WO2021179527A1 - 一种粉葛单芽茎段增殖及一步成苗培养方法 - Google Patents

一种粉葛单芽茎段增殖及一步成苗培养方法 Download PDF

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WO2021179527A1
WO2021179527A1 PCT/CN2020/107676 CN2020107676W WO2021179527A1 WO 2021179527 A1 WO2021179527 A1 WO 2021179527A1 CN 2020107676 W CN2020107676 W CN 2020107676W WO 2021179527 A1 WO2021179527 A1 WO 2021179527A1
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culture
seedling
bud
seedlings
sterile
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严华兵
肖亮
曾文丹
尚小红
曹升
陆柳英
赖大欣
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广西壮族自治区农业科学院
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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
    • A01H4/001—Culture apparatus 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
    • A01H4/008—Methods for regeneration to complete plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/40—Afforestation or reforestation

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  • the invention belongs to the technical field of in vitro culture and propagation of plant tissues, and specifically relates to a method for multiplying single bud stem segments of Pueraria lobata and one-step seedling cultivation.
  • Pueraria thomsonii Benth is one of the more abundant resources in the legume Pueraria DC. It is a dual-purpose plant for medicine and food recognized by the Ministry of Agriculture and the National Health and Family Planning Commission. , The reputation of "Asian Ginseng". Pueraria lobata is rich in starch, and the starch content in fresh pueraria is about 15% to 32%. It is an important raw material for starch industry and can be used as a new energy source for non-grain biomass. At the same time, Pueraria lobata root tuber contains isoflavones such as puerarin and daidzein and more than 10 kinds of trace elements necessary for the human body.
  • Pueraria lobata is mainly distributed in the area south of the Yangtze River. Guangxi is rich in germplasm resources and has a wide distribution area. The annual planting area of Pueraria lobata is more than 200,000 mu, ranking first in the country. Among them, Heping Town, Teng County, Wuzhou City, Guangxi is the famous "Hometown of Pueraria lobata" in China. Since the 1990s, the planting of Pueraria lobata has been developed on a large scale. Therefore, the development of large-scale pueraria lobata seedling technology is of great significance to the healthy development of pueraria lobata industry.
  • Pueraria lobata seedlings mainly adopt cutting and tissue culture in vitro propagation.
  • Chinese patent CN102144555B is a method for rapid propagation of kudzu root, which is to sterilize kudzu root shoots and inoculate them on a prepared medium, germinate and transfer them to a prepared differentiation medium for cultivation. Seeing that the young leaves are pulled out and the stems are elongated , Cut and connect to the above-prepared differentiation medium for propagation;
  • the rapid propagation method of Chinese patent application CN106172004A Pueraria lobata root is to select the shoot tip of the pueraria root as the explant, use MS medium as the basic medium for the bud induction medium, and add 0.25 ⁇ 0.35 mg/L 2,4-dichlorophenoxyacetic acid.
  • the rooting medium uses 1/2MS medium as the basic medium, add 0.25-0.3 mg/L naphthaleneacetic acid and 0.1-0.2 mg/L gibberellin, and the strong seedling medium uses 2/3MS medium as the basic medium.
  • Chinese patent application CN108064699A is a method for tissue culture propagation of Pueraria lobata root plant in vitro, using thin slices of pueraria root plant stems as explants, through callus induction, adventitious bud induction, adventitious bud proliferation, rooting culture and optional strong The seedling cultivation step is completed.
  • the above studies have disclosed the tissue culture and propagation methods of Pueraria lobata plants in vitro, but they have a common feature. Their seedling propagation methods are through the proliferation of cluster buds, and then through adventitious bud rooting culture, strong seedlings and transplanting to survive. This technology The route has weak seedling growth, and the quality of tissue culture seedlings needs to be improved by replacing the medium for strong seedling culture or separately performing strong seedling, rooting culture and other technical links.
  • the purpose of the present invention is to provide a method for multiplication of single buds of Pueraria lobata and one-step seedling cultivation, which not only improves the quality of tissue culture seedlings and shortens the culture period, but also can provide multiple species and hairy roots of Pueraria lobata Genetic transformation technology provides good receptor materials.
