CN103651127A - In-vitro conservation method for chirita longgangensis - Google Patents
In-vitro conservation method for chirita longgangensis Download PDFInfo
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- CN103651127A CN103651127A CN201310608468.4A CN201310608468A CN103651127A CN 103651127 A CN103651127 A CN 103651127A CN 201310608468 A CN201310608468 A CN 201310608468A CN 103651127 A CN103651127 A CN 103651127A
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Abstract
The invention provides an in-vitro conservation method for chirita longgangensis. The in-vitro conservation method comprises the following steps: selecting out aseptic chirita longgangensis seedling materials: inoculating a part of the test materials to different culture media; inoculating a part of the test materials to culture media with different inhibitors; putting a part of the test materials in light with four illumination intensities; summing up the optimum condition for the in-vitro conservation of the chirita longgangensis. The in-vitro conservation method provides a new technical support for the germplasm resource conservation of the chirita longgangensis, which is an endangered species, as well as an effective path for sustainable utilization of the chirita longgangensis.
Description
Technical field
The present invention relates to a kind of Vitro Plant store method, particularly hilllock, a kind of lane lip post lettuce tongue in-vitro conservation method.
Background technology
Do hilllock lip post lettuce tongue, for Gesneriaceae Chirita plant does hilllock lip post lettuce tongue (Chirita longgangensis W. T. Wang in Guihaia), perennial herb.For the distinctive Gesneriaceae medicinal plant in Guangxi, be only distributed in the ground such as day grades, Daxin County, its root-like stock can be used for traumatic injury, rheumatic arthritis, is one of the raw material of the patent medicine " sweet osmanthus cream " of Tiandeng County, Guangxi production.
Do hilllock lip post lettuce tongue corolla lavender, cylinder white is to lavender, there is higher ornamental value, because its growing environment requires very harsh, suitable raw temperature range, humidity range and soil acidity or alkalinity scope are smaller, and seed is tiny, breeding difficulty, introduce a fine variety difficulty and survive, within 2004, be incorporated into < < Chinese Plants Red Data Book > >.Adopting method of the present invention is to realize the effective way that endangered species is done hilllock lip post lettuce tongue resource conservation and sustainable use.
Summary of the invention
The object of this invention is to provide hilllock, a kind of lane lip post lettuce tongue in-vitro conservation method, it can be preserved and do hilllock lip post lettuce tongue germ plasm resource.
The present invention achieves the above object by the following technical programs:
Hilllock, a lane lip post lettuce tongue in-vitro conservation method, comprises the following steps:
(1) choosing Medicinal Garden Of Guangxi Zhuang Autonomous Region preserves Ku Zhongnong hilllock lip post lettuce tongue test-tube plantlet in vitro and on propagating culture medium, cultivates Multiple Buds that 20d obtains as test material.
(2) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated on experimental scheme MS, 1/2MS, tri-kinds of medium of 1/4MS, inoculates 10 bottles, every bottle of 5 strain simple buds.The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d.
(3) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated into interpolation variable concentrations chlormequat (CCC), abscisic acid (ABA), paclobutrazol (PP
333) medium on, inoculate 10 bottles, every bottle of 5 strain simple buds.The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d.
(4) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated into 3 level design orthogonal experiment medium with sucrose, mannitol, sorbierite, inoculates 10 bottles, every bottle of 5 strain simple buds.The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d.
(5) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated into propagating culture medium, design 0,800 lx, and 1600 lx, 2,400 tetra-kinds of lx intensities of illumination, every kind of light intensity is inoculated 10 bottles, every bottle of 5 strain simple buds.The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, illumination 12-14 h/d.
(6) the growth recovery situation of preserving Nong hilllock lip post lettuce tongue test-tube plantlet is in vitro investigated: robust growth Nong hilllock lip post lettuce tongue test-tube plantlet simple bud is cut to be inoculated into and on best Storaged media, preserve the Nong hilllock lip post lettuce tongue lettuce tongue test-tube plantlet of surviving after 300d and be inoculated on MS+, every 30d subculture 1 time, after subculture 3 times, take plant height, form, leaf is long, leaf is wide, bud propagation multiple, average life rate, individual plant rooting rate are that index is investigated the recovery situation of doing hilllock lip post lettuce tongue test-tube plantlet.
By choosing, do hilllock lip post lettuce tongue aseptic seedling material; Test material is inoculated on different medium, test material is placed under four kinds of intensities of illumination, sum up and do the in vitro optimum condition of preserving of hilllock lip post lettuce tongue.
