Search

Cotyledon segments derived from zygote embryos of mango (Mangifera indica L. cv. Zihua) were cultured on agar medium for 28 days. Depending on different pre-treatments with plant growth regulators, two distinct patterns of adventitious roots were observed. A first pattern of adventitious roots was seen at the proximal cut surface, whereas no roots were formed on the opposite, distal cut surface. The rooting ability depended on the segment length and was significantly promoted by pre-treatment of embryos with indol-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for 1 h. A pre-treatment with the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) completely inhibited adventitious root formation on proximal cut surfaces. A second pattern of roots was observed on abaxial surfaces of cotyledon segments when embryos were pre-treated with 2,700 μM 1-naphthalenacetic acid (NAA) for 1 h. Histological observations indicated that both patterns of adventitious roots originated from parenchymal cells, but developmental directions of the root primordia were different. A polar auxin transport assay was used to demonstrate transport of [3H] indole-3-acetic acid (IAA) in cotyledon segments from the distal to the proximal cut surface. In conclusion, we suggest that polar auxin transport plays a role in adventitious root formation at the proximal cut surface, whereas NAA levels (influx by diffusion; carrier mediated efflux) seem to control development of adventitious roots on the abaxial surface of cotyledon segments.

For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl−1 (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl−1 (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

In this paper, we described the direct somatic embryogenesis from both immature cotyledon cuts and nucelli in the same mango cultivar (Mangifera indica L. var Zihua), studied the effect of growth conditions of embryogenic cultures (EMs) on cryopreservation and compared the cryopreservation response of EMs induced from these two different explants. Histological studies demonstrated that EMs derived from nucelli could be induced directly from epidermal cells of both sides of nucelli, whereas EMs derived from cotyledon cuts were induced only from epidermal cells of the adaxial side of the cotyledons. EMs from either nucelli or cotyledon cuts could be maintained in liquid medium or on solid medium and cryopreserved using a vitrification procedure. Success of cryopreservation of EMs depended on the dehydration treatment and the defined growth conditions during culture but not on their origins. When EMs were sampled during their exponential growth phase in liquid medium and dehydrated with PVS(3) solution for 5 min, survival of the EMs induced from cotyledon cuts and nucelli reached 77.7 and 80%, respectively, after cryopreservation in liquid nitrogen for 24 h. Furthermore, when dehydrated with PVS(3) solution for 30 min, all EMs induced from cotyledon cuts and 96.7% of EMs induced from nucelli could survive after cryopreservation. Cryopreservation did not affect the plant regeneration potential of EMs through somatic embryogenesis. The protocols of somatic embryogenesis and cryopreservation of mango EMs established in this study may offer potential ways to improve mango germplasm conservation and genetic improvement.

Changes in the levels of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) and polyamines were simultaneously investigated during the early phases of alfalfa somatic embryogenesis. These included the period of induction and subculture of callus, and 3- and 7-day suspension cultures for the induction of somatic embryogenesis. The polyamines contained in the embryogenic callus were found to include putrescine (Put), spermidine (Spd) and spermine (Spm), but the level of Spm was much less than that of Put and Spd. There was a dramatic increase in MACC after induction of embryogenesis, and ACC levels were lower in somatic embryos than in embryogenic callus. Induction of embryogenesis for 3 days increased the levels of ACC and polyamines to a maximum level, and these then reduced as the embryogenesis proceeded. The ratios of Put/Spd and ACC/MACC were decreased during the induction. This indicated that both high levels of ACC and polyamines might be a prerequisite for early differentiation during the induction of the embryogenesis. Thus, there appears not to be competition between polyamine biosynthesis and ethylene biosynthesis at least during the induction of somatic embryogenesis, because both the polyamines and ACC were simultaneously increased during the induction period. Conversion of ACC into MACC and the maintenance of a relatively high level of polyamines, especially Spd, appear to be important for further development of the embryos. When aminooxylvinylglycine (AOA) was added at the initiation of the callus subculture, it had no significant effect on the callus growth, the ethylene production and ACC level of the callus. However, AOA increased the numbers of the embryos accompanying an increase in Spd level and S-adenosylmethionine decarboxylase (SAMDC) activity. Thus, the AOA effect could be associated with Spd increase rather than with the effect of ethylene biosynthesis.

The histological origin of organogenesis was studied during plant regeneration of banana (Musa AAA cv. Williams) via direct organogenesis from micro-cross-sections of the rhizome in modified Murashige–Skoog (MS) medium (including 10 µm 6-BAP, 1 µm IAA and 50 µm KN). The frequency of the organogenesis was dependent on the position from where the explant was cut. Generally, more adventitious shoots were obtained from explants cut from regions close to apical meristem. The origin of adventitious shoots was multicellular, from cell layers with active cell division in the epidermis of the rhizome axis. After 24 h of culture, a layer of epidermal cells close to the leaf sheath base was actively dividing, and as the time in culture increased, regions containing actively dividing cells became enlarged. By the fifth day of culture, regions of actively dividing cells covered the whole peripheral zone of cortex. Several meristemoid structures were formed from the peripheral zone of cortex after 7 days of culture. These structures developed into adventitious shoots by the ninth day of culture. Vigorous plantlets could be regenerated from the shoots when they were transferred to rooting medium containing MS basal salts supplemented with 30 g L–1 sucrose, 6.5 g L–1 agar, a vitamin/amino acid mixture, 1 µm NAA and 1 µm 6-BAP.