CN105706900B - Sterile seeding and seedling raising method for hybrid orchid and Tibet cymbidium hybrid seeds - Google Patents

Abstract

The invention provides a sterile seeding and seedling raising method for hybrid orchid and Tibet sansevieria hybrid seeds, which takes the hybrid orchid Miss Taibei as a female parent and Tibet sansevieria as a male parent, obtains hybrid capsules through artificial pollination, and obtains hybrid progeny groups through the propagation steps of pod pretreatment, seed sowing, seed germination, synchronous propagation and differentiation of rhizomes, strong seedling rooting, seedling hardening, transplanting and the like of mature capsules. The invention has the advantages of high seed germination rate, more seedling formation, short seedling formation time, good seedling quality and the like, the survival rate of transplanting reaches 97.8 percent after 60 days, and a large number of obtained progeny plants lay a material foundation for breeding new excellent species (lines) of hybrid orchid and provide an effective new way for the production of hybrid orchid seedlings.

Description

Sterile seeding and seedling raising method for hybrid orchid and Tibet cymbidium hybrid seeds

Technical Field

The invention relates to a distant hybridization breeding and a method for sterile seeding and breeding of hybrid seeds, in particular to a method for sterile seeding and breeding of hybrid seeds of cymbidium hybridum and cymbidium tibetanum.

Background

The Hybrid Cymbidium (Hybrid Cymbidium) is a new orchid type cultivated by interspecific hybridization of Cymbidium and Cymbidium hybridum, integrates the characteristics of small and exquisite plants, fragrance and elegance of the Cymbidium hybridum, large flowers and pure and beautiful colors of the Cymbidium hybridum and the like, has long flowering period, flower-looking flowers and leaves-free flowers and higher ornamental and economic values, becomes a new favorite in the late years and daily consumption markets, the consumption share increases year by year, and the market potential is huge.

Sansevieria trifasciata (Cymbidium tracynum L.) is a perennial herb of the genus Cymbidium of the family Orchidaceae. The native products of southwest of Guizhou, Sichuan, Yunnan southwest to southeast and southeast of Tibet are distributed, Burma and Thailand are also distributed, and the Burma and the Thailand are widely cultivated at present. The leaves are long and dark green, the flower appearance is rough and spacious, the color is bright, the flower type is neat and the texture is firm, the leaves are not withered, the fragrance is provided, the stress resistance is strong, and the orchid is a famous new orchid in the world.

The interspecific distant hybridization of Cymbidium (Cymbidium) has affinity obstacle, interspecific hybrid embryos are usually poor in development and have no endosperm, hybrid seeds are difficult to germinate under natural conditions and are not easy to grow into seedlings, and a large number of young plants can be obtained in a short period through aseptic seeding and artificial culture. However, the germination difference of different hybrid embryos is large, and the distant hybridization breeding efficiency between orchid species is directly influenced. In recent years, part of intercropping hybridization studies of cymbidium have been reported in China, but the established technology has poor repeatability. Therefore, the deep research on the germination and development characteristics of the interspecific hybrid seeds of different types of cymbidium plants lays a foundation for further developing new cymbidium species cultivation.

In view of the pursuit of consumers for new and excellent varieties of orchid with 'new, odd, special and fragrant', the cultivation of more new varieties of orchid with independent intellectual property rights is a hot topic of research in the plant breeding community.

Disclosure of Invention

The invention aims to solve the technical problem of providing a sterile seeding and seedling raising method for hybrid orchid and Tibet sansevieria hybrid seeds.

