Mutagenesis of tissue cultures

Spontaneous variation found in regenerated plants has been termed "somaclonal variation" and is a common phenomenon in plants. We have been intending to enhance the variation in several elite corn inbred lines through the application of mutagens to maize tissue cultures using EMS, sodium azide, UV-light, X-ray and others. Friable, embryogenic calli of A619, B73 and Mo17 were initiated from immature embryos on MS medium supplemented with either 2,4-D (1.5-4 mg/l) or dicamba (1-8 mg/l), and maintained on the same medium plus proline (10-40 mM) and casein hydrolysate (100-500 mg/l) with a subculture interval of 10 to 14 days. These calli have been maintained for more than ten months.

EMS treatment: A619, B73 and Mo17 embryogenic calli were dispersed in liquid MS medium prior to EMS treatment. EMS concentrations of 0.1 to 0.2% (v/v) were applied to the calli for one to four hours. The treated calli were washed three times with fresh liquid medium. Percent survival was assessed under a dissecting microscope. An evaluation of EMS concentration and treatment duration was conducted for B73 only (Fig. 1). It appeared that the percent survival of B73 callus decreases with increasing EMS concentration and treatment duration, and that 0.2% EMS was the most appropriate concentration for maize tissue culture. Therefore, 0.2% EMS was applied to A619 and Mo17 calli (Fig. 2). The results also indicated that an EMS treatment duration of one to two hours is suitable for maize tissue culture. The sensitivity of maize calli to EMS differed among the genotypes and cell lines within a particular genotype. Mo17 callus was more sensitive to EMS treatment than A619 and B73. Regeneration of the treated calli is under way.

Sodium azide treatment: A619, B73 and Mo17 embryogenic calli were dispersed in sterilized 0.1 M phosphate buffer at pH 3.0 with or without added azide. After treatment, calli were washed with liquid MS medium and transferred to maintenance medium. The effect of azide concentration and treatment duration on B73 callus is shown in Figs. 3 and 4. Percent survival decreased with increasing azide concentration and treatment duration. Less than 5% survival was observed in B73 and Mo17 calli treated with 2 mM azide for longer than one hour. Phosphate buffer alone seemed to slightly reduce survival of B73 and Mo17 calli. Sensitivity of maize callus to azide was similar to EMS treatment; i.e., it was genotype and cell line dependent, and Mo17 callus was less sensitive to azide than A619 and B73.

A total of 83 R0 plants were regenerated from B73 calli treated either with azide or phosphate buffer alone and grown to maturity in a greenhouse. A hand microscope (Nikken, Tokyo) was used to determine pollen sterility. R0 regenerants with at least 5% pollen sterility were recorded (Table 1). The results indicated that the frequency of R0 regenerants with pollen sterility is higher for those plants from calli treated with either sodium azide or phosphate buffer alone than those plants from control calli. A total of 63 R0 plants produced enough seed (at least 15 seeds) for evaluation of variant segregation in R1 progeny tests. Chlorophyll variants were predominant, although other variants such as a white kernel mutant and some with altered vigor and earliness were also observed among the R1 progenies. R0 regenerants derived from calli treated with phosphate buffer alone produced as many variants as those from azide treated calli, and the frequency of variants from both treatments was higher than the control (Table 1). Therefore, it is not clear if a low pH value (pH 3.0), phosphate buffer, or sodium azide cause pollen sterility and mutation in the R0 regenerants.

UV irradiation: Prior to irradiation, B73 friable, embryogenic callus was dispersed in liquid MS medium and forced through a 600 µm screen. Cell clumps smaller than 600 µm were evenly spread on MS solid medium and irradiated with UV for 0.5 to 8 hours. A 15-W General Electric germicidal lamp emitting at approximately 2600 A with an energy output of 25 ergs/mm2/sec was used. The results indicated that percent survival of the irradiated callus decreased with increasing UV doses (Fig. 5). Calli irradiated with UV longer than 3 minutes became watery and died one month after irradiation. To date, only a few plants have been regenerated, and one yellow-green variant and one dwarf variant were detected in R1 progeny rows. The progenies segregated 3:1 for the respective characteristics indicating a single recessive mutation. Large-scale plant regeneration is under way.

X-ray irradiation (in collaboration with David Cheng and Tracy Yang): Macerated B73 friable embryogenic callus (smaller than 400 µm in diameter) was uniformly spread over a 60 x 20 mm petri dish containing 25 ml of maintenance medium and irradiated with 11 doses (0 to 8.4 kilorads (kR) ) of X-ray. Calli irradiated with doses higher than 2.7 kR died one month after irradiation. Three irradiated calli (0.8, 1.3 and 2.7 kR) showed significant increases in growth rate, and one (0.8 kR) showed an increase in embryogenic activity six months after irradiation.

A total of 230 R0 plants were regenerated for evaluation. Pollen fertility and seed set decreased with increasing X-ray dosage (Table 2). Of 230 R0 plants, 83 produced enough seeds (at least 15 seeds) for evaluation of mutant segregation in Rl progeny tests. The mutation rate increased with higher doses of X-ray.

Traits such as virescence, defective kernel, striate, white seedling, zebra band, viviparous, wilted, shrunken endosperm and vigorous plants have been observed in R1 progeny rows. Most of the variants segregated 3:1 for the respective traits, indicating a single recessive mutation. The degree of chromosomal alteration increased with X-ray dosage. Several sporocytes were collected and analysed. A deficiency of the proximal portion of the long arm of the chromosome, a trisomic-7, and translocations involving two or three chromosomes have been detected.

Figures 1 and 2.

Figures 3, 4, 5 and Table 1.

A.S. Wang, M.D. Hollingworth and J.B. Milcic
 
 


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