In Vitro Cell. Dev. Biol.—Plant 40:284–289, May– June 2004 q 2004 Society for In Vitro Biology 1054-5476/04 $18.00+0.00
DOI: 10.1079/IVP2003524
IN VITRO PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS THROUGH CELL SUSPENSION CULTURES OF HORSEGRAM [MACROTYLOMA UNIFLORUM (LAM.) VERDC.] S. VARISAI MOHAMED1*, C. S. WANG1, M. THIRUVENGADAM2, 1
AND
N. JAYABALAN2
Laboratory of Molecular Genetics, Division of Agronomy, Agricultural Research Institute, Council of Agriculture, Wufeng, Taichung 413, Taiwan, R.O.C. 2 School of Life Sciences, Bharathidasan University, Tiruchirappalli-620 024, India (Received 4 February 2003; accepted 3 November 2003; editor P. Ozias-Akins)
Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 mM 2,4dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 mM 2,4-D. Cotyledonary – torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 mM 2,4-D, 3.0% sucrose, 40 mg l21 L -glutamine, and 1.0 mM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development. Key words: liquid medium; calluses; differentiation; maturation; germination; hardening. suspension cultures has great potential to aid crop improvement through clonal propagation, in vitro selection, genetic transformation, and synthetic seed production. In the present study, we report regeneration of horsegram plants for the first time from embryogenic suspension cultures. The ontogeny of somatic embryos and the various factors affecting induction, maturation, and germination of somatic embryos have also been studied.
Introduction Horsegram [Macrotyloma uniflorum (Lam.) Verdc.] is an important drought-resistant pulse crop mainly grown in India, Africa, Australia, Burma, Malaysia, Mauritius, and the West Indies (Jeswani and Baldev, 1990). In peninsular India, it is cultivated on half a million hectares and accounts for more than 40% of the total area under pulse cultivation. Horsegram seeds are rich in protein, constituting an important part of vegetarian diets. During the past few decades, there has been no significant increase in the production of horsegram due to its susceptibility to viral yellow mosaic disease, pests, salinity (Williams et al., 1968; Muniyappa et al., 1975), indeterminate growth habit, photosensitivity, late maturity, and poor harvest index (Sreekataradhya et al., 1974). Improvement in its production through classical breeding has met with limited success due to the limited genetic variability in germplasm. Transfer of desirable genes from other sources through biotechnological approaches is an alternative method for crop improvement. An efficient regeneration system, a prerequisite for gene transfer, has not been established in this crop, with the exception of direct shoot organogenesis from shoot tip and cotyledonary node explants (Sounder Raj et al., 1989; Varisai Mohamed et al., 1998, 1999). However, there is no report on regeneration of plants from callus and suspension cultures via somatic embryogenesis. The establishment of embryogenic
Materials and Methods Plant material. Seeds of horsegram var. CO-1 were obtained from the National Pulses Research Centre, Vamban, Pudukkottai, Tamil Nadu, India. Seeds were disinfected with serial immersion in 6% Clorox (2% sodium hypochlorite) for 5 min, 70% ethanol (v/v) for 2 min, and 0.1% HgCl2 (w/v) for 7 min. After three rinses with sterile distilled water, seeds were germinated on MS medium (Murashige and Skoog, 1962) containing 3.0% sucrose (w/v) and 0.8% agar (w/v) (type-1, Hi-media Co., Mumbai, India) at 25 ^ 28C in the dark for the first 2 d and then transferred to a 16-h photoperiod of cool-white fluorescent light (120 mmol m22 s21). The pH of all the media was adjusted to 5.8 prior to autoclaving at 1.4 kg cm22 for 20 min. Callus induction. Primary leaves were excised from 7-d-old seedlings, cut into 0.3 –0.5 cm2 segments and cultured on 15 ml MS medium with 3% sucrose, 0.8% agar, and different concentrations of 2,4dichlorophenoxyacetic acid (2,4-D; 2.3–13.5 mM) for embryogenic callus induction. The culture tubes were capped with sterilized cotton plugs. The cultures were incubated at 25 ^ 28C under a 16 h light/8 h dark photoperiod with a light intensity of 120 mmol m22 s21. Cell suspension culture and somatic embryogenesis. Two-weekold, greenish white, friable calluses (approximately 750 mg fresh mass) derived from leaf segments were aseptically transferred to a 250-ml Erlenmeyer flask containing 30 ml of liquid MS medium supplemented with
