Silver nitrate and aminoethoxyvinylglycine promotein vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

J. Plant Physiol. 160. 423 – 430 (2003)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.) Soumendra K. Naik, Pradeep K. Chand* Plant Cell and Tissue Culture Facility, Post-Graduate Department of Botany, Utkal University, Bhubaneswar-751004, Orissa, India

Received June 24, 2002 · Accepted September 13, 2002

Summary A protocol is presented for direct adventitious shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 µmol/L benzyladenine (BA), 5.4 µmol/L naphthaleneacetic acid (NAA) and 10 % coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10 – 40 µmol/L) and aminoethoxyvinylglycine (AVG) (5–15 µmol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 µmol/L) or AVG (10 µmol/L) induced adventitious shoot buds from 57% or 53 % of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 µmol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1⁄2 MS) containing 0.54 µmol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil. Key words: Adventitious shoot organogenesis – ethylene inhibitor – fruit tree – Punica granatum L. Abbreviations: AVG = aminoethoxyvinylglycine. – BA = N6-benzyladenine. – CEPA = 2-chloroethylphosphonic acid. – CW = coconut water. – IBA = indole-3-butyric acid. – MS = Murashige and Skoog (1962) medium. – NAA = 1-naphthaleneacetic acid

Introduction Genetic improvement of fruit trees, in general, by conventional breeding methods is a slow and difficult process due to * E-mail corresponding author: [email protected]

their long life cycles. In this respect, the introduction of agronomically important traits by genetic transformation could be a rapid alternative method with minimum destruction of genetic integrity of the elite commercial cultivars. A desirable requirement for successful genetic transformation is the development of an efficient method to regenerate plants 0176-1617/03/160/04-423 $ 15.00/0

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adventitiously in a reasonably high frequency from explants such as leaf, stem, hypocotyl or cotyledon segments. In vitro plantlet regeneration through adventitious shoot formation from explants has been reported in a number of fruit species (Zimmerman and Swartz 1994). Punica granatum L. (pomegranate) is an important fruit tree, which is regarded considerably for its delicious fruits. Besides, the tree is greatly valued for its pharmaceutical relevance (Anonymous 1982). Shoot organogenesis has been documented in pomegranate from callus derived from anther wall (Moriguchi et al. 1987) or leaf segments (Omura et al. 1987). To date, however, direct plant regeneration through adventitious shoot formation has not been reported for this fruit tree. The result of our preliminary experiments (when we used only different phytohormones) together with previous findings (Moriguchi et al. 1987, Omura et al. 1987) indicated that pomegranate was, indeed, a recalcitrant species at least for in vitro regeneration through organogenesis. Of several factors responsible for in vitro recalcitrance of a plant species, production of ethylene in in vitro culture is a key factor. Ethylene (C2H4), a gaseous plant hormone, plays an important role in plant growth and development (Yang and Hoffman 1984). Ethylene is produced by cultured plant cells (LaRue and Gamborg 1971, Pua and Chi 1993) and possibly by the gelling agent like agar, in culture medium (Mensuari-Sodi et al. 1992). Several reports indicated that ethylene produced in culture might play a role in plant cell differentiation in vitro. The inhibition of ethylene action or its production using silver nitrate (AgNO3) or aminoethoxyvinylglycine (AVG) respectively has shown stimulatory effects on plant regeneration in Brassica species (Chi and Pua 1989, Chi et al. 1991, Palmer 1992), Helianthus annuus (Chraibi et al. 1991) and Nicotiana plumbaginifolia (Purnhauser et al.1987). However, its response needs to be addressed with each plant genotype, species and/or type of cultured cells or explant types. The present study was designed to examine the influence of ethylene inhibitors (AgNO3 and AVG) on adventitious shoot organogenesis of pomegranate. In the present communication, we report for the first time the stimulatory effect of these two ethylene inhibitors on adventitious shoot organogenesis leading to high frequency plant regeneration from cotyledon segments derived from axenic seedlings of pomegranate.

