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Plantlet Regeneration via Somatic Embryogenesis in Four Species of Crocus Roya Karamian Department of Biology, Faculty of Science Bu-Ali Sina University Hamadan Iran Keywords: meristems, micropropagation, plant growth regulators, somatic embryos Abstract

Somatic embryogenesis was initiated in four species of Crocus, C. sativus, C. cancellatus, C. michelsonii and C. caspius using shoot meristem culture on LS medium containing 4 mg/l NAA and 4 mg/l BA or 1 mg/l 2,4-D and 4 mg/l kinetin. Somatic embryogenesis was asynchronous in all of four species and various stages of somatic embryo development were observed when embryogenic calli with globular somatic embryos were transferred into half strength MS medium containing 1 mg/l abscisic acid. Maturated embryos could be germinated on half strength MS medium supplemented with 25 mg/l GA3. Complete plantlets were obtained by transferring of germinated embryos into half strength MS medium supplemented with 1 mg/l NAA and 1 mg/l BA at 20 ºC under a 16/8 h (light/dark) cycle. INTRODUCTION

Crocus is an important genus belonging to Iridaceae. It has nine species in Iran, many of which are valuable and horticulturally important. Like other ornamental monocotyledonous species with corms, they are generally propagated vegetatively. Numerous studies have appeared regarding in vitro propagation of Crocus sativus (George et al., 1992; Ebrahimzadeh et al., 1996; Ebrahimzadeh et al. 2000) but other members of the genus have received little attention (Ebrahimzadeh et al., 1996). The present study reports how regeneration of plantlets was achieved via somatic embryogenesis from shoot meristems culture in C. sativus, C. cancellatus, C. michelsonii and C. caspius. MATERIALS AND METHODS

Corms were collected from different areas of Iran during two annual flowering seasons. Sprouted corms were utilized as source of explants. Bulblets with small portion of corm attached were separated and washed with tap water and surface sterilized in 0.15% HgCl2 solution for 10 min followed by rinsing 3 times with sterile distilled water. Shoot meristems were dissected and cultured on LS medium (Linsmaier and Skoog, 1965) containing 3% sucrose and different concentration of cytokinins (BA, Kn) and auxins (2,4-D, IAA, NAA) (Tables 1-3). All media were solidified by 0.7% agar after adjusting the pH to 5.8 and autoclaved for 15 min at 121 °C at 104 kPa. Cultures were incubated in dark at 20 °C. The experiment was repeated twice using 20 explants per treatment. The data for callus initiation were scored after 6 weeks of culture. Callus and embryogenic callus induction frequencies were calculated as the percent of cultured shoot tips producing callus and embryogenic callus respectively. Data from a total of two replications were statistically analyzed using a SAS program and separated by DMRT. Embryogenic nature of cultures was maintained by visual identification and selection of embryogenic sectors and removal of soft and translucent nonembryogenic portions at the time of subculturing. Embryogenic calli with globular embryos were transferred to a half-strength liquid MS medium (Murashige and Skoog, 1962) without growth regulator or with 1 mg/l ABA for maturation of somatic embryos. Maturated somatic embryos were transferred to half strength MS medium containing 25 mg/l GA3 for germination. For plant regeneration, germinated somatic embryos were transferred to half strength MS

Proc. Ist IS on Saffron Eds: J.-A. Fernández & F. Abdullaev Acta Hort 650, ISHS 2004

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medium supplemented with 1 mg/l NAA and 1 mg/l BA and incubated under a 16/8 h (light/dark) photoperiod with a temperature of 20°C. RESULTS AND DISCUSSION

After 6-8 weeks of culture, shoot meristems on LS medium containing different growth regulators formed calli. Frequency of callus initiation, embryogenic callus production and morphogenetic nature of callus varied with kind of growth regulator used (Tables 1-3). In the studied species, the highest frequency of callus initiation and embryogenic callus induction were recorded on LS medium with NAA and BA or 2,4-D and kin. During initial stages of callus development yellowish and soft calli grew from cultured explants, which after 3-5 transfers were visually distinguishable to be of two types: yellow to brownish friable and nodular embryogenic calli with shining globular structures and off-white to yellow soft calli (Figures 1-4). Production of embryogenic calli increased considerably with careful selection of embryogenic regions and frequent subculturing on the maintenance MS medium. Somatic embryo development proceeded through various stages of globular, heart-shaped and bipolar embryos were observed in half strength liquid MS basal medium growth regulator free or with 1 mg/l abscisic acid. Somatic embryos showing bipolarity could be germinated on half strength MS medium containing 25 mg/l GA3. Germinated somatic embryos transferred into half strength MS medium containing 1 mg/l of each NAA and BA resulted in shoot and root development and plantlet regeneration after 8-10 weeks of inoculation under a 16/8 h (light/dark) photoperiod with a temperature of 20 ºC (Figures 1-4). Some plantlets were left to grow in culture under the same conditions without further subcultures produced corm. Then regenerated plantlets were kept on a medium with low nutrient levels to harden them before transfer to pots.

