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Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine
Scientia Horticulturae 106 (2005) 593–602 www.elsevier.com/locate/scihorti
Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine D.S. Vijaya Chitra, G. Padmaja * Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Received 20 May 2003; received in revised form 22 February 2005; accepted 4 May 2005
Abstract A brief culture of mulberry leaves for 8–10 days on MS medium with 18.17 mM TDZ followed by transfer to 8.88 mM BAP supplemented medium triggered high frequency shoot organogenesis (77.6– 89.2%) and favoured shoot elongation in Morus spp. Shoot proliferation was highest in the presence of 2.22 mM BAP with induction of 9.4–10.6 shoots per culture. High frequency of root induction (76.0– 86.6%) was observed on medium supplemented with 0.49 mM IBAwhereas increase in the level of IBA (4.92 mM) resulted in induction of roots along with developmentof callus from the base of the shoots. The regenerated plants established in soil at higher frequency in rainy season compared towinter and summer. # 2005 Elsevier B.V. All rights reserved. Keywords: Direct shoot organogenesis; Leaf explants; Morus indica L.; Morus alba L.; Thidiazuron; 6benzylaminopurine
1. Introduction Mulberry is of great economic importance in the sericulture industry because its foliage is used as food for silkworms. The dioecious nature of mulberry is a major barrier for genetic improvement by conventional hybridization. Moreover, the perennial and highly Abbreviations: BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962) medium; NAA, a-naphthalene acetic acid; TDZ, thidiazuron (1-phenyl-3- [1,2,3-thiadiazol-5-yl] urea); Kn, kinetin * Corresponding author. Tel.: +91 40 23134586; fax: +91 40 23130120. E-mail address: [email protected] (G. Padmaja). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.05.008
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heterozygous nature of the plant coupled with a prolonged juvenile period limits the speed of improvement using traditional methods (Ravindranan and Lakshmi Sita, 1994). Further, in vegetatively propagated plants like mulberry it takes many years to evolve a desirable clone from an economic and commercial point of view by routine propagation methods. Therefore, application of biotechnological tools for genetic improvement of mulberry attains greater significance, which in turn depends upon the availability of an efficient regeneration system. The in vitro regeneration of plantlets from the adventitious buds formed on the leaf explants derived from aseptically grown shoots (Oka and Ohyama, 1981; Mhatre et al., 1985), cultured embryos (Kim et al., 1985) or axillary buds (Yamanouchi et al., 1999; Vijayan et al., 2000) has been reported in different species of Morus. Most of the regeneration work from leaf explants dealt with temperate varieties, which are nonadaptive to tropical environments, and also there are limited reports available on regeneration from leaf explants of tropical varieties (Vijayan et al., 2000). Kapur et al. (2001) obtained high percentage of regeneration from leaf explants of mulberry on MS medium supplemented with 11.41 mM IAA, 4.99 mM TDZ and 11.77 mM AgNO3. In the present study, an efficient protocol for inducing direct shoot organogenesis from leaf explants and subsequent recovery of plants of economically important cultivars of mulberry is presented.
2. Materials and methods Shoot cultures were established by culturing nodal explants obtained from 3-year-old plants of Morus indica L., cultivars M-5, S-36 and S-13, and Morus alba L., cultivar China White. The excised nodal explants with axillary buds were surface sterilized in 70% alcohol for 1 min followed by 0.1% mercuric chloride (HgCl2) for 15 min. They were rinsed four to five times in sterile distilled water with 5 min duration each. The sterilized explants of M-5, S-36, S-13 and China White cultivars were cultured on MS (Murashige and Skoog, 1962) medium with 3% sucrose and 0.8% agar. The culture media were supplemented with 1.36 mM 2,4-D for inducing axillary bud sprouting in M-5, S-36 and S13 cultivars whereas those of China White cultivar were cultured on the same basal medium with a supplement of 9.29 mM Kn (Chitra and Padmaja, 2002). Shoot multiplication was achieved by culture of shoot tips on MS medium with 2.22 mM BAP. Leaves without petiole derived from in vitro multiplied shoots were used for induction of shoot organogenesis. Two leaf explants were cultured in sterile culture tubes (25 mm 150 mm) containing 15 ml of MS basal medium with 3% sucrose, 0.8% agar (Agar Powder, Extra pure, Hi-Media, Mumbai, India). The effect of BAP and TDZ on induction of direct organogenesis from leaf explants was tested by supplementation into MS medium in varied concentrations. In another experiment, the leaf explants were precultured on MS medium containing TDZ (9.08, 13.62, 18.17 and 22.7 mM) for different durations and subsequently transferred to the medium supplemented with 8.88 mM BAP for induction of direct organogenesis. Leaves of different sizes were cultured either with the abaxial or adaxial side in contact with the medium. Each treatment was repeated thrice at different times having at least 20 explants per treatment. The appearance of shoot buds
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from leaves was taken into consideration for calculating the shoot organogenesis from leaves. 2.1. Multiplication of shoots Shoots of 2–3 cm derived from leaves were cultured on MS medium supplemented individually with either BAP (2.22 and 4.44 mM) or TDZ (2.27 and 4.54 mM) for induction of multiple shoots. The appearance of shoots from the base of the explant and also from the axils of leaves was taken into consideration for calculating the multiplication frequencies. The average number of shoots induced per explant was recorded after 30 days of culture. 2.2. Root induction from shoots Healthy shoots derived from leaves or multiple shoots were transferred to MS medium supplemented individually with IAA (0.57 and 5.7 mM), IBA (0.49 and 4.9 mM), NAA (0.54 and 5.4 mM) or 2,4-D (0.45 mM and 4.5 mM) for root induction. The observations on the duration for root induction, nature of roots induced and the frequency of root induction were recorded. For all the experiments on shoot induction, shoot multiplication and root induction from shoots, the cultures were kept at 25 2 8C under a 16-h photoperiod with a photosynthetic photon flux density (PPFD) of 83.6 mE m2 s1 provided by white fluorescent tubes. 2.3. Acclimatization of regenerated plants Regenerated plants having well developed roots were removed from culture bottles and washed free of agar. They were transferred to plastic pots containing soil and organic manure (3:1) and kept in a net house under shade for 15–20 days. In the first week of transfer, the plantlets were covered with polythene covers to maintain humidity. After 15– 20 days of acclimatization, the plantlets were transferred to earthen pots and planted out in field. The percentage survival was recorded after 6 weeks of transfer to field.
