The effect of varying light regimes on in vitro rooting ofmicrocuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy-to-root ...
Plant Cell, 7issue and Organ Culture 41: 139-143, 1995. (~) 1995KluwerAcademic Publishers. Printedin the Netherlands.
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Light effects on in vitro rooting of pear cultivars of different rhizogenic ability G i a n p a o l o B e r t a z z a , R i t a Baraldi* & S t e f a n o Predieri Istituto di Ecofisiologia delle Piante Arboree da Frutto, Consiglio Nazionale delle Ricerche, via Gobetti 101, 40129 Bologna, Italy (* requests for offprints)
Received22 June 1994;acceptedin revisedform23 January1995 Key words: auxin, light, micropropagation, Pyrus communis, phytochrome, rhizogenesis.
Abstract
The effect of varying light regimes on in vitro rooting ofmicrocuttings of two pear (Pyrus communis L.) cultivars was investigated. Cultures of the easy-to-root 'Conference' and the difficult-to-root 'Doyenne d'Hiver' were incubated for 21 days with or without indole-3-butyric acid (IBA) in the medium in darkness or under continuous far-red (8 /~.mol m -2 s -1 , blue, white or red (15 or 36 #mol m -2 s -~) light. 'Conference' rooted without IBA when exposed to red, blue or white light while no rooting was observed under far-red light and in darkness. The high rooting efficiency under red and, by contrast, the inhibition under far-red light and darkness suggest the involvement of the phytochrome system in rhizogenesis. The addition of IBA to the culture medium enhanced root production under all light regimes in both cultivars. Red light, especially at the lower photon fluence rate, had a positive effect by increasing root extension (number x length of roots) and stimulating secondary root formation. Abbreviations: IBA - Indole-3-butyric acid, R - red light, B - blue light, FR - far-red light, W - white light, D -
darkness, Pfr - active (far-red light absorbing) form of phytochrome, Ptot - total phytochrome, B A - benzyl-adenine Introduction
The formation of adventitious roots by shoot microcuttings is a phenomenon under the control of various endogenous and external factors, including hormones, growth regulators (Fellenberg 1976; Batten & Goodwin 1978) and light acting through the phytochrome system (Reddy et al. 1975; Pfaff & Schopfer 1980; Rossi et al. 1993). The involvement of phytochrome in hormone production during plant morphogenesis is fairly well established (Behringer et al. 1992; Buhlet et al. 1978; Wareing & Thompson 1976) and it is also been suggested for light-induction of rhizogenesis (Pinker et al. 1989). The effect of light on the initiation of adventitious roots varies with species and cultivar. Root induction in darkness increased the number of roots formed in apple and peach (Zimmerman 1984; Hammerschlag et al. 1987), whereas for mung bean and Sequoia induction in the light led to higher
root number (Jarvis & Shaheed 1987; Walker et al. 1987). A linear relationship between in vitro rooting enhancement and increasing values of the phytochrome photoequilibrium (Pfr/Ptot) in microcuttings of easy-to-root plants such as Prunus rootstock GF 655-2 has been reported (Rossi et al. 1993). The different uptake and metabolism of applied auxin during rooting in darkness by two pear cultivars with different rhizogenic ability (Baraldi et al. 1993) underline the role of exogenous growth regulators in rhizogenesis. The present study attempts to elucidate the effect of light and auxin on in vitro root formation and development of two pear cultivars, the easy-to-root 'Conference' and the difficult-to-root 'Doyenne d'Hiver'. A better understanding of growth substances/light interactions could have both physiological and practical implications for propagation of woody species.
140 Materials and methods
Plant material culture conditions and measurements Shoot cultures of 'Conference' and 'Doyenne d'Hiver' pears were grown initially on proliferation medium containing MS salts (Murashige & Skoog 1962), LS (Linsmaier & Skoog 1965) vitamins, 4.4 ?tM benzyladenine (BA), 0.49 #M indolebutyric acid (IBA), 2% sucrose (w/v) and 0.65% (w/v) agar (B&V, Parma, Italy). The pH of the medium was adjusted to 5.7 with 0.1 M KOH before autoclaving for 20 rain at 120°C. Cultures were incubated in walk-in chambers at 25 ± I°C and 16-h photoperiod. The light was provided by cool white fluorescent lamps at a photon fluence rate of 60 #mol m -2 s -1 at plant height. Shoots, 15 mm long, were taken from 30-day-old proliferating cultures and placed in 500 ml glass jars with glass covers and wrapped in plastic foil (Bormioli, Parma, Italy). Each jar contained 150 ml of rooting medium, which was the same as the proliferation medium except for the exclusion of BA and for reduction of mineral salts to one-half. This rooting medium contained or didn't 1.5 pM IBA. Cultures were incubated for 21 days in a walk-in chamber at 25 ± l°C under varying light regimes. Rooting percentages, the number of primary and secondary roots, length of primary roots, and root extension (number x length of roots) were determined after 21 days.