Outstanding advantages of the present invention is:
(1) doing in vitro preservation of hilllock lip post lettuce tongue is to be embodied as the effective way that endangered species is done hilllock lip post lettuce tongue resource conservation and sustainable use.
(2) under normal temperature, on 1/2MS Storaged media, add the chlormequat (CCC) of 0.5-1.5mg/L and can preserve for a long time hilllock, lane lip post lettuce tongue test-tube plantlet, can control plant and extend, seedling strain poor growth, plant is short strong, well developed root system.
Under normal temperature, in 1/2MS Storaged media, add sucrose 30-60g/L and can preserve for a long time hilllock, lane lip post lettuce tongue test-tube plantlet, can control plant and extend, seedling strain poor growth, plant is short strong, well developed root system.
Under normal temperature, in 1/2MS Storaged media, add agar 4.0-5.0g/L and can preserve for a long time hilllock, lane lip post lettuce tongue test-tube plantlet, can control plant and extend, seedling strain poor growth, plant is short strong, well developed root system.
Under normal temperature, in 1/2MS Storaged media, add sorbierite 3-7 g/L and can preserve for a long time hilllock, lane lip post lettuce tongue test-tube plantlet, can control plant and extend, seedling strain poor growth, plant is short strong, well developed root system.
Under normal temperature, light intensity 800-1600lx, 12 h/d can preserve for a long time and do hilllock lip post lettuce tongue test-tube plantlet, and can control plant and extend, seedling strain poor growth, plant is short strong, well developed root system.
(3) adopt in-vitro conservation method of the present invention, obtain Nong hilllock lip post lettuce tongue aseptic seedling through mode of appearance, grow all with not through in vitro preservation processing material without significant difference; The germ plasm resource that shows hilllock, the applicable lane of this store method lip post lettuce tongue is preserved for a long time.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described further.
Hilllock, a lane lip post lettuce tongue in-vitro conservation method, comprises the following steps:
(1) choosing Medicinal Garden Of Guangxi Zhuang Autonomous Region preserves red luxuriant half capsule lettuce tongue test-tube plantlet in storehouse in vitro and on propagating culture medium, cultivates Multiple Buds that 20d obtains as test material.
(2) the in vitro impact of preserving of the horizontal Dui Nong of mineral salt hilllock lip post lettuce tongue test-tube plantlet: it is MS medium that the explant that step (1) is obtained is done the applicable minimal medium of hilllock lip post lettuce tongue tissue-culturing rapid propagation, experimental scheme MS, 1/2MS, tri-kinds of inorganic salt concentration levels of 1/4MS, investigate the impact of each inorganic salt concentration on test material upgrowth situation, the experiment material survival rate of usining is not less than the evaluation index that holding time of 50% preserves as germplasm.The agar of 25-30g/L sucrose and 3.8-4.8g/L, the pH value of medium is 5.8,10 bottles of every processing inoculations, every bottle of 5 strain simple buds.25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d.(note: MS medium is Murashige and Skoog bis-people design in 1962, for using at present the most general medium.)
The impact of table 1 medium Dui Nong hilllock lip post lettuce in vitro holding time of tongue
| Medium | Holding time (d) | Upgrowth situation |
| MS | 332 | Bud strain well-grown, plant is light green, starts death, medium approach exhaustion after 260d after 5 months |
| 1/2MS | 310 | Well-grown, plant is light green, starts death, medium approach exhaustion after 260d after 4 months |
| 1/4MS | 203 | Bud strain poor growth, material withered the dying that hop to it after 3 months, medium approach exhaustion after 200d |
(3) the in vitro impact of preserving of plant growth inhibitor Dui Nong hilllock lip post lettuce tongue test-tube plantlet: the test-tube plantlet obtaining in step (1) is inoculated into chlormequat (CCC), abscisic acid (ABA), the paclobutrazol (PP that adds variable concentrations
333) in the 1/2MS medium of three plant growth regulators, investigate the impact of each factor on experiment material upgrowth situation, the experiment material survival rate of usining is not less than the evaluation index that holding time of 50% preserves as germplasm.The agar of 25-30g/L sucrose and 3.8-4.8g/L, the pH value of medium is 5.8,10 bottles of every processing inoculations, every bottle of 5 strain simple buds.25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d.Factor level is in Table 1.
