Callus Induction and Regeneration using Different Explants of Sorghum

Transcription

1 Callus Induction and Regeneration using Different Explants of Sorghum Intrduction In recent years, biotechnology is emerging as one of the latest tools of agricultural research. One of the areas of plant biotechnology involves the delivery, integration and expression of defined genes into plant cells, which can be grown in artificial culture media to regenerate plants. Thus biotechnological approaches have the potential to complement conventional methods of breeding by reducing the time taken to produce cultivars with improved characteristics. Tissue culture is defined as the aseptic in vitro regeneration of plants from organs, tissues, cell or protoplasts under controlled conditions using artificial culture medium (Bhojwani and Razdan, 1996). It relies on the totipotency and plasticity of plant cells for whole plants to be regenerated from the small starting material. The application of genetic engineering in crop improvement is dependent upon efficient tissue culture protocols for callus induction and plant regeneration (Pola et al., 2008, Jogeswar et al., 2007). A prerequisite for transformation of any plant species is an efficient tissue culture system for the regeneration of whole plants from single cell. Embryogenic callus induction represent an ideal target tissue for cereal transformation but the establishement of embryogenic callus induction in cereal crops has proven more difficult to compared dicotyledonous species (Vasil, 1994; Sato et al., 2004). One of the challenges for monocots is the identification of suitable explants from which to establish embryogenic callus induction. These challenges could be addressed by the use of genetic engineering strategies to complement conventional breeding techniques. Sorghum is repeatedly the most 71

2 recalcitrant of the cereals for tissue culture manipulation (Gurel et al., 2009; Raghuwanshi and Birch, 2010). In Sorghum callus induction and plant regeneration have been reported using a number of explants including mature embryo, immature inflorescence, immature embryo, shoot tips, anthers and leaves (Maheswari et al., 2006). Despite this, there are still many challenges including genotypic specificity, growth condition sensitivity, phenolic production and difficulties in regeneration and acclimatization. Various strategies have been employed to counteract these challenges. These include the use of specific media components for different genotypes, investigation of different explants for establishing callus induction, use of absorbents and / or antioxidants to obviate the effects of phenolic production, optimization of growth conditions of explants donor plants and screening for superior genotypes (Bhaskaran and Smith, 1990; Sato et al., 2004; Maheswari et al., 2006, Gurel et al., 2009; Raghuwanshi and Birch, 2010). Sorghum recalcitrance is reportedly due to genotype-dependent responses, production of phenolics, lack of regeneration in long term in vitro cultures, low frequency and prolonged phase of somatic embryo s conversion into plantlets and problems in acclimatization has been found. (Brettell et al., 1980; Cai and Butler, 1990; Murty et al., 1990 a, b; Nahdi and De Wet, 1995; Kaeppler and Pedersen, 1996; Sargent, 2002; Sato et al., 2004; Bhaskran et al., 2005; Gupta et al., 2006, Maheswari et al., 2006, Jogeswar et al., 2007, Visarada and Kishore, 2007; Pola et al., 2008; Raguwanshi and Birh, 2010). Despite these problems, there are reports of successful callus induction and subsequent plant regeneration of Sorghum from various explants such as immature inflorescence 72

3 (Brettel et al., 1980; Boyes and Vasil, 1984; Cai and Butler, 1990; Eapen and George, 1990; Murty et al., 1990 a; Gupta et al., 2006; Pola et al., 2006, Jogeswar et al., 2007), Immature zygotic embryos (Gamborg et al., 1977; Thomas et al., 1977; Dunstan et al., 1978; Dunstan et al., 1979; Ma et al., 1987; Nguyen, 1999; Oldach et al., 2001), Mature embryos (Thomas et al., 1977; Botti and I.K., 1983; MacKinnon et al., 1986; Cai et al., 1987), seedlings (Masteller and Holden, 1970; Smith et al., 1983), leaf fragments (Wernicke and Brettel, 1980) and shoot tips or meristems (Bhaskaran et al., 1992; Zhong et al., 1998 b; Maheswari et al., 2006). The work presented in this chapter aim to differentiate the six varieties of Sorghum, based on their callus induction and regeneration response and standardizing the most favorable conditions for callus induction, growth of callus and regeneration of plantlets from different explants i.e., immature inflorescence, immature embryo and mature seed. 73

4 RESULTS Callus initiation The explants started exhibiting the callus development from the 3 rd day after inoculation. Callus initiation occurred on the surface or cut ends of the explants from 7-14 days after inoculation in immature inflorescence, immature embryo and mature seed cultures, bulging of the explants was observed from third day of inoculation (Plate No. 1, Plate No. 2 and Plate No. 3). A week after culturing, a dark brown and purple colour pigment began exuding from the cut ends of the explants into the culture medium. To control this phenol secretion, the explants present in liquid MS medium were kept in a shaker at 200 rpm for 24 hours. Then phenol secretion was released into the liquid MS medium and also activated charcoal (1%) or citric acid was added in culture medium to inhibit pigment formation. Frequent sub culturing was also done to overcome this problem of pigmentation in the medium. Callus induction in immature inflorescence: Immature inflorescence explants were cultured on MS medium supplemented with 2, 4-D and Kinetin (KN) at different concentrations to study their response for callus induction. Callus initiated by 7-14 days after inoculation from the inflorescence axis and spikelet s. The primary callus was non-embryogenic, loose, white and yellowish but differentiated rapidly into a pale green, nodular and friable embryogenic and only a very minor portion remained watery, yellow and soft, which was non-embryogenic, separated out during subculture. 74

