Commiphora Wightii Through Secondary Somatic Embryogenesis

Published Date: 02 Nov 2017

Somatic embryogenesis in Commiphora wightii was obtained using 5–8 mm long embryos from fruits (65–75 days old) collected during January to June. The embryos gave rise to embryogenic mass on media containing MS minerals and vitamins supplemented with 2,4–D (4.40µM), sucrose (3.0% w/v) and agar (0.6% w/v), when incubated under dark condition within 55–65 days. Further, these embryogenic mass induced somatic embryos within 40–45 days after transferring to light on media devoid of 2,4–D. These studies were detailed in our earlier report (S Kumar et al. 2009). The somatic embryos were asynchronous with certain level of abnormalities. The abnormalities observed were different sizes of globular embryos, single cotyledonary and fasciated somatic embryos. In order to circumvent these abnormalities and to achieve synchronized induction, studies were carried out to induce secondary somatic embryos. The induction of secondary somatic embryos was achieved on MS and SH media from primary cotyledonary somatic embryos. Secondary somatic embryos were induced from the edge of cotyledon and the tip of hypocotyl, and were 1.00–3.00 mm in length. A total of 74% of two cotyledonary embryo induced from secondary embryogenesis system against 24% induced from primary somatic embryogenesis system. The details are described in the paper.

Key words: Somatic embryo, morphological abnormality, synchronization, secondary somatic embryo, Commiphora wightii

Abbreviations: 2,4-D – 2,4-dichlorophenoxy-acetic acid, MS – Murashige Skoog’s (1962) medium, SH – Schenk and Hildebrand’s (1972) medium

Introduction

Commiphora wightii (Arnott) Bhandari, commonly called Guggul, is a major gum resin producing plant in India. It is a small, woody perennial and slow growing, medicinal plant found in arid and semi arid regions of India (Rajasthan, Gujarat, Maharashtra, Karnataka and Madhya Pradesh), Pakistan, Afghanistan and few other gulf countries. Guggul has become endangered, due to its slow growing nature, poor seed setting & germination, excessive & unscientific tapping for its gum resin and also because it has never been brought under cultivation. This plant has been included in Red Data Book of International Union for Conservation of Nature (IUCN) and assigned under "data deficient" category.

The plant has significant medicinal and pharmaceutical value due to the presence of medicinally important steroids, guggulsterone E and Z in gum resin, which makes it an economically important plant. Due to high market value of gum resin, increasing attention needed to be given towards its domestication and cultivation. Although this plant has many excellent traits, adequate attention has not been focused on its conservation and improvement. No selected germplasm is available for guggul, since, it is a wild and not domesticated. Systematic breeding programs are lacking due to the absence of cultivation and conservation programs. Conventional propagation through stem cuttings, air layering and seed are in place but have many limitations and are unable to meet the existing demand of high quality planting material. Therefore, an efficient regeneration system needs to be established for the improvement of this species through in vitro system for harnessing maximum benefits from this pharmaceutically important plant.

To date in C. wightii somatic embryogenesis from immature green fruits (Kumar et al., 2003), immature zygotic embryos (Kumar et al., 2004; Kumar et al. 2003; Singh et al., 1997) and juvenile immature ovules (Kumar et al., 2003) , 2–3 mm embryo (Singh et al., 1997) have been reported. However, all these reports showed low frequency of plantlet regeneration. In order to develop efficient and reproducible protocol for in vitro plantlet formation, the standardization of somatic embryogenesis was carried out.

Plant propagation through somatic embryogenesis have definite advantages, the most important being the rate of plantlets produced per explant is highest among all other in vitro regeneration systems. Besides, somatic embryo is a bipolar structure and there is no need for additional step of rooting / shooting, as required in other in vitro regeneration systems. In the previous report we reported the explant dependent high frequency and repetitive somatic embryogenesis in C. wightii with a very low conversion frequency i.e. 16%.

This report describes the protocol for the plant regeneration and development through primary and secondary SE where the total two cotyledonary SE obtained through primary somatic embryogenesis is 23.50% and that of secondary somatic embryogenesis is 73.97%. Amongst 23.50% normal two cotyledonary primary somatic embryos, when subjected to conversion gave rise to 83.94% conversion and that of secondary somatic embryos it is 60.78%. Thus, the effective conversion values of primary somatic embryos are 19.73% and that of secondary somatic embryos is 44.95%. It also describes the development of synchronized system through secondary somatic embryogenesis.

