NL2030708B1 - Tissue culture method of ancient tree - Google Patents

Tissue culture method of ancient tree Download PDF

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NL2030708B1
NL2030708B1 NL2030708A NL2030708A NL2030708B1 NL 2030708 B1 NL2030708 B1 NL 2030708B1 NL 2030708 A NL2030708 A NL 2030708A NL 2030708 A NL2030708 A NL 2030708A NL 2030708 B1 NL2030708 B1 NL 2030708B1
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rinsing
huang
ancient tree
medium
seedlings
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Zhao Xiulian
Chang Ermei
Jia Zirui
Liu Jianfeng
Jiang Zeping
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Res Institute Of Forestry Chinese Academy Of Forestry
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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
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • 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
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • A01H7/00—Gymnosperms, e.g. conifers

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  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

UITTREKSEL The present disclosure provides a tissue culture method, of an ancient tree Platycladus orientalis (L.) Franco. The method mainly comprises the selection, preparation and disinfection of the materials of ancient tree planted by Huang Di, the screening of 5 sugar sources, antioxidants and adsorbents in the medium, the selection of medium, the ratio of auxin and cytokinin, and induction of formation of adventitious buds in an appropriate ratio. The culture medium suitable for the rooting of the adventitious buds of ancient tree planted by Huang Di is WPM, and 10 the tissue culture seedlings of the ancient tree planted by Huang Di are obtained by hardening seedling after transplanting into the matrix. (+ Fig. l) 15

Description

P1060 /NLpd
TISSUE CULTURE METHOD OF ANCIENT TREE
TECHNICAL FIELD
The present disclosure relates to the technical field of plant tissue culture, particularly to a tissue culture method of an ancient tree.
BACKGROUND ART
At present, in the tissue culture process of the ancient tree planted by Huang Di, only the proliferation formula of new buds are obtained, while no rooted seedling of tissue culture is obtained. In this research, the branches of the ancient tree planted by Huang Di are used as explants to screen the medium of different tissue culture stages. Therefore, it is of great signif- icance to establish an ancient tree Platycladus orientalis (L.)
Franco plant regeneration system with short cycle, high regenera- tion rate and good repeatability.
SUMMARY
The object of the present disclosure is to provide a tissue culture method of ancient tree Platycladus orientalis (L.) Franco, to realize the tissue culture seedlings of the ancient tree plant- ed by Huang Di and other ancient Platycladus orientalis (L.) Fran- co.
To achieve the above object, the present disclosure provides a tissue culture method of an ancient tree Platycladus orientalis (L.) Franco, comprising the following steps: (1) Preparation and disinfection of raw materials
Choosing robust, disease-free and 1-2 year-old new shoots from the ancient tree planted by Huang Di in Huangdi Mausoleum in
Shaanxi Province, cutting the explants into 5x5mm stem segments with a scalpel, disinfecting the surface with 5% NaClO for 5 min, rinsing with tap water 5 times, soaking in a detergent for 5 min, and rinsing with running tap water for 2 h; then repeatedly rins- ing twigs 5 times with sterile water on a clean bench, soaking in
75% alcohol for 30 s, and then rinsing 5 times with sterile water; after sterilization with 0.3% HgCl2, rinsing 5 times with sterile water. (2) Sugar source screening of culture medium
The sugar source is 3% sucrose. {3) screening of anti-browning substances
Activated carbon (AC) can prevent browning, with a concentra- tion of 5.00 g/L. (4) Primary culture
Inoculating sterilized stem segments of explants into a pri- mary induction medium, performing primary induction culture for 40-55 days until new buds emerge. (5) Proliferation culture
Inoculating the obtained new buds into a secondary prolifera- tion culture medium, performing secondary proliferation culture for 40-51 days until adventitious buds extend to 2-3 cm. (6) Rooting cultivation
Cutting the adventitious buds from the base and inoculating them into a rooting cultivation medium, performing culture for 35- 45 days until young roots extend to 2-3 cm. (7) Hardening seedlings and transplanting test-tube seed- lings
Opening the bottle cap 3 days before transplanting, such that the rooted seedling gradually expose to the external environment; 3 days later, taking out the seedlings with tweezers, washing off the culture medium on the roots, transplanting into the matrix, and observing the growth conditions of seedlings every day.
