CN112273229B - One-step seedling method for hydrangea - Google Patents
One-step seedling method for hydrangea Download PDFInfo
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- CN112273229B CN112273229B CN202011171693.2A CN202011171693A CN112273229B CN 112273229 B CN112273229 B CN 112273229B CN 202011171693 A CN202011171693 A CN 202011171693A CN 112273229 B CN112273229 B CN 112273229B
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to the technical field of plant rapid breeding, and particularly discloses a tissue culture method for one-step seedling formation of hydrangea.
Description
Technical Field
The invention relates to the technical field of plant rapid breeding, in particular to a seedling method of hydrangea macrophylla.
Background
Hydrangea (Hydrangea macrophylla) is deciduous shrub of Hydrangea (Hydrangea) of Saxifragaceae (saxfragaceae), native to japan and one of sichuan origin. Gardens and people in various parts of China are often cultivated, and varieties are numerous. In summer, inflorescences of the hydrangea macrophylla are spherical, the flower colors of the hydrangea macrophylla change along with the change of soil pH, the flowers are rich in color, and the hydrangea macrophylla is common ornamental flowers and trees and has high development and utilization values. The traditional propagation method of the eight flowers mainly comprises cuttage, plant division and layering, has the problems of long breeding period, poor stability of progeny characters, lack of a directional improvement system and the like, and cannot meet the requirements of markets for seedlings.
The genetic improvement and the clone propagation of the hydrangea are always paid attention by decision departments of forestry of various countries and are paid particular attention by ornamental plant breeding workers and gardening operators, the in vitro regeneration has the characteristics of high speed, short period, high efficiency, stability and the like, and the dawn is brought to the rapid asexual propagation and the industrialized operation of the hydrangea. Tissue culture methods related to hydrangea have been reported. Yangbaoming and the like adopt the eight immortal flower petioles to carry out in vitro culture and research (the eight immortal flower petioles are cultured in vitro and the plant regeneration technology is researched [ J ]. Shanxi agricultural science, 2018,46(02): 159-. Zhaoyengying, etc. induced regeneration research is carried out by adopting hydrangea leaves (hydrangea leaf induced regeneration technology [ J ] Hunan agricultural science, 2015(04):81-84+ 87.). Rayleigh et al (research on stem tissue culture technique of hydrangea macrophylla [ J ]. proceedings of northwest college of forestry 2008(04): 101-. Therefore, the plant tissue culture technology can establish a vegetative propagation system of plants in a short time, obtain a large number of seedlings, is not influenced by external conditions, has strong controllability and can realize annual production. However, the reported tissue culture methods all need to respectively induce callus in multiple steps, adventitious bud differentiation and root formation are realized, the time for obtaining the seedlings is still long, and the propagation coefficient is low.
At present, no successful report about the method for one-step seedling formation of the regeneration of the hydrangea in vitro is reported.
Disclosure of Invention
In order to relieve the commercial demand condition of the hydrangea and reduce the waste of resources, the invention further improves the in vitro rapid propagation system of the hydrangea, perfects the tissue culture method for one-step seedling formation of the tissue culture seedling of the hydrangea and obtains a regenerated plant with higher frequency. The invention is improved by the following steps: firstly, the stem section of the hydrangea is selected as a starting material, the selected material is easy to be used as the starting material for one-step seedling formation, and researches show that the stem section is superior to leaves or petioles, and theories speculate that the reason that the nutrition basis is possibly better than the leaves and the petioles is possible; and secondly, the induction and rooting can be completed in one step by improving the MS culture medium as a basic culture medium and matching with a proper hormone proportion (only one culture medium is needed).
The invention provides a tissue culture method for one-step seedling formation of hydrangea macrophylla, which comprises the following steps:
(1) cutting off the hydrangea stem segment as an initial explant;
(2) inoculating the explant obtained in the step (1) into a culture medium added with 0.1-0.5 mg/L of NAA and 0.5-3.0mg/L of BA;
(3) continuously culturing until adventitious buds forming at the wound part of the stem section are induced to 1-2 cm;
(4) and (4) continuously culturing the tissue culture seedlings obtained in the step (3) until complete regeneration seedlings with buds and roots are formed.
In a more preferred embodiment, the amount of NAA added is 0.1 to 0.2mg/L and BA 0.5-2.0mg/L, more preferably NAA 0.2mg/L and 6-BA 1.0 mg/L.
In specific embodiments, the basal medium of the medium is MS medium, or a medium in which the MS basal is modified by adding NH thereto4N03By (NH)4)2SO4And Ca (N0)3)2·4H2O。
More specifically, the basic medium composition of the medium is as follows:
in particular embodiments, the specific formulation process may be performed with reference to MS medium. Wherein the culture medium is prepared by sterilizing at 121 ℃ for 15-25 minutes at the pH of 5.8.
