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Kudzu root Induction of in vitro_4151

By Nicholas Cole,2014-11-24 16:14
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Kudzu root Induction of in vitro_4151

Kudzu root Induction of in vitro

     Author: Fan Zhang, Jian-Jun Qi, Li-Li Zhou, QU Yan-Ying, Li

    Xianen

    [Abstract] Objective To establish a kudzu-free vaccine under the conditions of

    cultivation and in vitro induced by in vitro root system. Ways to kudzu seeds as

    explants, MS medium as the basic medium, culture conditions of 25 ? , 2000 ~ 3000 Lx

    light, 12h / d. The results of mechanical damage kinds of skin to be effective in breaking seed dormancy. Remove the terminal bud is conducive to the occurrence of kudzu in vitro multiple shoot clumps. Rooting of plantlets have been transferred to root induction medium, the roots became thicker, forming the tube kudzu root, the best formula for the medium: MS 6-BA 2mg / L NAA 0.1mg / L sucrose 5%. Conclusion

    aseptic in vitro conditions in vitro can be induced in kudzu root.

    [Key words] kudzu root induction in vitro techniques

     Abstract: ObjectiveTo establish the sterilized seedling culture and the test tube earthnut induction system.MethodsThe seed of Pueraria thomsonii Benth.was cultured in MS culture medium as explant under the light length of 2000 ~ 3000Lx 12 h / d at 25 ? . ResultsMechanical break could efficiently smash the dormancy of seed. The shearing away of the top sprout would contribute to the germination of fascicled bud. When the plantlet with induced roots were shifted to tuberous root inducing culture medium, the root obviously augmented which consequently led to the formation of test tube tuberous root. The optimized medium was MS 6-BA2mg / L NAA 0.1 mg / L

    sucrose 5%. ConclusionTest tube tuberous root of Pueraria thomsonii Benth.could be induced in sterile conditions in vitro.

     Key words: Pueraria thomsonii Benth.; Test tube tuberous root; Induction

    technology

     Kudzu Pueraria thomsonii Benth. Is a bean is a perennial vine Kege, the main producing south of the Yangtze, in the 'Compendium of Materia Medica Shen Nong Ben Cao Jing''' in both its medicinal effects documented. Master Jin Ye Ge bleeding

    sores; Gehua to dealcoholic Xingpi; Ge Valley (seeds) rule diarrhea, hangover drugs; Pueraria Bu Xin Qingfei played chi, hangover drug. Its root hypertrophy, rich in starch and a variety of isoflavones. Modern science proves that puerarin in Pueraria

    flavonoid, has obvious anti-coagulation, inhibit platelet aggregation, reduce blood viscosity, such as the role of protease activated fibers [1].

     Multi-purpose production of kudzu asexual reproduction. Years of vegetative propagation, easily leading to degradation of germplasm, were as follows: root thinning, lower yields and quality. At present, the shoot-tip detoxification is to prevent

    viruses and solve common method of germplasm degradation [2], while kudzu shoot-tip

    on the ear piece more and villi density were carried out when the material sterile tissue culture is relatively difficult to get kudzu Shoot-tip there is a certain degree of difficulty

    there is virus-free seedlings; and virus-free seedling propagation coefficient is small, the

    survival rate is low, breeding cycle is long, very inconvenient transportation, which greatly limits the production promotion. In recent years, potatoes, Pinellia, Rehmannia and other crops are cultivated in vitro roots (tubers), attempt to try to root instead of in

    vitro virus-free seedlings in production applications. Among them, the potato in vitro culture techniques are maturing, can replace the test-tube seedlings as the supply of

    goods [3]. At present, the kudzu root in vitro studies at home and abroad not been

    reported, this study was carried out kudzu root induction of in vitro studies. Kudzu root formation induced in vitro, but also for kudzu root of the formation mechanism of research materials.

     1 Materials and methods

     1.1 Material of Hunan born the year the seeds of wild kudzu.

     1.2 Methods

     1.2.1 The selection of media and culture conditions MS basic medium, the basic concentration of 3% sucrose, solidified with 0.8% agar, pH value of 5.8 ~ 6.0,121 ?

    damp-heat sterilization 15 min, materials, placed in light training after inoculation box in order to develop conditions: 25 ? , 2000 ~ 3000 Lx light, 12 h / d.

     1.2.2 No access to vaccine will be kudzu seeds cut with scissors broken seed coat, with running water 20 min, moved into ultra-clean table, with 75% alcohol for surface

    disinfection of 30 s, then 0.1% of the health of mercury seed disinfection 10 min, sterile water rinse 5 ~ 6 times, the seeds will be sterilized with moist two-layer filter paper

    placed in the flask, in the light incubator 10 h. 10 h after smoke rose out of the seeds with 0.1% of the health of mercury for the first 2 disinfection, sterilization time of 6 min, sterile water rinse after 5 ~ 6 times the seeds were sterilized again moved to 1/2MS

    basic medium, in the light incubator for culture.

     1.2.3 seedling seed germination to be the proliferation of cultured non-vaccine for

    as long as about 2 cm from the roots, when cut, respectively, and do not remove the terminal bud terminal bud two kinds of treatment, go to the MS 6-BA 2 mg / L NAA

    0.1 mg / L medium, in the light incubator for the proliferation of cultured seedlings. Reposted elsewhere in the paper for free download http://

     1.2.4 seedling roots when the proliferation of cultured kudzu seedlings for as long

    as 2 ~ 3 cm when cut from the seedling base, go to 1/2MS IBA 1 mg / L for rooting culture medium.