As described above, the present disclosure provides a tissue culture method of an ancient tree Platycladus orientalis (L.)
Franco, and the disinfection effect of 0.3% HgCl2 on the ancient tree planted by Huang Di is better. The sugar source and anti- browning agent of the culture medium for the ancient tree planted by Huang Di are screened. The primary and proliferation basal me- dia of the ancient tree planted by Huang Di are both 1/2MS, the basal medium of the rooting culture is WPM. After hardening seed- lings, the tissue culture seedlings of the ancient tree planted by
Huang Di are ultimately obtained.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG.1 Morphology of the old tree planted by Huang Di at dif- ferent stages of tissue culture
A. Browning of explants, B. Activated carbon treatment, C.
Proliferation culture, D. Rooting Cultivation, E-F,. Developing status of roots of tissue culture seedlings, G-H, Transplant of rooted seedlings
DETAILED DESCRIPTION OF THE EMBODIMENTS
Example 1
A tissue culture method of ancient tree Platycladus oriental- is (L.) Franco comprised the following steps: 1.1 Establishment of explants
Explants were collected from robust, disease-free and 1-2 year-old new shoots from the ancient tree planted by Huang Di in
Huangdi Mausoleum in Shaanxi Province in May 2014. Three repli- cates were established for each treatment and 20 explants were collected for each replicate. 1.2 Test method 1.2.1 Establishment of a sterile system of explants of the ancient tree planted by Huang Di
The new shoots of the ancient tree planted by Huang Di col- lected were disinfected with 5% NaClO for 5 min, rinsed with tap water 5 times, soaked in a detergent for 5 min, and rinsed with running tap water for 2 h; then twigs were rinsed repeatedly with sterile water on a clean bench, soaked in 75% alcohol for 30 s, and then rinsed 5 times with sterile water; after sterilization with 0.3% HgCl2, rinsed 5 times with sterile water. The times of sterilization with 0.3%HgCl2 were 6, 8, 10, 12 min, respectively, which was used to compare the effect of different sterilizing time on the survival rate of tissue culture. After water was blown off, the part at the lower end that was in contact with the disinfect- ant was cut, and used for the subsequent experiment. After 30 days, the contamination rate, death rate and survival rate were counted. 1.2.2 Preparation of basal media
The preparation of basal media (MS, 1/2MS, B5, WPM, DKW) used in the present disclosure: the medium powder was purchased from
Shanghai Hengyuan Biotechnology Co., Ltd. 4.74 g of the purchased
MS medium was dissolved in 1,000 mL of sterile water, then 20 g/L sucrose and 7 g/L agar were added, and pH of the solution was ad- justed to 5.8, autoclaved at 121°C for 30 min, for later use. 1.2.3 The culture room conditions:
The temperature was 25+1°C, the light was 12-14 h, and the illumination intensity was 2,000-2,500 Lx. 1.2.2 Sugar source screening 3% glucose and 3% sucrose were added to the medium of ex- plants of the ancient tree planted by Huang Di. 1.2.4 Anti-browning
The activated carbon (AC), ascorbic acid (VC) and polyvi- nylpyrrolidone (PVP) with mass concentrations of 0, 1.00, 3.00, 5.00 g/L were added to the culture medium of explants of ancient tree planted by Huang Di, in 14 days after inoculation, the brown- ing rates were counted. 1.2.5 Primary culture
A suitable basal medium (MS, 1/2MS, B5, WPM, DKW) of the an- cient tree planted by Huang Di was screened, and 6-BA 0.1 mg/L+NAA 0.2 mg/L+ activated carbon 3.00 g-L-1 were added during culture.
New shorts were cut into small segments of 2-3 cm for inoculation, 45 days later, the contamination rate, death rate and survival rate were counted.
