Plant Cell Reports

Plant Cell Reports (1996) 16:215-218

© Springer-Verlag 1996

In vitro plant regeneration from leaf callus in Piper colubrinum Link S . M . Kelkar, G. B. Deboo, and K. V. Krishnamurthy Division of Plant Tissue Culture, National Chemical Laboratory, Pune - 411 008, India Received 31 January 1996/Revised version received 10 June 1996 - Communicated by G. C. Phillips

Abstract. Callus-mediated shoot regeneration from leaf explants o f Phytophthora resistant pepper (Piper colubrinum Link.) is described. The effect o f basal media composition and growth regulators on in vitro response o f explants was evaluated. Shoot buds were induced and elongated on half-strength MS medium containing 2.0 m g 1-I B A and 0.5 mg 11 N A A , as well as 1.0 m g 1-1 B A and 0.5 nag 1"1 2,4-D. The shoots were rooted in half-strength MS medium with or without I A A or IBA, and then were transferred to soil with 100% survival.

Piper colubrinum can serve as a potential donor o f various resistance traits in a black pepper improvement programme. The wild and cultivated species o f pepper are known to show graft (Alconero et al. 1972) as well as sexual incompatibility (Nambiar and Sarama 1977). These barriers potentially can be overcome by asymmetric hybridization and direct D N A transfer methodologies. However, an efficient in vitro regeneration system is a major prerequisite for such studies. This paper presents the first report o f in vitro plantlet regeneration from leaf explants o f P. colubrinum.

Abbreviations: BA, Nt-Benzyladenine; 2,4-D, 2,4dichlorophenoxy-acetic acid; IAA, Indole-3-acetic acid; IBA, Indole-3-butyric acid; Kin, kinetin; N A A , ocNaphthaleneacetic acid

NIaterials and methods Plant material. Greenhouse grown 6-8 year old plants of Piper eolubrinum were used as source plants. Fully opened, green leaves

Introduction Disease resistance is a desirable trait which is often more prevalent in wild plants than cultivated plants belonging to the same taxon. Such wild plants are often exploited as potential donors o f resistance traits in various crop improvement programmes involving conventional or modern techniques. Piper colubrinum Link. is a multiple disease and pest resistant species occurring wild in Brazil. It is reported to be resistant to fungal pathogens Phytophthora capsici Leonian and Fusarium solani f piperi as well as to plant parasitic nematodes Radopholus similis Chitwood and Meloidogyne incognita (Cobb) Thorne. (Purseglove et al. 1981). The economically important species Piper nigrum L., black pepper, is susceptible to these pathogens which cause considerable yield loss every year.

Correspondence to: K. V. Krishnamurthy

were used as explants. The leaves were thoroughly washed with detergent water and treated with 70% ethanol for 1 rain followed by rinsing several times with sterile distilled water. The leaves were surface sterilised in 0.1% mercuric chloride for 5 rain and washed with sterile distilled water several times. Sterilised leaves were cut lengthwise into 0.5 cm2 pieces and cultured in petridishes of 55 mm diameter (Laxbro, India) containing MS (Murashige and Skoog 1962) medium at full- and half-strength (containing half-strength major salts). A total of 100 explants were cultured per treatment. The explants were re-sterilised in first two subcultures to minimise contamination.

Callus initiation. Half-strength MS medium was supplemented with eytokinins BA or kinetin (0.5, 1.0, 2.0, 3.0 mg ll) alone or in combination with the auxins NAA or 2,4-D (0.5, 1.0 mg ll). Ten sterile explants recovered after re-sterilisation were cultured per treatment, and the experiment was repeated thrice in addition to a pilot experiment. Thus the results presented in Table 1 and 2 are for a total of 40 explants. Shoot induction. The calli induced on half-strength MS were subcultured onto fresh media of the same composition for the induction of shoot buds. Further subcultures occurred at 3 week intervals. Shoot buds elongated into shoots on the same medium after 2-3 subcultures.