Light treatments Shoots were kept in darkness or under continuous irradiation at varying photon fluence rates with blue, red, far-red or white light. A 12-h FR-pretreatment of shoots before being placed in darkness was done to transform the physiologically active Pfr into the inactive species Pr (Mohr, 1972). White light was provided by Philips TLD 40W 33RS cool white fluorescent tubes (Philips, The Netherlands). Blue light was produced by filtering the same light source through a Lee 141 cinemoid filter (Lee Filters LTD, Hants, UK), while red light was produced via a Lee 106 filter. Far-red light was provided by Linestra incandescent tubes filtered through Plexiglas PG 501/3 (Impla spa, Italy) and KG 3/2 mm glass filters (Schott & Geinz, Mainz, Germany). The spectral emission curves and photon fluence rates of these light sources were measured with a calibrated LiCor 1800 Spectroradiometer. Photon fluence rates at microshoot level were 15 or 36/~mol m-2
s- ~ for W (400-700 nm), B (450-460 nm) and R (656666 nm) and 8 pmol m -2 s-l for FR (726-736 nm). Emission spectra of these light sources are reported in Rossi et al. (1993). The calculated phytochrome photoequilibrium values (Mancinelli 1989) were 0.004 for FR, 0.4 for B, 0.7 for W and 0.8 for R.
Experimental design The statistical design was completely randomized. For both cultivars six jars, each containing 10 shoots, were used for each light treatment and IBA concentration. The experiment was conducted three times. Differences referred to as significant had a P value of less than 0.05 as determined by analysis of variance using the Proc GLM procedure of PC SAS version 6.04 (SAS Institute Inc. Cary, NC, USA). Percentage data were subjected to arcsine transformation prior to analysis.
R~ults
'Conference' No root emergence was observed without IBA from microshoots incubated in darkness and under continuous FR light, whereas rhizogenesis was increased by exposure to B, W and R (Table 1). Rooting was higher with 15 #tool m -2 s-l R but significantly decreased at 36 #mol m -2 s -1. Rooting rate varied between 30% and 40% under W and B light at 15 #tool m -2 s - l ; the higher photon fluence rate of both light sources decreased rooting. The addition of IBA to the culture medium enhanced rooting regardless of light regime. Rooting was maximum in darkness and under 15 #mol m -2 s - l of R, and significantly decreased under FR and low photon flux of W and B. The higher photon fluence rates of W, B and R reduced root production compared to lower levels of light. Without IBA root number per rooted shoot was equivalent under W, B and R, regardless of fluence rate. The longest roots were recorded under B and at the high levels of W and R. Root extension did not differ among light treatments. When IBA was added to the medium the highest root number was recorded under FR and the lower level of R. The other light regimes did not significantly affect root number per rooted shoot in comparison to darkness. The low level of R resulted in a significant increase in the number of secondary roots per microcutting, compared to all
141
Table 1. Effects of tested light regimes on rooting percentage, number of primary and secondary roots per plant, root length and extension of 'Conference' in vitro with or without 1.5/~M IBA after 21 days of culture. Treatments IBA (tzM)
Light D FR R W B
D FR R
1.5
W B
Photon flux (tzmol m -2 s -1) 0 8 15 36 15 36 15 36 0 8 15 36 15 36 15 36
Percent rooting 0e 0e 63bc 20d 30 cd 23 d 40 c 17 d 100 a 73 b 90a 72 b 73 b 47c 70 b 23 d
Root no. Primary
Root no. Secondary
Root length (mm)
Root extension (ram)
0e 0e 3.7cd 2.9cd 3.6 cd 3.1 cd 2.1 d 1.9 d
0d 0d 0d 0d 0d 0d 0d 0d
Of Of 23.9 bc 42.1a 24.2 bc 32.9 ab 35.9 a 41.4 a
0e 0e 74.1 bc 98.2 bc 73.7 bc 84.8 bc 76.7 bc 83.4 bc
5.9 b 9.7 a 8.7a 5.5 bc 5.9 b 6.1b 4.8 bc 5.1 bc
0d 0d 48.9a 13.6 b 5.7 c 0d 1.1 d 0.9 d
5.9 e 3.7 e 16.1d 12.9 d 17.2 cd 14.5d 23.9 bc 23.8 bc
32.9 d 36.5 d 140.9 a 63.8 c 86.1 bc 76.2 bc 105.9 b 108.4 ab
Values are means of three experiments. Means followed by a different letter are significantly different based on the PDIFF option for the least-squares means presented here (SAS Institute, lnc., Rel.6.04).
Table 2. Effects of tested light regimes on rooting percentage, root number per plant, root length and root extension of 'Doyenne d'Hiver' with 1.5 ttM IBA after 21 days of culture. Treatments IBA (~M) 1.5
Light D FR R W B
Photon flux (/~mol m-2 s - l ) 0 8 15 36 15 36 15 36
Percent rooting
Root no.
