CN114080962B - Bletilla striata seedling growth promoting method based on Piriopsis cumingii - Google Patents
Bletilla striata seedling growth promoting method based on Piriopsis cumingii Download PDFInfo
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- CN114080962B CN114080962B CN202111361245.3A CN202111361245A CN114080962B CN 114080962 B CN114080962 B CN 114080962B CN 202111361245 A CN202111361245 A CN 202111361245A CN 114080962 B CN114080962 B CN 114080962B
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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
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
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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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
Abstract
The invention discloses a bletilla striata seedling growth promoting method based on Piriosa indicum, which comprises the following steps: will be used for transplanting and domesticating bletilla striata seedlingsSterilizing the substrate to obtain a sterilized seedling substrate; the concentration is 25-50 g.L ‑1 Is prepared into Pityrosporum indicum liquid with concentration of 1-3×10 8 cfu/mL of Pityrosporum indicum spore liquid; soaking or irrigating the bottle-out seedlings of the bletilla striata with the pyriform liquid or the pyriform sporium indicum liquid; and transplanting the bletilla striata to a substrate, and planting until the bletilla striata is out of the nursery. The method can shorten the domestication period of bletilla striata tissue culture seedlings and lead the bletilla striata seedlings out of the nursery in advance.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a bletilla striata seedling growth promoting method based on Pityrosporum indicum.
Background
Rhizoma bletillae (Bletilla striata (Thunb.) Reichb.f.), rhizoma Bletilla, herba Hedyotidis Diffusae, gan Gen, rhizoma Bletilla, orchidaceae, perennial herb, flower mauve or pink not only has very high ornamental value, but also is one of the traditional Chinese medicines in China, and is recorded in the pharmacopoeia of the people's republic of China, and the rhizoma bletillae can be harvested after being planted for 4-5 years mainly by taking dry tubers as medicines.
The rhizoma bletillae dry tuber mainly contains bibenzyls, phenanthrenes and derivatives thereof, has the effects of astringing to stop bleeding, detumescence, promoting granulation and the like, and can be used for externally treating wound bleeding, scalding, treating sores, internally treating hematemesis, lung diseases, hemoptysis, chronic gastric ulcer, tumors and the like in medical clinic. Modern medicine proves that rhizoma bletillae also has strong antioxidation and anti-aging effects, and can be used for health care, skin care and beauty maintenance. Besides, the bletilla striata can be used as a paste for preparing cigarette butts of high-grade cigarettes, is industrially used as raw materials such as sizing silk, sizing yarn or paint, etc., for brewing wine, etc., and the bletilla striata polysaccharide gum can be used as a natural coating preservative for fruit and vegetable preservation and used as a natural food thickener for food processing.
The bletilla striata has wide application, but wild resources are endangered due to long-term mining, and the second-level protection plants in the national important protection wild plant directory are listed in the year 2021, 9 and 7, and meanwhile, the bletilla striata is also one of varieties which are mainly cultivated in the 'Chun Liuwei' and the 'qu Liuwei'. Because the bletilla striata wild resources are listed in the second-level protection plants of the national important protection wild plant directory, the artificial cultivation of the bletilla striata seedlings is impossible depending on digging the wild resources, the traditional seed-dividing propagation coefficient is low, the variety degradation is easy to cause, the seedling production can only depend on seed propagation or tissue culture, the stem tip tissue culture is long in period and high in cost, the bletilla striata seedlings are produced mostly by adopting a seed sterile propagation mode, but the problems of long production period (usually more than 6 months), weak seedling growth, slow growth, environmental sensitivity, small flowers, flowering delay and the like of the bletilla striata tissue culture seedlings are found in the artificial cultivation, and the industrialization of the bletilla striata seedlings and the rapid development of the artificial cultivation are severely restricted. Aiming at the problems, the invention provides a bletilla striata seedling growth promoting method based on Pityrosporum indicum, so as to solve the problem of bletilla striata seedling production by artificial cultivation and promote the healthy development of the bletilla striata seedling industry.
