Indian Journal of Biotechnology Vol 15, April 2016, pp 261-265
Effect of genotypes and activated charcoal on high frequency in vitro plant regeneration in sugarcane Pallavi Mittal1, Ruma Devi2* and S S Gosal3 1
School of Agricultural Biotechnology, 2Department of Vegetable Science and 3Directorate of Research Punjab Agricultural University, Ludhiana 141 004, India Received 24 October 2014; revised 2 March 2015; accepted 15 May 2015
A series of experiments were conducted to explore the effect of activated charcoal on the in vitro morphogenic response of 3 sugarcane varieties, viz., CoJ 83, CoJ 88 and CoJ 64. The analysis of variance at 5% level of significance indicated that the differences among different varieties and media composition containing different concentrations of activated charcoal were statistically significant. The MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 mg/L), kinetin (Kin; 0.5 mg/L) and activated charcoal (2.0 g/L) recorded maximum percentage of somatic embryogenesis in CoJ 83 (76.01), followed by CoJ 88 (72.56) and CoJ 64 (64.59). While the best plant regeneration was observed on MS medium supplemented with 6-benzylaminopurine (BAP; 0.5 mg/L) and activated charcoal (2.0 g/L) in CoJ 83 (82.32%), followed by CoJ 88 (73.35%) and CoJ 64 (69.20%). Further, maximum percentage of shoot elongation was observed in CoJ 83, followed by CoJ 88 and CoJ 64, on ½ MS medium supplemented with activated charcoal 2.0 g/L. At the same time, it was also noticed that activated charcoal promoted the shoot length in all the varieties and reduced albinism significantly in micropropagated plants. Thus activated charcoal can be added in the plant regeneration media to enhance somatic embryogenesis and plant regeneration. It can efficiently be also used to improve the frequency of shoot multiplication and transformation. Keywords: Albinism, charcoal, plant regeneration, Saccharum officinarum, somatic embryogenesis
Introduction Sugarcane (Saccharum officinarum L.; 2n=80-205) is an important sugar crop of India, ranks among the world’s top ten food crops and annually provides 60% of the total sugar produced worldwide. The genetic complexity, low fertility and large genotypeenvironment interactions make traditional sugarcane breeding and genetic studies arduous. Further, an efficient photosynthetic rate and higher biomass production make this an excellent target material for industrial processing and a valuable alternative for animal feeding. Because of its global importance, much research has focused on sugarcane crop improvement through plant breeding, and more recently through biotechnological applications. Effective utilization of biotechnological approaches, such as, isolation of somaclonal variants, protoplast fusion and genetic transformation rely on efficient and reliable regeneration systems1-5. Sugarcane tissue culture was first initiated in Hawaii in 1961. Since then plant regeneration has —————— *Author for correspondence: Mobile: 91-9878399555
[email protected]
been reported from young leaves1-4, immature inflorescences5 and apical meristems1, while organogenesis from young leaves 3,6. In most of these studies, regeneration frequencies from the calli are reported to be very low. Moreover, many times due to excessive micro-tillering in vitro, problem of crowding is witnessed, which inhibits subsequent normal shoot elongation, thereby resulting in decreased rate of multiplication of sugarcane. Besides, albinism of regenerated shoots has been reported to be one of the major bottlenecks in sugarcane micropropagation7. Further, during genetic transformation studies, use of antibiotics, such as, carbenecillin, hygromycin and kanamycin, add to the production of weak shoots and sometimes albino shoots also, ultimately resulting into a very low frequency of transformation8,9. However, use of activated charcoal has been reported to enhance the plant regeneration, shoot elongation and decrease the frequency of albino plants in sugarcane10 and cotton11. Therefore, an investigation was carried to determine the impact of charcoal on the somatic embryogenesis, plant regeneration, shoot elongation, length of elongated shoots and occurrence of frequency of
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albino shoots in 3 commercially important varieties of sugarcane, i.e., CoJ 83, CoJ 88 and CoJ 64
values at 5% level of significance were calculated and interpretations were made accordingly.
