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Plant Growth Regul (2007) 51:271–281 DOI 10.1007/s10725-007-9171-5

ORIGINAL PAPER

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (Catharanthus roseus (L.) G. Don) at morphological and biochemical levels A. Junaid Æ A. Mujib Æ M. P. Sharma Æ Wei Tang

Received: 23 June 2006 / Accepted: 26 January 2007 / Published online: 22 February 2007 Springer Science+Business Media B.V. 2007

Abstract An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l–1) and BA (1.5 mg l–1). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryoderived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and A. Junaid A. Mujib (&) M. P. Sharma Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi 110 062, India e-mail: [email protected] W. Tang Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA

the role of plant growth regulators in two modes of somatic embryo formation have been discussed. Keywords Amino acid Catharanthus roseus Protein Somatic embryogenesis Sugars Abbreviations ANOVA Analysis of variance BA N6-benzyladenine 2,4-D 2,4-dichlorophenoxyacetic acid 2,4,5-T 2,4,5-trichlorophenoxyacetic acid CPA Chlorophenoxyacetic acid GA3 Gibberellic acid HEC Hypocotyl-derived embryogenic callus HECSE HEC-derived somatic embryo IAA Indole-3-acetic acid NAA a-naphthaleneacetic acid MS Murashige and Skoog’s (1962) medium PCSE Primary cotyledonary somatic embryo PCSEC PCSE-derived secondary embryogenic callus SE Somatic embryogenesis

Introduction Madagaskar periwinkle (Catharanthus roseus (L.) G. Don) has been studied extensively for its

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Plant Growth Regul (2007) 51:271–281

were collected in the beginning of August 2003 and surface-disinfested by immersing in 70% ethanol for 10 min followed by three-times washing with sterile double distilled water. Seeds were isolated from fruits inside a laminar hood, treated with 0.5% mercuric chloride for 2 min followed by 1.0% (w/v) H2O2 for 3 min, and finally rinsed with sterile double distilled water.

anticancerous property (Van der Heijden et al. 1989). It has been reported that more than 100 phytochemicals can be produced in Catharanthus roseus, of which vincristine and vinblastine are the most important indole alkaloids (Miura et al. 1988; Moreno et al. 1995). These two alkaloids have been used as therapeutic agents to treat a number of cancers (Mukherjee et al. 2001). However, the yield of these compounds is notably very low. A comprehensive multidisciplinary approach has been integrated in order to improve the alkaloid contents (Verpoorte et al. 1993; Moreno et al. 1995; Mujib et al. 2003). Different tissues of this plant were used to establish cell cultures, and alkaloids contents were quantitatively analyzed (Hirata et al. 1990; Marfori and Alejar 1993; Mujib et al. 1995). In this process, various factors which influence in vitro biosynthesis of alkaloids have also been described (Goddijn et al. 1995; Garnier et al. 1996; Carpine et al. 1997). Although somatic embryogenesis (SE) has been reported in a wide variety of plant genera (Thorpe 1995; Mujib and Samaj 2006), information on SE has been relatively new in Catharanthus roseus (Junaid et al. 2004, 2006a), and it has never been used in alkaloid enrichment programme. Earlier, a preliminarily study on plant regeneration from immature zygotic embryos was reported in Catharanthus (Kim et al. 2004). The advantage of somatic embryogenesis is that the initial cell population can be used as a single cellular system, and their genetic manipulation appears to be easy. In this study, an efficient somatic embryogenesis and rapid plant regeneration system have been established from hypocotyl- and primary cotyledonary somatic embryo-derived calluses. Biochemical differences and the role of plant growth regulators in somatic embryo proliferation, maturation and germination into plantlets have also been discussed.

For induction of embryogenic callus, hypocotyls were cultured on MS medium supplemented with different auxins (2,4-D, CPA, 2,4,5-T, NAA and IAA) alone (0.25–2.0 mg l–1) or in combination with BA (0.5–2.0 mg l–1). This type of embryogenic callus, referred to as hypocotyl-derived embryogenic callus (HEC), was maintained with periodic subculturing at an interval of four weeks. The HEC and somatic embryos derived from it (referred to as HECSEs) were used for maturation, germination and plant conversion studies. All cultures were incubated under a 16 h photoperiod (100 lmol m–2 s–1 PFD) from cool-white fluorescent lamps (F40 T12/CW/EG, Phillips, New Delhi, India) at 25C.