Materials and methods

Plant material

Explant used for induction of primary somatic embryos (PSE)

The fruits of C. wightii were found throughout the year, which is the only source of immature tissue i.e. embryos (Immature Embryos, IE). In view of these considerations, the embryos (IE) were selected as the explant and hence fruits were collected throughout the year. The collection of explant and the size and age classification were reported earlier (S Kumar et al., 2009).

Explant used for induction of secondary somatic embryos (SSE)

Primary cotyledonary SE of size ranging between 1.00–9.00 mm were observed in 40–45 d culture. The fully developed cotyledonary stage SE having two cotyledons were described as normal, which were 3.00–9.00 mm long and were categorized into Type I (3.01–5.00 mm long), Type II (5.01–7.00 mm long) and Type III (7.01–9.00 mm long) and used for further experimentations. Size less then 3.00 and up to 1.00 mm long showed precocious conversion and were eliminated from the experiments. Therefore the explant used for the induction of SSE was the primary two cotyledonary SE ranging between the sizes of 3.00–9.00 mm.

Surface sterilization

The fruits were surface sterilized as per the method described earlier (S Kumar et al., 2009).

Media

Media for primary somatic embryogenesis

Media for embryogenic mass induction – CIM2 [MS minerals and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v) + 4.4 µM 2,4–D].

Media for primary SE induction – CIM [MS minerals and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v) devoid of growth regulators].

Media for secondary somatic embryogenesis

Four different media for the induction of SSE were formulated, as given below:

CIM [MS minerals and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v)].

½ CIM [1/2 MS minerals and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v)].

SH [(Schenk and Hildebrand's mineral and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v)].

½SH [(1/2 Schenk and Hildebrand's mineral and vitamins supplemented with sucrose 3% (w/v) + agar 0.6 % (w/v)].

The pH of the media was adjusted to 6.00±0.3 with 1N NaOH/1N HCl before autoclaving and the media was autoclaved under 15 psi at 121°C for 15 min. The pH meter used was of Thermo Orion Three StarTM (Thermo Electron Corporation, USA). The autoclave make was of NAT steel company (Nat Steel Equipment Pvt. Ltd. India, Model – 20 HA/E, Serial number–990).

The cultures were incubated under dark condition at 25±1°C and light condition under photoperiod of 8 h light/16 h dark under fluorescent light (irradiance 80 µmol m-2 s-1), at 25±1°C, throughout the experiment.

Microscopic examination and measurement of size of primary and secondary somatic embryos

All the stages of somatic embryos viz. globular, heart, torpedo and cotyledonary were visible on calli during 40–45 days of incubation under 8 h light/16 h dark under fluorescent light (irradiance 80 µmol m–2 s–1), at 25±1°C on CIM media. The primary cultures were asynchronous and have certain levels of abnormalities (different size of globular embryos, single cotyledonary and fasciated somatic embryos). The different sizes of the somatic embryos were measured under the microscope (Leica StereoZoom S8APO, Switzerland) calibrated with ocular (Part number– 10446447, Switzerland) and stage micrometer (Part number–10310345, Switzerland). The images were captured with the help of digital camera (Canon PowerShot, S80), attached to the microscope. The interphase software of digital camera and microscope was ZoomBrowser. The data was collected from 30 embryos at each stage collected randomly. The average measurement was given in Table 1 and 2, respectively for primary globular and cotyledonary somatic embryos & Table 4 and 5, respectively for secondary globular and cotyledonary somatic embryos. Primary globular stage somatic embryos were between 25–30 in number per culture and were of different sizes ranging between 0.11–3.00 mm in diameter. Primary heart stage somatic embryos were between 15–25 in number per culture and were ranging between 0.3–0.8 mm in length. Those of primary torpedo stage somatic embryos were between 35–40 in number per culture and were ranging between 0.8–3.05 mm in length. The numbers of primary cotyledonary stage somatic embryos were very less i.e. between 10–15 in number per culture and were ranging between 1.00–9.00 mm in length. The number of primary heart stage somatic embryos was less as compared to primary torpedo stage somatic embryos in the culture it could be due to low transformation rate of globular to heart and high transformation rate from heart to torpedo stage somatic embryos respectively (S Kumar et al. 2009).

