AU2021100612A4 - A method for preparation of scions - Google Patents
A method for preparation of scions Download PDFInfo
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- AU2021100612A4 AU2021100612A4 AU2021100612A AU2021100612A AU2021100612A4 AU 2021100612 A4 AU2021100612 A4 AU 2021100612A4 AU 2021100612 A AU2021100612 A AU 2021100612A AU 2021100612 A AU2021100612 A AU 2021100612A AU 2021100612 A4 AU2021100612 A4 AU 2021100612A4
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- culture
- scions
- pecan
- axillary
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G2/00—Vegetative propagation
- A01G2/30—Grafting
- A01G2/35—Cutting; Inserting
-
- 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
- A01H4/002—Culture media 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Health & Medical Sciences (AREA)
- Physiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention refers to method of preparation of scions. Selecting stem segments with axillary
buds of three-year-old grafted seedlings as explants, cutting the stem segments into stem
segments after cleaning and disinfection, inoculating the stem segments into an axillary bud
induction culture medium for culture, transferring the generated axillary buds into subculture,
and selecting strong rootless plantlets without browning phenomenon, with stem length of 2-4
cm as scions.
Description
A method for preparation of scions
The invention refers to a method for preparation of scions which belongs to the technical
field of biological technology.
[Caryaillinoinensis(Wangenh.)K.Koch]also known as pecan or hickory, it is a hickory plant
of Juglandaceae.It originated in the United States and northern Mexico and is one of the
famous dried fruit trees in the world. Its nuts are large in size, thin in shell, high in kernel
rate, easy in kernel taking and high in yield. Its kernel is nice looking and delicious, without
astringency and rich in nutrition, so it is an ideal additive material for health food or bread,
candy and other foods. Pecan is also an important woody oil plant. Its kernel oil content is
as high as 70%, among which unsaturated fatty acid content is as high as 97%. It is the
first-class cooking oil and salad oil. Pecan is also an excellent tree species for timber and
garden greening. Pecan can not only provide nutritious dried fruits, high-quality wood and
other forest products for the market, but also afforest barren hills and wastelands, beautify
the environment, integrating economic, ecological and social benefits. Its diverse products
have become an important material foundation for improving people's diet structure and
Chinese national economic construction.
China has introduced pecan for more than 100 years, but it has not yet formed a large
scale commercial production. The traditional propagation method of pecan is mainly
sowing, but it takes 10-15 years for the seedlings to bear fruit normally. It takes a long time
from sowing to bearing seedling age, which puts great pressure on the growers, thus affecting the enthusiasm of the pecan growers. Moreover, it is difficult to keep its excellent characteristics in seedling propagation, and the offspring are prone to character segregation. Therefore, the propagation method of pecan must rely on asexual propagation.
At present, the asexual propagation method of pecan is mainly grafting, and the immature
grafting technology and low grafting survival rate are the main factors that limit the
industrialization development of Pecan. (Peng Fangren, 2012)
In recent years, through the unremitting efforts of Chinese scientific and technical
workers, the grafting technology of pecan has made a breakthrough. The grafting survival
rate of pecan can reach more than 60% by adopting the improved growing season square
bud grafting technology (Zhang et al, 2015), but there are still widespread problems such
as low grafting survival rate and obvious differences in grafting survival rate among
different varieties. (Weng Chunyu et al, 2012)
The purpose of the invention is to solve the problem of low grafting survival rate of pecan
in the existing technologies, and offer a micro-grafting method of Pecan.
Technical scheme:
The micro-grafting method of Pecan, including the following steps:
(1) Preparation of scions
In the fine variety of pecan, stem segments with axillary buds of three-year-old grafted
seedlings are selected as explants, which are washed and soaked in 70-75v% ethanol
solution for 20-30s, wash with sterile water, disinfect with 0.05-0.lwt% HgCl2 solution for 10-15min, then wash with sterile water, cut into lcm long stem segments, and inoculate to axillary bud induction medium for culture, 15-20 days later, transfer the generated axillary buds into a subculture medium for culture to obtain rootless plantlets, and select rootless plantlets with no pollution or browning phenomenon, and with 2-4cm stem length, leaves and good growth condition as scions.