Material and methods

Somatic embryo proliferation and secondary somatic embryogenesis

Plant material Unripe fruits (follicles) of Catharanthus roseus (L.) G. Don naturally grown in Jamia Hamdard (Hamdard University, New Delhi) herbal garden

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In vitro germination of seeds Surface-disinfested seeds (12–25) of Catharanthus roseus were placed in GA-7 Magenta vessels (Sigma, St. Louis, MI, USA) containing 50 ml MS (Murashige and Skoog 1962) medium without organic compounds and growth regulators. All media were adjusted to pH 5.7 and sterilized at 121C for 15 min. Germinating seedlings were grown until they reached 2 cm in length. They were removed from the culture vessels, and the hypocotyls were excised. Callus induction from hypocotyls

Somatic embryos were proliferated from friable calluses (40–50 mg) on MS medium supplemented with NAA (1.0 mg l–1) and different concentrations of BA (0.5–2.0 mg l–1). Secondary calluses, induced on primary cotyledonary-type


somatic embryos (developed from HEC), were further used for proliferation in the same medium to compare the differences in their rate of proliferation, if any. Secondary somatic embryos were also obtained from primary cotyledonary somatic embryos (PCSEs) on the same proliferation medium within 10–12 weeks. The number of secondary somatic embryos per primary somatic embryo was recorded. Scanning electron microscopy For scanning electron microscopy, embryogenic callus was fixed in 2% glutaraldehyde adjusted to pH 6.8 in 0.1 M phosphate buffer for 24 h at 4C. The tissue was washed in the buffer, postfixed for 2 h in similarly buffered 1% osmium tetroxide, dehydrated in a graded ethanol series and finally coated with gold palladium. The prepared samples were examined and photographed in a LEO 435 VP (Zeiss, Oberkochen, Germany) scanning electron microscope operating at 15–25 kV. Suspension culture For establishing suspension culture, secondary calluses were dissected from primary cotyledonary somatic embryos and cultured in liquid MS medium supplemented with 2,4-D (1.0 mg l–1) or NAA (1.0 mg l–1). Cultures were placed on a rotary shaker at 120 rpm at 25 ± 2C. After four days (10 h day–1), the suspension was filtered in a laminar hood with sterile Whatman filter paper No. 2. Secondary callus (40–50 mg) was similarly cultured on proliferation medium supplemented with NAA (1.0 mg l–1) and BA (0.5– 2.0 mg l–1), where a heterogeneous masses of somatic embryos were produced. This type of callus derived from PCSE was referred to as PCSEC. The embryogenic competence and subsequent regeneration ability of PCSEC was monitored and compared with that of HEC. Before maturation and germination, percent somatic embryogenesis and number of somatic embryos at different stages, viz., globular, heart, torpedo and cotyledonary were recorded.

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Maturation and conversion of somatic embryos For maturation, white opaque cotyledonary somatic embryos from both HEC and PCSEC were cultured on MS medium supplemented with GA3 (1.0 mg l–1) and 30 g l–l maltose or glucose since these two carbon sources were earlier found to be very effective in maturation of somatic embryos in Catharanthus roseus (Junaid et al. 2006a). The percent maturation of somatic embryos and their growth were recorded from 5th week onwards. Matured somatic embryos (25 per culture) from both HEC and PCSEC were cultured on MS medium containing BA (0.5 mg l–1) and 60 g l–1 maltose for ensuing quick germination, as reported earlier (Junaid et al. 2006a). Growth features, like rooting, shoot development and conversion to plantlets were recorded at periodic intervals. Acclimatization Converted plantlets were removed from culture vessels, transplanted in micro-plastic pots containing sterile soil rite, thoroughly covered with perforated polythene bags and grown for one month at 25 ± 2C under a 16 h photoperiod (100 lmol m–2 s–1 PFD). Plantlets were then transferred to pots containing 1:1 soil rite and sand for another 2–3 weeks at room temperature, and finally planted in 100% soil under natural conditions. Estimation of protein For protein estimation, 0.5 g tissue was ground in a mortar and pestle with 1.0 ml (0.1 M) phosphate buffer (pH 7.0), placed on ice and centrifuged at 5,000 rpm for 10 min. With 0.5 ml TCA, the sample was again centrifuged at 5,000 rpm for 10 min. The supernatant was discarded, and the pellet was dissolved in 1.0 ml of 0.1 N NaOH after washing with double distilled water. After adding 5.0 ml of Bradford reagent (Bradford 1976), the optical density was measured at 595 nm as described above.