Materials and Methods Explant preparation Seeds of pomegranate (Punica granatum L. cv. Ganesh) were collected from fully ripe fruits and washed free of juicy testa. They were kept under running tap water for 1h followed by a 3 min treatment with a 5 % (v/v) aqueous solution of Teepol (Reckitt’s Colman, India) and rinsed 5 – 6 times with distilled water. Seeds were surface sterilized for 2 min with a 0.01 % (w/v) aqueous solution of HgCl2 (BDH, India) and finally rinsed (5 – 6 changes) with autoclaved distilled water. The surfa-

ce – sterilized seeds were germinated in 30 mL screw-capped glass tubes containing Murashige and Skoog’s (1962) basal medium at half salt-strength, 50 mg/L myo-inositol and 15 g/L sucrose (1⁄2 MS) gelled with 8 g/L agar (Hi-media, India). Six to nine day old seedlings were used as explant source. The cotyledons without proximal ends were transversely cut into two halves (5–7 mm2), and hypocotyls were cut into 3 – 5 mm segments and placed horizontally on the medium.

Culture medium and growth conditions For shoot regeneration studies, Murashige and Skoog’s (1962) medium (MS) was used containing 30 g/L sucrose and 100 mg/L myo-inositol. The medium was further supplemented with 2.2–13.3 µmol/L benzyladenine (BA) alone or in combination with 2.7– 5.4 µmol/L naphthaleneacetic acid (NAA). Coconut water (CW) from fresh green fruits was collected and added to the nutrient medium at different concentrations (5–15 % v/v). The pH of the medium was adjusted to 5.8 before gelling with 8 g/L agar (Hi-media, India). All experiments were conducted using 300 mL screw-capped glass jars each containing 40 mL medium. Cultures were maintained at 25 ± 1 ˚C, with 35 µmol m – 2 s –1 photon flux density provided by cool white fluorescent tubes (Philips, India) and 60 % relative humidity.

Experimental set-up for studying the influence of AgNO3 and AVG The effect of two ethylene inhibitors, AgNO3, an inhibitor of ethylene action and AVG, an inhibitor of ethylene production, on shoot regeneration was investigated. The shoot regeneration medium (MS + 8.9 µmol/L BA + 5.4 µmol/L NAA + 10 % CW) was supplemented with a range of concentrations of AgNO3 (10 – 50 µmol/L) or AVG (5–15 µmol/ L). Following shoot bud differentiation, the explants along with the shoot clumps were transferred to the same medium but devoid of AgNO3 or AVG. In addition, cotyledon explants were grown on regeneration medium supplemented with optimal concentrations of AgNO3 or AVG in the absence or presence of 20 – 60 µmol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound, to investigate the interaction between the ethylene inhibitor and the promoter. AgNO3, AVG and CEPA were prepared separately, filter sterilized, and then added to the autoclaved medium.

Rooting of shoots Shoots, 2.0 – 2.5 cm in length, were excised and transferred to halfstrength MS medium (1⁄2 MS) supplemented with 0.054 – 5.4 µmol/L NAA or 0.049 – 4.9 µmol/L IBA. The medium was gelled with 0.15 % phytagel (Sigma, USA). After 5–7 days of root initiation the rooted shoots were transferred to an auxin-free half-strength MS medium (1⁄2 MS) for further elongation of roots as well as the shoots.

Acclimatization of plantlets Plantlets with well-developed roots were removed from the culture medium and after washing the roots gently under running tap water, plantlets were transferred to plastic pots (7.5 cm diameter) containing autoclaved vermi-compost (Ranjan’s Agrotech, Bhubaneswar) and

Stimulation of adventitious regeneration of pomegranate using AgNO3 and AVG were covered with polyethylene bags to maintain high humidity. The potted plantlets were kept in a culture room at 25 ± 1˚C and a photon flux density of 50 µmol m – 2 s –1. After 3 weeks, plantlets were transferred to larger pots (18 cm) containing soil : compost (1 : 1) and kept under shade for another 2 weeks and finally transferred to outdoors under full sun.

Statistical analysis of data In the shoot development experiment, each treatment consisted of ten replicates (culture jar) and the experimental unit was three explants per jar. Data on percentage of explants forming shoots, shoot number and shoot length were collected after 90 days, and the means were calculated. Duncan’s Multiple Range Test (DMRT) was used to compare the means. For rooting experiments, each treatment consisted of 20 replicates (culture tubes) and one explant per experimental unit. Root number and length data were collected after 30 days. All the experiments were repeated twice.