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Rooting. The callus derived shoots, 3-4 cm in height, were rooted in half-strength MS medium with or without auxins IAA or IBA (0.110.0 mg 1"1). Five plantlets were used per treatment and the experiment was repeated twice. Media and culture conditions. All media contained 3% sucrose and 0.6% agar (Qualigens, India). The pH was adjusted to 5.8 before autoclaving. All the plant growth regulators, except for IAA and IBA, were autoclaved with the medium, whereas filter sterilized IAA and IBA were added to the medium after autoclaving. All the cultures were incubated at 25 +_ I°C and at a light intensity of 35 gEm2s 1 and 16 h photoperiod. Acclimatization. Sixty rooted plantlets were transferred to small 5 cmz pots containing sterilized soil : vermiculite mixture in 1:1 proportion. Plantlets were hardened at 25 +- I°C for 7 d at 35gEm2s~ and then transferred to the greenhouse. Histology. For histological observations, calli showing formation of shoot buds were fixed in formalin : glacial acetic acid : alcohol (1:1:20) for 3 d. Following dehydration in t-butanol, the material was embedded in paraffin wax (melting point: 58-60°C). Serial sections of 10 gm thickness were cut using a rotary microtome (Reichert Jung, 2050 Supercut, Germany). The sections were dehydrated through a xylene-alcohol series, stained with 1% haematoxylin and counterstained with 1% eosin, and mounted in D.P.X. mountant. Results and Discussion The systemic presence o f endophytic bacteria in Piper cultures (Aminnddin et al. 1992; Phillip et al. 1992; Bhat et al. 1992; Meyer et al. 1992; Fitchet 1990) caused serious problems in establishment o f sterile cultures: 76% o f 500 explants used in this study had bacterial contamination. Re-sterilization o f explants with 0.1% mercuric chloride in the first two subcultures, as described by Bhat et al. (1992), was found to be useful in reducing the contamination. About 50% o f the re-sterilised explants remained sterile throughout the culture period.

Effect o f basal m e d i u m The mineral salt composition o f the basal medium used is known to affect the regeneration capacity in various species (Sato 1995; Preece 1995). In the present study,

the concentration o f mineral salts influenced the related phenomena o f callus formation and the browning o f explants. Using full- strength MS medium 83.5% o f the explants became brown and only 14.4% o f the explants formed callus. The use o f modified medium containing major elements at half-strength successfully reduced browning o f explants to 17.5% and increased the percentage o f explants forming callus to 66%. For this reason, half strength MS was used in further experiments. In contrast, previous reports on tissue culture o f the genus Piper (Mathews and Rao 1984; Phillip et al. 1992; Bhat et al. 1992; Aminuddin et al. 1992) used full-strength MS basal medium.

Effect o f growth regulators The type o f growth regulator, its concentration, and the combination o f growth regulators determined the frequency o f callus response, morphology o f the callus, induction and elongation o f shoot buds, and rooting o f the shoots. Both the cytokinins, B A and kinetin, supported callus induction in the leaf explants at all concentrations tested (Table 1). The media containing kinetin invariably produced white or whitish green callus at the cut ends o f the explants, whereas in presence o f BA the whole explant callused producing white, whitish green or compact, intense green callus (Table 1). The callusing response followed a definite pattern with respect to cytokinin concentrations when used alone in the medium. The percentage o f explants forming callus decreased with increase in cytokinin concentration (Table 1). These observations are in agreement with those o f Mathews and Rao (1984) in that higher concentrations o f cytokinins (2.0, 5.0 mgl "1) lead to blackening o f the explants. All the calli recovered on various m e d i a combinations (Table 1) were subcultured onto the petridishes containing fresh medium for shoot induction at intervals o f 3 weeks. After 2-3 subcultures, only the compact, intense green calli (Fig. la) induced on four media

Table 1. Effect of different concentrations and combinations of the plant growth regulators on percentage callus induction (mean +_S.E) and callus morphology in leaf explants of P. colubrinum. Cytokinin Conc Auxin (rag l"l) (mg 1"l) 0 2,4-D NAA 0.5 1.0 0.5 1.0 BA 0.5 90.0 + 0.0 GW 70.0 +_11.1 W 60.0 + 0.0W 40.0 _+0.0W 100.0 + 0.0GW Kin. 0.5 75.0_+ 5.5 W 20.0~3.5 W 50.0_+ 6.1W 60.0 + 7.9W 50.0 + 7.9W BA 1.0 75.0 + 7.5 GW 100.0 _+0.0 G 80.0 + 3.5G 100.0 + 0.0GW 40.0 + 3.5GW Kin. 1.0 60.0+ 0.0W 60.0 +_3.5 W 50.0 + 16.5W 20.0 + 6.1W 25.0 + 7.5W BA 2.0 75.0 + 2.5 GW 100.0 + 0.0 G 73.0 + 5.4GW 100.0 + 0.0G 50.0 + 0.0GW Kin. 2.0 45.0 + 5.5 GW 50.0+8.6 W 50.0 + 6.1W 20.0 +_3.5GW 50.0 _+3.5GW BA 3.0 67.0+7.3 GW 47.0±2.1 GW 100.0+_0.0 80.0 +-4.1W 80.0 +-0.0W Kin. 3.0 20.0 +-3.5 W 10.0+_ 5.0W 0.0 + 0.0 50.0 _+6.1GW 0.0 +-0.0 G: Green, W'. White, GW: Greenish white. Data were obtained using a total of 40 explants in 4 repeated experiments.