The results reported here showed that two types of calli grew from cultured shoot meristems. Production of embryogenic callus was initially slow but careful selection of embryogenic regions and frequent subculturing on the maintenance MS medium resulted in vigorous proliferation of embryogenic callus. Somatic embryo development in all of studied species was asynchronous and different stages could be observed simultaneously in the same embryogenic callus. This protocol agrees with general mode of plant regeneration through somatic embryogenesis in number of plants. It was observed that 20ºC temperature was suitable for the regeneration of calli. This result agrees with earlier reports on this genus (Ebrahimzadeh et al., 2000). Many aspects can affect the maturation and germination process, such as temperature and light condition, age of explants and concentration of growth regulators (Firoozabady and DeBoer, 1993). The present study where callus cultures derived from meristems, facilitates the proliferation of embryogenic callus and subsequent regeneration of plantlets from somatic embryos and proved to be a useful alternate method for micropropagation. Literature Cited Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R. 1996. Regeneration of

neoformant organs from organ parts of some species of Crocus. J. Sci. I. R. Iran 7: 65-76.

Ebrahimzadeh, H., Karamian, R. and Noori-Daloii, M.R. 2000. Somatic embryogenesis and plantlet regeneration in saffron, Crocus sativus L. J. Sci. I. R. Iran 11: 169-173.

Firoozabady, E. and DeBoer, D.L. 1993. Plant regeneration via somatic embryogenesis in many cultivars of cotton (Gossypium hirsutum L.). In Vitro Cell. Dev. Biol. 29P: 166-173.

George, P.S., Visvanath, S., Ravishankar, G.A. and Ventkataraman, L.V. 1992. Tissue culture of saffron (Crocus sativus L.): Somatic embryogenesis and shoot regeneration. Food Biotechnol. 6: 217-223.

Linsmaier, E.M. and Skoog, F.1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18: 100-127.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays

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with tobacco tissue cultures. Physiol. Plant. 15: 473-493. Tables Table1. Morphogenic response of shoot meristem culture of C. sativus Growth regulators Auxin + Cytokinin

No. of Responsive explants

% Callus Initiation

% Embryogenic callus initiation

Morphogenic nature

BA + NAA 4 4

4 1

1 4

35 26 24

87.5a 65ab 60b

40a

32.5ab 35ab

Compact,nodular embryogenic

BA + 2,4-D 4 4 4 1 1 4

20 13 11

50bc 32.5d 27.5de

32.5ab 27.5b 30b

Yellowish soft friable and compact nodular

BA + IAA 4 4 4 1 1 4

3 5 3

7.5e

12.5e 7.5e

0d 0d 0d

Very slow growing compact callus with

growing buds

Kn + NAA 4 4 4 1 1 4

14 26 16

35d 60b 40cd

20b

17.5c 15c

Compact hard callus with green buds

Kn + 2,4-D 4 4 4 1 1 4

21 19 8

52.5bc 47.5c 20de

25bc 25bc

22.5bc

Brownish soft friable and compact nodular

Kn + IAA 4 4 4 1 1 4

10 10 6

25de 25de 15de

15c

12.5c 15c

Whitish hard callus with growing buds

Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 20 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p

�≤0.05.

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Table 2. Morphogenic response of shoot meristem culture of C. cancellatus Growth regulators Auxin + Cytokinin

No. of Responsive explants

% Callus initiation

% Embryogenic callus

initiation

Morphogenetic nature

2,4-D + BA 4 4 1 4 4 1

6 5 4

30de 25de 20de

5c 5c 5c

Yellow to brownish, compact, nodular

IAA + BA 4 4 1 4 4 1

2 3 1

10e 15de 5e

0c 5c 0c

Off-white to yellow, compact , nodular

NAA + BA 4 4 1 4 4 1

16 13 11

80a 65ab 55bc

10c 10c 5c

Yellowish, soft callus

2,4-D + Kn 4 4 1 4 4 1

13 12 8

65ab 60abc 40cd

40ab 45a 30b

Off-white, friable, nodular embryogenic

callus

IAA + Kn 4 4 1 4 4 1

5 4 3

25de 20de 15de

15c 10c 5c

White, soft callus with leaf-like structures

NAA + Kn 4 4 1 4 4 1

5 8 5

25de 40cd 25de

0c 0c 0c

White, soft callus

Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 20 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p

�≤0.05.

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Table 3. Morphogenic response of shoot meristem culture of C. michelsonii Growth regulators Auxin + Cytokinin

No. of Responsive explants

% Callus Initiation

% Embryogenic callus initiation

Morphogenetic nature

BA + NAA 4 4

4 1

1 4

12 5

15

30b 8c

37.5b

8.3cd

0d 0d

Off-white, soft callus

BA + 2,4-D 4 4 4 1 1 4

10 8

12

25b 15b 30b

10c

12.5c 0d

Brownish, compact , nodular callus

BA + IAA 4 4 4 1 1 4

0 0 0

0c 0c 0c

0d 0d 0d

�

Kn + NAA 4 4 4 1 1 4

20 18 18

50ab 45ab 45ab

20b

16.7bc 0d

Off-white, soft, friable, nodular callus

Kn + 2,4-D 4 4 4 1 1 4

26 20 25

65a 50ab 62.5a

27b 40a 20b

Yellow to brownish, friable, nodular

embryogenic callus

Kn + IAA 4 4 4 1 1 4

5 0 0

8c 0c 0c

0d 0d 0d

Brownish, soft callus

Explants were cultured on LS medium supplemented with growth regulators in different combinations. Value is the mean for two experiments, 20 explants in each experiment. Data were taken 6 weeks after cultures. Mean separation within rows by DMRT, p

�≤0.05.

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Fig. 1. Plantlet regeneration via somatic embryogenesis in C. sativus.

Fig. 2. Plantlet regeneration via somatic embryogenesis in C. cancellatus

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.

Fig. 3. Plantlet regeneration via somatic embryogenesis in C. michelsonii.

Fig. 4. Plantlet regeneration via somatic embryogenesis in C. caspicus.