Results

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Table 1. Adventitious shoot regeneration from cotyledon and hypocotyls explantsa. Growth regulators (µmol/L)

% explants forming shoots

Shoots/ explants

Shoot length (cm)

C

H

C

H

C

H

MS Basal

–

–

–

–

–

–

BA 2.2 4.4 8.9 13.3

– 6.66 c 10.0 c –

– 3.33 e 6.66 de –

– 1.00 f 1.33 e –

– 1.00 c 1.11 c –

– 1.88 e 2.01 d –

– 1.82 d 1.84 d –

BA + NAA 2.2 + 2.7 4.4 + 2.7 8.9 + 2.7 2.2 + 5.4 4.4 + 5.4 8.9 + 5.4

– 13.33 c 23.33 b – 26.66 ab 33.33 a

– 10.00 cd 13.33 bc – 16.66 b 23.33 a

– 1.47 d 1.76 c – 2.12 b 2.65 a

– 1.30 bc 1.73 abc – 1.93 ab 2.18 a

– 2.24 c 2.47 ab – 2.41 b 2.56 a

– 2.08 c 2.25 b – 2.19 b 2.44 a

Means within a column for BA or for BA + NAA treatments having same letter are not significantly different at P < 0.05 according to Duncan’s multiple range test (DMRT). C: Cotyledon; H: Hypocotyl; – : No response.

a

Influence of growth regulators on adventitious organogenesis Neither cotyledons nor hypocotyls responded to a growth regulator-free MS medium. Addition of BA either singularly or in combination with NAA to the medium was essential to induce shoot bud differentiation from the explants. Twenty-three percent of the hypocotyl segments exhibited shoot bud initiation on a medium containing 8.9 µmol/L BA, 5.4 µmol/L NAA and 10 % CW within 8 – 9 weeks and from each explant ca. 2.2 shoots developed in 90 days averaging 2.4 cm in length (Table 1). In case of cotyledon, 33 % of the explants exhibited adventitious shoot bud initiation within 7– 8 weeks of culture in the above medium. The average number of shoots per explant was 2.6, and they had an average length of 2.5 cm in 90 days (Fig. 1 a, Table 1). The initial response of the explants was swelling followed by enlargement in size within 2 weeks of culture on the regeneration medium. In case of cotyledons the shoot buds differentiated from the margin as well as from the surface of the explant, whereas in hypocotyls shoots developed only from the upper protruding surface. Interestingly, a feature common to both the type of explants was that a few shoots also developed from the surface of some of the explants, which was in contact with the medium. Also, shoot bud differentiation was accompanied by the formation of a small amount of green, compact and nodular callus in most of the explants. Differentiation of isolated leaves from cotyledons was also a common feature along with shoot bud differentiation.

Influence of AgNO3 and AVG In this study, the presence of ethylene inhibitors namely AgNO3 (20 µmol/L) or AVG (10 µmol/L) in the shoot regenera-

tion medium (MS + 8.9 µmol/L BA + 5.4 µmol/L NAA + 10 % CW) was found beneficial as they significantly enhanced the percentage shoot regeneration and number of regenerated shoots per cotyledon explant (Figs. 2, 3). However, neither AgNO3 nor AVG had any promotive effect on adventitious shoot regeneration from hypocotyl segments (data not shown). At the best tested concentration of AgNO3 (20 µmol/L) or AVG (10 µmol/L) 57 % or 53 % of the cotyledon explants showed shoot bud regeneration respectively (Fig. 2). No difference was detected in the duration of shoot bud initiation between the control medium and that augmented with either of the ethylene inhibitors. These shoot buds developed into shoots within 35 days upon transfer to a medium free of AgNO3 or AVG. The average number of shoots per explant were 5.3 or 4.4 in regeneration media fortified with optimal levels of AgNO3 or AVG respectively (Figs. 1c, b, 3). The effect of ethylene on shoot regeneration was further investigated by an exogenous application of CEPA, an ethylene releasing compound. Results showed that the promotive effect of ethylene inhibitors on shoot regeneration was reversed by CEPA. On the AVG medium, the frequency of shoot regeneration from cotyledon explants declined sharply even at 20 µmol/L CEPA, regeneration being reduced by 23 % (Fig. 4). On the other hand, shoot regeneration that had been enhanced by AgNO3 became less affected by CEPA, the latter causing only about 10 % reduction even at a higher concentration, i.e. 40 µmol/L (Fig. 4).

