CN112042525B - Method for cultivating pentaploid seedless momordica grosvenori - Google Patents
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Abstract
The invention discloses a method for cultivating pentaploid seedless momordica grosvenori, which comprises the following steps: a cultivation method of a pentaploid seedless momordica grosvenori comprises the following steps: the diploid momordica grosvenori is subjected to double induction treatment to obtain tetraploid momordica grosvenori, and the tetraploid momordica grosvenori is hybridized with the triploid momordica grosvenori subjected to double induction treatment to obtain the pentaploid momordica grosvenori; the condition is that diploid and triploid momordica grosvenori is pre-cultured before the double induction treatment. Compared with triploid seedless momordica grosvenori, the fruit of the polyploid seedless momordica grosvenori is larger than that of the triploid seedless momordica grosvenori, higher in glycoside content than that of the diploid momordica grosvenori, and higher in economic value. The method for planting the polyploid seedless momordica grosvenori according to the invention can produce more momordica grosvenori sweet glycosides V per mu, reduce the production cost of the momordica grosvenori sweet glycosides V and provide theoretical and practical basis for the later polyploid breeding.
Description
Technical Field
The invention relates to the technical field of plant cultivation, in particular to a cultivation method of pentaploid seedless momordica grosvenori.
Background
Momordica grosvenori, a fruit of perennial vine of the Cucurbitaceae family. Siraitia grosvenori, its leaf-heart shape, heterosexual plant, blossoming in summer and bearing fruit in autumn. Fructus momordicae is also a medicine and food dual-purpose material, and has the main effects of relieving cough and reducing sputum. The fructus Siraitiae Grosvenorii has high nutritive value, and is rich in vitamin C, and glycoside, fructose, glucose, protein, lipid, etc. In recent years, the mogroside V in the grosvenor momordica fruit has high sweetness and good mouthfeel, particularly does not contain heat, does not cause the increase of blood sugar and obesity, and has high economic value.
The momordica grosvenori is a diploid plant, fruits contain a large number of seeds, the seeds account for about one third of the whole fruits in volume, and the seeds do not contain sweet glycoside substances, so that the whole content of the sweet glycosides in the momordica grosvenori is low. Because the commercial value of the momordica grosvenori is reduced due to the existence of a large number of seeds, the cultivation of the seedless momordica grosvenori is an important way for obtaining the high-sweet-glycoside momordica grosvenori because the seedless momordica grosvenori does not contain seeds, but the volume of the triploid seedless momordica grosvenori is nearly half of that of the diploid momordica grosvenori, and the total sweet-glycoside yield is not actually improved. The pentaploid seedless momordica grosvenori has no seeds and is large in size, the content of the mogrosides is obviously improved, and the problems of low mogroside content and high commercial cost of the momordica grosvenori are solved.
Patent CN101120653A discloses a breeding method of triploid seedless momordica grosvenori, comprising the following steps: 1) taking diploid momordica grosvenori female and male plant explants to breed tissue culture seedlings; 2) taking tissue culture subculture seedlings, and inducing chromosome doubling; 3) cutting different parts of the female and male plants after induction treatment for differential culture; 4) cutting stem tip and stem segment of the bud to perform bud growth culture; 5) carrying out chromosome number detection on the cultured complete plant, screening a tetraploid plant, and carrying out rooting culture and seedling hardening; 6) planting tetraploid plants and diploid plants according to a conventional technology, and carrying out artificial pollination and hybridization during blooming to obtain hybrid seeds; 7) propagating the hybrid seeds into complete plants, and detecting the number of chromosomes; screening out triploid plants, carrying out rooting culture and hardening seedlings; 8) and (3) planting the triploid plant and the diploid plant, carrying out artificial pollination on a triploid female plant by using a diploid male plant during flowering, and obtaining the seedless momordica grosvenori after the female plant bears fruits.
Patent CN103210835A discloses a technique for breeding seedless luo han guo by crossing triploid male plant and quadruplicate female plant of luo han guo; CN102812902A discloses a technique for cultivating triploid Momordica grosvenori, which is to cross a stable tetraploid female plant and a diploid male plant to obtain fruits, after breeding, planting the fruits as virus-free seedlings of the triploid female plant by using a flow cytometer and RFLP analysis, then crossing the fruits with the diploid male plant, and obtaining triploid Momordica grosvenori without seeds after fruiting. CN103299899A discloses a cross breeding technique of fructus momordicae, which is to cross a diploid with a hexaploid to breed a tetraploid fructus momordicae variety with high yield and high resistance.
