A method for forming regenerated plants through direct somatic embryogenesis of konjac bulbil

Abstract

The invention provides a method for forming a regenerated plant by a direct somatic embryogenesis pathway of a pearly konjac, collecting the germination of unopened spathe flames of a pearly konjac class yellow konjac, using tender female inflorescences as explants, screening and optimizing the female inflorescences of the pearly konjac to induce direct somatic embryogenesis, maturation and plant regeneration conditions, and preliminarily establishing an efficient plant regeneration system. Under suitable temperature, light and culture conditions, the somatic embryo directly germinates into seedlings and transplants well. The invention provides a theoretical basis and technical methods for the in vitro preservation of germplasm resources of pearly konjac in Hainan hot zone, rapid breeding of seedlings and large-scale promotion, and the construction of a genetic transformation receptor system regeneration system of pearly konjac, which is of great significance to the sustainable development of the pearly konjac industry in Hainan hot zone.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and relates to a method for forming regenerated plants through direct somatic embryogenesis of bulbil konjac. Background Art

[0002] Konjac (Amorphophallus konjac) is a perennial herb common in subtropical areas. It is a plant of the Araceae family and the genus Amorphophallus. The tuber is flat and spherical, and is a semi-shade plant. It likes a warm environment and avoids high temperature exposure; it likes fertilizer and avoids drought, and is afraid of strong winds. It grows well in sandy loam with good water quality and rich organic matter. Konjac tubers are rich in glucomannan and have important edible value and medicinal value. With the development of my country's economy, the market demand for konjac products is expanding, and the industry prospect is relatively broad. However, due to the few excellent germplasms, high price of seed taro, backward planting technology methods, lack of infrastructure, and low technical content of agricultural product processing, konjac is currently seriously diseased, poor in quality, and low in planting efficiency, making it difficult to enter foreign markets and obtain rich income. So far, flower konjac has the largest planting area in my country, followed by white konjac, but most konjac cultivars belong to local group species, and the differences between varieties such as disease resistance and high yield are relatively large. The traditional method of konjac cultivation usually uses small konjac corms growing laterally from underground konjac corms for reproduction. The konjac reproduction coefficient is low, the incidence of diseases is high, and the yield and quality are seriously reduced, which has a great negative impact on the konjac industry (Zhang Donghua, Wang Qingping. High-yield technology of foliar corms of bulbil konjac [J]. New Rural Technology, 2020(02):8-10.).

[0003] Amorphophallus bulbiferi is a domesticated species of wild konjac in my country. Compared with the other konjac varieties mentioned above, its seed tubers are larger, the water content is lower, the dry slice flour yield is higher, and some indicators of fine flour are better than other varieties. Amorphophallus bulbiferi is a type of konjac that can grow aerial bulbils on the leaf surface. Its bulbils can be used as planting materials. It can also grow asexually without pollination after flowering to obtain mature seeds, avoiding the obstacle of extremely weak sexual reproduction ability of traditional cultivated varieties. The unique reproduction and pollination method of Amorphophallus bulbiferi gives it a great advantage in konjac reproduction and production. Although the yellow konjac of bulbils can be asexually induced to bloom and set seeds, not all perennial konjac corms can naturally bloom and reproduce. Especially when the phenomenon of flower and leaf co-growth occurs, with the vigorous growth of leaves, the nutrients of the corms are gradually consumed, resulting in slow seed growth, even difficulty in fruiting and maturing, and a reduction in growth and reproduction rate (Zhang Donghua, Zhou Fan, Zhao Jianrong, etc. Artificial induction flowering asexual breeding method of bulbil konjac [P]. Yunnan Province: CN200510048637.9, 2008-10-01.). Therefore, the method of artificial induction must be used for konjac corms to obtain large-scale plants and breeding seeds.