5 Embryogenic callus was separated from the primary culture and subcultured onto fresh medium at regular intervals for prolonged periods for further proliferation and development, which formed globular structures. These globular structures developed into somatic embryos on the surface of the callus when maintained on the same MS medium for 2-3 weeks. The frequency of embryogenesis increased with consecutive subcultures. The embryogenic calli on transfer to regeneration medium produced whitish embryoids, which later differentiated into plantlets on the same medium. The plantlets rooted 1-2 weeks later on MS basal medium and were then transferred to jiffy cups for hardening for a week. The plants were then transferred to glasshouse and were grown to maturity. Effect of PGRs concentrations on callusing Different concentrations of 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l of 2, 4-D, IAA, IBA, NAA, KN and ZN were used for their effect on callus formation. While callus initiation occurred in all these concentrations of 2, 4-D, IBA, KN there was no callus initiation at lower concentration of IAA, NAA and ZN. Callus induction frequency varied among the different concentrations. It reached a maximum of 84% at 2.0 mg/l 2, 4-D (Table.3). It was followed by 2.5 mg/l of IBA, IAA and NAA each with 72% induction frequency. Callus induction frequency increased in a combination of 2.0 mg/l 2, 4 D and 0.5 mg/l of KN in all the varieties by 8-16%. The variety IS 3477 showed maximum callus induction frequency (100%) shown in (Plate No. 1, C), followed by IS (92%), IS 7005 (76%) IS 2898 (72%), IS 7155 (60%), and IS 1202 (52%) was shown in (Table 4). 75

6 Growth rate of total callus, embryogenic callus weights of immature inflorescence The callus growth rate of immature inflorescence was given in the (Fig. 3 and 4). Weights of total callus and embryogenic callus data were also collected at intervals of 3 rd, 6 th, 9 th and 12 th weeks here in immature inflorescence also the varieties of IS 3477, IS 33095, IS 7005 (non pigmented varieties) gave the highest response in terms of both callus weights of total and embryogenic, followed by IS 2898, IS 7155, IS 1202 (pigmented varieties). The least response was found with IS These calli weights growth were tested by analysis of variance and genotypic differences were found to be significant (p<0.05) (Table 5 and 6). Total callus volume and embryogenic callus volume The total volume of the callus was highest in the IS 3477, followed by IS 33095, IS 7005, IS 2898, IS 7155 and IS The average quantity of total callus volume per explant ranged from a minimum of 20 mm to a maximum 89 mm (Fig. 5). The results of these initial studies indicated that the embryogenic and non embryogenic could be distinguished by the end of three weeks after their culture inoculation. The embryogenic callus volume growth rate was highest in the variety IS 3477 followed by IS 33095, IS 7005, IS 2898, IS 7155 and IS The average quantity of embryogenic (E) callus volume per explant ranged from a minimum of 18.5 mm to a maximum of 37.5 mm. The average volume of E callus volume in different varieties at 3 rd, 6 th, 9 th and 12 th week intervals is summarized in (Fig. 6). 76

7 Table 3. Effect of plant growth regulators (PGRs) on callus induction in immature inflorescence (25 explants per treatment) Concentrations of PGRs No. of Explants responded 2,4 D 2,4,5- T IBA IAA NAA KN ZN With E.Calli E. Calli freq (%)

8 Table 4 Comparative effect of 2,4-D and KN and their combination in callus induction frequency from immature inflorescence explants Varieties IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 PGR concentration mg/l Total no of explants inoculated No. of explants responded with E. Calli E. Calli frequency 2,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l ,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l ,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l ,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l ,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l ,4-D 2mg/l KN 0.5mg/l ,4-D 2mg/l+KN 0.5mg/l

9 Fig. 3 Total callus weight of Immature inflorescence AVARAGE WEIGHT OF TOTAL CALLUS weight in mg IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 3rd week 6th week 9th week 12th week Fig. 4 Embryogenic callus weight of Immature inflorescence AVARAGE WEAGHT OF EMBRYOGENIC CALLUS weight im mg rd w eek 6th w eek 9th w eek 12th w eek 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 79

10 Fig.5 Total callus volume of Immature inflorescence Callus volume in mm IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 3rd week 6th week 9th week 12th week Fig. 6 Embryogenic callus volume of Immature inflorescence Callus volume in mm rd week 6th week 9th week 12th week 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 80

11 Table 5 Analysis of variance test for total callus weight of Immature inflorescence (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): 2.90 Table 6. Analysis of variance test for Embryogenic callus weight of Immature inflorescence (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS * Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): Callus induction in immature embryo: The callus formed from the immature embryo were usually appeared as a mixture of two types-embryogenic and non-embryogenic. The embryogenic callus was opaque, white or light yellow in colour, more compact, globular with smooth surface and morphogenic in nature. Non-embryogenic calli were unorganized, soft, loosely packed, pale yellow or dull creamy in colour. Generally embryogenic calli showing globular structure are viable after days of explant inoculation. They were easily separated from each other with increase in the number of subcultures, callus morphology was 81

