摘要
从超甜玉米的花药培养物中,筛选出28个具有高度胚胎发生能力的类型Ⅱ愈伤组织系,这种愈伤组织质地松散,易于分散,可以长期进行继代培养。将3个月龄的类Ⅱ愈伤组织接种在液体培养基中进行振荡培养,建立起分散程度很好的8个悬浮细胞系。实验结果说明,培养基中加入酪蛋白水解物(CH)和2-(N-吗啉代)乙基磺酸(MES)对悬浮细胞的生长是有利的。培养基的 pH 迅速下降吋,细胞的增殖十分缓慢,当培养基的 pH 值开始升高时,细胞的增殖也显著加快。利用滋养培养法,可以使悬浮细胞的愈伤组织形成率大大提高(比对照高11.8—19.7倍),并在低密度下达到较高的(41.3%)植板率。染色体计数表明,继代培养6个月之后,88.0%的悬浮细胞和95.2%的再生植株的染色体数保持着2n=x=10的单倍性水平。
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 al- lele.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 po- lyhaploids that carry more than one set of chromosomes.The successful application of hap- loids for mutant selection requires the establishment of cell susoension 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 ha- ploid 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 2mg/1 2,4-D.Some embryos developed and germinated subse- quently into complete plantlets when the embryogenic callus was transferred on to the regene- ration medium with 0.01mg/l 2,4-D and 2mg/l zeatin.Eight suspension cell lines were estab- lished from 3-month-old embryogenic friable callus in liquid medium.The medium which con- tained the mineral salts of N6 medium,vitamins and inositol of MS medium and was supple- mented with 2mg/l,2,4-D,200mg/l casein hydrolysat (CH),1.0g/1 2-(N-morpholino)-ethanesul- fonic 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 mo- re colony formation frequency,than that on filters without feeder layer.The data in the Ta- ble 1 show that the plated supersweet maize cells were nursed most effectively by the paren- tal supersweet maize suspension cultures.The suspension cells and the regenerated plantlets were cytologically stable.Among 390 suspension cells examined,343 (88.0%)had the ha- ploid chromosomes (2n=10),45(11.5%) had an aneuploid chromosome number,amongst them 24 ceils with 11 chromosomes (2n=10+1) 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).
关键词
玉米
悬浮细胞培养
愈伤组织
Maize
Suspension cell culture
Type Ⅱ callus
Haploid
Chromosome stability