[0004] In addition, compared with other konjac qualities, yellow konjac with bulbils is suitable for semi-shade economic crops in tropical and subtropical low-altitude areas. It is currently the most suitable cultivated variety for the climatic conditions in Hainan. It has the characteristics of high glucomannan content, short growth cycle, and long growth period. It is suitable for the high temperature and high humidity climate environment in low-altitude areas such as Hainan. It has good product planting and market development potential, but currently the amount of seeds used in cultivation is relatively large (Mao Dajian, Wang Xian'an. Thoughts on the breeding of konjac seed taro and the selection of improved varieties [J]. Agriculture and Technology, 2013, 33(10):123.), the method of propagation by cutting bulbs is not enough to meet the growing demand for seeds, and the strains are impure (Liu Haoxia and Zhang Shenglin. Research progress in konjac breeding [J]. Southern Agriculture, 2007(02):37-40.), and the quality differences between strains are large (Shi Xinlong. Research on the effect of artificial regulation on the production of multiple leaves in konjac [D]. Southwest University, 2011.), and tissue culture technology is urgently needed to solve the problem of seed demand and strain purification. Summary of the invention

[0005] The invention aims to provide a method for forming regenerated plants by directly generating somatic embryos of konjac bulbil, and optimizes in vitro tissue culture conditions by inducing the ovary and other main organs in the female inflorescence to directly generate somatic embryos, thereby laying a good foundation for the establishment of a konjac detoxification tissue culture seedling breeding system and gene genetic transformation.

[0006] A method for forming regenerated plants by direct somatic embryogenesis of bulbil konjac, comprising:

[0007] 1) selecting the 10-15 cm flower buds that have not been opened before planting of the 2nd-3rd generation bulbil-type yellow konjac seed taro, collecting the unopened spathes, and cutting the tender female inflorescences as explants;

[0008] 2) The explant material was first soaked in 70%-75% alcohol for 45 seconds, then rinsed with sterile water, and then soaked in 2.0%-2.5% sodium hypochlorite for disinfection for 10-15 minutes; then cut into (0.4±0.1) cm×(0.4±0.1) cm in size, and inoculated in the culture medium to achieve primary induction, proliferation and germination regeneration; the primary somatic embryo (with pearl bud structure) induction medium was MS+0.5mg / L 6-BA+1mg / L NAA+3mg / L 2,4-D+30g / L sucrose+2.3g / L plant gel, and the pearl bud embryo proliferation medium was MS+6-BA 3.0mg / L+NAA0.5mg / L+30g / L sucrose+2.3g / L plant gel; the pearl bud embryo plant regeneration medium was: MS+6-BA 0.5mg / L+NAA 0.1mg / L+30g / L sucrose+2.3g / L plant gel.

[0009] Preferably, step 1) further comprises: harvesting 2-3 year old bulbil konjac corms adapted for planting in Hainan in December, selecting corms weighing 1000g-1500g, free from pests and diseases, and free from mechanical damage (more preferably with brown epidermis, oblate and plump corms, and plump flower buds), preliminarily selecting corms with flower buds, smearing them with straw ash and storing them in a cool and dry place; in February or March of the following year, when the corms gradually sprout terminal buds to a length of 4 cm, placing the corms with sprouted terminal buds (flower buds) on river sand, spraying water every day to accelerate germination, and collecting unopened spathes when the flower buds sprout and grow to form flower stalks and spathes to a length of 10cm-15cm.

[0010] Preferably, the specific explant organs for inducing direct somatic embryogenesis are mainly ovaries, etc. Preferably, in step 2), the culture conditions for inducing direct somatic embryogenesis with bulbil structure in the primary generation are: after inoculation, the material is placed in a dark environment at (25±2)°C for 2 weeks, and then transferred to (25±2)°C, 1000lx~1500lx light conditions for culture, 12h of light per day, and cultured for 10d~15d.

[0011] Preferably, in step 2), the direct induction culture conditions for somatic embryos with bulbil structures are: the material is cultured at (27±2)°C and 2000lx of light, with 12h of light per day for 25d to 30d.

[0012] Preferably, in step 2), the somatic embryo seedling regeneration culture conditions are: the material is cultured at (27±2)°C, 1000 lx light conditions, 12 hours of light per day, and cultured for 20 to 25 days.

[0013] More preferably, the somatic embryo seedling regeneration culture further comprises: picking the bulbil somatic embryos into single ones or cutting out tissue blocks containing several bulbil somatic embryos, and inoculating them into plant regeneration culture medium.

