Plant Cell, Tissue and Organ Culture 35: 157-163, 1993. © 1993 Kluwer Academic Publishers. Printed in the Netherlands.

Effect of a-naphthaleneacetic acid and sucrose levels on the development of cultured embryos of coconut G.R. Ashburner I'*, W.K. Thompson & J.M. Burch 2

Department of Agriculture, Institute of Plant Sciences, P.O. Box 174, Ferntree Gully, Victoria 3156, Australia (1present address: Department of Agriculture, Institute of Plant Sciences, Swan Street, Burnley Vic 3121, Australia; 2Faculty of Biological and Behavioural Sciences, The University of New South Wales, P.O. Box 1, Kensington NSW 2037, Australia) (*requests for offprints) Received 3 November 1992; accepted in revised form 14 May 1993

Key words: auxin, Cocos nucifera, embryo culture, NAA Abstract

Mature coconut embryos were germinated in a modified Murashige and Skoog medium and then cultured on BMY 3 medium incorporating sucrose in the range of 4 to 8%. a-Naphthaleneacetic acid (NAA) was added into the medium at concentrations ranging from 0 to 800 txM for periods of 4 to 24 weeks. Application of N A A for 4 weeks stimulated shoot growth, whereas application periods greater than 4 weeks had no significant effect. NAA in the range of 100-300 ~M stimulated elongation of the primary root and the optimum concentration increased with increases in sucrose levels. Production of adventitious roots was stimulated by the addition of NAA with levels of 200 p~M and above being the most effective. Increasing the sucrose concentration from 4% through to 8% stimulated root elongation in the absence of NAA and inhibited shoot growth whether NAA was present or absent.

Abbreviations: IAA - indole-acetic acid, NAA - a-naphthaleneacetic acid

Introduction

Embryo culture of the coconut palm (Cocos nucifera L.) has the potential to be useful in the transportation of germplasm for breeding programs by lowering costs, overcoming storage problems and satisfying most phytosanitary requirements that presently preclude any germplasm transfer (Ashburner & Thompson, in press). Embryo culture is also the basis for rescuing potentially abortive embryos such as the 'Makapuno' type (Guzman & Rosario 1964) and for in vitro screening, for drought tolerance (Karunaratne et al. 1991) and for Coconut Cadang-Cadang Viroid resistance (Rillo et al. 1988). Forming a root system capable of sustaining a

plantlet during post-culture acclimatisation has been identified as a problem (Guzman et al. 1971; Gupta et al. 1984; Assy Bah et al. 1987). However, some studies have shown that enhanced root growth may be achieved either by increasing the sugar content of the growth medium (Guzman et al. 1971; Assy Bah et al. 1989) by the incorporation of activated charcoal (Guzman & Manuel 1977). Assy Bah et al. (1987) also claimed that N A A enhanced root growth. The aim of this work was to investigate further the concentration and timing of N A A needed to optimise the rooting response, to explore the interaction of sucrose and N A A on the root response and to evaluate the form of this response in terms of primary and adventitious root production and shoot growth.

158 Materials and methods

General Mature coconuts, commercially imported from Tonga or the Solomon Islands, were used as a source of zygotic embryos. Coconuts used for any one experiment were from the same shipment. Embryos were excised and surface disinfested using the method of Ashburner et al. (1991), then placed into 10 ml of sterilised germination medium. The germination medium used for initial culture in each experiment consisted of MS salts (Murashige & Skoog 1962) with the vitamins of Morel & Wetmore (1951), 6% (w/v) sucrose and 0.2% (w/v) activated charcoal (Sigma). The growth medium used in the post-germination culture consisted of BMY 3 (Eeuwens 1978) containing 0.2% (w/v) activated charcoal, 0.8% (w/v) agar (Difco-Bacto) and concentrations of sucrose and NAA as required for each experiment. The medium was dispensed into either 80 mm x 25 mm or 105 mm x 42 mm cylindrical polycarbonate jars depending on the medium volume, and autoclaved at 121°C for 20min. Cultures were grown at 30-31°C with a 16-h photoperiod provided by cool-white fluorescent tubes (90~mol m-2s -l) for 24 to 28 weeks. Cultures were sub-cultured from germination medium to 10 ml of growth medium at germination as indicated by the emergence of the plumule, or at week 8 in the case of recalcitrant embryos. The embryos were then sub-cultured to fresh media at weeks 12 and 20 when the volume of the media was increased to 20 and 40ml respectively to allow for natural development of the embryo.

