CN110558229A - Seedling hardening method for remarkably improving transplanting survival rate of cassava tissue culture seedlings - Google Patents

Abstract

the invention discloses a seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings, which comprises the following steps: maintaining mature stem sections of the cassava leaves, and subculturing the cassava leaves in a seedling culture medium for 33-37 days to obtain tissue culture seedlings; taking out the tissue culture seedlings, cleaning, clamping stems at positions 0.5cm higher than root systems of the tissue culture seedlings by using planting cotton, and transplanting the tissue culture seedlings to peat soil pits for filling soil and planting; covering the potted plant with a freshness protection bag, shading the whole process, taking down the bag after preserving soil moisture for a plurality of days, and performing the planting process and the planting in a room 6-12 hours after the planting; removing the bags, culturing for 12-16 days, transferring cassava plants to formula soil, taking down the planting cotton, culturing for 14-21 days, and hardening seedlings, wherein the formula soil is 4: 1: 1: 1: 1 peat soil, humus soil, vermiculite, perlite and laterite. The method comprises the steps of firstly carrying out subculture controllable culture on mature stem sections of cassava leaves, then planting in peat soil by using planting cotton, and then transferring into soil with a specific formula to cultivate strong seedlings with good quality, wherein the survival rate is remarkably improved from 50% to 95%.

Description

seedling hardening method for remarkably improving transplanting survival rate of cassava tissue culture seedlings

Technical Field

The invention relates to the field of agricultural technology and tissue culture, in particular to a seedling hardening method capable of remarkably improving the transplanting survival rate of cassava tissue culture seedlings.

Background

Cassava is a plant belonging to the genus cassava of the family euphorbiaceae, is drought-resistant and barren-resistant, is an important starch crop, is widely planted in multiple countries and regions such as africa, america and asia, is one of three potato crops in the world, and is known as an underground granary and a starch king. The cassava has wide application, can be used as grains such as cassava rice, and also can be used as important industrial raw materials such as cassava root starch, dry flake modified starch, alcohol, sorbitol and the like, and the products are all important raw materials in the industries such as food, medicine, textile, paper making and the like. However, the average yield of cassava is low, and the technical researches on breeding, popularization and high-yield cultivation of high-yield stable-yield high-starch improved varieties must be enhanced to develop the large-scale cassava planting industry.

Cassava is a vegetative propagation crop, and the traditional method adopts the mature lignified seed stem of the cassava as the main propagation mode. In recent years, the tissue culture rapid propagation technology and genetic engineering breeding of cassava are receiving increasing attention. The tissue culture preservation and propagation of improved cassava seed seedlings are the modern breeding trend, and the tissue culture seedling hardening-off technology is the key step for the promotion of improved cassava seeds. However, the survival rate of the cassava tissue culture seedlings in field transplantation is low, and the growth potential is poor, so that the cassava tissue culture seedlings become a restriction factor of modern breeding. The hardening and transplanting is to move the cassava tissue culture seedlings from a culture medium to outdoor soil, and the process has the transformation from heterotrophic culture to autotrophic culture and needs gradual exercise adaptation. In the prior art, the roots of the cassava tissue culture seedlings are long and thin and are easy to coil, the roots are easy to break in the transplanting process, and the stems are easy to fall down; the plant grows slowly, the transplanting period is long, the transplanting survival rate is not high, and the survival rate is only 50%. The establishment of perfect tissue culture seedling breeding and transplanting technology is the technical guarantee of molecular breeding and is a key link of the industrial production of cassava seedlings.

Disclosure of Invention

In view of the above, the invention provides a seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings, and solves the problem of low survival rate caused by root system damage, stalk lodging, poor soil adaptability and the like in the existing transplanting process of the cassava tissue culture seedlings.