[0014] Preferably, the method further comprises: moving the bulbil-like yellow konjac somatic embryo seedlings obtained after somatic embryo germination and rooting to natural light and room temperature for a few days, transplanting the seedlings into a plug tray filled with a sterilized matrix, and maintaining water balance during the growth of the seedlings.

[0015] More preferably, the ratio of river sand to humus soil in the sterilized matrix is ​​1:1.

[0016] The beneficial effects of the present invention are:

[0017] The invention performs operations such as primary start-up culture, differentiation and proliferation of somatic embryos or buds, plant regeneration and transplanting seedlings on different explant parts of the bulbil-like yellow konjac, selects tender inflorescences of the bulbil-like yellow konjac as explants, and has higher bud differentiation and proliferation efficiency. A complete in vitro rapid propagation system of the bulbil-like yellow konjac comprising explant disinfection and screening, induction differentiation and proliferation of somatic embryos or buds, somatic embryo seedling plant regeneration and transplanting is preliminarily established. Among them, a way of directly generating bulbil somatic fine embryos from cells in the ovary part of the tender female inflorescence to form a regenerated plant is adopted, the process is simpler, the primary induction time is significantly shortened, the proliferation rate is higher, and the system has the advantages of faster speed and stronger genetic stability.

[0018] The present invention provides a theoretical basis and technical methods for the in vitro preservation of germplasm resources of the bulbil-type yellow konjac in Hainan tropical zone, rapid propagation of seedlings and large-scale promotion, and construction of a genetic transformation receptor system regeneration system for the bulbil-type yellow konjac. It has a positive effect on solving the supply and demand problems of seed taro and disease problems in the production process of the bulbil-type yellow konjac, and is of great significance for promoting the sustainable development of the bulbil-type yellow konjac industry in Hainan tropical zone. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The process from the germination of bulbil-type yellow taro to the development of new plants; A. Bulbil-type yellow taro plants under rubber forests; B. Aerial bulbils on the leaves; C. 2-3 year-old bulbil-type yellow taro globular rhizomes; D. Select bulbil-type yellow taro corms with plump flower buds; E. Flower buds germinate to form unopened spathes; F. Leaf buds germinate and sprout to form sturdy compound leaves.

[0020] Figure 2 Schematic diagram of different parts of the young inflorescence explant of Amorphophallus taro; A. The terminal bud of the bulb with flower buds grows into a flower stalk to form an unopened spathe; B. Different parts of the young spathe are shown after the bracts are peeled off.

[0021] Figure 3 Dedifferentiation induction and bud proliferation of different explants of bulbil-like Amorphophallus; A. Appearance of explants in different developmental parts of tender inflorescence (i. tender inflorescence, ii. terminal bud of flower bud with growth point); B. Somatic embryo induction of bulbil in organs such as ovary of tender female inflorescence for 30 days; C. Induction of the growth point of terminal bud of flower bud for 30 days; D. Somatic embryo proliferation and somatic embryo morphology of bulbil of tender female inflorescence; E. Bud proliferation and bud morphology at the growth point of flower bud.

[0022] Figure 4 Induced differentiation, proliferation, regeneration of somatic embryo seedlings and plant transplantation of bulbil somatic embryos in young female inflorescence of Amorphophallus taro; A. Differentiation of somatic embryos in bulbil of young female inflorescence; B. Proliferation of somatic embryos in bulbil; C. Germination of somatic embryos in bulbil to form monopolar buds; Plant regeneration of DE somatic embryo seedlings; F. Transplantation of regenerated plants; G. Bulbous somatic embryo seedlings 2 months after transplantation; H. Growth of globular rhizomes of single somatic embryo seedlings 2 months after transplantation.

