Introduction

Present era of biotechnology based agricultural science has emerged in response to the limitations of conventional plant breeding techniques.  Somatic embryos in view of the single cell origin may be used as “Target Tissue” in genetic transformation studies.  However, a reproducible method of induction of somatic embryos and  an efficient and reproducible  regeneration protocol from the Somatic embryos is a prerequisite  in utilizing the power and potential of this  technology. Although, excellent progress has been made in obtaining transgenic plants from most of the oil seed crop plants, no significant progress has been made in this direction in safflower, most likely due to non availability of an efficient regeneration protocol from the target tissue i.e somatic embryos. According to Nikam & Shitole (1999), regeneration frequency and rooting capacity of safflower is very low.  However, protocols of plantlet regeneration from cotyledonary explants for Indian (George & Rao, 1982; Tejovathi & Anwar, 1987, 1993) Turkish (Dilek Ba alma et al, 2008)   and American safflower cultivars (Charleen & William, 1996) have been developed. Besides, protocol for in vitro induction of capitulum (Tejovathi & Anwar, 1984; Yashodra et al, 1993) and in vitro induction of androgenic haploids (Prasad et al, 1990, 1991) for Indian safflower cultivars have also been developed. In addition, somaclonal variation (Seeta et al, 2000)   and in vitro pollen (Seeta et al, 1999) as novel sources of genetic variability in safflower have also been reported.

Present study describes in detail induction of somatic embryos  and plantlet regeneration from somatic embryos.

Materials and Method

Plant material

Seeds of ten different Genotypes viz. Manjira, A-1, HUS-305, Tara, GMU-826, GMU-827, APRR-3, Bhima, Co -1, KAS-1 of safflower (Carthamus tinctorius L) were procured from the Directorate of oil seed Research (I.C.A.R.), Rajendranagar, Hyderabad, grown and maintained in the Plant Genetics experimental farm, Department of Genetics, Osmania University were used in the present study.

Surface sterilization and inoculation

Seeds of ten different genotypes were surface sterilized with 0.1% Hgcl₂ in sterile flasks under aseptic conditions for 8-10 minutes and were washed thoroughly with sterile distilled water for three times each of 5 minutes duration. Seeds were then germinated on sterilized wet filter paper bridges. Cotyledon, hypocotyls and root of seven days old seedlings were used as explants for the induction of somatic embryogenesis.  The explants were cut into small pieces and about 4-5 explants/tube were inoculated onto  MS and   B5   medium supplemented with different phytohormones viz: 2,4-D, 2,4,5-T, NAA and IAA at different concentrations (0.5 mg^-1 to  2.0 mg^-1)  for the induction of somatic embryogenesis.  Cytokinins such as 6-Benzylaminopurine (BAP) at four different concentrations (0.5, 1.0, 1.5 and 2.0 mg^-l) and kinetin (KN) at two different concentrations 0.1 mg^-l and 0.5 mg^-l were incorporated in the MS medium. Amino acids viz: proline, Glutamine, Serine and Asparagine at two different concentrations 0.1 mg^-l and 0.5 mg^-l  were also incorporated into the medium to observe the embryogenic response.    All the experiments were carried out in a sterile chamber and the culture tubes were incubated under cool, white and continuous light at 25 ± 1°C with 50-60% relative humidity. Fluorescent tubes of 2000-2500 lux provided the light. All the cultures were maintained in a minimum of three replicates.

To observe the morphogenic response, fully developed globular to torpedo shaped somatic embryos were transferred into medium, supplemented with different concentrations of auxins and cytokinins. Shoot elongation was achieved by regular sub culturing of the shoot buds into the same medium and  for rhizogenesis elongated shoots were transferred to   MS half strength medium supplemented with different concentrations of indole-3-butyric acid (IBA) for rooting.

Regenerated plants obtained from the somatic embryo of Manjira, A-1 and HUS-305 with well-developed roots were taken out of the medium, washed with water to remove agar medium and transferred to pots containing vermiculite. Potted plants were kept at a temperature of 25-26°C. the plants thus established were grown to maturity.

Data  Analysis
The experimental results were calculated according to the following formulae.

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Statistical Analysis

Statistical analysis is done according to the Snedecor and Cochran (1980). Analysis of Variance (two ways, three ways, and four ways) is calculated as per the requirement.

F- test of significance:

If the calculated f value is >= f-tabulated value, it is inferred that significant difference exists. Comparison of means between treatments is done with the help of critical difference.

CD =’t’ table value at Err.df X Ö 2x EMSS/n.

