In vitro culture of 0.15, 0.25 mm immature embryos. I. Picloram effects

--M. D. Garcia, M. del C. Molina and O. Caso

Plants have been obtained from isolated immature embryos cultured on nutrient media in the absence of any plant growth regulator (Haagen-Smit et al., Science 101:243, 1945; Sheridan et al., MNL 52:88-90, 1978; Van Lammeren, AAM, Acta Bot. Neerl. 37:49-61, 1988). However, no plants were regenerated from embryos smaller than 0.3 mm in length (about 9 days after pollination). Picloram and kinetin combinations increased the growth opportunity of 0.3 to 0.6 mm length maize embryos excised and cultured in vitro (Garcia et al., unpublished).

The aim of this study was to obtain maize plants through 0.15 to 0.2 mm length. Three different Picloram concentrations (in combination or not with kinetin) were evaluated.

Plants of cv. Ever Green were grown under field conditions and auto- or sib-pollinated in the summer of 1989-1990. Embryos were excised from caryopses between 7 and 9 days after pollination (0.15 to 0.25 mm length) and cultured on 6 differed solidified nutrient media. All of them contained the inorganic components of N6 medium (Chu, Proc. Symp. Plant Tissue Culture, 43-45, 1978) with added 0.025 mg/l Na2MoO4, 0.025 mg/l CuSO4, 0.025 mg/l CoCl, vitamins of Haagen-Smit (1945), 1500 mg/l asparagine, 5% sucrose and 6 different combinations of Picloram and kinetin (Table 1).

The embryos and subsequent seedlings were incubated at 28-30 C with a 16 hour photoperiod from cool white fluorescent lights with an intensity of 2500 Lx. Plant frequency (P.F.) obtained in every culture medium was calculated as No. of embryos which formed plants/total no. of embryos x 100. After a month in culture, plants were transferred to pots with a mixture of soil-sand (2:1), placed in the greenhouse and grown to maturity.

Table 1. Plant growth regulator concentrations of culture media
 
Plant growth regulator (mg/l) Kinetin (0.05) Without kinetin
Picloram 0.05 G J
Picloram 0.1 H K
Picloram 0.2 I L

Embryos in culture showed different responses (Table 2), but most of them germinated prematurely and formed plants (Graphic 1).

Table 2. Abnormalities observed on embryo germination.
 
Culture media % of non-growing embryos % of callus % of plants with radicular callus % embryos producing root development % of growing scutellums without germination
G 0 0 0 0 23.5
H 5.5 0 16.6 0 5.5
I 5.8 17.6 23.53 0 0
J 0 0 13.3 0 0
K 15 0 10 5 0
L 0 21.7 21.7 0 0
The embryos on medium J germinated at about 5 days in culture, before the other ones, and showed the least number of abnormalities. However, plants obtained from these were small, not very vigorous, and old leaves got necrotic before a month in culture. 15.79% of these embryos died after germination.

Embryos on medium G formed the largest frequency and the most vigorous plants. Plants obtained on media H, I and L showed very curled leaves.

The largest and more heterogeneous scutellum growth before germination was on media with kinetin (Table 3).

In conclusion, the results demonstrate that through the use of a low concentration of Picloram and kinetin (0.05 mg/l of each one) in the culture medium it is possible to obtain a high maize plant frequency from embryos less than 0.3 mm length through premature germination.

Table 3. Average of scutellum growth before germination.
 
Culture media Average of scutellum growth (mm)
G 3.34
H 3.53
I 3.34
J 2.04
K 1.96
L 1.79

Graphic 1.  Plant frequency obtained from embryos on different culture media.


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