The invention is realized by the following steps: a method for aseptically sowing and raising seedlings of hybrid orchid and Tibet cymbidium hybrid seeds comprises the following steps:

(1) artificial pollination: hybridizing by taking Miss taibei as a female parent and Tibet sansevieria as a male parent, taking flowers of 2-4 days after the female parent blooms, removing pollen blocks on a lipflap and a stigma by using sterilized forceps, and taking pollen blocks of flowers of the male parent which are completely opened to be placed in a stamen cavity of the stigma of the female parent; shearing the mature capsule which is not cracked after pollination for 205d, and performing aseptic seeding;

(2) pod pretreatment and seed sowing:

pretreating mature capsules, and then sowing seeds of the mature capsules in a germination culture medium; the germination culture medium is 1/2MS +6-BA1.0mg/L + NAA0.5mg/L + sucrose 20g/L + active carbon 1.0g/L + agar 5.8 g/L;

(3) seed germination: inoculating seeds into the germination culture medium for culture, wherein the culture temperature is 25 +/-2 ℃, the illumination intensity is 1200Lux +/-100, and the illumination time is 12 h/d;

(4) proliferation and differentiation culture of rhizome: selecting rhizomes formed by germination of seeds with similar growth states, cutting the rhizomes into segments with the length of 0.6-0.8 cm under the aseptic condition, inoculating the segments into proliferation and differentiation culture media of 1/2MS +6-BA2.0mg/L + NAA1.0mg/L + banana puree 80g/L + sucrose 20g/L + active carbon 1.5g/L + agar 5.8g/L, and culturing at the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500Lux +/-100 and the illumination time of 12 h/d;

(5) strong seedling and rooting culture: carefully cutting off 2.5-3.5 cm seedlings formed by differentiating rootstocks, cutting off old roots after sorting, only leaving 1-2 short and tender new roots, and inoculating the seedlings into a rooting and seedling strengthening culture medium comprising MS +6-BA1.0mg/L + NAA2.0mg/L + sucrose 25g/L + banana mud 100g/L + active carbon 2.5g/L + agar 5.8g/L, and culturing at the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500Lux +/-100 and the illumination time of 12 h/d;

(6) hardening and transplanting seedlings: when the rooted seedlings grow to 6.0-7.0 cm, 3-4 blades and 3-4 roots, moving the bottle seedlings from the culture room to a greenhouse, hardening the seedlings, and continuing to grow for 30 days, wherein the shading rate of the greenhouse is 60%, the temperature is kept at 25 +/-2 ℃, and the air humidity is kept at 60% -70%;

3-5 days before planting, opening a bottle cover to adapt to the external environment, taking out seedlings, cleaning a root culture medium, soaking for 3min by using 1000-time diluted solution of 50% carbendazim wettable powder, naturally airing in a shade, planting in a fermented matrix, and sprinkling irrigation on plants and plant materials by using hymexazol solution; watering after 2d, spraying water and 0.1% carbendazim bactericide on the leaf surface according to a preset period.

Further, in the step (2), the specific method for pretreating the mature capsule is as follows:

washing mature capsule under running water for 3min, brushing epidermis with toothbrush stained with detergent, removing stigma, residual withered valve and epidermoid lesion with scalpel, and wiping with 75% alcohol; soaking in 1% NaClO solution for 15min, shaking with ultrasonic cleaner for 5-10 min, transferring to a hyperstatic workbench, taking out capsule with sterilized forceps, and cleaning with sterile water for 2 times; the capsule is dipped in 75% alcohol and then ignited to sterilize for 10 s.

Further, in the step (2), the specific operation method for sowing the seeds of the mature capsule in the germination medium is as follows:

pinching the stem of the mature capsule with fingers to enable the capsule to face the air port and the hand to be behind, holding the disinfected scalpel with the other hand to cut the fruit pod open the fruit peel, exposing the seeds, directly clamping the seeds with tweezers, and uniformly spreading the seeds on the surface of a germination culture medium; or transferring the sterilized seeds into a culture bottle with sterile water, shaking up, and sucking 1ml of shaking-up suspension by using a sterile pipette and uniformly dripping the suspension onto a germination culture medium surface.

Further, the matrix comprises a mixture of 2: 1 bark and crushed stone.