  • a method for multiplying and cultivating single bud stem segments of Pueraria lobata single bud and one-step seedling formation specifically includes the following steps:
  • Explant treatment select young vines that grow robustly and are free of diseases and insect pests, soak and disinfect, then rinse with sterile water, and finally use sterile filter paper to absorb the moisture on the surface of the stem for use;
  • Aseptic seedling culture Under aseptic conditions, cut the S1 treated vine into budded stems, and insert the lower end of the biology into the primary culture medium for cultivation to obtain aseptic seedlings;
  • the primary culture medium is: MS basic medium+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar, pH is 5.8;
  • Single-bud stem proliferation and rooting induction under aseptic conditions, cut the aseptic seedling obtained in the previous step into single-bud stems with an axillary bud and transfer to the intermittent immersion culture device for subsequent proliferation and rooting culture;
  • the formula of the subculture rooting medium is: modified MS+NAA 0.02 ⁇ 0.05mg/L+IBA 0.01 ⁇ 0.02mg/L+PVP0.02% ⁇ 0.05%+1-aminocyclopropanecarboxylic acid 2 ⁇ 4mg/L+sucrose 30g /L;
  • Tissue cultured seedlings are transplanted and survived:
  • the sterile bottle seedlings with a height of 3 ⁇ 4cm, strong growth and well-developed roots are transferred from the cultivation room to the greenhouse for smelting, then the seedlings are taken out, the attached liquid medium is washed, and the nutrients are planted. In the bowl.
  • the immersion disinfection described in S1 of the present invention is then rinsed with sterile water by first immersing and disinfecting with 0.1% mercury liters for 6-10 minutes, and then rinsing with sterile water for 4 to 5 times.
  • the biological lower end is vertically inserted into the primary culture medium for culture, and the culture conditions are: temperature 25 ⁇ 1°C, light 1500-2000lx, light time 16h/d, and culture period of 25-30d.
  • the sub-generation rooting culture described in S3, the culture conditions are: temperature 25 ⁇ 1°C, light 1500-2000lx, light time 16h/d; the intermittent immersion culture condition is 2min culture every 4h.
  • tissue cultured seedlings of Pueraria lobata root obtained by sub-generation and rooting culture described in S3 can also be directly rooted, and the rooting rate is over 99%.
  • the present invention has the following advantages:
  • the method for tissue culture of Pueraria lobata generally includes the treatment and disinfection of explants, callus induction, adventitious bud induction, adventitious bud proliferation, rooting culture and optional strong seedling culture, etc.
  • the seedling proliferation The method is to multiply by clumping buds, and then go through the steps of adventitious bud rooting culture, strong seedlings and transplanting to survive. This technical route has weak seedling growth, and needs to replace the medium for strong seedling culture or separate strong seedling, rooting culture, etc.
  • the method of the present invention is an efficient and simple technical method for in vitro propagation of Pueraria lobata, which adopts the method of single bud subgeneration and one-step seedling formation. It is not only simple to operate, but also has high robust rooting rate and high rooting rate of tissue cultured seedlings obtained. It is 99.6%-100%, the plant root system is vigorous, the transplanting survival rate is high, and the transplanting survival rate of the method of the present invention is as high as 99% or more.
  • the culture method of the secondary proliferation and rooting culture of the present invention is intermittent immersion culture. Compared with the existing Pueraria lobata tissue culture method, the rooting rate can be significantly increased, and the root system is developed, which improves the transplantation of tissue culture seedlings. The survival rate of planting, while avoiding the step of cleaning the culture medium during transplanting, reduces the workload and improves the work efficiency.
  • 1-aminocyclopropane carboxylic acid is a non-protein amino acid contained in pear and apple juice. It is a kind of cyclic amino acid. Plant tissues generally have the ability to perform under aerobic conditions. The activity of decomposing ACC to produce ethylene.
  • adding an appropriate concentration of ACC can significantly increase the rooting rate of Pueraria lobata tissue cultured seedlings, and at the same time achieve the effect of strong seedlings and increase the survival rate of transplanting;
  • Polyvinylpyrrolidone (PVP) can effectively inhibit the cultivation of Pueraria lobata tissue cultured seedlings. During the process, browning caused slow growth of plants and reduced growth vigor and other unfavorable factors.