3. Results 3.1. Induction of direct shoot organogenesis from leaves Plant regeneration via direct shoot organogenesis has been achieved from the cultured leaf explants of all four cultivars. The size and the orientation of leaves remarkably influenced the response for direct organogenesis. Leaves of an intermediate size (1–4 cm) responded efficiently for shoot organogenesis whereas newly developed leaves (smaller than 1 cm) and very old leaves (greater than 4 cm) did not exhibit any response. Shoot bud induction was observed from leaves cultured with the abaxial surface in contact with the medium. Direct shoot organogenesis was achieved with a frequency of 30.1–46.7% and 13.6– 32.5% from leaves of different cultivars cultured on medium with 8.88 mM and 13.31 mM BAP, respectively (Table 1). The number of shoots induced per explant (5.3–6.4) and the
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Concentration of cytokinin (mM)
M-5 cultivar Shoot regeneration (%)
No. of shoots/ explant
8.88 BAP 13.31 BAP 17.75 BAP 22.19 BAP 9.08 TDZ 13.62 TDZ 18.17 TDZ 22.70 TDZ
46.7 2.2 32.5 3.7 18.5 1.0 8.7 1.9 12.7 3.3 27.5 3.0 84.1 2.1 64.8 1.0
6.4 0.4 2.6 0.2 1.3 0.1 0.4 0.1 0.5 0.0 0.7 0.0 2.8 0.3 2.2 0.1
c d e f ef d a b
S-36 cultivar
a b c d d d b b
S-13 cultivar
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.6 0.1a 0.8 0.0 c 0.6 0.0.d 0.5 0.0 d 0.5 0.0 d 0.5 0.0 d 1.2 0.2 b 0.7 0.1 c
44.4 4.4 28.4 3.0 8.3 1.4 6.1 0.8 20.4 1.6 32.2 2.9 87.1 1.6 65.9 1.4
5.9 0.2 3.1 0.3 1.4 0.1 0.6 0.0 0.5 0.0 0.5 0.0 2.7 0.1 2.1 0.1
c d f f e d a b
a b d e e e c d
China White cultivar
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.7 0.1 0.8 0.1 0.5 0.0 0.5 0.0 0.4 0.0 0.4 0.0 1.5 0.2 1.2 0.1
43.7 1.9 22.7 1.6 14.3 2.8 11.0 1.2 11.3 1.0 20.4 1.9 80.9 3.9 48.9 1.6
5.7 0.3 2.2 0.1 1.6 0.1 0.4 0.1 0.4 0.0 0.5 0.1 2.7 0.1 1.5 0.1
a c c c c c a b
b c d d d c a b
a c d e e e b d
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.8 0.1 a 0.8 0.1bc 0.5 0.0 d 0.5 0.0 d 0.4 0.0 d 0.4 0.0 d 0.9 0.1 b 0.6 0.0 cd
30.1 1.5 13.6 1.8 0.0 0.0 7.8 1.1 16.2 2.6 79.0 0.9 45.6 2.9
5.3 0.2 1.6 0.1 0.0 0.0 0.4 0.0 0.5 0.1 1.8 0.2 1.0 0.1
c de
e d a b
Average length of shoots (cm) a bc
d d b c
0.9 0.1 0.5 0.1 0.0 0.0 0.4 0.0 0.5 0.0 0.5 0.1 0.5 0.0
a b
b b b b
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Table 1 Effect of BAP and TDZ on direct shoot organogenesis from leaves of mulberry
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average length of shoots (0.9–1.8 cm) were higher in the presence of 8.88 mM BAP in different cultivars. The frequency of shoot organogenesis, the number of shoots as well as the average length of shoots induced per explant decreased with the increase in the concentration of BAP (17.75 and 22.19 mM) in M-5, S-36 and S-13 cultivars whereas in China White cultivar, no shoot regeneration was observed. Of the different concentrations of TDZ tested, high frequency of shoot organogenesis (79.0–87.1%) was observed in the presence of 18.17 mM TDZ from leaves of different cultivars (Table 1). A large number of shoot buds proliferated from the basal portion of the leaves cultured on medium containing 18.17 and 22.7 mM TDZ. However, only few buds developed into shoots of 0.5–1.5 cm at the end of 30 days and growth of the rest of the adventitious shoot buds remained arrested. Thus further experiments were carried out by culture of leaf explants for different durations on TDZ followed by transfer to BAP supplemented medium. Efficient shoot development was observed from leaves previously cultured for 8–10 days on TDZ supplemented medium and then transferred to medium with 8.88 mM BAP, in comparison to those cultured for 30 days on TDZ supplemented medium. The concentration of TDZ used in the induction medium influenced the regeneration capacity of leaf explants (Table 2). The frequency of shoot regeneration from leaf explants of different cultivars was low (6.8–33.4%) on media containing 9.08 and 13.62 mM TDZ. The best results were obtained when leaf explants were cultured on 18.17 mM TDZ and subsequently transferred to 8.88 mM BAP supplemented medium that induced high frequency shoot regeneration (77.6–89.2%) and facilitated rapid shoot development with induction of 16.8–18.1 shoots per explant in different cultivars. A further increase in the concentration of TDZ (22.7 mM) in the induction medium decreased the shoot regeneration (43.9–67.7%) as well as the number of shoots induced per explant that ranged from 10.3 to 11.9 in different cultivars. 