Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus

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1 Scientia Horticulturae 98 (2003) Direct somatic embryogenesis and plant regeneration from leaf cultures of ornamental species of Dianthus Aparna Pareek, S.L. Kothari * Department of Botany, University of Rajasthan, Jaipur , India Accepted 21 February 2003 Abstract Protocol for direct somatic embryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoog s (MS) liquid medium supplemented with 2,4-D (1 mg/l) was used for direct induction of somatic embryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf explants in liquid medium. Development of embryos to heart and torpedo stages was achieved in the liquid medium incorporated with polyethylene glycol (PEG 6000) at a concentration of 2.5%. Embryo maturation was further promoted by addition of casein hydrolysate (CH) (200 mg/l) in MS liquid medium. Embryos germinated to form plantlets on solid MS medium supplemented with GA 3 (1 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions. # 2003 Elsevier B.V. All rights reserved. Keywords: Leaf explants; Somatic embryogenesis; Regeneration; Dianthus caryophyllus; Dianthus chinensis; Dianthus barbatus 1. Introduction The genus Dianthus includes 300 species of which D. caryophyllus, D. barbatus and D. chinensis are cultivated commercially for cut flowers. The techniques of plant tissue culture and genetic engineering could provide an alternative and complementary tool to improve Dianthus and develop novel varieties. However, successful application of tissue culture Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; BAP, 6-benzylamino-purine; cv., cultivar; CH, casein hydrolysate; FAA, formalin acetic acid alcohol; GA 3, gibberellic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; Kn, kinetin; PEG, polyethylene glycol; S.D., standard deviation * Corresponding author. Tel.: þ ; fax: þ address: slkothari@lycos.com (S.L. Kothari) /$ see front matter # 2003 Elsevier B.V. All rights reserved. doi: /s (03)

2 450 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) technique in breeding programs, transformation experiments or other investigations presupposes the development of a reproducible in vitro plant regeneration system with appropriate culture conditions. Although in the genus Dianthus plant regeneration from a variety of explants has been reported that include shoot tips (Earle and Langhans, 1975), stem pieces (Frey and Janick, 1991; Nugent et al., 1991; Watad et al., 1996), leaf segments (Miller et al., 1991; Jethwani and Kothari, 1996; Kantia and Kothari, 2002), petals (Kakehi, 1979; Gimelli et al., 1984; Nugent et al., 1991; Nakano et al., 1994), anthers (Villalobos, 1981) and ovules (Demmink et al., 1987) but somatic embryogenesis has been reported only in D. caryophyllus (Frey et al., 1992; Nakano and Mii, 1993; Sankhla et al., 1995; Yantcheva et al., 1998). Embryogenesis induced from mature somatic tissue is the desired approach of rapid and fast vegetative propagation (Ammirato, 1983; Janick et al., 1989). This procedure provides cytological and genetic stability of regenerated plants (Pedroso and Pais, 1995). The power of somatic embryogenesis as a propagation tool becomes especially apparent when the goal is to generate large number of propagules of an ornamental that is either rare or has some unusual feature that makes clonal multiplication highly desirable. Embryogenic cultures of ornamental plants are increasingly finding application as a source of material amenable to production of genetically transformed plants. A vigorous and efficient embryogenic system besides providing a framework for genetic manipulation could also facilitate development of compact storage, packaging and distribution methods for elite varieties through the formation of synthetic seeds. In the present study a reproducible regeneration protocol for efficient plant recovery via somatic embryogenesis in three ornamental species of Dianthus in liquid media has been described which is characterized by well-defined stages of embryo development, maturation, germination and plant regeneration. 2. Materials and methods 2.1. Plant materials and culture conditions Seeds of D. caryophyllus cv. Chabaud, D. barbatus cv. Tall Double Mix and D. chinensis cv. Fire Carpet were surface sterilized with 0.1% HgCl 2 solution for 3 4 min and rinsed three times with sterile distilled water. Seeds were cultured on half strength MS medium (Murashige and Skoog, 1962) containing agar (0.8%) for germination. First four pairs of leaves were taken from aseptically raised seedlings and cut into small pieces (3 4 mm). The distal half of the leaf was removed and only proximal part was cultured into 100 ml Erlenmeyer flasks in liquid medium (40 ml volume). Fifteen explants were cultured per flask. Liquid culture was maintained for 28 days on a rotary shaker (120 rpm) in the growth chamber at C. Similar procedure was followed for all the three species of Dianthus Composition of the media Induction of embryogenesis Somatic embryos were induced in liquid MS medium supplemented with 2,4-D (0.5, 1, 2, 5 mg/l), 2,4-D (1 mg/lþþbap (0.2, 0.4, 0.6, 0.8, 1 mg/l) in combinations, 2,4-D