  • a method for multiplying and cultivating single bud stem segments of Pueraria lobata single bud and one-step seedling formation specifically includes the following steps:
  • Explant treatment select young vines that grow robustly and are free of diseases and insect pests, first soak and disinfect with 0.1% mercury liters for 10 minutes, then rinse with sterile water for 5 times, and finally use sterile filter paper to absorb moisture on the surface of the stem for use;
  • Aseptic seedling culture Under aseptic conditions, cut the S1 treated vine into shoots with buds, and insert the lower end of the biology into the primary culture medium for cultivation.
  • the culture conditions are: temperature 25 ⁇ 1°C ,Illumination of 1500lx, illumination time of 16h/d, culture period of 25 days, to obtain sterile seedlings;
  • the primary culture medium is: MS basic medium+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar, pH is 5.8;
  • Single bud stem proliferation and rooting induction under aseptic conditions, cut the sterile seedlings obtained in the previous step into single bud stems with an axillary bud and transfer them to the intermittent immersion culture device for subsequent proliferation and rooting culture;
  • the conditions are: temperature 25 ⁇ 1°C, light 2000lx, light time 16h/d; intermittent immersion culture conditions are 2min every 4h;
  • the formula of the subculture rooting medium is: modified MS+NAA 0.03mg/L+IBA 0.01mg/L+PVP 0.02%+1-aminocyclopropane carboxylic acid 3mg/L+sucrose 30g/L;
  • Tissue cultured seedlings are transplanted and survived: the sterile bottle seedlings with a plant height of 3 to 4 cm, strong growth and well-developed roots are transferred from the culture room to the greenhouse for 5 days, then the seedlings are taken out, and the attached liquid medium is washed and planted
  • a method for multiplying and cultivating single bud stem segments of Pueraria lobata single bud and one-step seedling formation specifically includes the following steps:
  • Explant treatment select young vines that grow robustly and are disease-free, first soak and disinfect with 0.1% mercury liters for 6 minutes, then rinse with sterile water 4 times, and finally use sterile filter paper to absorb moisture on the surface of the stem for use;
  • Aseptic seedling culture Under aseptic conditions, cut the S1 treated vine into shoots with buds, and insert the lower end of the biology into the primary culture medium for cultivation.
  • the culture conditions are: temperature 25 ⁇ 1°C ,Illumination 2000lx, illumination time 16h/d, culture period of 30 days, to obtain sterile seedlings;
  • the primary culture medium is: MS basic medium+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar, pH is 5.8;
  • Single bud stem proliferation and rooting induction under aseptic conditions, cut the sterile seedlings obtained in the previous step into single bud stems with an axillary bud and transfer them to the intermittent immersion culture device for subsequent proliferation and rooting culture;
  • the conditions are: temperature 25 ⁇ 1°C, light 1800lx, light time 16h/d; intermittent immersion culture conditions are 2min culture every 4h;
  • the formula of the subculture rooting medium is: modified MS+NAA 0.05mg/L+IBA 0.02mg/L+PVP 0.05%+1-aminocyclopropane carboxylic acid 4mg/L+sucrose 30g/L;
  • Tissue cultured seedlings are transplanted and survived: the sterile bottle seedlings with a plant height of 3 to 4 cm, strong growth and well-developed roots are transferred from the culture room to the greenhouse for 7 days, then the seedlings are taken out, the attached liquid medium is washed, and the seedlings are planted.
  • a method for multiplying and cultivating single bud stem segments of Pueraria lobata single bud and one-step seedling formation specifically includes the following steps:
  • Explant treatment select young vines that grow robustly and are disease-free, first soak and disinfect with 0.1% mercury liters for 8 minutes, then rinse with sterile water 4 times, and finally use sterile filter paper to absorb moisture on the surface of the stem for use;
  • Aseptic seedling culture Under aseptic conditions, cut the S1 treated vine into shoots with buds, and insert the lower end of the biology into the primary culture medium for cultivation.