*Author to whom correspondence should be addressed: Email
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SOMATIC EMBRYOGENESIS OF HORSEGRAM 7.9 mM 2,4-D alone or in combination with 0.4 mM 6-benzylaminopurine (BA) or 0.5 mM kinetin (KIN). Cultures were agitated on a gyratory shaker at 130 rpm, 25 ^ 28C, under a 16 h light/8 h dark photoperiod of 2.5 mmol m22 s21. A 15 ml aliquot of the cell suspension was replaced with fresh medium at 7-d intervals. Cultures were filtered through 125-mm stainless steel sieves to separate individual cells and small cell clumps. Cell suspension cultures were observed under a microscope during the culture period, and the growth was measured for the first 12 d by determining the packed cell volume (PCV) of the samples from 10 replicates. PCV was measured after centrifugation of the suspension at 500 £ g for 5 min in a graduated centrifuge tube (Hall, 1991). MS basal medium was used as a control. Embryos at different stages of development were separated manually and subcultured in liquid MS medium containing different concentrations of 2,4D. After 28 d of culture, cotyledonary–torpedo-shaped embryos were transferred to hormone-free one-fourth, half-, or full-strength MS liquid medium, MS supplemented with 7.9 mM 2,4-D alone or in combination with 0.4–1.9 mM abscisic acid (ABA) or 0.3 –1.4 mM gibberellic acid (GA3) or 0.5–2.5 mM 2-isopentenylamine (2-ip) for maturation and germination. The germinated embryos were transferred to agar-solidified MS basal medium for further growth and development.
Effect of media, carbohydrates, auxins, cytokinins, amino acids, and additives in suspension culture. Two-week-old, greenish white, friable calluses (750 mg fresh mass) from leaf segments were cultured in liquid on different basal media [one-fourth, half-, and full-strength MS, B5 (Gamborg et al., 1968), MS salts þ B5 vitamins, or LS (Linsmaier and Skoog, 1965)], carbohydrates (1–5% sucrose, glucose, fructose, or 1–4% maltose), auxins [2.9–11.4 mM IAA, 1.1 –13.5 mM 2,4-D, 2.7 –10.8 mM NAA, 2.5– 9.8 mM indole-3-butyric acid (IBA)], cytokinins [2.5–9.8 mM 2-ip individually or in combination with 0.5 –2.2 mM BA or 0.5– 2.4 mM KIN], amino acids (10–50 mg l21 L -glutamine, proline, or alanine), 1.4–5.4 mM adenine sulfate, 1.0–3.8 mM ABA, 2.5–10 mM polyvinylpyrrolidone, 0.005– 0.02 mM casein hydrolyzate, or 141.8–567 mM ascorbic acid to determine their efficiency on the induction of somatic embryogenesis. The frequency of embryo induction and stages of somatic embryos were recorded. Transplantation. The plantlets that developed from germinated embryos on solid MS medium were transferred to 16-cm plastic pots containing a vermiculite, sand, and red soil mixture (1:1:1). Each pot was covered with a polythene bag to ensure high humidity for the initial 15 d, and then the humidity was gradually reduced by making holes in the polythene bags to harden the plants. The hardened plantlets were nourished with half-strength MS nutrient solution. The hardened plants were established in soil and grown to maturity in a plant growth chamber (Saveer Biotech, New Delhi, India) under a 16 h photoperiod at 28 ^ 28C. Statistical analysis. For callus induction, at least 50 explants were used and each experiment was repeated three times. Samples of suspension cultures were taken randomly at the end of each subculture, and the number of embryos was counted using a microscope. Counts were made from 20 independent samples, and the percentage of embryos was calculated on the basis of the total number of cell clusters present in the field. A completely randomized design was used in all experiments and analysis of variance and mean separation were carried out using Duncan’s multiple range test and significance was determined at the 5% level (Gomez and Gomez, 1976).
