WO2023109320A1 - 一种获得四倍体艾纳香不定芽的方法 - Google Patents

一种获得四倍体艾纳香不定芽的方法 Download PDF

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WO2023109320A1
WO2023109320A1 PCT/CN2022/127259 CN2022127259W WO2023109320A1 WO 2023109320 A1 WO2023109320 A1 WO 2023109320A1 CN 2022127259 W CN2022127259 W CN 2022127259W WO 2023109320 A1 WO2023109320 A1 WO 2023109320A1
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tetraploid
root
buds
medium
ainaxiang
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PCT/CN2022/127259
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French (fr)
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陈晓鹭
王丹
于福来
元超
杨永
肖永锋
罗沁
黎玉兰
陈振夏
陈英桦
于平
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 JP2023565397A priority Critical patent/JP2024513535A/ja
Publication of WO2023109320A1 publication Critical patent/WO2023109320A1/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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • 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
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/12—Leaves

Definitions

  • the invention belongs to the technical field of plant polyploid breeding, and in particular relates to a method for obtaining adventitious buds of tetraploid Aina.
  • 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. Plants, whose extracts are the main raw materials of various Chinese patent medicines, are widely used in food, cosmeceutical and other market fields, and have good market prospects.
  • Ainaxiang is a medicinal plant, and its medicinal ingredient, L-borneol, belongs to its secondary metabolites. Polyploid breeding is beneficial to promote the accumulation of its secondary metabolites, including L-borneol. important direction of breeding.
  • the axillary buds are treated, that is, the buds treated are already existing and have completed bud differentiation. Therefore, in the process of artificially inducing their chromosome doubling, it can only act on cells that are undergoing mitosis. Cells that have completed mitosis cannot be stimulated to double their chromosomes without continuing mitosis. Therefore, the situation of incomplete processing can be caused, that is, all cells of the whole treatment object cannot be fully doubled, and there are both doubled tetraploid cells and non-doubled diploid cells in the obtained buds, that is, Chimera. During the growth and reproduction of chimeric plants, it is easy for polyploid plants to degenerate into diploid plants due to the difference in growth and development trends between polyploid cells and diploid cells. Naxiang's breeding needs.
  • leaf explants to first induce diploid callus using a medium, and then artificially induce the diploid callus cells to double into tetraploid callus Tissues were then transferred to another medium to induce tetraploid callus to differentiate into tetraploid adventitious shoots.
  • this technique there is no document reporting the successful application of this technique in the polyploid breeding of Aenarci.
  • artificial mutagenesis is carried out at the callus stage, but because some of the unsuccessfully mutated cells may also differentiate to form adventitious shoots, there is also the problem of chimerism in this process.
  • this technique needs to go through three steps, prepare at least three kinds of media, inoculate at least three times, and cultivate at least three stages, so not only the process is cumbersome, but also there is a certain probability of non-purpose variation that occurs occasionally in the callus stage , interfere with the results of mutagenesis or genetic transformation.
  • the purpose of the present invention is to only select the diploid plant with bud stem section (mainly utilizing existing terminal bud and axillary bud) in order to overcome the existing Ainaxiang tetraploid technology as explant, by stimulating fixed bud (terminal bud) and axillary bud) cell doubling to obtain tetraploid insufficiency, providing a method of using diploid Ainaxiang root as explant material to induce root cell doubling and direct differentiation to produce adventitious buds.
  • the present invention does not need to rely on existing fixed buds (terminal buds or axillary buds), but directly doubles and differentiates into adventitious buds from somatic cells.
  • each cell can form a bud independently and directly, and will not produce a large number of chimeras mixed with heterogeneous cells, thereby improving the accuracy of directed mutagenesis or genetic transformation, not only to obtain complete tetraploid It not only provides a technical basis for the artificial mutation and transgenic breeding of Ainaxiang, but also provides technical reference.
  • the first object of the present invention is to provide a method for inducing diploid Ainarka somatic cells to simultaneously double into tetraploid and directly differentiate to produce tetraploid adventitious buds.
  • the second object of the present invention is to provide diploid Ainaxiang root tissue without passing through the callus stage, and directly differentiate into the application in the technology of tetraploid adventitious buds.
