Method for forming regenerated plant by means of direct generation of amorphophallus bulbifer somatic embryo

Abstract

The invention provides a method for forming a regenerated plant through a direct generation way of a amorphophallus bulbifer somatic embryo, which comprises the following steps: collecting germinated non-opened Buddhist flame of amorphophallus bulbifer, taking a tender female inflorescence as an explant, screening and optimizing amorphophallus bulbifer female inflorescence to induce the direct generation, maturation and plant regeneration conditions of the somatic embryo, and obtaining the regenerated plant through the direct generation way of the amorphophallus bulbifer somatic embryo. And an efficient plant regeneration system is preliminarily established. Under proper temperature, illumination and culture conditions, the direct germination and seedling formation of the somatic embryo and the transplanting condition are good. The method provides a theoretical basis and a technical method for in-vitro preservation of germplasm resources of the amorphophallus bulbifer in the Hainan hot area, rapid propagation and large-area popularization of seedlings and construction of a genetic transformation receptor system regeneration system of the amorphophallus bulbifer, and is of great significance in assisting sustainable development of the amorphophallus bulbifer industry in the Hainan hot area.

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 a direct somatic embryogenesis pathway of konjac bulbil. Background Art

[0002] Konjac (Amorphophallus konjac) is a perennial herb common in subtropical areas, is Araceae, Amorphophallus plant, tuber oblate, is semi-shade plant, likes warm environment, avoids high temperature exposure; like fertilizer avoids drought, is afraid of strong wind attack, and grows well in sandy loam with good water quality and abundant organic matter. Konjac tuber is rich in glucomannan, has important edible value and medicinal value. Along with my country's economic development, market is expanding the product demand of konjac, and industry prospect is wider, but because good germplasm is few, seed taro price is high, planting technology method is backward, lacks infrastructure, agricultural product processing technology content is lower and other reasons, konjac disease is serious at present, quality is poor, planting efficiency is low, is difficult to enter foreign market, obtains rich income. So far, flower konjac is the largest in my country's cultivated area, and next is white konjac, but most konjac cultivars belong to local group species, and between variety, character such as disease resistance, high yield are bigger. The traditional konjac cultivation method 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 konjac bulbils [J]. New Rural Technology, 2020(02):8-10.).

[0003] Amorphophallus bulbiferi is a domesticated species of wild konjac in my country. Compared to the other konjac varieties mentioned above, its seed tubers are larger, have a lower water content, a higher dry sheet flour yield, and some of the fine flour indicators are superior to other varieties. Amorphophallus bulbiferi is a type of konjac that can grow aerial bulbils on its leaves. Its bulbils can be used as planting material and can also reproduce asexually after flowering without pollination to produce mature seeds, circumventing the extremely weak sexual reproduction ability of traditional cultivated varieties. The unique reproduction and pollination method of Amorphophallus bulbiferi gives it a significant advantage in konjac reproduction and production. Although the yellow konjac of bulbil can carry out asexual flowering and setting seed, it is not that any perennial konjac corm all can naturally bloom and breed, especially when the phenomenon of mosaic and leaf coexistence occurs, along with the vigorous growth of blade, corm nutrient consumes gradually, causes seed growth slow, even is difficult to set solid maturity, and growth and reproduction rate reduces (Zhang Donghua, Zhou Fan, Zhao Jianrong, etc. bulbil konjac artificial induction flowering asexual breeding method [P]. Yunnan Province: CN200510048637.9,2008-10-01.).Therefore, the konjac corm must be used the method for artificial induction, could obtain large-scale plant, breeding and use seed.

[0004] In addition, compared with other konjac varieties, 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 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 propagation of konjac seed taro and the selection of improved varieties [J]. Agriculture and Technology, 2013, 33(10):123.), the 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 of 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 problems of seed demand and strain purification. Summary of the Invention

[0005] The present invention aims to provide a method for forming regenerated plants by directly generating somatic embryos of konjac bulbil. The method comprises inducing the ovary and other major organs in the female inflorescence to directly generate somatic embryos, optimizing in vitro tissue culture conditions, and laying a good foundation for the establishment of a virus-free tissue culture seedling breeding system and genetic transformation of konjac.