Results

It was observed that all the IE ranging between 1–12 mm long in size collected during January to December, were green in color at the time of inoculation, gradually turned white; the surface of the cotyledon turned rough and gave rise to callus, which started emerging from the abaxial surface of the cotyledon as a smooth shiny mass, under dark condition within 10–15 d. These calli were further kept and grown for 6–7 weeks and transferred to the Callus Induction Media (CIM) under photoperiod conditions.

Under light condition some of the callus mass gave rise to brown pigmentation and some remained non–pigmented. It was observed that the pigmented callus gave rise to SE. On the other hand non–pigmented callus never induced SE. It was found that not all the pigmented population, but only a certain percentage of pigmented calli was induced towards embryogenic line. Hence, after 15 d (70–80 d from explant inoculation) of incubation under photoperiod conditions three different embryogenic calli line were separated and maintained, these were as below,

Calli line 1: Calli Pigmented Induced (75DCPI)

Calli line 2: Calli Pigmented Non-Induced (75DCPNI)

Calli line 3: Calli Non-Pigmented Non-Induced (75DCNPNI)

The globular stage SE stared appearing (visible manifestation of SE were marked by appearance of green coloration over brown pigmentation) (Fig. 1) within 10–20 d from EM developed from IE collected during the months from January to June and 20–25 d for the fruits collected during the months from July to December. The cultures were further incubated on CIM media under photoperiod conditions for the induction of different stages of SE (globular to torpedo shape), which were confirmed with stereomicroscope.

Induction of Primary Somatic Embryos

All the stages of SE viz. globular, heart, torpedo and cotyledonary were visible on calli during 30–45 d of incubation under light condition on CIM media (Fig. 1). In one culture all the stages (i.e. globular, heart, torpedo and cotyledonary) (Fig. 1) appeared indicating the asynchronous nature of the culture. Besides asynchronous induction, certain levels of abnormalities were also detected, particularly; different sizes of globular embryos (Fig. 2), single cotyledonary embryos (Fig. 3a) and fasciated (fused) embryos (Fig. 3b).

In 30–45 d culture, different sizes of globular stage embryos ranging from 0.11–3.00 mm were present (Fig. 2) which were classified into 6 different types based on its size, ranging form 0.11–3.00 mm (Table 1). SE of size between 0.11–0.33 mm transformed into its next stage i.e., heart stage. Size of primary globular stage SE more than 0.33 mm did not transformed into heart stage embryos, but only increased in size and were described abnormal sized globular embryos. Amongst the primary globular stage SE, different percentage size wise were calculated, which are detailed in Table 1.

Table 1: Percent of different sizes of globular somatic embryos in the culture within 40–45 days of culture

Size of globular embryo (mm)

Percent globular embryo

0.11–0.50

46.62±2.40

0.51–1.00

19.33±1.70

1.01–1.50

10.49±1.10

1.51–2.00

09.39±1.20

2.00–2.50

08.28±0.80

2.51–3.00

03.86±0.12

The sizes were of globular embryos presented in mm taking average from 30 globular embryos of each size range. Globular embryos were presented in percent. Data: Mean±SD, n= 3.

Different sizes of heart stage were observed and hence, abnormalities in terms of different sizes of heart stage were also detected in 30–45 d culture (data not given). It was realized that, these abnormalities were carried further and appeared in cotyledonary embryos.

Beside normal SE (two cotyledonary SE), abnormal SE (single cotyledonary and fasciated SE) were also observed in 30–45 d culture. Thus, there were three different types of cotyledonary SE obtained, which were as follows,

Single cotyledonary somatic embryos (Fig. 4a)

Fasciated somatic embryos (Fig. 4b)

Two cotyledonary somatic embryos (Fig. 4c)

The detail percent of single cotyledonary and fasciated SE in 30–45 d culture respectively is detailed in the Table 2.