3.2. Multiplication of the shoots Multiple shoots were induced from shoot tips differentiated from leaves at a high frequency of 78.7–93.3% on MS medium with 2.22 mM BAP (Table 3). The emergence of multiple shoots occurred in 9–10 days and the average length of shoots was 2.4–3.6 cm after 30 days of culture. Thidiazuron at 2.27 mM supported induction of 1.5–1.7 shoots, with a frequency of 72.0–76.0% and shoot length of 1.3–1.6 cm in different cultivars. The shoots produced on thidiazuron containing medium were fasciated and exhibited stunted growth with development of dark green leaves. The shoot multiplication rate decreased with the increase in the concentration of BAP (4.44 mM) and TDZ (4.54 mM). 3.3. Root induction from the shoots The frequency as well as the nature of roots induced from leaf regenerated shoots varied with the type and concentration of auxin used. Efficient root induction from shoots was observed on medium containing low levels of IBA (0.49 mM), 2,4-D (0.45 mM), IAA (0.57 mM) and NAA (0.54 mM) compared to higher concentrations tested. Healthy, thick roots were induced in 12–14 days with a frequency of 86.6, 85.2, 80.2, 76.0% in M-5, S-36,
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Concentration M-5 cultivar of cytokinins Shoot No. of (mM) regeneration shoots/ (%) explant
S-36 cultivar Average Shoot length of regeneration shoots (cm) (%)
S-13 cultivar No. of shoots/ explant
Average Shoot length of regeneration shoots (cm) (%)
China White cultivar No. of shoots/ explant
Average Shoot length of regeneration shoots (cm) (%)
No. of shoots/ explant
Average length of shoots (cm)
4.8 0.3 d
1.0 0.2 b
9.08 TDZ # 8.88 BAP
13.3 3.4 d 6.5 0.3 d
1.3 0.1 c
19.3 1.9 d 6.1 0.2 d
1.1 0.2 c
11.4 0.8 d 5.7 0.4 d
1.0 0.1 c
6.8 3.5 d
13.62 TDZ # 8.88 BAP
27.4 4.7 c
8.6 0.2 c
2.4 0.3 b
33.4 3.4 c
8.6 0.4 c
2.4 0.3 b
21.0 2.8 c
8.5 0.4 c
1.7 0.2 b
17.3 2.8 c
7.6 0.2 c
0.9 0.0 b
18.17 TDZ # 8.88 BAP
84.9 3.7 a
17.5 0.4 a
3.7 0.2 a
89.2 3.7 a
18.1 0.2 a
3.7 0.1a
80.6 5.2 a
17.0 0.3 a
3.2 0.2 a
77.6 3.5 a
16.8 0.4 a
2.4 0.1 a
22.70 TDZ # 8.88 BAP
64.7 3.4 b 11.9 0.3 b 2.8 0.2 b
67.7 3.4 b 11.4 0.3 b 2.0 0.2 b
48.1 3.2 b 10.3 0.4 b 1.6 0.1 b
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
43.9 3.7 b 10.4 0.6 b 1.2 0.1 b
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Table 2 Direct shoot organogenesis from leaves of different cultivars cultured for 8–10 days on medium with TDZ followed by transfer to 8.88 mM BAP
Concentration M-5 cultivar of cytokinin Shoot No. of (mM) induction shoots (%) 2.22 4.44 2.27 4.54
BAP BAP TDZ TDZ
93.3 1.9 82.6 1.7 76.0 1.3 61.1 1.1
a 10.3 0.4 a b 5.2 0.2 b c 1.6 0.1 c d 1.6 0.1 c
S-36 cultivar Average Shoot length of induction shoots (cm) (%) 3.6 0.1 2.1 0.2 1.6 0.1 0.6 0.1
a b c d
82.9 1.6 74.5 1.2 73.6 3.0 61.9 1.6
S-13 cultivar No. of shoots
a 10.6 0.2 a b 5.0 0.3 b b 1.7 0.1 c d 1.5 0.0 c
Average Shoot length of induction shoots (cm) (%) 3.4 0.1 2.0 0.2 1.6 0.1 0.5 0.1
a b c d
78.7 1.8 75.0 1.9 72.1 0.8 57.8 1.2
China White cultivar No. of shoots
a 10.5 0.4 a ab 4.1 0.3 b b 1.5 0.1 c c 1.3 0.1 c
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
Average Shoot length of induction shoots (cm) (%) 3.0 0.1 2.0 0.2 1.5 0.1 0.5 0.1
a b b c
81.1 1.8 70.5 1.1 72.0 1.7 47.9 0.7
No. of shoots a b b c
9.4 0.3 2.4 0.1 1.5 0.1 1.2 0.1
Average length of shoots (cm) a b c c
2.4 0.1 1.7 0.1 1.3 0.1 0.5 0.1
a b c d
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Table 3 Effect of BAP and TDZ on multiple shoot induction from shoots regenerated from leaves
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600
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S-13 and China White cultivars, respectively on medium containing 0.49 mM IBA that enabled better survival upon transfer to soil. High levels of IBA (4.92 mM) decreased the frequency of root induction (43.8–64.3%) along with callus development from the base of the cultures in all the cultivars. Incorporation of 0.45 mM 2,4-D triggered root induction with a frequency of 72.6, 78.7, 68.0, 60.3% in M-5, S-36, S-13 and China White cultivars, respectively. Slender, thin roots were induced from shoots cultured on medium supplemented individually with IAA (0.57 mM) or NAA (0.54 mM) at a frequency of 41.7–67.1 and 13.5–33.6%, respectively in different cultivars. 3.4. Establishment of micropropagated plants in field The regenerated plants of different cultivars survived with a high frequency of 70.7– 80.3% in rainy season than in winter (66.4–75.4%) and summer seasons (56.5–67.1%). Lowest value for plant establishment was observed in the summer season in the China White cv. (56.5 1.7%).
