J Integr Plant Biol. ›› 1989, Vol. 31 ›› Issue (10): -.

• Research Articles •    

Establishment and Characterization of Haploid Suspension Cells in Supersweet Maize

Sun Jing-san (C.S.Sun), Lu Tie-gang and M. R. Sondahl   

Abstract: Maize haploids contain only one set of chromosomes (monohaploids or true haploids), there is no opposition of dominant and recessive alleles. No gene is masked by a dominant allele. With these characteristics, haploid cell lines of maize offer an ideal system of isolating recessive mutants. It is easier to detect mutations in this system than those in diploids or polyhaploids that carry more than one set of chromosomes. The successful application of haploids for mutant selection requires the establishment of cell suspension cultures. Although a numerous studies on the haploid production from anther culture of maize have been reported during the last decade, the establishment of embryogenic cell suspension cultures from haploid callus of maize has not yet been documented. In this paper authors describe the selection of haploid friable embryogenic callus lines from some commercial important cultivars of supersweet maize and the establishment of haploid embryogenic cell suspension cultures. Twenty eight haploid highly embryogenic friable callus (Type Ⅱ) lines were selected from gametophytic callus of commercial supersweet maize. This callus produced a large number of globular somatic embryos on its surface on the callus maintenance medium containing 2 mg/l 2,4-D. Some embryos developed and germinated subsequently into complete plantlets when the embryogenic callus was transferred on to the regeneration medium with 0.01 mg/l 2,4-D and 2 mg/l zeatin. Eight suspension cell lines were established from 3-month-old embryogenic friable callus in liquid medium. The medium which contained the mineral salts of N6 medium, vitamins and inositol of MS medium and was supplemented with 2 mg/l, 2,4-D, 200 mg/l casein hydrolysat (CH), 1.0 g/l 2-(N-morpholino)-ethanesulfonic acid (MES) and 3% sucrose, was optimal for growth of the suspension cells. Addition of CH and MES into the liquid medium was beneficial for the growth of the suspension cells. When the conditions were optimal, the suspension cells doubled in dry weight after about 3 days. An exponential increase in dry weight of the suspension cells occurred between 7 and 8 day following subculture (Fig. 2). Culture medium pH decreased from an initial pH of 5.6 after subculture to pH 4.1 within 2 days culture. There was a direct relationship between the growth rate of suspension cells and medium pH changes (Fig. 2). Suspension cells which were plated at low densities on filters directly over a feeder layer of nurse cells exhibited more colony formation frequency than that on filters without feeder layer. The data in the Table 1 show that the plated supersweet maize cells were nursed most effectively by the parental supersweet maize suspension cultures. The suspension cells and the regenerated plantlets were cytologically stable. Among 390 suspension cells examined, 343 (88.0%) had the haploid chromosomes (2n= 10), 45(11.5%) had an aneuploid chromosome number, amongst them 24 cells with 11 chromosomes (2n=10+l) and 21 cells with 12 chromosomes (2n=10+2), only two cells (0.51%) had a diploid chromosome number (2n=20) (Table 2). Examination of 21 regeneration plants derived from suspension cells revealed that all of them were haploid (2n=10) except one (2n=20+1).

Key words: Maize, Suspension cell culture, Type Ⅱ callus, Haploid, Chromosome stability

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