~（60）Co-γ射线、NaN_3对小水榕生物学诱变效应的研究
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摘要
小水榕是观赏水草的一种,叶子和株型都较细小,叶面平滑而深绿,造景可塑性好。小水榕的传统繁殖靠根茎侧芽分株而获得小苗,繁殖速度极慢,且品种单一,抗冷性差。因此,本试验以小水榕为试材,研究适宜诱导愈伤组织及增殖、分化的培养基,探讨60Co-γ射线对愈伤组织的诱变剂量及NaN3对试管苗的诱变浓度,并对诱变后的愈伤组织及试管苗的生长势、细胞畸变等进行初步研究;旨在优化小水榕的快速繁殖技术,并为改良小水榕繁育技术提供理论依据。
主要研究结果如下:
1.以小水榕腋芽为外植体,用1:100的84消毒液浸泡10min后,0.1%HgCl2消毒15min,愈伤组织诱导的适宜培养基为:MS+0.5mg/L 2,4-D+0.1mg/L CPPU。愈伤组织增殖分化的适宜培养基为:MS+0.5mg/L 6-BA+0.1mg/L NAA。
2.固体培养更适合愈伤组织的分化,而液体培养则适于愈伤组织的增殖。黄白色及红色紧致型愈伤组织及黑黄色紧致型愈伤组织易分化。
3.低剂量60Co-γ射线(<30Gy)对小水榕愈伤组织分化有刺激作用;辐照剂量高于80Gy以上,60Co-γ射线对愈伤组织的损伤严重,甚至产生致死作用。10Gy的60Co-γ射线辐照后小水榕愈伤组织的分化率最高(88%),为适宜剂量;30Gy为半致死剂量。小水榕愈伤组织经60Co-γ射线诱变后,与对照相比,分化产生试管苗的株高、净光合速率降低,而微核率及染色体畸变率升高;叶形指数及叶绿素含量没有明显的规律。在5Gy的辐照剂量诱变下,分化出较对照苗叶片狭长的幼苗,可能为变异苗,需对其做进一步鉴定。
4.试管苗经NaN3诱变后,出现了明显的白化现象,且随浓度的增加,白化苗越多。NaN3诱变小水榕的半致死浓度为9mmol/L。与对照相比,成活率、株高、叶绿素含量、净光合速率下降,SOD、POD、CAT酶活性、微核率及染色体畸变率略有增高;叶形指数与对照相似。试管苗矮化现象明显,利于增大植株表面积,促进植株与水体中环境的物质气体交换。除矮化现象以外,未观察到其他外形变化。
5.核型分析以小水榕根尖为材料,16:00取材,饱和对二氯苯水溶液预处理2h,卡诺固定液固定12h,1mol/L盐酸60℃水浴中解离3~4min,并以卡宝品红染色效果较好。小水榕核型为2n=2X=30=12m+14sm+4st,属于“2B”类型。
Anubias barteri is a kind of aquarium plant. It depends on roots and division of caudexes’side buds for propagation. Because of singleness of variety and poor resistance to cold, the rate of propagation is terribly low. In this experiment, appropriate medium of the callus induction, proliferation and bud differentiation for Anubias barteri were studied. In order to obtain superior mutated callus, the effects of 60Coγray and NaN3 on Anubias barteri were discussed after the determination of suitable dosage of 60Coγray and optimum conditions of NaN3. As a result, the rapid propagation system was optimized, and the main results are as follows:
1. The buds was used as explants, which was sterilized with 0.1% HgCl2 for 15min after dipped in 84 disinfection solution with concentration 1:100(84 disinfection solution:H2O) for 10min before cultured with medium. And the appropriate composition for the medium is MS ++0.5mg/L 2,4-D+0.1mg/L CPPU, which is better and appropriate for induction of proliferation and differentiation for callus by adding in 0.5mg/L 6-BA and 0.1mg/L NAA.
2. The callus differentiation prefers to grow in solid culture. And the callus proliferation prefers to grow in liquid culture. Three types callus are found easy to differentiate.
3. The effect of 60Coγray on callus for Anubias barteri has been tested, and the median lethal dose is equal to 30Gy. The callus will be damaged or deadly destroyed if it was treated with the radiation dosage above 80Gy. The stimulating effects of lower dose(<30Gy) were found and the highest rate(88%) of callus proliferation occurred when treated with 10Gy radiation dosage which is suitable for the induction of mutant. In contrast to the control, the height and the net photosynthetic rate of the mutant is low, and the frequence of micronucleus and chromosomal distortion are rised. As a result, a superior mutant with with long and narrow leaves is obtained by 5Gy.
4. The results showed that NaN3 can cause albinism at seedling stage of Anubias barteri. And the 9mmol/L NaN3 is the median lethal concentration. In contrast to the control, survival rate, seedling height, the chlorophyll content and net photosynthetic rate of the mutant are significantly decreased, and the POD activity, SOD activity, CAT activity, and the frequence of micronucleus and chromosomal distortion of the mutant increased with irradiation dose. As a result, the Anubias barteri are obviously dwarf, which is helpful to enlarge the superficial area and promote gas exchange environment between plant material and water.