  • the culture conditions are: temperature 25 ⁇ 1°C ,Illumination of 1800lx, illumination time of 16h/d, culture period of 28 days, to obtain sterile seedlings;
  • the primary culture medium is: MS basic medium+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar, pH is 5.8;
  • Single bud stem proliferation and rooting induction under aseptic conditions, cut the sterile seedlings obtained in the previous step into single bud stems with an axillary bud and transfer them to the intermittent immersion culture device for subsequent proliferation and rooting culture;
  • the conditions are: temperature 25 ⁇ 1°C, light 1500lx, light time 16h/d; intermittent immersion culture conditions are 2min every 4h;
  • the formula of the subculture rooting medium is: modified MS+NAA 0.02mg/L+IBA 0.02mg/L+PVP 0.03%+1-aminocyclopropanecarboxylic acid 2mg/L+sucrose 30g/L;
  • Tissue cultured seedlings are transplanted and survived:
  • the sterile bottle seedlings with a plant height of 3 to 4 cm, strong growth and well-developed roots are transferred from the culture room to the greenhouse for 6 days, then the seedlings are taken out, the attached liquid medium is washed, and the seedlings are planted.
  • step S3 the subgeneration rooting medium formula is: MS+IBA0.02mg/L+NAA0.02mg/L, the rest is the same as in Example 3.
  • step S3 the subgeneration rooting medium formula is: MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.03%, the rest is the same as in Example 3.
  • step S3 the subculture rooting medium formula is: MS+IBA0.01mg/L+NAA0.02mg/L+1-aminocyclopropanecarboxylic acid 2mg/L, the rest is the same as in Example 3.
  • step S3 the formula of the subgeneration rooting medium is: modified MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.03%+1-aminocyclopropanecarboxylic acid 6mg/L, the rest are the same Example 3.
  • step S3 the following generation rooting medium formula is: modified MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.08%+1-aminocyclopropanecarboxylic acid 2mg/L, the rest is the same Example 3.
  • step S3 the influence of different subgeneration rooting medium formulations on the rooting rate of Pueraria lobata is shown in Table 1:
  • ACC 1-aminocyclopropane carboxylic acid
  • PVP polyvinylpyrrolidone
  • step S3 the effects of different induction methods on the regeneration cycle and transplant survival rate of Pueraria lobata root tissue culture seedlings are shown in Table 2:
  • the method of the present invention greatly shortens the cultivation period through the innovation of the cultivation method and the cultivation method, shortening the cultivation period from 120-160 days of the conventional method to 50-55 days; and the operation is simple and the cost is low.
  • the tissue culture seedlings obtained are robust and have a high rooting rate, up to 100%.
  • the plant has a vigorous root system and a high survival rate after transplanting.

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Abstract

一种粉葛单芽茎段增殖及一步成苗培养方法，具体包括如下步骤：S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，浸泡消毒然后用无菌水冲洗，最后用无菌滤纸吸干茎段表面水分备用；S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，获得无菌苗；S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生根培养；S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗，然后取出幼苗，洗净附着的液体培养基，栽入营养钵中。

Description

一种粉葛单芽茎段增殖及一步成苗培养方法
本申请要求于2020年03月13日提交中国专利局、申请号为202010175228.X、发明名称为“一种粉葛单芽茎段增殖及一步成苗培养方法”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
本发明属于植物组织离体培养繁殖技术领域，具体涉及一种粉葛单芽茎段增殖及一步成苗培养方法。
背景技术
粉葛(Pueraria thomsoniiBenth)为豆科葛属(Pueraria DC.)植物中资源较为丰富的一类，是国家农业部和卫计委共同认定的药食两用植物，素有“北参南葛”、“亚洲人参”之美誉。粉葛富含淀粉，鲜葛根中淀粉含量约为15％～32％，是重要的淀粉工业原料，可作为非粮生物质新能源来源。同时，粉葛块根中含有葛根素、大豆苷等异黄酮类物质和10多种人体所必需的微量元素，不仅具有清热解毒、生津透疹、升阳止泻的作用，而且在心血管系统、抗癌、降血糖等方面显示了多种药理活性，是新兴的绿色保健食品和出口紧销产品。随着大健康理念的产生及产业发展，药食同源的粉葛越来越受到人们的重视。
粉葛主要分布在长江以南地区，广西粉葛种质资源丰富，分布面积广，年种植葛根面积超过近20万亩，居全国首位。其中，广西梧州市藤县和平镇是中国著名的“葛根之乡”，从20世纪90年代以来就大规模发展粉葛种植。因此，开展野葛规模化育苗技术，对葛根产业的健康发展意义重大。
现有研究中，粉葛育苗主要采用扦插和组织离体培养繁殖两种方式。例如：
中国专利CN102144555B一种葛根的快速繁殖方法，是将葛根幼芽消毒后接种到配制好的培养基上萌动后转接到配制好的分化培养基上培养，见有幼叶抽出，茎节拉长，剪断接入上述配制好的分化培养基上进行扩繁；
中国专利申请CN106172004A葛根的快速繁殖方法，是选用葛根茎尖作为外植体，芽诱导培养基以MS培养基为基本培养基，添加0.25～0.35 毫克/升2，4-二氯苯氧醋酸，生根培养基以1/2MS培养基为基本培养基，添加0.25-0.3毫克/升萘乙酸以及0.1-0.2毫克/升赤霉素，壮苗培养基以2/3MS培养基为基本培养基，添加0.3-0.5毫克/升萘乙酸；茎尖的萌发率均达92％以上，生根率可达91％以上；
中国专利申请CN108064699A一种葛根植物的组织离体培养繁殖方法，利用葛根植物茎段的薄切片为外植体，通过愈伤组织诱导、不定芽诱导、不定芽增殖、生根培养和可选的壮苗培养步骤完成。以上研究公开了葛根植物的组织离体培养繁殖方法，但是有一个共同特点，其种苗增殖方式都是通过丛生芽增殖，再经过不定芽生根培养、壮苗并移栽成活环节，这一技术路线存在苗长势弱，需要通过更换培养基进行壮苗培养或者单独进行壮苗、生根培养等技术环节来提高组培苗的质量。
发明内容
针对上述问题。为了克服以上缺点，本发明目的在于提供一种粉葛单芽茎段增殖及一步成苗培养方法，既提高粉葛组培苗质量和缩短培养周期，又能够为粉葛多倍体育种和毛状根遗传转化技术提供良好的受体材料。
本发明的目的可以通过以下技术方案实现：
一种粉葛单芽茎段增殖及一步成苗培养方法，具体包括如下步骤：
S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，浸泡消毒然后用无菌水冲洗，最后用无菌滤纸吸干茎段表面水分备用；
S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，获得无菌苗；
初代培养基为：MS基本培养基+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L蔗糖+6.0g/L琼脂，pH为5.8；
S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生根培养；
继代增殖生根培养基配方为：改良MS+NAA 0.02～0.05mg/L+IBA 0.01～0.02mg/L+PVP0.02％～0.05％+1-氨基环丙烷羧酸2～4mg/L+蔗糖30g/L；
S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗，然后取出幼苗，洗净附着的液体培养基，栽入营养钵中。