Table 2: Percentage of different size and type of cotyledonary somatic embryos on induction media after 40–45 days

Size of somatic embryos (mm)

Percent Single cotyledonary (SC)

Percent Double cotyledonary (DC)

Percent Fasciated embryos (FE)

1.00 – 2.00

1.75 ± 0.02

1.32 ± 0.01

1.12 ± 0.03

2.01 – 3.00

4.37 ± 0.12

2.65 ± 0.06

2.79 ± 0.07

3.01 – 4.00

8.29 ± 0.10

7.96 ± 0.09

8.37 ± 0.11

4.01 – 5.00

11.35 ± 0.13

11.06 ± 0.08

11.73 ± 0.07

5.01 – 6.00

15.28 ± 0.18

13.71 ± 0.06

15.08 ± 0.03

6.01 – 7.00

17.46 ± 0.20

16.81 ± 0.10

17.31 ± 1.21

7.01 – 8.00

19.91 ± 1.68

19.91 ± 1.57

20.67 ± 0.19

8.01 – 9.00

21.83 ± 2.41

26.54 ± 0.75

22.90 ± 1.25

Three different types of PSEs were observed on CIM media based on number of cotyledon (Single Cotyledonary (SC), Double Cotyledonary (DC) and Fasciated (FE)), after 30–45 days. The sizes were of PSEs were presented in mm taking average from 30 globular embryos of each size range. Size of PSEs were given in mm. Data: Mean±SD, n= 3.

Out of all the three types of cotyledonary stage somatic embryos, the embryo with two cotyledons (double cotyledonary) were considered normal and used for further conversion studies, and were size wise classified into 3 sub–categories (Fig. 5a, b and c), given in Table 3. Cotyledonary somatic embryos less than 3.00 mm and up to 1.00 mm in length showed precocious germination and eliminated from the experiments.

Table 3: Size and type classification of different primary cotyledonary stage somatic embryos

Type

Size of PSE (mm)

I

3.01 – 5.00

II

5.01 – 7.00

III

7.01 – 9.00

Different types of primary cotyledonary stage somatic embryo which were classified based on the size, Type I (Immature), Type II (Pre-mature) and Type III (Mature). Size of different types of somatic embryos was given in mm.

Induction of Secondary Somatic Embryos

Four different media, as described earlier (Materials and Methods), were used for the induction of SSE. The response of type I and II primary cotyledonary SE was better than those from type III primary cotyledonary SE on all the media tested (i.e., CIM, ½ CIM, SH and ½ SH). The full and half strength CIM and half strength SH media gave rise to direct SE. During which the induction of embryos started appearing from cotyledon, especially towards the edge of cotyledon and tip of the hypocotyl (Fig. 6a and b). The SSE induction on full strength SH media was indirect, through callus intervention. The entire PSE got converted into calli within 5–8 d and latter gave rise to globular embryos. The calli were brown in color during the induction of SSE (Fig. 6c). The induction of SSE was independent of auxin. Since, the inoculum used was primary embryos which formed on auxin free media. Further, the SSE induced was synchronized. The induction time pattern of SSE was comparable with that of PSE i.e. 10–15 d was required for the visible appearance of globular embryos, after transfer to light. These globular embryos formed gave rise to cotyledonary SE during 25–30 d (i.e. 35–45 d after inoculation of primary somatic embryos) after visible appearance of globular somatic embryos. The major advantages being the 2,4–D devoid media on which the secondary embryos emerged. Different percent of globular somatic embryos induced were detailed in the Table 4.

Although, the globular stage secondary somatic embryo formation was observed from all PSE viz. Type I, Type II and Type III, the percent induction was different. Overall 80 to 92% embryo induction was observed from Type I and Type II primary cotyledonary SE. The percent induction of secondary globular embryos was considerably lower from the type III primary cotyledonary embryos (Table 5). Amongst these, abnormalities in terms of various different sizes of secondary globular somatic embryos were observed and grouped as,

0.11–0.50 mm globular embryos

0.51–1.00 mm globular embryos

Interestingly, the embryos (secondary globular embryo) with size 0.51–1.00 mm were less in frequency compared to that of 0.11–0.50 mm (Table 4). The secondary globular somatic embryos of size 0.11–0.33 mm converted entirely to the next step, i.e., heart stage. However, those globular embryos of size greater than 0.33 mm enlarged further and did not developed into next stage.