  • the present invention claims to protect a method for obtaining adventitious buds of tetraploid A. sinensis, which utilizes root explants of diploid A. sinensis to directly induce doubling and differentiate to form tetraploid adventitious buds.
  • the source of the tissue culture method to obtain the regeneration buds is the root of diploid Ainaxiang as explants; the tissue culture method only prepares a medium, inoculates it once, and cultivates a cycle to obtain the tetraploid regeneration buds.
  • Ai Naxiang's somatic cell differentiation is indeterminate budding process, achieving the effect of chromosome doubling.
  • the method comprises the following steps: taking the root of diploid Ainaxiang as an explant, cutting the root into sections, and containing 0.025 ⁇ 0.1mg/L NAA, 1.0 ⁇ 2.0mg/L 6-BA and 90 ⁇ 150mg/L
  • the doubling medium of L colchicine was cultured.
  • the doubling medium is that 0.025 ⁇ 0.1mg/L NAA, 1.0 ⁇ 2.0mg/L 6-BA, 90 ⁇ 150mg/L colchicine, 10 ⁇ 70g/L sucrose and 2 ⁇ 8g/L agar.
  • the roots include definite or adventitious roots.
  • the culture time is 20-60 days.
  • the length of the root segment is greater than 2 cm.
  • the length of the root segment is 4-8 cm.
  • the efficiency of inducing adventitious buds is low.
  • the efficiency of inducing adventitious buds is higher. But when the length is too long, the number of explants that can be obtained by the same number of roots is on the low side.
  • the basal medium is 1/2 MS medium or MS medium.
  • the doubling medium is basal medium supplemented with 0.05 mg/L NAA, 1.0 mg/L 6-BA, 120 mg/L colchicine, 30 g/L sucrose and 6 g/L agar.
  • the present invention has the following beneficial effects:
  • the present invention overcomes the shortcomings of only selecting the budding stem section as the explant before, proliferating and inducing doubling through fixed buds (terminal buds and axillary buds), and provides a kind of root with Aina Xiang as the explant material,
  • the one-step method of inducing root cells to directly double and differentiate to produce tetraploid adventitious buds fills in the gap that the root of Aina radicis is used as an explant material for polyploid breeding of Aina radiata.
  • the application results of the present invention show that the root of Ainaxiang not only can be used as an explant for multiplication and doubling, but also can be used as a better option for explant selection.
  • the use of root explants increases the number of cultivable explant material objects, that is, increases the source of effective explants in the biotechnology breeding process of Ainaxiang.
  • the present invention can directly double the diploid Aina root cells and simultaneously differentiate into tetraploid adventitious buds, which is simpler and quicker than the prior art, and reduces the generation of chimeras;
  • 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 effect diagram of the induction of tetraploid adventitious buds of Aina radix explants.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials 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.
  • Effective root section refers to the unpolluted root section in the embodiment;
  • Chimera rate (%) (total number of chimera adventitious buds/total number of adventitious buds) ⁇ 100%.
  • Embodiment 1 A method for obtaining tetraploid Ainaxiang adventitious buds
  • the culture medium for aseptic sowing uses 1/2 MS as the base medium, and also contains 0.01-0.5 mg/L NAA, 20-50 g/L sucrose and 3-7 g/L agar.
  • the doubling medium is MS-based medium containing 0.05mg/L NAA, 0.1mg/L 6-BA, 120mg/L colchicine, 30g/L sucrose and 6g/L agar.
  • Induction rate of tetraploid buds number of tetraploid adventitious buds/number of explants.
  • Embodiment 2 A method for obtaining tetraploid Ainaxiang adventitious buds
  • each bud-bearing stem segment is 2-3 cm, and each stem segment has 2-3 living terminal buds or axillary buds;
  • the adventitious root induction medium is based on 1/2 MS, and also contains 0.01-0.5 mg/L NAA, 20-50 g/L sucrose and 3-7 g/L agar.
  • the doubling medium is based on 1/2MS, and also contains 0.1mg/L NAA, 2.0mg/L6-BA, 120mg/L colchicine, 70g/L sucrose and 6g/L agar.
  • Induction rate of tetraploid buds number of tetraploid adventitious buds/number of explants.