12 occurred in both embryogenic and non-embryogenic calli. The embryogenic callus became opaque, white and sometimes yellowish, compact and nodulated. Even after becoming yellowish, they showed proliferation (Plate No. 2 C). They became gradually cup or dish and club shaped structures. In contrast to this, non-embryogenic callus turned brown in colour and became more unorganized. Effect of PGRs concentration on callusing The different concentrations viz., 1.0, 1.5, 2.0, 2.5, and 3.0 mg/l of 2, 4-D, IBA, IAA, BAP, NAA, KN and ZN were tested for their effect on callus formation from the immature embryo explants of the six Sorghum varieties. Callus initiation occurred in all the concentrations of all PGRs. However, callus induction frequency varied from concentration to concentration. Callus induction frequency in immature embryo Callus induction frequency were ranged between 40% and 88% in immature embryo of the different PGR types used, 2,4-D at a concentration to mg/l produced the highest frequency 76-88% (Table.7). The next best were IAA 2.0 mg/l, IBA 2.0 mg/l which gave 72% frequency (Table. 7). Addition of a KN 0.5mg/L combination with 2, 4-D 2.5 mg/l to the callus induction medium was more effective in that it enhanced the callus formation frequency by 4-15% (Plate 2). At this combination, the highest frequency in the variety IS 3477 (92%) followed by IS (76%), IS 7005 (72%), IS 2898 (60%), IS 7155 (56%) and IS 1202 (48%) (Table 8). The embryogenic callus obtained at this combination was white, compact and globular. Hence, further 82

13 studies on callus induction and callus growth were done with this combination of 2, 4-D 2.5 mg/l + KN 0.5 mg/l only. Weights of total and embryogenic callus of immature embryos Weights of total callus and embryogenic callus data were also collected at intervals of 3 rd, 6 th, 9 th and 12 th weeks. Both total and embryogenic callus weights were the highest in IS 3477, followed by IS 33095, IS 7005, IS 2898, IS 7155 and IS 1202 (Fig. 7 and 8). There was an increase in the weight of the callus with increasing subcultures. However, the quantity of increase was different in different varieties. The variety differences in the total callus weight and embryogenic callus weights were statistically significant (p<0.05) (Table 9 and 10). Volume of total callus and embryogenic callus The quantity of the callus formed, varied from variety to variety. To quantify these differences in the callusing ability of the different accessions, data were recorded at intervals of 3 rd, 6 th, 9 th and 12 th weeks after inoculation. Total callus volume embryogenic and non embryogenic types and embryogenic callus volume (E) were measured. The data were obtained from a minimum of 50 explants i.e. 5 each from 10 explants for each PGR treatment in all the six varieties and two replications were used for each treatment. The total callus volume was highest in the variety IS lowest in the IS 1202 (Fig 9). Whereas embryogenic callus was highest in IS 3477 lowest in IS The average volume of total callus per explant ranged between a low of 14 mm to a high of 79 mm in IS and the embryogenic callus volume was the highest in IS 3477, followed by IS 33095, IS 7005, IS 2898, IS It ranged from a minimum of 9 mm to a maximum 57 mm per explant (Fig. 10). 83

14 Table 7. Effect of PGRs on callus induction in immature embryo culture (25 explants per treatment) Concentrations of PGRs No. of Explants responded 2,4 D IBA IAA NAA BAP KN ZN With E.Calli E. Calli frequency (%)

15 Table 8. Comparative effect of 2, 4-D and KN and their combination in callus induction frequency from immature embryo explants Varieties PGR concentration mg/l Total no of explants inoculated No. of explants with E. Calli E.Calli frequency IS 3477 IS IS 7005 IS 2898 IS 7155 IS ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l ,4-D 2.5mg/l KN 0.5mg/l ,4-D 2.5mg/l+KN 0.5mg/l

16 Fig. 7 Total callus weight of immature embryo AVARAGE WEIGHT O F TO TAL CALLUS weight in mg rd w eek 6th w eek 9th w eek 12th w eek 50 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties Fig. 8 Embryogenic callus weight of immature embryo AVARAGE WEIGHT OF EMBRYOGENIC CALLUS weight in mg IS 3477 IS IS 7005 IS 2898 IS 7155 IS rd week 6th week 9th week 12th week Different varieties 86

17 Fig. 9 Total callus volume of Immature embryo Callus volume in mm IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 3rd week 6th week 9th week 12th week Fig. 10 Embryogenic callus volume of Immature embryo Callus volume in mm rd w eek 6th w eek 9th w eek 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 87

18 Table 9. Analysis of variance test for total callus weight of Immature embryo (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS * Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): 2.90 Table 10. Analysis of variance test for Embryogenic callus weight of Immature embryo (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS * Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): 2.90 Callus induction in Mature seed: Callus initiated from mature seed after 10 days of inoculation from the scutellum. Initially the callus was friable and non embryogenic. After subcultures at weekly intervals, the compact nodular embryogenic callus from friable callus could be differentiated. 88