Root length (mm)
Root extension (mm)
77ab 68 b 99a 55 bc 33 cd 28 d 71 ab 41 cd
3.8a 2.9 b 3.8a 2.9 b 3.5 ab 2.4 b 3.1 ab 3.3 ab
9.6d 6.4 e 26.1a 12.5 d 20.8 abc 20.1 bc 24.1 ab 17.9 c
35.1 bc 14.6 d 104.1 a 30.8 c 45.4 bc 31.2 bc 43.8 b 36.9 bc
Values are means of three experiments. Means followed by a different letter are significantly different based on the PDIFF option for the least-squares means presented here (SAS Institute, Inc., Rel.6.04).
o t h e r l i g h t r e g i m e s . B l u e l i g h t s t i m u l a t e d r o o t elong a t i o n m o r e t h a n R a n d t h e l o w fluence rate o f W. M i c r o s h o o t s in F R a n d d a r k n e s s h a d the s h o r t e s t roots. R o o t e x t e n s i o n w a s g r e a t e s t u n d e r R at 15 # t o o l m - 2 s - ~, f o l l o w e d b y B a n d W at b o t h p h o t o n flux density. T h e h i g h e r l e v e l o f R r e s u l t e d in less r o o t d e v e l o p m e n t c o m p a r e d to l o w e r p h o t o n fluence rate o f R.
'Doyenne d'Hiver' ' D o y e n n e d ' H i v e r ' did n o t r o o t w i t h o u t I B A in the m e d i u m r e g a r d l e s s o f t h e l i g h t r e g i m e ( d a t a n o t reported), w h e r e a s roots m e r g e d u n d e r all l i g h t t r e a t m e n t s w h e n I B A w a s in the c u l t u r e m e d i u m ( T a b l e 2). T h e m a x i m u m r o o t i n g w a s r e c o r d e d u n d e r l o w R, d a r k n e s s a n d l o w B. R o o t i n g w a s significantly l o w e r u n d e r 36 # m o l m - 2 s -1 R t h a n u n d e r the l o w e r p h o t o n fluence
142 rate of R. The lowest rooting was under W light and high levels of B and R. The number of roots per rooted shoot formed in darkness and under low R was significantly higher compared with FR and 36 #mol m -2 s -1 of R and W. Longer roots were formed under R and B at 15 #mol m -2 s - l than at the higher level of these two wave bands. Dark, FR and high R did not promote root elongation. Low R fluence rate was the most effective regime for root extension and FR greatly inhibited it.
Discussion In vitro adventitious root formation and development
of 'Conference' and 'Doyenne d'Hiver' microcuttings were differently affected by light and exogenous auxin. For the difficult-to-root 'Doyenne d'Hiver' microshoots, the IBA supply was essential for the achievement of consistent rooting. Light alone without IBA was not capable of stimulating rhizogenesis regardless of the spectrum and the photon fluence rates. On the contrary, under favourable light regimes the easy-to-root 'Conference' rooted even without the exogenous auxin. In 'Conference' the absence of rooting observed in conditions of low phytochrome photoequilibria (D and FR), and the great root emergence at higher levels (B, W and R) support the involvement of phytochrome in rooting of microcuttings, as reported elsewhere (Rossi et al. 1993). On the other hand, the rooting differences found under the different light regimes are unlikely to be related to differences in photosynthetic carbon fixation since under our experimental conditions there was an exogenous carbohydrate supply, low photon fluxes and lack of CO2 enrichment (Rossi etal. 1993). The phytochrome induction of rhizogenesis may occur in part through modulation of auxin levels as reported for phytochrome regulation of stem growth (Behringer et al. 1992). The addition of IBA to the medium sharply increased rhizogenesis in darkness and modified responses of both cultivars to light regimes. The inhibition of rooting observed with higher photon fluxes compared to the lower ones is a quite common response for many woody species, for which a dark period is often required for rooting (Rugini et al. 1988). In both the tested pear cultivars, incubation of the shoots in the light did not result in a decrease in root number in comparison to incubation in the dark, which had been reported by others (Van der Krieken et al.
1992; Zimmerman 1984; Hammerschlag et al. 1987). All light regimes stimulated root elongation as compared to darkness, excepting FR in 'Conference' and FR and high R in 'Doyenne d'Hiver'. Microcuttings appeared particularly affected by red light. Without IBA, the low level of R resulted in the highest rooting percentage. The R effect on rooting, similar to the effect of an auxin application, may involve changes in endogenous auxin in response to the R signal (Pinker et al. 1989). With IBA, R, especially at the low photon fluence rate, had positive effects, extending root system development and stimulating lateral root differentiation. This inducing of microcuttings to produce a good root system is important to enhancing survival and growth during the difficult phase of acclimatization. The correlations of rooting to phytochrome photoequilibrium in 'Conference' suggest that in vitro cultured cuttings of this genotype may represent a suitable model for further physiological investigations on the phytochrome mechanism in the rooting of woody species.
Acknowledgement The authors wish to thank M. Govoni and U. Daghia for their valuable technical assistance.
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