CN104585038B - A kind of abductive approach of Lignum Santali Albi triploid - Google Patents
A kind of abductive approach of Lignum Santali Albi triploid Download PDFInfo
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- CN104585038B CN104585038B CN201510058720.8A CN201510058720A CN104585038B CN 104585038 B CN104585038 B CN 104585038B CN 201510058720 A CN201510058720 A CN 201510058720A CN 104585038 B CN104585038 B CN 104585038B
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Abstract
The invention discloses the abductive approach of a kind of Lignum Santali Albi triploid.It is by after the sterilization of Lignum Santali Albi mature seed, Lignum Santali Albi seed 20～24h after aseptically sterilizing by the gibberellins aqueous solution soaking of 250～500mg/L, then take out seed sterile distilled water to rinse, then episperm is peeled off, remaining seed is seeded on inducing culture, is placed in intensity of illumination 40～50 μm ol m‑2s‑1, light application time 12～16h/d, cultivation temperature 26 ± 2 DEG C is cultivated, and induces adventitious bud, and adventitious bud is seeded on identical inducing culture, is placed in intensity of illumination 40～50 μm ol m‑2s‑1, light application time 12～16h/d, cultivation temperature 26 ± 2 DEG C carries out successive transfer culture, evoking adventive bud differentiation and growth, then carries out polyploid screening with identifying and obtain Lignum Santali Albi triploid.The present invention becomes triploid by induction Lignum Santali Albi seed, doubles research for carrying out Lignum Santali Albi polyploid, it is thus achieved that excellent Lignum Santali Albi new varieties, and development Lignum Santali Albi industry provides one of effective technical way.The present invention has that operation is simple, using value advantages of higher.
Description
Technical field:
The invention belongs to field of plant reproduction, be specifically related to the abductive approach of a kind of Lignum Santali Albi triploid.
Background technology:
Lignum Santali Albi (Santalum album L.) is that Santalaceae (Santalaceae) Lignum Santali Albi belongs to (Santalum) semiparasite aiphyllium,
For famous and precious, rare plant, it is distributed mainly on India, Indonesia, Australian and more Pacific archipelagos.Mankind's profit
By its history of existing thousands of years, the most famous because its timber can be used for carving and refine Oleum Santali albi, have " green gold "
Title.Sandalwood is the raw material extracting Oleum Santali albi, and it contains the compositions such as α and β santalol, and it is mainly used in cosmetics, perfume (or spice)
The raw material of material, medical material and advanced processes carving etc.；Antibacterial and the antineoplastic component that it contains, as Chinese medicine, can be used for
Calm the nerves, pure air and treat some intractable diseases.
For many years, people want to develop sandalwood industry in artificial culture mode always, it is achieved higher economy return.State
Inside and outside research worker carries out substantial amounts of research to the tissue-culturing rapid propagation of Lignum Santali Albi.Research data shows: in Fiji, Lignum Santali Albi and Fiji
Sexual hybridization kind F of Lignum Santali Albi1Obvious hybrid vigor is revealed in representative, and its growth potential is higher by 300% than maternal Fiji Lignum Santali Albi, compares male parent
Lignum Santali Albi is the highest by 32%；Edgeworthia chrysantha Lindl. aspect, Lignum Santali Albi needs 10 years, and hybrid Lignum Santali Albi then needs 6-7；In terms of heartwood growth,
General every strain Lignum Santali Albi needs to plant 25-30 heartwood and reaches 40-80 kilogram, and hybrid Lignum Santali Albi F1 only need to plant 15-20.By
This is visible, and cultivating Lignum Santali Albi new lines is one of effective way improving its economic worth.At present, China Lignum Santali Albi belong to the screening of kind of source,
Cross-breeding, excellent strain the aspect such as innovation the most blank, as carried out germ plasm resource and technological innovation not in time, certainly will restrict
The development of China's Lignum Santali Albi industry, it is impossible to break away from the passive situation being completely dependent on import.Therefore, develop Lignum Santali Albi industry in China and seek
Look for other cost-effective method imperative with the economic benefit improving Lignum Santali Albi.
Polyploid (polyploid) means in organisms cell containing having more than 2 genomic individualities.Usually, many times
The merits such as body plant is thick compared with the nutrition organs of diplont, nutrient substance more abundant, strong stress resistance.At present, may be used
Cultivated by endosperm dedifferentiation, two kinds of approach of Cross between diploid and tetraploid obtain triploid.Domestic, existing Citrus, Herba Marsileae Quadrifoliae
Really, Fructus Lycii, Fructus actinidiae chinensis, Semen Maydis, the report of the induced triploid plant such as Fructus Hordei Vulgaris, but there is no the phase of Lignum Santali Albi triploid induction
Close report.
