WO2023109318A1 - 一种一步诱导艾纳香根细胞分化产生不定芽的培养方法 - Google Patents

一种一步诱导艾纳香根细胞分化产生不定芽的培养方法 Download PDF

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WO2023109318A1
WO2023109318A1 PCT/CN2022/127164 CN2022127164W WO2023109318A1 WO 2023109318 A1 WO2023109318 A1 WO 2023109318A1 CN 2022127164 W CN2022127164 W CN 2022127164W WO 2023109318 A1 WO2023109318 A1 WO 2023109318A1
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root
adventitious
adventitious bud
bud induction
induction medium
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PCT/CN2022/127164
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French (fr)
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陈晓鹭
于福来
肖永锋
罗沁
黎玉兰
陈振夏
陈英桦
于平
元超
王丹
官玲亮
张影波
黄梅
谢小丽
王凯
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中国热带农业科学院热带作物品种资源研究所
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Priority to JP2023563181A priority Critical patent/JP7486683B2/ja
Publication of WO2023109318A1 publication Critical patent/WO2023109318A1/zh

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce

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  • the invention relates to the technical field of plant tissue culture, in particular to a culture method for one-step induction of Aina radix root cell differentiation to produce adventitious buds.
  • Ainaxiang (Blumea balsamifera (L.) DC.) is a perennial herb of the genus Ainaxiang in the family Asteraceae. It is distributed in Yunnan, Guizhou, Hainan, Taiwan and other places in China. , its extract is the main raw material of many kinds of Chinese patent medicines, and is widely used in food, cosmeceutical and other market fields, and has a good market prospect.
  • the planting technology of Ainaxiang has been relatively backward, and the production is mainly based on seed propagation and root division propagation.
  • Seed propagation belongs to sexual reproduction, and the disadvantage is that the emergence rate is low, and the traits of offspring are separated, and the original seed cannot be maintained; split-root propagation belongs to asexual reproduction, and the disadvantage is that the pathogens of the original plant cannot be eliminated, and the efficiency is low; Multiplication is the main difficulty in Ainaxiang planting technology. Therefore, it is necessary to carry out research on the in vitro propagation of Ainaxiang seedlings.
  • Predecessors have carried out a variety of technical attempts on in vitro rapid propagation of Ainaxiang, including inducing axillary bud proliferation, inducing leaf explants to produce callus first, and then inducing callus differentiation to obtain adventitious buds, etc., such as patent CN102550421A " "Rapid Breeding and Cultivation Method of Fufang Ainaxiang Seedlings” discloses the cultivation method of using the axillary bud stem section of a heterogeneous plant of the same genus Fufang Ainaxiang as an explant to induce clustered buds, such as the patent CN103250645A "Diangui The Rapid Propagation Method of Ainaxiang Medicinal Material” discloses the use of the stem tip, tender stem, old stem, leaves, flower buds and seeds of Ainaxiang, another heterogeneous plant of the same genus, to induce callus, and then A culture method for induction of test-tube plantlets.
  • these technologies are difficult to meet the current
  • inducing the proliferation of axillary buds is the most commonly used rapid propagation technology for Ainaxiang seedlings. It achieves the purpose of proliferation by stimulating the growth of the original lateral bud growth points of the plant, and does not involve the process of cell dedifferentiation. If you try to carry out artificial mutagenesis or transgene in this way, the probability of getting a chimera will be very high, because it is difficult for the multiple cells that make up the original bud to mutate or be transformed, and the two kinds of cells will be mixed together to produce a chimera. fit. During the growth process of chimera, the problem of character instability and character disappearance will occur, which leads to the failure of breeding work.
  • a newer technique is to use leaf explants, first use one medium to induce callus, and then transfer to another medium to induce callus differentiation to form adventitious shoots.
  • Usually artificial mutagenesis is carried out in the callus stage, but because some of the unsuccessfully mutated cells may also differentiate to form adventitious buds, there is also the problem of chimerism in this process.
  • this technique needs to go through two steps, prepare at least two kinds of medium, inoculate at least twice, and cultivate at least two stages, so not only the process is cumbersome, but also there is a certain probability of non-purpose in the callus stage Variation, interference with mutagenesis or genetic transformation results.