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Estimation of free amino acids Free amino acids were estimated by the method of Lee and Takahashi (1966). In brief, 0.1 g tissue was incubated overnight in 70% ethanol followed by washing with double distilled water. Then 1.5 ml of 55% glycerol and 0.5 ml ninhydrin solution were added, boiled at 100C for 20 min and cooled down. The final volume was made up to 6 ml with double distilled water, and the optical density was measured at 570 nm as described above.


medium supplemented with NAA (1.0 mg l–1) plus BA (1.5 mg l–1) or 2,4-D (1.0 mg l–1) alone. A maximum of 85% callusing was recorded with 2,4-D. However, the calluses induced from other sources (leaf, stem and root) were nonembryogenic characterized by their compact and nodular appearance. Beside 2,4-D, other synthetic auxins, such as 2,4,5-T and CPA were also effective in callus induction, but with low to moderate intensity. High embryogenic callus induction was achieved by continuous subculturing on fresh nutrient medium for two years; the callus was friable, light yellow and fast-growing.

Estimation of total sugars Total sugars in developing somatic embryos and different parts of somatic embryo-derived plantlets were estimated according to Dey (1990). All samples (0.5 g) were extracted twice with 90% ethanol, and the extracts were pooled. The final volume of the pooled extract was made up to 25 ml with double distilled water. To an aliquot of the extract, 1.0 ml of 5% phenol and 5.0 ml concentrated analytical-grade sulphuric acid were added, and the final volume was made up to 10 ml with double distilled water. The optical density was measured at 485 nm as described above. A solution containing 1.5 ml of 55% glycerol, 0.5 ml ninhydrin and 4.0 ml double distilled water was used as a calibration standard. Statistical analysis The data on the effects of growth regulators on different stages of primary and secondary embryogenesis and other parameters were analyzed by one-way analyses of variance (ANOVAs). Values are means of five replicates from two experiments, and the presented mean values were separated using Duncan’s Multiple Range Test (DMRT) at P £ 0.05.

Results and discussion Callus induction The embryogenic callus of Catharanthus roseus (Fig. 1A) was initiated from hypocotyls on MS

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Somatic embryo initiation and proliferation from hypocotyl callus Various concentrations of auxins (0.25–2.0 mg l–1) were used to induce embryogenic callus on MS medium. However, somatic embryos developed rapidly on medium containing 1.0 mg l–1 NAA. Somatic embryogenesis could be further improved when BA (0.5–2.0 mg l–1) was added to the NAA-medium (Fig. 2). Table 1 summarizes the response, which shows that 1.0 mg l–1 NAA plus 1.5 mg l–1 BA had a maximum favourable effect on somatic embryogenesis. Secondary somatic embryogenesis and proliferation of somatic embryos Hypocotyl-derived somatic embryos were maintained on the same proliferation medium by repeated subculturing. Three distinct responses were observed when somatic embryos, especially the cotyledonary-type, were cultured on NAA (1.0 mg l–1)- and BA (0.5–2.0 mg l–1)-containing proliferation media: (a) secondary somatic embryogenesis, (b) secondary callusing (Fig. 1b), and (c) secondary callusing plus secondary somatic embryogenesis (Table 2). Scanning electron microscopic observations showed that secondary somatic embryos were formed at the base of the primary somatic embryos. The induction of secondary callus and its embryogenic response were thoroughly evaluated and compared with that of primary callus induced from hypocotyls. Morphologically, secondary callus was compact, hard, yellowish, and grew very