Until now, the culture method of polyploid momordica grosvenori is mainly focused on the culture of triploid and tetraploid, and the applicant searches that a mature, stable and efficient culture method for obtaining the polyploid momordica grosvenori without seeds is not found. The difficulty in cultivating the pentaploid seedless momordica grosvenori is that the polyploid survival rate is low and the breeding efficiency is low when polyploid induction is carried out, so that the commercial application prospect is poor. At present, the cultivation method of the pentaploid seedless momordica grosvenori is blank. The momordica grosvenori fruits obtained by the breeding method are large in size and high in stevioside content, the bottleneck problem of popularization of the momordica grosvenori glycosides caused by high cost is solved, the production cost and the production period of the momordica grosvenori glycosides can be reduced, benefits are provided for application and popularization of the momordica grosvenori fruits in the fields of food, medicine, health care and the like, and economic benefits are considerable.
Disclosure of Invention
In order to solve the problems of the prior art that a stable, reliable and efficient cultivation method of the polyploid seedless momordica grosvenori is lacked, the mogroside content is low, and the production cost is high, the invention provides a cultivation method of the polyploid seedless momordica grosvenori, which comprises the following steps: the diploid momordica grosvenori is subjected to double induction treatment to obtain tetraploid momordica grosvenori, and the tetraploid momordica grosvenori is hybridized with the triploid momordica grosvenori subjected to double induction treatment to obtain the pentaploid momordica grosvenori; the condition is that diploid and triploid momordica grosvenori is pre-cultured before the double induction treatment.
Preferably, the pre-culture is to culture the disinfected fructus momordicae seeds for 5 to 9 days at 30 to 40 ℃ under the dark condition under the aseptic condition; more preferably, the culture is carried out at 35-40 ℃ for 1-3 days and then at 25-28 ℃ for 2-8 days; most preferably, the culture is carried out at 36-38 ℃ for 1-2 days and then at 26 ℃ for 4-5 days. The aseptic conditions are well known in the art, as long as an aseptic environment suitable for culturing the seeds is achieved, and in one embodiment of the invention, 8-10 layers of neutral sterile filter paper are added to a sterile petri dish, sterile water is added until complete submersion and no water flows properly, and the Lo Han Guo seeds are sealed on the filter paper.
The double induction treatment is to insert momordica grosvenori seeds into MS liquid culture medium of colchicine for treatment.
Further, the induction treatment conditions are 25-28 ℃, 16-20 hours of illumination per day, 3000lux of illumination, and 4-8 hours of dark culture; furthermore, the concentration of the colchicine is 3-5 mg/L; the MS liquid culture medium is MS culture medium plus 25-35g/L sucrose, and the pH value is 5.5-6.
The germination of the momordica grosvenori seeds is very difficult, pretreatment is not carried out, the embryos are easy to die without beginning to germinate by directly using colchicine for induction, the efficiency of directly inducing the buds or tissue culture seedlings is low, and polyploid plants are difficult to obtain. The method activates the embryo of the seed after the pre-culture treatment of the seed at high temperature (35-40 ℃) and then normal temperature (25-28 ℃), the embryo of the seed begins to sprout to emit a bud point, the vitality of the growing point emitted by the embryo is stronger than that of the growing point emitted by the embryo without sprouting, the tolerance to colchicine is higher, and the colchicine induction treatment is carried out on the seed at the moment, so that the vitality of the bud is improved. The higher the concentration of colchicine, the greater the chance of inducing polyploidy, but too high a concentration can lead to death of the plant. The pre-cultured momordica grosvenori seeds can be treated on the premise of properly increasing the concentration of colchicine without causing death of germinated new buds of embryos, so that the mutagenesis rate of polyploidy is further increased.
Preferably, the cross is a tetraploid male flower pollinating a hexaploid female flower.
Triploid momordica grosvenori can be obtained by self-breeding, and can also be obtained by the methods described in patents CN101120653A, CN103210835A, and CN 102812902A.
Preferably, the triploid Lo Han Guo is obtained by crossing diploid and tetraploid Lo Han Guo. More preferably, the tetraploid momordica grosvenori is obtained by performing double induction treatment on the diploid momordica grosvenori seeds; more preferably, the cross is a diploid male flower pollinating a tetraploid female flower.