[0023] Figure 5 The female flower explants of Amorphophallus konjac form regenerated plants through direct organogenesis; A. Young inflorescence explant material of Amorphophallus konjac (the red framed part is the female inflorescence); B. Single female flower on the female inflorescence induces meristematic cell formation (the female flower consists of a flat spherical wide disc-shaped ovary and a stigma, and the stigma is sessile); C. Female flowers directly differentiate into bulbil somatic embryos (indicated by the red arrow); D. Somatic embryos proliferate and form dicotyledonous bulbil somatic embryos at the same time; E. The plumule and radicle of mature bulbil somatic embryos occur synchronously to form bipolar regenerated plants; F. Transplantation of regenerated plants; G. Transplanted surviving female inflorescence bulbil somatic embryos regenerated plants. DETAILED DESCRIPTION

[0024] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0025] Example

[0026] In order to establish an efficient and stable plant regeneration system for bulbil-type yellow taro, the present application targets the shortage of seedlings of bulbil-type yellow taro in Hainan Province, collects the sprouted but unopened spathes of bulbil-type yellow taro, uses the sprouting growth points and tender inflorescences as explants, screens and optimizes the primary induction, proliferation and sprouting regeneration of bulbil-type yellow taro, and preliminarily establishes an efficient plant regeneration system.

[0027] 1. Materials and Methods

[0028] 1.1 Time and place of collection of bulbil-like yellow konjac explants

[0029] The inflorescence explants of yellow konjac bulbils were collected from the konjac plantation in Danzhou, Hainan.

[0030] In the present embodiment, the 2-3 year old bulbil konjac corms adapted to the planting in Hainan area were harvested in December, and the corms of about 1000g-1500g, brown epidermis, no pests, no mechanical damage, oblate and full corms, and full flower buds were selected, and the corms with flower buds were initially selected and stored in a cool and dry place after being smeared with straw ash. The terminal bud (flower bud) of the flower bud corm has a thick and full bud body, which is spherical and obviously stretches out of the corm bud nest, while the terminal bud (leaf bud) of the leaf bud corm has a relatively small bud, which grows in the bud nest of the corm. In February and March of the following year, the terminal buds of the bulbs will sprout one after another. When they are about 4 cm long, the bulbs with terminal buds (flower buds) are placed on river sand and sprayed with water every day to promote germination. When the flower buds sprout and form flower stalks and spathes, which are about 10cm to 15cm long, the unopened spathes can be collected and the tender female inflorescences can be cut off as explants (the ovary organ is mainly used, the flower of the bulbil is composed of ovary, style and stigma).

[0031] The process from the germination of yellow konjac to the development of new plants Figure 1 As shown, different parts of the explants Figure 2 shown.

[0032] 1.2 Test methods

[0033] 1.2.1 Collection of budded and unopened spathe inflorescences

[0034] On a sunny morning, after the dew has dried, select the unexpanded sprouts of the second-generation bulbil yellow konjac seed taro before planting, and cut healthy and plump sprout explant materials. Wrap the collected sprouts with clean wet gauze, place them in an ice box for refrigeration, and bring them back to the laboratory for explant disinfection and inoculation.

[0035] 1.2.2 Explant sterilization and inoculation

[0036] The outermost leaf sheath of the bud is peeled off and cut into several centimeters long segments. After soaking in 75% alcohol for 45 seconds, rinse with sterile water, and then disinfect the surface of the flower bud with 2% sodium hypochlorite for 10 minutes, and rinse with sterile water 5 times. The bud and inflorescence explants are marked. The bud growth point is cut into four pieces longitudinally and inoculated into the culture medium longitudinally; the tender inflorescence is cut into 0.5cm×0.5cm in size and inoculated in the culture medium flatly. After 10 days of inoculation, the contamination and start-up of the explants are counted, among which the explant contamination rate (%) = (the number of contaminated explants / the total number of inoculations) × 100%.

[0037] 1.2.3 Screening and optimization of bud induction and proliferation culture media

[0038] After 35 days of inoculation, the medium suitable for the introduction of bulbil-like yellow konjac explants in Hainan was preliminarily screened according to the induction, proliferation and growth morphology of the buds or bulbils somatic embryos. The induction rate of buds or bulbils somatic embryos (%) = (the number of explants induced to sprout or bulbil somatic embryos / the number of explants inoculated originally) × 100%, and the proliferation coefficient = (the number of new sprouts or bulbil somatic embryos / the number of explants inoculated originally). At the same time, the differentiation and growth of the inflorescence and growth point buds were described according to the actual situation.