  • the present invention proposes a one-step culture method for inducing the differentiation of Aina root cells to produce adventitious buds. It is not necessary to induce explants to dedifferentiate to form callus and then differentiate to form adventitious buds, but to directly differentiate from somatic cells For adventitious buds, the generation of chimeras is reduced, and it provides a technical basis for artificial mutation and transgenic breeding of Ainaxiang.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • adventitious bud induction medium comprises: 0.01 ⁇ 0.5mg/L NAA, 0 ⁇ 1.0mg/L 6-BA, 10 ⁇ 70g/L sucrose and 2 ⁇ 8g/L agar and basic medium;
  • step S3 Inducing adventitious buds: inserting the root segments of step S1 into the adventitious bud induction medium prepared in step S2 for cultivation to obtain adventitious buds of Ainaxiang.
  • the root is a main root or a lateral root, and the length of the root segment is 1-25 cm.
  • the root is an adventitious root.
  • the length of the root segment is 3 to 4 cm; if the length of the root segment is too short, the efficiency of inducing adventitious buds will be low; if the length is too long, the amount of root segment culture materials will be too small, resulting in a waste of root segment resources; When the length is 3-4cm, the efficiency of inducing adventitious buds is high, and more root culture materials can be obtained, making full use of the root of Aina.
  • the adventitious bud induction medium is the addition of 0.025-0.1 mg/L NAA, 1 mg/L 6-BA, 10-70 g/L sucrose and 2-8 g/L agar to the basal medium.
  • the adventitious bud induction medium further includes: 0-100 mg/L vitamin C and 0-100 mg/L polyvinylpyrrolidone.
  • the adventitious bud induction medium is 0.025 ⁇ 0.1mg/L NAA, 1mg/L 6-BA, 10 ⁇ 70g/L sucrose, 2 ⁇ 8g/L agar, 0.01 ⁇ 10mg/L L vitamin C and 0.01 ⁇ 10mg/L polyvinylpyrrolidone.
  • the adventitious bud induction medium is the addition of 0.05mg/L NAA, 0.1mg/L6-BA, 30g/L sucrose and 2-8g/L agar to the basal medium.
  • the adventitious bud induction medium is to add 0.05mg/L NAA, 1mg/L 6-BA, 30g/L sucrose, 6g/L agar, 10mg/L vitamin C and 10mg/L poly Vinylpyrrolidone.
  • the culture condition is 25-28°C.
  • the culturing time is 10-90 days, and the optimum culturing time is 20 days.
  • the present invention provides a kind of with the root of Ai Naxiang as explant material, the culture method that one step induces root cell direct differentiation to produce adventitious bud, fills up the blank of Ai Naxiang's root as explant material, increases
  • the explant material objects that can be cultivated have increased the source of effective explants in the process of propagation and proliferation of Ainaxiang, and the cultivation effect of using the roots of Ainaxiang as explants is better, which overcomes the need to select budded stems. Segments or leaves serve as explants, through proliferation or callus regeneration in the absence of adventitious shoots.
  • the adventitious bud induction medium is designed for the root of Aina, which can directly differentiate the cells of the root of Aina to form adventitious buds, without having to go through the link of forming callus, which is simpler than the prior art It is fast and can reduce the chimeras produced by later biotechnology breeding.
  • the present invention saves the cost of consumables, time and manpower, improves the accuracy of directed mutagenesis or genetic transformation of A. chinensis, and provides a technical basis for the artificial mutagenesis and cell engineering breeding of A. chinensis.
  • Fig. 1 is the adventitious buds directly differentiated from the explants of the root segment of the present invention.
  • test methods used in the examples of the present invention are conventional methods unless otherwise specified.
  • the sterile seedlings used in the present invention are all derived from the tissue culture seedlings of Ainaxiang cultivated in the South Medicine Tissue Culture Room of the Variety Resources Institute of Danzhou Tropical Crops Academy of Sciences, Hainan Republic from September 2019 to October 2020.