  • Induction rate number of tetraploid adventitious buds/number of explants.
  • MS Using MS as the basal medium, add 6-BA at a concentration of 1.0mg/L, 120mg/L colchicine, 30g/L sucrose, 6g/L agar and different concentrations of NAA solutions, respectively 0mg/L and 0.025mg /L, 0.05mg/L, 0.075mg/L, 0.1mg/L, take 0mg/L as the control group, and prepare culture medium respectively, insert the segmented root tissue (the length of the root segment is about 4.0cm), 60d Afterwards, the chromosome ploidy of adventitious buds was identified, and the number of tetraploid adventitious buds was counted to calculate the induction rate.
  • 6-BA a concentration of 1.0mg/L, 120mg/L colchicine, 30g/L sucrose, 6g/L agar and different concentrations of NAA solutions, respectively 0mg/L and 0.025mg /L, 0.05mg/L,
  • NAA concentration (mg/L) Induction rate% 0 0
  • Induction rate number of tetraploid adventitious buds/number of explants.
  • MS Using MS as the basal medium, add 6-BA, 0.05mg/LNAA, 30g/L sucrose, 6g/L agar at a concentration of 1.0mg/L, and then add different concentrations of colchicine, respectively 50mg/L, 70mg/L L, 90mg/L, 120mg/L, 150mg/L, and be prepared into culture medium respectively, insert the root tissue of segment (the length of root section is about 4.0cm), carry out chromosomal ploidy identification to the adventitious bud that produces after 60d, Count the number of tetraploid adventitious buds and calculate the doubling induction rate.
  • 6-BA 6-BA
  • 0.05mg/LNAA 30g/L sucrose
  • 6g/L agar at a concentration of 1.0mg/L
  • different concentrations of colchicine respectively 50mg/L, 70mg/L L, 90mg/L, 120mg/L, 150mg/L
  • Induction rate number of tetraploid adventitious buds/number of explants.

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Abstract

本发明公开了一种获得四倍体艾纳香不定芽的方法，即选用二倍体艾纳香的根切段作为外植体，以含有0.025～0.1mg/L NAA、1.0～2.0mg/L 6-BA和90～150mg/L秋水仙素的加倍培养基进行培养，诱导外植体细胞同时进行染色体加倍和分化不定芽。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和生物技术育种过程中有效外植体来源。更重要的是，本发明使艾纳香的根细胞直接在染色体加倍的同时分化形成不定芽，不必经过形成愈伤组织的过程，由此简化了技术环节，减少了再生芽的变异和嵌合体的产生；本发明应用于艾纳香多倍体育种，将可显著节省时间和人力成本，提高工作效率。