The invention provides a seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings, which comprises the following steps:

(1) And (3) breeding: cutting the cassava tissue culture seedlings growing for 50-60 days into 1.5-2.5 cm stem sections, reserving leaves on the stem sections, and subculturing in a seedling culture medium for 33-37 days to obtain cassava tissue culture seedlings suitable for transplanting;

(2) Planting: after the cassava transplanting tissue culture seedlings are taken out, removing culture medium attached to roots, fixing planting cotton with the thickness of 1cm above the root systems by about 0.5cm, putting the planting cotton into a pot and presetting pits, and then transplanting the tissue culture seedlings fixed with the customized cotton into the pits for burying;

(3) Preserving soil moisture: immediately covering the cassava pot with a preservation bag after field planting, shading the whole process, opening one corner of the preservation bag after preserving soil moisture for 4 days, and completely taking down the bag after 2 days;

(4) Soil replacement: and after the bags are removed, continuously culturing in the original peat soil for 12-16 days, then taking down the planting cotton, transferring the cassava plants into the formula soil, culturing for 14-21 days, and completing hardening and transplanting. The formula soil comprises the following components in percentage by volume of 4: 1: 1: 1: 1 peat soil, humus soil, vermiculite, perlite and laterite.

Preferably, in the step (1), the seedling culture medium is an MS minimal medium containing 0.1mg/L paclobutrazol.

Preferably, in the step (1), the average seedling height of the cassava tissue culture seedlings is 5-8 cm, the root length is 1.5-5 cm, and 3-7 seedlings are rooted.

preferably, in the step (2), before transplanting, the peat soil is watered with water permeable boiling water for scalding soil for disinfection and sterilization or is sterilized by an oven at 120 ℃ for 1h, and the temperature is naturally reduced to room temperature for standby.

Preferably, in the step (2), the transplanting and field planting process is carried out indoors, the transplanting and field planting process is carried out only indoors for 6-12 hours, the indoor temperature is 24-28 ℃, the indoor humidity is 70-80%, and the rest seedling hardening process can be carried out outdoors.

By adopting the method provided by the invention, the mature stem section of the cassava reserved leaf is subjected to subculture controllable culture to obtain a material which is short and strong in root system and suitable for transplanting; then fixedly planting the seedlings in peat soil, strengthening the fixation of the seedlings and maintaining stable growth; and transferring the cassava leaves into specific formula soil to create suitable environments for different growth stages, gradually enhancing the adaptability of roots to the soil and finally obtaining cassava plants with good growth vigor. Under the technology, the seedling hardening survival rate of the cassava reaches up to 95 percent, and the survival rate of the cassava after transplanting to a field reaches 100 percent.

Furthermore, the mature stem section with the reserved leaves is adopted for subculture, the chlorophyll content of the leaves is improved and the aging process of the leaves is delayed under a seedling culture medium, so that a photosynthetic product is provided for the germination of axillary buds on the stem section, and the method has important significance for accelerating the early growth of tissue culture seedlings.

Furthermore, pp333 is added into the MS culture medium, on one hand, the green return of the leaves is promoted, the stress resistance of the plants is increased, the stomatal resistance of the leaves is improved, and the transpiration effect of the leaves is reduced, so that the adaptability of the transplanted seedling leaves to the environment is enhanced; on the other hand, the method is beneficial to forming a short and thick root system, improving the respiratory strength of the root system and reducing the damage of the root during transplanting. The tissue culture seedling obtained by the seedling culture medium has strong and developed root system and strong and flourishing leaf color.

Furthermore, the cassava is fixed by planting cotton, so that the cassava tissue culture seedlings are beneficial to directional growth, wind prevention and lodging resistance are realized, and the stable growth of root systems is facilitated. In addition, the transplanting process is carried out indoors, the seedlings are placed indoors for 6-12 hours after being transplanted, the temperature is required to be 24-28 ℃, the indoor humidity is 70% -80%, the rest seedling hardening processes can be carried out outdoors, the seedling hardening process is not limited to a greenhouse, the seedling hardening cost is greatly reduced, and the survival rate of the tissue culture seedlings of the cassava is improved to 95%.

Furthermore, peat soil is used for field planting in the initial stage, and the soil is loose and breathable and is beneficial to the relaxation of the root system of the tissue culture seedlings; the formulated soil is adopted in the later period, the nutrient is sufficient, and the root strengthening and water retention effects are strong. The two soil matching designs can create suitable environments for different growth stages, gradually enhance the adaptability of roots to soil, and realize the effects of high survival rate of cassava tissue culture seedlings and good seedling quality.