The invention has the advantages that: the seed germination is fast, the germination rate is high, the time for the rhizome to differentiate into seedlings is short, and a large number of test-tube seedlings can be quickly obtained; the seedling quality is high, the survival rate of the seedling transplantation reaches 97.8 percent, and an effective new way is provided for the large-scale production of the hybrid orchid seedlings.

Detailed Description

The invention adopts hybrid orchid Miss Taibei as a female parent and wild domesticated Tibet sansevieria as a male parent to perform interspecific hybrid seed sterile seeding and seedling propagation, and the specific steps are as follows:

(1) artificial pollination: hybridizing by taking Miss taibei as a female parent and Tibet sansevieria as a male parent, taking flowers 2-4 d after the female parent blooms when the female parent and the Tibet sansevieria are simultaneously bloomed in the beginning of 12 months, removing pollen blocks on lipflaps and stigma by using sterilized tweezers, and placing pollen blocks of flowers completely opened by the male parent in a stigma cavity of the female parent; after pollination, a label is hung on the female parent plant, and the pollination date, the hybrid male parent and the number of the pollinated flowers are marked on the label;

(2) pod pretreatment and seed sowing: shearing mature capsules which are not cracked after pollination for 205d, carefully brushing epidermis by dipping the surface of the capsules with detergent, removing chapiters, residual withered petals and insect spots of the epidermal disease by using an operating knife, and wiping the capsules by using 75% alcohol; soaking in 1% NaClO solution for 15min, shaking with ultrasonic cleaner for 5-10 min, transferring to a hyperstatic workbench, taking out capsule with sterilized forceps, and cleaning with sterile water for 2 times; igniting and disinfecting the clamped capsule on an alcohol lamp for 10s after the clamped capsule is dipped in 75% alcohol, pinching a fruit stem with fingers to enable the capsule to face an air port and to be behind the hand, horizontally cutting the capsule by using another scalpel which is disinfected by holding the hand to open fruit peel, exposing seeds, and directly clamping the seeds by using tweezers and uniformly scattering the seeds on the surface of a germination culture medium; or transferring the sterilized seeds into a culture bottle with a small amount of sterile water, shaking up, sucking 1ml of shaking up suspension liquid by using a sterile straw, dropping the suspension liquid onto a germination culture base surface, and uniformly distributing the suspension liquid; the germination culture medium comprises: 1/2MS +6-BA1.0mg/L + NAA0.5mg/L + sucrose 20g/L + active carbon 1.0g/L + agar 5.8g/L, and the pH value is 5.6.

(3) Seed germination: inoculating the seeds into a germination culture medium for culture, wherein the culture temperature is 25 +/-2 ℃, the illumination intensity is 1200Lux +/-100, and the illumination time is 12 h/d; culturing for 35 days, swelling to form white spherical embryo in about 60 days, continuously culturing to form white protocorm, converting into green protocorm under light, and culturing for 100 days to obtain germinated protocorm-like body to form white-light green rhizome;

(4) proliferation and differentiation of rhizomes: selecting rhizomes formed by germination of seeds with similar growth states, cutting the rhizomes into segments with the length of 0.6-0.8 cm under the aseptic condition, inoculating the segments to proliferation and differentiation culture media of 1/2MS +6-BA2.0mg/L + NAA1.0mg/L + banana puree 80g/L + sucrose 20g/L + active carbon 1.5g/L + agar 5.8g/L, and culturing at the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500Lux +/-100 and the illumination time of 12 h/d; the multiplication coefficient of the rhizome reaches 5.16 after 70 days of culture, and strong seedlings with roots can be obtained after 125 days of culture, and the seedling rate reaches 90.8%.