Experimental treatments The effect of altering the NAA concentration of the medium during the germination and growth stages of embryo development were investigated in a factorial experiment with three NAA concentrations (0, 100 and 200 IxM) in the germination media combined with the same three concentrations in the growth media (Experiment 1). Initially the sucrose concentration of the growth medium was 8% (w/v) but was reduced to 6%

(w/v) at week 12. The effect of both NAA and sucrose concentration in the growth stage was examined in Experiment 2. Germinated embryos were cultured on growth medium with the factorial combination of four N A A concentrations (0, 100, 200 or 300 IxM) with three sucrose levels (4, 6 or 8% w/v). The effect of NAA application for a shorter duration was examined by applying 0, 200, 400 or 8001xM NAA to the growth medium containing 4% (w/v) sucrose between week 20 and week 24 of the culture period (Experiment 3).

Analyses Data for shoot length, primary root length and secondary and adventitious root numbers (Fig. 1), as measured at week 28, were analysed by analysis of variance. For Experiments 2 and 3, a polynomial analysis of variance was used to identify any quadratic effects in the NAA response. In Experiment 2, the relationship between root length and treatment was further examined by fitting orthogonal polynomials. Binary data relating to the proportion of plants producing adventitious roots were analysed using a generalised linear model employing a logit link function. All analyses were processed using a Genstat 5 (Lawes Agricultural Trust, Rothamsted) statistical package.

Results

The effect of NAA on plant growth during the germination and growth stages are presented in Table 1. The apparent trend of shoot depression with increasing NAA levels in either stage was not statistically significant. Primary root length and secondary root number was significantly reduced by 200 ~M NAA in the germination medium but was significantly enhanced by the same concentration in the growth medium. NAA induced plants to form adventitious roots (Table 1), and there was a significant interaction between the NAA levels in the germination and growth media that resulted in maximum adventitious root number when the NAA concentration was maintained at 200 ~M in both stages (Fig. 2). The effect of NAA on plant growth also

159 between sucrose and NAA concentrations on primary root growth as shown in Fig. 3. In the absence of NAA, root growth increased with increasing sucrose concentration. When NAA was present, the optimum concentrations were in the range of 175-200txM at the lower sucrose levels (4-6%); however, with 8% sucrose, root length did not peak and continued to increase with increasing levels of NAA. The proportion of plants producing adventitious roots generally increased with increasing NAA concentrations, but the response to a low concentration of NAA was greatest at 4% sucrose (Fig. 4). Similarly, the number of adventitious roots per plantlet was also increased by high levels of NAA and the greatest response was obtained at the 4 and 6% sucrose levels. (Fig. 5).

Discussion

Fig. 1. The coconut plantlet after 24 weeks of embryo culture. (a) photosynthetic leaves (eophylls); (b) scale leaves; (c) cotyledonary sheath; (d) undeveloped cotyledon; (e) primary root (radicle); (f) secondary roots; (g) adventitious root. Scale bar represents 10 mm.

depends on its timing and duration of exposure (Table 2). An increase in NAA concentration between weeks 20 and 24 caused a linear enhancement in shoot and all aspects of root growth. The proportion of plants producing adventitious roots increased ten-fold when 800 IxM NAA was used. Increasing the sucrose content of the growth medium significantly depressed shoot growth in a linear fashion and mean values for shoot length at week 28 were 126 mm for 4% sucrose, 76 mm for 6% sucrose and 56 mm for 8% sucrose. The response of root growth to sucrose content was greatly dependent on the NAA concentration of the medium. There was a significant interaction

The response of shoot and root development was highly dependent on both the concentration of sucrose and NAA used and on the period and timing of exposure to NAA. Increasing the sucrose concentration of the basal medium had a negative effect on shoot elongation and, in the absence of NAA, a positive effect on root elongation. This differen~ tial effect on shoot and root growth has previously been recorded in Cocos nucifera L. (Guzman et al. 1971; Rosario & Guzman 1976; Karunaratne et al. 1985) as well as in other members of the Arecaceae (Buffard-Morel 1968; Ko, pers. comm.). Plants frequently respond to water stress by increasing the root to shoot ratio (Turner & Begg 1981) and it has been proposed by Borchert (1973) that this response maintains a balance between the absorptive surface for water uptake and the transpirational leaf surface area. It is possible that in the present experiments a water stress was induced in the developing plantlet by increasing the sucrose concentration and, therefore, the osmolarity of the basal medium. Karunaratne et al. (1991) found similar results when using other osmotica. The response of root elongation to increasing sucrose concentration may not be solely due to a change in the root: shoot ratio, since a similar response was evident