The patent 201510921991.1 provides a method for rapidly producing rhizoma bletilla seedlings, which comprises the steps of carrying out liquid co-culture on rhizoma bletillae seeds and rhizoma bletillae mycorrhizae fungi until the rhizoma bletillae seeds germinate, and carrying out rhizoma bletillae seedling culture on the germinated rhizoma bletillae seeds, wherein the rhizoma bletillae mycorrhizae fungi are mycorrhizae fungi strain YY-51 of the order of Ceripola (Sebacinales sp.), the rhizoma bletillae mycorrhizae fungi are preserved in China general microbiological culture center (China general microbiological culture Collection center) at 31 of the month of 2015, and the preservation address is Beijing No.1, xiyun No. 3, national academy of sciences of China, and the preservation number is GCMCC No.11104. The bletilla striata artificial seeds or seedlings manufactured by the method are easy to preserve and transport, the problems that the operation process is complex, the technical requirements are high, a large amount of manpower and material resources are required in the repeated subculture, transplanting or field planting process in the traditional tissue culture process are avoided, and the production cost is low. Although only mycorrhizal fungi are cultured and co-cultured with bletilla striata seeds, artificial seeds are embedded and the like, aseptic operation is needed, but the pretreatment procedure of the bletilla striata seeds is complex, the risk of pollution of the seeds is increased, and the liquid culture of the bletilla striata seeds and the bletilla striata mycorrhizal fungi is more polluted than the solid culture.
Patent 201410265619.5 provides a culture method of oncidium: (1) culturing the culture seedlings of the oncidium by adopting an MS culture medium; (2) Culturing Pityrosporum indicum in solid PDA culture medium; (3) co-cultivation of the oncidium with the Pityrosporum indicum; and (4) taking out the culture of the culture seedlings of the oncidium. The culture method reduces the disease rate of the transplanted seedlings of the oncidium, and improves the stress resistance.
Disclosure of Invention
The invention aims to solve the technical problem of providing a bletilla striata seedling growth promotion method based on Pityrosporum indicum, which can shorten the domestication period of bletilla striata tissue culture seedlings and lead the bletilla striata seedlings to be out of nursery in advance.
In order to solve the technical problems, the invention provides a bletilla striata seedling growth promoting method based on Pityrosporum indicum, which comprises the following steps:
(1) Sterilizing a substrate for transplanting and domesticating rhizoma bletillae seedlings to obtain a sterilized seedling substrate;
sub-packaging the sterilized seedling substrate into plastic small square basins with the caliber of 7cm for later use;
(2) The concentration is 25-50 g.L -1 Is prepared into Pityrosporum indicum liquid with concentration of 1-3×10 8 cfu/mL of Pityrosporum indicum spore liquid for later use;
(3) Any one of the following modes is selected:
method A: cleaning the root agar of the bottle-out bletilla striata seedlings, absorbing the moisture on the root surface, soaking the bottle-out bletilla striata seedlings in the Pityrosporum indicum liquid or Pityrosporum indicum liquid for 24+/-2 hours (so that the root of the seedlings is fully contacted with Pityrosporum indicum), taking out the Pityrosporum indicum liquid or Pityrosporum indicum liquid adhered on the root surface, cleaning the bottle-out Pityrosporum indicum liquid or Pityrosporum indicum liquid with clear water, and planting the bottle-out Pityrosporum indicum liquid or Pityrosporum indicum liquid in a sterilized seedling culture matrix;
method B: cleaning the root agar of the bottle-out bletilla striata seedlings, sucking the water on the root surface, directly planting the bottle-out bletilla striata seedlings in a sterilized seedling substrate, and irrigating with prepared Pityrosporum indicum liquid or Pityrosporum indicum liquid, wherein 10+/-1 mL of Pityrosporum indicum liquid or Pityrosporum indicum liquid is irrigated for each plant;
(4) After the bletilla striata is transplanted to the substrate, planting (watering and fertilizing according to the conventional mode) until the bletilla striata is out of the nursery.
