Effect of Growth Regulators Concentrations on in Vitro Multiplication of Three Elite Sugarcane (Saccharum Ocinarum L.) Genotypes using Shoottip Culture

: Conventional vegetative propagation of sugarcane generally has low multiplication rate and allows distribution of diseases. Micropropagation is the only practical means of achieving rapid, large-scale production of disease-free quality planting material. Experiments on shoot tip culture initiation and shoot multiplication were laid out in completely randomized design with 2x3x3 and 4x5x3 factorial treatment arrangements respectively. Data was subjected to analysis of variance (ANOVA) and significant means were separated using Duncan's multiple range tests. With regard to shoot multiplication, genotype Q200 showed a maximum of 13.59 shoots per explant with 5.83cm shoot length on a medium fortified with 2 mg/l BAP alone, while genotype Q217 produced a maximum of 15.28 shoots per explant with 5.37cm mean shoot length on a medium supplied with 2.0 mg/l BAP and 0.25 mg/l kinetin. Likewise, Co-0238 produced maximum of 13.56 shoots per explant with mean shoot length 6.50 cm on medium fortified with 1.5 mg/l BAP + 0.5mg/l kinetin

Rapid clonal propagation of sugarcane planting materials depends on the genotype and the plant growth regulators combinations used and needs to develop plant growth regulators combinations for each genotype (Singh et al., 2006, Melaku et al., 2016. Plant growth regulators necessities for in vitro propagation responses differ from cultivar to cultivar in sugarcane (Pathak et al., 2009). It is necessary that an efficient protocol is needed for every new variety or clone of sugarcane to get rapid shoot initiation and shoot multiplication. Thus, this research work was designed with the objective to study effect of growth regulators concentrations on in vitro multiplication of sugarcane genotypes (Q200, Q217 and Co-0238) using shoot tip explants

Materials and methods
The study was conducted at Holeta National Agricultural Biotechnology Laboratory of the Ethiopian Institute of Agricultural Research. Three elite sugarcane genotypes (Q-200, Q-217 and Co-0238) were used for the study. They were obtained from the Ethiopian Sugar Corporation, Research and Training Division. The Sugar Corporation imported Q-200 and Q-217varieties from Australia and Co-0238 variety from India. These genotypes are among the selected ones to be commercialized by the Ethiopian Sugar Corporation for their cane yield performance and sugar quality.

Culture Media Preparation and Sterilization
The basal medium employed for the culture is MS medium (Murashige and Skoog, 1962).
The required amount of macronutrient, micronutrient, vitamins from their respective stock solution were dissolved in double distilled water along with 30 g/L sucrose (for initiation and multiplication) The final volume was made up to the required level with double distilled water and then divided into required volume of treatments, to which amount of PGRs from stock solution were added in combinations at different concentrations. The pH was adjusted in all cases to 5.8 by using 1N NaOH and/or 1N HCL before gelling with agar. Agar-Agar type I (4.5 g/l for culture initiation) and Phytagel (2.0 g/l for multiplication) were added to the nutrient and heated on magnetic hot plate till it was completely melted. Before autoclaving, the media was poured into washed and dried test tubes of 30 ml volume for initiation and 50 ml volume of culture jars for multiplication, then, capped and labeled properly. The dispensed medium was then autoclaved at 121º C for 15 minutes at 15-psi pressure.

Explant collection and sterilization
The intact leaves were removed, and the segments with leave sheath were taken to the laboratory for surface sterilization and explant preparation.

Data Analysis
The analysis of variance for different variables was performed by GenStat Version 15.1, (GenStat15.1 (64-bit Edition) 2012, VSN International Ltd.) and for significantly different treatments, mean separation was done with Duncan's multiple range tests at or below the probability level of 5%

Statement on Plant Guidelines
This study was performed in accordance with the relevant institutional, national, and international guidelines and legislation.

Culture Initiation
The shoot tip explants on full MS media lacking of plant growth regulator (control) failed to grow. However, shoot initiation was observed in MS media supplemented with PGR. It is evident from Table 2  2.0 mg/L BAP alone was found to be the best for sugarcane genotype Q217 and Q200, respectively. Comparison of the three genotypes showed that Q217 was a better responsive than Q200 and Co-0238 for in vitro multiplication in full MS medium.

Conclusions
From the result obtained in the study, it is concluded that the developed protocol is helpful for rapid in vitro propagation of the sugarcane planting materials and hence enhance the availability of healthy and true to type planting materials in Ethiopian sugarcane plantations