4. Discussion The size and the orientation of the leaves were determining factors affecting induction of direct shoot organogenesis from leaves of mulberry. The best response of shoot organogenesis was observed from leaves of 1–4 cm that were cultured with the abaxial side in contact with the medium. Yamanouchi et al. (1999) reported stable regeneration of plantlets from immature leaves isolated from winter buds of field-grown mulberry. It is inferred from these studies that the size of the leaf plays an important role in its endogenous potential to regenerate adventitious buds. Our results also confirm observations by Mhatre et al. (1985) who concluded that the upright position of leaf explants is favourable for maximum induction of shoot buds in Morus indica L. Kumar et al. (2003) observed enhanced shoot bud development from cotyledonary node explants of mungbean (Vigna radiata L.) after 8 days exposure of explants to 2 mM TDZ. They further reported that continuous exposure of explants to TDZ resulted in the complete loss of regeneration ability and high callus growth of explants. Bhatnagar et al. (2001) achieved 50% regeneration from hypocotyl and 70% from cotyledon explants in Morus indica when cultured on 5 mM TDZ for 10 days. Thereafter, subculture on TDZ free medium lead to the formation of multiple shoots. Our results confirm these observations since a twostage culture procedure consisting of culture of leaves on 18.17 mM TDZ for 8–10 days followed by a secondary medium consisting of 8.88 mM BAP was found to be beneficial for promoting shoot elongation. Debnath (2005) reported that moderate concentrations (1– 5 mM) of TDZ supported bud and shoot regeneration from in vitro derived Lingonberry leaves, but strongly inhibited shoot elongation. TDZ initiated cultures produced usable shoots after one additional subculture on medium containing 1–2 mM zeatin. These reports emphasize the importance of double stage treatment particularly when TDZ is employed for improving the shoot morphogenesis and thus can be attempted in other plant species. The type of cytokinin used in the medium had a marked effect on the rates of shoot proliferation with better results obtained with BAP in relation to TDZ. Furthermore, in
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presence of the TDZ shoots were fused and appeared fasciated with development of thick dark green leaves. The formation of stunted or the fasciation of the shoots on TDZ supplemented medium has been reported in Rhododendron (Preece and Imel, 1991). Variation in the activity of different cytokinins can be explained by their differential uptake rate reported in different genomes (Blakesey, 1991), varied translocation rates to meristematic regions and metabolic processes, in which the cytokinin may be degraded or conjugated with sugars or amino acids to form biologically inert compounds as reported by Tran Thanh Van and Trinh (1990), and Kaminek (1992). The stimulatory effect of auxins in the root formation from the shoots depends partly on the type of auxin employed in Morus alba L. (Anuradha and Pullaiah, 1992). Kim et al. (1985) observed substantial amount of callus formation at the base of the leaf regenerated shoots on 4.92–24.6 mM IBA supplemented medium whereas in the presence of 0.49 mM IBA, few but healthier and more vigorously growing roots were formed directly at the base of the shoots. The presence of callus between the root and shoot resulted in poor vasculature connection, which made field survival of the plantlets difficult. Hossain et al. (1992) observed root formation from the shoots excised from the nodal explants on 4.92– 5.37 mM IBA or NAA in Morus laevigata Wall. Bhau and Wakhlu (2001) reported that IBA was the most effective auxin (2.46 mM) for root induction from shoots regenerated from the callus of Morus alba L. whereas higher levels of auxins encouraged callus formation from the cut ends of the explants. Our results confirm these observations since IBA at 0.49 mM was found to be the best for root induction for the leaf regenerated shoots of all four cultivars compared to 2,4-D, IAA or NAA. In the present study, a two-step approach involving short incubation of leaves for 8–10 days on TDZ supplemented medium followed by transfer to medium with BAP for promoting shoot development from leaves of four elite cultivars has been developed. Shoot proliferation rates were high on medium containing 2.22 mM BAP. Efficient rooting from the shoots of all four cultivars was observed on medium containing 0.49 mM IBA.
Acknowledgements We gratefully acknowledge University Grants Commission, New Delhi for providing financial assistance under unassigned grant scheme to carry out this work. DSVC is thankful to University Grants Commission, New Delhi for the award of a Junior Research Fellowship and Senior Research Fellowship. We also acknowledge the Department of Science and Technology, New Delhi for rendering assistance to the Department in the form of fund for Infrastructure in Science and Technology and the facilities established under this programme have been utilized for the research work.
References Anuradha, M., Pullaiah, T., 1992. Micropropagation of mulberry (Morus alba L.). Annali Di Botanica 15, 35–41. Bhatnagar, S., Kapur, A., Khurana, P., 2001. TDZ-mediated differentiation in commercially valuable Indian mulberry, Morus indica cultivars K2 and DD. Plant Biotech. 18, 61–65.
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Bhau, B.S., Wakhlu, A.K., 2001. Effect of genotype, explant type and growth regulators on organogenesis in Morus alba. Plant Cell Tiss. Org. Cult. 66, 25–29. Blakesey, D., 1991. Uptake and metabolism of 6-benzyladenine in shoot proliferation of Musa and Rhododendron. Plant Cell Tiss. Org. Cult. 25, 69–74. Chitra, D.S.V., Padmaja, G., 2002. Seasonal influence on axillary bud sprouting and micropropagation of elite cultivars of mulberry. Sci. Hort. 92, 55–68. Debnath, S.C., 2005. A two-step procedure for adventitious shoot regeneration from in vitro derived Lingonberry leaves: shoot induction with TDZ and shoot elongation using zeatin. HortScience 40, 189–192. Hossain, M., Rahman, S.M., Zaman, A., Joader, O.I., Islam, R., 1992. Micropropagation of Morus laevigata Wall from mature trees. Plant Cell Rep. 11, 522–524. Kaminek, M., 1992. Progress in cytokinin research. TIBTECH 10, 159–162. Kapur, A., Bhatnagar, S., Khurana, P., 2001. Efficient regeneration from mature leaf explants of Indian mulberry via organogenesis. Sericologia 41, 1–8. Kim, H.R., Patel, K.R., Thorpe, T.A., 1985. Regeneration of mulberry plantlets through tissue culture. Bot. Gaz. 146, 335–346. Kumar, P., Srivastava, G.C., Panwar, J.D.S., Chandra, R., Raghuveer, P., 2003. Efficiency of thidiazuron on in vitro shoot regeneration from cotyledonary node explant in mungbean. Indian J. Plant Physiol. 8, 398–401. Mhatre, M., Bapat, V.A., Rao, P.S., 1985. Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.). Plant Cell Rep. 4, 78–80. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Oka, S., Ohyama, K., 1981. In vitro initiation of adventitious buds and its modification by high concentration of benzyl adenine in leaf tissues of mulberry (Morus alba L.). Can. J. Bot. 59, 68–74. Preece, J.E., Imel, M.R., 1991. Plant regeneration from leaf explants of Rhododendron P. J. H. Hybrids. Sci. Hort. 48, 159–170. Ravindranan, R., Lakshmi Sita, G., 1994. Micropropagation of difficult-to-root elite cultivars and induction of embryogenesis in mulberry (Morus indica L.). In: Tandon, P. (Ed.), Advances in Plant Tissue Culture in India. Proceedings of the XIII Plant Tissue Culture Conference, Pragati Prakashan, India, pp. 155–169. Tran Thanh Van, K., Trinh, T.H., 1990. Organogenic differentiation. In: Bhojwani, S.S. (Ed.), Plant Tissue Culture. Application and Limitations. Elsevier, Amsterdam, pp. 34–53. Vijayan, K., Chakraborti, S.P., Roy, B.N., 2000. Plant regeneration from leaf explants of mulberry: influence of sugar, genotype and 6-benzyladenine. Ind. J. Exp. Biol. 38, 504–508. Yamanouchi, H., Koyama, A., Machii, H., 1999. Effect of medium conditions on adventitious bud formation in immature mulberry leaves. JARQ 33, 267–274.