Summary of the invention:
It is an object of the invention to provide one simply, easily operate, the abductive approach of Lignum Santali Albi triploid can be obtained.
The abductive approach of the Lignum Santali Albi triploid of the present invention, it is characterised in that comprise the following steps:
By Lignum Santali Albi (Santalum album L.) mature seed sterilize after, aseptically with 250～500mg/L gibberellins
Lignum Santali Albi seed 20～24h after aqueous solution soaking sterilization, then takes out seed sterile distilled water and rinses, then peel episperm off,
Remaining seed is seeded on inducing culture, is placed in intensity of illumination 40～50 μm ol m-2s-1, light application time 12～16h/d, cultivate
Temperature (26 ± 2 DEG C) is cultivated, and induces adventitious bud, and adventitious bud is seeded on identical inducing culture, is placed in illumination strong
Degree 40～50 μm ol m-2s-1, light application time 12～16h/d, cultivation temperature (26 ± 2 DEG C) carries out successive transfer culture, and it is indefinite to induce
Bud polarization and growth, then carry out polyploid screening with identifying and obtain Lignum Santali Albi triploid；
Described inducing culture every liter is containing 6-BA 0.2～0.5mg, sucrose 20～30g and the agar of constant, and surplus is MS
Culture medium, pH 5.5-5.8.
Described sterilization is preferably Lignum Santali Albi seed the most first with volume fraction 75% alcohol water blend soaking disinfection 1
Min, then with mass fraction 0.1%HgC1 solution soaking sterilize 12min, sterile distilled water rinse 3-5 time.
Described taking-up seed sterile distilled water rinses, and preferably rinses 3～5 times.
The agar of described constant is the amount making inducing culture become solid, and its amount belongs to this area Conventional wisdom, as every liter lures
Lead the agar that culture medium contains 7.5g.
Described polyploid screening is that the adventitious bud induced carries out breaking up and growing unrooted seedling with qualification, and unrooted seedling grows tall
During to 3～about 4cm, using the diploid Seedling of the external stem section culture of Lignum Santali Albi as comparison, compare Preliminary screening by morphological observation
Go out the plant that the diploid Seedling of stem section culture external from Lignum Santali Albi is different, through the method for stem tip chromosome tabletting counting, again identify tool
The Seedling of slender blade is triploid.
Described MS culture medium is international culture medium, and its composition and collocation method refer to books (Tan
Wen Cheng, Dai Cegang edit. ornamental plant tissue culture technique. and Beijing: China Forestry Publishing House, 1991.).
The present invention is inoculated into the inducing culture containing the low concentration basic element of cell division through the Lignum Santali Albi seed of transmission from one meridian to another high concentration gibberellins pretreatment
In, so that it may so that its direct evoking adventive bud, through 2 successive transfer culture and obtain Lignum Santali Albi triploid, save substantial amounts of people
Power and material resources, the breeding for Lignum Santali Albi hexaploid provides technical support.It is expected to obtain the heartwood of higher yield, obtains higher economy
Income, and offer reference for the breeding in other Lignum Santali Albi kind source.
Therefore, the present invention becomes triploid by induction Lignum Santali Albi seed, doubles research for carrying out Lignum Santali Albi polyploid, it is thus achieved that excellent
Lignum Santali Albi new varieties, development Lignum Santali Albi industry provides one of effective technical way.The present invention has that operation is simple, using value
Advantages of higher.
Accompanying drawing illustrates:
Fig. 1 is Lignum Santali Albi liploid plant；
Fig. 2 is Lignum Santali Albi triploid, and blade is elongated；
Fig. 3 is Lignum Santali Albi metaphase chromosome A, diploid 2n=20；B, triploid 3n=30, scale=5 μm.
Detailed description of the invention:
Following example are to further illustrate the present invention rather than limitation of the present invention.
Embodiment 1:
The abductive approach of Lignum Santali Albi triploid, comprises the following steps:
(1) outer implant selects and sterilization
Take the ripe fresh fruit of South China Botanical Garden introducing and planting 20 age Lignum Santali Albi (Santalum album L.), peel sarcocarp off by hand,
The clean seed of distilled water flushing, then first uses volume fraction 75% alcohol water blend soaking disinfection 1 on superclean bench by seed
Min, then with mass fraction 0.1%HgC1 solution soaking sterilization 12min, sterile distilled water flushing 3 times, finally incite somebody to action after sterilizing
Seed be placed on the aseptic filter paper on superclean bench and dry up.
