In vitro adventitious shoot formation on petioles of commercial cultivars of delphinium

Scientia Horticulturae 90 (2001) 143±150

In vitro adventitious shoot formation on petioles of commercial cultivars of delphinium Keizo Hosokawa*, Hisae Koiwa, Saburo Yamamura Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan Accepted 15 November 2000

Abstract Culture conditions of plant growth regulators were examined for promoting ef®cient plant regeneration from petiole explants of commercial cultivars of delphinium. The optimal combination of a cytokinin and an auxin was determined using petiole explants of delphinium cv. Blue Springs. Concentrations of individual cytokinins for the optimal regeneration of shoots were 10 mg l 1 for 6benzylaminopurine, 1 mg l 1 for N-phenyl-N0 -1,2,3-thiadiazol-5-yl urea and 5±10 mg l 1 for zeatin, in combination with 2,4-dichlorophenoxyacetic acid at 1 mg l 1. We applied these optimized conditions for regeneration of adventitious shoots to petiole explants of 18 other commercial cultivars. Adventitious shoots regenerated from 14 cultivars out of the 19 ones tested at frequencies of 2±53%. Shoots of cv. Blue Springs that had regenerated in vitro rooted in a hormonefree medium and ®nally were transferred to soil. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Adventitious shoot; Delphinium; Petiole

1. Introduction The genus Delphinium is a member of the family Ranunculaceae and consists of over 300 species. Delphinium includes some horticulturally Abbreviations: MS medium, Murashige and Skoog's medium (Murashige and Skoog, 1962); BA, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, 1-naphthaleneacetic acid; TDZ, N-phenyl-N0 -1,2,3-thiadiazol-5-yl urea * Corresponding author. Present address: Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan. Tel.: ‡81-298-38-0572; fax: ‡81-298-38-0575. E-mail address: [email protected] (K. Hosokawa). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 4 9 - 1

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important species such as D. elatum L., D. cheilanthum Fisch., D. formosum Boiss. and Heut., and D. hybridum hort. (Royal Horticultural Society, 1949). Plants of D. hybridum hort. are especially of ornamental importance, and are cultivated for cut ¯owers and as garden plants. The potential for molecular breeding is very high, although classical breeding has developed many cultivars of delphinium. Genetic engineering techniques could be useful in the creation of new breeding approaches to produce plant varieties with novel characteristics (Mol et al., 1995). However, adventitious shoot or embryo regeneration systems are a prerequisite for these techniques and, to our knowledge, no such systems have been reported in delphinium. Tissue culture techniques in delphinium are mainly used for micropropagation through shoot tip culture (Pryce et al., 1993). We attempted to induce the regeneration of shoots from leaf, petiole and root explants of delphinium cv. Blue Springs on medium supplemented with various combination of cytokinins and auxins. However, the regeneration was mainly observed on petiole explants and occurred at low frequencies on leaf and root explants (15 and 7% utmost, respectively). Then, we determined the optimal conditions for regeneration of plants from the petiole explants of cv. Blue Springs and regenerated shoots at various frequencies from petiole explants of 18 other commercial cultivars of delphinium under similar conditions. This regeneration system is therefore expected to pave the way for the molecular-biology-based breeding of delphinium. 2. Materials and methods 2.1. Plant materials Seeds of 11 cultivars of delphinium (Blue Springs; Magic Fountain series (Dark Blue, Lavender, Lilac Pink, Sky Blue and White) and Paci®c Giant series (Blue, Deep Blue, Light Blue, Lilac and White)) were obtained from Sakata Seed (Kanagawa, Japan). Seeds of 8 cultivars of delphinium (Beverly Hills series (Salmon Shade, Scarlet and Yellow Shade); Belladonna series (Belladonna Imp., Bellamosum Imp., Cardinale, Casablanca); and Dwarf Butter¯y) were obtained from Daiichi Seed (Tokyo, Japan). Seeds of all cultivars were surface-disinfected with 1% sodium hypochlorite solution for 15 min and then the seeds were rinsed three times in sterile distilled water. The disinfected seeds were allowed to germinate on modi®ed MS medium, which included half-strength macronutrients (KNO3, NH4NO3 and CaCl2), 30 g l 1 sucrose and 3 g l 1 gellan gum (Wako Pure Chemical Industries, Osaka, Japan). Seedlings were cultured aseptically under 16 h of light per day from ¯uorescent lamps (50 mmol m 2 s 1) at 208C.