In a more specific embodiment of the present invention, the cultivation method of the pentaploid momordica grosvenori without seeds comprises the following steps:
(1) peeling, cleaning and disinfecting momordica grosvenori seeds, pre-culturing, performing multiple induction treatment and culture, and performing chromosome multiple identification after the momordica grosvenori seeds germinate and grow;
(2) carrying out tissue culture propagation culture and rooting culture on identified tetraploid momordica grosvenori seedlings;
(3) after the rooted tetraploid momordica grosvenori seedlings are hardened, the diploid momordica grosvenori and the tetraploid momordica grosvenori are hybridized, and the produced fruits are triploid momordica grosvenori;
(4) peeling seeds of triploid fructus momordicae, cleaning, disinfecting and pre-culturing; performing multiple induction treatment and culture, and performing chromosome multiple identification after the fructus momordicae seeds germinate and grow;
(5) placing the identified six-ploid Grosvenor momordica fruit seedlings in an MS proliferation culture medium for tissue culture propagation culture and rooting culture;
(6) transplanting the tetraploid momordica grosvenori seedlings in the step (2) and the hexaploid momordica grosvenori seedlings in the step (5) into a field for hybridization, wherein the obtained fruits are the pentaploid momordica grosvenori fruits; taking seeds in the fruit of the polyploid momordica grosvenori to germinate and then cultivating the seeds in a field, remaining female plants after blooming, removing male plants, pollinating the polyploid momordica grosvenori by diploid pollen, and obtaining the polyploid seedless momordica grosvenori after fruiting.
Preferably, the pre-culture in the step (1) and/or the step (4) is to activate the disinfected fructus momordicae seeds for 1 to 3 days at 35 to 40 ℃ in the dark under the aseptic condition and then culture the fructus momordicae seeds for 3 to 8 days at 25 to 28 ℃; most preferably, the activation is carried out at 36-38 deg.C for 1-2 days, and then the culture is carried out at 25-26 deg.C for 4-5 days. The aseptic conditions are well known in the art, as long as an aseptic environment suitable for culturing the seeds is achieved, and in one embodiment of the invention, 8-10 layers of neutral sterile filter paper are added to a sterile petri dish, sterile water is added until complete submersion and no water flows properly, and the Lo Han Guo seeds are sealed on the filter paper.
According to the invention, researches show that the induction rate of polyploidy can be obviously improved by performing pre-culture treatment on the momordica grosvenori seeds and then treating the bud points of the seeds which just germinate by colchicine.
Preferably, in the step (1), the momordica grosvenori seeds are selected from momordica grosvenori varieties with larger fruits and higher content of the sweet glycosides, and more preferably diploid momordica grosvenori cultivars. The average weight of the bigger fruits is 70-80g of fresh momordica grosvenori fruits, and the higher content of the stevioside refers to the average content of 0.4-0.5 wt% of the stevioside.
Preferably, in the step (1), the peeling, cleaning and disinfecting are to peel off the seeds, wash the pulp on the surfaces of the seeds with tap water, dry the pulp in the shade, place the seeds in a triangular flask after peeling, wash the seeds with sterile water for 3 to 5 times until the sterile water is clear, add 75% alcohol for disinfection for 1 to 2 minutes, rapidly pour off the alcohol, add 30 to 45% sodium hypochlorite and place the seeds in a shaking table, disinfect the seeds at 150 plus 200rpm for 20 to 30 minutes, finally wash the seeds with sterile water for 3 to 5 times, and pour off the water for later use.
Preferably, in the step (1), the multiple induction treatment is carried out by immersing the momordica grosvenori seeds in the MS liquid medium of colchicine.
Further, the induction treatment conditions are 25-28 ℃, 16-20 hours of illumination per day, 3000lux of illumination, and 4-8 hours of dark culture; furthermore, the concentration of the colchicine is 3-5 mg/L; the MS liquid culture medium is MS culture medium plus 25-35g/L sucrose, and the pH value is 5.5-6.
Preferably, after the momordica grosvenori seeds germinate and grow in the step (1), the bud characteristics are observed when the momordica grosvenori seeds grow to 5-8 cm later, and chromosome fold identification is carried out on the momordica grosvenori seedlings with thick stems and wide leaves. Chromosome ploidy identification is well known in the art, and is performed by taking the root tip of momordica grosvenori and performing routine tabletting identification.