The impact of table 2 hormone Dui Nong hilllock lip post lettuce tongue holding time
| Hormone | Concentration (mg/L) | Holding time (d) | Upgrowth situation |
| CCC | 0.5 | 251 | Bud strain well-grown, plant is light green, starts dead after 4 months |
| CCC | 1.0 | 317 | Bud strain well-grown, plant is light green, starts dead after 8 months |
| CCC | 1.5 | 295 | Bud strain well-grown, plant is light green, starts dead after 5 months |
| PP 333 | 0.5 | 131 | Bud strain well-grown, plant is light green, starts dead after 3 months |
| PP 333 | 1.0 | 164 | Bud strain well-grown, plant is light green, starts dead after 4 months |
| PP 333 | 1.5 | 201 | Bud strain well-grown, plant is light green, starts dead after 5 months |
| ABA | 0.5 | 168 | Bud strain well-grown, plant is light green, starts dead after 3 months |
| ABA | 1.0 | 205 | Bud strain well-grown, plant is light green, starts dead after 5 months |
| ABA | 1.5 | 201 | Bud strain well-grown, plant is light green, starts dead after 5 months |
(4) the in vitro impact of preserving of osmotic pressure Dui Nong hilllock lip post lettuce tongue test-tube plantlet: with 3 level design orthogonal experiments of sucrose, mannitol, sorbierite, investigate the impact of each factor on experiment material upgrowth situation, the experiment material survival rate of usining is not less than the evaluation index that holding time of 50% preserves as germplasm.The agar of 25-30g/L sucrose and 3.8-4.8g/L, the pH value of medium is 5.8,10 bottles of every processing inoculations, every bottle of 5 strain simple buds.Illumination 14～16 h, light intensity 1600 lx, 25 ± 1 ℃ of room temperatures.
Table 3 osmotic pressure orthogonal experiment L
9(3
4) Dui Nong hilllock lip post lettuce tongue preserves impact in vitro
Note: K1 is the numerical value sum of " 1 " corresponding holding time of level, and K2, K3 are respectively the numerical value sum of " 2,3 " corresponding holding time of level.
(5) the in vitro impact of preserving of light intensity Dui Nong hilllock lip post lettuce tongue test-tube plantlet: doing the applicable illumination condition of hilllock lip post lettuce tongue tissue-culturing rapid propagation is 1600 lx, experimental scheme 0,800 lx, 1600 lx, 2,400 tetra-kinds of lx intensities of illumination, investigate the impact of each light intensity on test material upgrowth situation.The experiment material survival rate of usining is not less than the evaluation index that holding time of 50% preserves as germplasm.The agar of 25-30g/L sucrose and 3.8-4.8g/L, the pH value of medium is 5.8,10 bottles of every processing inoculations, every bottle of 5 strain simple buds.Illumination 14～16 h, 25 ± 1 ℃ of room temperatures.
The in vitro impact of preserving of table 4 intensity of illumination Dui Nong hilllock lip post lettuce tongue
| Intensity of illumination (lx) | Holding time (d) | Upgrowth situation |
| 0 | 48 | The plant death of gradually wilting |
| 800 | 187 | Plant is light green, starts dead after 4 months |
| 1600 | 332 | Bud strain well-grown, plant is light green, starts dead after 8 months |
| 2400 | 221 | Plant vitrifying gradually in 2 months, starts dead after 5 months |
(6) the growth recovery situation of preserving Nong hilllock lip post lettuce tongue test-tube plantlet is in vitro investigated: robust growth Nong hilllock lip post lettuce tongue test-tube plantlet simple bud is cut to be inoculated into and on best Storaged media, preserve the Nong hilllock lip post lettuce tongue lettuce tongue test-tube plantlet of surviving after 300d and be inoculated on MS+, every 30d subculture one 1 times, after subculture 3 times, take plant height, form, leaf is long, leaf is wide, bud propagation multiple, average life rate, individual plant rooting rate are that index is investigated the recovery situation of doing hilllock lip post lettuce tongue test-tube plantlet.
The test-tube plantlet recovering after the in vitro preservation of table 3 and the comparison of preserving front test-tube plantlet
| Material | Average life rate | Breeding rate | Rooting rate | Plant height/cm |
| Normally | 7.15±0.32 | 7.11±0.57 | 99.6±0.31 | 8.42±0.48 |
| Preserve | 6.85±0.29 | 6.67±0.33 | 99.1±0.27 | 7.74±0.43 |
Conclusion: normal temperature Xia Nong hilllock lip post lettuce tongue preserves optimal medium in vitro and is: MS+ sucrose 60g/L+ agar 4.0g/L+ sorbierite 5 g/L+CCC1.0mg/L; Best preservation condition is light intensity 1600lx, 12 h/d.