Table 4: Percent secondary globular somatic embryos induced within 10–15 days on from primary somatic embryos

Type of primary cotyledonary SE

Size of secondary globular embryo induced (mm)

Percent secondary globular stage embryos

CIM

(direct SE)

½ CIM

(direct SE)

SH

(indirect SE)

½ SH

(direct SE)

Type I

0.11 – 0.50

83.33 ± 2.87

86.95 ± 3.50

83.33 ± 5.24

85.71 ± 5.81

Type II

88.23 ± 4.89

90.90 ± 8.54

92.59 ± 7.41

91.30 ± 2.21

Type III

62.50 ± 2.87

57.14 ± 1.25

70.00 ± 5.12

66.66 ± 1.47

Type I

0.51 – 1.00

37.5 ± 2.47

42.86 ± 5.41

30.00 ± 2.47

33.33 ± 2.58

Type II

16.66 ± 0.14

13.04 ± 1.10

16.66 ± 2.27

14.28 ± 1.44

Type III

11.76 ± 0.21

09.09 ± 1.10

07.40 ± 1.41

08.69 ± 1.11

Four different media, CIM, ½ CIM, SH and ½ SH were used for the induction of secondary embryos. Three different types (Type I, II and III) of primary somatic embryo were used for the induction of secondary somatic embryos. Data was collected after 15 days of inoculation of primary somatic embryos. Data: Mean±SD, n= 3.

The secondary cotyledonary SE were developed on both full and half strength CIM & SH, after 35–45 d of incubation under light condition. The secondary cotyledonary SE, were of different percent and sizes in the culture. Different percent of secondary cotyledonary SE which were observed on type I, II and III primary cotyledonary SE were detailed in Table 5. Secondary cotyledonary SE were comparatively smaller in size (i.e. 1.00–3.00 mm long), as compared to primary cotyledonary SE (3.00–9.00 mm long). They were classified into two categories, in 35–45 d old culture (Fig. 7a and b), they were,

1.01–2.00 mm cotyledonary SE

2.01–3.00 mm cotyledonary SE

The synchronized induction of secondary globular somatic embryos was reflected during the development of cotyledonary embryos and were observed on all the three different types of primary cotyledonary SE (Type I, II and III). By and large, 30–47% secondary cotyledonary SE of 1.01–2.00 mm long, 20–34% secondary cotyledonary SE of 2.01–3.00 mm long were observed in 35–45 d old culture (Table 5).

Table 5: Percent secondary cotyledonary somatic embryos induced from primary cotyledonary somatic embryos

Stage of primary cotyledonary somatic embryos

Size of secondary somatic embryo induced (mm)

Percentage of different sizes of cotyledonary somatic embryos

CIM

(direct sec. SE)

½ CIM

(direct sec. SE)

SH

(indirect sec. SE)*

½ SH

(direct sec. SE)

Type I

1.01–2.00

45.45±6.11

40.00 ± 0.63

42.85 ± 0.47

46.66 ± 0.78

Type II

36.36 ±1.18

40.00 ± 2.46

35.71 ± 0.88

37.14 ± 2.13

Type III

30.30±2.78

37.77 ± 4.11

38.63 ± 0.56

33.33 ± 1.23

Type I

2.01–3.00

24.24 ±3.11

33.33 ± 0.47

32.14 ± 2.46

20.00 ± 2.51

Type II

21.21 ± 0.41

28.88 ± 0.89

25.00 ± 0.89

28.57 ± 0.24

Type III

24.24 ± 0.88

26.00 ± 0.57

27.27 ± 0.33

37.25 ± 0.11

Different percent of secondary cotyledonary somatic embryos were induced from three different types of primary cotyledonary somatic embryos (Type I, II and III). Sizes were given in mm. Three different media used were CIM, ½ CIM, SH and ½ SH. Data was collected from 35–45 days old culture. Data: Mean±SD, n= 3.

*Induction of secondary embryos through callus intervention.

Overall, in case of primary somatic embryogenesis, percent induction of two cotyledonary SE was less as compared to secondary two cotyledonary SE, the detail of which are given in Table 6.

Table 6: Percent normal and abnormal somatic embryos during primary and secondary somatic embryogenesis

Somatic embryos

Single cotyledonary embryos

(%)

Fasciated embryos

(%)

Double cotyledonary embryos (%)

Primary somatic embryos

42.33±0.32

34.17±0.87

23.50±0.34

Secondary somatic embryos

10.53± 0.61

15.50±0.37

73.97±0.22

Percent induction of normal (double cotyledonary) and abnormal (single and fasciated) somatic embryos during primary and secondary somatic embryogenesis. Values are given in percent. Data was collected from 35–45 days old culture. Data: Mean±SD, n= 3.

It was observed that the percent induction of single cotyledonary embryos reduced from 42.33% in PSE to 10.53% in SSE. Similarly, it reduced from 34.17% in PSE to 15.50% in SSE for fasciated embryos. The induction of normal (double cotyledonary) somatic embryos increased form 23.50% in PSE to 73.97% in SSE.