本发明S1所述的浸泡消毒然后用无菌水冲洗，是先用0.1％升汞浸泡消毒6～10min，然后用无菌水冲洗4～5次。
S2所述的将生物学下端垂直竖插于初代培养基中培养，培养条件为：温度25±1℃，光照1500～2000lx，光照时间16h/d，培养周期为25～30d。
S3所述的继代增殖生根培养，培养条件为：温度25±1℃，光照1500～2000lx，光照时间16h/d；所述的间歇浸没培养条件为每4h培养2min。
S3所述的继代增殖生根培养得到葛根组培苗也可直接生根，其生根率为99％以上。
S4所述的从培养室移入温室大棚炼苗，是从培养室移入温室大棚炼苗5～7d；所述的营养钵，其中的混合基质是泥炭土：珍珠岩＝3:1的体积比。
与现有技术相比，本发明具有如下优点：
1、现有技术中粉葛组培方法一般包括外植体的处理和消毒、愈伤组织诱导、不定芽诱导、不定芽增殖、生根培养和可选的壮苗培养等步骤，其种苗增殖方式都是通过丛生芽增殖，再经过不定芽生根培养、壮苗并移栽成活环节，这一技术路线存在苗长势弱，需要通过更换培养基进行壮苗培养或者单独进行壮苗、生根培养等技术环节来提高组培苗的质量；而本发明通过单芽茎段增殖及一步成苗方法，既提高了粉葛组培苗质量和缩短了培养周期，又能够为粉葛多倍体育种和毛状根遗传转化技术提供良好的受体材料。
2、本发明方法是一种高效、简易的粉葛离体繁育技术方法，采用单芽继代增殖及一步成苗的方法，不仅操作简单，所获得的组培苗健壮生根率高，生根率为99.6％～100％，植株根系旺盛，移栽成活率高，本发明方法的移栽成活率高达99％以上。
3、本发明所述继代增殖生根培养的培养方式为间歇式浸没培养，与现有的粉葛组培培养方式相比，可以显著提高生根率，且根系发达，提高了组培苗的移栽成活率，同时避免了移栽时清洗培养基的步骤，减轻工作 量，提高工作效率。
4、本发明方法中1-氨基环丙烷羧酸(ACC)为含于梨及苹果果汁中的非蛋白性质的氨基酸，为环状氨基酸的一种，植物组织一般都具有在有氧的条件下使ACC分解生成乙烯的活性。本实验通过添加适宜浓度的ACC可显著提高葛根组培苗的生根率，同时达到了壮苗的效果，提高了移栽成活率；聚乙烯吡咯烷酮(PVP)可有效抑制粉葛组培苗在培养过程中因褐化导致植株生长缓慢、降低生长势等不利因素。
具体实施方式
以下结合实施例对本发明的具体实施方式做进一步说明。
实施例1
一种粉葛单芽茎段增殖及一步成苗培养方法，具体包括如下步骤：
S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，先用0.1％升汞浸泡消毒10min，然后用无菌水冲洗5次，最后用无菌滤纸吸干茎段表面水分备用；
S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，培养条件为：温度25±1℃，光照1500lx，光照时间16h/d，培养周期为25天，获得无菌苗；
初代培养基为：MS基本培养基+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L蔗糖+6.0g/L琼脂，pH为5.8；
S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生根培养；培养条件为：温度25±1℃，光照2000lx，光照时间16h/d；间歇浸没培养条件为每4h培养2min；
继代增殖生根培养基配方为：改良MS+NAA 0.03mg/L+IBA 0.01mg/L+PVP 0.02％+1-氨基环丙烷羧酸3mg/L+蔗糖30g/L；
S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗5d，然后取出幼苗，洗净附着的液体培养基，栽入混合基质是泥炭土：珍珠岩＝3:1的体积比的营养钵中。
实施例2
一种粉葛单芽茎段增殖及一步成苗培养方法，具体包括如下步骤：
S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，先用0.1％升汞浸泡消毒6min，然后用无菌水冲洗4次，最后用无菌滤纸吸干茎段表面水分备用；
S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，培养条件为：温度25±1℃，光照2000lx，光照时间16h/d，培养周期为30天，获得无菌苗；
初代培养基为：MS基本培养基+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L蔗糖+6.0g/L琼脂，pH为5.8；
S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生根培养；培养条件为：温度25±1℃，光照1800lx，光照时间16h/d；间歇浸没培养条件为每4h培养2min；
继代增殖生根培养基配方为：改良MS+NAA 0.05mg/L+IBA 0.02mg/L+PVP 0.05％+1-氨基环丙烷羧酸4mg/L+蔗糖30g/L；
S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗7d，然后取出幼苗，洗净附着的液体培养基，栽入混合基质是泥炭土：珍珠岩＝3:1的体积比的营养钵中。
实施例3
一种粉葛单芽茎段增殖及一步成苗培养方法，具体包括如下步骤：
S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，先用0.1％升汞浸泡消毒8min，然后用无菌水冲洗4次，最后用无菌滤纸吸干茎段表面水分备用；
S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，培养条件为：温度25±1℃，光照1800lx，光照时间16h/d，培养周期为28天，获得无菌苗；
初代培养基为：MS基本培养基+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L蔗糖+6.0g/L琼脂，pH为5.8；