19 The embryogenic callus was compact, highly nodular, yellowish white while non embryogenic callus was friable and brown. The embryogenic calli on transfer to regeneration medium produced whitish embryoids, which later differentiated into plantlets on same medium. After 1-2 weeks, the plantlets rooted on MS basal medium. The regeneration with well developed roots was removed from the magenta boxes, washed free of agar to grow in jiffy cups. The completely acclimatized plants were transferred to pots in green house and allowed to grow to maturity (Plate No. 3). Effect of PGRs concentration on callusing Five different concentrations of 1.0, 1.5, 2.0, 2.5 and 3.0 mg/l 2, 4-D, IBA, IAA, NAA, BAP, KN and ZN have been tested for their effects on callus initiation. Through callus initiation was observed in all the auxins concentrations, its callus induction frequency varied from concentration to concentration. All types of auxins tested in this experiment, 2, 4-D gave maximum response at 2.5 mg/l in all the varieties tested (Table 11). Callus induction frequency in mature seed Callus induction frequencies were ranged from 36% and 76% in the varieties studied. The frequency was maximum in the variety IS 3477 (76%), followed by IS (68%), IS 7005 (64%), IS 2898 (52%), IS 7155 (40%) and IS 1202 (36%) at 2.5 mg/l of 2, 4-D. It increased in combination with 1.0 mg/l of BAP in all the varieties by 8-24%. The variety IS 3477 gave maximum callus induction frequency (76%) in response to the combination of 2, 4-D + BAP, least response was found in IS 1202 (36%) (Table 12). 89

20 Growth rate of total and embryogenic callus weights of mature seed For the study of growth rate of total and embryogenic callus weights were gathered at 3 rd, 6 th, 9 th and 12 th week intervals. In general, maximum growth rate in terms of both total and embryogenic callus weights was highest in the variety IS 3477, followed by IS 33095, IS 7005, IS 2898, IS 7155 and IS 1202 (Fig. 11 and 12). The general pattern of growth rate in all the varieties was an increase in the weight of the callus with increasing subcultures. However, the quantity of increase was different in different varieties. The variety differences in the total callus weight and embryogenic callus weights were statistically significant (p<0.05) (Table 13 and 14) Total callus volume and embryogenic callus volume The total volume of the callus was highest in the variety IS 33095, followed by IS 3477, IS 7005, IS 2898, IS 7155, IS The average quantity of total callus volume per explant ranged from a minimum of 13.5 mm to a maximum of 65.5 mm. The results of these initial studies indicated that E and NE could be distinguished by the end of three weeks after their culture inoculation. The average total callus volume in different varieties at 3 rd, 6 th, 9 th and 12 th week intervals were shown in (Fig. 13) The embryogenic callus volume growth rate was highest in the variety IS 3477, followed by IS 33095, IS 7005, IS 2898, IS 7155 and IS The average quantity of embryogenic callus volume per explant ranged from a minimum of 6 mm to a maximum of 23.3 mm. The average volume of E callus volume in different varieties at 3 rd, 6 th, 9 th and 12 th week intervals was summarized in (Fig. 14) 90

21 Table 11. Effect of PGRs on callus induction in mature seed (25 explants per treatment) Concentrations of PGRs No. of Explants responded 2,4 D 2,4,5- T IBA IAA NAA KN ZN With E.Calli E. Calli freq (%)

22 Table 12. Comparative effect of 2, 4-D and BAP and their combination in callus induction frequency from mature seed explants Varieties IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 PGR concentration mg/l Total no of explants inoculated No. of explants responded with E. Calli E. Calli frequency 2,4-D 2.5 mg/l BAP 0.5mg/l ,4-D 2.5 mg/l + BAP 0.5 mg/l ,4-D 2.5 mg/l BAP 0.5mg/l ,4-D 2.5mg/l + BAP 0.5mg/l ,4-D 2.5 mg/l BAP 0.5 mg/l ,4-D 2.5 mg/l + BAP 0.5mg/l ,4-D 2.5 mg/l BAP 0.5 mg/l ,4-D 2.5 mg/l + BAP 0.5 mg/l ,4-D 2.5 mg/l BAP 0.5mg/l ,4-D 2.5 mg/l + BAP 0.5 mg/l ,4-D 2.5 mg/l BAP 0.5 mg/l ,4-D 2.5 mg/l+ BAP 0.5 mg/l

23 Fig. 11 Total callus weight of Mature seed AVARAGE WEIGHT OF TOTAL CALLUS Weight in mg rd week 6th week 9th week 12th week 50 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties Fig. 12 Embryogenic callus weight of Mature seed AVARAGE WEIGHT OF EMBRYOGENIC CALLUS 300 Weight in mg rd week 6th week 9th week 12th week 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 93

24 Fig. 13 Total callus volume of Mature seed 70 Callus volume in mm rd week 6th week 9th week 12th week 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties Fig. 14 Embryogenic callus volume of Mature seed 25 Callus volume in mm rd week 6th week 9th week 12th week 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 94

25 Table 13. Analysis of variance test for total callus weight of Mature seed (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS *Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): 2.90 Table 14. Analysis of variance test for Embryogenic callus weight of Mature seed (ANOVA) Source of variation Sum of squares Degrees of freedeom Mean sum of squares Variance ratio TSS CSS RSS ESS * Showing significant difference; P (0.05; 3; 15): 3.29; P (0.05; 5; 15): 2.90 Regeneration Regeneration in immature inflorescence The compact pale green colored embryogenic calli were separated from the explants and cut into small pieces and transferred to regeneration medium. Plant regeneration was improved by exposing the organized callus to low levels of cytokinins, when the cultures transferred into high concentrations of cytokinins, the cultures underwent necrosis. BAP 1.5 mg/l concentration was optimum for shoot development 95