  • the number of effective root segments in the embodiments refers to unpolluted root segments.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • the aseptic seedlings of Ainaxiang are healthy Ainaxiang seeds
  • aseptic sowing is carried out, and it is obtained by culturing for 12 days;
  • the aseptic sowing medium is 1/2MS as the basal medium, and 0.01-0.5mg/L NAA, 20-50g /L sucrose and 3 ⁇ 7g/L agar;
  • step S3 Inoculate the root segments from step S1 into the adventitious bud induction medium prepared in step S2, culture them at 25-28° C. for 20 days, and use the adventitious bud induction medium to cultivate segmented roots to obtain adventitious buds.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • step S3 Inoculate the root segment from step S1 into the adventitious bud induction medium prepared in step S2, culture it at 25-28° C. for 20 days, and use the adventitious bud induction medium to culture segmented root tissues to obtain adventitious buds.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • each section is 25cm long, sterilized, and get the root segment;
  • the adventitious root is obtained by inoculating the explant into the adventitious root induction medium for differentiation and cultivation for 30 days;
  • the adventitious root induction medium is prepared with 1 /2MS as the basic medium, add 0.01-0.5mg/L NAA, 20-50g/L sucrose and 3-7g/L agar to the basic medium;
  • step S3 Inoculate the root segment from step S1 into the adventitious bud induction medium prepared in step S2, culture at 25-28° C. for 90 days, and use the adventitious bud induction medium to cultivate segmented adventitious roots to obtain adventitious buds.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • the sterile seeding medium is 1/2MS as the base medium, and in the base medium, 0.01 ⁇ 0.5mg/L NAA, 20 ⁇ 50g/L sucrose and 3 ⁇ 7g/L agar are added;
  • step S3 Inoculate the budded stem section in step S1 into the adventitious bud induction medium prepared in step S2, cultivate at 25-28°C for 20 days, and use the adventitious bud induction medium to cultivate the budded stem section to obtain adventitious buds.
  • a culture method for one-step induction of Aina root cell differentiation to produce adventitious buds comprising the following steps:
  • the aseptic seeding medium is based on 1/2MS, in which 0.01-0.5mg/L NAA, 20-50g/L sucrose and 3-7g/L agar are added;
  • step S3 Inoculate the leaves of step S1 into the adventitious bud induction medium prepared in step S2, culture at 25-28°C for 20 days, and cultivate the leaves with the adventitious bud induction medium, but no adventitious buds were obtained.
  • the rate of shoot acquisition from root explants was higher (number of shoots/source of explant, 127%) than when only stem segments with buds were used as explants ( 108%).
  • the purpose of cultivation is to multiply, using both budding stem segments and aseptic seedlings to induce aseptic adventitious roots as explants can obviously achieve the effect described in the description of the present invention, that is, "increased Ainaxiang in the expansion Effective source of explants in the process of proliferation", the shoot acquisition rate was the highest (155%).
  • Experimental method take MS as the basic medium, add 0.05mg/L NAA, 30g/L sucrose, 2-8g/L agar, 10mg/L vitamin C and 10mg/L polyvinylpyrrolidone; then add different concentrations of 6- BA, respectively 0mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L, with 0mg/L as the control group, and respectively prepared as a culture medium, inoculated into segmented root tissue (the length of the root segment is about 3.0 ⁇ 4.0cm), after 30 days, the polluted root explants were excluded and the number of adventitious buds was counted to calculate the induction rate.
  • Experimental method set the amount of hormones added with different concentrations of NAA, use MS as the basal medium, add 1.0mg/l 6-BA, 30g/L sucrose, 2-8g/L agar, 10mg/L vitamin C and 10mg/L Polyvinylpyrrolidone, and then add different concentrations of NAA solutions, respectively 0mg/L, 0.025mg/L, 0.05mg/L, 0.075mg/L, 0.1mg/L, with 0mg/L as the control group, and respectively prepared
  • the culture medium was inserted into segmented root tissue (the length of the root segment was about 3.0-4.0 cm), the contaminated root explants were excluded after 30 days, the number of adventitious buds was counted, and the induction rate was calculated.