[0006] A method for forming regenerated plants by directly generating somatic embryos from konjac bulbil, comprising:

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

[0008] 2) Explants were soaked in 70%-75% alcohol for 45 seconds, rinsed with sterile water, and then disinfected by soaking in 2.0%-2.5% sodium hypochlorite for 10-15 minutes. The explants were then cut into (0.4±0.1) cm × (0.4±0.1) cm pieces and plated in culture medium for primary induction, proliferation, and germination regeneration. The induction medium for primary somatic embryos (with pearl bud structures) consisted of MS + 0.5 mg / L 6-BA + 1 mg / L NAA + 3 mg / L 2,4-D + 30 g / L sucrose + 2.3 g / L phytagel. The pearl bud embryo proliferation medium consisted of MS + 3.0 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 2.3 g / L phytagel. The pearl bud embryo plant regeneration medium consisted of MS + 0.5 mg / L 6-BA + 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 cultivation in Hainan in December, selecting corms weighing 1000g-1500g, free of pests and diseases, and free of 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 successively 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 into flower stalks and spathes to a length of 10cm-15cm.

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

[0011] Preferably, in step 2), the direct induction culture conditions for somatic embryos with pearl bud structure are: culturing the material at (27±2)°C and 2000lx illumination, with 12 hours of illumination per day for 25 to 30 days.

[0012] Preferably, in step 2), the somatic embryo seedling regeneration culture conditions are: the material is cultured at (27±2)°C, 1000 lx illumination, 12 hours of illumination 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 the blocks into plant regeneration culture medium.

[0014] Preferably, the method further comprises: placing the bulbil-like yellow konjac somatic embryo seedlings obtained after somatic embryo germination and rooting under natural light and room temperature for a few days, transplanting the seedlings into a plug tray filled with a sterilized substrate, 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 generation initiation culture, differentiation and proliferation of somatic embryos or buds, plant regeneration and transplanting seedlings on different explant parts of the bulbil-like yellow konjac, screens out that the tender inflorescence of the bulbil-like yellow konjac is used as the explant, and has higher bud differentiation and proliferation efficiency. A complete in vitro rapid propagation system of the bulbil-like yellow konjac, comprising disinfection and screening of explants, induction and proliferation of somatic embryos or buds, regeneration of somatic embryo seedlings and transplanting, is preliminarily established. Specifically, a method of directly generating bulbil somatic fine embryos from cells in the ovary part of the tender female inflorescence to form regenerated plants has a simpler process, significantly shortens the primary generation induction time, has a higher proliferation rate, and 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 pearly bud yellow taro in Hainan tropical zone, rapid propagation of seedlings and large-scale promotion, and the construction of a genetic transformation receptor system regeneration system for the pearly bud yellow taro. It has a positive effect on solving the supply and demand problems of seed taro and disease problems in the production process of the pearly bud yellow taro, and is of great significance for promoting the sustainable development of the pearly bud yellow taro 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 a new plant; A. Bulb-type yellow taro plants under rubber forests; B. Aerial bulbils on the leaves; C. 2-3 year-old bulbil-type yellow taro bulbous rhizomes; D. Selected bulbil-type yellow taro bulbs 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 bulbilii; 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 the tender inflorescence (i. tender inflorescence, ii. terminal bud of flower bud with growing point); B. Somatic embryo induction of bulbils in organs such as the ovary of tender female inflorescence for 30 days; C. Induction of the growing point of the terminal bud of flower bud for 30 days; D. Somatic embryo proliferation and somatic embryo morphology of bulbils in tender female inflorescence; E. Bud proliferation and morphology at the growing point of flower bud.

[0022] Figure 4 Induced differentiation, proliferation, regeneration of somatic embryos from bulbils of tender female inflorescences of Amorphophallus taro; A. Differentiation of somatic embryos from bulbils of tender female inflorescences; B. Proliferation of somatic embryos from bulbils; C. Germination of somatic embryos from bulbils to form monopolar buds; DE somatic embryo seedling regeneration; F. Transplantation of regenerated plants; G. Somatic embryo seedlings from bulbils 2 months after transplantation; H. Growth of globular rhizomes from single somatic embryo seedlings 2 months after transplantation.

