We have obtained friable, non-mucilaginous embryogenic callus from cultured immature embryos of an elite maize inbred line derived from Iowa stiff stalk synthetic germplasm. This callus was used to generate embryogenic cell suspensions containing compact clumps of densely cytoplasmic cells. Plants were readily regenerated from the suspensions, which were also an excellent source of protoplasts. The suspension cultures maintained their plant regeneration capacity through many months. The cultures were cryopreserved in liquid nitrogen and embryogenic callus and suspension cultures were routinely recovered from cryopreserved cells. Protoplasts were isolated in large numbers from both primary and cryopreserved suspension cultures. Cultured protoplasts divided and formed embryogenic callus both when plated in agarose and on nurse cultures. Hundreds of plants were regenerated from protoplast-derived calli. Plants with viable progeny were obtained although many of the regenerates showed morphological and reproductive abnormalities.

Regeneration of fertile plants from protoplasts of elite inbred maize

We report plant regeneration from maize (Zea mays L.) protoplasts with recovery of fertile plants from a Cateto inbred line adapted to tropical regions. Protoplasts were isolated from embryogenic cell suspension cultures capable of regenerating fertile plants. Cell colonies and embryogenic calli were obtained at high frequencies when protoplasts were cultured either in a thin layer of liquid medium or on cellulose-nitrate filters placed on a feeder layer of maize cells. High plating efficiencies were obtained with both culture methods using either a modified KM-8p or a modified N6 culture media. The results confirmed that maize protoplasts maintain the regeneration capacity of donor cells. We also demonstrated that high plating efficiencies and plant regeneration from maize protoplasts can be achieved with different culture methods. Furthermore, we recovered fertile plants with viable progenies from maize protoplasts.

Plant Regeneration and Recovery of Fertile Plants from Protoplasts of Maize (Zea Mays L.)

Regenerants from a 30-month-old haploid and a 10-month-old diploid tissue culture were cross-pollinated to generate a synthetic genotype (HE/89) with improved competence for maintenance of totipotency in various cultured expiants. The HE/89 zygotic embryos developed friable, embryogenic cultures in the commonly used MS-and N6-based media without the addition of L-proline. By optimalization and changing the culture conditions, we were able to regulate the maintenance of the earlier, more synchronous (Type II) and the later, asynchronous (Type I) in vitro embryogenesis, as well as the shift between different ontogenic stages. Within 70 days after the inoculation of immature embryos a relatively homogeneous, early-embryogenic suspension culture usable for protoplast isolation was established from the initially surface-grown cultures. Using modified solutions for protoplast isolation and culture, viable protoplasts were reproducibly obtained from which plants were regenerated via defined ontogenic steps. Despite the long in vitro history of the parental genotypes, 60–70% of the more than 500 plants derived from the HE/89 protoplasts set seeds following self or sib-pollination.

An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture.

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley ( Hordeum vulgare L.)

In order to evaluate the impact of ethylene in maize tissue culture, silver nitrate has been used as an inhibitor of ethylene action. Type II callus initiation rate was improved when immature embryos were cultured on a modified Murashige & Skoog medium containing various concentrations of silver nitrate (5, 10, 20 mgl-1). Regeneration ability of calli initiated and maintained in presence of silver nitrate was enhanced. No modification of callus growth rate neither of ethylene production has been detected.

Enhancement of production and regeneration of embryogenic type II callus in Zea mays L. by AgNO3

Maize (Zea mays L) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos The somatic embryos can be induced to form roots and small leaf-like structures The genotype was the hybrid A188xBlack Mexican Sweet Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos The basal medium for the suspension culture was N6 (CC Chu et al, 1975, Scientia Sinica 18, 659–668) The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 40 mgl Protoplasts had to be cultured in a low-osmoticum medium (03 M mannitol) for subsequent cell divisions to occur The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S)

K. L. Chang

Papers

Embryogenic callus formation from maize protoplasts