Description

一种获得四倍体艾纳香不定芽的方法
本申请要求于2021年12月14日提交中国专利局、申请号为202111522569.0、发明名称为“一种获得四倍体艾纳香不定芽的方法”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
本发明属于植物多倍体育种技术领域，具体涉及一种获得四倍体艾纳香不定芽的方法。
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、中国台湾等地，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，良种种苗扩繁是主要难点。艾纳香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。因此，有必要开展艾纳香种苗的离体扩繁研究。艾纳香作为药用植物，其药效成分左旋龙脑属于其次生代谢物，多倍体育种有利于促进其次生代谢物包括左旋龙脑的积累，因此多倍体育种是艾纳香生物技术育种的重要方向。
前人对艾纳香染色体加倍技术进行多种尝试，主要是基于诱导腋芽染色体加倍。但是，该技术存在根本性的缺陷。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。
在现有技术中，处理的是腋芽，即处理的芽是已经存在的、已完成芽分化的腋芽，因此在人工诱导其染色体加倍的过程中，只能作用于正在有丝分裂的细胞，对前期已经完成了有丝分裂的细胞，如果其不继续有丝分 裂，就不能刺激其染色体加倍。因此，会造成处理不完全的情况，即不能使整个处理对象的所有细胞完全加倍，所获得的芽体中既有加倍的四倍体细胞，又有未加倍的二倍体细胞的情况，即嵌合体。嵌合体植株在成长和繁殖过程中，很容易因为多倍体细胞和二倍体细胞生长发育趋势差异而出现多倍体植株退化成二倍体植株的情况，因此，现有技术难以满足目前艾纳香的育种需求。
另一种可能尝试的技术，是使用叶片外植体，先使用一种培养基诱导产生二倍体愈伤组织，然后人工诱导技术，诱导二倍体愈伤组织细胞加倍为四倍体愈伤组织，再转移到另一种培养基，诱导四倍体愈伤组织分化形成四倍体不定芽。目前尚未有文件报道这种技术在艾纳香多倍体育种方面的成功应用。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，这种技术需要经过三个步骤，配制至少三种培养基，接种至少三次，培养至少三个阶段，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，目前仍缺乏一种不会产生嵌合体或嵌合体率较低，且过程简便的获得四倍体艾纳香不定芽的组织培养方法。
发明内容
本发明的目的是为了克服现有艾纳香四倍体技术仅选择二倍体植株带芽茎段(主要利用其中已存在的顶芽和腋芽)作为外植体，通过刺激定芽(顶芽和腋芽)细胞加倍获得四倍体不足，提供一种用二倍体艾纳香的根作为外植体材料，诱导根细胞同时加倍和直接分化产生不定芽的方法。本发明不需要借助已有的定芽(顶芽或腋芽)，而是由体细胞直接加倍并分化为不定芽。本发明的好处是每一个细胞都可以独立直接地形成一个芽，不会产生大量异质细胞混杂的嵌合体，从而提高了定向诱变或遗传转化的准确性，不仅是获得完全四倍体的技术基础，而且为艾纳香人工诱变、转基因等育种工作提供技术参考。
本发明的第一个目的是提供一种诱导二倍体艾纳香体细胞同时加倍为四倍体并直接分化产生四倍体不定芽的方法。
本发明的第二个目的是提供二倍体艾纳香根部组织在不经过愈伤组 织阶段，直接分化为四倍体不定芽的技术中的应用。
为了实现上述目的，本发明是通过以下方案予以实现的：
本发明要求保护一种获得四倍体艾纳香不定芽的方法，利用二倍体艾纳香的根外植体直接诱导加倍并分化形成四倍体不定芽。该组培方法获得再生芽的来源是作为外植体的二倍体艾纳香根部；该组培方法只配制一种培养基，接种一次，培养一个周期即可获得四倍体再生芽，完成艾纳香的体细胞分化不定芽过程，达到染色体加倍的效果。
该方法包括以下步骤：以二倍体艾纳香的根为外植体，将根切成段后，以含有0.025～0.1mg/LNAA、1.0～2.0mg/L 6-BA和90～150mg/L秋水仙素的加倍培养基进行培养。
优选的，所述加倍培养基为在基础培养基中添加了0.025～0.1mg/L NAA、1.0～2.0mg/L 6-BA、90～150mg/L秋水仙素、10～70g/L蔗糖和2～8g/L琼脂。
优选的，所述根包括定根或不定根。
优选的，培养时间为20～60d。
优选的，根切成段后，根段的长度大于2cm。
