A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots.

Factors influencing induction, propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

Conventional plant breeding is based on genetic variation and selection of the desired genotypes. The availability of genetic diversity and genetic variation is the starting point of any breeding programme. In most crops, sufficient genetic variation is present among land races, cultivars and their wild relatives. In conventional plant breeding programmes this is followed by several years of selection and field evaluation before a desired genotype is released as an improved cultivar. In many sexually propagated crops, e.g., rice, wheat, maize, barley, the genetic variability is usually recombined through hybridization. The desired recombinants are then selected from the segregating populations in the subsequent generations which are tested, multiplied and released as improved cultivars. However, a number of important crops such as banana, plantain, date palm, citrus, potato, sweet potato, pineapple, apple, and pear are propagated from vegetative parts, and are not amenable to improvement in the same manner as sexually propagated plants. Many of these plants are complex polyploids, take several years to flower and fruit, some are self-incompatible. In others, such as banana and plantain, there is little or no seed formation. Often the propagule size is too big and bulky to handle and to grow large populations. Hence, it is not possible to produce sufficiently large populations required to obtain the desired recombinant in a short duration. In these plants, mutation induction offers the possibility to alter a few characters without disrupting the genome while retaining all the other characters of clones.

In-vitro Techniques and Mutagenesis for the Improvement of Vegetatively Propagated Plants

High-frequency somatic embryogenesis was achieved from an embryogenic cell suspension culture of Acanthopanax koreanum Nakai. Stem segments were cultured on Murashige and Skoog (MS) medium containing auxins and cytokinins. Opaque and friable embryogenic callus formed on MS medium with 4.5 µm 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 µm kinetin or zeatin, but was highest on medium containing 4.5 µm 2,4-D alone. Embryogenic calli were transferred to MS liquid medium containing 4.5 µm 2,4-D and maintained by subculture at 2-week intervals. Initiation of somatic embryogenesis and development up to the globular stage from embryogenic cell clumps occurred in medium containing 0.45 µm 2,4-D, whereas maturation and germination of somatic embryos occurred in MS medium lacking 2,4-D. Cytokinin treatment suppressed the normal growth of embryos, but stimulated secondary somatic embryogenesis from the surfaces of primary embryos. Plants from somatic embryos were acclimatized in a greenhouse.

Somatic embryogenesis and plant regeneration from suspension cultures of Acanthopanax koreanum Nakai.

SummaryEstablishment of fast-growing, highly regenerable callus cultures was examined in Muscari armeniacum Leichtl. ex Bak. in order to develop an efficient genetic transformation system. High-frequency callus formation was obtained from leaf explants of cv. Blue Pearl on media containing 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC). Fast-growing, yellowish nodular callus lines and white friable callus lines containing a few somatic embryos were established on initiation medium supplemented with 4.5 μM 2,4-D and with 54 μM NAA, respectively. The yellowish nodular calluses vigorously produced shoot buds after transfer to media containing 0.44–44 μM 6-benzyladenine (BA), whereas the white friable calluses produced numerous somatic embryos upon transfer to plant growth regulator-free (PGR-F) medium. Histological observation of shoot buds and somatic embryos indicated that the former consisted of an apparent shoot meristem and several leaf primordia, and the latter had two distinct meristematic regions, corresponding to shoot and root meristems. Both shoot buds and somatic embryos developed into complete plantlets on PGR-F medium. Regenerated plants showed no observable morphological alterations. High proliferation and regeneration ability of these calluses, were maintained for over 2 yr.

Organogenesis and somatic embryogenesis from callus cultures in Muscari armeniacum Leichtl. ex Bak. In Vitro Cell Dev

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l−1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l−1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l−1 glycine, 690 mg l−1 proline, and 1.0 g l−1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l−1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l−1 6-benzylaminopurine; addition of 2.0 mg l−1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l−1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.

Jae Whune Kim

Papers

Plant regeneration through somatic embryogenesis from suspension cultures of Allium fistulosum L.