3 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) (1 mg/lþþbap ð0.2 mg/lþþch (100, 200, 500, 750, 1000 mg/l) in combinations. Fifteen explants were incubated into embryo induction medium (EIM) in 100 ml Erlenmeyer conical flasks (Borosil). Five flasks were prepared for each treatment. Cultures were incubated for 4 weeks in the dark on a rotary shaker (120 rpm) in the growth chamber at C Subculture Liquid cultures were subcultured weekly and fresh medium was added. Globular stage embryos were subcultured to embryo development medium after 4 weeks of culture. The heart and torpedo stage embryos were subcultured to embryo maturation medium after 4 weeks of culture. After 3 weeks the cotyledonary stage embryos were cultured for germination to various hormone supplemented media Development of somatic embryos to heart and torpedo stages Globular stage embryos formed in all the three species of Dianthus were cultured for 21 days in liquid MS medium supplemented with polyethylene glycol (PEG MW 6000) at concentrations of 0.5, 0.75, 1, 2.5, 5 and 10% for further development to heart and torpedo stages. Culture vessels and culture conditions were same as for primary cultures Maturation of somatic embryos formation of cotyledons Following liquid media were used for maturation of somatic embryos: (a) MS þ CH (100, 200, 500, 750, 1000 mg/l). (b) MS þ CH ð100; 200; 500; 750; 1000 mg/lþþbap (0.5, 1, 2 mg/l) in various combinations. (c) MS þ CH (200 mg/lþþbap (0.5 mg/lþþaba (0.2, 0.4, 0.6, 0.8, 1 mg/l) in various combinations. (d) MS þ BAP ð0:5; 1; 2; 5 mg/lþþaba (0.2, 0.4, 0.6, 0.8, 1 mg/l) in various combinations. (e) MS þ BAP ð0:5 mg/lþþga 3 ð0:5; 1; 2; 5 mg/lþþaba (0.2 mg/l) in various combinations Plantlet formation Individual cotyledonary somatic embryos were cultured on MS solid medium containing different concentrations of GA 3 (0.5, 1, 2, 5 mg/l) in D. caryophyllus; liquid MS þ 2,4-D ð0:5; 1; 2 mg/lþþbap (0.5, 1, 2 mg/l) in D. barbatus and solid MS þ BAP (0.5, 1, 2 mg/l) in D. chinensis. Fifty embryos were cultured in each medium. Various other hormonal combinations (viz. cotyledonary embryos were also cultured on MS, 1/2 MS, 1/3 MS, MS supplemented with GA 3 (1 mg/lþþiba (1 mg/l), IBA (0.5, 1, 2, 5 mg/l), IAA (0.5, 1, 2, 5 mg/l), GA 3 (1 mg/lþþkn ð0:5 mg/lþþbap (0.5 mg/l), Kn (0.5, 1, 2, 3, 5 mg/l), ABA (0.2, 0.4, 0.6, 0.8, 1 mg/l), BAP (0.5 mg/l), GA 3 (1 mg/lþþkn (0.5 mg/l)) were also tried for germination of somatic embryos. The effect of phytagel (0.2%) was also observed on germination.

4 452 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Root development and field transfer The regenerated plantlets (3 4 cm long) of Dianthus spp. were cultured further on rooting media MS þ NAA (1 mg/l). After plantlets with both shoots and roots had grown to 8 cm, these rooted plantlets, after removing the agar under running tap water, were individually transferred to earthen pots containing a mixture of organic manure and soil. Each pot was covered with a polyethylene bag for 4 5 days initially to maintain humidity Histological analysis For histological examination leaf explants with initiated structures were fixed in FAA (formalin:acetic acid:ethanol 1:1:18) dehydrated through a tertiary butyl alcohol xylol series and embedded in paraffin wax (Johansen, 1940). Serial sections (10 12 mm) were cut with a rotary microtome and stained with safranine Statistical analysis The mean and standard deviations for each embryo-counts were computed from five random samples of 1 ml suspension of each concentration. The whole experiment was repeated twice. Presented results summarize the data of two independent experiments with five replicates in each run, which were evaluated by one way analysis of variance (ANOVA) and comparisons between the mean values of treatments were made by the least significant difference (LSD) test. 3. Results Somatic embryos were formed after 3 4 weeks of incubation in embryo induction medium MS þ 2,4-D (1 mg/l) in about 85% of the cultures in all the three species of Dianthus. The embryos were formed all over the surface of explants but appeared mainly on the cut edges (Fig. 1(1)). It was observed that bigger explants produced lesser number of embryos and explants that were too small turned black and failed to form any embryos. Embryos callused if the concentration of 2,4-D was increased (5 mg/l) in D. caryophyllus and fewer embryos were induced in D. barbatus and D. chinensis (Table 1). In many cases, somatic embryos formed embryogenic callus or secondary embryos. Therefore, somatic embryos of Dianthus were also used to induce embryogenic callus. In all the three species the embryos were also induced in MS medium supplemented with 2,4-D þ BAP and 2,4- D þ BAP þ CH, but the number of embryos formed was less in comparison to that formed on 2,4-D supplemented medium. Individual globular embryos formed in liquid medium along with those attached with the leaf tissue for their further development to heart and torpedo stages were cultured in the embryo development medium (liquid MS supplemented with PEG (2.5%) for 2 weeks). The medium had a positive effect on the development of somatic embryos, 75% developing heart and torpedo stages. Absence or low level of PEG (0.5%) in the medium lead to the formation of callus from globular embryos in D. caryophyllus and D. barbatus. Higher levels of PEG (10%) arrested embryo development (Table 2).