The propagation expanding culture in the step (2) and the step (5) is carried out for 25-30 days in an MS proliferation culture medium, wherein the MS proliferation culture medium is obtained by adding 6-BA 0.15-0.25mg/L + TDZ 0.2-0.3mg/L +25-35g/L sucrose +6-9g/L agar powder into an MS minimal medium, and the pH value is 5.8; culturing at 25-28 deg.C under illumination of 3000lux for 16-20 hr per day and illumination of 2000-; the rooting culture is carried out in an MS rooting culture medium, wherein the MS rooting culture medium is an MS basic culture medium added with NAA 0.1-0.2mg/L +25-35g/L sucrose +6-9g/L agar powder, and the pH value is 5.8; the culture temperature is 25-28 ℃, the illumination is 16-20 hours per day, the illumination is 2000-3000lux, the dark culture is carried out for 4-8 hours, and the culture is carried out for 25-30 days.
The propagation culture is 2-3 generations, and each seedling can propagate to 30-60 plants.
In the step (3), the diploid momordica grosvenori and the tetraploid momordica grosvenori are hybridized, namely, a diploid male flower is used for pollinating a tetraploid female flower; in the step (6), the tetraploid and the hexaploid are hybridized to pollinate female flowers of the hexaploid by male flowers of the tetraploid, and the obtained fruits are the pentaploid momordica grosvenori fruits.
The inventors have found that in a cross where high ploidy female flowers are pollinated with low ploidy male flowers, the higher the pollination efficiency, the higher the sweet glycoside content. Namely, when the diploid and the tetraploid are hybridized, the diploid is a male flower, and the tetraploid is a female flower; when the tetraploid and the hexaploid are crossed, the tetraploid is a male flower, and the hexaploid is a female flower.
The peeling, cleaning, disinfecting, pre-culturing, multi-time induction processing and culturing steps in the step (4) are consistent with the conditions in the step (1).
The triploid seedless momordica grosvenori has no seeds, and the fruits have no auxin for promoting the growth of the seeds in the growth process, so that the obtained fruits are smaller than the fruits with the conventional diploid size. However, compared with the triploid seedless momordica grosvenori, the cultivated polyploid seedless momordica grosvenori has higher times of chromosomes, the obtained momordica grosvenori is larger, and the content of the main component, namely the glycoside V, in the momordica grosvenori is higher. The pentaploid seedless momordica grosvenori cultivated by the method has higher economic value, and provides theoretical and practical basis for polyploid breeding in the future.
Drawings
FIG. 1 is an HPLC plot of the pentaploid mogroside V obtained in example 1.
FIG. 2 is an HPLC chromatogram of the diploid seeded Lo Han Guo mogroside V obtained in example 1.
FIG. 3 is an HPLC plot of the triploid Lo Han Guo mogroside V obtained in example 1.
Fig. 4 is a comparison graph of diploid seeded luo han guo, triploid seedless luo han guo and pentaploid seedless luo han guo from bottom to top, respectively.
Detailed Description
The cultivation method of the present invention will be further described with reference to the following examples.
The Momordica grosvenori seeds were obtained from seedling farms of the Cheng Bio Inc. of Huacheng, Hunan.
Example 1
Selecting a momordica grosvenori variety with larger fruits (70-80 g of single fruits) and the sweet glycoside V content of about 0.46%, peeling 120 seeds, cleaning the momordica grosvenori variety in tap water, putting the momordica grosvenori variety in a container after peeling, washing the momordica grosvenori variety with sterile water for 3 times until liquid is clear, adding 75% alcohol for disinfection for 2 minutes, adding 40% sodium hypochlorite after pouring off the alcohol, putting the momordica grosvenori variety in a shaking table for disinfection at 150rpm for 20 minutes, finally washing the momordica grosvenori variety with sterile water for 5 times, and pouring off the water for later use.
Placing 8-10 layers of neutral sterile filter paper in a sterile culture dish, adding sterile water until the sterile filter paper is completely immersed and no water flows properly, placing the sterilized fructus Siraitiae Grosvenorii seeds on the filter paper, covering the filter paper with a cover, sealing, culturing at 36-38 deg.C in dark for 2 days, and culturing at 25-26 deg.C for 5 days.
100 seeds from which embryos started to germinate during pre-culture were selected, placed in a sterile petri dish with sterile filter paper, and cultured for 24 hours in MS liquid medium with colchicine concentration of 4 mg/L.
After the culture is finished, the seeds are taken out of the culture dish and then are inserted into a triangular flask added with an MS basic culture medium for culture, the endosperm is downwards inserted into the culture medium, only the embryo is exposed on the culture medium, the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
And observing bud characteristics when the momordica grosvenori seeds germinate to 5-8 cm, taking the momordica grosvenori seedlings with thick stems and wide leaves for counting the survival rate, and taking young roots grown by tissue culture for chromosome multiple identification by adopting a tabletting method.