  • NAA concentration (mg/L) Induction rate (%) 0 45.0 0.025 57.5 0.05 82.5 0.075 55.0 0.1 47.5
  • the promotion effect is gradually enhanced; when the NAA concentration is higher than 0.05mg/L, the total number of adventitious buds obtained decreases with the increase of NAA concentration, and the NAA concentration is added at a concentration of 0.025-0.1mg/L, and the number of buds per unit root segment is high
  • the control group shows that the combined use of NAA and other components in the medium of this application can improve the effect of inducing adventitious bud regeneration.
  • Experimental method Set up 3 levels of MS medium, namely MS, 1/2MS, 3/4MS, and add 0.05mg/LNAA, 1.0mg/L 6-BA, 30g/L sucrose, 2 ⁇ 8g/L Agar, 10mg/L vitamin C, and 10mg/L polyvinylpyrrolidone were prepared into medium respectively, and inserted into segmented root tissues (the length of the root segment was about 3.0-4.0cm). After 30 days, the number of adventitious buds was counted, and the induced Rate.
  • the results are shown in Table 4.
  • the present invention uses MS as the basic medium, and the number of sprouts per unit root segment is the largest, and the effect of inducing root explant dedifferentiation to produce adventitious buds is good, and the induction rate is significantly higher than 1/2MS and 3/ 4MS basic medium.

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Abstract

本发明提供一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，选用艾纳香的根段作为外植体进行培养，并设计不定芽诱导培养基，仅使用该培养基便可诱导外植体直接分化不定芽；本发明填补了艾纳香根作为外植体的空白，增加了艾纳香在扩繁增殖过程中有效外植体来源。此外，本发明使艾纳香的根细胞直接分化形成不定芽，不必经过形成愈伤组织的过程，由此简化了技术环节，并减少了再生芽的变异和嵌合体的产生，提高了艾纳香定向诱变或遗传转化的准确性，可应用于艾纳香育种，将可显著节省时间和人力成本，提高工作效率。

Description

一种一步诱导艾纳香根细胞分化产生不定芽的培养方法
本申请要求于2021年12月14日提交中国专利局、申请号为202111522786.X、发明名称为“一种一步诱导艾纳香根细胞分化产生不定芽的培养方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及植物组织培养技术领域，特别涉及一种一步诱导艾纳香根细胞分化产生不定芽的培养方法。
背景技术
艾纳香(Blumea balsamifera(L.)DC.)是菊科艾纳香属多年生草本植物，分布于我国云南、贵州、海南、台湾等地，是中药艾片(左旋龙脑)的唯一来源植物，其提取物是多种中成药的主要原料，并广泛应用于食品、药妆等市场领域，具有良好的市场前景。
长期以来，艾纳香种植技术相对落后，在生产上以种子繁殖和分根繁殖为主。种子繁殖属于有性繁殖，缺点在于出苗率低，且后代性状分离，无法保持原有种性；分根繁殖属于无性繁殖，缺点在于无法排除原有植株的病原体，且效率低下；良种种苗扩繁是艾纳香种植技术的主要难点。因此，有必要开展艾纳香种苗的离体扩繁研究。