26 (Table. 15). The number of shoots was maximum in non pigmented varieties IS 3477 (22), IS (20), IS 7005 (17) and pigmented varieties IS 2898 (15), IS 7155 (14), IS 1202 (12) per culture shown in (Fig. 15) In regeneration medium the somatic embryos gradually changed from white to green colour shoot buds (Plate No.1). The shoot buds proliferated to shoot within 6-18 days. The levels of cytokinins used for plant regeneration appeared to be critical in producing normal vigorous shoots in large numbers. Initially for regeneration, different concentrations 1.0 to 3.0 mg/l of BAP, KN, ZN, TDZ, GA3 and IAA were used, but shoot formation was optimum at 1.5 mg/l BAP concentration (Plate No. 1 E). Elongated and well developed shoots were transferred to rooting medium containing NAA at 1 mg/l (Plate No. 1). Root initiation was observed within two weeks after culture. The number of roots was maximum with IS 3477 (47), followed by IS (37), IS 7005 (33), IS 2898 (30), IS 7155 (27) and IS 1202 (25) per culture (Fig. 15) Regeneration in immature embryo At the end of 3 rd week, the embryogenic and non embryogenic calli were cut into small pieces and transferred to regeneration medium. The non embryogenic callus underwent necrosis after transferring onto regeneration medium. In all the explants embryoids formation and regeneration occurred on the embryogenic calli only. Embryogenic calli after transferring to regeneration medium, embryoid formation occurred within nine days after culture transfer. After days, clusters of rounded or oval structures were observed with their basal ends embedded in the callus mass. These 96

27 structures turned into green coloured shoot buds and later developed into shoots in the presence of light (Plate No. 2 E). Effect of PGR on regeneration Different concentrations of cytokinins were tested for their effects on regeneration. High frequency of regeneration was observed on the regeneration medium with 2.0 mg/l BAP (Table 16). In general, regeneration on media containing higher concentrations of hormones was less prominent in appearance and turned yellow without further development. When TDZ, a phenyl urea compound was used in the medium, it inhibited callus formation, and germination occurred in those embryos without any callus formation or changing the medium. The number of shoots was maximum in non pigmented varieties IS 3477 (20), IS (18), IS 7005 (16) and pigmented varieties IS 2898 (13), IS 7155 (10), IS 1202 (8) per culture (Fig. 16). Shoots of 2-4 cm in height were transferred to a rooting medium containing half strength MS medium with 1 mg/l NAA and 2% sucrose. Initially different concentrations of 0.5 mg/l NAA gave good response. Root number was approximately proportional to the shoot number in all the varieties. The number of roots was maximum in the variety IS 3477 (39), followed by IS (32), IS 7005 (31), IS 2898 (29), IS 7155 (26) and IS 1202 (22) per culture (Fig.16). Regeneration in Mature seed The embryogenic callus was compact, highly nodular, yellowish white. After transfer to regeneration medium, shoot formation from embryogenic calli occurred in about days (Plate No. 3 E). Optimum response was with ZN at 1.5 mg/l (Table 97

28 17). The number of shoots was maximum in the variety IS 3477 (11) followed by IS (10), IS 7005 (8), IS 2898 (7), IS 7155 (6), IS 1202 (4) (Fig. 17). Well developed and elongated shoots were transferred to rooting medium containing NAA at 1.0 mg/l. Root initiation occurred within 9-13 days. The number of roots was maximum in the non pigmented varieties IS 3477 (26), IS (25), IS 7005 (23) and pigmented varieties IS 2898 (20), IS 7155 (19), IS 1202 (16) per culture (Fig 17). Shoot and Root lengths Shoot and root lengths were taken of these three explants at week intervals 3 rd, 6 th, 9 th weeks (Tables 18 and 19). The growth of the shoot was initially slow during the 3 rd week, later increased during the subculture period. Of all the varieties, shoot and root growth was highest in the non pigmented variety of IS 3477 and IS Acclimatization and transfer of plantlets to soil The regenerated plantlets of six varieties of Sorghum with healthy rooted shoots were collected from different explants and then washed with distilled water to remove all traces of the medium from the roots. They were then transferred into small pots or plastic cups containing sterilized soil. The pots were then covered with polythene bags to maintain high humidity. The plantlets were watered weekly thrice with sterile water. Initially the cultures were maintained for one week in culture room and then they were transferred to greenhouse. After two weeks, the polythene bags were removed and plantlets were transferred into pots with normal soil and kept under diffuse sunlight for about ten days in green house. Later, they were grown under full sunlight like the normal seedlings. The potted plants produced new leaves and showed healthy growth. Morphologically there was no detectable variation between in vitro raised and naturally 98

29 grown plants. The regenerated plantlets that have grown into complete plants in greenhouse were later transferred to field for further development Table 15. Effect of different PGRs concentrations on regeneration from Immature inflorescence BAP KN ZN TDZ GA 3 Explants responded SHOOTS ROOTS 99