The identification result is that 54 plants are survived together, and 19 plants are identified to be tetraploid momordica grosvenori.
And placing identified tetraploid momordica grosvenori seedlings in an MS proliferation culture medium comprising an MS culture medium, 1 mg/L6-BA, 0.2mg/L, 30g/L sucrose and 8g/L agar powder for tissue culture and propagation for 2 generations to obtain 132 strains, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours per day, the illumination is 2000lux, and the dark culture is 8 hours.
And (3) placing the expanded tetraploid momordica grosvenori seedlings in an MS rooting culture medium comprising an MS culture medium, 0.1mg/L NAA, 0.5mg/L IBA, 30g/L sucrose and 8g/L agar powder for rooting culture, wherein the culture conditions comprise that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
After the tetraploid momordica grosvenori seedlings which have rooted are hardened, the seedlings are transplanted to a field for planting, diploid male flowers are also transplanted on the same day, and after the diploid male flowers bloom, diploid pollen is used for pollination, and the fruits which are obtained are triploid momordica grosvenori fruits.
Selecting triploid fructus Siraitiae Grosvenorii fruits after pollination, peeling off seeds, cleaning in tap water, peeling, placing in a container, washing with sterile water for 3 times until the liquid is clear, adding 75% alcohol for disinfection for 2 minutes, pouring off alcohol rapidly, adding 40% sodium hypochlorite, placing in a shaking table, sterilizing at 150rpm for 20 minutes, washing with sterile water for 5 times, and pouring off water for later use.
Placing 8-10 layers of neutral sterile filter paper in a sterile culture dish, adding sterile water until the culture dish is completely soaked and no water flows properly, placing the sterilized fructus Siraitiae Grosvenorii seeds on the filter paper, sealing, culturing at 36-38 deg.C in dark for 2 days, and culturing at 25-26 deg.C for 5 days.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, selecting 100 seeds of which the embryos start to germinate after being pre-cultured, putting the seeds into the sterile culture dish, and adding an MS liquid culture medium with colchicine concentration of 4mg/L for culturing for 24 hours.
Taking out the seeds from the culture dish, inserting the seeds into a triangular flask added with an MS basic culture medium for culture, inserting endosperm downwards into the culture medium to only expose the embryos on the culture medium, and carrying out dark culture for 8 hours under the culture conditions that the temperature is 25-28 ℃, the illumination is 16 hours every day, and the illumination is 2000 lux.
Observing bud characteristics when the Momordica grosvenori grows to 5-8 cm after germination, and taking the Momordica grosvenori seedling with thick stem and wide leaf, and performing chromosome multiple identification by conventional tabletting method to obtain the hexaploid Momordica grosvenori seedling.
The result of the identification is 47 strains which survive together, wherein 11 strains are identified as hexaploid.
Placing the identified six-ploid momordica grosvenori seedlings in an MS proliferation culture medium of an MS culture medium, 1 mg/L6-BA, 0.2mg/L, 35g/L sucrose and 7g/L agar powder for tissue culture and propagation for 1 generation to obtain 97 strains, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours per day, the illumination is 2000lux, and the dark culture is 8 hours.
Hardening the tetraploid momordica grosvenori seedlings and the hexaploid momordica grosvenori seedlings, and transplanting the seedlings into a field.
And (3) pollinating the hexaploid female flowers with the tetraploid male flowers, and reserving the seeds after fruit bearing is mature as the pentaploid momordica grosvenori seeds.
And (3) germinating the remained pentaploid momordica grosvenori seeds in a thermostat, transplanting the seeds with diploid female plants, diploid male plants and triploid female plants to a field, pollinating the female plants with diploid pollen on the day of flowering, and obtaining the pentaploid seedless momordica grosvenori, diploid seeded momordica grosvenori and triploid seedless momordica grosvenori after fruiting. After the fruit stem turns yellow and matures, 1-2 fruits with better maturity are selected from different plants of each variety, and 100 fruits are selected from each variety.
The content of the sweet glycoside V in the pentaploid seedless momordica grosvenori, the diploid seedless momordica grosvenori and the triploid seedless momordica grosvenori are respectively detected by an HPLC method (see the figures 1 to 3). Fig. 4 is a comparison graph of diploid seeded luo han guo, triploid seedless luo han guo and pentaploid seedless luo han guo from bottom to top, respectively.