In a preferred mode, the explant in the step (1) is a stem section obtained from 1-2cm long sterile seedlings of hydrangea.
In a specific embodiment, the culturing time in step (3) is 12-30 days, the culturing condition is 28 +/-2 ℃ and the illumination is 80 mu mol.m-2. s-116 h/d.
In another embodiment, said step (4) is performed for 60-90 days under the conditions of 28. + -. 2 ℃ and 80. mu. mol.m-2. s-116 h/d light.
Preferably, the method further comprises the following seedling exercising steps:
(5) transferring the regenerated plantlet obtained in the step (4), the culture medium and a culture bottle together into a culture condition of 28 +/-2 ℃ and illumination of 80 mu mol.m-2. s-116 h/d for tolerance culture for 1-2 weeks, then opening the cover of the culture bottle, injecting 10-20ml of sterile water or deionized water, and hardening the plantlet for 5-8 days.
Further preferably, the method further comprises a transplanting step:
(6) transplanting the regenerated plant after seedling hardening in the step (5) into a matrix of peat, vegetable garden soil and perlite, and growing in a greenhouse with natural illumination at 28 +/-2 ℃.
The invention provides a tissue culture method for tissue culture seedlings of hydrangea by one step, which takes hydrangea stem segments as starting materials, can generate hydrangea regeneration plants by one step through culture without inducing the formation of adventitious buds and roots step by step through multiple culture mediums, greatly reduces the seedling culture period of hydrangea, reduces the resource waste of breeding seeds, can stably provide excellent seedlings with stable characters and uniform quality for markets, and experiments show that the explant of each hydrangea initiation seedling can generate up to 12 regeneration plants and has high phenotype consistency with parents. Moreover, the generation of the seedlings is not limited by seasons, and the seedlings can be produced all the year round. By establishing a regeneration system of one-step seedling of hydrangea, researches on development of new species (line) cultivation and high-quality seedling propagation of ornamental shrubs represented by hydrangea and the like are conducted, and the method has profound significance on sustainable development of forestry economy and reduction of breeding cost.
Drawings
FIG. 1 Stem segment starting explants taken from sterile shoots.
FIG. 2 adventitious buds of multiple shoots induced at the wound site of the stem segment.
FIG. 3 the base of the hydrangea stem segment is enlarged.
FIG. 4 shows the better root system after the induction of the cluster buds of hydrangea.
FIG. 5 hormone ratios: rooting under NAA 0.2mg/L and 6-BA 2.0 mg/L.
FIG. 6 hormone ratios: rooting under NAA 0.2mg/L and 6-BA 1.0 mg/L.
FIG. 7 regenerated plantlets at the acclimatization stage.
FIG. 8 regeneration of transplanted hydrangea macrophylla plants.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.
(1) Preparation of hydrangea media (BXH): a minimal medium was prepared according to Table 1, pH5.8, sterilized at 121 ℃ for 15-25 minutes. The medium was used for induction, elongation and rooting of adventitious buds. The invention adoptsThe culture medium is improved compared with MS culture, and NH in the common MS culture medium4N03By (NH)4)2SO4And Ca (NO)3)2·4H2O to increase NO in the culture medium3 -，Ca+And SO4 2-Content, thereby being beneficial to inducing differentiation elongation and rooting.
TABLE 1 summary of the composition of the culture Medium for hydrangea (BXH)
(2) Material taking: stem segments (1.5cm) were taken from sterile seedlings of hydrangea as starting explants (FIG. 1).
(3) Cutting the hydrangea stem segments obtained in the step (2) and inoculating the cut hydrangea stem segments to BXH culture media respectively added with NAA (0, 0.1, 0.2, 0.3, 0.5mg/L), BA (0, 0.5, 1.0, 2.0, 3.0mg/L) and NAA and 6-BA in different concentration ratios (shown in Table 1). Each treatment was transferred to 10 adventitious shoots, 3 replicates were set. After 2-4 weeks of culture, new bud formation was induced at the wound site of the stem segment (FIG. 2). At this time, the base of hydrangea macrophylla is enlarged and no root system occurs (FIG. 3).
(4) The culture was continued for 4-8 weeks with formation of roots. Wherein fig. 4 shows the better rooting situation, fig. 5 and fig. 6 show the hormone ratios respectively: NAA 0.2mg/L and 6-BA 2.0mg/L, and the hormone ratio: NAA 0.2mg/L and 6-BA 1.0 mg/L. The first one has callus formed in the base part and relatively low rooting rate, and the second one has high rooting rate and great rooting amount, i.e. the second one is superior. And after 90 days of culture, counting the germination rate and the one-step seedling rate.
The culture conditions of the steps (3) and (4) are as follows: culturing under illumination (60 μmol. m-2.s-1) for 16h/d at 25 + -2 deg.C.