[0039] 1.2.4 Culture conditions

[0040] (1) Culture medium

[0041] Orthogonal experiment of primary induction of konjac: MS + 30g / L sucrose + 2.3g / L plant gel (Sigma, Phytagel т M P8169-1KG) was used as the basic culture medium.

[0042] Table 1L9(3 3 ) Factors and levels of orthogonal experiments

[0043]

[0044] Table 2L9(3 3 ) Hormone concentration ratio of orthogonal experimental culture medium

[0045]

[0046] Konjac proliferation medium: MS + 6-BA 3.0 mg / L + NAA 0.5 mg / L + 30 g / L sucrose + 2.3 g / L plant gel as the basic culture medium.

[0047] (2) Culture conditions

[0048] Initial induction: After inoculation, the material was cultured in a dark environment at 25°C for 2 weeks, then transferred to 25°C and cultured under the conditions of 1000lx~1500lx of light, with daily light exposure of about 12h. After 10d, the contamination rate of the explants and the induction rate of bud or bulbil embryos were counted.

[0049] Bud or bulbil somatic embryo induction: the material was cultured at 27°C and 2000 lx of light, with about 12 hours of light per day. After 30 days, the induction of the bulbil-like yellow konjac bud growth points and inflorescence explants was counted.

[0050] Plant regeneration induction: Cut the induced buds at the growth point into single buds, pick the bulbil embryos into single ones or cut the tissue blocks containing several bulbil embryos, inoculate them into plant regeneration medium, and culture the materials at 27℃ and 1000lx light conditions, with about 12h of light per day. After 20d, count the number of regenerated plants with roots and the growth of the plants.

[0051] 1.2.5 Statistical analysis

[0052] Excel 2017 and DPS7.5 software were used for data statistics and analysis, and Duncan's new multiple range method was used for multiple comparisons.

[0053] 2. Results and Analysis

[0054] 2.1 Primary bud induction

[0055] Using growth points and inflorescences as explants is an effective way to obtain plant virus-free tissue culture seedlings. They have fast growth rates, high reproduction rates, and are not prone to mutation. They can maintain the genetic characteristics of the plant, maintain the characteristics of excellent varieties, and greatly improve the reproduction efficiency. The flower bud growth points and unopened spathe tender inflorescences of the bulbil-like yellow konjac were used as explants and cultured in the primary induction basic culture medium. Twelve culture media were used for each, and three replicates were set up to calculate the induction contamination rate and bud induction rate. The experimental results showed that M3 culture medium was the optimal primary induction culture medium. As shown in Table 3, compared with the flower bud growth point, the bud induction contamination rate of the tender inflorescence was significantly lower than that of the explants at the growth point, and its bud induction rate was also significantly higher than that of the bud induction rate at the growth point. Figure 3 As shown in B and C, multiple somatic embryos can be directly induced around the ovary and other organs in the young female inflorescence. Some of them are spherical embryos, some of them have one end that gradually protrudes into a nearly pear-shaped embryoid body, and more of them develop into bulbil embryos with double bud bracts, which are similar to the development of konjac zygotic embryos. The bulbil embryos are white and dense, and the bud bracts grow slightly irregularly, while the buds growing from the growing points are relatively few. In the primary induction, a piece of explant material can only induce 1 or 2 buds.

[0056] In summary, the differentiation of the initial generation of bulbil-like yellow konjac using young female inflorescences as explants is more efficient, and the somatic embryo buds at the growing points are relatively strong.

[0057] Table 3 Induction of flower bud growth points and tender female inflorescence explants of yellow konjac bulbils

[0058]

[0059] Note: Different lowercase letters in the same column of numbers in the table indicate significant differences (P<0.05).