Materials and Methods Primary explants, i.e., spindles (5-6 cm length and about 1.5 cm diam) were derived from the healthy, field grown apical stem portions of sugarcane varieties CoJ 83, CoJ 88 and CoJ 64 by removing the outer mature leaves. Spindles were first washed with TeepolTM for 5-10 min to remove dirt and debris, followed by washing in running tap water for 10-15 min to remove the adhered detergent. Subsequently, spindles were surface sterilized with 0.1% (w/v) HgCl2 for 9-10 min with gentle agitation and then rinsed thrice with sterile distilled water in a laminar airflow cabinet. The nodal portions of the spindles were discarded and outer 2 or 3 leaf whorls were removed using a sharp scalpel blade. The innermost leaf whorls were cut obliquely into about 1.0 cm pieces, which were wounded at 2-3 places and cultured on MS medium12 containing 3% (w/v) sucrose, solidified with 0.8% (w/v) agar and supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 4.0 mg/L) and kinetin (Kin; 0.5 mg/L). The cultures were incubated in dark at a temperature of 25±2ºC. The calli obtained on callus induction medium were transferred to multiplication medium, followed by regeneration and elongation medium (Table1) containing different concentration of activated charcoal. Plants obtained from embryogenic calli and young leaf rolls (direct shoots regeneration) were transferred on rooting medium [MS+NAA (3.0 mg/L)+IBA (2.0 mg/L)+sucrose (7%)]. Plants with freshly developed roots were thoroughly washed in running tap water and were kept in ½ MS (without sugar and vitamins) in open test tubes under high light intensity for 7-10 d in the incubation room for elongation of roots and hardening of plants. The hardened and fully grown in vitro rooted plantlets were transferred to polythene bags containing field soil with farm-yard manure and kept in the polyhouse for 6-8 wk in the month of February-March and September-October under the temperature 30±2ºC and humidity 70-80%.
Results and Discussion The analysis of variance for the experimental design revealed the significance of mean squares due to variety (g) and charcoal concentrations (c) for all the five parameters studied, i.e., somatic embryogenesis, plant regeneration, shoot elongation, shoot length and albinism (Table 2). This indicated that the in vitro morphogenic response in sugarcane was variety dependent and differed significantly with the concentration of charcoal added to the media.
Statistical Analysis
The observations were recorded on somatic embryogenesis, plant regeneration, shoot elongation, length of shoots and albinism per culture. The data were analyzed following the computer software package CPCS-I using factorial CRD design. CD
Table 1—Different media compositions used during plant regeneration studies in sugarcane Callus induction medium
C0
MS+2,4-D (4.0 mg/L)+Kin (0.5 mg/L)
Multiplication medium
M1
MS+2,4-D (2.5 mg/L)+Kin (0.5 mg/L) MS+2,4-D (2.5 mg/L)+Kin (0.5 mg/L)+charcoal (1.0 g/L) MS+2,4-D (2.5 mg/L)+Kin (0.5 mg/L)+charcoal (2.0 g/L) MS+2,4-D (2.5 mg/L)+Kin (0.5 mg/L)+charcoal (3.0 g/L) MS+BAP (0.5 mg/L) MS+BAP (0.5 mg/L)+charcoal (1.0 g/L) MS+BAP (0.5 mg/L)+charcoal (2.0 g/L) MS+BAP (0.5 mg/L)+charcoal (3.0 g/L) 1/2 MS 1/2 MS+charcoal (1.0 g/L) 1/2 MS+charcoal (2.0 g/L) 1/2 MS+charcoal (3.0 g/L) MS+NAA (3.0 mg/L)+IBA (2.0 mg/L)+sucrose (7.0%)
M2 M3 M4 Shoot regeneration medium
S1 S2 S3 S4
Elongation medium
Rooting medium
E1 E2 E3 E4 R0
Table 2—Analysis of variance (mean square values) for per cent somatic embryogenesis, shoot regeneration, shoot elongation, shoot length (cm) and albinism in relation to varieties and media compositions Source
Somatic Shoot Shoot Shoot Albinism embryogenesis regeneration elongation length (%) (%) (%) (cm) (%)
Variety (g) 473.541* Charcoal 237.67* conc. (c) Interaction 10.12* (g×c) Error 2.86 *Significant at P=0.05
488.46*
376.01* 10.69* 111.499*
41.15*
226.54* 21.12* 531.90*
3.20
5.37*
0.67
5.76
2.31
1.99
0.57
4.81
MITTAL et al: IN VITRO PLANT REGENERATION IN SUGARCANE
Callus Induction and Somatic Embryogenesis
The initiation of primary calli was observed 8-12 d after incubation from the cut ends of the explants as these tissues absorb more nutrients leading to rapid cell division and subsequent callus formation (Fig. 1A). After 30 d of incubation, the maximum percentage of callus induction was recorded in CoJ 83 (94.73%) (Fig. 1B), followed by CoJ 64 (82.45%) and CoJ 88 (46.15%). It was also observed that variety CoJ 83 and CoJ 64 formed creamish white, nodular, friable and compact calli, whereas variety CoJ 88 formed watery and sticky calli. This shows that genotypic differences existed with regards to quantity and quality of the callus induction among the varieties. Earlier, similar genotypic differences on callus induction have been reported in sugarcane6,13,14. The primary calli obtained on induction medium when sub-cultured on multiplication medium supplemented with different concentration of activated charcoal significantly enhanced the somatic embryogenesis in all the varieties of sugarcane after 15 d of sub-culture. On multiplication medium, the highest somatic embryogenesis recorded was 76.01, 72.56 and 64.59% in CoJ 83, CoJ 88 and CoJ 64,
Fig. 1 (A-G)—High frequency in vitro plant regeneration in sugarcane: (A) Callus initiation and browning of the media and explants after 12 d of incubation; (B) Callus after 30 d of incubation; (C) Stereomicrograph (16×) showing shoot regeneration from embryogenic calli after 15 d of culture; (D) Effect of charcoal on shoot regeneration after 15 d of incubation; (E) Effect of charcoal on shoot elongation after 15 d of incubation; (F) Hardening of regenerated plantlets in test tubes; & (G) Establishments of plants under field conditions in polybags.