前人对艾纳香离体快繁开展了多种技术尝试，其中包含诱导腋芽增殖、诱导叶片外植体先产生愈伤组织，再诱导愈伤组织分化得到不定芽等技术，如专利CN102550421A《馥芳艾纳香种苗快速繁育与栽培方法》公开了利用艾纳香的一种同属异种植物馥芳艾纳香腋芽茎段作为外植体诱导丛生芽的培养方法，如专利CN103250645A《滇桂艾纳香药材的快速繁殖方法》公开了利用艾纳香的另一种同属异种植物滇桂艾纳香的茎尖、嫩茎、老茎、叶片、花苞和种子进行愈伤组织的诱导，再进行试管苗的诱导的培养方法。但是，这些技术难以满足目前艾纳香的育种需求。
首先，诱导腋芽增殖是最常用的艾纳香种苗快繁技术，其通过刺激植株原有的侧芽生长点生长，达到扩繁的目的，不涉及细胞脱分化过程。如果试图用此方式进行人工诱变或转基因，得到嵌合体的几率会很高，因为， 构成原有的芽的复数细胞很难全部变异或得到转化，两种细胞将混杂在一起，从而产生嵌合体。嵌合体在生长过程中，会发生性状不稳定和性状消失的问题，导致育种工作失败。
较新的技术是使用叶片外植体，先使用一种培养基诱导产生愈伤组织，然后再转移到另一种培养基，诱导愈伤组织分化形成不定芽。通常人工诱变在愈伤组织阶段进行，但由于部分未成功诱变的细胞同样可能分化形成不定芽，因此该过程同样存在嵌合体的问题。此外，这种技术需要经过两个步骤，配制至少两种培养基，接种至少两次，培养至少两个阶段，因此不仅过程繁琐，而且还存在一定几率、偶发于愈伤组织阶段的非目的性变异，干扰诱变或遗传转化结果。
鉴于此，目前仍缺乏一种减少嵌合体产生，且过程简便的一步获得艾纳香不定芽的组织培养方法。
发明内容
鉴以此，本发明提出一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，不需要诱导外植体脱分化形成愈伤组织后再分化形成不定芽，而是由体细胞直接分化为不定芽，减少了嵌合体的产生，为艾纳香人工诱变、转基因等育种工作提供技术基础。
本发明的技术方案是这样实现的：
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.切取艾纳香的根，再将根切段，消毒，得根段；
S2.配制不定芽诱导培养基，所述不定芽诱导培养基包括：0.01～0.5mg/L NAA、0～1.0mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂和基础培养基；
S3.诱导不定芽：将步骤S1的根段接入步骤S2配制的不定芽诱导培养基中进行培养，得艾纳香不定芽。
优选的，所述根为主根或侧根，根段的长度为1～25cm。
优选的，所述根为不定根。
优选的，所述根段的长度为3～4cm；根段长度偏短，诱导产生不定芽的效率会较低；长度太长，根段培养材料的数量偏少，造成根段资源的浪费；在长度为3～4cm，诱导产生不定芽的效率高，且能够获得更多根 段培养材料，充分利用了艾纳香根。
优选的，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂。
优选的，所述不定芽诱导培养基还包括：0～100mg/L维生素C和0～100mg/L聚乙烯吡咯烷酮。
优选的，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖、2～8g/L琼脂、0.01～10mg/L维生素C和0.01～10mg/L聚乙烯吡咯烷酮。
优选的，所述不定芽诱导培养基为在基础培养基中加入0.05mg/L NAA、0.1mg/L6-BA、30g/L蔗糖和2～8g/L琼脂。
优选的，所述不定芽诱导培养基为在基础培养基中加入0.05mg/L NAA、1mg/L 6-BA、30g/L蔗糖、6g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮。
优选的，所述培养的条件为25～28℃。
优选的，所述培养的时间为10～90天，最佳培养的时间为20天。
与现有技术相比，本发明的有益效果是：
(1)本发明提供一种用艾纳香的根作为外植体材料，一步诱导根细胞直接分化产生不定芽的培养方法，填补了艾纳香的根作为外植体材料的空白，增加了可培养的外植体材料对象，即增加了艾纳香在扩繁增殖过程中有效外植体来源，而且使用艾纳香的根作为外植体的培养效果更佳，克服了选择带芽茎段或叶片作为外植体，通过增殖或愈伤组织再生不定芽的不足。
(2)本发明的培养方法，针对艾纳香根设计了不定芽诱导培养基，可以使艾纳香根细胞直接分化形成不定芽，不必经过形成愈伤组织的环节，比现有技术更简便快捷，且可以降低后期生物技术育种所产生的嵌合体。
(3)本发明节省了耗材、时间和人力成本，并提高了艾纳香定向诱变或遗传转化的准确性，为艾纳香人工诱变、细胞工程育种提供技术基础。
说明书附图
图1为本发明艾纳香根段外植体直接分化的不定芽。
具体实施方式
为了更好理解本发明技术内容，下面提供具体实施例，对本发明做进 一步的说明。本发明所述实施例只用于解释本发明，并非用于限定本发明的范围。
本发明实施例中所使用的试验方法如无特殊说明，均为常规方法。