(2) Preparation of rootstock
Washing mature Pecan seeds with tap water and soak for 3 days, then soak in 70
v% ethanol solution for 20-30s in a sterile room, washing with sterile water and
disinfecting with 0.05-0.lwt% HgCl2 solution for 10-15min, washing with sterile water,
then place them on an ultra-clean workbench to peel off the shells, and put the peeled
seeds into 0.1% HgCl2 solution for disinfection. Inserting it into embryo induction culture
medium vertically, culturing at 21-25°C, keeping air relative humidity at 7 0 - 80 %, and
completely dark for 25-30 days to obtain tissue culture seedlings as rootstock seedlings.
(3) Micro-grafting
Putting the rootstock seedlings under fluorescent lamp for 5-10 days, and graft them
when the light red tender stems turn green and the top leaves spread out. Take out the
rootstock seedlings, cut off the upper part of the embryo, keep the part 2cm above the
hypocotyl as the rootstock, cut down vertically in the center of the transverse section, split
the rootstock with a cut depth of 0.5-1cm, and symmetrically cut the two sides of the lower
end of the scion into a wedge shape with a cut length of 0.5-1cm. Then insert the scion into
the incision of the rootstock and wrap it with plastic wrap to complete the grafting. After
grafting, the grafted seedlings are transferred to the grafted seedling culture medium for culture, and after the grafted seedlings are cultured for 25 days, unbound them, and the seedlings are transplanted into the cultivation substrate after one week of seedling hardening.
In step (1), the formula of the axillary bud induction medium is as follows: Basic
medium + 6-BA 2.0-3.Omg/L + IBA 0.01-0.02mg/L + Sucrose 30g/L + Agar 5.8g/L. The
pH value is 5.8-6.0. The basic medium is selected from WPM, MS or DKW.
In step (1), the formula of the subculture medium is as follows: Improved DKW
+ 1.Omg/L 6-BA + O.Olmg/L IBA + 5.8g/L Agar + 30g/L Sucrose + 0.1-1.Og/L Anti
browning agent. The pH value is 5.8-6.0. The formula of the improved DKW is:
Ammonium nitrate 1416.0, boric acid 4.8, anhydrous calcium chloride 112.5, calcium
nitrate 1367.0, copper sulfate pentahydrate 0.25, Na2-EDTA 45.4, ferric sulfate
heptahydrate 33.8, magnesium sulfate 361.49, manganese sulfate monohydrate 33.5,
sodium molybdate 0.39 and nickel sulfate hexahydrate 0.005, potassium dihydrogen
phosphate 265.0, potassium sulfate 1559.0, zinc nitrate hexahydrate 17.0, thiamine
hydrochloride 5.22, inositol 100, nicotinic acid 1.0. (mg/L).
Furthermore, in step (1), 0.1wt% HgCl2 solution is preferably used for disinfection
treatment for 10 min. at this time, the pollution rate is the lowest and the germination rate
is the highest, up to 77%.
Furthermore, in step (1), the optimum medium for axillary bud induction is MS + 6
BA 2.Omg/L + IBA 0.02mg/L + Sucrose 30g/L + Agar 5.8g/L, the pH value is 5.8-6.0. The
germination rate is the highest.
Furthermore, in step (1), the culture conditions are as follows: Temperature is 25 °C±
2°C, light intensity is 2400-26001x, use fluorescent lamp as light source, illumination time
is 14h/d and relative humidity is 70-80%.
Furthermore, in step (1), in the subculture medium formula, the anti-browning agent
is selected from any one of activated carbon, polyvinylpyrrolidone or vitamin C.
Polyvinylpyrrolidoneis preferably used which has the lowest browning rate.
Furthermore, in step (2), the formula of embryo induction medium is: Improved DKW
+ KT 2mg/L + 6-BA 1mg/L + IBAO.Olmg/L, the pH value is 5.70-5.85. At this time the
germination rate of embryo is the highest and the contamination rate is the lowest. The
formula of the improved DKW is: Ammonium nitrate 1416.0, boric acid 4.8, anhydrous
calcium chloride 112.5, calcium nitrate 1367.0, copper sulfate pentahydrate 0.25, Na2
EDTA 45.4, ferric sulfate heptahydrate 33.8, magnesium sulfate 361.49, manganese sulfate
monohydrate 33.5, sodium molybdate 0.39 and nickel sulfate hexahydrate 0.005,
potassium dihydrogen phosphate 265.0, potassium sulfate 1559.0, zinc nitrate hexahydrate
17.0, thiamine hydrochloride 5.22, inositol 100, nicotinic acid 1.0. (mg/L).
Furthermore, in step (3), the formula of the grafted seedling culture medium is: MS +
6-BA 1mg/L + IAA 3.mg/L, the pH value is 5.70-5.85.