Results Callus induction. Primary leaf explants from 7-d-old seedlings produced greenish white friable calluses (Fig. 1a) on 2,4-Dcontaining medium within 10 – 12 d of culture. Initially the callus showed highly vacuolated cells and later certain cells of these calluses become embryogenic, containing dense cytoplasm, small vacuoles, and large nuclei with deeply stained nucleoli (Anbazhagan and Ganapathi, 1999). The maximum proliferation of greenish white and friable calluses was obtained on 9.0 mM 2,4-D, while minimal response was noted at 2.2 mM (Table 1). Cell suspension culture and embryogenesis. Two-week-old leafderived greenish white friable calluses were subcultured in liquid MS medium containing different concentrations of 2,4-D. After
5 – 7 d of culture on MS medium supplemented with 7.9 mM 2,4-D, cell division and proliferation was observed. The cultures became thick, mucilaginous, and brown in color after culture for 12 d in the same medium; therefore, it was necessary to transfer the cells to fresh medium at weekly intervals. Two weeks after initiation of suspension culture, cells differentiated to form somatic embryos. Microscopic observation of suspension cultures revealed that initial spherical cells were embryogenic, containing visible dense cytoplasm, large nucleus with small vacuoles, and deeply stained nucleolus. These spherical cells were embryogenic and divided transversely resulting into two, four, and subsequently to a group of cells, that was considered to be the pro-embryo. The pro-embryo further divided and formed globular (Fig. 1b, c), heart (Fig. 1d), and cotyledonary – torpedo-staged embryos (Fig. 1e– h). The torpedoshaped embryos recallused on 2,4-D-containing medium. Cotyledonary and torpedo stages were transferred to fresh liquid medium containing 3% sucrose, together with 1.0 mM ABA or 0.3 mM GA3 or 0.5 mM 2-ip for complete maturation (data not shown). Germination of the embryos and transplantation. After transfer of torpedo- and cotyledonary-stage embryos from MS liquid to solid medium, the embryos germinated into tiny plantlets (Fig. 1i, j), with a frequency of 4 – 5%. The plantlets were hardened and established in the soil (Fig. 1 l). Media optimization. The effect of different concentrations of 2,4-D (1.1 –13.5 mM) in liquid MS medium was assessed on induction of somatic embryogenesis (Fig. 1k). The frequency of somatic embryogenesis increased with an increase in the concentration of 2,4-D from 0.0 to 7.9 mM (Table 2). Further increase in 2,4-D concentration resulted in a decrease in embryo induction frequency and recallusing of embryos. Addition of BA and/or KIN to the 2,4-D medium suppressed somatic embryogenesis and produced greenish compact callus. The highest frequency of somatic embryos was achieved in full-strength MS medium containing 7.9 mM 2,4-D, followed by half- and one-fourth strength MS medium. Somatic embryos were not obtained in B5 and LS medium (Table 3). Sucrose at 3% was found to be the most effective carbohydrate for the induction and further development of the somatic embryos (Table 4). Among the additional compounds tested, ABA significantly increased the induction frequency of torpedo-shaped embryos and their maturation (data not shown), while adenine sulfate, polyvinylpyrrolidone, casein hydrolyzate, and ascorbic acid were not so effective in inducing the frequency of somatic embryo production or maturation. Glutamine at 40 mg l21 enhanced the induction, growth, and maturation of somatic embryos (Table 5). However, addition of proline or alanine reduced the frequency of somatic embryo induction and maturation. Discussion The establishment of embryogenic suspension cultures for the regeneration of plants is an ideal tool for the efficient in vitro selection and production of transgenic plants (Finer and McMullen, 1991; Christou, 1997). Embryogenic suspension cultures have been established in only a few grain legumes, including Glycine max (Finer and Nagasawa, 1988), Vigna aconitifolia (Kumar et al., 1988), Vigna unguiculata (Kulothungan et al., 1995), and Cajanus cajan (Anbazhagan and Ganapathi, 1999). In the present study, embryogenic suspension cultures for regeneration of horsegram plants have been established for the first time.