[0060] 2.2 Bud proliferation

[0061] During the proliferation of the bulbil-like yellow konjac buds, a spherical bud is formed on the side of the explant, which is similar to the mother body, but it does not immediately separate from the mother body. Instead, it is connected to the mother body and continues to receive nutrients from the mother body. This is an asexual reproduction method and is of great significance to plant growth and reproduction. The culture of the flower bud tender female inflorescence and growth point is placed in a proliferation medium, using 12 culture media each, setting up three groups of repetitions, and calculating their proliferation coefficients respectively. Figure 3 As shown in D and E, the young female inflorescence and the bud growth point have completed proliferation. The number of bulbil embryos produced by the proliferation of the young inflorescence is large, the proliferation speed is fast, and the proliferation coefficient is large. A piece of explant material can induce multiple proliferating bulbil embryos, and the proliferation efficiency is very high; but the number of proliferating buds at the growth point is small, the proliferation time is long, and the proliferation coefficient is small. One bud can only proliferate 2 to 3 buds. According to statistics, in the first proliferation subculture, the proliferation coefficient of the young inflorescence is 3 to 4 times that of the growth point, as shown in Table 4.

[0062] In summary, young inflorescence buds have higher proliferation efficiency and can be used as excellent explant materials for bud induction.

[0063] Table 4 Proliferation and growth of primary buds from different explant sources of Amorphophallus konjac

[0064]

[0065] Note: Different lowercase letters in the same column of numbers in the table indicate significant differences (P<0.05).

[0066] The above results show that using the tender flower buds of bulbil-like yellow konjac as explants, regenerated plants can be formed through adventitious bud induction or bulbil somatic embryogenesis induction, especially the bulbil somatic embryos induced by tender female inflorescence have a high reproduction coefficient. The optimal primary bud induction medium is MS+0.5mg / L 6-BA+1mg / L NAA+3mg / L 2,4-D+30g / L sucrose+2.3g / L plant gel, and the proliferation medium is MS+6-BA 3.0mg / L+NAA 0.5mg / L+30g / L sucrose+2.3g / L plant gel.

[0067] 2.3 Plant regeneration and transplanting

[0068] Cytokinins mainly activate cell division and promote differentiation and growth of various tissues, such as inducing bud formation and promoting bud growth, which helps to increase the proliferation coefficient and maintain the cell meristem state, while auxins promote growth by accelerating cell elongation, which helps cell differentiation and tissue maturation. The coordinated effect of the two is very important for growing and developing tissues or organs. In the plant regeneration morphological establishment stage of the bulbil embryo, adding low-concentration cytokinins can maintain the cells in a certain division and immature state. At the same time, adding appropriate concentrations of auxins can promote the root differentiation of monopolar bulbil embryos and the maturation of bipolar embryos, forming complete bipolar embryo seedlings and improving the survival rate of regenerated plants after transplantation. The plant regeneration medium of the bulbil embryo of the tender female inflorescence of the bulbil konjac is best when adding low-concentration 6-BA, combined with NAA and IBA. MS+6-BA 0.5mg / L+NAA0.1mg / L+30g / L sucrose+2.3g / L plant gel is the most suitable. Move the bulbil-type yellow konjac obtained after rooting to natural light and room temperature for a few days, and transplant the seedlings into a plug tray filled with a sterilized substrate (river sand: humus soil = 1:1) to keep the moisture suitable and balanced during the growth of the seedlings. Choosing the right planting medium can prevent excessive humidity and other factors that can easily cause fungi to grow. The temperature during the planting of the seedlings should be suitable, and daily maintenance should be carried out regularly. Only in this way can it be conducive to rooting and promote root development, improve the survival rate, and ultimately achieve a good transplanting effect. Figure 4As shown, the differentiation efficiency of the tender female inflorescence of the bulbil-like yellow taro is very high, the inflorescence bulbil somatic embryos proliferate and grow rapidly, the small buds gradually germinate into seedlings, the seedlings have thick rhizomes, a large number of roots, and lateral roots. In the growth process after transplanting, the rhizomes continuously absorb nutrients and grow stronger. About 2 months after transplanting, the bulbil somatic embryo tissue culture seedlings have a spherical rhizome diameter of about 1.5 cm to 3.0 cm visible to the naked eye, and the growth condition is good. The vascular bundles of the bulbil somatic embryos are not connected to the vascular bundles of the explant mother material, and it is easy to obtain virus-free and disease-free somatic embryo seedlings and regenerated plants, which can be promoted as seedlings for large-scale planting production in the future; in addition, the spherical micro-rhizomes of the somatic embryo seedlings can also be used as artificial seeds for planting.