30 Table 16. Effect of different PGRs concentrations on regeneration from Immature Embryo BAP KN ZN TAD GA3 SHOOTS ROOTS

31 Table 17. Effect of different PGRs concentrations on regeneration from Mature seed BAP KN ZN TAD GA3 No. of shoots and roots SHOOTS ROOTS

32 Fig. 15 Number of shoots and roots in Immature inflorescence SHOOTS AND ROOTS IN IMMATURE INFLORESCENCE No.of shoots and roots IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties shoots roots Fig. 16 Number of shoots and roots in Immature embryo SHOOTS AND ROOTS IN IMMATURE EMBRYO 40 No.of shoots and roots shoots roots 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 102

33 Fig. 17 Number of shoots and roots in Mature seed SHOOTS AND ROOTS IN MATURE SEED 30 No.of shoots and roots shoots roots 0 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 Different varieties 103

34 Table 18. Shoot length in different varieties Varieties SHOOT AGE IMMATURE INFLORESCENCE IMMATURE EMBRYO MATURE SEED IS rd Week 1.91± ± ±0.02 IS rd Week 1.31± ± ±0.01 IS rd Week 1.61± ± ±0.03 IS rd Week 1.42± ± ±0.04 IS rd Week 1.45± ± ±0.09 IS rd Week 1.30± ± ±0.02 IS th Week 3.42± ± ±0.10 IS th Week 2.66± ± ±0.05 IS th Week 3.27± ± ±0.05 IS th Week 3.24± ± ±0.03 IS th Week 3.42± ± ±0.08 IS th Week 3.21± ± ±0.02 IS th Week 6.58± ± ±0.04 IS th Week 5.95± ± ±0.09 IS th Week 5.67± ± ±0.07 IS th Week 4.30± ± ±0.09 IS th Week 4.41± ± ±0.09 IS th Week 4.16± ± ±

35 Table 19. Root length in different varieties Varieties Root Age Immature Immature Mature seed inflorescence embryo IS rd Week 3.28± ± ±0.09 IS IS 7005 IS 2898 IS 7155 IS 1202 IS 3477 IS IS 7005 IS 2898 IS 7155 IS 1202 IS 3477 IS IS 7005 IS 2898 IS 7155 IS rd Week 3.01± ± ± rd Week 2.98± ± ± rd Week 3.41± ± ± rd Week 2.96± ± ± rd Week 2.98± ± ± th Week 6.70± ± ± th Week 6.09± ± ± th Week 6.66± ± ± th Week 5.78± ± ± th Week 6.28± ± ± th Week 4.67± ± ± th Week 11.06± ± ± th Week 11.02± ± ± th Week 12.02± ± ± th Week 10.77± ± ± th Week 9.86± ± ± th Week 8.38± ± ±

36 DISCUSSION In the present study, the results revealed to improve the tissue culture response of Sorghum by investigating the use of pigmented and non pigmented varieties under different growth conditions and using various explants. Sorghum is notorious for variety specific responses in tissue culture and, as a result, it is extremely challenging to obtain consistent tissue culture responses from the different Sorghum varieties available. As such, the approach of screening a number of genotype to identify elite varieties for best embryogenic callus establishment and regeneration frequencies is favored (Ma et al., 1987; Bhaskaran and Smith, 1988; Rao et al., 1995; 1999; Carvalho et al, 2004; Sai Kishore et al, 2006; Pola et al., 2008; Raghuwansi and Birch, 2010). The ability to produce somatic embryos and their differentiation into green plantlets through somatic embryogenesis was found to be influenced by several factors, such as the genotype, organ from which it is derived, the physiological state of the explants and its size. Orientations of the explants on the medium, its density and the period of incubation may also affect shoot bud differentiation. The physiological stage of explants is affected by the age of the donor plant, which has a direct approach on the regeneration ability of the explants. The use of immature embryo and immature inflorescence tissue enabled high embryogenic callus induction frequency, as well as the regeneration frequency, while mature seed explants failed to show such effect response. Bhojwani and Razdon (1996) reported that this ability is especially true for cereals and tree species 106

37 A previous work on in vitro aspects in cereals revealed that, of the various media used viz., MS (Murashige and Skoog 1962), B5 (Gamborg et al., 1977), LS (Ketchum et al., 1987), N6 (Chu et al., 1975) and I6 medium (Cai and Butler 1990). MS has been the most frequently used medium for Sorghum tissue culture. Hagio (1994) recommended that, MS medium was effective for Sorghum tissue culture. He reported that, in most of the tissue culture works on Sorghum were performed by using MS medium. For this reason, in the our study MS medium was used as a basal medium. In the present investigation, among the six varieties, the three explants studied, and the most suitable explants to produce maximum embryogenic callus was that of immature embryo and immature inflorescences. Of the six varieties, IS 3477, IS and IS 7005 (Non pigmented varieties) showed higher values in terms of frequency of embryogenic callus, quantity of embryogenic callus, growth rate, regeneration frequency, number of regenerated plantlets per explants, and number of roots per culture. While IS 2898, IS 7155 and IS 1202 showed lower values for most of the characters. Such genotypic differences were also observed by Hagio (1994) and Rao et al., (2000), Sairam et al., (2000), and Pola et al., (2006) in Sorghum. Several authors reported that the size of the explants is critical for callus initiation; Rathus et al., (2004) observed good response with 0.8 to 1 mm long embryos. Serhantova et al., (2004) found small size immature embryo (0.5 mm to 1.5 mm) had 100% of callus formation and a high efficiency of plant regeneration than large size embryos. George and Eapen et al., (1989) reported that frequency of somatic embryogenesis and plant regeneration from mm long immature inflorescence. Hasegawa et al., (1995) used 0.5 to 2 cm long immature inflorescence. Rao et al., (2000) 107