Measuring the number of each plant by using 30 plants, calculating the total output per mu according to the number of 120 plants per mu, measuring the average weight of 100 fruits of each variety, cutting each fruit, uniformly mixing one eighth of the cut fruits, crushing the mixture, extracting 200g of the mixture, and detecting the stevioside V. The measurement results are respectively as follows:
example 2
Selecting a momordica grosvenori variety with larger fruits and the content of the stevioside V of about 0.45 percent, peeling 120 seeds, cleaning the momordica grosvenori variety in tap water, placing the momordica grosvenori variety in a container after peeling, washing the momordica grosvenori variety with sterile water for 3 times until the liquid is clear, adding 75 percent alcohol for disinfection for 2 minutes, adding 40 percent sodium hypochlorite after pouring off the alcohol, placing the momordica grosvenori variety in a shaking table, disinfecting the momordica grosvenori variety with 150rpm for 20 minutes, finally washing the momordica grosvenori variety with the sterile water for 5 times, and pouring off the water for later use.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, adding sterile water until the sterile filter paper is completely immersed and no water flows properly, putting the sterilized momordica grosvenori seeds on the filter paper, covering the filter paper with a cover, sealing, culturing for 2 days under a dark condition at 38-40 ℃, and culturing for 5 days at 25-26 ℃.
100 seeds from which embryos started to germinate during pre-culture were selected, placed in a sterile petri dish with sterile filter paper, and cultured for 12 hours in MS liquid medium with colchicine concentration of 5 mg/L.
After the culture is finished, the seeds are taken out of the culture dish and then are inserted into a triangular flask added with an MS basic culture medium for culture, the endosperm is downwards inserted into the culture medium, only the embryo is exposed on the culture medium, the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
And observing bud characteristics when the momordica grosvenori seeds germinate to 5-8 cm, taking the momordica grosvenori seedlings with thick stems and wide leaves for counting the survival rate, and taking young roots grown by tissue culture for chromosome multiple identification by adopting a tabletting method.
The identification result is 37 plants which survive together, and 16 plants are identified as tetraploid momordica grosvenori.
And placing identified tetraploid momordica grosvenori seedlings in an MS proliferation culture medium comprising an MS culture medium, 1 mg/L6-BA, 0.2mg/L, 25g/L sucrose and 8g/L agar powder for tissue culture and propagation for 2 generations to obtain 97 strains, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours per day, the illumination is 2000lux, and the dark culture is 8 hours.
And (3) placing the expanded tetraploid momordica grosvenori seedlings in an MS rooting culture medium comprising an MS culture medium, 0.1mg/L NAA, 0.5mg/L IBA, 25g/L sucrose and 8g/L agar powder for rooting culture, wherein the culture conditions comprise that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
After the tetraploid momordica grosvenori seedlings which have rooted are hardened, the seedlings are transplanted to a field for planting, diploid male flowers are also transplanted on the same day, and after the diploid male flowers bloom, diploid pollen is used for pollination, and the fruits which are obtained are triploid momordica grosvenori fruits.
Selecting triploid fructus Siraitiae Grosvenorii fruits after pollination, peeling off seeds, cleaning in tap water, peeling, placing in a container, washing with sterile water for 3 times until the liquid is clear, adding 75% alcohol for disinfection for 2 minutes, pouring off alcohol rapidly, adding 40% sodium hypochlorite, placing in a shaking table, sterilizing at 150rpm for 20 minutes, washing with sterile water for 5 times, and pouring off water for later use.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, adding sterile water until the sterile culture dish is completely soaked and no water flows properly, placing the sterilized momordica grosvenori seeds on the filter paper, sealing, culturing for 2 days at 38-40 ℃ in the dark, and culturing for 5 days at 25-26 ℃.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, selecting 100 seeds of which the embryos start to germinate after being pre-cultured, putting the seeds into the sterile culture dish, and adding an MS liquid culture medium with colchicine concentration of 5mg/L for culturing for 12 hours.
Taking out the seeds from the culture dish, inserting the seeds into a triangular flask added with an MS basic culture medium for culture, inserting endosperm downwards into the culture medium to only expose the embryos on the culture medium, and carrying out dark culture for 8 hours under the culture conditions that the temperature is 25-28 ℃, the illumination is 16 hours every day, and the illumination is 2000 lux.
Observing bud characteristics when the Momordica grosvenori grows to 5-8 cm after germination, and taking the Momordica grosvenori seedling with thick stem and wide leaf, and performing chromosome multiple identification by conventional tabletting method to obtain the hexaploid Momordica grosvenori seedling.