本发明所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
本发明所使用的无菌苗均来源于2019年9月～2020年10月份在海南省儋州市热带作物科学院品种资源研究所南药组培室中所培育的艾纳香组培苗。
实施例中有效根段数指的是未污染的根段。
诱导率是指接种30d后成功诱导根段分化形成的不定芽总数占外植体总数的百分比，计算公式为诱导率(％)＝不定芽总数÷外植体总数×100。
实施例1
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.取艾纳香无菌实生苗，取主根和侧根，将根切段，每段长1～4cm，消毒，得根段；所述艾纳香无菌实生苗为将健康艾纳香种子进行常规灭菌后进行无菌播种，培养12天所得；所述无菌播种培养基为以1/2MS为基础培养基，在基础培养基中，加入0.01～0.5mg/L NAA、20～50g/L蔗糖和3～7g/L琼脂；
S2.以MS为基础培养基，在基础培养基中加入0.05mg/L NAA、0.1mg/L 6-BA、30g/L蔗糖、6g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮，得不定芽诱导培养基；
S3.将步骤S1的根段接种到步骤S2配制的不定芽诱导培养基中，在25～28℃培养20天，利用不定芽诱导培养基培养分段的根得到不定芽。
实施例2
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.选择健康无病虫害植株，对土壤进行灌根消毒3天后，不破坏根表皮的情况下挖取艾纳香根部组织，用毛笔小心去除表面土壤颗粒，自来 水冲洗3遍、无菌水冲洗3遍，在超净台中进行常规灭菌后，将根分段，每段长3～6cm；
S2.以1/2MS为基础培养基，在基础培养基中加入0.025mg/L NAA、0.1mg/L 6-BA、10g/L蔗糖和6g/L琼脂，得不定芽诱导培养基；
S3.将步骤S1的根段接种到步骤S2配制的不定芽诱导培养基中，在25～28℃培养20天，利用不定芽诱导培养基培养分段的根部组织得到不定芽。
实施例3
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.取艾纳香不定根分段，每段长25cm，消毒，得根段；所述不定根为将外植体接种到不定根诱导培养基中分化培养30天所得；所述不定根诱导培养基以1/2MS为基础培养基，在基础培养基中，加入0.01～0.5mg/L NAA、20～50g/L蔗糖和3～7g/L琼脂；
S2.配制不定芽诱导培养基以MS为基础培养基，在基础培养基中加入0.1mg/L NAA、2.0mg/L 6-BA、70g/L蔗糖、8g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮；
S3.将步骤S1的根段接种到步骤S2配制的不定芽诱导培养基中，在25～28℃培养90天，利用不定芽诱导培养基培养分段的不定根得到不定芽。
对比例1
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.取艾纳香无菌实生苗，取带芽茎段，消毒；所述艾纳香无菌实生苗为将健康艾纳香种子进行常规灭菌后进行无菌播种，培养12天所得；所述无菌播种培养基为以1/2MS为基础培养基，在基础培养基中，加入0.01～0.5mg/L NAA、20～50g/L蔗糖和3～7g/L琼脂；
S2.以MS为基础培养基，在基础培养基中加入0.05mg/L NAA、0.1mg/L 6-BA、30g/L蔗糖、6g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮，得不定芽诱导培养基；
S3.将步骤S1的带芽茎段接种到步骤S2配制的不定芽诱导培养基中，在25～28℃培养20天，利用不定芽诱导培养基培养带芽茎段得到不定芽。
对比例2
一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，包括以下步骤：
S1.取艾纳香无菌实生苗，摘取叶片，消毒；所述艾纳香无菌实生苗为将健康艾纳香种子进行常规灭菌后进行无菌播种，培养12天所得；所述无菌播种培养基为以1/2MS为基础培养基，在基础培养基中，加入0.01～0.5mg/L NAA、20～50g/L蔗糖和3～7g/L琼脂；
S2.以MS为基础培养基，在基础培养基中加入0.05mg/L NAA、0.1mg/L 6-BA、30g/L蔗糖、6g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮，得不定芽诱导培养基；
S3.将步骤S1的叶片接种到步骤S2配制的不定芽诱导培养基中，在25～28℃培养20天，利用不定芽诱导培养基培养叶片，未得到不定芽。
统计实施例1～3和对比例1～2诱导培养后的不定芽数，计算不定芽诱导率，结果见表1-1：
表1-1不定芽诱导效果
Figure PCTCN2022127164-appb-000001