Drawings

FIG. 1 is a schematic diagram of the breeding of tissue culture seedlings in step (1) of the present invention, wherein a and b are a full plant map and a root system map of tissue culture seedlings in MS culture medium, and c and d are a full plant map and a root system map of tissue culture seedlings in seedling culture medium (with pp333 added);

FIG. 2 is a schematic diagram of the field planting process of the tissue culture seedling in step (2) of the present invention;

FIG. 3 is a schematic view of the soil moisture conservation of the tissue culture seedling in step (3) of the present invention;

fig. 4 is a schematic diagram of the result of the hardening-seedling transplanting of the cassava tissue culture seedlings (the left diagram is a schematic diagram of the result of the hardening-seedling transplanting of the cassava tissue culture seedlings in the first comparative example, and the right diagram is a schematic diagram of the result of the hardening-seedling transplanting of the cassava tissue culture seedlings in the first embodiment).

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

The MS basic culture medium (product number is HB8469) is purchased from Haibo biotechnology limited, and peat soil, humus soil, vermiculite, perlite and laterite can be purchased and obtained.

the first embodiment is as follows: a seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings comprises the following steps: (1) and (3) breeding: cutting the cassava tissue culture seedlings growing for 50-60 days into stem sections of test-tube seedlings of 1.5-2.5 cm, reserving leaves on the stem sections, and subculturing the stem sections in a seedling culture medium (MS basic culture medium +0.1mg/L paclobutrazol pp333) for 33-37 days to obtain the cassava tissue culture seedlings suitable for transplanting. The tissue culture seedlings have the average height of 5.24cm, good growth vigor, dark green leaves, thick and strong stems, short and thick roots, the average root length of 2.7cm and the average root division of 5.45.

(2) Planting: taking out the cassava tissue culture seedling in the step (1) from a tissue culture bottle, and cleaning a culture medium without damaging a root system. Then, cylindrical planting cotton with the diameter of 4.5cm and the thickness of 1cm is used for clamping the root of the cassava tissue culture seedling stem at the position 0.5cm higher than the root hair. Digging a round pit with the depth of 3.5cm and the size suitable for the fixed planting cotton, placing the cassava tissue culture seedling fixed with the fixed planting cotton at the pit, filling soil to be as high as the surrounding soil, paying attention not to break roots, and performing the planting process as shown in figure 2. The peat soil is poured thoroughly with boiling water in advance, or sterilized in an oven at 120 ℃ for 1 hour, subjected to soil scalding sterilization, and naturally cooled to room temperature for later use. And ensuring sufficient water content of peat soil during transplanting, and uniformly stirring. The transplanting and field planting process is carried out indoors, the transplanting and field planting process is carried out only after the transplanting and field planting process is carried out indoors for 6-12 hours, the temperature is 24-28 ℃, the humidity is 70% -80%, and the rest seedling hardening process can be carried out outdoors.

(3) Preserving soil moisture: immediately covering the transplanted seedlings with freshness protection bags after field planting, supporting the seedlings with wood rods, placing the seedlings in a sand box under a sunshade as shown in figure 3, shading the seedlings in the whole process, preserving soil moisture for 4 days without watering, lifting one corner of the freshness protection bag to enable the freshness protection bag to be communicated with external air, and gradually adapting to the conversion from autotrophy to heterotropy. After 2 days, the freshness protection package is completely taken down, and observation is carried out after 3 hours. If the transplanted cassava seedlings have wilting signs, 50ml of water can be poured, and the freshness protection bags are covered again for seedling revival.

(4) soil replacement: pouring water once every two days after bag removal, continuously culturing for 12-16 days, transferring the cassava transplanting seedlings into formula soil, taking down the planting cotton, continuously culturing for 14-21 days to finish acclimatization and transplanting, and observing whether the cassava transplanting seedlings survive and calculating the survival rate as shown in the right side of figure 4, wherein the formula soil is a mixed matrix of peat soil, humus soil, vermiculite, perlite and laterite (the volume ratio is 4: 1: 1: 1).

