酸浆和小酸浆的组织培养研究
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摘要
酸浆属植物酸浆和小酸浆具有重要的药用价值,特别是酸浆不仅具有药用价值,而且具有重要的食用和观赏价值,拥有广阔的开发利用前景。本研究以河南省宝天曼国家级自然保护区的野生酸浆和小酸浆为实验材料,研究了不同的外植体类型、不同的激素类型及浓度配比对植物细胞脱分化和再分化的影响,探讨了组织培养过程中防止污染及褐化的技术措施,初步建立了酸浆和小酸浆的组织培养体系。
实验结果表明:
(1)酸浆叶片用0.1 %的升汞消毒7 min效果最好,存活率可高达90.8 % ;叶柄和茎段对消毒剂较敏感,消毒时间要适当的缩短,最佳的升汞消毒时间都是5 min;子叶、胚轴由于接触外界的时间较短,3 min即可达理想的消毒效果。
(2)酸浆虽然为多年生草本植物,但其茎木质化较为严重,在初代培养中外植体褐化严重,不利于组织培养体系的建立。培养基中加入0.5 g/L的PVP,可以在一定程度上抑制外植体褐化。
(3)酸浆诱导不定芽最佳的外植体类型为叶柄,最佳的生长素类型为IAA,最佳的激素浓度组合为MS+2.0 mg/L 6-BA+ 0.5 mg/L IAA,不定芽分化率可达62.5 %。
(4)继代培养中不定芽增殖的最佳培养基为MS+2.0㎎/L 6-BA+0.05㎎/L IAA,不定芽增殖倍数可达5.0。
(5)在酸浆试管内生根实验中,生长素IAA和培养基中低的营养物质浓度都有利于根的形成,但以低的营养物质浓度更有利于根的生成,生根率可达91.7 %。
(6)在小酸浆叶片、叶柄、茎段、膨大花萼四种外植体类型中,可以产生两种类型的愈伤组织,一种为膨大肿胀型,以膨大花萼易于形成此类愈伤组织,一种为疏松脆型,以培养初期的茎段、叶柄及叶片的切口处易于形成此类愈伤组织。两种类型的愈伤组织中,以疏松脆型的愈伤组织生长较快,不定芽分化率较高。四种外植体中以茎段的愈伤组织诱导率最高,可达100 %,且形成时间较早。在茎段细胞脱分化形成愈伤组织的同时,有时伴随有不定芽直接由茎段外植体产生。总之,茎段为小酸浆组织培养较为适合的外植体类型。
(7)生长素为小酸浆愈伤组织诱导必需的植物激素,但其浓度变化对愈伤组织诱导率没有显著影响。愈伤组织诱导不定芽最佳的激素配比为MS+2.0 mg/L 6-BA+ 0.05 mg/L NAA或MS+3.0 mg/L 6-BA,诱导率都可达100 %。
(8)小酸浆试管苗增殖适合的6-BA浓度范围为0.05 -0.5 mg/L,最高增殖率可达4.28,过高的细胞分裂素浓度抑制试管苗增殖。
(9)低浓度的细胞分裂素有利于小酸浆花芽分化及开花,其中以0.05 mg/L 6-BA的诱导作用较显著。随着6-BA浓度的提高,其促进作用逐渐转变为抑制作用,当6-BA浓度达2.0 mg/L时,就可以严重抑制花芽分化。
(10)试管苗生根较适合的培养基为:MS+0.05 mg/L NAA。根浓密粗壮,易于移栽成活。
Physalis alkekengi L. and P. minima L. have important medicinal values. P. alkekengi is also of great culinary and ornamental potentials. In this study, P. alkekengi and P. minima plants were collected from Baotianman National Nature Reserve, Henan. They were experimentally manipulated to examine effects of explant type, hormone type and concentration on cell de-differentiation and re-differentiation. Techniques for preventing contamination and browning in tissue culture were discussed. A preliminary tissue culture system was established.
The following results were obtained:
(1) Optimal sterilization durations using 0.1 % HgCl2 were 7 minutes for P. alkekengi leaves, 5 minutes for stems and petioles, and 3 minutes for cotyledons and hypocotyls, respectively.
(2) Despite being a perennial herbaceous plant, P. alkekengi has highly lignified stems, which lead to severe browning in first generation explants, seriously hampering the establishment of stable tissue culture systems. This problem can be meliorated by adding 0.5 g/L PVP to the culture medium.
(3) For inducing adventitious buds, petiole was the optimal explant, IAA was the best growth hormone, and MS+2.0 mg/L 6-BA+0.5 mg/L IAA was the ideal hormone mixture.
(4) MS+2.0 mg/L 6-BA+0.5 mg/L IAA was the ideal hormone mixture for inducing second generation adventitious buds.
(5) IAA and low nutrient concentrations were conducive to root development.
(6) P. minima explants generated two types of calluses: swelling and hard ones generated by Calyx explants, loose and brittle ones generated by stem, petiole and leaf explants. The latter type grew faster and had higher adventitious bud induction rates. Stem explants had the highest callus induction rate (100 %), and sometimes produced adventitious buds. In summary, the stem was a suitable explant type.
(7) Auxin was necessary for inducing callus formation. Its concentration however did not significantly affect callus induction rate. The optimal hormone mixture for callus induction was MS+2.0 mg/L 6-BA+0.05 mg/L NAA or MS+3.0 mg/L 6-BA.
(8) The optimal 6-BA concentration for test tube P. minima seedling propagation was 0.05-0.5 mg/L. Excessive concentrations of cytokinin inhibited seedling propagation.