38 used cm long immature inflorescences. Pola et al., (2006), Sarada et al., (2003) and Jogeswar et al., (2007) used 1-4 cm long immature inflorescence. Callus induction and regeneration from different explants used in Sorghum has been reported by Bhaskaran and Smith (1988), Cai et al., (1987), Harshavardhan et al., (2002), Murthy et al., (1990 a), Nahdi and De wet (1995), Zhong et al., (1998), Syamala and Devi (2003). Hence in all the six varieties studied in the present study, suitable size and developmental stage is require to produce maximum embryogenic callus i.e., immature embryos of mm, immature inflorescences of 1-4 cm and whole mature seeds produced maximum embryogenic callus. A distinct feature of callusing in cereals and grasses is the formation of two different types of calli viz., embryogenic and non embryogenic calli which differ markedly in their regenerative potential. Often, both types were found to develop from the same explant segment and differed morphologically in their appearance (Vasil and Vasil 1982). Formation of embryogenic and non embryogenic type calli from same explant has been observed in all the explants and genotypes tested in this study. A clearcut difference between the two callus types could be made morphologically by the end of days after explant inoculation. The possibilities of such early distinction have some benefit for their separation quite at an early stage before the embryogenic type is out grown by the non embryogenic type. Based on the explant response, callus type, plant regeneration and genetic stability, Morrish et al., (1987) recognized six different callus types in Graminaceae and Cai and Butler (1990) Lusardi and Lupotto (1990) and Rao et al., (2000), (Pola et al., 108

39 2006; Sarada et al. 2003; Pola et al., 2009) recognized two types of calli and the embryogenic callus of the present study look like to match up to their observation. For varieties IS 2898, IS 7155 and IS 1202, Immature inflorescence and Immature embryo derived callus produced large amounts of phenolics during the 6 th and 9 th week on regeneration media when shoots and roots being to develop. Since phenolics have a detrimental effect on plant growth to regeneration media and two concentrations each of PVP, Ascorbic acid and activated charcoal were investigated for reducing phenolic production. Although the activated charcoal and ascorbic acid treatments were effective in reducing phenolic production, none of the treatments showed any reduction in the frequencies of necrosis or improvements in the percentage of explants regenerating or the number of plantlets in regeneration reported in Sorghum (Kaeppler and Pedersen, 1996; Carvalho et al., 2004; Sato et al., 2004; Sai Kishore et al., 2006; Raghuwanshi and Birch, 2010). In the present study, we have used different chemicals for phenolic secretion in culture media i.e., PVP, Ascorbic acid, and activated charcoal. The difficulty of using chemicals to reduce phenolic production in Sorghum cultures has been reported previously by Wernicke et al., (1982), ascorbic acid, glutathione, cysteine, activated charcoal and Polyclar AT (insoluble polyvinylpyrrolidone) to negate the accumulation of toxic phenolics in tissue culture of Sorghum. Only a slight inhibition of the pigments was observed by the addition of 200 mg/l Polyclar AT. Phenolic suppressing basal callusing and causing vitrification (ascorbic acid) or adsorbing non-phenolic medium components PVP, activated charcoal (Walter and Purell, 1979; Sharma and Chandel, 1992; Thomas, 2008; Mederos-Molina, 2009). 109

40 Low level of 2, 4-D has been the most commonly used callus inducing hormone in the cereals, Hagio (1994), Manjula et al., (2000) and Pola et al., (2006) also reported that cereals in general require 2, 4-D to initiate the callus cultures and its higher concentrations have been found to be less effective in the formation of embryogenic callus (Lu et al., 1983). George and Eapen (1989) and Murthy et al., (1990) obtained high frequency of embryogenic callus from the explants of immature inflorescence on MS medium supplemented with 2 and 2.5 mg/l 2, 4-D respectively. Rao et al., (2000) reported high frequency of callus induction by using 1.5 mg/l of 2, 4-D. Mythili et al., (2001) obtained embryogenic callus by using 2 mg/l 2, 4-D in the callus induction medium. Arti et al., (1994) reported 81% callus induction frequency and % regeneration frequency from immature inflorescences cultures. They reported that the optimum concentration for callus induction was 2.5 mg/l mg/l IAA + 2 mg/l NAA and 0.2 mg/l KN for regeneration. A similar trend was observed in the present study i.e., 2.5 mg/l 2, 4-D was optimum concentration to obtain high frequency of embryogenic calli in immature embryo, 2.0 mg/l 2, 4-D for immature inflorescence cultures when combination with 0.5 KN mg/l Where as in mature seed cultures 2.5 mg/l 2, 4-D combination with BAP 1.0 mg/l was the optimum concentration to obtain high frequency of embryogenic callus. Generally, 2, 4-D alone has been used for callus induction in cereals. However, there have been some instances where kinetin has also been used in conjunction with 2, 4-D for callus induction from these three explants of Sorghum. Cai and Butler (1990) investigated callus induction from using explants high tannin Sorghum cultivars and observed that kinetin increased the frequency of embryogenic callus 3-4 fold in two 110