Claims (1)
- The in-vitro conservation method of 1.Yi Zhongnong hilllock lip post lettuce tongue, is characterized in that the method comprises the following steps:(1) choosing Medicinal Garden Of Guangxi Zhuang Autonomous Region preserves Ku Zhongnong hilllock lip post lettuce tongue test-tube plantlet in vitro and on propagating culture medium, cultivates Multiple Buds that 20d obtains as test material;(2) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated on experimental scheme MS, 1/2MS, tri-kinds of medium of 1/4MS, inoculates 10 bottles, every bottle of 5 strain simple buds; The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d;(3) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated on the medium that adds variable concentrations chlormequat, abscisic acid, paclobutrazol, inoculates 10 bottles, every bottle of 5 strain simple buds; The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d;(4) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated into 3 level design orthogonal experiment medium with sucrose, mannitol, sorbierite, inoculates 10 bottles, every bottle of 5 strain simple buds; The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, light intensity 1600 lx, illumination 12-14 h/d;(5) 1-2cm step (1) being obtained does the aseptic Multiple Buds of hilllock lip post lettuce tongue and is inoculated into propagating culture medium, design 0,800 lx, and 1600 lx, 2,400 tetra-kinds of lx intensities of illumination, every kind of light intensity is inoculated 10 bottles, every bottle of 5 strain simple buds; The agar of 25-30g/L sucrose and 3.8-4.8g/L in medium, the pH value of medium is 5.8,25 ± 1 ℃ of cultivation temperature, illumination 12-14 h/d;(6) the growth recovery situation of preserving Nong hilllock lip post lettuce tongue test-tube plantlet is in vitro investigated: robust growth Nong hilllock lip post lettuce tongue test-tube plantlet simple bud is cut to be inoculated on best Storaged media and preserve after 300d, survival Nong hilllock lip post lettuce tongue lettuce tongue test-tube plantlet is inoculated on MS+, every 30d subculture 1 time, after subculture 3 times, take plant height, form, leaf is long, leaf is wide, bud propagation multiple, average life rate, individual plant rooting rate are that index is investigated the recovery situation of doing hilllock lip post lettuce tongue test-tube plantlet.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104430306A (en) * | 2014-11-10 | 2015-03-25 | 中国科学院昆明植物研究所 | Gesneriaceae plant cryopreservation method |
| CN104756864A (en) * | 2014-12-09 | 2015-07-08 | 广西壮族自治区药用植物园 | In-vitro conservation method for Hemiboea cavaleriei var. paucinervis |
| CN104756865A (en) * | 2014-12-09 | 2015-07-08 | 广西壮族自治区药用植物园 | In-vitro conservation method for Hemiboea cavaleriei Levl |
| CN105265319A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Isolated preservation method for illicium difengpi B.N Chang et al. |
| CN106417010A (en) * | 2016-08-29 | 2017-02-22 | 中国科学院昆明植物研究所 | Tissue culture method of gesneriaceae plant |
| CN110574670A (en) * | 2019-09-26 | 2019-12-17 | 北京林业大学 | Method for controlling flowering character of primulina plant by chlormequat chloride root irrigation |
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| CN101965800A (en) * | 2010-11-19 | 2011-02-09 | 中国科学院华南植物园 | Tissue culture breeding method for metabriggsia ovalifolia |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104430306A (en) * | 2014-11-10 | 2015-03-25 | 中国科学院昆明植物研究所 | Gesneriaceae plant cryopreservation method |
| CN104756864A (en) * | 2014-12-09 | 2015-07-08 | 广西壮族自治区药用植物园 | In-vitro conservation method for Hemiboea cavaleriei var. paucinervis |
| CN104756865A (en) * | 2014-12-09 | 2015-07-08 | 广西壮族自治区药用植物园 | In-vitro conservation method for Hemiboea cavaleriei Levl |
| CN104756864B (en) * | 2014-12-09 | 2017-04-26 | 广西壮族自治区药用植物园 | In-vitro conservation method for Hemiboea cavaleriei var. paucinervis |
| CN105265319A (en) * | 2015-11-18 | 2016-01-27 | 广西壮族自治区药用植物园 | Isolated preservation method for illicium difengpi B.N Chang et al. |
| CN106417010A (en) * | 2016-08-29 | 2017-02-22 | 中国科学院昆明植物研究所 | Tissue culture method of gesneriaceae plant |
| CN110574670A (en) * | 2019-09-26 | 2019-12-17 | 北京林业大学 | Method for controlling flowering character of primulina plant by chlormequat chloride root irrigation |
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