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respectively in Ludhiana (Fig. 2). Addition of activated charcoal could promote the production, maturation and germination of somatic embryos11. The best medium for the differentiation and germination of somatic embryos was MS with 0.1 mg/L activated charcoal. Darkening of the culture medium was observed in the cultured explants of all the varieties due to release of some phenolic substances from the cut ends of the explants (Fig. 1A). Darkening is considered an undesirable phenomenon as it affects the nutrient uptake by blocking the cut ends of explants thereby decrease callusing efficiency13,15,16. The percentage increase in somatic embryogenesis was higher in CoJ 88 as compared to CoJ 83 and CoJ 64 owing to adsorption properties of activated charcoal as this variety released higher amount of phenolics and resulted in higher media darkening. Activated charcoal (AC) is composed of carbon, arranged in a quasigraphitic form of small particle size. In Vitro Shoot Regeneration
The data presented in Fig. 3 shows the effect of charcoal on plant regeneration. The maximum plant regeneration was recorded in variety CoJ 83 (82.32%), followed CoJ 88 (73.35) and CoJ 64 (69.20) on the regeneration medium supplemented with 2.0 g/L charcoal (Figs 1C & D). Further increase or decrease beyond this concentration of charcoal, considerable decrease was observed in the percentage of plant regeneration. Charcoal has been reported to increase the plant regeneration by many workers 10,11,17-19.
Fig. 2—Effect of medium composition containing different concentrations of charcoal on somatic embryogenesis in three commercial varieties of sugarcane.
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Fig. 3—Effect of medium composition containing different concentrations of charcoal on plant regeneration in three commercial varieties of sugarcane.
Fig. 5—Effect of media composition containing different concentrations of charcoal on shoot length in three commercial varieties of sugarcane.
Fig. 6—Effect of media composition containing different concentrations of charcoal on albinism in three commercial varieties of sugarcane. Fig. 4—Effect of media composition containing different concentrations of charcoal on percentage shoot elongation in three commercial varieties of sugarcane. In Vitro Shoot Elongation
The shoot elongation in the different varieties of sugarcane on the elongation medium supplemented with activated charcoal is shown in Fig. 4. The maximum percentage of shoot elongation was observed in CoJ 83 (98.53%), followed by CoJ 88 (92.57%) and CoJ 64 (85.09%) on the elongation medium supplemented with 2.0 g/L activated charcoal. It was also recorded that charcoal significantly promoted the shoot length in all the varieties of sugarcane (Fig. 5). With the addition of 2.0 g/L charcoal, the maximum shoot length of 10.15 cm was achieved in variety CoJ 83, followed by CoJ 88 (8.42 cm) and CoJ 64 (7.54 cm) (Fig. 1E).
The addition of charcoal in the culture medium also boosted up the photosynthetic machinery of the sugarcane plants, thereby reduced the frequency of albinism in micropropagated plants. The data presented in Fig. 6 reveals that minimum albinism was recorded in CoJ 83 (18.43%), followed by Co J 64 (19.88%) and Co J 88 (23.60%) owing to genetic differences. The plants were healthier and dark green in appearance in the charcoal supplemented medium (Figs 1D & E). Use of charcoal in the culture medium promoted somatic embryogenesis, shoot regeneration and shoot elongation, even from vitrified shoot clumps, and thus improved the rate of shoot multiplication through micropropagation in sugarcane. Activated charcoal has earlier been reported to enhance plant regeneration, shoot
MITTAL et al: IN VITRO PLANT REGENERATION IN SUGARCANE
elongation and decrease the frequency of albino plants in sugarcane10 and cotton11,20.
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Rooting and Acclimatization
After 15 d on elongation medium, plantlets were transferred onto the rooting medium. Plantlets were then thoroughly washed in running tap water and kept in ½ MS (without sugar and vitamins) in open test tubes under high light intensity for 7-10 d in the incubation room for elongation of roots and hardening of plants. The hardened and fully-grown in vitro rooted plantlets were transferred to polythene bags containing field soil with farm-yard-manure and kept in the polyhouse for the field establishment (Figs 1F & G). The plantlets transferred to soil exhibited good survival rate ranging from 85-90%. Thus it is concluded that an optimum level of charcoal is helpful in boosting up of in vitro shoot regeneration by improving somatic embryogenesis and plant regeneration system in sugarcane.
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