5 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Fig. 1. (1) Leaf explants of D. caryophyllus showing globular embryoids. Arrow indicates leaf explant (bar ¼ 250 mm). (2) Various stages of embryogenesis, viz. globular, heart and torpedo shapes (bar ¼ 500 mm). (3) Magnified view of the above stages (bar ¼ 250 mm). (4) Heart and torpedo stage embryos in a section (bar ¼ 125 mm). (5) Rooted plantlet of D. caryophyllus. (6) Acclimatized plants of D. chinensis growing in pot under field conditions.

6 454 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Table 1 Results of induction of embryogenesis from leaf explants of Dianthus spp. cultured in liquid MS medium with various media supplements (culture period: 4 weeks) a Media supplements (mg/l) Mean no. of embryoids/ml S.D. D. caryophyllus D. barbatus D. chinensis 2,4-D ac a a b b b a c c c d a 2,4-D þ BAP 1.0 þ a a a 1.0 þ b b b 1.0 þ b b b 1.0 þ b b b 1.0 þ b b c 2,4-D þ BAP þ CH 1.0 þ 0.2 þ a a a 1.0 þ 0.2 þ b b b 1.0 þ 0.2 þ c c c 1.0 þ 0.2 þ c c d 1.0 þ 0.2 þ d d 0 e a Means followed by same letters are not significantly different at 0.05 level of significance. Various concentrations of CH (100, 200, 500, 750, 1000 mg/l) and BAP (0.5, 1, 2, 5 mg/l) were tried for maturation of embryos, i.e. for formation of cotyledons (Table 3). Presence of BAP was necessary for normal development of cotyledons. In the absence of BAP few somatic embryos became abnormal, having fused cotyledons. The greatest mean number of embryos which developed cotyledons was obtained on MS medium containing CH Table 2 Effect of PEG on maturation, viz. development to heart and torpedo stages of somatic embryos (culture period: 4 weeks) a,b Medium Mean no. of embryoids/ml developed to heart and torpedo stages S.D. D. caryophyllus D. barbatus D. chinensis MS 0a 0a 0a MS þ PEG (0.5%) 0 a 0 a b MS þ PEG (0.75%) b b c MS þ PEG (1%) b b d MS þ PEG (2.5%) c c e MS þ PEG (5%) 0 a d 0 a MS þ PEG (10%) 0 c a 0 c a 0 c a a Means followed by same letters are not significantly different at 0.05 level of significance. b Percent maturation was assessed from a minimum of 20 randomly selected embryos 4 weeks after transfer to embryo maturation medium. c Indicates callusing from somatic embryos.