     1.2.5 kudzu root induction of in vitro when the kudzu root length of seedlings to about 1cm, the timely and transferred to root induction medium in vitro for kudzu root induction in vitro cultivation. Root induction medium was: MS, MS 6-BA 1 mg / L

    NAA 1.0 mg / L, MS 6-BA 2 mg / L NAA 0.1 mg / L, MS 6-BA 2 mg / L NAA 1.0 mg / L,

    MS 6-BA 3 mg / L NAA 0.1 mg / L, which were 5% sucrose concentration.

     1.2.6 medium of different sucrose concentration on root induction of the basic medium was used: MS 6-BA 2 mg / L NAA 0.1 mg / L, respectively, in the basic

    medium with different sucrose concentrations: 3%, 5 %, 8% and 12%, and root length

    of about 1 cm test-tube seedlings of kudzu moved into the medium, the effects of different sucrose concentration on in vitro root growth.

     2 Results and analysis

     2.1 Establishment of culture system free vaccine because of kudzu seeds with

    dormancy, seed germination after sterilization directly into bed is not germinate, while the seed coat by mechanical destruction of the method to be effective in breaking seed dormancy, seed germination rate of 98.5%. In addition, the kudzu seed easily and

    thoroughly disinfected, after a sterile seed contamination rate as high as 85.4%, while the two disinfection methods can greatly reduce the contamination rate during seed germination, so that sterile seed germination rate of more than 45%.

     2.2 The proliferation of cultured proliferation of cultured seedlings 15 d after the seedlings are two kinds of processing multiple shoot clumps grow. Remove the terminal bud of the kudzu seedlings will grow about 8 about 2 cm propagation of adventitious

    buds, not to remove the terminal bud of the kudzu seedlings sprouting around 4 cluster shoots about 2 cm experiment proved beneficial to remove the terminal bud powder Ge multiple shoot clumps from happening.

     2.3 of plant hormones on thickening of adventitious roots and seedling roots with adventitious root formation in roots of seedlings moved into induction medium, 30 d after the observation of kudzu plants in a variety of the growth medium. The results showed that: In the blank MS medium culture kudzu seedlings, 30 d after the roots to continue elongation (5 cm or so), no obvious main root, root there is no sign of thickening, its diameter is less than 0.1 cm; in the rooting medium grown kudzu seedlings, the roots continue to elongate (8 cm or so) there is an obvious main root, the main root diameter of about 0.15 cm; in the MS 6-BA 2 mg / L NAA 0.1 mg / L

    medium, the seedlings given birth to a small amount of uncertainty bud, root elongation was not obvious (2.5 cm or so), and no longer as new growth of adventitious

    roots, but root thickening apparent diameter of about 0.8 cm can be achieved (Figure 1). When the NAA concentration in the medium is high, the plant base callus of serious, seedlings tend to glass, and its root no obvious thickening phenomenon. Medium

increased the concentration of 6-BA, seedlings easy to form buds from birth. Choose

    which induce root contains a lower concentration of 6-BA and NAA medium more

    appropriate.

     2.4 medium sucrose concentration on root formation in adventitious roots of the

    seedlings with the inflow of different sucrose concentrations of the medium observed after 10d, in the sucrose concentration of 3% of the medium, adventitious root thickening is not obvious, sucrose concentration of 5 % and 8% of the medium became

    thicker roots can be seen in the sucrose concentration of 8% of the high sugar content medium, the seedlings than the small, dark green leaves, plants in the growth inhibitory state. The results show that a high sugar content is conducive to the formation of kudzu

    root, but the high sugar content can inhibit the growth of seedlings, while the sugar content of 5% of the medium is more suitable for the formation of kudzu root in vitro.

     3 Discussion

     Kudzu seeds have dormancy, experiments show that mechanical destruction of seed coat is an effective way to break the seed dormancy, indicating kudzu seed coat impermeability of the gas-tight and affecting the main reason for kudzu seed

    germination. In terms of production, artificial removal of terminal buds used to eliminate the top edge, can promote lateral bud growth, increase the number of branches, this study also proved beneficial to remove the terminal bud kudzu seedlings in vitro propagation of adventitious buds from happening.

     At present, the impact of kudzu root on the formation of domestic and international factors and physiological mechanism have not been reported on the physiological mechanism of the formation of potato tubers studies have shown that,

    GA, and ABA and the formation of potato tubers, GA inhibited the formation of tubers, while the ABA on tuberization have a facilitating role. Tuber formation has also been a number of other factors, including light, temperature, and under the conditions

    of in vitro culture medium, sucrose concentration of tuber formation of the main factors [4]. This study also demonstrates the high sucrose concentration in culture medium conducive to the formation of kudzu root in vitro, GA, ABA pairs of kudzu

    root in vitro the role and light, temperature on the impact of kudzu root in vitro needs further study.

    [References]

     [1] Fengrui Zhi, Chen Bizhu. Kudzu Resources Survey [J]. Chinese Pharmaceutical Journal, 1993,28 (5): 273.

     [2] Zhou Yan, Qing-Lin Liu, Jia-Shu Cao, et al. Plant Cell Cell Engineering

    Theory and Technology [J]. Beijing: China Agricultural University Press, 2001:90.

     [3] LIU Jun, Xie from China. Potato tuber development mechanism and gene

    expression [J]. Botany Bulletin, 2001,18 (5): 531.

     [4] Cheng Long-jun, Guo-Ping, Hong-Juan Ge. Sweet potato tuber-specific

    protein-Sporamin Research progress [J]. Botany Bulletin, 2001,18 (6): 672. Reposted elsewhere in the paper for free download http://www.hi138. com

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