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Fig. 1 Primary and secondary somatic embryogenesis in Catharanthus roseus. (A) Hypocotyl-derived friable embryogenic callus (HEC). (B) Primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). (C) Shoots differentiating on PCSEC during germination of somatic embryos. (D) Somatic embryos proliferating on PCSEC. (E) PCSECderived somatic embryos maturing on medium containing 1.0 mgl–1 GA3. (F) A converted plantlet showing root and shoot development. (G) Transplanted plantlets growing ex vitro

slowly compared with hypocotyl-induced primary callus (HEC), which was friable, transparent to watery at initiation stage and relatively fastgrowing. The hypocotyls responded quickly and within 7–10 days callusing occurred compared with PCSE-induced secondary callus, where a minimum of two to three weeks’ incubation was

necessary for callus induction. PCSE-derived secondary calluses were isolated (Fig. 1B, C) and transferred to liquid MS medium supplemented with 2,4-D or NAA (1.0 mg l–1) for faster embryogenic callus growth. Secondary callus masses (40–50 mg) were cultured on the same somatic embryo induction and proliferation

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Somatic embryogenesis (%)

80 HEC

70

a

PCSEC

a

b

60

b

50

d c

40 30

c

d

d e

d

e

20 10

media containing various concentrations of NAA and BA. Within 2–3 weeks, secondary calluses proliferated and produced somatic embryos of different morphological stages that could be easily isolated. A comparative analysis involving HEC and PCSEC systems revealed that somatic embryogenesis in terms of both percentage and number of somatic embryos proliferated was higher in HEC than in PCSEC (Fig. 1D).

0 0.0

0.5

1.0

1.5

1.75

2.0

-l

Maturation of somatic embryos

BA (mgl )

Fig. 2 Effect of BA on primary and secondary somatic embryogenesis in Catharanthus roseus. The basal medium is based on MS medium supplemented with 1.0 mg l–1 NAA. Means with common letters are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT). HEC = Hypocotyl-derived embryogenic callus and PCSEC = Primary cotyledonary somatic embryo-derived secondary embryogenic callus

For maturation, morphologically advanced white opaque cotyledonary somatic embryos were individually isolated from each stock, and were cultured on maturation medium, which primarily contained GA3 (1.0 mg l–1) with maltose or glucose (30 g l–1). In contrast to proliferation, PCSEC-derived somatic embryos matured faster

Table 1 Effect of BA on somatic embryogenesis from two different callus lines after seven weeks’ culture on MS medium supplemented with 1.0 mg l–1 NAA BA (mg l–1)

Total number of somatic embryos/culture HEC

0.0 0.5 1.0 1.5 1.75 2.0

A

21.0eB 38.8d 82.5b 99.3a 46.3d 64.8c

Number of somatic embryos Globular

Heart

Torpedo

Cotyledonary

PCSEC

HEC

PCSEC

HEC

PCSEC

HEC

PCSEC

HEC

PCSEC

17.3 e 25.4de 63.4b 81.4a 31.7d 52.3c

13.8e 18.3de 54.0b 61.5a 21.5d 39.3c

11.3e 14.0de 44.6b 53.3a 16.4d 31.7c

5.0a 12.0c 18.5b 22.5a 18.5b 12.2c

4.8d 7.1c 11.0b 17.3a 10.4b 9.6b

2.3c 6.3b 7.0b 9.0a 4.0c 10.2a

1.2d 4.6c 5.4bc 6.4b 3.9c 8.8a

0.0d 2.3c 3.0b 6.3a 2.3c 3.0b

0.0d 0.0d 2.2b 4.3a 1.0c 2.1b

A

HEC = Hypocotyl-derived embryogenic callus; PCSEC = Primary cotyledonary somatic embryo-derived secondary embryogenic callus

B Means with common letters within a column are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT)

Table 2 Effect of BA on secondary callusing and somatic embryogenesis from primary cotyledonary somatic embryos (PCSEs) after six weeks’ culture on MS medium supplemented with 1.0 mg l–1 NAA BA (mg l–1)

Secondary callusing (%)

Secondary somatic embryogenesis (%)

Secondary callusing + secondary somatic embryogenesis (%)

Number of secondary somatic embryos/PCSE

0.0 0.5 1.0 1.5 1.75 2.0

0.0eA 20.7a 15.2b 5.2c 3.7d 0.0e

0.0 d 31.9a 28.3a 20.6b 9.3c 0.0d

4.4e 32.6d 40.2c 72.1b 84.4a 31.8d

1.9e 3.5c 4.6b 6.4a 3.3c 2.6d

A

Means with common letters within a column are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT)

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juvenile shoots without any roots, and (c) only roots without any shoots. Plantlet-conversion, in terms of not only conversion rate but also shoot and root growth, was much higher for somatic embryos developed from PCSEC (Fig. 1F; Fig. 3B). When the plantlets with well-developed roots and shoots were finally transferred to the field, about 100% survival occurred and they flowered normally (Fig. 1G).