Furthermore, in step (3), the cultivation substrate is river sand or a mixture of perlite,
vermiculite and humus soil with a weight ratio of 1:1:1.
The beneficial effects of the invention are as follows: The invention completes the
sterilization and aseptic culture of the explants of the stem segments with axillary buds of
the variety seedlings, which is a breakthrough point in the tissue culture process of the
Pecan. In the previous studies, the stem segments of the seedlings were used as the explant
materials, and there was no precedent for the successful tissue culture of the stem segments
of the variety seedlings, because as the material of asexual propagation, it must come from
the improved variety of clones to be valuable. Moreover, the invention completes the
micro-grafting technology in the test tube, and the grafting and transplanting is successful.
Comparing with the existing grafting method of Pecan, the micro-grafting method of Pecan
has the advantages of high grafting survival rate, low cost, small occupation area, and can
be manipulated effectively.
The technical scheme of the invention is described in detail in combination with
specific embodiments. In the following embodiments, the formula of the improved DKW
is: Ammonium nitrate 1416.0, boric acid 4.8, anhydrous calcium chloride 112.5, calcium
nitrate 1367.0, copper sulfate pentahydrate 0.25, Na2-EDTA 45.4, ferric sulfate
heptahydrate 33.8, magnesium sulfate 361.49, manganese sulfate monohydrate 33.5,
sodium molybdate 0.39 and nickel sulfate hexahydrate 0.005, potassium dihydrogen
phosphate 265.0, potassium sulfate 1559.0, zinc nitrate hexahydrate 17.0, thiamine
hydrochloride 5.22, inositol 100, nicotinic acid 1.0. (mg/L).
Embodiment 1
A micro-grafting method of Pecan, including the following steps:
(1) Preparation of scions
The tender stems with axillary buds of three-year-old grafted seedlings of Pecan
(Pawnee) are used as explants, and soaked in detergent for 20min, wash with tap water for 2h, soak in 70-75v% alcohol for 30s in sterile operating table, wash with sterile water for 4 times, disinfect with 0.1wt% HgCl2 solution for 10mmin, then wash with sterile water for 6 times, and cut into lcm long stems, then inoculate into axillary bud induction culture medium(MS + 6-BA 2.Omg/L + IBA 0.02mg/L + Sucrose 30g/L + Agar 5.8g/L, and pH value is 5.8-6.0) for culturing. After 15-20 days of culture, the induction of axillary buds is counted (the germination rate of axillary buds is 81.9%), and the axillary buds are transferred to subculture medium (Improved DKW + 1.Omg/L 6-BA + 0.0lmg/L IBA
+ 5.8g/L Agar + 30g/L Sucrose + 0.g/Lpolyvinylpyrrolidone, and pH value is 5.8-6.0) for
culture (Culture condition: The temperature is 25°C± 2C, the light intensity is 2400
26001x, the fluorescent lamp is used as the light source, the illumination time is 14h/d,
and the relative humidity of the air is 70-80%). Then the rootless plantlets (with browning
rate of 25%) is obtained. The rootless plantlets without pollution and browning, with 2
4cm length and leaves and good growth status are selected as scions.
(2) Preparation of rootstock
Taking the seeds of mature Pecan (Pawnee), wash with tap water and soak for 3
days, then soak in 70- 75v% ethanol solution for 30s in a sterile room, rinse with sterile
water, disinfect with 0.1wt% HgCl2 solution for 10mmin, rinse with sterile water, and then
place on an ultra-clean workbench to peel off the shells. Seed kernels are sterilized in
0.1% HgCl2 solution for 5min. The embryos were cut from sterilized seed kernels and
inoculate into embryo induction medium vertically. (The formula of the embryo
induction medium is: Improved DKW + KT 2mg/L + 6-BA 1mg/L + IBA0.Olmg/L), the
depth of inoculation is one-third of the medium. The culture is carried out under the conditions of 21-25°C, relative humidity of 70-80% and full darkness. After 30 days of culture, tissue culture seedlings are obtained and used as rootstocks.