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Fig, 1. in vitro plant regeneration from leaf explaJats of wild pepper, Piper colubrinum, (A) Callus initiation, (B) Shoot bud formation.(C) Shoot elongation. (D) Root formation. (E) Hardening of rooted plants.

Fig. 2. Histological preparations showing different stages of shoot bud formation from leaf derived callus in P. colubrinum. (A) Meristematic pocket (m) with surrounding callus cells indicating initiation of differentiation. Bar = 0.1 mm. (B) Protrusion of meristematic cells forming shoot bud(s) with peripheral callus cells. Bar = 0.1 ram. (C) Differentiated shoot bud (s) with two leaves (1). Bar = 0.2 mm

218 combinations (Table 2) formed shoot buds (Fig. lb), while all other media combinations supported only callus proliferation. T a b l e 2. Effect of growth regulators on shoot bud induction and

shoot elongation in 8-9 weeks old callus cultures ofP. colubrinum. 1/2 MS + % Shoot bud No. of Shoots/ Growth Regulators Formation explant (rag l"1) Mean _+ S.D Mean + S.D BA 2.0 72_+5 7.0_+2.8 NAA 0.5 BA 1.0 90_+9 4.6_+1.7 2,4-D 0.5 BA 1.0 50 _+8 0 2,4-D 1.0 BA 2.0 52 +_9 0 2,4-D 0.5 Data were obtained using a total of 40 explants in 4 repeated experiments.

The elongation of shoot buds into shoots after 2-3 subcultures (Fig. lc) was restricted to two media combinations, i.e. 2.0 mg 11 BA + 0.5 mg 1-1 NAA, and 1.0 mg 1l BA + 0.5 mg 1-1 2,4-D. The differences between these two treatments were found to be insignificant when analysed by student's T test (Wardlaw 1985). Even though four media combinations supported shoot bud formation (Table 2), shoot buds induced on media combinations 1.0 mg 1"1 BA + 1.0 mg1-1 2,4-D and 2.0 mg 11 BA + 0.5 mg 11 2,4-D failed to elongate after 7-8 subcultures, but dedifferentiated into white friable callus. The callus proliferated for another two subcultures, then turned brown and eventually died.

(10-100%). However, callus formation occurred at the base of shoots in presence of auxin, making the origin of the roots obscure. Half-strength MS medium without growth regulators was found to be optimum with 100% rooting frequency, with no callus formation at the base of shoots (Fig. ld). The survival rate of rooted plantlets after hardening (Fig. le) was 100% and 60 hardened plants were successfully transferred to pots.

Histology The origin of shoot buds from callus was examined by histological preparations. The calli showed asynchronous development of shoot buds, that is, shoot buds at more than one stage of differentiation were observed in the same callus. Meristematic pockets of small cells with dense cytoplasm and darkly stained nuclei (Fig. 2a) scattered in callus tissue indicated initial stages of differentiation. The presence of small protrusions of meristematic cells at the periphery of callus indicated the initiation of shoot buds (Fig. 2b). The anatomy of differentiated shoot bud along with leaves (Fig. 2c) originating from callus confirms the organogenic pathway of morphogenesis. This standardised protocol of regeneration of plants from leaf explants of P. colubrinum should be useful in marker-assisted asymmetric hybridization programme involving P. colubrinum and P. nigrum. Acknowledgments. We thank Dr. (Mrs.) S. Hazra for her valuable help. We also acknowledge assistance in photography by Mr. Parag Akkadkar. The Junior Research Fellowship awarded by the C.S.I.R., Government of India, New Delhi to S.M.K. is acknowledged.

T a b l e 3. Effect of growth regulators on rooting response of in vitro

produced shoots of P. colubrinum. Auxin Concentration % Rooting Callus (mg 1l ) Mean ± S.D. IBA 0.1 10 + 0 + 1.0 60 +_ l + 5.0 40 _+ 1 + 10.0 60 _+0 + IAA 0.1 20 _+0 + 1.0 100 _+0 + 5.0 80 _+ 1 + 10.0 20 +_0 + NONE 100 _+0 + : present ; - : absent Data were obtained using a total of 10 replicates for each treatment in two repeated experiments.

The shoots, 3-4 cm in height, were excised from the callus and transferred to half-strength MS basal medium with or without IAA and IBA at different concentrations (0.1-1.0 mg 1-1). Both IAA and IBA induced rooting at all concentrations tested (Table 3) at varying frequency

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