表1-2
Figure PCTCN2022127164-appb-000002
Figure PCTCN2022127164-appb-000003
结果表明，本发明使用根段进行不定芽诱导的，不定芽诱导率虽然不高于带芽茎段诱导不定芽的诱导率，但是高于叶片诱导不定芽的诱导率。
虽然通过单位外植体获得芽数量(芽数/统计的外植体，芽诱导率42％～58％)比较低，但是，由于每棵来源植株可获得的根外植体数量[(48/20＝)2.4～(100/20＝)5.0个外植体/株]显著多于可获得的带芽茎段的数量[((60/40＝)约1.5个/株)，即使去除污染的外植体，每棵植株可获得的根外植体数量仍比较高[(40/20＝)2～(52/20＝)2.6个外植体/株)。当使用无菌苗作为来源植株时，根外植体的芽获取率较高(芽数/外植体来源，127％)高于仅以带芽茎段为外植体时的芽获取率(108％)。当培养目的为扩繁的时候，同时使用带芽茎段和无菌苗诱导获得无菌不定根作为外植体，显然能达到本发明说明书所描述的效果，即“增加了艾纳香在扩繁增殖过程中有效外植体来源”，芽获取率最高(155％)。
在生物育种过程中，面对所得到的特殊种质，往往需要利用有限的来源植株尽可能多地获取和利用有价值的外植体，而不是把根部作为不值得培养的部分直接抛弃，方可达到最高的芽获取率。在这种情况下，本发明技术的创新尤为具有意义。实际上，其他专业技术人员正是由于无法从根外植体获得不定芽，所以才大多选择了以芽繁芽的方式诱导不定芽，即，其他专业技术人员的经验和认识是：以根为外植体进行培养的不定芽诱导效率为0％。
实施例4不同6-BA浓度对艾纳香根细胞脱分化产生不定芽的影响
实验方法：以MS为基本培养基，添加0.05mg/L NAA、30g/L蔗糖、2～8g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮；再分别添加不同浓度的6-BA，分别是0mg/L，1.0mg/L，1.5mg/L，2.0mg/L，以0mg/L为对照组，并分别配制成培养基，接种分段的根部组织(根段长约3.0～4.0cm)，30d后排除被污染的根外植体后统计不定芽发生数，计算诱导率。
表2不同6-BA浓度对艾纳香根细胞脱分化产生不定芽的影响
6-BA浓度(mg/L) 诱导率(％)
0 64.6
1.0 77.5
1.5 55.0
2.0 41.7
结果如表2所示，当6-BA浓度为1.0mg/L的时候，单位根段出芽数最多，诱导艾纳香根外植体体细胞脱分化出不定芽的效果最佳。当6-BA浓度为1.5mg/L时，单位根段出芽数55.0％，而当6-BA浓度为2.0mg/L时，单位根段出芽数降至41.7％，均低于对照组。
实施例5不同NAA浓度对艾纳香根细胞脱分化产生不定芽的影响
实验方法：设置不同浓度NAA的激素添加量，以MS为基础培养基，添加1.0mg/l的6-BA、30g/L蔗糖、2～8g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮，再添加不同浓度的NAA溶液，分别是0mg/L，0.025mg/L，0.05mg/L，0.075mg/L，0.1mg/L，以0mg/L为对照组，并分别配制成培养基，接入分段的根部组织(根段长约3.0～4.0cm)，30d后排除被污染的根外植体，统计不定芽发生数，并计算诱导率。
表3不同NAA浓度对艾纳香根细胞脱分化产生不定芽的影响
NAA浓度(mg/L) 诱导率(％)
0 45.0
0.025 57.5
0.05 82.5
0.075 55.0
0.1 47.5
结果如表3所示，当NAA浓度为0.05mg/L时，单位根段出芽数最多，诱导不定芽再生的效果最佳。从总体来看，5种NAA浓度以0.05mg/L为临界点，当NAA浓度低于0.05mg/l时，获得不定芽总数随NAA浓度的增长而增多，说明此时NAA对不定芽分化起促进作用且效果逐渐增强；当NAA浓度高于0.05mg/L时，获得不定芽总数随NAA浓度的增长而减少，而且NAA浓度添加浓度为0.025～0.1mg/L，单位根段出芽数均高于对照组，说明NAA与本申请培养基中的其他成分合用，可以提高诱导不定芽再生的效果。
实施例6不同MS培养基条件对艾纳香根细胞脱分化产生不定芽的影 响
实验方法：设置3个等级的MS培养基，分别是MS、1/2MS、3/4MS，并添加0.05mg/LNAA、1.0mg/L的6-BA、30g/L蔗糖、2～8g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮，分别配制成培养基，接入分段的根部组织(根段长约3.0～4.0cm)，30d后统计不定芽发生数，并计算诱导率。
表4不同MS培养基条件对艾纳香根细胞脱分化产生不定芽的影响
基础培养基 诱导率(％)
1/2MS 47.5
3/4MS 60.4
MS 90.0
结果如表4所示，本发明以MS作为基本培养基，有效单位根段出芽数最多，诱导根外植体脱分化产生不定芽的效果佳，且诱导率显著高于1/2MS和3/4MS基本培养基。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (18)

