Development of In Vitro Callus Induction and Regeneration Protocol for Newly Released Sugarcane (Saccharum officinarum L.) Genotypes from Inner Leaf Sheath

Abstract

Improvement of sugarcane through conventional breeding takes longer time (8-10 years) to release new improved genotypes. Callus induction and regeneration protocol is a prerequisite for genetic improvement through genetic engineering. Hence, this study was initiated to develop a protocol for in vitro callus induction and regeneration of three sugarcane genotypes (C86-56, C132-81 and SP70-1284) using immature young leaf as a source of explants. Seed cane of 30 cm size having 2 nodes each were obtained from Metehara Research and Development, operating under Ethiopian Sugar Corporation. They were then brought to the National Agricultural Biotechnology Research center and planted in the greenhouse to serve as sources of explants. The treatments were arranged in a factorial experiment laid out in a completely randomized design. For callus induction MS medium supplemented with five levels of 2,4-D (2.0, 2.5, 3.0, 3.5 and 4.0 mg/l) was used. Immature leaves were collected from the green house grown plants 3 months after planting and were cultured on callus induction medium after sterilizing with 5% berekina for 15 minutes and then followed by 70% alcohol for 30 seconds under the biological safety cabinet. For plant regeneration four levels of BAP (0.5, 1.0, 1.5 and 2.0 mg/l) were used in combination with four levels of IBA (0.0, 0.25, 0.5 and 0.75 mg/l). Callus induction experiment was replicated four times and each replicate was represented by four explants per a culture jar and shoot regeneration experiment was replicated three times, 2 embryogenic calli per jar each for regeneration. Data were recorded for all parameters and analyzed using Statistical Analysis System (SAS) Software version 9.3. Means were separated using least significant differences (LSD) at 0.001 probability level. Analyses of variance indicated that days to callus initiation, frequency of callus induction and callus weight were affected significantly (p≤0.001) by genotype, 2, 4-D levels and their interaction. Genotype C132-81 was identified as the best for early callus initiation, high frequency of callus and callus weight. The highest percentage (92.31%) of callus initiation was recorded on medium supplemented with 3.0 mg/l of 2,4-D with higher callus weight (454.5 mg). Regeneration percentage, number of shoots, shoot length and number of leaves per shoot were also significantly (p≤0.001) affected by genotype, BAP, IBA and their interaction effect. Genotype C132-81 also recorded significantly higher number of shoots (43.44±0.5) on MS medium supplemented with 1 mg/l BAP in combination with 0.5 mg/l IBA. Genotype C86-56 gave higher shoot length (6.38±0.08) cm while C132-81 gave the higher number of leaves (8.24±0.06) at 2.0 mg/l BAP+0.25 mg/l IBA. In conclusion, 3.0 mg/l and 3.5 mg/l 2,4-D concentrations were found optimum for callus induction and development, while 1.5 mg/l BAP+0.5 mg/l IBA was the best for plant regeneration.

Keywords: genotype, explant, callus induction, regeneration

DOI: 10.7176/JBAH/11-3-05

Publication date: February 28^th 2021