IN VITRO PLANT REGENERATION, SOMATIC EMBRYOGENESIS AND MOLECULAR DIVERSITY OF ETHIOPIAN POTATO [Plectran-thus edulis (Vatke) Agnew]

Abstract

Ethiopian potato (Plectranthus edulis) is an indigenous, underutilized tuber crop which has received very little attention in research, development, conservation, production and utiliza-tion. Considering the immense potential of biotechnological methods for conservation and im-provement of Ethiopian potato, the present study was designed to develop in vitro protocols for plant regeneration and somatic embryogenesis as well as to assess its genetic diversity. The study was conducted at National Agricultural Biotechnology Research Center, Holleta. Three commonly grown genotypes namely Unnuka, Lofuwa and Chankua were used for the tissue culture study. Surface sterilization of the respective explant for each tissue culture experiment was optimized using various levels of NaOCl at different time. Shoot induction and multiplica-tion of nodal culture were evaluated with application of BAP/ KN alone and with 0.1 mg/L GA3. The effect of IBA and IAA as well as media strength were tested on rooting of nodal culture derived microshoots. Different potting substrates were assessed for acclimatization of plantlets. Meristem initiation was evaluated at various levels of BAP alone and with GA3. Subsequent shoot multiplication was performed with application of BAP/ KN alone and with IBA. Half and full strength MS media containing IBA and NAA separately were used for rooting of mericlones. In somatic embryogenesis, the effect of 2, 4-D alone and with KN/ BAP and IAA with KN/ BAP were tested for callus induction. Successively embryogenic calli were transferred to KN medium alone and with IAA for embryo development and maturation. Cytokinins (BAP, KN and TDZ) with GA3 were evaluated for embryo conversion. The tissue culture parts of experiments were done using CRD in factorial arrangement with four replications. Twenty EST-SSR markers de-veloped from P. barbathus ESTs were studied for their amplification in Ethiopian potato. Among the 20 SSR markers, 15 were transferable to Ethiopian potato and used for diversity analysis of 130 genotypes. Surface sterilization with 1.5% NaOCl for 10 minutes was effective for nodal explant while 1% NaOCl for10 minutes for shoot tip and leaf explants. In nodal cul-ture, early shoot initiation and the highest shoot induction frequency were recorded in MS me-dia containing 1 mg/L BAP and 1 mg/L BAP + 0.1 mg/L GA3 regardless of genotypes. Shoot multiplication medium containing 1 mg/L BAP + 0.1 mg/L GA3 for Unnuka and Lofuwa and 2 mg/L BAP + 0.1 mg/L GA3 for Chankua were most suitable, produced optimum shoot number. In terms of shoot length, PGRs free medium in lofuwa and 0.5 mg/L KN + 0.1 mg/L GA3 in Chankua and Unnuka resulted in the highest shoot length. Earlier root initiation of nodal de-rived shoots was observed in half strength media with 1 mg/L IBA for Chankua and Unnuka while for Lofuwa earlier shoot initiation observed on half strength media with 0.5 mg/L IBA. The highest rooting frequency was achieved at 1 mg/L IBA followed by 1 mg/L IAA irrespective of the genotypes. Half strength media with 1 mg/L IBA showed highest rooting performance for XXII Unnuka and Chankua. The same media strength with 1 mg/L IAA was effective for Lofuwa rooting. River sand alone was the best acclimatizing substrate followed by sand + soil in 2:1 ratio for all genotypes. In meristem culture initiation, Lofuwa initiate shoots faster at BAP 1 mg/L + 0.2 mg/L GA3 followed by Unnuka and Chankua at 0.5 mg/L BAP+ 0.2 mg/L GA3. Genotypes Unnuka and Lofuwa showed highest initiation frequency in media with 1 mg/L BAP + 0.2 mg/L GA3, whereas genotype Chankua, showed the best result in 1.5 mg/L BAP + 0.2 mg/L GA3. Media with 0.75 mg/L BAP + 0.25 mg/L IBA was the most suitable for shoot multi-plication of Unnuka and Chankua which showed best performances for most of the parameters. Lofuwa gave best result at 1 mg/L BAP + 0.25 mg/L IBA. The maximum shoot length was measured on PGRs free medium for the three genotypes. In rooting of mericlones half strength MS media with 0.5 mg/L IBA was the optimum for genotype Chankua and Lofuwa with respect to all rooting parameters whereas, highest rooting for Unnuka recorded at full strength MS media with 1mg/L IBA. Calli were induced in all media, except PGRs free media. For embryo-genic callus formation, media with 1-1.5 mg/L 2, 4-D + 0.5 mg/L KN was effective for Unnuka and Lofuwa, whereas in Chankua the highest percentage of embryogenic callus was obtained at 1 mg/L IAA + 0.5 mg/L KN. Half strength MS media with 0.2 mg/L KN + 0.1 mg/L IAA gave the highest mean number of somatic embryo for genotype Unnuka and Lofuwa. whereas for Chankua the same media containing 0.4 mg /l KN+ 0.1 mg/L IAA induced highest number of somatic embryos. Media with 1 mg/L BAP + 0.5 mg/L GA3 resulted in the highest conversion rate, shoot number and number of shoots with roots in genotype Unnuka. For Lofuwa and Chankua medium with 0.5 mg/L TDZ + 0.5 mg/L GA3 was effective for embryo conversion. Out of the 15 transferable EST-SSR primers 14 were polymorphic. The number of alleles per locus ranged from 4-10 with average of 7.2 alleles. Allele frequency ranged from 0.23 to 0.54 with mean value of 0.40. The PIC values showed high genetic diversity. The Intra-population allelic variations contributed 97% of genetic diversity while only 3% was due to variation among populations. The 130 genotypes were clustered into three major clusters irrespective of geo-graphical location. In general, the results obtained from the study could be used in management and improvement the crop for better utilization.