Plant Biotechnology Institute

Microsurgery of immature embryonic axis and recovery of plants through in vitro culture

--V. R. Bommineni*

*University of Western Ontario, London, Ontario, Canada

We reported the recovery of mature, fertile plants from the in vitro culture of shoot apical meristem for a variety of genotypes (MNL 63:87-88, and 64:79; Plant Cell Tissue Organ Cult. 19:225-234, 1989). In these dissections, the shoot apical meristem dome was exposed by removing the leaf primordia layers without disturbing the apical meristem cells. This report considers a culture technique to recover the plantlets from bisected embryonic axes of immature embryos.

As reported earlier, 11 to 14 days after pollination (DAP) ears of A188 were surface sterilized with 10% 'Javex' for 20-25 min, rinsed three times with sterile water, and embryos were dissected in the culture hood under the microscope. A single median longitudinal incision was made through the embryonic axis by using a sterile razor blade. The two halves of each embryo were separated and placed on MS medium with no plant hormones (MNL 63:87-88, and 64:79). In a second series, the median longitudinal incision was made in such a way that the bisected embryonic axis was kept intact at the basal surface of the scutellum (referred to as graft sections in Table 1). Two weeks after our standard in vitro culture, the recovered plantlets were transplanted into soil (pots) and grown in the glasshouse.

Data on the recovery of plants and their maturity are summarized in Table 1. No difference was noticed between explants of separated, or grafted sections. A high percentage (50%-75%) of bisected explants developed into two plants, but only 10-15% of these plants grew to maturity. Some abnormal plants were noticed from this experiment (Table 1).

Table 1. Recovery of plants from bisected longitudinal sections of immature embryonic axes of maize (A188).
  a* b   c   d e**
    (b1) (b2) (c1) (c2)    
Grafted sections (placed together) 64 30 33(52) 35 6***(18) 9(19) 6(13)
Separated sections 79 17 61(77) 48 5****(8) 6(10) 14(24)
Control (embryos) 14 14 0 13 0 0 0
*a = number of embryos cut in half (longitudinal); b = number of plants transferred to peat pots - (b1) number of single plants, and (b2) number of paired plants (percent of paired plants - b2 x 100/a); c = number of plants matured - (c1) number of single plants, and (c2) number of paired plants (percent of paired plants - c2 x 100/b2); d = number of terminal node tassel/ear plants (percent of tassel/ear plants - d x 100/c); e = number of Abphyll plants (percent of Abphyll plants - e x 100/c).

**Origin of Abphyll ranges from 2nd to 5th node from top of the plants.

***Of the six, two are terminal node tassel/ear plants and two others are Abphyll plants.

****Of the five, two are terminal node tassel/ear plants and one other is Abphyll plant.

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors

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