Example two: the difference from the first embodiment is that the concentration of paclobutrazol in the breeding step (1) is MS, MS +0.05mg/L, MS +0.15mg/L, MS +0.2mg/L, MS +0.25mg/L, MS +0.3mg/L respectively, and the result is shown in table 1, in the concentration range of 0-0.25 mg/L, along with the increase of PP333 concentration, the leaves become thicker, the leaf color deepens, the plant height obviously shortens, the root system becomes thicker and stronger, and the root number obviously increases; when the concentration of the PP333 is in the range of 0.25 mg/L-0.35 mg/L, the leaves are dark green, the leaves are thick, but some leaves can be curled and shrunk. The result shows that the tissue culture seedling grows strongly with the increase of the concentration of the PP333, but the health state of the tissue culture seedling is influenced by the over-high concentration. It can be seen from the table that the addition of PP333 with a concentration of 0.1mg/L can most effectively improve the differentiation rate of callus and the rate of green seedling formation, as shown in fig. 1, if the tissue culture seedling does not produce bulk callus and the rooting is successful, it indicates that the callus tissue at the root is successfully differentiated into root system, on the contrary, it is failed, if the tissue culture seedling grows new leaves and the original leaves are not faded and yellowed, it indicates that the tissue culture seedling forms green seedling, and the callus differentiation rate and the green seedling formation rate are counted by counting 20 experimental plants. Therefore, the preferable selected tissue culture seedlings for transplanting are 5-8 cm high on average, good in growth vigor, thick and green in leaves, thick and strong in stems, 1.5-5 cm long in root system and 3-7 in root division.

TABLE 1 Effect of different concentrations of paclobutrazol on cassava tissue culture seedlings

Example three: the difference from the first embodiment is that the thicknesses of the planted cotton in the step (2) are respectively 3cm, 2cm, 1.5cm, 0.75cm and 0cm, and the experimental results show that the planted cotton is too thick (more than 1.5 cm), so that the contact part of the cassava seedling and the planted cotton is rotten, leaves are wilted, and the planted cotton finally dies; the field planting cotton is too thin (less than 0.75 cm), cannot play a role in fixing, and tissue culture seedlings are easy to fall down, leaves are wilted, stems are bent, roots are not firm, the seedling revival period is delayed, and even death is caused; the fixed planting cotton is 1cm, the fixing effect is good, the leaf is upright, the root is stable to grow, the seedling revival period is greatly shortened, and the fixed planting cotton has strong adaptability and stress resistance to the environment.

TABLE 2 influence of different planting cotton thickness on the transplantation of tissue culture seedlings of cassava

example four: the difference from the first embodiment is that the components and the proportion of the soil used for field planting in the step (2) and soil replacement in the step (3) are different, 20 plants in each group are used as an experimental group, the experimental result is shown in table 3, peat soil is selected for transplanting and field planting in the step (2), and the volume ratio of the selected peat soil in the step (3) is 4: 1: 1: 1: 1, peat soil: humus soil: perlite: vermiculite: the survival rate of the red soil exercising seedlings reaches 95 percent. The peat soil is selected at the initial stage to loosen and ventilate the soil, so that the relaxation of the root system of the tissue culture seedling is facilitated, the damage of the root system is effectively avoided, and the formula soil is adopted at the later stage, so that the peat soil is sufficient in nutrients and has strong root strengthening and water retention effects. The two soil matching designs can create suitable environments for different growth stages, gradually enhance the adaptability of roots to soil, and realize the effects of high survival rate of cassava tissue culture seedlings and good seedling quality.

TABLE 3 influence of different soils on the survival rate of tissue culture seedling transplantation

Comparative examples one to two: according to the prior art (research on cassava test-tube seedling hardening and transplanting technology, Guangdong agricultural science 2011, 11 th stage), in a first comparative example, the tissue culture seedlings in the step (1) are flushed with tap water to remove a culture medium, a direct transplanting mode is adopted, a preservative film is covered on a transplanting container to keep humidity, the container is uncovered after 7 days, and the seedlings are hardened after continuous culture for 30 days, as shown in the left side of FIG. 4; and (2) cleaning the culture medium of the tissue culture seedlings, putting the tissue culture seedlings in a tissue culture bottle containing tap water in a water culture hardening and transplanting mode, covering the tissue culture bottles without screwing, removing the cover after 3 days, transplanting the tissue culture seedlings into soil after culturing for 4 days, and finishing hardening the seedlings after continuously culturing for 30 days.

TABLE 4 influence of different seedling hardening and transplanting modes on the transplanting survival rate of tissue culture seedlings of cassava

in conclusion, by adopting the hardening-seedling method for improving the transplanting survival rate of the tissue culture seedlings of the cassava provided by the invention, the mature stem sections of the leaves of the cassava are reserved for subculture controllable culture, then the seedlings are planted in peat soil and transferred into soil with a specific formula to produce strong seedlings, the hardening-seedling survival rate is up to 95%, and the survival rate of the seedlings transplanted to the field is 100%.