[0069] The main process of the method of the present invention is: tender female inflorescence - inducing the unpollinated ovary epidermal cells of female flowers to form embryonic cells - directly differentiating somatic embryoids - somatic embryo proliferation - mature somatic embryos germinating embryos and roots - forming somatic embryos to regenerate plants. Figure 5 The present invention utilizes the unpollinated ovary of the tender female inflorescence as an explant to form a regenerated plant through direct somatic embryogenesis. The process is simpler and the primary induction time is significantly shortened. It has the advantages of faster speed, stronger genetic stability, higher proliferation rate and higher disease-free degree of seedlings.

[0070] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A method for directly generating regenerated plants from somatic embryos of konjac bulbil, comprising: 1) selecting the 10-15 cm flower buds that have not been opened before planting of the 2nd-3rd generation bulbil-type yellow konjac seed taro, collecting the unopened spathes, and cutting the tender female inflorescences as explants; 2) The explant material was first soaked in 70%-75% alcohol for 45 seconds, then rinsed with sterile water, and then soaked in 2.0%-2.5% sodium hypochlorite for disinfection for 10-15 minutes; then cut into (0.4±0.1) cm×(0.4±0.1) cm in size, and inoculated in the culture medium to achieve primary induction, proliferation and germination regeneration; the primary somatic embryo induction medium was MS+0.5 mg / L6-BA+1 mg / L NAA+3 mg / L 2,4-D+30 g / L sucrose+2.3 g / L plant gel, the pearl embryo proliferation medium was MS+6-BA3.0 mg / L+NAA 0.5 mg / L+30 g / L sucrose+2.3 g / L plant gel, and the pearl embryo plant regeneration medium was: MS+6-BA0.5 mg / L+NAA 0.1mg / L+30g / L sucrose+2.3g / L plant gel.

2. The method according to claim 1, characterized in that Step 1) also includes: harvesting 2-3 year old bulbil konjac corms suitable for planting in Hainan in December, selecting corms weighing 1000g-1500g, free of pests and diseases, and free of mechanical damage, preliminarily selecting corms with flower buds, smearing them with straw ash and storing them in a cool and dry place; in February or March of the following year, when the corms gradually sprout terminal buds to a length of 4cm, placing the corms with terminal buds on river sand, spraying water every day to accelerate germination, and collecting unopened spathes when the flower buds sprout and grow to form flower stalks and spathes to a length of 10cm-15cm.

3. The method according to claim 1, characterized in that The specific explant organ for inducing direct somatic embryogenesis is mainly the ovary.

4. The method according to claim 1, characterized in that: In step 2), the initial initiation and induction of direct somatic embryogenesis with pearl bud structure culture conditions are as follows: after inoculation, the material is placed in a dark environment at (25±2)°C for 2 weeks, then transferred to (25±2)°C, cultured under light conditions of 1000lx~1500lx, 12h of light per day, and cultured for 10d~15d.

5. The method according to claim 1, characterized in that In step 2), the direct induction culture conditions for somatic embryos with bulbil structures are as follows: the material is cultured at (27±2)°C and 2000lx of light, with 12 hours of light per day for 25 to 30 days.

6. The method according to claim 1, characterized in that In step 2), the somatic embryo seedling regeneration culture conditions are: the material is placed at (27±2)°C, 1000lx light conditions, 12h of light per day, and cultured for 20d to 25d.

7. The method according to claim 6, characterized in that The somatic embryo seedling regeneration culture also includes: picking the bulbil somatic embryos into single ones or cutting out tissue blocks containing several bulbil somatic embryos, and inoculating them into the plant regeneration culture medium.

8. The method according to any one of claims 1 to 7, characterized in that: Also includes: The bulbil-like yellow konjac somatic embryo seedlings obtained after somatic embryo germination and rooting were moved to natural light and room temperature for a few days, and the seedlings were transplanted into a plug tray filled with a sterilized substrate to maintain water balance during the growth of the seedlings.

9. The method according to claim 8, characterized in that The ratio of river sand to humus soil in the sterilized matrix is ​​1:1.

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