41 cultivars, had a modest (25-30%) increase in five cultivars while one cultivar did not respond at all. In addition to the genotypic response in callus formation, an increase in pigment formation was also observed with the use of kinetin. A genotypic effect of kinetin for increasing the production of somatic embryos in immature inflorescence derived callus of three Sorghum cultivars was also reported by Jogeswar et al., (2007). Results on the growth rate of callus as reflected by the increase in total and embryogenic weights upto 12 weeks indicated significant difference among the genotypes and auxin concentrations. Higher concentrations of auxins in the callus induction medium appear to be unfavorable for the faster growth rate of embryogenic callus. The culture response was greatly influenced by the genotypes in all types of explants. Genotype effects on callusing ability from Sorghum were reported previously by Cai and Butler (1990). They reported that genotypic differences in pigment production, embryogenic callus formation and plant regeneration. Effect of genotype on callus induction and plant regeneration were evident from seed explant sources were also reported in Sorghum (Bhaskaran et al., 2005). A plant growth hormone like those of BAP, KN, ZN and TDZ also has been tried in several cereals and grass systems with varying effects. Arti et al., (1994) used 0.2 mg/l KN for culturing immature inflorescence, Kuruvinashetti et al., (1998) obtained regeneration from organized callus by using mg/l BAP. Murthy et al., (1990) reported high frequency of regeneration from different explants of Sorghum by using 2.0 mg/l IAA and 0.1 mg/l BAP were most suitable for shoot induction and 0.1 mg/l of BAP and 1 mg/l NAA for root initiation, they reported efficient regeneration in sweet Sorghum varieties. Shan et al., (2000) recommended that, TDZ has the potential for enhancing the regeneration of other cereals and grass species. He reported efficient 111

42 regeneration by using with 0.2 mg/l TDZ in both barely and wheat derived callus cultures. Shan et al., (2000) found 0.2 mg/l TDZ as most potential for enhancing the regeneration of barley and wheat. The regeneration of the four types of explants of the present study was found to be influenced by the presence of 1.0 mg/l to 2 mg/l concentration of BAP, ZN and TDZ along with 100 mg/l L-proline and L-asparagine in the regeneration medium. Syamala et al., (2003) reported that 2 mg/l BAP+ 0.5 mg/l 2, 4-D are suitable to obtain maximum percentage of regeneration in Sorghum. Rathus et al., (2004) observed that, the combination of 1 mg/l IAA and 1 mg/l ZN gave the best results for shoot regeneration. While Manjula et al., (2000) reported shoot initiation on hormone free medium, Rathus et al., (2004) observed deleterious effect of various concentrations of cytokinins on both callus induction and subsequent regeneration. By contrast, the present study found that the addition of cytokinins to the callus induction medium resulted in a better embryogenic callus frequency and regeneration. Eudes et al., (2003) reported that, regeneration of differentiated cereal plant cell from callus remains a major limiting step in obtaining high number of clones or independent transgenic cereal lines. Gao et al., (2005) expressed that Sorghum is one of the most difficult plant species to manipulate through tissue culture. Visarada et al., (2003), Prathibha et al., (2001), Rathus et al., (2004), Bhaskran et al., (2005), Maheswari et al., (2006), Jogeswar et al., (2007) Gurel et al., (2009); Raghuwanshi and Birch, (2010) and reported using different concentrations of cytokinins like BAP, KN, ZN for regeneration. In the present study achieved efficient callus induction and enhanced regeneration from different explants of Sorghum by using appropriate culture conditions like selection of genotype, explant size, culture medium, PGR type and concentration and other environmental factors like temperature and light intensity. 112

43 Another finding from this study was that the use of immature inflorescence as explants for callus induction in Sorghum has advantages over immature embryos. The advantages of using immature inflorescences as opposed to immature embryos are that the former are easier to isolate, do not require elaborate sterilization protocols, withstand physical manipulation (even some physical damage ) better and require less time for material to be ready since planting. Further, the use of immature inflorescence avoids the need to bag panicles to avoid cross pollination and provides more flexibility in harvesting time as immature inflorescences of varying sizes are equally responsive while immature embryos have to be harvested days post anthesis and mm in size. These advantages have also been highlighted by other workers (Gupta et al., 2006; Kishore et al., 2006). Embryogenic callus in plants can be obtained from various explants by manipulation of the exogenous growth regulator concentrations. However, the range of tissue that can be used for tissue culture of Sorghum is limited due to endogenous levels of plant growth regulators, genetic factors, the extent of tissue differentiation and phenolic production (Wernicke et al., 1982; Bhaskaran and Smith, 1988; Bhaskran and Smith 1990). Wernicke et al., (1982) postulated that tissues of different maturities exist within explants which have varying degrees of totipotencies. They suggested that the more immature tissue, the more meristematic cells amenable to embryogenesis were present. They showed that increasing the concentration of 2, 4-D could induce more mature tissues to embryogenic pathway. However, there was limit to this, as cells which matured beyond a threshold were not amenable to callus formation. Thus, the physiology of the Sorghum explants coupled with genotype specificity would determine which explants are amenable to tissue culture. 113