The pp333 is added into the MS culture medium, so that on one hand, the greening of the leaves is promoted, the stress resistance of the plants is increased, the stomatal resistance of the leaves is improved, the transpiration of the leaves is reduced, and the adaptability of the transplanted seedling leaves to the environment is enhanced; on the other hand, the method is beneficial to forming a short and thick root system, improving the respiratory strength of the root system and reducing the damage of the root during transplanting. The tissue culture seedling obtained by the seedling culture medium has strong and developed root system and strong and flourishing leaf color.

The fixation of the field planting cotton is beneficial to the directional growth of the tissue culture seedlings of the cassava, the wind prevention and lodging resistance are realized, the stable growth of root systems is facilitated, the moisture preservation can be effectively matched, the transpiration effect of the seedlings is inhibited, and the resistance of the seedlings is enhanced.

Peat soil is selected for planting in the initial stage, the soil is loose and breathable, the relaxation of the root system of the tissue culture seedlings is facilitated, and the formula soil is adopted in the later stage, so that the nutrient is sufficient, and the effects of root strengthening and water retention are strong. The two kinds of soil are matched to create suitable environments for different growth stages, the adaptability of roots to the soil is gradually enhanced, and the effects of high survival rate of cassava tissue culture seedlings and good seedling quality are achieved.

In addition, the transplanting and field planting process is carried out indoors, the cassava tissue culture seedling survival rate is improved to 95%, the transplanting and field planting process can be carried out indoors only for 6-12 hours after the transplanting and field planting, the temperature is required to be 24-28 ℃, the indoor humidity is 70% -80%, the rest seedling hardening processes can be carried out outdoors without being limited to a greenhouse, the seedling hardening cost is greatly reduced, and the survival rate of the cassava tissue culture seedlings is improved to 95%.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (6)

1. a seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings is characterized by comprising the following steps:

(1) And (3) breeding: cutting the cassava tissue culture seedlings growing for 50-60 days into 1.5-2.5 cm stem sections, reserving leaves on the stem sections, subculturing on a seedling culture medium, and culturing for 33-37 days to obtain transplanted tissue culture seedlings;

(2) Planting: taking out the transplanted tissue culture seedlings, cleaning, fixing the field planting cotton at a position 0.5cm higher than the root systems of the transplanted tissue culture seedlings, transplanting the seedlings into peat soil pits in a pot, filling soil and field planting to obtain cassava pot plants;

(3) preserving soil moisture: covering the cassava pot with a freshness protection bag, shading the whole process, lifting a corner of the bag after preserving soil moisture for 4 days, and completely taking down the freshness protection bag after 2 days;

(4) Soil replacement: removing the bag, culturing in original peat soil for 12-16 days, taking down the planting cotton, transferring the cassava plant to formula soil, culturing for 14-21 days, and then completing hardening seedling and transplanting, wherein the formula soil comprises the following components in a volume ratio of 4: 1: 1: 1: 1 peat soil, humus soil, vermiculite, perlite and laterite.

2. The seedling hardening method for remarkably improving the transplanting survival rate of the cassava tissue culture seedlings according to claim 1, wherein in the step (1), the seedling culture medium is an MS minimal medium containing 0.1mg/L paclobutrazol.

3. The seedling hardening method for remarkably improving the transplanting survival rate of the tissue culture seedlings of cassava according to claim 1, wherein in the step (1), the average seedling height of the tissue culture seedlings of cassava is 5-8 cm, the root length is 1.5-5 cm, and 3-7 seedlings are rooted.

4. The seedling hardening method for remarkably improving the transplanting survival rate of the tissue culture seedlings of cassava according to claim 1, wherein in the step (2), before transplanting, the peat soil is watered thoroughly with boiling water to perform soil scalding sterilization or is sterilized by an oven at 120 ℃ for 1h, and then is naturally cooled to room temperature for later use.

5. A seedling hardening method for remarkably improving the transplanting survival rate of cassava tissue culture seedlings according to claim 1, wherein in the step (2) and the step (3), the field planting process and the field planting are carried out indoors for 6-12 hours, the indoor temperature is 24-28 ℃, and the humidity is 70-80%.

6. The seedling hardening method for remarkably improving the transplanting survival rate of the cassava tissue culture seedlings according to claim 1, wherein in the step (2), the planted cotton is 4.5cm in diameter and 1cm in thickness.