S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生 根培养；培养条件为：温度25±1℃，光照1500lx，光照时间16h/d；间歇浸没培养条件为每4h培养2min；
继代增殖生根培养基配方为：改良MS+NAA 0.02mg/L+IBA 0.02mg/L+PVP 0.03％+1-氨基环丙烷羧酸2mg/L+蔗糖30g/L；
S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗6d，然后取出幼苗，洗净附着的液体培养基，栽入混合基质是泥炭土：珍珠岩＝3:1的体积比的营养钵中。
对比例1
除步骤S3中，继代增殖生根培养基配方为：MS+IBA0.02mg/L+NAA0.02mg/L之外，其余同实施例3。
对比例2
除步骤S3中，继代增殖生根培养基配方为：MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.03％之外，其余同实施例3。
对比例3
除步骤S3中，继代增殖生根培养基配方为：MS+IBA0.01mg/L+NAA0.02mg/L+1-氨基环丙烷羧酸2mg/L之外，其余同实施例3。
对比例4
除步骤S3中，继代增殖生根培养基配方为：改良MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.03％+1-氨基环丙烷羧酸6mg/L之外，其余同实施例3。
对比例5
除步骤S3中，继代增殖生根培养基配方为：改良MS+IBA0.02mg/L+NAA0.02mg/L+PVP0.08％+1-氨基环丙烷羧酸2mg/L之外，其余同实施例3。
不同继代增殖生根培养基配方对粉葛生根率的影响对比结果如下：
在步骤S3中，不同继代增殖生根培养基配方对粉葛生根率的影响参见表1：
表1不同继代增殖生根培养基配方对粉葛生根率的影响
Figure PCTCN2020107676-appb-000001
表1实验结果表明：
通过添加适应浓度的1-氨基环丙烷羧酸(ACC)和聚乙烯吡咯烷酮(PVP)可有效抑制粉葛组培苗在培养过程中因褐化等原因导致植株生长缓慢，降低生长势等不利因素，显著提高了粉葛组培苗的生根率，同时达到了壮苗的效果，显著提高了移栽成活率。
不同诱导方法对葛根组培苗再生周期及移栽成活率的影响对比结果如下：
在步骤S3中，不同诱导方法对葛根组培苗再生周期及移栽成活率的影响参见表2：
表2不同诱导方法对葛根组培苗再生周期及移栽成活率的影响
Figure PCTCN2020107676-appb-000002
表2实验结果表明：
本发明方法通过培养方式和培养方法的创新，大大缩短了培养周期，由常规方法的120～160d缩短为50～55d；且操作简单，成本低廉。同时获得的组培苗健壮、生根率高，最高可达100％。植株根系旺盛，移栽成活率高。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同 等变化或修饰变更，均应属于本发明所涵盖专利范围。

Claims (6)

  1. 一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：具体包括如下步骤：
    S1.外植体处理：选取生长健壮、无病虫害的幼嫩藤蔓，浸泡消毒然后用无菌水冲洗，最后用无菌滤纸吸干茎段表面水分备用；
    S2.无菌苗培养：在无菌条件下，将经过S1处理的藤蔓切成带芽的茎段，将生物学下端垂直竖插于初代培养基中培养，获得无菌苗；
    初代培养基为：MS基本培养基+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L蔗糖+6.0g/L琼脂，pH为5.8；
    S3.单芽茎段增殖生根诱导：在无菌条件下，将上步骤所获得无菌苗切成带一个腋芽的单芽茎段转接到间歇式浸没培养装置进行继代增殖生根培养；
    继代增殖生根培养基配方为：改良MS+NAA 0.02～0.05mg/L+IBA 0.01～0.02mg/L+PVP0.02％～0.05％+1-氨基环丙烷羧酸2～4mg/L+蔗糖30g/L；
    S4.组培苗移栽成活：将株高3～4cm、生长健壮、根系发达的无菌瓶苗从培养室移入温室大棚炼苗，然后取出幼苗，洗净附着的液体培养基，栽入营养钵中。
  2. 根据权利要求1所述的一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：S1所述的浸泡消毒然后用无菌水冲洗，是先用0.1％升汞浸泡消毒6～10min，然后用无菌水冲洗4～5次。
  3. 根据权利要求1所述的一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：S2所述的将生物学下端垂直竖插于初代培养基中培养，培养条件为：温度25±1℃，光照1500～2000lx，光照时间16h/d，培养周期为25～30d。
  4. 根据权利要求1所述的一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：S3所述的继代增殖生根培养，培养条件为：温度25±1℃，光照1500～2000lx，光照时间16h/d；所述的间歇浸没培养条件为每4h培养2min。
  5. 根据权利要求1所述的一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：S3所述的继代增殖生根培养得到葛根组培苗能直接生根。
  6. 根据权利要求1所述的一种粉葛单芽茎段增殖及一步成苗培养方法，其特征在于：S4所述的从培养室移入温室大棚炼苗，是从培养室移入温室大棚炼苗5～7d；所述的营养钵，其中的混合基质是泥炭土：珍珠岩＝3:1的体积比。
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