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Segments of petioles (approximately 7 mm in length) were excised aseptically from 10-week-old seedlings of cv. Blue Springs to establish optimal conditions for regeneration of adventitious shoots. Petiole explants of 18 other cultivars were also excised aseptically for use in the regeneration experiments. 2.2. Media and culture conditions Modi®ed MS medium supplemented with various plant growth regulators, under various concentrations, was used. The pH of the media was adjusted to 5.7± 5.8 with 1 N KOH prior to autoclaving. For determinations of optimum concentrations of each cytokinin and of each auxin, we cultured petiole explants of cv. Blue Springs on modi®ed MS medium with various concentrations of each cytokinin (BA, TDZ and zeatin; 0±20 mg l 1) and auxin (2,4-D and NAA; 0± 1 mg l 1). The petiole explants of 18 other commercial cultivars of delphinium were cultured on three different media, namely, modi®ed MS medium with three combinations of plant growth regulators, as follows: medium A, 10 mg l 1 BA plus 1 mg l 1 2,4-D; medium B, 1 mg l 1 TDZ plus 1 mg l 1 2,4-D; and medium C, 10 mg l 1 zeatin plus 1 mg l 1 2,4-D. The regeneration of adventitious shoots was monitored as shoot regeneration frequency and numbers of shoots produced per regenerating explant. All explants were incubated under the same cultural conditions mentioned above. Regenerated shoots were transferred to modi®ed MS medium for inducing adventitious roots and were cultured at 208C with 16 h of light per day from ¯uorescent lamps (50 mmol m 2 s 1) for 4 weeks. Regenerated plants with a well-established root system were rinsed carefully to remove solidi®ed medium and transferred to pots that contained vermiculite. The pots were placed in a container that was covered with polyvinylidenechloride ®lm (Kure Wrap; Kureha Chemical Industry, Tokyo, Japan) to avoid desiccation. Between the 2nd and 4th week after the start of culture in vermiculite, the ®lm was gradually removed. Plants in pots were transferred to a greenhouse after completion of acclimatization. Finally, plants of cv. Blue Springs were cultivated in a ®eld under a polyethylene tunnel. 3. Results 3.1. Effects of plant growth regulators on shoot regeneration from petiole explants In order to establish optimum conditions for regeneration of adventitious shoots from petiole explants, we tested three cytokinins (BA, TDZ and zeatin) in

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Table 1 Effects of cytokinins (BA, TDZ and zeatin), in combination with auxins (NAA and 2,4-D) on regeneration of adventitious shoots from petiole explants of delphinium cv. Blue Springsa Cytokinin (mg l 1)

Percentage of explants producing shoots 2,4-D (mg l 1) 0

NAA (mg l 1) 0.1

1

0.1

1

BA 0 1 5 10 20

0 0 0 0 0

0 7 7 0 47

0 20 20 52 20

0 0 7 23 20

0 0 7 7 17

TDZ 1 5 10 20

0 0 0 0

7 0 0 0

47 40 14 20

7 7 17 3

7 3 0 0

Zeatin 1 5 10 20

0 0 0 0

5 10 30 20

45 55 55 45

5 10 10 10

10 15 37 50

a

Cultures were scored after 8 weeks of culture; 20±30 explants were examined in each case.