7 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Table 3 Effect of media supplements on maturation, viz. development of cotyledons of somatic embryos (culture period: 3 weeks) a,b Media supplements (mg/l) Mean number of somatic embryos matured S.D. D. caryophyllus D. chinensis D. barbatus CH þ BAP 200 þ a a a 200 þ a b b 200 þ b c c 200 þ cd d d 500 þ d b 6.4 1b 750 þ c c c 1000 þ c 0 c 0 c CH a a a a a a a c a c a c a c a c a c b c a c ab c CH þ BAP þ ABA 200 þ 0.5 þ a a a 200 þ 0.5 þ a a a 200 þ 0.5 þ ab a c 4.6 2a 200 þ 0.5 þ a a c a 200 þ 0.5 þ ac a a BAP þ ABA 0.5 þ a a a 0.5 þ a a b 1 þ a ab b 2 þ ab 4.6 2bc b BAP þ GA 3 þ ABA 0.5 þ 0.5 þ a a a 0.5 þ 1 þ a b b 0.5 þ 5 þ b c a a Percent maturation was assessed from a minimum of 20 randomly selected embryos 3 weeks after transfer to embryo maturation medium. b Means followed by same letters are not significantly different at 0.05 level of significance. c Indicates callusing of the embryos. (200 mg/l) and BAP (0.5 mg/l) where 75% germination was observed (Fig. 1(2) and (3)). However, there were no clear optima with respect to the concentrations of BAP. Higher levels of CH (500, 750, 1000 mg/l) resulted in hindrance of embryo maturation and caused callusing of embryos. In the absence of BAP cotyledons remained undeveloped. The efficiency of conversion of such structures to plantlets was relatively low. Mature somatic embryos were isolated for further development while the remaining tissue with embryos at early stages of development were re-cultured on fresh maturation medium. Histological observations of thin sections of leaf explants indicated that embryos were formed directly

8 456 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Table 4 Effect of various supplements on germination of somatic embryos of D. caryophyllus Media Response MS Rhizogenesis 1/2 strength MS Rhizogenesis 1/3 strength MS Rhizogenesis MS þ GA 3 (0.5, 1, 2, 3, 5 mg/l) Plantlet with both well-developed shoots and roots formed on MS þ GA 3 (1 mg/l) MS þ phytagel (0.2%) Greening of the somatic embryos MS þ GA 3 (1 mg/l) þ IBA (1 mg/l) Greening of somatic embryos, formation of cotyledonary leaves and roots MS þ IBA (0.5, 1, 2, 5 mg/l) Greening of the embryos MS þ IAA (0.5, 1, 2, 5 mg/l) Rhizogenesis MS þ GA 3 (1 mg/l) þ Kn (0.5 mg/l) þ BAP (0.5 mg/l) Greening of the embryos MS þ Kn (0.5, 1, 2, 3, 5 mg/l) Greening of the embryos MS þ ABA (0.2, 0.4, 0.6, 0.8, 1 mg/l) Rhizogenesis MS þ BAP þ GA 3 þ Kn (0.5, 1, 0.5 mg/l) Greening of the embryos Table 5 Effect of various supplements on germination of somatic embryos of D. chinensis Media MS MS þ IBA (0.5, 1, 2, 5 mg/l) MS þ BAP (2 mg/l) Response Rhizogenesis Greening of the embryos Plantlet with both well-developed shoots and roots formed without a callus phase. They grew on the surface of the explants without any vascular connection with the maternal tissue. Later on they were able to form their own anatomically independent vascular system. Heart and torpedo stage embryos were also seen in the sections (Fig. 1(4)). Randomly picked cotyledonary somatic embryos were cultured on medium supplemented with different growth regulators for germination (Tables 4 6). For the majority of the hormonal combinations tried, rhizogenesis of the embryos was observed. Somatic embryo germination varied greatly. MS þ phytagel (0.2%) led to the greening of the somatic embryos. The inclusion of GA 3 (0.5, 1, 2, 5 mg/l) in the germination medium increased and optimized percent germination. Germination was preceded by greening of the embryos within 2 3 days of transfer to light and accompanied by further elongation and expansion of the cotyledons. This was rapidly followed by the development of leaves and Table 6 Effect of various supplements on germination of somatic embryos of D. barbatus Media Response MS Rhizogenesis 1/2 strength MS Rhizogenesis MS þ 2,4-D (0.5 mg/l) þ BAP (1 mg/l) Plantlet with both well-developed shoots and roots formed