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FIG . 1. In vitro regeneration of horsegram plants via embryogenic suspension culture: a, embryogenic leaf callus; b, globular embryo; c, matured globular; d, heart-shaped embryo; e, early torpedo-shaped embryo; f, matured torpedo-shaped embryo; g, early cotyledonarystage embryo; h, matured cotyledonary-stage embryo; i, germination of embryo showing root and shoot poles; j, young plant from somatic embryo in vitro; k, microscopic view of different stages of somatic embryos; l, hardened plant.
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SOMATIC EMBRYOGENESIS OF HORSEGRAM TABLE 1
TABLE 3
THE RESPONSE OF LEAF EXPLANTS WITH RESPECT TO CALLUS INDUCTION AND NATURE OF THE CALLUS ON 2,4-D-CONTAINING MS SOLID MEDIUM
EFFECT OF DIFFERENT BASAL MEDIA CONTAINING 7.9 mM 2,4-D ON INDUCTION OF SOMATIC EMBRYOGENESIS IN HORSEGRAM Different stages of somatic embryosz
2,4-D (mM)
Callus induction (%)
0.0 2.2 4.5 6.7 9.0 11.2 13.5
0.0 4.7 22.0 36.3 48.6 25.7 11.0
Callus nature Medium type
0.0 YF YF GWF GWF GWF WYF
ef cd b a c e
Globular (%)
1 4MS 1 2MS
MS B5 L6 MS-B5
Values with the same letter are not significantly different according to Duncan’s multiple range test (P ¼ 0.05). Each value represents the treatment means of 10 independent replicates. YF, yellow friable; GWF, greenish white friable; WYF, white-yellow friable.
The choice of initial explant is a critical factor for embryogenic callus induction and initiation. In the majority of legumes, immature zygotic embryos, young cotyledons, or vegetative shoot apices have been the most responsive explants for the induction of somatic embryogenesis (Hartweck et al., 1988). However, in the present study, leaf segments were found to produce somatic embryos. The acquisition of embryogenic potential under auxin stimulus in such explants is manifested through a callus phase. Among different auxins tested, 2,4-D at 7.9 –9.0 mM was most effective for inducing somatic embryogenesis in a liquid medium. IAA and NAA failed to induce somatic embryogenesis, indicating that leaf segments have different sensitivity to various auxins and
Heart (%)
5.4 e 18.6 33.2 12.4 1.8 17.6
b a d f c
3.4 c
1.6 c
n.d.
8.2 b 16.2 a n.d. n.d. 4.2 c
2.4 b 5.4 a n.d. n.d. 1.2 b
Means within a column having the same letter are not statistically significant (P ¼ 0.05) according to Duncan’s multiple range test. Each value represents the treatment means of 10 replicates. z Embryo percentage denotes the number of embryos relative to the total number of embryos formed per flask. n.d., not determined due to nil response.