Sterile new shorts were inoculated into the primary culture medium. Orthogonal experimental design was used to screen the op- timal primary culture medium of the ancient tree planted by Huang
Di. The concentrations of added 6-BA were 0.05, 0.10, 0.50 mg/L respectively, the concentrations of added NAA were 0.05, 0.10, 0.20 mg/L respectively, and the concentration of added activated carbon was 3.00 g-L-1. 45 days later, the average growth, the av- erage number of proliferating buds and the growth situation were counted. 1.2.6 Proliferation culture
The buds of tissue culture seedlings cultured on the primary culture medium were cut into small segments for proliferation cul-
ture. A suitable basal medium (MS, 1/2MS, B5, WPM, DKW) for pro- liferation culture of the ancient tree planted by Huang Di was screened; NAA 0.1 mg/L, IBA 0.05 mg/L and 6-BA 0.05 mg/L were add- ed during the culture, 45 days later, the average growth and 5 growth situation were counted. 0.02, 0.05, 0.10 mg/L of 6-BA, 0.05, 0.10, 0.20 mg/L of KT, and 0.05, 0.10, 0.20 mg/L of NAA were added into the screened suitable basal medium. Orthogonal experimental design was used for screening. The buds of tissue culture seedlings cultured on the primary culture medium were cut into small segments, 45 days lat- er, the average growth and growth situation were counted. 1.2.7 Rooting cultivation
The tissue culture seedlings of proliferation culture were cultured into the three basic media (1/2MS, WPM and DKW) to screen suitable basal medium for the rooting of ancient tree planted by
Huang Di; 30 days later, the rooting rate and rooting situation were counted.
The rooting cultivation was performed for the tissue culture seedlings of proliferation culture. Orthogonal experimental design was adopted. The concentration gradients of the hormones NAA and
IBA were 0.00, 0.20, 0.50, 1.00 mg/L and 0.50, 1.00, 1.50 mg/L, respectively. 40 days later, the rooting rate and rooting situa- tion were counted. 1.2.8 Hardening seedlings and transplanting test-tube seed- lings
The bottle cap was opened 3 days before transplanting so that the rooted seedlings gradually exposed to the external environ- ment. After 3 days, the seedlings were taken out with tweezers, and the medium on the roots was washed off, then seedlings were transplanted into the matrix (turfy soil: vermiculite: per- lite=1:1:1), watered and placed in an incubator. The growth condi- tions of seedlings were observed every day.
2. Results and analysis 2.1 Effect of different sterilizing time on sterilizing ef- fect of explants of the ancient tree planted by Huang Di
Table 1 Effect of different sterilizing time on sterilizing effect of explants of the ancient tree planted by Huang Di
Sterili- Contaminati- Death rate ‚ . zing time ij . Survival rate (%) on rate (£) (%) (min) 6.00 40.00 16.72 43.34 8.00 25.81 19.45 54.82 10.00 24.55 20.64 54.98 12.00 24.21 44.88 31.22
As shown in Table 1, after 30 days of culture in the culture medium, the contamination rate after disinfection with 0.3%HgCl2 for 6 min was the highest (Figure A). When disinfected for 12 min, the contamination rate was the lowest but the survival rate was also the lowest, and the survival rate was only 31.22%. Survival rates after 8 and 10 min of disinfection were higher than those of other treatments, which were 54.82% and 54.98% respectively. Over- all, the too long or too short sterilizing time was not suitable.
A sterilizing time of 10 min was more suitable for the ancient tree planted by Huang Di. 2.2 Sugar source screening
The medium was supplemented with 3% glucose and 3% sucrose, and the browning rates of the ancient tree planted by Huang Di were 78.07% and 64.85%, respectively. Based on the results in Ta- ble 2, different sugar sources in the medium had a certain effect on the browning of tissue culture of explants. The effect of su- crose addition was better than glucose addition for the ancient tree planted by Huang Di.