Results and discussion

Successful induction of somatic embryos either directly or indirectly and subsequent plantlet regeneration depends upon the composition of the nutrient media, choice of the explants besides genotype (Buckley et al, 2000). Among the two different media, B5  medium showed relatively low frequency of embryogenesis while  MS medium showed  maximum embryogenic response. Superiority of MS medium over B₅ used in the study may be attributed to the higher nitrate content in the MS medium. The source and amount of total nitrogen in the basal medium in particular are the critical factors for in vitro response of the explant (Dilek Ba alma et al, 2008). The highest rate of embryogenesis was observed in groundnut in the modified MS medium (Sabitha & Reddy, 1996). Somatic embryogenesis in groundnut was also induced directly without any intervening callus phase on MS medium (Venkatchalam et al, 1997).  Somatic embryogenesis in Niger was induced on LS medium (Venkatesham & Reddy, 1996). However, the degree of response varied with the genotype and the explants.

Among the ten genotypes tested HUS-305 exhibited the maximum response followed by Tara, Manjira and A-1 where as, other genotypes showed only callus induction without any differentiation into somatic embryos.  Observations made in this study clearly suggest that the genotypes used critically influenced the in vitro response towards somatic embryogenesis. Somatic embryogenesis in Soybean is strongly genotype dependent and has been linked to the genetic background of the cultivars. Of the 20 lines of northern adopted cultivars of soybean tested for the induction of somatic embryos, it was noticed that although most cultivars have the ability to regenerate in vitro, many cultivars have a very poor response (Buckley et al, 2000). Genotypic differences in the induction of somatic embryos have also been reported in groundnut (Sabitha & Reddy 1996).

Of the three different explants viz: Cotyledon, Hypocotyl and Root used, It is obvious from the data recorded (Table-1 & 1a) that the percent induction of somatic embryos was maximum (84.85%) with the hypocotyl explant. whereas with cotyledonary explants, % frequency of induction of somatic embryos was 23.48%,  histological studies of these embryos revealed all the stages globular, heart and torpedo shaped structures. (Fig-1D)  while, the root explants completely failed to respond. The analysis of variance (ANOVA) revealed that the variation associated with the explants, genotypes and their interaction is highly significant.   Successful induction of somatic embryos depends on the explants as well as on the nutritional and hormonal components in the culture media (Sabitha & Reddy 1996). In Niger, immature leaves were the most responsive towards the induction of somatic embryos (Venkatesham & Reddy, 1996) while cotyledonary explant yielded maximum frequency of somatic embryos in soybean (Buckley et al, 2000). In sunflower, immature embryo was found to be efficient in the induction of somatic embryos (Finer, 1987).

Table 1  Role of genotypes and explants for the induction of somatic embryogenesis in Safflower.

Table 1a. Analysis of variance of the role of genotype and explant  for induction of  somatic embryogenesis in safflower.

Table 2: Role of explants & auxins for the induction of somatic  embryogenesis in different genotypes of Safflower.

NAA & IAA showed least  embryogenic response, hence not included in the data.

Table 3: Effect of Aminoacids for the induction of somatic embryogenesis in different genotypes of Safflower.

Table 4: Morphogenic  response of somatic embryos in MS medium with various concentrations of Auxins and Cytokinin.

Table 5:  Rhizogenic response of regenerated shoots in different genotypes of safflower.

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Figure 1: A.B.C.D   Click here to View figure

The role of four different auxins viz: 2,4-D, 2,4,5-T, NAA and IAA in different concentrations ranging from 0.5 mg^-1 to 2.0 mg^-1 on the induction of somatic embryogenesis was also evaluated. Among the four auxins tested, 2,4,5-T showed the highest embryogenic efficiency followed by 2,4-D. Hypocotyl explants of the genotype HUS-305 with 1.5 mg^-1 2,4,5-T showed the maximum (84.85%) embryogenic response (Fig-1A) followed by Tara, (Fig-1B) Manjira  and A-1.  Increasing or decreasing concentrations of 2,4,5-T    decreased the embryogenic response (Table-2 & 2a). ANNOVA revealed that variation associated with the effects of auxin, its concentration and their interaction is highly significant at 1% level. These observations are in agreement with the findings in chickpea (Sagare et al, 1993).   Other auxins (NAA and IAA at all concentrations) used in this study failed to induce somatic embryos. However, their higher concentrations (1.5 mg^-1 and 2.0 mg^-1) resulted in the formation of embryogenic calli and/or rooting.