(Fig. 1E) than HEC-derived ones. In both systems, the maturation of somatic embryos was higher in maltose-medium than that in glucosemedium (Fig. 3A). The somatic embryos developed from PCSEC were larger in size, and their growth was found to be maintained even after nine weeks of growth period (Table 3). Germination and conversion of somatic embryos

Biochemical analysis at different stages of somatic embryogenesis

Dark-green matured somatic embryos from both HEC and PCSEC were similarly cultured on MS medium supplemented with 0.5 mg l–1 BA for germination and conversion to plantlets. Table 4 shows three types of responses and their growth: (a) plantlets with both shoots and roots, (b)

(A)120

5th week

100

7th week

a

As the morphology and embryogenic response of HEC and PCSEC were different, biochemical analyses were carried out in both the types of somatic embryogenesis systems. In PCSEC, there

9th week

b

c

Maturation (%)

c

80

a

a

b

c

60 40 20

b b

a

c

a

c

b

0 HEC

PCSEC

HEC

PCSEC

Glucose

Maltose -l

Carbon source (30 g l )

(B)

90

b

80

a

b

70

Conversion (%)

a

60

a

50 40

b

30

c

20

c

c c

c

c d

d

10

d

d

0 HEC

PCSEC

PCSEC

HEC

Plant

Fig. 3 Factors affecting the maturation and conversion of somatic embryos in Catharanthus roseus. (A) Effects of carbon sources on the maturation of somatic embryos developed from two different types of calluses; the maturation medium is based on MS medium supplemented with 1.0 mg l–1 GA3. (B) Conversion of somatic embryos, developed from two different types of calluses, into roots,

Shoot

HEC

PCSEC Root

shoots and plantlets; the conversion medium is based on MS medium supplemented with 0.5 mg l–1 BA plus 60 g l–1 maltose. Means with common letters are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT). HEC = Hypocotyl-derived embryogenic callus and PCSEC = Primary cotyledonary somatic embryo-derived secondary embryogenic callus

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different embryogenic calluses, viz., primary hypocotyl-callus and secondary embryogenic callus derived from primary cotyledonary somatic embryos, produced repetitive somatic embryos in culture. These two processes collectively produced a large number of somatic embryos within a limited time and space. In this system, secondary somatic embryogenesis was also operative where adventive somatic embryos regenerated directly from primary cotyledonary somatic embryos. The development of secondary somatic embryos (on primary somatic embryos) is, however, not uncommon in tissue cultures (Fernandez-Guijarro et al. 1995; Raemakers et al. 1995; Iantcheva et al. 2001; Zegzouti et al. 2001; Barbulova et al. 2002). We found that a range of combinations of BA and NAA was useful for secondary somatic embryogenesis. The same combinations of growth regulators were earlier reported to be very effective in primary somatic embryogenesis in Catharanthus roseus (Junaid et al. 2006a). In the present study, the proliferation, maturation and germination of somatic embryos were different in two systems involving separate tissue sources. The proliferation of somatic embryos was much higher in hypocotyl-callus, while the maturation and germination were higher in callus derived from primary cotyledonary somatic embryos. Although two callus-types were different biochemically, their differential responses to somatic embryogenesis (process) might be due to variation in the level of endogenous plant growth regulators. The difference in callus morphology and subsequent

Table 3 Effect of two different carbon sources on the maturation of somatic embryos (length, mm) on MS medium supplemented with 1.0 mg l–1 GA3 Growth period (weeks)

Maltose (30 g l–1)

Glucose (30 g l–1)