[0023] Figure 5 Regeneration of female flower explants of Amorphophallus konjac through direct organogenesis; A. Young inflorescence explant material of Amorphophallus konjac (the red frame is the female inflorescence); B. Meristematic cell formation is induced in a single female flower on the female inflorescence (the female flower consists of a flattened, broadly disc-shaped ovary and a stigma, and the stigma is sessile); C. Direct differentiation of somatic embryos from female flowers into bulbils (indicated by red arrows); D. Somatic embryos proliferate and simultaneously form dicotyledonous bulbil somatic embryos; E. Synchronous development of plumules and radicles of mature bulbil somatic embryos to form bipolar regenerated plants; F. Transplantation of regenerated plants; G. Plants regenerated from bulbil somatic embryos of surviving transplantation. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the present invention, 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 following specific description 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 the bulbil-type yellow devil, this application targets the shortage of seedlings of the bulbil-type yellow devil in Hainan Province, collects the germinated unopened spathes of the bulbil-type yellow devil, uses the germination growth points and tender inflorescences as explants, screens and optimizes the primary induction, proliferation and germination regeneration of the bulbil-type yellow devil buds, and preliminarily establishes an efficient plant regeneration system.

[0027] 1. Materials and Methods

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

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

[0030] In the present embodiment, December gathers the 2~3 year raw bulbil konjac corms of adaptation Hainan area plantation, choose the corm of about 1000g~1500g of size, brown epidermis, no disease and insect, no mechanical damage, bulb oblate full, full flower bud, tentatively select the bulb of tool flower bud, smear the back in cool and dry place and store with straw ash.The terminal bud (flower bud) of flower bud corm, bud body is thick and full, is spherical, obviously stretches out outside the corm bud nest, and the terminal bud (leaf bud) of leaf bud corm, bud is relatively little, grows in the bud nest of corm. From February to 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 as explants (the ovary organ is mainly used, and the flower of the konjac is composed of ovary, style and stigma).

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

[0032] 1.2 Test methods

[0033] 1.2.1 Collection of budded but unopened spathe inflorescences

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

[0035] 1.2.2 Explant disinfection and inoculation

[0036] Peel off the outermost leaf sheath of the bud and cut into several centimeters long segments. Soak the buds in 75% alcohol for 45 seconds, rinse once with sterile water, then disinfect the bud surface with 2% sodium hypochlorite for 10 minutes and rinse five times with sterile water. Mark the bud and inflorescence explants. Cut the bud growth point into four pieces longitudinally and inoculate them into the culture medium; cut the young inflorescence into 0.5 cm x 0.5 cm pieces and inoculate them flatly into the culture medium. After 10 days of inoculation, count the explant contamination and priming status. Explant contamination rate (%) = (number of contaminated explants / total number of inoculations) × 100%.

[0037] 1.2.3 Screening and Optimization of Bud Induction and Proliferation Culture Media

[0038] Thirty-five days after inoculation, a preliminary screening of suitable culture media for the introduction of bud-forming yellow konjac explants into Hainan was conducted based on the induction, proliferation, and growth morphology of bud or bulbil somatic embryos. Here, the bud or bulbil somatic embryo induction rate (%) = (number of explants induced to produce buds or bulbil somatic embryos / number of original inoculated explants) × 100%, and the proliferation coefficient = (number of newly produced buds or bulbil somatic embryos / number of original inoculated explants). Simultaneously, the differentiation and growth of inflorescence and growing point buds were described based on actual conditions.

