In Vitro PROTOCOL DEVELOPMENT FOR SOMATIC EMBRYOGENESIS, MERISTEM AND ANTHER CULTURE OF ANCHOTE [Coccinia abyssinica (Lam.) Cong.]

Abstract

Anchote is one of the most important but underutilized indigenous root tuber crops in Ethiopia. The scarcity of quality planting materials and improved varieties are the most important factors limiting production of the crop. Considering the huge importance of plant biotechnological tools in the improvement of anchote, experiments were conducted to develop in vitro protocols for somatic embryogenesis, meristem culture and anther culture of two anchote accessions (KUWE and accession number 223098), each from different genotype. Optimization of surface sterilization for each explant source was done prior to culture establishment. The concentration of 6-Benzylamimopurine (BAP) with and without Gibberellic Acid (GA3) and Naphthalene Acetic Acid (NAA) for meristem establishment while BAP and Kinetin (Kn), singly and in combination, for shoot multiplication were evaluated. Level of Indol Butyric Acid (IBA) and Indol Acetic Acid (IAA), each on both full and half nutrient-strength of Murashige and Skoog medium (FMS and HMS), were tested for root induction. Concentration and combination of 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and BAP were evaluated for callus induction from leaf and anther explants. Level of BAP alone and with 2, 4-D was evaluated, on both FMS and HMS, in somatic embryo development. Level of BAP was evaluated, on both FMS and HMS media with and without activated charcoal (AC), in somatic embryo conversion. The concentration of BAP, on FMS + AC, was tested for shoot regeneration from anther callus. About 81% of meristems were found clean in shoot buds surface sterilized with 1% NaOCl for 10 minutes. Leaf explants of 80% in genotype 223098 and 76% in genotype KUWE were found clean in leaves surface sterilized with 1 and 0.5% NaOCl, respectively each for 10 minutes. The highest percent (85.94%) of clean anthers was recorded from flower buds surface sterilized with 1% NaOCl for 5 minutes. About 80% of meristems shoot induction frequency was recorded, in genotype 223098, at 1 mg/L BAP while about 71% of meristems, in genotype KUWE, induced shoot at 0.5 mg/L BAP. The highest shoot number (6.52), in genotype 223098, was recorded from meristem shoot multiplication medium supplemented with 0.5 mg/L BAP while, in genotype KUWE, 0.5 mg/L BAP + 0.5 xvii mg/L Kn produced 6.32 shoots. FMS + 0.5 mg/L IBA resulted in 100% root induction frequency of meristem derived shoots with no sign of callus development. About 58% of plantlets, in genotype KUWE, and 42% of plantlets, in genotype 223098, survived after a month of acclimatization. Leaf explants of both genotypes supplemented with combinations of 2, 4-D and BAP resulted in 100% callus induction frequency. Higher levels of 2, 4-D (4 and 8 mg/L) combined with BAP promoted embryogenic callus development; 8 mg/L 2, 4-D + 1 mg/L BAP being the best. The highest number (36.25) of somatic embryos was recorded from genotype 223098 on HMS + 2 mg/L BAP while FMS + 0.5 mg/L BAP produced 34 somatic embryos in genotype KUWE. Somatic embryo conversion was achieved only on FMS medium amended with AC. The highest number of total shoots (5.75), 4.00 of them with root, was recorded, in genotype 223098, at 2 mg/L BAP. A maximum number of shoots (4.75), in genotype KUWE, was also recorded at 2 mg/L BAP while the maximum number of plantlets (3.75) at 1 mg/L BAP. The survival rates of plantlets obtained from somatic embryos were 42% and 34%, in genotype KUWE and 223098, respectively. The highest percent (84.37%) of anthers, in genotype KUWE, formed calli at 1 mg/L of each 2, 4-D and BAP while a maximum of 62.50% of anthers, in genotype 223098, induced callus at 1 mg/L 2, 4-D + 2 mg/L BAP. Lower levels of 2, 4-D (1 and 2 mg/L) + BAP induced massive, greenish-white, morphogenic callus in earlier days of anther culture. The highest shoot number (4.25) in anther culture was recorded, in genotype KUWE, at 2 mg/L BAP while a maximum of 3.75 shoots was obtained, in genotype 223098, at 4 mg/L BAP. Out of 10 sample plantlets, three in genotype 23098 and five in genotype KUWE, were found haploids. After a month of acclimatization, the survival rate of anther-derived plantlets were 32% and 28% for genotype KUWE and 223098, respectively. In general, in vitro protocols for somatic embryogenesis, meristem culture and anther culture of both KUWE and 223098 genotypes of anchote successfully developed in this study.