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Figure 1. a. Adventitious shoot regeneration from a cotyledon explant in the regeneration medium (MS + 8.9 µmol/L BA + 5.4 µmol/L NAA + 10 % CW). b. Adventitious shoot regeneration from a cotyledon explant in the regeneration medium following transfer from a AVG (10 µmol/L) supplemented medium. c. Adventitious shoot regeneration from a cotyledon explant in the regeneration medium following transfer from a AgNO3 (20 µmol/L) supplemented medium. d. A well rooted shoot in 1⁄2 MS. e. A 4 months-old plant established in garden soil.

Stimulation of adventitious regeneration of pomegranate using AgNO3 and AVG

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Figure 2. Influence of AgNO3 and AVG on percentage shoot regeneration from cotyledon explants in MS medium augmented with 8.9 µmol/L BA + 5.4 µmol/L NAA + 10 % CW.

Figure 3. Influence of AgNO3 and AVG on number of shoots regenerated per cotyledon explants in MS medium augmented with 8.9 µmol/L BA + 5.4 µmol/L NAA + 10 % CW.

Rooting of in vitro regenerated shoots The excised shoots failed to produce roots in 1⁄2 MS without any auxin supplement even at day 30 of culture. Addition of

an auxin to the medium was essential to induce rooting in the regenerated shoots. About 90 % of the excised shoots developed roots within 10–15 days of culture in 1⁄2 MS containing NAA at an optimal concentration of 0.54 µmol/L. The roots as

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Figure 4. Effect of 2-chloroethylphosphonic acid (CEPA) on adventitious shoot regeneration from cotyledon explants grown at optimal level of AgNO3 (20 µmol/L) or AVG (10 µmol/L).

well as shoots showed rapid elongation following transfer to a hormone-free 1⁄2 MS. In 30 days, an average of 7.4 roots developed from each shoot with a root length averaging 4.4 cm (Fig. 1d).

Establishment of plants in soil Plantlets obtained in vitro with fully expanded leaves and well developed roots were successfully acclimatized in vermicompost and eventually in soil (Fig. 1 e). Sixty-two percent of plantlets survived following transfer to vermi-compost and 84 % of these plants transferred to soil survived.

Discussion We have been successful in developing a protocol for adventitious plant regeneration from seedling derived explants of pomegranate which differs from earlier organogenesis reports in which the explants as well as the cultivars were different (Omura et al. 1987, Moriguchi et al. 1987, Kantharajah et al. 1998). Most importantly in those reports, organogenesis involved an intermediate callus phase which is not a desirable

feature for plant transformation. Also, the percentage regeneration and the average number of shoots per explant were very low compared to the results of this study. Additionally, in the present investigation, the multiplication of pre-existing axillary buds (meristem) and their possible role on shoot bud formation was ruled out as both the proximal ends of cotyledons as well as the attached embryonic axis were eliminated. In the present study a cytokinin (BA) alone could induce adventitious shoot buds, but at a frequency which was unacceptably low. An auxin (NAA) supplement was beneficial and a combination of 8.9 µmol/L BA and 5.4 µmol/L NAA along with 10 % CW was proven to be the best recipe for shoot organogenesis. NAA and BA combinations were also rewarding in many fruit tree species (Zimmerman and Swartz 1994). The experimental results of this study involving permutations and combinations of growth regulators, together with observations on callus mediated organogenesis as reported earlier (Moriguchi et al. 1987, Omura et al. 1987) indicated that pomegranate is, indeed, a difficult species, if not a recalcitrant one, at least in respect of in vitro regeneration through organogenesis. Of several causal factors the production of ethylene, a gaseous plant hormone, by plant tissue cultures has received considerable attention as a possible factor for culture recalcitrance (Benson 2000). Tissue culture can promote inhibitory interactive effects between exogenous and endogenous hormones and recalcitrance can be associated with the overproduction and accumulation of ethylene in the culture vessel. The interaction of ethylene with other plant growth regulators is highly complex and is still little understood. The synthesis and activities of auxins, cytokinins and ethylene are thought to be closely related (Klee and Romano 1994). Interestingly, ethylene also shares a common precursor pathway with polyamine biosynthesis via s-adenosylmethionine (SAM). The exogenous application of auxins and cytokinins (Gude and van der Plas 1985) as well as gelling agents like agar in the culture medium (Mensuari-Sodi et al. 1992) can stimulate the production of ethylene in tissue culture. There are many contrasting examples which show that the regulation of ethylene levels in tissue cultures can have both positive and negative effects on in vitro morphogenesis and proliferative growth. Ethylene was shown to inhibit shoot regeneration in callus cultures of sunflower and tobacco (Huxter et al. 1981, Paterson-Robinson and Adams 1987). Using an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), the negative effect of ethylene on morphogenesis in maize callus culture was demonstrated (Songstad et al. 1988). Moreover, as reported in several recalcitrant plant species such as chinese cabbage (Chi et al. 1991), mustard (Pua and Chi 1993), rice (Adkins et al. 1993) and wheat (Purnhauser et al. 1987), there was a marked enhancement in growth and differentiation of cultured cells when the ethylene production was inhibited or its action was interfered using various ethylene inhibitors. These experimental observations suggest that, the regeneration recalcitrance may be, at least in part, due to the presence of high levels of ethylene in culture. This