更优选的，根切成段后，根段的长度为4～8cm。长度偏短的时候，诱导产生不定芽的效率较低。长度较长的时候，诱导产生不定芽的效率较高。但是长度太长的时候，同样数量的根能得到的外植体数量就偏少。
优选的，所述基础培养基为1/2MS培养基或MS培养基。
更优选的，所述加倍培养基为在基础培养基中添加了0.05mg/LNAA、1.0mg/L6-BA、120mg/L秋水仙素、30g/L蔗糖和6g/L琼脂。
与现有技术相比，本发明具有以下有益效果：
(1)本发明克服了此前仅选择带芽茎段作为外植体，通过定芽(顶芽和腋芽)增殖并诱导加倍的不足，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加倍并同时分化产生四倍体不定芽的方法，填补了艾纳香的根作为艾纳香多倍体育种外植体材料的空白。本发明的应用结果显示，艾纳香的根不仅可以作为增殖和加倍的外植体，甚至还是作为更好的外植体选择可选项。使用根外植体，增加了可培养的外植体材料对象，即增加了艾纳香在生物技术育种过程中有效外植体来源。
(2)本发明可以使二倍体艾纳香根细胞直接加倍并同时分化形成四倍 体不定芽，比现有技术更简便快捷，且减少了嵌合体的产生；
(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为艾纳香根外植体诱导四倍体艾纳香不定芽效果图。
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。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；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。
实施例中有效根段指的是未污染的根段；诱导率是指诱导形成的四倍体不定芽总数占二倍体外植体总数的百分率，计算公式为诱导率(％)＝(四倍体不定芽总数/二倍体外植体总数)×100％。嵌合体率(％)＝(嵌合体不定芽总数/不定芽总数)×100％。
实施例1一种获得四倍体艾纳香不定芽的方法
S1.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，得到艾纳香无菌实生苗，取其根部组织；
S2.将得到的根部组织分段，根段长1～8cm；
S3.利用加倍培养基培养分段的根部组织20天，得到不定芽；
其中，无菌播种的培养基以1/2MS为基础培养基，还含有0.01～0.5mg/LNAA、20～50g/L蔗糖和3～7g/L琼脂。
加倍培养基以MS为基础培养基，含有0.05mg/LNAA、0.1mg/L 6-BA、120mg/L秋水仙素、30g/L蔗糖和6g/L琼脂。
S4.对得到的不定芽进行染色体倍性鉴定，统计四倍体芽数量，计算四倍体芽诱导率。
结果显示当二倍体根段长度为1～2cm的时候，几乎无法得到四倍体芽(根段长度为1cm时无法得到，根段长度为2cm时，96个二倍外植体 只得到1个四倍体芽)，因此如果根据常规操作把根段分割为1～2cm长，并在加倍培养基上进行培养，将几乎无法达到加倍目的，获得多倍体的植株。只有当二倍体根段长度达到或超过4cm的时候，能得到加倍后的四倍体不定芽。
表1不同长二倍体根外植体诱导四倍体不定芽的诱导率
Figure PCTCN2022127259-appb-000001
四倍体芽诱导率：四倍体不定芽数/外植体数。
实施例2一种获得四倍体艾纳香不定芽的方法
S1.利用不定根诱导培养基培养外植体分化得到不定根，培养外植体30天；
S2.将不定根分段，每段长6cm；
S3.利用传统艾纳香组培技术培养得到带芽茎段，每个带芽茎段长度2～3cm，每个茎段带有活的顶芽或腋芽2～3个；
S4.利用加倍培养基培养分段的不定根和带芽茎段，培养60天，得到不定芽；
其中，不定根诱导培养基以1/2MS为基础培养基，还含有0.01～0.5mg/LNAA、20～50g/L蔗糖和3～7g/L琼脂。
加倍培养基以1/2MS为基础培养基，还含有0.1mg/LNAA、2.0mg/L6-BA、120mg/L秋水仙素70g/L蔗糖和6g/L琼脂。
S5.对得到的不定芽进行染色体倍性鉴定，统计二倍体、四倍体和嵌合体芽数量，计算四倍体芽诱导率和嵌合体率。
结果显示以根为外植体诱导加倍得到的四倍体的技术，与已有技术相比不定芽效率较高，且嵌合体比例低。
表2不同类型外植体诱导四倍体芽的诱导率
外植体类型 外植体数 四倍体芽诱导率(％) 嵌合体率(％)
根段 96 10.42 30.00
带芽茎段 96 6.25 83.33
四倍体芽诱导率：四倍体不定芽数/外植体数。
实施例3不同6-BA浓度的影响
一、实验方法
