NAA greatly enhanced root number

r o o t i n g % I 0 0 -

9 0 -

80 - -[ ]

7 0 -

6 0 -

5 0 -

4 0 -

20

0 ~ 7 14 21

WHITE

I

28 7 1 4 21 28

RED

I - NAA

7 iT

7 14 21 2 8 7 14

BLUE FAR

+ NAA

I -1 - 21 2B 7 14 21 2 8 days

RED DARK

Fig. 3. Rooting percentages in the absence or presence of t).5 txM NAA after various periods of different spectral quality light. Data collected at the end of light period of each treatment.

148

100

90

80

70

60

50

40

30

20

10

0

r o o t i n g %

7 14 21 28

WHITE

7 14 21 28

RED

T

7 14 21 28

BLUE

I I I I

7 14 21 28 28 d a y s

FAR RED DARK

I l- NAA E~]+ NAA

Fig. 4. Root ing percentages in the absence or presence of 0.5 IzM N A A on day 28. Plantlets were grown in W, R, B or FR using the light regimes in Fig. 1.

r o o t n u m b e r 4 -

3 -

2 -

0 7 14 21 28

WHITE

I

7 14 21 28

RED

~ - ] - NAA

14 21 28

BLUE

JJ l 7 14 21 28

FAR RED

~ J ~ + NAA

,J 28

DARK

days

Fig. 5. Root number per plant in the absence or presence of 0 . 5 / z M N A A on day 28. Plantlets were grown in W, R, B or FR using the light regimes in Fig. 1.

in dark grown shoots (Fig. 5), from Fig. 6 it is apparent that root elongation required light. Both R and B were more effective in producing root elongation then when in combination with auxin.

Discussion

A previous study on shoot proliferation of the same species (Baraldi et al. 1988) demonstrated that BA action can be expressed only in presence

149

Root e x t e n s i o n ( m m ) 100 -

80- ]-

6 0 -

4 0 -

E 0 -

0 -

W H I T E

7

RED BLUE FAR RED

t l - NAA ~ + NAA

DARK

Fig. 6. Root extension (number of roots × root length) per plant in the absence or presence of 0.5/xM NAA after 28 days. The data refer to the light treatments e) (28 days of light) and a) (28 days of darkness) as in Fig. l.

of light, showing that both cytokinins and light are indispensable factors in this developmental process. The present experiments, in which auxin and light are shown to act independently on in vitro rooting, confirm the complexity of the regulation of growth and differentiation pro- cesses. This underscores the need to determine the combination of environmental plus hormonal factors that control such processes as cell prolif- eration, shoot elongation, rooting and root elon- gation in order to fully understand these pro- cesses.

An unambiguous interpretation of the results presented in this paper as action and interaction of phytochrome and NAA depends on our cul- tures making carbohydrate from the sucrose sup- plied on the media rather than via photosyn- thetic carbon fixation. If these cultures were able to make net gains in photosynthetic carbon fixa- tion, the growth differences reported could be a function of carbohydrate supply dependent upon different efficiencies of B, R and W in stimula- tion of photosynthesis. However, it has been shown that carbon fixation on in vitro cultures is enhanced by the use of high fluence rates, CO 2 enrichment of the head space of the culture containers and a reduction in the sucrose content

of the media supplied (Neuman & Bender 1987; Langford & Wainwright 1987; Bender 1985; Fu- jiwara et al. 1989; Kozai et al. 1988; Infante et al. 1989; Righetti et al. 1990), and in the experi- ments reported here none of these conditions are met. Cultures were grown on 2% sucrose media in gas-tight jars and lacked CO 2 enrichment. In addition, low photon fluence rates (36/xmol

- 2 --I m s ) were used for these experiments. Under these conditions, any photosynthetic fixa- tion would be minimal and the difference in efficiency between the light regimes would also be low in absolute terms. In a photosynthetic study with closely related plant material (Prunus avium), Righetti et al. (1990) reported that in cultures grown for 30 days on a medium contain- ing 2% sucrose under low fluence radiation, CO 2 accumulates in the head space of the flask throughout the experiment. In consequence, the differences in rooting observed in the experi- ments here are unlikely to be due to differences in photosynthetic CO 2 fixation.

Long term irradiations with FR light have, in the past, been associated with the high irradiance response (HIR) but Beggs et al. (1980) have shown that the HIR is lost during the process of de-etiolation, and some possible examples of

150

HIR in green tissue appear in the literature (Morgan et a1.1980; Lercari 1982; Lercari & Deitzer 1987; Carr-Smith et al. 1989).

In the majority of work carried out with green tissue, FR is thought to produce only low levels of Pfr. Work from various laboratories (Smith & Whitelam 1990) now supports this view since the phytochrome present in green plants is signifi- cantly different from that found in etiolated tissue.

We have, therefore, been able to correlate the rooting responses of our Prunus shoot cultures to the phytochrome photoequilibrium induced by the light treatments used. The data presented in Fig. 7 are derived from the rooting percentage and the total root length measured on day 28 after the shoots had received various light re- gimes. These data are presented in a log-linear from after the style of Morgan & Smith (1976), and show a good linear relationship indicating that rooting response to light is under the control of phytochrome. The deviation of the data de- rived from the B treatments may indicate that although phytochrome is the prime pigment con- trolling the rooting response, interference may arise from simultaneous stimulation of a specific B-absorbing photoreceptor.

A comparison of Fig. 3 and Fig. 4 shows that almost the maximum percentage of rooting is obtained during the first seven days of R, and this does not significantly increase during a sub- sequent 21 days in the dark. A short period of R

2 l o g r o o t i n g ~. l o g r o o t extens ion ...... ~.- 12

1 , 5 . . . . . . . . . . . . . . . ~ " ~ . . . : ' = ' " " A - 1 , 5

1 . . . . . 1

0,5 0,5

0 L i i i i i L f 0

o,t 0,z o,a 0,4 0,5 0,8 0,7 0,8 0,9 photoequil ibriurn

• - ~ ' " r o o t i n g ~ ~ r o o t extens ion

Fig. 7. Logarithm of the rooting percentage after 7 days of light and of the total root extension as a function of the calculated phytochi'ome photoequilibrium. The reported val- ues of photoequilibrium are: 0.04 for FR, 0.4 for B, 0.7 for W, 0.86 for R.

irradiation would consequently be capable to accelerating rhizogenesis. Results with NAA- enriched media indicate that this auxin promotes the initiation of rooting, even in the dark, after an initial lag period. The pattern of root forma- tion resulting from the presence of the growth regulator (Fig. 3) appears to dominate develop- ment under W and FR. Illumination of the cul- tures with R is as affective as treating the cul- tures with N A A and indeed, it is more effective in stimulating rooting in the short term than is NAA.

The results indicate a complex interrelation- ship between light and plant growth regulators, and rule out the simple linear situation with light acting to increase hormone level, suggested by the rooting response to NAA in the dark.

While B is almost as active as W in promoting rooting in absence of auxin, B also appears to inhibit the promotion of rooting by N A A . The possible inhibitory effect of high fluence rate B, acting via specific photoreceptor on rooting in the presence of auxin is presently under investi- gation in this laboratory.

Acknowledgements

We would like to thank Dr. T. H. Attridge, N.E. London Polytechnic, U.K., Prof. R. H. Zim- merman and Dr. J. Pernise Slovin, U.S.D.A., Beltsville, U.S.A., for helpful discussion and encouragement to publish these data. We are also grateful to A. Mazza and M. Govoni for their technical assistance. This work was funded by the Italian National Research Council and by M.U.R.S.T. 40%.

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