(2) Seeds preprocess
With the aseptic gibberellins aqueous solution soaking pretreatment seed 24h of 250mg/L under aseptic condition, the seed after process is in ultra-clean
Use aseptic water washing 3 times on workbench, be then placed on aseptic filter paper, peel off hard with sharp aseptic operation blade
Episperm, remaining seed is stand-by.
(3) embryo inducing culture
Inducing culture, formula is every liter and contains 6-BA 0.5mg, sucrose 30g, agar 7.5g, and surplus is MS culture medium,
pH 5.7.According to above-mentioned formula, after its component mix homogeneously, adjust pH 5.7.Fill inducing culture 25ml in every bottle again,
Sterilizing is standby.The seed (remaining seed) that step (2) is peelled off episperm is seeded on inducing culture, 5/bottle, and totally 30
Bottle, is placed in intensity of illumination 50 μm ol m-2s-1, light application time 14h/d, cultivation temperature (26 ± 2 DEG C) is cultivated, and cultivates 4
Week, the bud point of outer implant visible green, continues to cultivate 1 week, lures followed by culture in identical culture medium (inducing culture)
Leading the differentiation of adventitious bud and grow unrooted seedling, condition of culture is intensity of illumination 50 μm ol m-2s-1, light application time 14h/d,
Cultivation temperature (26 ± 2 DEG C).
(4) screening of polyploid and qualification
As unrooted seedling length up to about the 3cm derived, (scheme with the diploid Seedling of the external stem section culture of Lignum Santali Albi for comparison
1), compare Preliminary screening by morphological observation and go out plant (three times of the Lignum Santali Albi that the diploid Seedling of stem section culture external from Lignum Santali Albi is different
Body, such as Fig. 2)；Through the method for stem tip chromosome tabletting counting, again identify that the Seedling of tool slender blade is triploid (Fig. 3).
Embodiment 2:
The abductive approach of Lignum Santali Albi triploid, comprises the following steps:
(1) outer implant selects and sterilization
Take the ripe fresh fruit of South China Botanical Garden introducing and planting 20 age Lignum Santali Albi (Santalum album L.), peel sarcocarp off by hand,
The clean seed of distilled water flushing, then first uses volume fraction 75% alcohol water blend soaking disinfection 1 on superclean bench by seed
Min, then with mass fraction 0.1%HgC1 solution soaking sterilization 12min, sterile distilled water flushing 5 times, finally incite somebody to action after sterilizing
Seed be placed on the aseptic filter paper on superclean bench and dry up.
(2) Seeds preprocess
With the aseptic gibberellins aqueous solution soaking pretreatment seed 20h of 500mg/L under aseptic condition, the seed after process is in ultra-clean
Use aseptic water washing 5 times on workbench, be then placed on aseptic filter paper, peel off hard with sharp aseptic operation blade
Episperm, remaining seed is stand-by.
(3) embryo inducing culture
Inducing culture, formula is every liter and contains 6-BA 0.2mg, sucrose 20g, agar 7.5g, and surplus is MS culture medium,
pH 5.5.According to above-mentioned formula, after its component mix homogeneously, adjust pH 5.5.Fill inducing culture 25ml in every bottle again,
Sterilizing is standby.The seed (remaining seed) that step (2) is peelled off episperm is seeded on inducing culture, 5/bottle, and totally 30
Bottle, is placed in intensity of illumination 40 μm ol m-2s-1, light application time 16h/d, cultivation temperature (26 ± 2 DEG C) is cultivated, and cultivates 4
Week, the bud point of outer implant visible green, continues to cultivate 1 week, lures followed by culture in identical culture medium (inducing culture)
Leading the differentiation of adventitious bud and grow unrooted seedling, condition of culture is intensity of illumination 40 μm ol m-2s-1, light application time 16h/d,
Cultivation temperature (26 ± 2 DEG C).
(4) screening of polyploid and qualification
As unrooted seedling length up to about the 4cm derived, with the diploid Seedling of the external stem section culture of Lignum Santali Albi for comparison, logical
Cross morphological observation to compare Preliminary screening and go out the plant that the diploid Seedling of stem section culture external from Lignum Santali Albi is different；Through stem tip chromosome pressure
The method of sheet counting, identifies that the Seedling of tool slender blade is triploid again.