As an improvement of the bletilla striata seedling growth promoting method based on Piriosa indicum of the invention, in the step (2):
the preparation method of the Pityrosporum indicum liquid comprises the following steps:
the mycelium blocks obtained after the Pityrosporum indicum is cultured on a PDB culture medium are scattered by adding water (sterilized water) to prepare the mycelium blocks with the concentration of 25-50 g.L -1 Is dispersed by a high-speed refiner;
the Pityrosporum indicum spore liquid is:
adding physiological saline into Pityrosporum indicum cultured in PDA culture medium to obtain extract with concentration of 1-3×10 8 cfu/mL spore fluid (spore concentration calculated with a cell counter).
As a further improvement of the bletilla striata seedling growth promoting method based on Pirio: in the step (4), fertilizer can be irrigated after the bletilla striata is transplanted to the substrate for 30d, and then fertilizer is irrigated once every 15d until the bletilla striata is transplanted to the nursery, wherein the fertilizer adopts N: p: k=20: 10:20, the irrigation concentration is 2 liters of water per 1 g, and the volume of each plant of the fertilizer is 40mL.
As a further improvement of the bletilla striata seedling growth promoting method based on Pirio: in the step (1), the seedling substrate is sterilized by high temperature of 121 ℃ for 60min; or by conventional disinfection methods using commercially available and well-known soil disinfectant dazomet.
In step (4) of the present invention: after the bletilla striata is transplanted into the matrix, leaf surface spraying or watering is required to be carried out within 15 days according to the dry and wet degree of the leaves and the matrix; after new roots grow out, fertilization can be performed, and fertilization is performed at intervals until the new roots leave the nursery, which is a conventional technology.
In general: the pseudobulb of the bletilla striata seedling is required to reach 5mm and the plant height reaches 8cm.
The invention realizes the growth promotion of rhizoma bletillae seedlings by adding Pityrosporum indicum, and the seedlings can be transplanted to the field after 120 days of transplanting domestication.
The invention adopts Pityrosporum indicum added with proper concentration to realize the growth promotion effect on rhizoma bletillae seedlings.
As preferable: the technical proposal of the invention uses 25 to 50 g.L -1 Soaking rhizoma Bletillae seedling in Pyricularia Indici solution for 24 hr or 2×10 8 L. -1 The concentration of the Pityrosporum indicum spore liquid is 10mL per plant, which is favorable for establishing symbiotic relation between Pityrosporum indicum and rhizoma bletillae seedlings, the fresh weight increase multiple and the pseudobulb diameter increase multiple of the cultivated 120d rhizoma bletillae seedlings reach 3.777+/-0.761+/-4.353 +/-0.112 and 1.522+/-0.384-1.627+/-0.208 respectively, the average height of the overground part of the cultivated 120d rhizoma bletillae seedlings reaches 21.10+/-0.69-21.15+/-1.66 cm, the average diameter of the pseudobulb reaches 15.582 +/-1.609-16.348 +/-0.790 mm, the average root number of the single plant reaches 12.6+/-0.7-12.8+/-1.8 root, and the standard of the quality classification of the rhizoma bletillae seedlings is reached by referring to DB 53/T901-2019, and the standard of the first-stage seedlings of the rhizoma bletillae seedlings (the diameter of the pseudobulb is not less than 15mm, the plant height is not less than 12cm, the robust root is complete and no mildew) is at least more than 180d conventional period of the rhizoma bletillae seedlings. The invention lays a good foundation for shortening the production period of the bletilla striata seedlings and improving the quality of the bletilla striata seedlings in the future.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
in the following examples:
the seedling substrate is as follows: model LM-2 Langbo seedling substrate manufactured by LAMBERT PEAT MOSS INC company.
