In vitro culture of immature embryos

In order to determine an adequate culture medium for obtaining the best growth of immature embryos, plants of sweet corn and Colorado Klein were grown in the greenhouse, out of the normal age for this species (for which embryos grew slowly). The embryos were excised 22 and 28 days post-pollination, at 1.2 to 2 and 2.2 to 4.0mm in length, respectively.

The culture media used contained mineral salts of N6 medium (Chu, Proc. Symp. Plant Tissue Cult., pp.43-50, Science Press, Peking, 1978), with the addition of (mgl-1) glycine, 3.0; thiamine, 0.75; ascorbic acid, 20.0; nicotinic acid, 1.0; vitamin B6, 0.2; adenine, 0.2; succinic acid, 25.0; pantothenic acid, 0.5; biotin, 0.003 and asparagine, 1500 (Haagen Smit et al., Science 101:243, 1945); agar, 0.8% and 4 different concentrations of sucrose, 2.5% 5.0% 10.0% 15.0%, resulting in 4 treatments labeled A, B, C and D respectively The media were adjusted to pH 5.8.

The embryos were isolated and placed on the culture media with the rounded scutellar side exposed and the flat plumule radicle axis side in contact with the medium. The embryos were maintained at 28C with a 16/8h photoperiod for 60 days. Then the plants were transplanted to pots with a sterile mixture of soil-vermiculite (1:1) and grown in the greenhouse. The plants were finally transplanted to soil 108 days after sowing.

The immature embryos on all media showed similar behavior at the beginning, independently of their initial size, developing normally up to a determined length, after which all of them germinated (Table 1).

The youngest embryos did not continue their growth on medium A. Almost all the embryos developed into plants on media B, C and D, with the greatest number developing on medium B. Concentrations of sucrose affected the survival of the youngest embryos more than the oldest ones (Table 2).

Table 2. Influence of culture medium and initial length of the embryo over the number of plants transplanted to pots.
Initial height of embryos (mm)
Average of transplantable plants
  Medium A Medium B Medium C Medium D
1.2 to 2.0 33.3 93.7 66.7  78.9
2.2 to 4.0 75.0 100 70.0 88.9

It seems likely that sucrose concentration increases osmotic value in the medium, which is necessary for growth induction in proembryos of several species, probably to allow an effective flow of metabolites (Raghavan and Srivastava, 9. Embryo Culture. In Experimental Embryology of Vascular Plants, Johri, ed., p.195, 1982).

In a short period after transplantation, 46.8% of plants died. Plants from medium B showed the largest percentage of survival (100%), while none of the plants from the smallest embryos growing on medium A was successfully transplanted. On the same culture medium, the number of plants arising from the largest embryos always exceeded the number of plants arising from the smallest ones (Table 3).

Plants began to flower about 135 days after embryo sowing and 20 days later all of them had flowered; 81% of plants showing abnormalities such as an ear developing where normally there is a tassel, tassel seed, and nonbranching tassel. Plants did not reach their normal height, showing differences related to the different types of flowering (Table 4). Notwithstanding growth abnormalities, cytological analysis revealed a somatic chromosome number of 2n = 20 in every tested plant.

Table 1. Growth of embryos before germination.

Table 3. Influence of culture medium and initial length of the embryo over the number of plants that reached maturity.

Table 4. Relationship between plant height and type of flowering.

Dina Garcia1 and Maria del C. Molina

(1) Fellow, Com. Inv. Cient. Prov. Buenos Aires

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

Return to the MNL 62 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page