Stimulation of adventitious regeneration of pomegranate using AgNO3 and AVG presumption was supported by another study, where the transgenic mustard plants, expressing antisense ACC oxidase RNA exhibited an inverse relationship between shoot regeneration and ethylene biosynthesis (Pua and Lee 1995). On the other hand, positive morphogenetic response of ethylene was reported in a few tree species. In Norway spruce (Kvaalen 1994) it enhanced the induction of embryogenic tissues, whereas in Pinus radiata, it promoted shoot bud differentiation in cotyledon explants (Kumar et al. 1987) and in eastern white cedar, axillary shoot elongation (Nour and Thorpe 1994). The above contrasting results show that the role of ethylene in in vitro morphogenesis, perhaps, vary from species to species and thus needs to be examined with each plant genotype, species and/or type of cultured cells. Keeping this in view, in the present study the effect of two ethylene inhibitors AgNO3 and AVG on adventitious shoot proliferation from cotyledon and hypocotyl tissues of pomegranate was investigated. AgNO3 is believed to inhibit ethylene action by competing with ethylene for binding sites located predominantly at the intracellular membrane (Beyer 1976, Veen and Overbeek 1989). AVG is a potent inhibitor of ethylene biosynthesis; it inhibits pyridoxial phosphate-dependent enzymes e.g. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (Yang and Hoffman 1984). In this study both AgNO3 and AVG appeared to be effective in promoting adventitious shoot regeneration from cotyledon tissues of pomegranate. There was a significant enhancement in the percentage of regeneration as well as number of shoots regenerated per cotyledon. However, the stimulatory effect of ethylene inhibitors were tissue specific or explant dependent as of the two types of explants used the promotive effect was observed only in case of cotyledons. Interestingly, neither of these could enhance the frequency of regeneration from hypocotyl segments. This type of variability in the regeneration response of different explants types of a given plant species has also been reported in Brassica sp. (Chi et al. 1990, Palmer 1992). However, the cause(s) for such a variation is not yet clearly understood. In the present study, although both the ethylene inhibitors promoted adventitious shoot regeneration from cotyledon explants, AgNO3 appeared to be more effective than AVG. The stimulation of shoot morphogenesis elicited by AgNO3 or AVG is in agreement with other reports on Brassica campestris (Chi and Pua 1989, Palmer 1992, Zhang et al. 1998), Brassica juncea (Pua and Chi 1993), Capsicum annuum (Hyde and Philips 1996), Manihot esculenta (Zhang et al. 2001), Triticum aestivum and Nicotiana plumbaginifolia (Purnhauser et al. 1987). The regulatory role of ethylene is further exemplified by the reversion of the promotive effect of AVG by exogenous application of 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. However, the regeneration capacity of explants grown on AgNO3 medium was only slightly affected by CEPA. This may be attributed to an interference of ethylene binding by Ag + which, as a result, prevents or reduces ethylene action (McKeon and Yang 1987). Although, it

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could not be possible to measure or monitor the ethylene levels due to lack of facility, from the results presented herein the response is assumed to be ethylene related. These results support the hypothesis that ethylene is a key factor in the down-regulation of cell differentiation and indicate that enhanced shoot regeneration from cotyledons of pomegranate is closely associated with reduced ethylene synthesis and/or action. In conclusion, this plant regeneration protocol using cotyledons derived from axenic seedlings presented herein could be useful for genetic improvement of this important fruit tree through introgression of useful genes mediated by Agrobacterium vectors. Acknowledgements. Financial assistance from C.S.I.R, New Delhi, India to S. K. Naik through a Senior Research Fellowship is gratefully acknowledged.

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