(5) Strengthening and rooting seedlings: 2.5-3.5 cm of base parts formed by differentiating rhizome are full, seedlings with leaf tips opening and heart leaves exposing are carefully cut off, old roots are cut off after sorting, only 1-2 short and tender new roots are left, and the seedlings are inoculated on a rooting and strong seedling culture medium of MS +6-BA1.0mg/L + NAA2.0mg/L + sucrose 25g/L + banana mud 100g/L + active carbon 2.5g/L + agar 5.8g/L for culture, the culture temperature is 25 +/-2 ℃, the illumination intensity is 1500Lux +/-100, and the illumination time is 12 h/d; the rooting rate of the seedlings can reach 98.4 percent after about 90 days of culture;

(6) hardening and transplanting seedlings: when the rooted seedlings in the bottle grow to 6.0-7.0 cm, 3-4 leaves and 3-4 roots, moving the bottle seedlings from the culture room to a greenhouse, hardening the seedlings, continuing growing for 30 days, keeping the shading rate of the greenhouse for hardening the seedlings at 60%, keeping the temperature at 25-28 ℃ and keeping the air humidity at 60% -70%; 3-5 d before the seedlings are taken out of the bottle and planted, opening the bottle cover to enable the seedlings in the bottle to adapt to the external environment; taking out plantlets, carefully cleaning root culture medium, soaking in 1000 times of 50% carbendazim wettable powder for 3min, naturally drying in the shade, planting in fermented matrix of bark and gravel (mass ratio of 2: 1), and sprinkling plant and plant material with hymexazol solution; 2d, watering thoroughly, spraying water to leaf surfaces and bactericide such as 0.1% carbendazim, and performing conventional cultivation management; after a series of careful management, the survival rate of test-tube plantlets transplanted for 60 days reaches 97.8 percent.

The invention has the following advantages:

(1) the invention utilizes the culture medium (1/2MS +6-BA2.0mg/L + NAA1.0mg/L + banana mud 80g/L + cane sugar 20g/L + active carbon 1.5g/L + agar 5.8g/L) with the same formula to realize synchronous proliferation and differentiation of the rhizome, and reduce the bottle transferring link from proliferation to differentiation, thereby not only improving the seedling rate and reducing the pollution rate, but also greatly reducing the workload, saving the economic cost and shortening the seedling time by about 30 days.

(2) The invention adds active carbon in the whole culture process, which can adsorb partial nutrient substances in the culture medium and waste generated in the metabolic process, and promote the morphogenesis and organ formation of seeds.

(3) The invention firstly smelts the seedlings in the greenhouse to continue growing for 30d before transplanting, is beneficial to forming bottle seedling false bulbs and thickening blades, and lays a solid foundation for improving the transplanting survival rate in the later period.

In a word, the method has the advantages of fast seed germination, high germination rate, short time for differentiating the rootstock into the seedlings and capability of fast obtaining a large number of test-tube seedlings; the quality of the seedlings is high, and the survival rate of the seedlings transplanted reaches 97.8 percent. Provides an effective new way for the large-scale production of the hybrid orchid seedlings.

The invention takes the Miss Taibei of the cultivated species of hybrid orchid as a female parent and takes the wild Tibet cymbidium as a male parent to develop hybrid breeding, solves the key technologies of sterile seeding and seedling raising of hybrid seeds, seed germination rate improvement, root multiplication and differentiation rate improvement, rooting and strong seedling culture, hardening and transplanting and the like, obtains a large number of hybrid progeny plants, not only lays a material foundation for breeding excellent new varieties with new flower colors, flower types and strong resistance, but also provides an effective new way for the production of hybrid orchid seedlings, promotes the sustainable development of industrialization of the hybrid orchid, and can further protect wild orchid resources.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Claims (2)

1. A method for aseptically sowing and raising seedlings of hybrid orchid and Tibet cymbidium hybrid seeds is characterized in that: the method comprises the following steps:

(1) artificial pollination: hybridizing by taking Miss taibei as a female parent and Tibet sansevieria as a male parent, taking flowers of 2-4 days after the female parent blooms, removing pollen blocks on a lipflap and a stigma by using sterilized forceps, and taking pollen blocks of flowers of the male parent which are completely opened to be placed in a stamen cavity of the stigma of the female parent; shearing the mature capsule which is not cracked after pollination for 205d, and performing aseptic seeding;