Both primary (3.01–9.00 mm) and secondary (1.00–3.00 mm) two cotyledonary embryos were used for conversion experiments. The conversion frequency reached up to 83.94 and 60.78% for primary and secondary SE.

Discussion

EM was proliferated within 6–7 weeks under dark condition. SE induction started with the appearance of light brown pigmentation in the smooth shiny callus within 10–20 d (i.e. 70–85 d after the inoculation of explant) after transfer to light condition and CIM media (MS media devoid of 2,4–D). Generally, a prolonged dark treatment is required to induce somatic embryogenesis in Helianthus annuus (Sujatha and Prabakaran, 2001; Vasic et al. 2001). Appearance of pigmentation of callus was considered to be the landmark and phenotypic marker to distinguish between embryogenic and non–embryogenic callus (Keng et al. 2009; Corredoira et al. 2002).

Maximum number of SE induced from embryonic callus of C. wightii when transferred to media containing 0.25 mg/l BA and 0.1 mg/l IBA (Kumar et al. 2003). Unlike this study, SE in the present study could be induced on the simplest minimal media without any hormones and 91.25% of EM, derived from 5–8 mm long embryos, induced somatic embryogenesis, globular stage within 10 days, and in less than a month’s time all the other stages appeared. Aberrations like different sizes of globular and single cotyledonal and fasciated SE in the primary culture; and somatic embryos in different developmental stages were present giving rise to asynchronous system.

Even though somatic embryogenesis offers great potential, it also has some limitations. Firstly, the development of SE tends to be non–synchronous (Zimmerman, 1993; Zegzouti et al. 2001) thus embryos of all stages can be present in one culture system. Maximova et al. (2002) classified induced somatic embryos into three categories based on morphology, normal embryos, fasciated embryos, and abnormal embryo–like structures. They also reported that, except the normal embryos, the other embryos did not germinated and gave rise to plantlets. The larger number of abnormal embryos produced during primary somatic embryogenesis could be the result of variation in the number of cells that participate in the formation of each individual, and the possible lack of effective spatial coordination during development (Buzzy et al. 2009). The induction of abnormal SE was reported by many workers, as in Aralia cordata (Lee and Soh 1994), Glycin max (Fernando et al. 2002), Different Cocoa Genotypes (Dwomo and Quainoo, 2012). Zakizadeh et al. (2008) has observed, that, the induction of abnormal SE was related to the friable EM, they reported abnormalities of SE, in terms of single cotyledonary and more than two cotyledonary SE. Similar results were demonstrated in Casava (Osorio et al. 2012), Onobrychis sativa (Mohajer et al. 2012), Cyclamen persicum (Hoenemann et al. 2010), sweet corn inbred line (Thobunluepop, 2009), Rosa hybrida (Zakizadeh et al. 2008), Feijoa sellowiana (Stefanelloa et al. 2005) and Eleutherococcus senticosus (Choi et al. 1999), where friable EM induced SE formation. Puhan and Rath (2012) reported that, friable EM dos not induced SE and remained non–embryogenic. In many studies, SE have been reported to be morphologically different compared to zygotic embryos (Choi et al. 1997). Abnormal morphology such as poor cotyledon development, callused embryos or malformed embryos has been frequently observed (Pence et al. 1980; Choi et al. 1997). High concentrations of cytokinins can also lead to formation of abnormal SE (Zakizadeh et al. 2008). In some species, use of 2,4–D resulted in a high frequency of morphologically abnormal embryos which failed to convert into plants (Jiménez 2001). However, abnormality of SE is common in many species (Dodeman et al. 1997) and is reported in many species as, Fraxinus mandshurica (Kong et al. 2012), Desmodium gangeticum (Puhan and Rath 2012), Capsicum chinense (López-Puc et al. 2006), and Sugarbeet (Tsai and Saunders 1995). Variations in embryo morphology, particularly number and morphology of cotyledons and its genetic studies have earlier been reported in Arabidopsis (Jürgens et al. 1991, Aida et al. 1997, Aida et al. 2002) and larch (Harrison and Von Aderkas 2004). In embryos of zygotic origin, embryo size, genetic heritability, phenotypic variation and gene expression seem to control cotyledon number (Aida et al. 2002). SE are more susceptible to variation in cotyledon number than their zygotic counterparts, this may be due to a number of factors like culture conditions, growth regulators and basal medium used (Bharathy and Agrawal 2008). Variability in cotyledon number in SE due to application of plant growth regulators like BA (von Aderkas 2002) & auxin and anti–auxin 2,3,5–triiodobenzoic acid (Choi et al. 1997) has been reported.