(5) And (3) transferring the rooted seedlings obtained in the step (4) together with a culture medium, a culture bottle and the like into a culture condition with the temperature of 28 +/-2 ℃ and the illumination (80 mu mol.m-2.s-1) for 16h/d for tolerance culture for 1-2 weeks, then opening the cover of the culture bottle, injecting 10-20ml of sterile water or deionized water, and hardening the seedlings for 5-8 days (figure 7).
(6) Transplanting the rooted plantlets into a matrix of peat, vegetable garden soil and perlite (3: 6: 1), and growing well in a greenhouse with natural illumination at 28 + -2 ℃ to obtain regenerated plantlets of hydrangea macrophylla consistent with the naturally grown plantlets (figure 8).
The experimental results are as follows: wherein, the rooting rate (%) is calculated as the number of explants inducing adventitious roots/the total number of explants inoculated x 100%. The result shows that the rooting rate can reach 100 percent (the regenerated seedling with good rooting is shown in figure 4), the budding number is 8-12 per explant, the formula is that NAA is added in 0.2mg/L and 6-BA is added in 1.0mg/L, the regenerated plant grows normally, and the transplanting survival rate is high. The induction effect of adventitious roots is inferior by adding NAA0.1mg/L and 1.0 mg/L6-BA, the rooting rate is 83.6 percent, and the bud number is 8. The overall data show that the whole one-step seedling induction effect is good, and the induction period is 70-90 days (see table 1).
TABLE 1 Effect of different hormone combinations on one-step shoot Induction
The experiment shows that the stem section is used as an initial explant, hormones NAA and BA are added into an improved MS culture medium, and the concentration of the phytohormones is adjusted, so that the method for one-step seedling formation of the hydrangea tissue culture method is realized, wherein the budding rate and the rooting quantity are obviously improved, the adventitious bud can be formed in one step in about 70-90d, the one-step seedling formation induction effect is good, the budding number is 8-12, the bud green is strong, the adventitious root grows robustly, the transplanting is easy to survive, and the optimal proportion of the hormones in the culture is as follows: 6-BA 1.0mg/L + NAA 0.2 mg/L.
Claims (9)
1. A tissue culture method for one-step seedling formation of hydrangea macrophylla is characterized by comprising the following steps:
(1) cutting off the hydrangea stem segment as an initial explant;
(2) inoculating the explant obtained in the step (1) into a culture medium added with 0.1-0.3 mg/L of NAA and 1.0-2.0 mg/L of BA; wherein the basic culture medium of the culture medium comprises the following components:
(3) Continuously culturing until the wound part of the stem section is induced to form adventitious buds of multiple buds;
(4) and (4) continuously culturing the adventitious buds in the step (3) until the plants form complete regeneration seedlings with buds and roots.
2. The tissue culture method of hydrangea macrophylla one-step seedling as claimed in claim 1, wherein the addition amount of NAA is 0.2 to 0.3 mg/L and the addition amount of BA is 1.0 to 2.0 mg/L.
3. The tissue culture method of hydrangea macrophylla one-step seedling as claimed in claim 1, wherein the addition amount of NAA is 0.2mg/L and the addition amount of BA is 1.0 mg/L.
4. The tissue culture method of hydrangea macrophylla one-step seedling as claimed in claim 1, wherein the pH of the culture medium is 5.8, and the culture medium is sterilized at 121 ℃ for 15-25 minutes.
5. The tissue culture method of one-step seedling of hydrangea as set forth in any one of claims 1 to 4, wherein the explant of the step (1) is a stem section obtained from 1-2cm long sterile seedling of hydrangea.
6. The tissue culture method of hydrangea macrophylla one-step seedling as claimed in claim 1, wherein the culturing time of step (3) is 12-30 days until adventitious bud is 1-2cm long, the culturing condition is 28 ± 2 ℃, and illumination is 80 μmol · m-2·s-1，16 h/d。
7. The tissue culture method of hydrangea macrophylla one-step seedling as claimed in claim 1, wherein the culture time of step (4) is 60-90 days, the culture condition is 28 ± 2 ℃ and the illumination is 80 μmol · m-2·s-1，16 h/d。
8. The tissue culture method for one-step seedling formation of hydrangea macrophylla as claimed in claim 1, further comprising the following seedling hardening-up step (5):
transferring the regenerated plantlet obtained in step (4) together with culture medium and culture bottle to 28 + -2 deg.C, and irradiating with light 80 μmol ·m-2·s-1After carrying out tolerance culture for 1 to 2 weeks under the culture condition of 16h/d, opening the cover of the culture bottle, injecting 10 to 20ml of sterile water or deionized water, and hardening the seedlings for 5 to 8 days.
9. The tissue culture method for one-step seedling of hydrangea macrophylla as claimed in claim 8, further comprising a transplanting step (6):
transplanting the regenerated plant after seedling hardening in the step (5) into a matrix of peat, vegetable garden soil and perlite, and growing in a greenhouse with natural illumination at 28 +/-2 ℃.
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