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

Abstract

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.

Maize Histone Deacetylasehda101Is Involved in Plant Development, Gene Transcription, and Sequence-Specific Modulation of Histone Modification of Genes and Repeats

“…The resulting cassettes were used to generate the pRpd3-53 and pRpd3-35 plasmids ( Figure 1A). These plasmids were employed to transform protoplasts from maize suspension cell line HE-89 (Morocz et al, 1990) using the polyethylene glycol method. Two weeks after protoplast transformation, the microcalli were plated on agar-solidified medium containing 200 mg/L DL-gluphosinate.…”

Coleoptilar node - A season-independent explant source for in vitro culture in maize (Zea mays L.)

“…Plant regeneration has also been reported from calli initiated from different explants, namely, anthers [2][3][4], glumes [5], immature inflorescence [6], immature tassels [7][8][9], leaf segments [10][11][12], protoplasts [13], seedling segments [14][15][16], shoot tips [17][18][19], shoot apical meristem [20], mature embryos [15,21,22], and immature embryos [23][24][25][26][27][28][29][30].…”

Improvement of Longevity and Viability of Sperm Cells Isolated from Pollen of Zea mays L.

“…In isolation and/or culture of Z. mays protoplasts, 0.6 or 0.7 M mannitol, 0.7 M sorbitol, or 0.06 M sucrose with 0.33 M mannitol were used (1,5,8,15,16). Furthermore, the use of several different sugars, including glucose, fucose, galactose, maltose, and sucrose, was also reported in the culture of Z. mays protoplasts isolated from an embryogenic suspension culture (18); reasons for choosing these sugars were not indicated. In our studies, use of either mannitol or sorbitol resulted in rapid cell lysis.…”