Shoot regeneration via direct organogenesis from in vitro derived leaves of mulberry using thidiazuron and 6-benzylaminopurine D.S. Vijaya Chitra, G. Padmaja * Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India Received 20 May 2003; received in revised form 22 February 2005; accepted 4 May 2005
Abstract A brief culture of mulberry leaves for 8–10 days on MS medium with 18.17 mM TDZ followed by transfer to 8.88 mM BAP supplemented medium triggered high frequency shoot organogenesis (77.6– 89.2%) and favoured shoot elongation in Morus spp. Shoot proliferation was highest in the presence of 2.22 mM BAP with induction of 9.4–10.6 shoots per culture. High frequency of root induction (76.0– 86.6%) was observed on medium supplemented with 0.49 mM IBAwhereas increase in the level of IBA (4.92 mM) resulted in induction of roots along with developmentof callus from the base of the shoots. The regenerated plants established in soil at higher frequency in rainy season compared towinter and summer. # 2005 Elsevier B.V. All rights reserved. Keywords: Direct shoot organogenesis; Leaf explants; Morus indica L.; Morus alba L.; Thidiazuron; 6benzylaminopurine
1. Introduction Mulberry is of great economic importance in the sericulture industry because its foliage is used as food for silkworms. The dioecious nature of mulberry is a major barrier for genetic improvement by conventional hybridization. Moreover, the perennial and highly Abbreviations: BAP, 6-benzylaminopurine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog (1962) medium; NAA, a-naphthalene acetic acid; TDZ, thidiazuron (1-phenyl-3- [1,2,3-thiadiazol-5-yl] urea); Kn, kinetin * Corresponding author. Tel.: +91 40 23134586; fax: +91 40 23130120. E-mail address: [email protected] (G. Padmaja). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.05.008
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heterozygous nature of the plant coupled with a prolonged juvenile period limits the speed of improvement using traditional methods (Ravindranan and Lakshmi Sita, 1994). Further, in vegetatively propagated plants like mulberry it takes many years to evolve a desirable clone from an economic and commercial point of view by routine propagation methods. Therefore, application of biotechnological tools for genetic improvement of mulberry attains greater significance, which in turn depends upon the availability of an efficient regeneration system. The in vitro regeneration of plantlets from the adventitious buds formed on the leaf explants derived from aseptically grown shoots (Oka and Ohyama, 1981; Mhatre et al., 1985), cultured embryos (Kim et al., 1985) or axillary buds (Yamanouchi et al., 1999; Vijayan et al., 2000) has been reported in different species of Morus. Most of the regeneration work from leaf explants dealt with temperate varieties, which are nonadaptive to tropical environments, and also there are limited reports available on regeneration from leaf explants of tropical varieties (Vijayan et al., 2000). Kapur et al. (2001) obtained high percentage of regeneration from leaf explants of mulberry on MS medium supplemented with 11.41 mM IAA, 4.99 mM TDZ and 11.77 mM AgNO3. In the present study, an efficient protocol for inducing direct shoot organogenesis from leaf explants and subsequent recovery of plants of economically important cultivars of mulberry is presented.
2. Materials and methods Shoot cultures were established by culturing nodal explants obtained from 3-year-old plants of Morus indica L., cultivars M-5, S-36 and S-13, and Morus alba L., cultivar China White. The excised nodal explants with axillary buds were surface sterilized in 70% alcohol for 1 min followed by 0.1% mercuric chloride (HgCl2) for 15 min. They were rinsed four to five times in sterile distilled water with 5 min duration each. The sterilized explants of M-5, S-36, S-13 and China White cultivars were cultured on MS (Murashige and Skoog, 1962) medium with 3% sucrose and 0.8% agar. The culture media were supplemented with 1.36 mM 2,4-D for inducing axillary bud sprouting in M-5, S-36 and S13 cultivars whereas those of China White cultivar were cultured on the same basal medium with a supplement of 9.29 mM Kn (Chitra and Padmaja, 2002). Shoot multiplication was achieved by culture of shoot tips on MS medium with 2.22 mM BAP. Leaves without petiole derived from in vitro multiplied shoots were used for induction of shoot organogenesis. Two leaf explants were cultured in sterile culture tubes (25 mm 150 mm) containing 15 ml of MS basal medium with 3% sucrose, 0.8% agar (Agar Powder, Extra pure, Hi-Media, Mumbai, India). The effect of BAP and TDZ on induction of direct organogenesis from leaf explants was tested by supplementation into MS medium in varied concentrations. In another experiment, the leaf explants were precultured on MS medium containing TDZ (9.08, 13.62, 18.17 and 22.7 mM) for different durations and subsequently transferred to the medium supplemented with 8.88 mM BAP for induction of direct organogenesis. Leaves of different sizes were cultured either with the abaxial or adaxial side in contact with the medium. Each treatment was repeated thrice at different times having at least 20 explants per treatment. The appearance of shoot buds
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from leaves was taken into consideration for calculating the shoot organogenesis from leaves. 2.1. Multiplication of shoots Shoots of 2–3 cm derived from leaves were cultured on MS medium supplemented individually with either BAP (2.22 and 4.44 mM) or TDZ (2.27 and 4.54 mM) for induction of multiple shoots. The appearance of shoots from the base of the explant and also from the axils of leaves was taken into consideration for calculating the multiplication frequencies. The average number of shoots induced per explant was recorded after 30 days of culture. 2.2. Root induction from shoots Healthy shoots derived from leaves or multiple shoots were transferred to MS medium supplemented individually with IAA (0.57 and 5.7 mM), IBA (0.49 and 4.9 mM), NAA (0.54 and 5.4 mM) or 2,4-D (0.45 mM and 4.5 mM) for root induction. The observations on the duration for root induction, nature of roots induced and the frequency of root induction were recorded. For all the experiments on shoot induction, shoot multiplication and root induction from shoots, the cultures were kept at 25 2 8C under a 16-h photoperiod with a photosynthetic photon flux density (PPFD) of 83.6 mE m2 s1 provided by white fluorescent tubes. 2.3. Acclimatization of regenerated plants Regenerated plants having well developed roots were removed from culture bottles and washed free of agar. They were transferred to plastic pots containing soil and organic manure (3:1) and kept in a net house under shade for 15–20 days. In the first week of transfer, the plantlets were covered with polythene covers to maintain humidity. After 15– 20 days of acclimatization, the plantlets were transferred to earthen pots and planted out in field. The percentage survival was recorded after 6 weeks of transfer to field.