(3) Micro-grafting
Putting the rootstock seedlings under fluorescent lamp for 10 days, and graft them
when the light red tender stems turn green and the top leaves spread out. Take out the
rootstock seedlings, cut off the upper part of the embryo, keep the part 2cm above the
hypocotyl as the rootstock, cut down vertically in the center of the transverse section, split
the rootstock with a cut depth of 0.5-1cm, and symmetrically cut the two sides of the lower
end of the scion into a wedge shape with a cut length of 0.5-1cm. Then insert the scion into
the incision of the rootstock and wrap it with plastic wrap to complete the grafting. After
grafting, the grafted seedlings are transferred to the grafted seedling culture medium (The
formula of the grafted seedling culture medium is: MS + 6-BA 1mg/L + IAA 3.Omg/L) for
culture, and after the grafted seedlings are cultured for 25 days, unbound them, and the
seedlings are transplanted into the cultivation substrate (A mixture of perlite, vermiculite
and humus with a weight ratio of 1:1:1) after one week of seedling hardening.
In Embodiment 1, the survival rate of grafting and transplanting are 86.7% and 92%
respectively.
Embodiment 2
In step (1), the axillary bud induction medium is as follows: WPM + 6-BA 2.mg/L
+ IBA 0.02mg/L + Sucrose 30g/L + Agar 5.8g/L, and pH value is 5.8-6.0. The results
shows that the germination rate of axillary buds is 80%.
The rest of the steps are the same as those of Embodiment 1.
Embodiment 3
In step (1), the subculture medium is as follows: Improved DKW +1.Omg/L 6-BA
+ 0.0lmg/L IBA + 5.8g/L Agar + 30g/L Sucrose + 0.lg/L Vitamin C, and pH value is 5.8
6.0. The browning rate is 30%.
The rest of the steps are the same as those of Embodiment 1.
Embodiment 4
In step (1), the subculture medium is as follows: Improved DKW +1.Omg/L 6-BA
+ 0.01mg/L IBA + 5.8g/L Agar + 30g/L Sucrose + 1.0g/L Activated carbon, and pH value
is 5.8-6.0. The browning rate is 35%.
The rest of the steps are the same as those of Embodiment 1.
Embodiment 5
In step (3), the days of seedling hardening is days, and the rest of the steps are the
same as those of Embodiment 1. The survival rates of grafting and transplanting are 80%
and 85.6%, respectively.
Embodiment 6
In step (3), the culture medium is river sand, and the rest of the steps are the same as
those of Embodiment 1. The survival rates of grafting and transplanting are 83.3% and
86.7%, respectively.
Claims (5)
1. A method for preparation of scions comprising the following steps:
In the fine variety of Pecan, stem segments with axillary buds of three-year-old
grafted seedlings are selected as explants, which are washed and soaked in 70-75v%
ethanol solution for 20-30s, wash with sterile water, disinfect with 0.05-0.lwt% HgCl2
solution for 10-15min, then wash with sterile water, cut into lcm long stem segments,
and inoculate to axillary bud induction medium for culture. Transferring the generated
axillary buds into a subculture medium for culture to obtain rootless plantlets, and
selecting rootless plantlets with no pollution or browning phenomenon, and with 2-4cm
stem length, leaves and good growth condition as scions.
2. A method according to Claim 1, wherein 0.1wt% HgCl2 solution is used for disinfection
for 10min.
3. A method according to Claim 1, wherein the formula of the axillary bud induction
medium is: MS + 6-BA 2.Omg/L + IBA 0.02mg/L + Sucrose 30g/L + Agar 5.8g/L, and pH
value is 5.8-6.0.
4. A method according to Claim 1, wherein the culture conditions are as follows:
Temperature is 25C ±2C, light intensity is 2400-26001x, use fluorescent lamp as light
source, illumination time is 14h/d and relative humidity is 70-80%.
5. A method according to Claim 1, wherein in the subculture medium formula, the anti
browning agent is selected from any one of activated carbon, polyvinylpyrrolidone or
vitamin C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021100612A AU2021100612A4 (en) | 2021-02-01 | 2021-02-01 | A method for preparation of scions |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021100612A AU2021100612A4 (en) | 2021-02-01 | 2021-02-01 | A method for preparation of scions |
Publications (1)
| Publication Number | Publication Date |
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| AU2021100612A4 true AU2021100612A4 (en) | 2021-04-15 |
Family
ID=75397146
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| Application Number | Title | Priority Date | Filing Date |
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| AU2021100612A Ceased AU2021100612A4 (en) | 2021-02-01 | 2021-02-01 | A method for preparation of scions |
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| AU (1) | AU2021100612A4 (en) |
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2021
- 2021-02-01 AU AU2021100612A patent/AU2021100612A4/en not_active Ceased
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