The identification result is 32 strains which survive together, wherein 12 strains are identified as hexaploid.
Placing identified six-ploid momordica grosvenori seedlings in an MS culture medium, 1 mg/L6-BA and 0.2mg/L MS proliferation culture medium for tissue culture and propagation for 1 generation to obtain 97 strains, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
Hardening the tetraploid momordica grosvenori seedlings and the hexaploid momordica grosvenori seedlings and transplanting the seedlings into a field.
And (3) pollinating the hexaploid female flowers with the tetraploid male flowers, and reserving the seeds after fruit bearing is mature as the pentaploid momordica grosvenori seeds.
And (3) germinating the remained pentaploid momordica grosvenori seeds in a thermostat, transplanting the seeds and diploid male plants to a field, pollinating female plants with diploid pollen on the day of blooming, and obtaining the pentaploid momordica grosvenori without seeds after fruiting. After the fruit stem turns yellow and matures, 1-2 fruits with better maturity are selected from different plants of each variety, and 100 fruits are selected from each variety
Respectively detecting the content of the glycoside V of the pentaploid seedless momordica grosvenori seeds by using an HPLC method. After 100 fruits of each variety are measured for average weight, cutting each fruit, taking one eighth of the fruits, uniformly mixing and crushing the fruits, taking 200g of the fruits for extraction, and detecting the stevioside V. The measured result is as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.57 |
| Average weight (g) of 100 fruits | 69.52 |
| Mu number yield (one) | 9747 |
| Sweet glycosides produced per mu V Total amount (g) | 3898.8 |
Example 3
Selecting a momordica grosvenori variety with larger fruits and the content of the stevioside V of about 0.45 percent, peeling 120 seeds, cleaning the momordica grosvenori variety in tap water, placing the momordica grosvenori variety in a container after peeling, washing the momordica grosvenori variety with sterile water for 3 times until the liquid is clear, adding 75 percent alcohol for disinfection for 2 minutes, adding 40 percent sodium hypochlorite after pouring off the alcohol, placing the momordica grosvenori variety in a shaking table, disinfecting the momordica grosvenori variety with 150rpm for 20 minutes, finally washing the momordica grosvenori variety with the sterile water for 5 times, and pouring off the water for later use.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, adding sterile water until the sterile filter paper is completely immersed and no water flows properly, putting the sterilized momordica grosvenori seeds on the filter paper, covering the filter paper with a cover, sealing, culturing for 2 days under the dark condition of 35-37 ℃, and culturing for 5 days at 25-26 ℃.
100 seeds from which embryos started to germinate during pre-culture were selected, placed in a sterile petri dish with sterile filter paper, and cultured for 48 hours in MS liquid medium with colchicine concentration of 3 mg/L.
After the culture is finished, the seeds are taken out of the culture dish and then are inserted into a triangular flask added with an MS basic culture medium for culture, the endosperm is downwards inserted into the culture medium, only the embryo is exposed on the culture medium, the culture conditions are that the temperature is 25-26 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
And observing bud characteristics when the momordica grosvenori seeds germinate to 5-8 cm, taking the momordica grosvenori seedlings with thick stems and wide leaves for counting the survival rate, and taking young roots grown by tissue culture for chromosome multiple identification by adopting a tabletting method.
The identification result is 49 plants which survive together, wherein 21 plants are identified to be tetraploid momordica grosvenori.
And placing identified tetraploid momordica grosvenori seedlings in an MS culture medium, 1 mg/L6-BA and 0.2mg/L MS proliferation culture medium for tissue culture and propagation for 2 generations to obtain 138 plants, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
And (3) placing the expanded tetraploid momordica grosvenori seedlings in an MS rooting culture medium of an MS culture medium, 0.1mg/L NAA and 0.5mg/L IBA for rooting culture, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
After the tetraploid momordica grosvenori seedlings which have rooted are hardened, the seedlings are transplanted to a field for planting, diploid male flowers are also transplanted on the same day, and after the diploid male flowers bloom, diploid pollen is used for pollination, and the fruits which are obtained are triploid momordica grosvenori fruits.
Selecting triploid fructus Siraitiae Grosvenorii fruits after pollination, peeling off seeds, cleaning in tap water, peeling, placing in a container, washing with sterile water for 3 times until the liquid is clear, adding 75% alcohol for disinfection for 2 minutes, pouring off alcohol rapidly, adding 40% sodium hypochlorite, placing in a shaking table, sterilizing at 150rpm for 20 minutes, washing with sterile water for 5 times, and pouring off water for later use.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, adding sterile water until the sterile culture dish is completely soaked and no water flows properly, placing the sterilized momordica grosvenori seeds on the filter paper, sealing, culturing for 2 days at 35-37 ℃ in the dark, and culturing for 5 days at 25-26 ℃.