[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 light exposure of about 12 hours per day. After 10 days, the contamination rate of the explants and the induction rate of buds or bulbils 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 growing point into single buds, pick the bulbil embryos into single ones or cut out tissue blocks containing several bulbil embryos, inoculate them into plant regeneration medium, and culture the materials at 27°C and 1000lx light conditions, with light exposure for about 12 hours per day. After 20 days, 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 a fast growth rate, a high reproduction rate, are not prone to mutation, 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-type yellow taro were used as explants and placed in the primary induction basic culture medium for culture. Twelve culture media were used for each, and three groups of repetitions were set up to statistically analyze 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 bud was significantly lower than that of the explant 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 gradually protruding into a nearly pear-shaped embryoid body, and more of them develop into pearly somatic embryos with double bud bracts, which are similar to the development of konjac zygotic embryos. The pearly somatic embryos are white and dense, and the bud bracts grow slightly irregularly. The buds growing from the growing point 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 primary generation of bulbil-like yellow taro is more efficient when using young female inflorescences as explants, and the somatic embryos 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 indicate significant differences (P<0.05).

[0060] 2.2 Bud proliferation

[0061] During the proliferation of the bulbil-like yellow konjac bud, a spherical bud is formed on the side of the explant, which bulges outward. The bud is similar to the mother plant, but it does not immediately separate from the mother plant. Instead, it is connected to the mother plant and continues to receive nutrients from the mother plant. This is a form of asexual reproduction 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 replicates, and calculating their proliferation coefficients. Figure 3 As shown in Figures D and E, both the young female inflorescence and the bud growth point have completed proliferation. The young inflorescence produced a large number of bulbil embryos, with a rapid proliferation rate and a large proliferation coefficient. Multiple bulbil embryos can be induced from a single explant, demonstrating high proliferation efficiency. However, the number of buds proliferating from the growth point was small, the proliferation time was long, and the proliferation coefficient was low, with only two to three buds proliferating from one bud. Statistics show that during the first proliferation subculture, the proliferation coefficient of the young inflorescence was 3 to 4 times that of the growth point, as shown in Table 4.

[0062] In conclusion, 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 Konjac konjac

[0064]

[0065] Note: Different lowercase letters in the same column 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, especially the bulbil somatic embryos induced by tender female inflorescences 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 phytagel, and the proliferation medium is MS+6-BA 3.0mg / L+NAA 0.5mg / L+30g / L sucrose+2.3g / L phytagel.

[0067] 2.3 Plant regeneration and transplanting

[0068] Cytokinins primarily activate cell division and promote the differentiation and growth of various tissues, such as inducing bud formation and growth, which helps increase proliferation coefficients and maintain cell meristems. Auxins, on the other hand, promote growth by accelerating cell elongation, aiding cell differentiation and tissue maturation. The coordinated action of these two factors is crucial for growing and developing tissues or organs. During the morphological establishment phase of plant regeneration from bulbil embryos, the addition of low concentrations of cytokinins can maintain a certain level of cell division and immaturity. Simultaneously, the addition of appropriate concentrations of auxins can promote root differentiation of monopolar bulbil embryos and maturation of bipolar embryos, leading to the formation of complete bipolar embryos and improving the survival rate of regenerated plants after transplantation. The best medium for plant regeneration from bulbil embryos in young female inflorescences of bulbil konjac is supplemented with low concentrations of 6-BA, combined with NAA and IBA. The most suitable medium is MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 2.3 g / L phytogel. Move the bulbils of yellow konjac obtained after rooting to natural light and room temperature for a few days, and then transplant the seedlings into a hole tray filled with a sterilized substrate (river sand: humus soil = 1:1), and 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 should be suitable during the planting process of the seedlings, 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 young female inflorescence of the bulbil-type yellow taro is very high, the somatic embryos of the inflorescence rapidly proliferate and grow, the small buds gradually germinate into seedlings, the rhizomes of the seedlings are thick, the number of roots is large, and there are lateral roots. During 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 visible spherical rhizome with a diameter of about 1.5 cm to 3.0 cm, and are in good growth condition. 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 to regenerate plants, which can be promoted as seedlings for large-scale planting production in the future. In addition, the spherical microrhizomes 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 germinate embryos and roots - forming somatic embryos to regenerate plants. Figure 5 The present invention utilizes the unpollinated ovary of the tender female inflorescence as the explant and forms regenerated plants 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 based on the above description. It is impossible to enumerate all implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection 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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