以MS为基本培养基，添加0.05mg/LNAA、120mg/L秋水仙素、30g/L蔗糖、6g/L琼脂和不同浓度的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6.0cm)，60d后排除被污染的根外植体后统计四倍体芽数，计算加倍效率。
二、实验结果
结果如表3所示，当培养基中添加的6-BA浓度为1.0mg/L时，诱导艾纳香根外植体体细胞加倍并脱分化出四倍体不定芽的效果最佳。
表3不同6-BA浓度对艾纳香体细胞脱分化产生不定芽的影响
6-BA浓度(mg/L) 诱导率(％)
0 0
1.0 7.75
1.5 5.5
2.0 4.17
诱导率：四倍体不定芽数/外植体数。
实施例4不同NAA浓度的影响
一、实验方法
以MS为基础培养基，添加浓度为1.0mg/L的6-BA、120mg/L秋水仙素、30g/L蔗糖、6g/L琼脂和不同浓度的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4.0cm)，60d后对不定芽进行染色体倍性鉴定，并统计四倍体不定芽发生数，计算诱导率。
二、实验结果
结果显示，NAA浓度为0.05mg/L时，加倍效果最佳。
表4不同NAA浓度的影响
NAA浓度(mg/L) 诱导率％
0 0
0.025 2.08
0.05 10.42
0.075 4.17
0.1 3.13
诱导率：四倍体不定芽数/外植体数。
实施例6不同秋水仙素浓度的影响
一、实验方法
以MS为基础培养基，添加浓度1.0mg/L的6-BA、0.05mg/LNAA、30g/L蔗糖、6g/L琼脂，再添加不同浓度的秋水仙素，分别是50mg/L、70mg/L、90mg/L、120mg/L、150mg/L，并分别配制成培养基，接入分段的根部组织(根段长约4.0cm)，60d后对产生的不定芽进行染色体倍性鉴定，统计四倍体不定芽数，并计算加倍诱导率。
二、实验结果
结果当秋水仙素浓度为120mg/L时，诱导二倍体艾纳香根外植体产生四倍体不定芽的效果最佳。
表6不同蔗糖浓度对艾纳香体细胞脱分化产生不定芽的影响
秋水仙素浓度(mg/L) 诱导率％
50 0
70 0
90 2.08
120 10.42
150 4.17
诱导率：四倍体不定芽数/外植体数。
以上所述仅为本发明的较佳实施例而已，并不用限制本发明，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 (12)

  1. 一种获得四倍体艾纳香不定芽的方法，其特征在于，包括以下步骤：以二倍体艾纳香的根为外植体，将根切成段后，以含有0.025～0.1mg/L NAA、1.0～2.0mg/L 6-BA和90～150mg/L秋水仙素的加倍培养基进行培养。
  2. 根据权利要求1所述获得四倍体艾纳香不定芽的方法，其特征在于，所述加倍培养基为在基础培养基中添加了0.025～0.1mg/L NAA、1.0～2.0mg/L 6-BA、90～150mg/L秋水仙素、10～70g/L蔗糖和2～8g/L琼脂。
  3. 根据权利要求1所述获得四倍体艾纳香不定芽的方法，其特征在于，所述根包括定根或不定根。
  4. 根据权利要求1所述获得四倍体艾纳香不定芽的方法，其特征在于，培养时间为20～60d。
  5. 根据权利要求1所述获得四倍体艾纳香不定芽的方法，其特征在于，根切成段后，根段的长度大于2cm。
  6. 根据权利要求5所述获得四倍体艾纳香不定芽的方法，其特征在于，所述根段的长度为4～8cm。
  7. 根据权利要求2所述获得四倍体艾纳香不定芽的方法，其特征在于，所述基础培养基为1/2MS培养基或MS培养基。
  8. 根据权利要求2或7所述获得四倍体艾纳香不定芽的方法，其特征在于，所述加倍培养基为在基础培养基中添加了0.05mg/LNAA、1.0mg/L 6-BA、120mg/L秋水仙素、30g/L蔗糖和6g/L琼脂。
  9. 一种用于获得四倍体艾纳香不定芽的加倍培养基，其特征在于，所述加倍培养基含有0.025～0.1mg/LNAA、1.0～2.0mg/L 6-BA和90～150mg/L秋水仙素。
  10. 根据权利要求9所述的加倍培养基，其特征在于，所述加倍培养基为在基础培养基中添加了0.025～0.1mg/L NAA、1.0～2.0mg/L 6-BA、90～150mg/L秋水仙素、10～70g/L蔗糖和2～8g/L琼脂。
  11. 根据权利要求9所述的加倍培养基，其特征在于，所述加倍培养基为在基础培养基中添加了0.05mg/LNAA、1.0mg/L 6-BA、120mg/L秋 水仙素、30g/L蔗糖和6g/L琼脂。
  12. 根据权利要求10或11所述的加倍培养基，其特征在于，所述基础培养基为1/2MS培养基或MS培养基。
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