160 Table 1. Main effects of initial and final NAA concentration on growth parameters at 24 weeks (Experiment 1). NAA concentration (IxM)

Shoot length (mm)

0 100 200 LSD p = 0.05

48.2 44.6 38.6 10.6

0 100 200 LSD p = 0.05

47.6 44.7 37.2 10.7

Primary root length (mm)

Secondary root number 1

Plants with adventitious roots (%)2

0.70 (5.0) 0.54 (3.4) 0.41 (2.6) 0.17

15 21 41 -

0.43 (2.7) 0.47 (3.0) 0.72 (5.3) 0.17

16 26 35 -

Initial (Germination medium) 33.5 31.2 23.8 6.4

Final (Growth medium) 23.8 29.1 34.9 6.4

~Logl0 transformation. Figures in parentheses are retransformed means. 2percentage data analysed using a generalised linear model and presented as predicted means. Significance obtained was: initial concentration p < 0.001, final concentration p < 0.05

.Q

E

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=o °O ~ t,.-

200

100

0

Initial NAA Concentration

~" (uM)~'¢

Fig. 2. The effect of initial and final NAA concentration on adventitious root number. Initial concentrations were applied to the germination medium and final concentrations to the growth medium.

in isolated root cultures of coconut (Fulford et al. 1981; Justin & Fulford 1980). The effect of NAA on shoot growth was variable. When NAA was present throughout much or all of the culture period, there was a trend for reduced shoot growth. However, when NAA exposure was limited to 4 weeks late in the growth period, shoot growth was stimulated even at the highest NAA concentration of 800 ~M.

Similarly, continuous NAA at 200-300 p,M inhibited primary root growth when used with a low level (4-6%) of sucrose, but when used for a 4-week pulse, high levels of NAA increased the number of plants forming abundant adventitious roots, with the highest level producing the greatest response. The sensitivity of in vitro coconut plantlets to NAA increases with decreasing sucrose content. This interaction may result from the higher sucrose medium causing a water stress that decreases endogenous auxin levels (Davies et al. 1986). This effect on adventitious root formation differed from that on root elongation where high NAA and high sucrose levels were the most effective treatments. Assy Bah et al. (1989) found that adventitious root production increased with sucrose concentration up to an optimal 9% (w/v) in the absence of NAA. Their results differ from those presented here and may be due to the source of the embryos used. In the present experiments the minimum period of N A A supply was 4 weeks. Fulford et al. (1981) reported that excised coconut roots grew better in vitro when the duration of NAA treatment was less than 3 days. Toruan (1978) reported a stimulation of both root and shoot growth following prolonged treatment with IAA. It is possible that prolonged IAA treatment was equivalent to using a short pulse of N A A , since IAA is rapidly metabolised in plant tissue (Ray 1958) and breaks down in culture medium (Dunlap et al. 1986). The optimum duration for auxin

161 Table 2. Effect of NAA application for 4 weeks (between weeks 20 and 24) on growth parameters measured at week 28.

NAA contration (IxM)

Shoot length (ram)

Root length (mm)

Secondary number'

Plants with adventitious roots (%)

Adventitious root number 1

0 200 400 800

110 132 150 166

32 34 42 53

0.66 (4.6) 1.0 (10.0) 1.0 (10.0) 1.43 (26.9)

8 29 55 83

0.6 (1.16) 0.15 (1.4) 0.35 (2.2) 0.68 (4.8)

0.01 NS

0.02 NS

0.01 NS

0.001 NS

0.001 NS

Significance Linear effect Quadratic effect

~Logl0 transformation. Values in parentheses are retransformed means. 70 8%

60 A E E

50

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40

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80

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200

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NAA concentration (uM)

0 0

100

200

300

NAA concentration (uM) Fig. 3. The effect of NAA concentration and sucrose concentration, during the growth stage, on mean primary root length at week 28. The predicted auxin response curve for each sucrose concentration was obtained by calculating linear and quadratic polynomials.