Table 2 Sugar source screening
Sugar sour- Basal NAA IBA Browning ce medium (mg/L) (mg/L) rate (%) 3% glucose 1/2MS 0.05 0.05 78.07 3% sucrose 1/2MS 0.05 0.05 64.85 2.3 Effect of anti-browning agents with different concentra- tion on browning of explants
As shown in Table 3, the addition of 3.00 g:L-1 activated carbon could significantly inhibit the browning of explants of the ancient tree planted by Huang Di (Figure B), and the browning rate was only 13.45 (P <0.05), but the highest occurrence rate of new buds treated with 5.00 g°L-1 activated carbon was 5.44. Therefore, adding 3.00 g-L-1 activated carbon to the culture medium could in- hibit the browning of explants of the ancient tree planted by
Huang Di.
Table 3 Effect of anti-browning agents with different concen- tration on browning of explants
Mass concen- Average bud Browning . number 6 tration/ rate (%)
Treatments / (pecs/explan { g/L) t s)
Control (ck) 0.00 1.72 25.72 . 1.00 3.45 15.72 ea 3.00 4.72 13.45 5.00 5.44 15.64 . . 1.00 3.64 22.64
Pe acid 3.00 3.81 18.88 5.00 3.45 15.72
Polyvinyl 1.00 2.45 21.45 pyrrolidone { 3.00 3.54 16.64
PVP) 5.00 3.938 18.88

Claims (2)

CONCLUSIESCONCLUSIONS 1. Weefselkweekmethode van oude bomen, omvattende de volgende stappen: (1) Bereiding en desinfectie van grondstoffen Het kiezen van robuuste, ziektevrije en 1-2 jaar oude nieuwe scheuten van de oude boom geplant door Huang Di in het Huangdi Mausoleum in de provincie Shaanxi, het snijden van de explantaten in 5x5 mm stengelsegmenten met een scalpel, het desinfecteren van het oppervlak met 53 NaClO gedurende 5 minuten, 5 keer spoelen met leidingwater, 5 minuten weken in een wasmiddel en 2 uur spoelen met stromend kraanwater; het vervolgens herhaaldelijk spoelen van de takjes 5 keer met steriel water op een schone bank, 30 s onder- dompelen in 75% alcohol en vervolgens 5 keer spoelen met steriel water; na sterilisatie met 0,3% HgCcl:, 5 keer spoelen met steriel water; (2) Suikerbronscreening van kweekmedium De suikerbron is 3% sucrose; (3) Screening van middelen tegen bruin worden Actieve kool (AC) kan bruin worden tegengaan, met een concentratie van 5,00 g/L; 14) Primaire kweek Het inoculeren van gesteriliseerde stengelsegmenten van explanta- ten in een primair inductiemedium 1/2MS+6-BA 0,10 mg/L+NAA 0,20 mg/L+ actieve kool 3,00 gL 7%; (5) Proliferatie kweek Het inoculeren van de verkregen nieuwe knoppen in een secundair kweekmedium 1/2MS+ 6-BA 0,05 mg/L + NAA 0,2 mg/L + KT 0,1 mg/L; (6) Wortelteelt Het snijden van adventieve knoppen van de basis snijden en het en- ten ervan in bewortelingsmedium WPM+ NAA 0,5 mg/L + IBA 0,5 mg/L; (7) Zaailingen harden en reageerbuiszaailingen verplanten 3 dagen voor het verplanten de dop van de fles openen, zodat de geroote zaailing geleidelijk wordt blootgesteld aan de externe om- geving; 3 dagen later, de zaailingen eruit halen met een pincet, het kweekmedium op de wortels afwassen, in de matrix overplanten en elke dag de groeiomstandigheden van zaailingen observeren.1. Tissue culture method of old trees, including the following steps: (1) Preparation and disinfection of raw materials Choosing robust, disease-free and 1-2 years old new shoots from the old tree planted by Huang Di in Huangdi Mausoleum in Shaanxi Province , cutting the explants into 5x5 mm stem segments with a scalpel, disinfecting the surface with 53 NaClO for 5 minutes, rinsing 5 times with tap water, soaking in detergent for 5 minutes, and rinsing with running tap water for 2 hours; then repeatedly rinsing the