3. Results 3.1. Induction of direct shoot organogenesis from leaves Plant regeneration via direct shoot organogenesis has been achieved from the cultured leaf explants of all four cultivars. The size and the orientation of leaves remarkably influenced the response for direct organogenesis. Leaves of an intermediate size (1–4 cm) responded efficiently for shoot organogenesis whereas newly developed leaves (smaller than 1 cm) and very old leaves (greater than 4 cm) did not exhibit any response. Shoot bud induction was observed from leaves cultured with the abaxial surface in contact with the medium. Direct shoot organogenesis was achieved with a frequency of 30.1–46.7% and 13.6– 32.5% from leaves of different cultivars cultured on medium with 8.88 mM and 13.31 mM BAP, respectively (Table 1). The number of shoots induced per explant (5.3–6.4) and the
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Concentration of cytokinin (mM)
M-5 cultivar Shoot regeneration (%)
No. of shoots/ explant
8.88 BAP 13.31 BAP 17.75 BAP 22.19 BAP 9.08 TDZ 13.62 TDZ 18.17 TDZ 22.70 TDZ
46.7 2.2 32.5 3.7 18.5 1.0 8.7 1.9 12.7 3.3 27.5 3.0 84.1 2.1 64.8 1.0
6.4 0.4 2.6 0.2 1.3 0.1 0.4 0.1 0.5 0.0 0.7 0.0 2.8 0.3 2.2 0.1
c d e f ef d a b
S-36 cultivar
a b c d d d b b
S-13 cultivar
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.6 0.1a 0.8 0.0 c 0.6 0.0.d 0.5 0.0 d 0.5 0.0 d 0.5 0.0 d 1.2 0.2 b 0.7 0.1 c
44.4 4.4 28.4 3.0 8.3 1.4 6.1 0.8 20.4 1.6 32.2 2.9 87.1 1.6 65.9 1.4
5.9 0.2 3.1 0.3 1.4 0.1 0.6 0.0 0.5 0.0 0.5 0.0 2.7 0.1 2.1 0.1
c d f f e d a b
a b d e e e c d
China White cultivar
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.7 0.1 0.8 0.1 0.5 0.0 0.5 0.0 0.4 0.0 0.4 0.0 1.5 0.2 1.2 0.1
43.7 1.9 22.7 1.6 14.3 2.8 11.0 1.2 11.3 1.0 20.4 1.9 80.9 3.9 48.9 1.6
5.7 0.3 2.2 0.1 1.6 0.1 0.4 0.1 0.4 0.0 0.5 0.1 2.7 0.1 1.5 0.1
a c c c c c a b
b c d d d c a b
a c d e e e b d
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
Average length of shoots (cm)
Shoot regeneration (%)
No. of shoots/ explant
1.8 0.1 a 0.8 0.1bc 0.5 0.0 d 0.5 0.0 d 0.4 0.0 d 0.4 0.0 d 0.9 0.1 b 0.6 0.0 cd
30.1 1.5 13.6 1.8 0.0 0.0 7.8 1.1 16.2 2.6 79.0 0.9 45.6 2.9
5.3 0.2 1.6 0.1 0.0 0.0 0.4 0.0 0.5 0.1 1.8 0.2 1.0 0.1
c de
e d a b
Average length of shoots (cm) a bc
d d b c
0.9 0.1 0.5 0.1 0.0 0.0 0.4 0.0 0.5 0.0 0.5 0.1 0.5 0.0
a b
b b b b
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Table 1 Effect of BAP and TDZ on direct shoot organogenesis from leaves of mulberry
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average length of shoots (0.9–1.8 cm) were higher in the presence of 8.88 mM BAP in different cultivars. The frequency of shoot organogenesis, the number of shoots as well as the average length of shoots induced per explant decreased with the increase in the concentration of BAP (17.75 and 22.19 mM) in M-5, S-36 and S-13 cultivars whereas in China White cultivar, no shoot regeneration was observed. Of the different concentrations of TDZ tested, high frequency of shoot organogenesis (79.0–87.1%) was observed in the presence of 18.17 mM TDZ from leaves of different cultivars (Table 1). A large number of shoot buds proliferated from the basal portion of the leaves cultured on medium containing 18.17 and 22.7 mM TDZ. However, only few buds developed into shoots of 0.5–1.5 cm at the end of 30 days and growth of the rest of the adventitious shoot buds remained arrested. Thus further experiments were carried out by culture of leaf explants for different durations on TDZ followed by transfer to BAP supplemented medium. Efficient shoot development was observed from leaves previously cultured for 8–10 days on TDZ supplemented medium and then transferred to medium with 8.88 mM BAP, in comparison to those cultured for 30 days on TDZ supplemented medium. The concentration of TDZ used in the induction medium influenced the regeneration capacity of leaf explants (Table 2). The frequency of shoot regeneration from leaf explants of different cultivars was low (6.8–33.4%) on media containing 9.08 and 13.62 mM TDZ. The best results were obtained when leaf explants were cultured on 18.17 mM TDZ and subsequently transferred to 8.88 mM BAP supplemented medium that induced high frequency shoot regeneration (77.6–89.2%) and facilitated rapid shoot development with induction of 16.8–18.1 shoots per explant in different cultivars. A further increase in the concentration of TDZ (22.7 mM) in the induction medium decreased the shoot regeneration (43.9–67.7%) as well as the number of shoots induced per explant that ranged from 10.3 to 11.9 in different cultivars. 