e x p o s u r e s a p p e a r s to d e p e n d on N A A c o n c e n tration and sucrose concentration and warrants further attention. T h e c o n c e n t r a t i o n o f N A A n e e d e d to achieve r e s p o n s e s in this s t u d y was m u c h g r e a t e r t h a n r e p o r t e d in e a r l i e r w o r k (Sajise & G u z m a n 1972). M u c h o f the auxin was p r o b a b l y a d s o r b e d b y t h e a c t i v a t e d c h a r c o a l in t h e m e d i u m ( W e a t h e r h e a d et al. 1978). F u l f o r d et al. (1979) f o u n d

/

Fig. 4. The effect of NAA concentration and sucrose concentration, during the growth stage, on the frequency of plantlets with adventitious roots. Data analysed using a generalised linear model and a significant (p <0.05) interaction was obtained for the interaction of sucrose and NAA.

that in a m e d i u m c o n t a i n i n g a c t i v a t e d c h a r c o a l , a o n e h u n d r e d - f o l d i n c r e a s e in N A A was n e e d e d for c o c o n u t r o o t s to a c h i e v e s i m i l a r results to t h o s e o b t a i n e d in a c h a r c o a l - f r e e m e d i u m . A n o t a b l e f e a t u r e o f t h e p r e s e n t results is t h e p r o s p e c t of m a n i p u l a t i n g t h e f o r m o f t h e c o c o n u t p l a n t l e t in vitro to o p t i m i s e p l a n t s u r v i v a l o n t r a n s f e r to free-living c o n d i t i o n s . T h e size o f t h e s h o o t can b e m a n i p u l a t e d b y v a r y i n g t h e s u c r o s e c o n c e n t r a t i o n a n d also, to a lesser e x t e n t , by varying NAA. Root elongation and adventitious r o o t p r o d u c t i o n can b e p r o m o t e d b y a c o n t i n u -

162

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3o)

.o

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1

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References

/ f--

¸¸

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300 200 100 0 NAA concentration (uM)

J

Fig. 5. The effect of NAA concentration and sucrose concentration, during the growth stage, on the mean number of adventitious roots per plantlet. Data analysed by analysis of variance using log10 transformation. Values presented are retransformed means.

o u s , o r p u l s e d , a p p l i c a t i o n of N A A , a l t h o u g h t h e r e s u l t is d e p e n d e n t on sucrose c o n c e n t r a t i o n . T h e t y p e of p l a n t l e t t h a t s h o u l d be p r o d u c e d to o p t i m i s e s u b s e q u e n t a c c l i m a t i s a t i o n r e m a i n s to b e i n v e s t i g a t e d , In vivo, t h e c o c o n u t p r o d u c e s multiple roots soon after germination (Davis 1968) with the r a d i c l e b e i n g r e p l a c e d b y a d v e n t i t i o u s r o o t s arising f r o m the o b c o n i c a l s e e d l i n g axis ( T o m l i n s o n 1990). W e c o n s i d e r that the m o s t d e s i r a b l e p l a n t l e t for a c c l i m a t i s a t i o n m a y b e o n e with a w e l l - d e v e l o p e d s h o o t a n d t h a t can d e v e l o p n u m e r o u s a d v e n t i t i o u s roots.

Acknowledgements

W e t h a n k M r P. F r a n z for statistical advice, M r D. R i c h a r d s for discussions r e g a r d i n g this w o r k a n d M s P. N i c h o l s for t y p i n g t h e m a n u s c r i p t . T h i s w o r k was s u p p o r t e d b y the A u s t r a l i a n C e n t r e for I n t e r n a t i o n a l A g r i c u l t u r a l R e s e a r c h in c o l l a b o r a t i o n with t h e C o c o a a n d C o c o n u t Research Institute, Papua New Guinea.

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Rosario AG del & Guzman EV de (1976) The growth of coconut 'makapuno' embryos in vitro as affected by mineral composition and sugar level of the medium during the liquid and solid cultures. Philipp. J. Sci. 105:215-22 Sajise SU & Guzman EV de (1972) Formation of adventitious roots in coconut 'makapuno' seedlings grown in medium supplemented with naphthalene acetic acid. Philipp. J Biol. 1:197-206 Tomlinson PB (1990) The Structural Biology of Palms. Oxford University Press, Oxford Toruan N (1978) Pertumbuhan dan perkembangan embrio kelapa dalam Kulturaseptik. Menara Perkebunan 46: 2136 Turner NC & Begg JE (1981) Plant-water relations and adaptation to stress. Plant Soil 58:97-131 Weatherhead MA, Burdon J & Henshaw GG (1978) Some effects of activated charcoal as an additive to plant tissue culture media. Z. Pflanzenphysiol. 89:141-7