The PDB medium is a conventional PDB medium (containing 200 g.L.) -1 Potato, sucrose 20 g.L -1 )，
PDA medium is conventional PDA medium (containing 200 g.L.) -1 Potato, sucrose 20 g.L -1 Agar 10 g.L -1 )，
The strain of Pityrosporum indicum can be used, for example, in the paper "Pityrosporum indicum induces disease resistance, growth promotion, stress resistance and preliminary study of its mechanism" (university of Zhejiang, shuoshi paper, sun Chao, 2010).
The culture dish is a conventional culture dish.
Example 1 addition of Alternaria alternata to the growth of tissue-cultured seedlings
(1) Sterilization of the transplanting matrix: sterilizing the seedling substrate at high temperature, cooling (namely sterilizing at 121 ℃ for 60min, cooling to room temperature), and sub-packaging in small square basin with caliber of 7 cm; as a seedling substrate after sterilization for standby;
(2) Preparation of Pityrosporum indicum liquid/spore liquid
(1) Preparation of Pityrosporum indicum liquid:
culturing Pityrosporum indicum strain in culture dish containing PDA solid medium at 25+ -2deg.C with photoperiod of 12h.d -1 10-15 days; punching the edge of the cultivated Pityrosporum indicum colony on PDA solid medium with a stainless steel puncher with diameter of 6mm to obtain Pityrosporum indicum block with diameter of 6mm containing vigorous hyphae.
300mL of PDB culture medium is filled in a culture container, 3 Pityrosporum indicum blocks with the diameter of 6mm are added, and the culture container is placed in a shaking table for shaking culture at the culture temperature of 27+/-1 ℃ and at 180rpm for 15-20 d, and the culture is carried out in the dark; centrifuging the bacterial liquid at 6000rpm for 5min, taking mycelium blocks at the lower layer obtained by centrifugation, adding sterilized water according to the feed liquid ratio of 25-50 g/L, and dispersing by a high-speed refiner to prepare bacterial liquid (namely, the bacterial liquid concentration is 25-50 g/L);
(2) preparation of Pityrosporum indicum spore liquid
Culturing Pityrosporum indicum strain in culture dish containing PDA solid medium at 25+ -2deg.C with photoperiod of 12h.d -1 And 10-15 days.
Then 10-15 mL of physiological saline containing 0.5% (volume%) Tween is added into the Pityrosporum indicum cultured in the PDA solid culture dish, and a medicine spoon with a thinner edge is used for scraping the colony surface so as to separate spores from hyphae; filtering with gauze for 20min after ultrasonic separation to remove impurities, calculating spore concentration with a cell counting plate, and diluting to required concentration according to requirements.
(3) Preparation of bletilla striata seedlings: cleaning root agar of rhizoma bletillae tissue culture seedlings with pseudobulb not less than 5mm and plant height not less than 8cm meeting bottle discharge standards, and sucking water on the root surface for later use;
description of the invention: the culture method of the bletilla striata tissue culture seedlings refers to published test of pseudobulb induction in bletilla striata bottle, and the formula of the culture medium is MS+30g.L -1 sucrose+6-BA 1.0mg.L -1 +KT 0.5mg·L -1 +NAA0.2rng·L -1 +agar until obtaining bletilla striata tissue culture seedlings (pseudobulb is more than or equal to 5mm, plant height is more than or equal to 8 cm) which can come out of the bottle, and the number of roots is generally 3-4.