3.2. Multiplication of the shoots Multiple shoots were induced from shoot tips differentiated from leaves at a high frequency of 78.7–93.3% on MS medium with 2.22 mM BAP (Table 3). The emergence of multiple shoots occurred in 9–10 days and the average length of shoots was 2.4–3.6 cm after 30 days of culture. Thidiazuron at 2.27 mM supported induction of 1.5–1.7 shoots, with a frequency of 72.0–76.0% and shoot length of 1.3–1.6 cm in different cultivars. The shoots produced on thidiazuron containing medium were fasciated and exhibited stunted growth with development of dark green leaves. The shoot multiplication rate decreased with the increase in the concentration of BAP (4.44 mM) and TDZ (4.54 mM). 3.3. Root induction from the shoots The frequency as well as the nature of roots induced from leaf regenerated shoots varied with the type and concentration of auxin used. Efficient root induction from shoots was observed on medium containing low levels of IBA (0.49 mM), 2,4-D (0.45 mM), IAA (0.57 mM) and NAA (0.54 mM) compared to higher concentrations tested. Healthy, thick roots were induced in 12–14 days with a frequency of 86.6, 85.2, 80.2, 76.0% in M-5, S-36,
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Concentration M-5 cultivar of cytokinins Shoot No. of (mM) regeneration shoots/ (%) explant
S-36 cultivar Average Shoot length of regeneration shoots (cm) (%)
S-13 cultivar No. of shoots/ explant
Average Shoot length of regeneration shoots (cm) (%)
China White cultivar No. of shoots/ explant
Average Shoot length of regeneration shoots (cm) (%)
No. of shoots/ explant
Average length of shoots (cm)
4.8 0.3 d
1.0 0.2 b
9.08 TDZ # 8.88 BAP
13.3 3.4 d 6.5 0.3 d
1.3 0.1 c
19.3 1.9 d 6.1 0.2 d
1.1 0.2 c
11.4 0.8 d 5.7 0.4 d
1.0 0.1 c
6.8 3.5 d
13.62 TDZ # 8.88 BAP
27.4 4.7 c
8.6 0.2 c
2.4 0.3 b
33.4 3.4 c
8.6 0.4 c
2.4 0.3 b
21.0 2.8 c
8.5 0.4 c
1.7 0.2 b
17.3 2.8 c
7.6 0.2 c
0.9 0.0 b
18.17 TDZ # 8.88 BAP
84.9 3.7 a
17.5 0.4 a
3.7 0.2 a
89.2 3.7 a
18.1 0.2 a
3.7 0.1a
80.6 5.2 a
17.0 0.3 a
3.2 0.2 a
77.6 3.5 a
16.8 0.4 a
2.4 0.1 a
22.70 TDZ # 8.88 BAP
64.7 3.4 b 11.9 0.3 b 2.8 0.2 b
67.7 3.4 b 11.4 0.3 b 2.0 0.2 b
48.1 3.2 b 10.3 0.4 b 1.6 0.1 b
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
43.9 3.7 b 10.4 0.6 b 1.2 0.1 b
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Table 2 Direct shoot organogenesis from leaves of different cultivars cultured for 8–10 days on medium with TDZ followed by transfer to 8.88 mM BAP
Concentration M-5 cultivar of cytokinin Shoot No. of (mM) induction shoots (%) 2.22 4.44 2.27 4.54
BAP BAP TDZ TDZ
93.3 1.9 82.6 1.7 76.0 1.3 61.1 1.1
a 10.3 0.4 a b 5.2 0.2 b c 1.6 0.1 c d 1.6 0.1 c
S-36 cultivar Average Shoot length of induction shoots (cm) (%) 3.6 0.1 2.1 0.2 1.6 0.1 0.6 0.1
a b c d
82.9 1.6 74.5 1.2 73.6 3.0 61.9 1.6
S-13 cultivar No. of shoots
a 10.6 0.2 a b 5.0 0.3 b b 1.7 0.1 c d 1.5 0.0 c
Average Shoot length of induction shoots (cm) (%) 3.4 0.1 2.0 0.2 1.6 0.1 0.5 0.1
a b c d
78.7 1.8 75.0 1.9 72.1 0.8 57.8 1.2
China White cultivar No. of shoots
a 10.5 0.4 a ab 4.1 0.3 b b 1.5 0.1 c c 1.3 0.1 c
Means followed by the same letter in a column are not significantly different ( p < 0.05) by Newman–Keul’s multiple range test.
Average Shoot length of induction shoots (cm) (%) 3.0 0.1 2.0 0.2 1.5 0.1 0.5 0.1
a b b c
81.1 1.8 70.5 1.1 72.0 1.7 47.9 0.7
No. of shoots a b b c
9.4 0.3 2.4 0.1 1.5 0.1 1.2 0.1
Average length of shoots (cm) a b c c
2.4 0.1 1.7 0.1 1.3 0.1 0.5 0.1
a b c d
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Table 3 Effect of BAP and TDZ on multiple shoot induction from shoots regenerated from leaves
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600
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S-13 and China White cultivars, respectively on medium containing 0.49 mM IBA that enabled better survival upon transfer to soil. High levels of IBA (4.92 mM) decreased the frequency of root induction (43.8–64.3%) along with callus development from the base of the cultures in all the cultivars. Incorporation of 0.45 mM 2,4-D triggered root induction with a frequency of 72.6, 78.7, 68.0, 60.3% in M-5, S-36, S-13 and China White cultivars, respectively. Slender, thin roots were induced from shoots cultured on medium supplemented individually with IAA (0.57 mM) or NAA (0.54 mM) at a frequency of 41.7–67.1 and 13.5–33.6%, respectively in different cultivars. 3.4. Establishment of micropropagated plants in field The regenerated plants of different cultivars survived with a high frequency of 70.7– 80.3% in rainy season than in winter (66.4–75.4%) and summer seasons (56.5–67.1%). Lowest value for plant establishment was observed in the summer season in the China White cv. (56.5 1.7%).