(9) Low concentrations of cytokinin stimulated flower bud differentiation and flowering, the optimal concentration was 0.05 mg/L 6-BA. Excessive concentrations of cytokinin inhibited flower bud differentiation.
(10) The optimal growth medium for root development was MS+0.05 mg/L NAA.
实验结果表明:
(1)酸浆叶片用0.1 %的升汞消毒7 min效果最好,存活率可高达90.8 % ;叶柄和茎段对消毒剂较敏感,消毒时间要适当的缩短,最佳的升汞消毒时间都是5 min;子叶、胚轴由于接触外界的时间较短,3 min即可达理想的消毒效果。
(2)酸浆虽然为多年生草本植物,但其茎木质化较为严重,在初代培养中外植体褐化严重,不利于组织培养体系的建立。培养基中加入0.5 g/L的PVP,可以在一定程度上抑制外植体褐化。
(3)酸浆诱导不定芽最佳的外植体类型为叶柄,最佳的生长素类型为IAA,最佳的激素浓度组合为MS+2.0 mg/L 6-BA+ 0.5 mg/L IAA,不定芽分化率可达62.5 %。
(4)继代培养中不定芽增殖的最佳培养基为MS+2.0㎎/L 6-BA+0.05㎎/L IAA,不定芽增殖倍数可达5.0。
(5)在酸浆试管内生根实验中,生长素IAA和培养基中低的营养物质浓度都有利于根的形成,但以低的营养物质浓度更有利于根的生成,生根率可达91.7 %。
(6)在小酸浆叶片、叶柄、茎段、膨大花萼四种外植体类型中,可以产生两种类型的愈伤组织,一种为膨大肿胀型,以膨大花萼易于形成此类愈伤组织,一种为疏松脆型,以培养初期的茎段、叶柄及叶片的切口处易于形成此类愈伤组织。两种类型的愈伤组织中,以疏松脆型的愈伤组织生长较快,不定芽分化率较高。四种外植体中以茎段的愈伤组织诱导率最高,可达100 %,且形成时间较早。在茎段细胞脱分化形成愈伤组织的同时,有时伴随有不定芽直接由茎段外植体产生。总之,茎段为小酸浆组织培养较为适合的外植体类型。
(7)生长素为小酸浆愈伤组织诱导必需的植物激素,但其浓度变化对愈伤组织诱导率没有显著影响。愈伤组织诱导不定芽最佳的激素配比为MS+2.0 mg/L 6-BA+ 0.05 mg/L NAA或MS+3.0 mg/L 6-BA,诱导率都可达100 %。
(8)小酸浆试管苗增殖适合的6-BA浓度范围为0.05 -0.5 mg/L,最高增殖率可达4.28,过高的细胞分裂素浓度抑制试管苗增殖。
(9)低浓度的细胞分裂素有利于小酸浆花芽分化及开花,其中以0.05 mg/L 6-BA的诱导作用较显著。随着6-BA浓度的提高,其促进作用逐渐转变为抑制作用,当6-BA浓度达2.0 mg/L时,就可以严重抑制花芽分化。
(10)试管苗生根较适合的培养基为:MS+0.05 mg/L NAA。根浓密粗壮,易于移栽成活。
Physalis alkekengi L. and P. minima L. have important medicinal values. P. alkekengi is also of great culinary and ornamental potentials. In this study, P. alkekengi and P. minima plants were collected from Baotianman National Nature Reserve, Henan. They were experimentally manipulated to examine effects of explant type, hormone type and concentration on cell de-differentiation and re-differentiation. Techniques for preventing contamination and browning in tissue culture were discussed. A preliminary tissue culture system was established.
The following results were obtained:
(1) Optimal sterilization durations using 0.1 % HgCl2 were 7 minutes for P. alkekengi leaves, 5 minutes for stems and petioles, and 3 minutes for cotyledons and hypocotyls, respectively.
(2) Despite being a perennial herbaceous plant, P. alkekengi has highly lignified stems, which lead to severe browning in first generation explants, seriously hampering the establishment of stable tissue culture systems. This problem can be meliorated by adding 0.5 g/L PVP to the culture medium.
(3) For inducing adventitious buds, petiole was the optimal explant, IAA was the best growth hormone, and MS+2.0 mg/L 6-BA+0.5 mg/L IAA was the ideal hormone mixture.
(4) MS+2.0 mg/L 6-BA+0.5 mg/L IAA was the ideal hormone mixture for inducing second generation adventitious buds.
(5) IAA and low nutrient concentrations were conducive to root development.
(6) P. minima explants generated two types of calluses: swelling and hard ones generated by Calyx explants, loose and brittle ones generated by stem, petiole and leaf explants. The latter type grew faster and had higher adventitious bud induction rates. Stem explants had the highest callus induction rate (100 %), and sometimes produced adventitious buds. In summary, the stem was a suitable explant type.
(7) Auxin was necessary for inducing callus formation. Its concentration however did not significantly affect callus induction rate. The optimal hormone mixture for callus induction was MS+2.0 mg/L 6-BA+0.05 mg/L NAA or MS+3.0 mg/L 6-BA.
(8) The optimal 6-BA concentration for test tube P. minima seedling propagation was 0.05-0.5 mg/L. Excessive concentrations of cytokinin inhibited seedling propagation.
(9) Low concentrations of cytokinin stimulated flower bud differentiation and flowering, the optimal concentration was 0.05 mg/L 6-BA. Excessive concentrations of cytokinin inhibited flower bud differentiation.
(10) The optimal growth medium for root development was MS+0.05 mg/L NAA.
引文
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