In vitro propagation of elite sugarcane (Saccharum officinarum L.) Genotypes in liquid media using shoot tips

Abstract

Conventional vegetative propagation of sugarcane generally has low multiplication rate and allows dissemination of diseases. This results in shortage of quality planting materials. In vitro propagation is emerging as powerful technique to alleviate such limitations. To date, there is no protocol developed for in vitro propagation of commercial sugarcane genotypes through liquid culture in Ethiopia. Therefore, the present study was conducted with the aim of optimizing the protocol for in vitro propagation of two sugarcane genotypes (N52 and N53) in liquid culture through shoot tip culture. Experiments on shoot multiplication, in vitro rooting, ex vitro rooting and acclimatization were laid out in completely randomized design with factorial treatment arrangements. For in vitro multiplication, different concentrations and combinations of BAP (0, 0.5, 1, 1.5 and 2 mg/l) and Kinetin (0, 0.5, 1 and 1.5 mg/l) were used. For in vitro root induction, ½ strength MS liquid medium supplemented with different concentrations and combination of Sucrose (0, 40, 50, 60 and 70 g/l ) and NAA (0,3,5 and 7 mg/l) were used. In ex vitro rooting, uniform micro-shoots were dipped in different concentrations of NAA (0, 10 20, 30 and 40 mg/l) at their basal ends and transferred onto tray filled with sand and soil substrate mixture in 2:1 ratio. For acclimatization, substrate mixtures of sand + soil + farmyard manure were used in six different ratios. Data was subjected to analysis of variance (ANOVA) and means were separated using REGWQ (Ryan-Einot-Gabriel-Welsch). With regard to shoot multiplication, genotype N52 showed a maximum of 6.95 ± 0.19 shoots per explant with 4.75 ± 0.06 cm shoot length on a medium fortified with 3% sucrose and 2 mg/l BAP + 0.5mg/l kinetin while genotype N53 produced a maximum of 6.30 ± 0.26 shoots per explant with 3.94 ± 0.03 average shoot length on a medium supplied with 3% sucrose and 1.5 mg/l BAP and 0.5 mg/l kinetin. Half MS liquid medium + 50 g/l sucrose + 3 mg/l NAA induced the highest rooting (100%) with an average root number per shoot of 23.5 ± 1.29 for N52. For N53, ½ MS liquid medium supplemented with 5 mg/l NAA + 50 g/l sucrose induced the highest rooting response of 100% with an average root number per shoot of 21.76 ± 0.57. In ex vitro rooting, 20 mg/l NAA was found optimal concentration with the highest (76%) rooting frequency with an average of 8.06 ± 0.13 root number per shoot for N52 whereas 30 mg/l NAA gave a maximum of 70% rooting frequency with 4.52 ± 0.19 average root number per shoot for N53. In acclimatization, best survival rate with vigorous growth was achieved on substrate mixtures containing sand + soil in 1:1: ratios in both N52 and N53. From the present results we can conclude that Ms + 2 mg/l BAP with 0.5 mg/l Kinetin was the best combination for shoot multiplication of N52, while MS + 1.5 mg/l BAP with 0.5 mg/l Kinetin was optimum for best multiplication of N53. For in vitro rooting of genotype N52 and N53, ½ MS liquid medium supplemented with 3 mg/l NAA + 50 g/l sucrose and 5 mg/l NAA + 50 g/l sucrose were the optimal combination, respectively. For ex vitro rooting of N52 and N53, 20 mg/l and 30 mg/l NAA were the best concentrations, respectively. Substrate mixture composed of sand + soil in 1:1 ratio found to be the best acclimatization media for both genotypes. Finally, it could be suggested that this protocol can be used for rapid in vitro propagation of these genotypes. Developing protocol for these genotypes using bioreactor and other PGRs types and combinations are the future line of work suggested.