4. Discussion The size and the orientation of the leaves were determining factors affecting induction of direct shoot organogenesis from leaves of mulberry. The best response of shoot organogenesis was observed from leaves of 1–4 cm that were cultured with the abaxial side in contact with the medium. Yamanouchi et al. (1999) reported stable regeneration of plantlets from immature leaves isolated from winter buds of field-grown mulberry. It is inferred from these studies that the size of the leaf plays an important role in its endogenous potential to regenerate adventitious buds. Our results also confirm observations by Mhatre et al. (1985) who concluded that the upright position of leaf explants is favourable for maximum induction of shoot buds in Morus indica L. Kumar et al. (2003) observed enhanced shoot bud development from cotyledonary node explants of mungbean (Vigna radiata L.) after 8 days exposure of explants to 2 mM TDZ. They further reported that continuous exposure of explants to TDZ resulted in the complete loss of regeneration ability and high callus growth of explants. Bhatnagar et al. (2001) achieved 50% regeneration from hypocotyl and 70% from cotyledon explants in Morus indica when cultured on 5 mM TDZ for 10 days. Thereafter, subculture on TDZ free medium lead to the formation of multiple shoots. Our results confirm these observations since a twostage culture procedure consisting of culture of leaves on 18.17 mM TDZ for 8–10 days followed by a secondary medium consisting of 8.88 mM BAP was found to be beneficial for promoting shoot elongation. Debnath (2005) reported that moderate concentrations (1– 5 mM) of TDZ supported bud and shoot regeneration from in vitro derived Lingonberry leaves, but strongly inhibited shoot elongation. TDZ initiated cultures produced usable shoots after one additional subculture on medium containing 1–2 mM zeatin. These reports emphasize the importance of double stage treatment particularly when TDZ is employed for improving the shoot morphogenesis and thus can be attempted in other plant species. The type of cytokinin used in the medium had a marked effect on the rates of shoot proliferation with better results obtained with BAP in relation to TDZ. Furthermore, in
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presence of the TDZ shoots were fused and appeared fasciated with development of thick dark green leaves. The formation of stunted or the fasciation of the shoots on TDZ supplemented medium has been reported in Rhododendron (Preece and Imel, 1991). Variation in the activity of different cytokinins can be explained by their differential uptake rate reported in different genomes (Blakesey, 1991), varied translocation rates to meristematic regions and metabolic processes, in which the cytokinin may be degraded or conjugated with sugars or amino acids to form biologically inert compounds as reported by Tran Thanh Van and Trinh (1990), and Kaminek (1992). The stimulatory effect of auxins in the root formation from the shoots depends partly on the type of auxin employed in Morus alba L. (Anuradha and Pullaiah, 1992). Kim et al. (1985) observed substantial amount of callus formation at the base of the leaf regenerated shoots on 4.92–24.6 mM IBA supplemented medium whereas in the presence of 0.49 mM IBA, few but healthier and more vigorously growing roots were formed directly at the base of the shoots. The presence of callus between the root and shoot resulted in poor vasculature connection, which made field survival of the plantlets difficult. Hossain et al. (1992) observed root formation from the shoots excised from the nodal explants on 4.92– 5.37 mM IBA or NAA in Morus laevigata Wall. Bhau and Wakhlu (2001) reported that IBA was the most effective auxin (2.46 mM) for root induction from shoots regenerated from the callus of Morus alba L. whereas higher levels of auxins encouraged callus formation from the cut ends of the explants. Our results confirm these observations since IBA at 0.49 mM was found to be the best for root induction for the leaf regenerated shoots of all four cultivars compared to 2,4-D, IAA or NAA. In the present study, a two-step approach involving short incubation of leaves for 8–10 days on TDZ supplemented medium followed by transfer to medium with BAP for promoting shoot development from leaves of four elite cultivars has been developed. Shoot proliferation rates were high on medium containing 2.22 mM BAP. Efficient rooting from the shoots of all four cultivars was observed on medium containing 0.49 mM IBA.
Acknowledgements We gratefully acknowledge University Grants Commission, New Delhi for providing financial assistance under unassigned grant scheme to carry out this work. DSVC is thankful to University Grants Commission, New Delhi for the award of a Junior Research Fellowship and Senior Research Fellowship. We also acknowledge the Department of Science and Technology, New Delhi for rendering assistance to the Department in the form of fund for Infrastructure in Science and Technology and the facilities established under this programme have been utilized for the research work.
References Anuradha, M., Pullaiah, T., 1992. Micropropagation of mulberry (Morus alba L.). Annali Di Botanica 15, 35–41. Bhatnagar, S., Kapur, A., Khurana, P., 2001. TDZ-mediated differentiation in commercially valuable Indian mulberry, Morus indica cultivars K2 and DD. Plant Biotech. 18, 61–65.
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Bhau, B.S., Wakhlu, A.K., 2001. Effect of genotype, explant type and growth regulators on organogenesis in Morus alba. Plant Cell Tiss. Org. Cult. 66, 25–29. Blakesey, D., 1991. Uptake and metabolism of 6-benzyladenine in shoot proliferation of Musa and Rhododendron. Plant Cell Tiss. Org. Cult. 25, 69–74. Chitra, D.S.V., Padmaja, G., 2002. Seasonal influence on axillary bud sprouting and micropropagation of elite cultivars of mulberry. Sci. Hort. 92, 55–68. Debnath, S.C., 2005. A two-step procedure for adventitious shoot regeneration from in vitro derived Lingonberry leaves: shoot induction with TDZ and shoot elongation using zeatin. HortScience 40, 189–192. Hossain, M., Rahman, S.M., Zaman, A., Joader, O.I., Islam, R., 1992. Micropropagation of Morus laevigata Wall from mature trees. Plant Cell Rep. 11, 522–524. Kaminek, M., 1992. Progress in cytokinin research. TIBTECH 10, 159–162. Kapur, A., Bhatnagar, S., Khurana, P., 2001. Efficient regeneration from mature leaf explants of Indian mulberry via organogenesis. Sericologia 41, 1–8. Kim, H.R., Patel, K.R., Thorpe, T.A., 1985. Regeneration of mulberry plantlets through tissue culture. Bot. Gaz. 146, 335–346. Kumar, P., Srivastava, G.C., Panwar, J.D.S., Chandra, R., Raghuveer, P., 2003. Efficiency of thidiazuron on in vitro shoot regeneration from cotyledonary node explant in mungbean. Indian J. Plant Physiol. 8, 398–401. Mhatre, M., Bapat, V.A., Rao, P.S., 1985. Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.). Plant Cell Rep. 4, 78–80. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Oka, S., Ohyama, K., 1981. In vitro initiation of adventitious buds and its modification by high concentration of benzyl adenine in leaf tissues of mulberry (Morus alba L.). Can. J. Bot. 59, 68–74. Preece, J.E., Imel, M.R., 1991. Plant regeneration from leaf explants of Rhododendron P. J. H. Hybrids. Sci. Hort. 48, 159–170. Ravindranan, R., Lakshmi Sita, G., 1994. Micropropagation of difficult-to-root elite cultivars and induction of embryogenesis in mulberry (Morus indica L.). In: Tandon, P. (Ed.), Advances in Plant Tissue Culture in India. Proceedings of the XIII Plant Tissue Culture Conference, Pragati Prakashan, India, pp. 155–169. Tran Thanh Van, K., Trinh, T.H., 1990. Organogenic differentiation. In: Bhojwani, S.S. (Ed.), Plant Tissue Culture. Application and Limitations. Elsevier, Amsterdam, pp. 34–53. Vijayan, K., Chakraborti, S.P., Roy, B.N., 2000. Plant regeneration from leaf explants of mulberry: influence of sugar, genotype and 6-benzyladenine. Ind. J. Exp. Biol. 38, 504–508. Yamanouchi, H., Koyama, A., Machii, H., 1999. Effect of medium conditions on adventitious bud formation in immature mulberry leaves. JARQ 33, 267–274.