9 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) Table 7 Effect of GA 3 on germination of somatic embryos in D. caryophyllus Germination medium MS þ GA 3 (mg/l) Response Germination (%) a 0.5 Greening, callusing of embryos 10 1 Greening, plantlets with well-developed shoots and roots 75 2 Rhizogenesis 5 5 Rhizogenesis 5 a Assessed from a minimum of 50 randomly selected embryos 4 weeks after transfer to embryo germination medium. roots. Further root and shoot development was achieved when somatic embryo-derived plantlets were transferred to MS basal medium. Plantlets with both well-developed shoots and roots were formed on MS medium supplemented with GA 3 (1 mg/l) in D. caryophyllus. GA 3 treatment (1 mg/l) rapidly induced germination of somatic embryos after 2 weeks. GA 3 (0.5 mg/l) resulted in callusing of the embryos while GA 3 (2, 5 mg/l) caused rooting (Table 7). In D. barbatus embryos germinated in MS liquid medium supplemented with 2,4-D (0.5 mg/lþþbap (1 mg/l). No result was observed at higher levels of BAP (2, 5 mg/l). In D. chinensis embryos germinated on MS solid medium supplemented with BAP (2 mg/l). Germinated embryos were transferred on MS medium for further growth. Germinated small plantlets were transferred for rooting on MS medium supplemented with NAA (1 mg/l) (Fig. 1(5)). Plantlets with both shoots and roots grown to about 8 cm were transferred to pots. Plants were covered with polythene bags initially to acclimatize before being exposed to field conditions (Fig. 1(6)). 4. Discussion Direct somatic embryogenesis obviating callus phase is a way to produce large number of somatic embryos from which clonal plants can be regenerated. This has been achieved in D. barbatus, D. caryophyllus and D. chinensis. In D. caryophyllus the somatic embryogenesis was obtained by (Frey et al., 1992; Nakano and Mii, 1993; Sankhla et al., 1995) through callus phase but Yantcheva et al. (1998) obtained somatic embryos without callus formation in D. caryophyllus by using virus free nodal cuttings and leaves as explants. The results obtained in the present study are nearly akin to those of Yantcheva et al. (1998) in D. caryophyllus. Our study extends the results to D. barbatus and D. chinensis.leaflets of in vitro raised seedlings of D. barbatus, D. caryophyllus and D. chinensis were utilized for somatic embryogenesis. The cultures were maintained for 28 days on a rotary shaker (120 rpm) in the dark in the growth chamber at C. The somatic embryos in all these species were formed from the cut edges of leaf explants. This may be due to increase in embryogenic competence of wounded tissue due to changes at the level of endogenous growth regulators (Ivanova et al., 1994). MS medium supplemented with 2,4-D (1 mg/lþþbap (0.2 mg/l) resulted in induction of somatic embryogenesis in D. caryophyllus (Yantcheva et al., 1998), 2,4-D alone

10 458 A. Pareek, S.L. Kothari / Scientia Horticulturae 98 (2003) could induce somatic embryogenesis without callus preformation in D. barbatus, D. caryophyllus and D. chinensis in the present study. Capability of somatic embryo induction by 2,4-D in the three species used presently may be due to the genotypic difference of D. caryophyllus, but somatic embryo induction in D. barbatus and D. chinensis by 2,4-D alone is significant and indicates the utility of this hormone in somatic embryogenesis in Dianthus species. This is evidenced further as 2,4-D alone induces a high number of somatic embryos, but the number reduced when used in combination with BAP and CH (Table 1). This indicates antagonistic action of BAP and CH in combination with 2,4-D. The most critical stage of somatic embryos is the transition from globular to torpedo stage. Few heart shaped and torpedo shaped embryos were formed in the induction phase, if cultures were kept for a longer time in the induction phase. The role of PEG as an osmoticum and dessicator is important in embryo development. Whereas in D. caryophyllus used by Yantcheva et al. (1998) the highest percentage of embryo maturation occurred in MS medium supplemented with PEG (1%), in all three species investigated in this study. PEG (2.5%) was ideal for maximum embryo maturation. This difference points to the genotypic difference of D. caryophyllus used by Yantcheva et al. (1998) and the present study. Of the various concentrations of CH and BAP used, CH (200 mg/l) and BAP (0.5 mg/l) were the best for subsequent embryo maturation. Whereas lower concentrations of these chemical combinations induced embryo rooting and hindered further embryo development, the higher concentrations resulted in impeded embryo maturation and the arrested embryos produced callus. In D. caryophyllus used by Yantcheva et al. (1998) NAA induced germination of somatic embryos, but in the present material NAA induced rooting. This differential response to NAA could be due to their genotypic differences in the two materials. These differences may be causative in the differential response to GA 3 treatments observed in the two materials. Though the population of somatic embryos increased at each harvest, their germination percentage and plant regeneration decreased in all the three species of Dianthus. Acknowledgements Financial support of CSIR in the form of award of JRF/SRF to AP is gratefully acknowledged. The work was also supported by the DBT funded project. References Ammirato, P.V., Embryogenesis. In: Evans, D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. I, pp Demmink, J.F., Custers, J.B.M., Bergervoet, J.H.W., Gynogenesis to by pass crossing barriers between diploid and tetraploid Dianthus species. Acta Hort. 216, Earle, E.D., Langhans, R.W., Carnation propagation from shoot tips cultured in liquid medium. HortScience 10,

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