combination with two auxins (2,4-D and NAA, Table 1). A regeneration frequency of about 50% was obtained with each of the three cytokinins in combination with 2,4-D at 1 mg l 1. The optimum concentrations of each cytokinin were 10 mg l 1 BA, 1 mg l 1 TDZ, and 5±10 mg l 1 zeatin (Fig. 1A). Furthermore, 2,4-D was more effective than NAA in stimulating the regeneration of adventitious shoots in almost all cases examined except for the case of 20 mg l 1 zeatin plus 1 mg l 1 NAA. 3.2. Regeneration of shoots from petiole explants of other cultivars We examined the regeneration of adventitious shoots from petiole explants of 19 commercial cultivars on media A, B and C (Table 2). Frequencies of regeneration of adventitious shoots reached 50% in only one cultivar, namely, cv. Blue Springs and a frequency of more than 40% was obtained in two cultivars (cv. White of Magic Fountain series and cv. White of Paci®c Giant series). No shoots regenerated in ®ve cultivars (cv. Cardinale of Belladonna series; cv. Salmon Shade, Scarlet and Yellow Shade of Beverly Hills series; and cv. Dwarf

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Fig. 1. Regeneration of plants from petiole explants of cv. Blue Springs. (A) Adventitious shoots regenerated from petiole explants after 8 weeks of culture on modi®ed MS medium supplemented with 10 mg l±1 zeatin and 1 mg l±1 2,4-D. (B) Development of adventitious roots on a regenerated shoot in modi®ed MS medium free of plant growth regulators after 4 weeks in culture. (C) Successfully acclimatized plantlet in a pot that contained vermiculite. (D) A regenerated plant ¯owering under ®eld conditions, 3 months after transplantation to the ®eld.

Butter¯y). The mean number of regenerated shoot was low and no signi®cant differences were observed among cultivars that regenerated shoots. 3.3. Morphological observations De novo regenerated shoots developed roots in all cases in the hormone-free medium within 4 weeks, and then the plants were acclimatized (Fig. 1B and C). In the case of cv. Blue Springs, 10 regenerated plants were grown in the ®eld under a polyethylene tunnel and all were phenotypically normal in terms of leaf shape, growth features, and the ¯ower shape and color (Fig. 1D).

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Table 2 Adventitious shoot regeneration from petiole explants of 19 cultivars of Delphinium spp. in culture on three types of mediaa Cultivar

Percentage of explants producing shoots Medium Ab

Medium Bb

Medium Cb

Blue Springs

52 …1:4  0:8†c

30 …1:4  0:5†

51 …1:5  0:8†

Magic Fountain Dark Blue Lavender Lilac Pink Sky Blue White

31 11 27 9 31

…1:4  0:6† …1:4  0:4† …1:9  0:5† …1:8  0:5† …1:3  0:4†

16 13 31 20 49

…1:7  0:7† …1:3  0:5† …1:6  0:5† …1:8  1:0† …1:5  0:5†

35 24 11 4 31

…1:6  0:6† …1:2  0:4† …1:6  0:5† …2:5  0:7† …1:1  0:3†

Pacific Giant Blue Deep Blue Light Blue Lilac White

11 27 11 20 11

…1:8  0:4† …1:3  0:4† …2:0  0:6† …1:6  0:7† …1:6  0:5†

13 9 0 31 42

…1:0  0:0† …1:3  0:5†

11 22 16 33 29

…2:6  0:8† …1:4  0:7† …1:0  0:0† …1:3  0:6† …1:8  0:5†

…2:3  0:8† …1:6  0:7†

Belladonna Belladonna Imp. Bellamosum Imp. Cardinale Casablanca

9 …1:3  0:5† 7 …1:0  0:0† 0 2 …1:0  0:0†

Beverly Hills Salmon Shade Scarlet Yellow Shade

0 0 0

0 0 0

0 0 0

Dwarf Butterfly

0

0

0

29 …1:6  0:7† 16 …1:7  0:6† 0 7 …1:7  0:6†

0 0 0 16 …1:6  0:7†

a

Cultures were scored after 8 weeks of culture; 20±45 explants were used for each experiment. Medium A, B and C were modi®ed MS medium that contained 10 mg l±1 BA and 1 mg l±1 2,4D, 1 mg l±1 TDZ and 1 mg l±1 2,4-D, and 10 mg l±1 zeatin and 1 mg l±1 2,4-D, respectively. c Parentheses indicate the average number of shoots S.D. produced per regenerating explant. b

