In vitro culture of 0.15-0.25 mm immature embryos. II. 2,4-dichlorophenoxy acetic acid (2,4-D) effects

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

2,4-D has been the most commonly used plant growth regulator to induce callus formation from immature embryos of maize and other Zea species. 2,4-D has frequently induced regeneration by organogenesis (Green and Phillips, Crop Sci. 15:417-420, 1975; Springer, et al., Protoplasma 101:269-281, 1979; Lowe et al., Plant Sci. 41:125-132, 1985) or somatic embryogenesis (Lu et al., Theor. Appl. Genet. 62:109-112, 1982; Vasil et al., Protoplasma 127:1-8, 1985; Franz and Schel,, JHN Acta Bot. Neerl. 36:247, 1987, among others). Regeneration ability depends mainly upon culture conditions, genotype (Tomes and Smith, Theor. Appl. Genet. 70:505-509, 1985) and embryo age (Rapela, MNL 59:59, 1985).

The aim of this work was to obtain a method of regenerating a high frequency of maize plants through organogenesis or somatic embryogenesis. This method could be used to get plants from difficult genotypes.

Plants of cv. Ever Green were grown under field conditions and auto- or sib-pollinated in the summer of 1989-1990. Embryos were excised and cultured on media as in the preceding article, with 6 different combinations of 2,4-D and kinetin (Table 1).

Table 1. Plant growth regulator concentration of culture media.
 
Plant growth regulators (mg/l) Kinetin (0.05 mg/l) Without kinetin
2,4-D 0.05 A D
2,4-D 0.1 B E
2,4-D 0.2 C F

The embryos and subsequent calli and plantlets were incubated at 28-30 C with a 16 hour photoperiod from cool white fluorescent lights calculated as No. of embryos which formed embryoids/total no. of embryos x 100. After two months in culture, regenerated plants were transferred to pots with a mixture of soil-sand (2:1), placed in the greenhouse and grown to maturity.

No plant regeneration was obtained through zygotic embryo germination. Some embryos developed roots or soft non-embryogenic calli near the coleorhiza. These calli turned yellow to brown and died or produced roots. A low number of embryos showed no growth (Table 2).

Table 2. Percentage of embryos producing non-embryogenic callus
 
Culture media % of non-growing embryos % of embryos forming roots or radicular callus
A 0 41.20
B 21.05 21.06
C 6.25 43.75
D 6.66 40.00
E 13.33 33.33
F 14.28 66.66

The other ones showed a compact, white scutellumgrowth. At the beginning of culture the surface was smooth. About 4 to 7 days in culture, swellings were seen along the scutellum and soft calli derived from the radicle embedded in the medium. White scutellum-like structures appeared at the surface after a week in culture. Green coleoptiles emerged from these structures. Scutellar notches were either median (like zygotic embryos) or terminal. Root growth was also observed and often covered somatic embryos.

Somatic embryos kept on 2,4-D containing medium either showed slow germination or fused together forming a white, compact callus. Further production of plants from this callus was by proliferation of adventitious shoots on media with 0.5 to 1 mg/l 2,4-D.

Regeneration was obtained only from the white, compact tissue that arose from scutellum, also called type I by Armstrong and Green (Planta 164:207-214, 1985).

Embryos on medium with 0.05 mg/l kinetin produced the largest frequency of somatic embryogenesis. Similar frequencies were obtained on media with 0.1 mg/l 2,4-D with or without kinetin (Graphic 1).

Somatic embryos germinated when they were transferred to a medium with 0.1 mg/l Picloram.

Graphic 1.  Somatic embryogenesis frequency induced by different 2,4-D concentrations.


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