5. Using the root-tip of Anubias barteri as materials, the suitable root-tip squashing method for chromosome preparation of Anubias barteri were as follows: sampling materials at 16:00pm and fixating with Carnoy’s ?, pretreating with saturated p-dichlorobenzene for 2h, dissociating with 1mol/L HCl at 60℃for 3-4min, and staining with carbon fuchsin. The karyotype of Anubias barteri belongs to“2B”of Stebbins: 2n=2x=30=12m+14sm+4st.
主要研究结果如下:
1.以小水榕腋芽为外植体,用1:100的84消毒液浸泡10min后,0.1%HgCl2消毒15min,愈伤组织诱导的适宜培养基为:MS+0.5mg/L 2,4-D+0.1mg/L CPPU。愈伤组织增殖分化的适宜培养基为:MS+0.5mg/L 6-BA+0.1mg/L NAA。
2.固体培养更适合愈伤组织的分化,而液体培养则适于愈伤组织的增殖。黄白色及红色紧致型愈伤组织及黑黄色紧致型愈伤组织易分化。
3.低剂量60Co-γ射线(<30Gy)对小水榕愈伤组织分化有刺激作用;辐照剂量高于80Gy以上,60Co-γ射线对愈伤组织的损伤严重,甚至产生致死作用。10Gy的60Co-γ射线辐照后小水榕愈伤组织的分化率最高(88%),为适宜剂量;30Gy为半致死剂量。小水榕愈伤组织经60Co-γ射线诱变后,与对照相比,分化产生试管苗的株高、净光合速率降低,而微核率及染色体畸变率升高;叶形指数及叶绿素含量没有明显的规律。在5Gy的辐照剂量诱变下,分化出较对照苗叶片狭长的幼苗,可能为变异苗,需对其做进一步鉴定。
4.试管苗经NaN3诱变后,出现了明显的白化现象,且随浓度的增加,白化苗越多。NaN3诱变小水榕的半致死浓度为9mmol/L。与对照相比,成活率、株高、叶绿素含量、净光合速率下降,SOD、POD、CAT酶活性、微核率及染色体畸变率略有增高;叶形指数与对照相似。试管苗矮化现象明显,利于增大植株表面积,促进植株与水体中环境的物质气体交换。除矮化现象以外,未观察到其他外形变化。
5.核型分析以小水榕根尖为材料,16:00取材,饱和对二氯苯水溶液预处理2h,卡诺固定液固定12h,1mol/L盐酸60℃水浴中解离3~4min,并以卡宝品红染色效果较好。小水榕核型为2n=2X=30=12m+14sm+4st,属于“2B”类型。
Anubias barteri is a kind of aquarium plant. It depends on roots and division of caudexes’side buds for propagation. Because of singleness of variety and poor resistance to cold, the rate of propagation is terribly low. In this experiment, appropriate medium of the callus induction, proliferation and bud differentiation for Anubias barteri were studied. In order to obtain superior mutated callus, the effects of 60Coγray and NaN3 on Anubias barteri were discussed after the determination of suitable dosage of 60Coγray and optimum conditions of NaN3. As a result, the rapid propagation system was optimized, and the main results are as follows:
1. The buds was used as explants, which was sterilized with 0.1% HgCl2 for 15min after dipped in 84 disinfection solution with concentration 1:100(84 disinfection solution:H2O) for 10min before cultured with medium. And the appropriate composition for the medium is MS ++0.5mg/L 2,4-D+0.1mg/L CPPU, which is better and appropriate for induction of proliferation and differentiation for callus by adding in 0.5mg/L 6-BA and 0.1mg/L NAA.
2. The callus differentiation prefers to grow in solid culture. And the callus proliferation prefers to grow in liquid culture. Three types callus are found easy to differentiate.
3. The effect of 60Coγray on callus for Anubias barteri has been tested, and the median lethal dose is equal to 30Gy. The callus will be damaged or deadly destroyed if it was treated with the radiation dosage above 80Gy. The stimulating effects of lower dose(<30Gy) were found and the highest rate(88%) of callus proliferation occurred when treated with 10Gy radiation dosage which is suitable for the induction of mutant. In contrast to the control, the height and the net photosynthetic rate of the mutant is low, and the frequence of micronucleus and chromosomal distortion are rised. As a result, a superior mutant with with long and narrow leaves is obtained by 5Gy.
4. The results showed that NaN3 can cause albinism at seedling stage of Anubias barteri. And the 9mmol/L NaN3 is the median lethal concentration. In contrast to the control, survival rate, seedling height, the chlorophyll content and net photosynthetic rate of the mutant are significantly decreased, and the POD activity, SOD activity, CAT activity, and the frequence of micronucleus and chromosomal distortion of the mutant increased with irradiation dose. As a result, the Anubias barteri are obviously dwarf, which is helpful to enlarge the superficial area and promote gas exchange environment between plant material and water.
5. Using the root-tip of Anubias barteri as materials, the suitable root-tip squashing method for chromosome preparation of Anubias barteri were as follows: sampling materials at 16:00pm and fixating with Carnoy’s ?, pretreating with saturated p-dichlorobenzene for 2h, dissociating with 1mol/L HCl at 60℃for 3-4min, and staining with carbon fuchsin. The karyotype of Anubias barteri belongs to“2B”of Stebbins: 2n=2x=30=12m+14sm+4st.
引文
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