4. Discussion Several factors are known to be important for shoot organogenesis (Brown and Thorpe, 1986). We observed pronounced differences in the frequencies of regeneration of adventitious shoots when we varied some of these factors. In particular, the type of explant, the presence of plant growth regulators (especially auxin) and the cultivar/genotype were all important factors for obtaining highfrequency regeneration of adventitious shoots. Each of the three cytokinins (BA, TDZ and zeatin), in combination with 2,4-D, had almost the same effects on regeneration of adventitious shoots. By contrast,

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2,4-D was more effective than NAA in the presence of each cytokinin at optimum concentration. An auxin appeared to be required for regeneration of adventitious shoots because no regeneration was observed on media supplemented only with cytokinins. The importance of cultivar/genotype in organogenesis has been reported in several species and, in particular, in some commercial cultivars (bell pepper, Ezura et al., 1993; subterranean clover, Heath et al., 1993; Gentiana, Hosokawa et al., 1996). Adventitious shoots were regenerated from petioles of plantlets of 14 of the 19 cultivars of delphinium tested. The extent of regeneration was dependent on the cultivar. A remarkable difference in frequency of regeneration of adventitious shoots was found among cultivars, and it seemed to be of the same importance as the combination of plant growth regulators used for regeneration. It was reported that Delphinium species was dif®cult to acclimatized ex vitro (Pryce et al., 1993). Although the survival after acclimatization was improved using the vessels with small apertures, the frequency of survival after acclimatization was low (Murphy et al., 1998). However, all delphinium plants acclimatized in these experiments survived and the plants in pots were successfully transferred to a greenhouse. In the present study, we described the ®rst successful regeneration of adventitious shoots from petiole explants of various commercial cultivars of delphinium. These regeneration systems that we developed in delphinium could be applicable to the molecular-biology-based breeding of delphinium. On the other hand, increase in the number of regenerated adventitious shoots will be needed for exploitation of our regeneration system for micropropagation and genetic transformation. We are now trying to develop a more ef®cient regeneration system in delphinium. References Brown, D.C.W., Thorpe, T.A., 1986. In: Vasil, I.K. (Ed.), Plant Regeneration by Organogenesis, Vol. 3, Academic Press, London, pp. 49±65. Ezura, H., Nishimiya, S., Kasumi, M., 1993. Ef®cient regeneration of plants independent of exogeneous growth regulators in bell pepper (Capsicum annumm L.). Plant Cell Rep. 12, 676±680. Heath, L.C., Chin, S., Spencer, C.D., Higgins, T.J.V., 1993. In vitro regeneration of commercial cultivars of subterranean clover. Plant Cell Tiss. Org. Cult. 35, 43±48. Hosokawa, K., Nakano, M., Oikawa, Y., Yamamura, S., 1996. Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana. Plant Cell Rep. 15, 578±581. Mol, J.N.M., Holton, T.A., Koes, R.E., 1995. Floriculture: genetic engineering of commercial traits. Trends Biotechnol. 13, 350±355. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473±497.

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Murphy, K.P., Santamaria, J.M., Davies, W.J., Lumsden, P.J., 1998. Ventilation of culture vessels. I. Increased growth in vitro and survival ex vitro of Delphinium. J. Horticult. Sci. Biotechnol. 73, 725±729. Pryce, S., Lumsden, P.J., Berger, F., Leifert, C., 1993. Effect of plant density and macronutrient nutrition on Delphinium shoot cultures. J. Horticult. Sci. 68, 807±813. Royal Horticultural Society, 1949. A Tentative Check-list of Delphinium Names. London, pp. 1±112.