Figure 2: A.B.C.D   Click here to View figure

Effect of combination of auxins (2, 4-D and 2, 4, 5-T) and cytokinin viz. kinetin on the induction of somatic embryogenesis was also studied. Different concentrations of 2, 4-D (0.5 mg^-1 to 2.0 mg^-1) and 2, 4, 5-T (0.5 mg^-1 to 2.0 mg^-1) in combination with kinetin (0.1 mg^-1 and 0.5 mg^-1) increased the callusing frequency with very low frequency of induction of somatic embryos. Results obtained clearly indicated that the presence of cytokinin inhibited the process of embryogenesis. Addition of cytokinin viz. kinetin with two different concentrations (0.1 mg^-1 and 0.5 mg^-1) was found to inhibit somatic embryogenesis in groundnut (Reddy & Reddy, 1993). For the induction of somatic embryos in sesamum 2,4-D alone was reported to be the most effective (Jaya & Balan, 1997) . In alfalfa, the inductive treatment is most commonly 2,4-D but other auxin such as 2,4,5-T was also effective (Lai & Mc Kersie, 1994). However, the highest rate of embryogenesis was observed in groundnut in the modified MS medium containing 2,4-D and KN (Sabitha & Reddy, 1996).

Organic components such as amino acids particularly proline present in the medium can also modulate the process of embryogenesis (Shetty & Mc Kersie, 1993). Keeping this in view, in the present study, effect of four different amino acids  viz. Proline, Glutamine, Asparagine and Serine, at two different concentrations (0.5 mgl^-1 and 1.0 mgl^-1)  was evaluated  to know their influence on embryogenic response using both cotyledon and hypocotyl explants.  It is obvious from the data shown (table-3), that both proline and glutamine at lower concentration increased the frequency of somatic embryos while Asparagine and Serine did not show much positive response. However among proline and glutamine, proline is more effective than glutamine. Another aspect of this study is that the positive response of both proline and glutamine in the induction of somatic embryogenesis is confined only to cotyledonary explants (Fig-1C). Addition of 0.5 and 1.0 mM of the amino acid in the medium increased the proliferation of embryogenic callus in Maize (Suprasanna et al, 1994).  However, no marked differences were observed between the basic medium and the medium containing 0.1mM amino acids  in triticum aestivum x Leymus angustus (Zohreh et al, 1993). Proline has been shown to improve somatic embryogenesis in maize (Armstrong & Green 1985).  Argenine and glutamic acid did not increase the formation of embryogenic callus in sugarcane (Ho & Vasil, 1983). In alfalfa, glutamine plays regulatory and nutritive role in somatic embryo maturation (Lai et al, 1992).

Since direct germination of somatic embryos into a plant was found to be poor, plantlet regeneration from somatic embryos was achieved in   a two step process: Shoot bud initiation from the somatic embryos is the first step followed by rhizogenesis of the well-developed shoots. Fully matured somatic embryos were isolated and were transferred to regeneration medium with different plant growth regulators viz. BAP (0.5 mg^-1 to 2.0 mg^-1) NAA and IAA (0.1mg^-1 and 0.5 mg^-1). Initiation of shoot bud (Fig.2A) was observed on MS medium supplemented with 0.5 mg^-1 BAP and 0.1mg^-1 NAA. The highest morphogenesis was observed in the genotype Manjira (84.71%) followed by A-1 (71.43%) HUS-305 (55%) and Tara (41.67%). Increasing concentrations of BAP 1.0 mg^-1 to 2.0 mg^-1 and NAA (0.1mg^-1 to 0.5 mg^-1) decreased the shoot regeneration frequency. When BAP (0.5 mg^-1) was supplemented with IAA, there was very poor shoot regeneration response in all the genotype tested (table-4). Shoot elongation was obtained by regular sub culturing the shoot buds on the same medium (Fig-2B).  In Arachis hypogea mature somatic embryos of the cultivar ICG 8123 were regenerated with a frequency of 4.2% on MS basal medium where as the other genotypes ICG 799 and ICG 1908 turned brown and failed to develop into complete plant-lets (Sabitha & Reddy, 1996).

In an another study in A. hypogea, germination of somatic embryos was observed on half-strength MS basal medium without growth regulators after two weeks of cultures (Reddy & Reddy, 1993). Somatic embryo maturation and germination was achieved on MS basal as well as MS supplemented with 1.0 mg^-1 Kinetin in Banium persicum (Wakhlu et al, 1990).

For rooting of the regenerated shoots of safflower, in the present study IBA was used with five different concentrations (1.0 mg^-1 to 5 mg^-1) to see its effect on rooting response.   The shoots of Manjira developed 6 to 8 elongated roots on the medium containing 5.0 mg^-1 IBA (Fig-2C).  The frequency of rooting was highest in the Genotype Manjira (38.09), followed by A-1 (34.62) and HUS-305 (26.67), while rooting response was completely absent in the genotype Tara.  Complete plantlets with well developed roots were removed from the tubes/bottles and were washed in the running tap water then these plantlets were transferred to plastic cups containing sterile peet math soil and later established into pots (Fig-2D) and were maintained in the green house.

Conclusion

Standardization of the protocol reported in the present study on induction of somatic embryogenesis and plantlet regeneration from somatic embryos will go a long way in the utilization of somatic embryos as target tissue keeping in view their single cell origin, in genetic transformation studies and in regeneration of transgenics (data under publication). 

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