twigs 5 times with sterile water on a clean bench, immersing 30 s in 75% alcohol and then rinsing 5 times with sterile water; after sterilization with 0.3% HgCl:, rinse 5 times with sterile water; (2) Sugar source screening of culture medium The sugar source is 3% sucrose; (3) Screening anti-browning agents Activated carbon (AC) can be anti-browning, with a concentration of 5.00 g/L; 14) Primary culture Inoculation of sterilized stem segments of explants in a primary induction medium 1/2MS+6-BA 0.10 mg/L+NAA 0.20 mg/L+ activated charcoal 3.00 gL 7%; (5) Proliferation culture Inoculating the obtained new buds in a secondary culture medium 1/2MS + 6-BA 0.05 mg/L + NAA 0.2 mg/L + KT 0.1 mg/L; (6) Rooting Cutting adventitious buds from the base cutting and grafting them into rooting medium WPM+ NAA 0.5 mg/L + IBA 0.5 mg/L; (7) Hardening seedlings and transplanting test tube seedlings Open the bottle cap 3 days before transplanting, so that the rooted seedling is gradually exposed to the external environment; 3 days later, take out the seedlings with tweezers, wash off the culture medium on the roots, transplant into the matrix, and observe the growing conditions of seedlings every day. 2. Weefselkweekmethode van oude bomen van Platycladus orientalis2. Tissue culture method of ancient trees of Platycladus orientalis (L.) Franco volgens conclusie 1, waarbij in stap (1) wordt uitge- voerd het kiezen van robuuste, ziektevrije en 1-2 jaar oude nieuwe scheuten van de oude boom geplant door Huang Di in het Huangdi Mausoleum in de provincie Shaanxi, het snijden van de explantaten met een scalpel in stengelsegmenten van 5 x 5 mm, het desinfecte- ren van het oppervlak gedurende 5 minuten met 5% Nacl0, het 5 keer spoelen met kraanwater, het gedurende 5 minuten onderdompelen in een wasmiddel, en het spoelen met stromend kraanwater gedurende 2 uur; het vervolgens herhaaldelijk met steriel water spoelen van de takjes 5 keer op een schone bank, 30 s in 75% alcohol onderdompe- len en vervolgens 5 keer spoelen met steriel water; na sterilisa- tie met 0,3% HgCl,, 5 keer spoelen met steriel water.(L.) Franco according to claim 1, wherein step (1) involves choosing robust, disease-free and 1-2 years old new shoots from the old tree planted by Huang Di in Huangdi Mausoleum in Shaanxi Province, cutting the explants with a scalpel into stem segments of 5 x 5 mm, disinfecting the surface with 5% NaClO for 5 minutes, rinsing 5 times with tap water, immersing in detergent for 5 minutes, and rinsing with running tap water for 2 hours; then repeatedly rinsing the twigs 5 times with sterile water on a clean bench, immersing 30 s in 75% alcohol and then rinsing 5 times with sterile water; after sterilization with 0.3% HgCl, rinse 5 times with sterile water.
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Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
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COLEMAN W K ET AL: "IN-VITRO CULTURE OF WESTERN RED-CEDAR THUJA-PLICATA PART 1 PLANTLET FORMATION", BOTANICAL GAZETTE, vol. 138, no. 3, 1977, pages 298 - 304, XP009539049, ISSN: 0006-8071 *
KONAR R.N ET AL: "Tissue culture as a method for vegetative propagation of forest trees", NEW ZEALAND JOURNAL OF FORESTRY SCIENCE, 1 January 1974 (1974-01-01), pages 279 - 290, XP055959720, Retrieved from the Internet <URL:https://www.scionresearch.com/__data/assets/pdf_file/0006/58731/NZJFS421974KONAR279_290.pdf> [retrieved on 20220912] *
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