HECA

PCSEC

HEC

PCSEC

5 7 9

9.7bB 11.5b 13.9b

10.0a 12.7a 15.5a

8.8d 10.7c 12.1c

9.1c 11.6b 14.1b

A


B

Means with common letters within a row are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT)

were more protein and amino acids than that in HEC. The amino acid and protein contents gradually increased with advancing stages of somatic embryogenesis (Table 5). In contrast, there was a decline in sugar content with increasing complexities in embryogenic process. Plantlets regenerated from two callus lines (HEC and PCSEC) via somatic embryogenesis were also characterized for biochemical changes. We observed that there were no major quantitative changes in protein and amino acid contents in plant parts (shoot, leaf and root) regenerated from HEC and PCSEC sources, but the leaves were biochemically more enriched with sugars and amino acids (data not shown). We report here a rapid plant regeneration system via primary and secondary somatic embryogenesis in Catharanthus roseus. Two

Table 4 Effect of BA (0.5 mg l–1) on the conversion of somatic embryos in liquid MS medium supplemented with 60 g l–1 maltose Growth period (weeks)

Plant-conversion (length, mm) Root HEC

5 7 9

Shoot A

9.5bB 13.8b 22.0b

Shoot-conversion (length, mm)

Root-conversion (length, mm)

PCSEC

HEC

PCSEC

HEC

PCSEC

HEC

PCSEC

12.5a 18.0a 25.6a

6.0c 6.5d 7.3c

6.6c 7.0c 7.8c

9.5b 14.1b 22.7b

12.5a 18.0a 25.7a

0.0d 6.9b 7.2c

0.0d 7.2c 7.7c

A


B Means with common letters within a row are not significantly different at P £ 0.05, according to Duncan’s Multiple Range Test (DMRT)

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279

Table 5 Biochemical characterization of two different embryogenic callus lines and somatic embryos at various developmental stages Parameters Embryogenic callus Proliferated somatic embryo Matured somatic embryo Germinated somatic embryo (mg g–1 FW) PCSEC HEC PCSEC HEC PCSEC HEC PCSEC HECA Protein Amino acid Sugar

2.3eB 1.0d 20.2a

3.0d 1.1cd 19.3ab

3.5d 1.2c 18.6b

4.3c 1.3bc 14.3d

4.0c 1.4b 15.7c

5.1b 1.5b 10.9e

4.7bc 1.5b 11.3e

6.2a 1.7a 9.7f

A


B Means with common letters within a row are not significantly different at p £ 0.05, according to Duncan’s Multiple Range Test (DMRT)

embryogenic competence was similarly observed in other plant systems (Wernicke and Milkovits 1986; Mujib et al. 1996). The morphology of somatic embryos and simultaneous accumulation of storage reserves have been shown to be a good indicator of their maturity and development (Merkle et al. 1995). It has also been demonstrated that this process is positively influenced by various compounds, like carbohydrates, sugar alcohols, PEG, etc. (Lipavska and Konra´dova´ 2004; Tang and Newton 2005; Junaid at al. 2006b). As a carbon source, the use of maltose and glucose for somatic embryo maturation has been reported in a number of studies (Alemanno et al. 1997; Xing et al. 1999), and, therefore, it is not surprising as to why we observed their promoting effect on the maturation process in the present study. In corroboration with previous information (Corredoira et al. 2003; Junaid et al. 2006a), maltose was found to be more beneficial in somatic embryo maturation, and BA-containing germination medium, especially in liquid phase, ensured maximum conversion to plantlets with well-developed shoots and roots; a few of them, however, had either only shoots or roots. The liquid BA-medium was earlier shown to be very effective in other plant systems (Afreen et al. 2002; Junaid et al. 2007). Somatic embryogenesis has several applications including mass propagation of plants. In Catharanthus roseus, we demonstrate a secondary somatic embryogenesis system, operative along with primary somatic embryogenesis originated from hypocotyls. Using both the methods, a large

number of somatic embryos and plantlets were obtained which could be used as medicinal raw material for producing alkaloids. Moreover, the present protocol may offer an efficient and ideal system for large-scale genetic transformation in Catharanthus roseus. The callus-mediated indirect somatic embryogenesis system of single-cell origin, which is operative in Catharanthus, may ensue the development of transgenics en masse as compared to other available systems. Besides, the embryogenic suspension culture can also be effectively used in target mutagenesis programme to produce cell lines capable of increased production of alkaloids. Acknowledgements We wish to thank two anonymous reviewers for their comments on the manuscript, and similarly we are highly grateful to the editor for his critical reading and editing.

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