(4) Addition of Pityrosporum indicum:
mode a (addition mode is soaking):
soaking the roots of the bletilla striata seedlings in the step (3) for a period of time (about 24 hours) by using the Pityrosporum indicum liquid or the spore liquid prepared in the step (2), then taking out the Pityrosporum indicum liquid or the spore liquid which is cleaned by clear water and stuck on the surfaces of the roots, and planting the Pityrosporum indicum liquid or the spore liquid in a sterilized seedling substrate;
pityrosporum indicum liquid concentrations are 25, 50 and 100 g.L respectively -1 The method comprises the steps of carrying out a first treatment on the surface of the Spore liquid concentration is 2×10 7 、2×10 8 、2×10 9 L. -1 ；
Mode B (irrigation mode):
directly planting the roots of the bletilla striata seedlings in the step (3) in a sterilized seedling substrate, and irrigating with the Pityrosporum indicum liquid or the spore liquid prepared in the step (2), wherein 10mL of Pityrosporum indicum liquid or the spore liquid is irrigated for each plant;
the concentration of Pityrosporum indicum liquid is 50g.L -1 Spore liquid concentration was 2×10 8 L. -1 。
(5) Transplanting, domesticating and managing bletilla striata seedlings:
and (3) performing the following conventional treatment on the bletilla striata seedlings transplanted into the seedling raising matrix, wherein the conventional treatment is performed on the bletilla striata seedlings obtained in the step (4):
in 15d, leaf surface spraying or watering is required according to the dry and wet degree of the leaves and the matrix; spraying or watering can be carried out by referring to published technical research on germination characteristics of bletilla striata source radix rehmanniae and hardening off of tissue culture seedlings;
after new roots grow out, fertilizer can be applied, and then fertilizer is applied at intervals until the new roots leave the nursery. In the step, fertilizer can be irrigated after the bletilla striata is transplanted to the substrate for 30d, and then fertilizer is irrigated once every 15d until the bletilla striata is transplanted to the nursery, wherein the fertilizer adopts N: p: the mass ratio of K is 20:10:20 The irrigation concentration is 2 liters of water per 1 gram, and the volume of each plant of the irrigation fertilizer is 40 mL/time.
(6) After the bletilla striata seedlings are transplanted for 120d, 15 plants are randomly selected for each group of treatment to measure indexes such as fresh weight, pseudobulb diameter, overground part height and root number, and the increase multiples of the indexes are calculated respectively. The results obtained are shown in Table 1 below.
TABLE 1
The invention takes the cultured bletilla striata tissue culture seedlings as materials, and promotes the growth of the bletilla striata seedlings by screening proper Piropora indica added concentration and mode. The addition concentration of bacterial liquid in Pityrosporum indicum addition mode A is 25-50g.L -1 According to the most suitable method, the fresh weight increase times and the pseudobulb diameter increase times of the bletilla striata tissue culture seedling transplanting domestication 120d reach 3.777+/-0.761-4.353 +/-0.112 and 1.522+/-0.384-1.627+/-0.208 respectively; spore liquid concentration 2×10 in Piropora indicum addition form B 8 L. -1 Irrigating rhizoma bletillae seedlings, wherein the fresh weight increase multiple and the pseudobulb diameter increase multiple of the rhizoma bletillae seedlings reach 4.299 +/-0.263 and 1.714+/-0.089 respectively, and adding the rhizoma bletillae seedlings and bacterial liquid at the concentration of 25-50 g.L -1 The growth promoting effect is equivalent; the diameter of the pseudobulb of the bletilla striata seedling cultivated by the method can reach 15mm, the plant height can reach more than 20cm, and the quality classification standard of the bletilla striata seedling is referred to DB 53/T901-2019, so that the standards of the first-stage seedlings of the bletilla striata seedling out of the nursery are all achieved (the diameter of the pseudobulb is more than or equal to 15mm, the plant height is more than or equal to 12cm, and the seedlings are robust, complete in root hair and free from mildew and rot).
Blank comparative example, growth of tissue-cultured seedlings without added Pityrosporum indicum
Rhizoma bletillae seedlings were immersed in sterile water for 24 hours as in example 1 and transplanted into sterilized seedling substrates, and acclimatization, management and index measurement were performed as in example 1, with the results shown in table 1 above.