Considering the advantages of SSE over PSE, the experiments were carried out to induce large number of SSE. The major advantages being the 2,4–D less media on which the secondary embryos emerged. Secondary somatic embryogenesis is a process whereby new SE are initiated from originally formed SE or PSE. The phenomenon of induction of secondary somatic embryogenesis has been described in at least 100 Gymnosperm and Angiosperm species (reviewed by Raemakers et al. 1995). The development of SSE is not uncommon in tissue cultures (Raemakers et al. 1995; Guijarro et al. 1995; Iantcheva et al. 2001; Zegzouti et al. 2001; Barbulova et al. 2002). Many authors have highlighted that, secondary somatic embryogenesis have supplementary advantages over primary somatic embryogenesis (Raemakers et al. 1995; Singh and Chaturvedi 2009). In some literature, it has been emphasized that, the frequency of induction of PSE from explant were less than that of SSE (Li et al. 2002; Da Silva and Yuffá 2003; Yang et al. 2012; Aboshama 2012). It was also reported in many plant species that, the frequency of conversion of SSE were higher than that of its PSE (Parra and Amo-Marco 1998; Nair and Gupta 2006; Aboshama 2011; Sundaram et al. 2011; Inpuay and Te-chato 2012).

During the present investigation it was noticed that fully formed cotyledonary primary SE were obtained on CIM media devoid of 2,4–D under light conditions, alternatively, SSE were obtained on full and half strengths CIM and SH, directly without auxin pre–incubation, as was done for the induction of PSE. Interestingly, during secondary somatic embryogenesis, the abnormalities like single and fused cotyledonary embryos and fasciated embryos were insignificant.

SSE’s were induced from the PSE when sub–cultured on the same SE induction media (Swamy et al. 2005; Lincy et al. 2009; Aquil et al. 2012). In many cases, in vitro developed SE are liable to form embryogenic callus or secondary embryos (Choi et al. 1999).

We used PSE as an explant to induce SSE. Primary and secondary SE collectively produced a large number of SE within a limited time and space. In this system, secondary somatic embryogenesis was induced through Type1, Type II and Type III PSE, where SE regenerated directly from primary cotyledonary SE. The induction of SSE was free from in born morphological aberration and was synchronized.

Synchronized induction of SSE was found to be more suitable for use as plant material for artificial seed production (Inpuay and Te-chato 2012). SSE have better conversion frequency than that of primary somatic embryos (Merkle et al. 1990; Weissinger and Parrott 1993; Choi et al. 1997; Das et al. 1997; das Neves et al. 1999; Giridhar et al. 2004; Chen and Chang 2004).

Somatic embryogenesis has been recorded from species across many genera and from a variety of plant tissues (reviewed by Williams and Maheswaran 1986; reviewed by Jiménez 2001; Kepczynska 2006). However, conversion of somatic embryos into emblings is a major problem (Helal 2011; Ravi and Anand 2012; Reddy et al. 2012). One difference between somatic and zygotic embryos is that, zygotic embryos after maturation undergoes a period of dormancy, whereas, somatic embryos continuous to grow. Some treatments successful in other species attempts to mimic the developmental cycle of the zygotic embryos, and include for example, a desiccation step prior to germination. In the present study, in order to convert somatic embryos, desiccation treatments of 1–12 days were given to primary and secondary cotyledonary somatic embryos separately. It was observed that the germination frequency of 7.01–9.00 mm long and 1.00–3.00 primary and secondary cotyledonary stage somatic embryos, respectively, have highest conversion frequency during 6 days desiccation treatment. The conversion of both primary and secondary somatic embryos was carried out using different concentration of osmoticum (polyethylene glycol, sucrose and mannitol) and desiccation, as preconversion treatments. The conversion frequency with 83.94 and 60.78% was achieved for primary and secondary somatic embryos respectively.

Acknowledgements

The explant was collected from Gujarat Ecological Education and Research (GEER) foundation. The work was funded both by the internal funding of the Puri foundation and external funding by Council of Scientific and Industrial Research & Programme support, Department of Biotechnology.