their concentration. Similar to earlier reports, addition of cytokinins to embryo induction medium inhibited somatic embryogenesis (Saunders et al., 1987; Tetu et al., 1990; Loiseau et al., 1995; Lakshmanan and Taji, 2000). In most legumes, optimal development or histo-differentiation requires removal or reduction of the TABLE 4 INFLUENCE OF CARBOHYDRATE TYPE AND CONCENTRATION ON SOMATIC EMBRYOGENESIS OF LEAF-DERIVED CALLUS OF HORSEGRAM ON MS LIQUID MEDIUM CONTAINING 7.9 mM 2,4-D Different stages of somatic embryosz
THE INFLUENCE OF 2,4-D CONCENTRATION ON INDUCTION OF SOMATIC EMBRYOGENESIS IN HORSEGRAM Carbohydrates 2,4-D (mM) 0.0 1.1 2.2 3.3 4.5 5.6 6.7 7.9 9.0 10.1 11.2 12.3 13.5
0.0 1.8 g 2.4f g 2.6 f 3.2 e 3.4 de 4.0 d 5.4 cd 5.6 c 6.6 bc 6.8 b 7.6 ab 8.2 a
Embryogenic cells at 7 d (%)z 0.0 8.0 12.4 29.8 37.6 45.2 53.4 65.6 62.4 48.2 39.2 26.8 19.2
g fg e d c b a a b d e f
Germination of embryo (%)
10.0 b 20.4 a 2.2 c n.d. 8.4 b
TABLE 2
Packed cell volume (PCV) at 5 d
Torpedo (%)
Different stages of somatic embryos (%)y Globular 0.0 1.2 3.2 8.6 12.4 17.6 21.2 33.2 18.4 15.2 7.4 3.6 1.4
i h f e cd b a c d fg g hi
Heart 0.0 0.0 2.8 4.0 6.2 9.4 13.6 20.4 11.2 7.6 3.4 1.8 1.2
f e d c b a b cd e fg g
Glucose
Torpedo 0.0 0.0 1.4 3.4 4.8 7.2 10.4 16.2 8.8 4.2 1.2 0.0 0.0
Sucrose f e d c b a c de f
Means within a column having the same letter are not statistically significant (P ¼ 0.05) according to Duncan’s multiple range test. Each value represents the treatment means of 10 independent replicates. z Percent of pro-embryogenic cells relative to the total number of callus cells present in the microscopic field. y The frequency of different stages of embryos was determined by taking the samples from 10 replicate flasks per 2,4-D treatment. Embryo percentage denotes the number of embryos relative to the total number of embryos formed per flask.
Fructose
Maltose
Concentrations (%)
Globular (%)
Heart (%)
Torpedo (%)
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4
2.2 c 5.6 c 11.8 b 12.4 ab 13.2 a 9.2 d 18.6 b 33.2 a 13.2 c 5.6 d 7.6 b 13.2 a 5.2 b 3.4 c 2.6 c 3.4 b 5.2 a 1.6 b n.d.
1.2 c 2.8 c 6.4 b 8.2 ab 8.8 a 4.4 d 12.8 b 20.4 a 7.6 c 3.4 d 3.2 b 5.8 a 2.8 bc 2.0 c 1.2 c 1.6 a 2.4 a n.d. n.d.
1.2 c 2.2 c 3.8 bc 4.6 b 5.4 a 2.6 c 8.4 b 16.2 a 1.8 cd 1.2 d 2.4 ab 3.6 a 2.2 b 1.8 bc 1.2 c n.d. 1.2 a n.d. n.d.
Means within a column for each carbohydrate treatment having the same letter are not statistically significant (P ¼ 0.05) according to Duncan’s multiple range test. Each value represents the treatment means of 10 replicates. z Embryo percentage denotes the number of embryos relative to the total number of embryos formed per flask. n.d., not determined due to nil response.
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VARISAI MOHAMED ET AL. TABLE 5
EFFECT OF AMINO ACIDS ON SOMATIC EMBRYOGENESIS IN CELL SUSPENSION CULTURE OF HORSEGRAM Different stages of somatic embryosz Amino acid type Glutamine
Proline
Alanine
Concentration (mg l21)
Globular (%)
Heart (%)
Torpedo (%)
10 20 30 40 50 10 20 30 40 50 10 20 30 40 50
28.2 c 33.4 b 34.2 b 36.2 a 24.6 d 10.2 b 20.6 a 7.4 c 2.8 d 1.2 d 2.8 a 3.2 a 1.2 b n.d. n.d.
26.2 c 30.4 b 32.2 ab 34.6 a 21.2 d 16.8 a 14.2 b 5.2 c 1.8 d 1.0 d 1.2 a 1.6 a n.d. n.d. n.d.
24.6 c 28.6 b 30.2 a 32.4 a 20.4 c 4.2 b 12.6 a 3.2 bc 1.2 c n.d. 1.0 a n.d. n.d. n.d. n.d.