The fresh weight increase multiple and the pseudobulb diameter increase multiple of the bletilla striata tissue culture seedling transplanting 120d are 2.351 +/-0.157 and 1.150+/-0.257 respectively, at the moment, the average height of the overground part is 15.88+/-0.53 cm, the average diameter of the pseudobulb reaches 11.800 +/-1.022 mm, the average root number of a single plant reaches 8.6+/-0.7, and the method is obviously lower than that of a treatment group added with Pityrosporum indicum.
Comparative example 1, step (4) of example 1 was modified to perform the addition of Pityrosporum indicum as follows:
directly planting the roots of the bletilla striata seedlings in the step (3) in a sterilized seedling substrate, and burying a 6mm Pityrosporum indicum block in the substrate at the position of 0.5-1 cm away from each root of the bletilla striata seedlings; the remainder was identical to example 1.
The obtained results showed that: the Pityrosporum indicum blocks are buried in the roots of the bletilla striata seedlings, the plant growth is equivalent to that of a blank control, and no obvious growth promoting effect is achieved.
From comparison with comparative example 1, it can be seen that: embedding Pityrosporum indicum blocks at the roots of bletilla seedlings is time-consuming and labor-consuming, and the bacterial liquid soaking or spore liquid pouring is convenient and quick, so that the method is convenient to operate in production; secondly, the components and the nutritional composition of the Langbo seedling substrate are possibly unsuitable for the growth of Pityrosporum indicum, and hyphae are difficult to contact with the roots of rhizoma bletillae, so that the growth promoting effect is not obvious.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (3)
1. The bletilla striata seedling growth promoting method based on Piriosa indicum is characterized by comprising the following steps of:
(1) Sterilizing a substrate for transplanting and domesticating rhizoma bletillae seedlings to obtain a sterilized seedling substrate;
(2) The concentration is 25 to ultra50 g·L -1 Is prepared into Pityrosporum indicum liquid or has concentration of 1-3×10 8 cfu/mLPityrosporum indicum spore liquid;
the preparation method of the Pityrosporum indicum liquid comprises the following steps:
mycelium blocks obtained after the Piriocella Indici is cultured on a PDB culture medium are dispersed by adding water to prepare the mycelium blocks with the concentration of 25-g.L -1 Is a bacterial liquid of (a);
the Pityrosporum indicum spore liquid is:
adding physiological saline into Pityrosporum indicum cultured in PDA culture medium to obtain extract with concentration of 1-3×10 8 cfu/mLSpore liquid;
(3) Any one of the following modes is selected:
method A: cleaning the root agar of the bottle-out bletilla striata seedlings, sucking the water on the root surface, and using 25-g.L -1 Soaking 24h in Pityrosporum indicum liquid, taking out Pityrosporum indicum liquid adhered to root surface, cleaning with clear water, and planting in sterilized seedling substrate;
method B: cleaning root agar of bottle-out rhizoma Bletillae seedling, absorbing water on root surface, directly planting in sterilized seedling substrate, and irrigating with prepared Pityrosporum indicum spore liquid with concentration of 10mL of 2×10 per plant 8 L. -1 Is a Pityrosporum indicum spore solution;
(4) And transplanting the bletilla striata to a substrate, and planting until the bletilla striata is out of the nursery.
2. The method for promoting the growth of bletilla striata seedlings based on Pityrosporum indicum according to claim 1, wherein: in the step (4), fertilizer is irrigated after the bletilla striata is transplanted into the substrate 30d, and then fertilizer is irrigated once every 15d until the bletilla striata is out of the nursery, wherein the fertilizer adopts N: p: k=20: 10:20, watering concentration is 2 liters of water for each 1 gram, and each plant is irrigated with 40 percent mL of fertilizer.
3. The method for promoting the growth of bletilla striata seedlings based on Pityrosporum indicum according to claim 2, wherein: in the step (1), the seedling substrate is sterilized at the high temperature of 121 ℃ for 60 min.
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