Embodiment 3:
The abductive approach of Lignum Santali Albi triploid, comprises the following steps:
(1) outer implant selects and sterilization
Take the ripe fresh fruit of South China Botanical Garden introducing and planting 20 age Lignum Santali Albi (Santalum album L.), peel sarcocarp off by hand,
The clean seed of distilled water flushing, then first uses volume fraction 75% alcohol water blend soaking disinfection 1 on superclean bench by seed
Min, then with mass fraction 0.1%HgC1 solution soaking sterilization 12min, sterile distilled water flushing 5 times, finally incite somebody to action after sterilizing
Seed be placed on the aseptic filter paper on superclean bench and dry up.
(2) Seeds preprocess
With the aseptic gibberellins aqueous solution soaking pretreatment seed 22h of 400mg/L under aseptic condition, the seed after process is in ultra-clean
Use aseptic water washing 4 times on workbench, be then placed on aseptic filter paper, peel off hard with sharp aseptic operation blade
Episperm, remaining seed is stand-by.
(3) embryo inducing culture
Inducing culture, formula is every liter and contains 6-BA 0.3mg, sucrose 25g, agar 7.5g, and surplus is MS culture medium,
pH 5.8.According to above-mentioned formula, after its component mix homogeneously, adjust pH 5.8.Fill inducing culture 25ml in every bottle again,
Sterilizing is standby.The seed (remaining seed) that step (2) is peelled off episperm is seeded on inducing culture, 5/bottle, and totally 30
Bottle, is placed in intensity of illumination 40 μm ol m-2s-1, light application time 12h/d, cultivation temperature (26 ± 2 DEG C) is cultivated, and cultivates 4
Week, the bud point of outer implant visible green, continues to cultivate 1 week, lures followed by culture in identical culture medium (inducing culture)
Leading the differentiation of adventitious bud and grow unrooted seedling, condition of culture is intensity of illumination 40 μm ol m-2s-1, light application time 12h/d,
Cultivation temperature (26 ± 2 DEG C).
(4) screening of polyploid and qualification
As unrooted seedling length up to about the 4cm derived, with the diploid Seedling of the external stem section culture of Lignum Santali Albi for comparison, logical
Cross morphological observation to compare Preliminary screening and go out the plant that the diploid Seedling of stem section culture external from Lignum Santali Albi is different；Through stem tip chromosome pressure
The method of sheet counting, identifies that the Seedling of tool slender blade is triploid again.
Claims (4)
1. the abductive approach of a Lignum Santali Albi triploid, it is characterised in that comprise the following steps:
After Lignum Santali Albi (Santalum album L.) mature seed is sterilized, Lignum Santali Albi seed 20～24h after aseptically sterilizing by the gibberellins aqueous solution soaking of 250～500mg/L, then take out seed sterile distilled water to rinse, then episperm is peeled off, remaining seed is seeded on inducing culture, is placed in intensity of illumination 40～50 μm ol m-2s-1, light application time 12～16h/d, cultivation temperature 26 ± 2 DEG C is cultivated, and induces adventitious bud, and adventitious bud is seeded on identical inducing culture, is placed in intensity of illumination 40～50 μm ol m-2s-1, light application time 12～16h/d, cultivation temperature 26 ± 2 DEG C carries out successive transfer culture, evoking adventive bud differentiation and growth, then carries out polyploid screening with identifying and obtain Lignum Santali Albi triploid；
Described inducing culture every liter is containing 6-BA 0.2～0.5mg, sucrose 20～30g and the agar of constant, and surplus is MS culture medium, pH 5.5-5.8.
Abductive approach the most according to claim 1, it is characterised in that described sterilization is that Lignum Santali Albi seed is the most first used volume fraction 75% alcohol water blend soaking disinfection 1min, then uses mass fraction 0.1%HgCl2Solution soaking sterilization 12min, sterile distilled water rinses 3-5 time.
Abductive approach the most according to claim 1, it is characterised in that described taking-up seed sterile distilled water rinses, and its washing time is 3～5 times.
Abductive approach the most according to claim 1, it is characterized in that, described polyploid screening with qualification is: the adventitious bud induced carries out breaking up and grow unrooted seedling, during unrooted seedling length up to 3～4cm, using the diploid Seedling of the external stem section culture of Lignum Santali Albi as comparison, compare Preliminary screening by morphological observation and go out the plant that the diploid Seedling of stem section culture external from Lignum Santali Albi is different, through the method for stem tip chromosome tabletting counting, again identify that the Seedling with slender blade is triploid.
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| CN101595824B (en) * | 2009-07-01 | 2011-01-12 | 广东省中药研究所 | Rapid in-vitro seedling raising method by utilizing sandalwood seed embryo |
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| CN101983557B (en) * | 2010-10-19 | 2012-10-03 | 东莞市睿绅生物技术有限公司 | In vitro quick breeding method of seedling stem of santal seed embryo |
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