Means within a column for each amino acid treatment having the same letter are not statistically significant (P ¼ 0.05) according to Duncan’s multiple range test. Each value represents the treatment means of 10 replicates. z Embryo percentage denotes the number of embryos relative to the total number of embryos formed per flask. n.d., not determined due to nil response.
auxin used for embryo induction (Rangasamy, 1986). In the present study, induction and development of embryos up to the torpedo– cotyledonary stage were achieved on the same auxin medium, indicating the relative insensitivity of the histo-differentiation to the inductive hormone in this species. However, auxin must be removed at a later stage to allow germination and plant development, which is in accordance with other reports on cowpea (Kulothungan et al., 1995) and pigeon pea (Anbazhagan and Ganapathi, 1999). Ontogeny of somatic embryo development has been studied only in a few legumes, i.e., Vigna species (Eapen and George, 1990; Kulothungan et al., 1995; Girija et al., 2000; Premanand et al., 2000), Glycine (Phillips and Collins, 1981; Finer and Nagasawa, 1988; Samoylov et al., 1998a, b), Arachis hypogaea (Ammirato, 1983; Eapen and George, 1993), Phaseolus (Martins and Sondahl, 1984; Kumar et al., 1988), and Cajanus cajan (Anbazhagan and Ganapathi, 1999). The present study reveals the development of pro-embryos through a division of spherical cells. In Vigna species, Eapen and George (1990) traced the development of pro-embryos from two distinct cells (round/oval and elongated) forming filamentous structures, which in turn produced single or multiple embryos. In cowpea (Premanand et al., 2000), the optimum conditions for maintenance of cell suspension cultures were the shaking speed and packed cell volume (inoculum per 25 ml medium). Similar results were observed in the present study where agitation of the cultures at 130 rpm was found to be optimal. Ontogeny of somatic embryo development in Cajanus cajan was reported by Anbazhagan and Ganapathi (1999). Similar results have also been obtained in the present study. In soybean, 18-mo.-old callus was used for induction and development of somatic embryos through cell suspension cultures (Phillips and Collins, 1981). On
the contrary, long-term callus was not found favorable for embryogenic cell suspension in horsegram. The mineral composition of the media and the type and concentration of carbohydrates and amino acids can play vital roles in somatic embryogenesis. Unfortunately, little effort has been extended to evaluate the effect of different basal nutrient formulations in somatic embryo induction in legumes (Merkle et al., 1995). Full-strength MS medium was found to be more effective than the other media used for induction and growth of somatic embryos. This may be due to the presence of a high level of nitrogen, particularly the reduced form (NH4þ), in MS medium. The type of carbon source has been found to affect the initiation of somatic embryos in horsegram, with sucrose inducing the highest frequency, followed by glucose and fructose. Maltose was completely ineffective for somatic embryogenesis in horsegram. Similar results were also obtained in peanut and soybean (Eapen and George, 1993; Samoylov et al., 1998a, b). Inclusion of glutamine or ABA in the regeneration medium greatly improved the frequency of normal embryo differentiation. In earlier reports, ABA was used to promote maturation of embryos (Ranch et al., 1985). Without ABA, immature somatic embryos precociously produced roots and never developed an apical shoot. In alfalfa, for instance, amino acid enrichment of the medium increases the number of embryos regenerated (Skokut et al., 1985) and embryo conversion into in vitro plantlets (Stuart and Strickland, 1984). Khlifi and Tremblay (1995) reported that in gymnosperms, glutamine is used to increase the number of embryos regenerated and encourage their development (Finer et al., 1989). This suggests that the exogenous supply of these substances plays a pivotal role in the physiological maturity of embryos. In conclusion, a protocol for somatic embryogenesis was found to be reproducible from embryogenic suspension cultures of horsegram. It was possible to produce somatic embryos within 2 mo. and to regenerate plants from mature embryos in 1 – 2 mo. Such protocols have great potential for improvement of this crop by biotechnological approaches such as in vitro selection, clonal propagation, genetic transformation, and production of synthetic seeds. Acknowledgments The senior author S.V.M. is grateful to CSIR, Government of India, New Delhi for financial support in the form of a senior research fellowship.
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