(2) pod pretreatment and seed sowing:

pretreating mature capsules, and then sowing seeds of the mature capsules in a germination culture medium; the germination culture medium is 1/2MS +6-BA1.0mg/L + NAA0.5mg/L + sucrose 20g/L + active carbon 1.0g/L + agar 5.8 g/L;

the specific method for pretreating mature capsules comprises the following steps:

washing mature capsule under running water for 3min, brushing epidermis with toothbrush stained with detergent, removing stigma, residual withered valve and epidermoid lesion with scalpel, and wiping with 75% alcohol; soaking in 1% NaClO solution for 15min, shaking with an ultrasonic cleaner for 5-10 min, transferring to an ultra-clean bench, taking out capsule with sterilized forceps, and cleaning with sterile water for 2 times; igniting and disinfecting the capsule on an alcohol lamp for 10s after dipping the capsule in 75% alcohol;

(3) seed germination: inoculating seeds into the germination culture medium for culture, wherein the culture temperature is 25 +/-2 ℃, the illumination intensity is 1200Lux +/-100, and the illumination time is 12 h/d;

(4) proliferation and differentiation culture of rhizome: selecting rhizomes formed by germinating seeds with similar growth states, cutting the rhizomes into segments with the length of 0.6-0.8 cm under the aseptic condition, inoculating the segments into proliferation and differentiation culture media of 1/2MS +6-BA2.0mg/L + NAA1.0mg/L + banana puree 80g/L + sucrose 20g/L + active carbon 1.5g/L + agar 5.8g/L, and culturing at the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500Lux +/-100 and the illumination time of 12 h/d;

(5) strong seedling and rooting culture: carefully cutting off 2.5-3.5 cm seedlings formed by differentiating rootstocks, cutting off old roots after sorting, only leaving 1-2 short and tender new roots, and inoculating the seedlings into a rooting and seedling strengthening culture medium comprising MS +6-BA1.0mg/L + NAA2.0mg/L + sucrose 25g/L + banana puree 100g/L + active carbon 2.5g/L + agar 5.8g/L, and culturing at the culture temperature of 25 +/-2 ℃, the illumination intensity of 1500Lux +/-100 and the illumination time of 12 h/d;

(6) hardening and transplanting seedlings: when the rooted seedlings grow to 6.0-7.0 cm, 3-4 blades and 3-4 roots, moving the bottle seedlings from the culture room to a greenhouse, hardening the seedlings, and continuing to grow for 30 days, wherein the shading rate of the greenhouse is 60%, the temperature is kept at 25 +/-2 ℃, and the air humidity is kept at 60% -70%;

3-5 days before planting, opening a bottle cover, taking out seedlings, cleaning a root culture medium, soaking the seedlings in 1000-time diluted solution of 50% carbendazim wettable powder for 3min, naturally drying the seedlings in the shade, planting the seedlings in a fermented matrix, and sprinkling the plants and the plant materials with hymexazol solution; 2d, watering thoroughly, and spraying water and 0.1% carbendazim bactericide on the leaf surfaces according to a preset period; the matrix comprises bark and gravel in a mass ratio of 2: 1.

2. The method for aseptically sowing and raising seedlings of hybrid orchid and sansevieria tibetana hybrid seeds as claimed in claim 1, which comprises the following steps: in the step (2), the specific operation method for sowing the seeds of the mature capsules in the germination culture medium is as follows:

pinching the stem of the mature capsule with fingers to enable the capsule to face the air port and the hand to be behind, holding the disinfected scalpel with the other hand to cut the fruit pod open the fruit peel, exposing the seeds, directly clamping the seeds with tweezers, and uniformly spreading the seeds on the surface of a germination culture medium; or transferring the sterilized seeds into a culture bottle with sterile water, shaking up, and sucking 1ml of shaking-up suspension by using a sterile pipette and uniformly dripping the suspension onto a germination culture medium surface.