  1. 一种一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，包括以下步骤：
    S1.切取艾纳香的根，再将根切段，消毒，得根段；
    S2.配制不定芽诱导培养基，所述不定芽诱导培养基包括：0.01～0.5mg/LNAA、0～1.0mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂和基础培养基；
    S3.诱导不定芽：将步骤S1的根段接入步骤S2配制的不定芽诱导培养基中进行培养，得艾纳香不定芽。
  2. 根据权利要求1所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述根为主根或侧根，根段的长度为1～25cm。
  3. 根据权利要求1所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述根为不定根。
  4. 根据权利要求3所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述不定芽诱导培养基替换为：以MS为基础培养基，在基础培养基中加入0.1mg/L NAA、2.0mg/L 6-BA、70g/L蔗糖、8g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮。
  5. 根据权利要求1或2所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述根段的长度为3～4cm。
  6. 根据权利要求1所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂。
  7. 根据权利要求1所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述不定芽诱导培养基还包括：0～100mg/L维生素C和0～100mg/L聚乙烯吡咯烷酮。
  8. 根据权利要求7所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖、2～8g/L琼脂、0.01～10mg/L维生素C和0.01～10mg/L聚乙烯吡咯烷酮。
  9. 根据权利要求1至8任一项所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述基础培养基为1/2MS、3/4MS或MS培养基。
  10. 根据权利要求1所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述培养的条件为25～28℃。
  11. 根据权利要求1或10所述的一步诱导艾纳香根细胞分化产生不定芽的培养方法，其特征在于，所述培养的时间为10～90天。
  12. 一种用于一步诱导艾纳香根细胞分化产生不定芽培养的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基包括：0.01～0.5mg/LNAA、0～1.0mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂和基础培养基。
  13. 根据权利要求12所述的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖和2～8g/L琼脂。
  14. 根据权利要求12所述的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.05mg/L NAA、0.1mg/L6-BA、30g/L蔗糖和2～8g/L琼脂。
  15. 根据权利要求12所述的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基还包括：0～100mg/L维生素C和0～100mg/L聚乙烯吡咯烷酮。
  16. 根据权利要求15所述的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.025～0.1mg/L NAA、1mg/L 6-BA、10～70g/L蔗糖、2～8g/L琼脂、0.01～10mg/L维生素C和0.01～10mg/L聚乙烯吡咯烷酮。
  17. 根据权利要求16所述的不定芽诱导培养基，其特征在于，所述不定芽诱导培养基为在基础培养基中加入0.05mg/L NAA、1mg/L 6-BA、30g/L蔗糖、6g/L琼脂、10mg/L维生素C和10mg/L聚乙烯吡咯烷酮。
  18. 根据权利要求12～17任一项所述的不定芽诱导培养基，其特征在于，所述基础培养基为1/2MS、3/4MS或MS培养基。
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