Putting 8-10 layers of neutral sterile filter paper into a sterile culture dish, selecting 100 seeds of which the embryos start to germinate after being pre-cultured, putting the seeds into the sterile culture dish, and adding an MS liquid culture medium with colchicine concentration of 3mg/L for culturing for 24 hours.
Taking out the seeds from the culture dish, inserting the seeds into a triangular flask added with MS basic culture medium for culture, inserting endosperm downwards into the culture medium to only expose the embryos on the culture medium, and carrying out dark culture for 8 hours under the culture conditions that the temperature is 34-36 ℃, the illumination is 16 hours every day, and the illumination is 2000 lux.
Observing bud characteristics when the Momordica grosvenori grows to 5-8 cm after germination, and taking the Momordica grosvenori seedling with thick stem and wide leaf, and performing chromosome multiple identification by conventional tabletting method to obtain the hexaploid Momordica grosvenori seedling.
The identification result is 53 strains which are survived together, wherein 15 strains are identified to be hexaploid.
Placing identified six-ploid momordica grosvenori seedlings in an MS culture medium, 1 mg/L6-BA and 0.2mg/L MS proliferation culture medium for tissue culture and propagation for 1 generation to obtain 97 strains, wherein the culture conditions are that the temperature is 25-28 ℃, the illumination is 16 hours every day, the illumination is 2000lux, and the dark culture is 8 hours.
Hardening the tetraploid momordica grosvenori seedlings and the hexaploid momordica grosvenori seedlings and transplanting the seedlings into a field.
And (3) pollinating the hexaploid female flowers with the tetraploid male flowers, and reserving the seeds after fruit bearing is mature as the pentaploid momordica grosvenori seeds.
And (3) germinating the remained pentaploid momordica grosvenori seeds in a thermostat, transplanting the seeds and diploid male plants to a field, pollinating female plants with diploid pollen on the day of blooming, and obtaining the pentaploid seedless momordica grosvenori, diploid seeded momordica grosvenori and triploid seedless momordica grosvenori after fruiting. After the fruit stem turns yellow and matures, 1-2 fruits with better maturity are selected from different plants of each variety, and 100 fruits are selected from each variety
And respectively detecting the content of the pentaploid seedless momordica glycoside V by using an HPLC method. After 100 fruits of each variety are measured for average weight, cutting each fruit, taking one eighth of the fruits, uniformly mixing and crushing the fruits, taking 200g of the fruits for extraction, and detecting the stevioside V. The measured result is as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.60 |
| Average weight (g) of 100 fruits | 67.82 |
| Mu number yield (one) | 9762 |
| Sweet glycosides produced per mu V Total amount (g) | 3972.4 |
Example 4
The other conditions and procedures were the same as in example 1 except that in the preculture after the initial sterilization of the momordica grosvenori seeds and the preculture after the sterilization of the triploid momordica grosvenori seeds, the preculture conditions were changed to dark conditions at 36 to 38 ℃ for 4 days, and then the preculture conditions were changed to dark conditions at 25 to 26 ℃ for 4 days. The field test was carried out under the same conditions, and the results were as follows:
example 5
The other conditions and procedures were the same as in example 1 except that in the preculture after the initial sterilization of the momordica grosvenori seeds and the preculture after the sterilization of the triploid momordica grosvenori seeds, the preculture conditions were changed to dark conditions at 33 to 35 ℃ for 2 days, and then to dark conditions at 25 to 26 ℃ for 5 days. The field test was carried out under the same conditions, and the results were as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.59 |
| Average weight (g) of 100 fruits | 66.49 |
| Mu number yield (one) | 9685 |
| Sweet glycosides produced per mu V Total amount (g) | 3799.3 |
Example 6
The other conditions and procedure were the same as in example 1 except that in the preliminary culture after sterilization of the original lo han guo seeds and the preliminary culture after sterilization of the triploid lo han guo seeds, the preliminary culture conditions were changed to dark conditions at 36-38 ℃ for 7 days. The field test was carried out under the same conditions, and the results were as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.56 |
| Average weight (g) of 100 fruits | 67.26 |
| Mu number yield (one) | 9847 |
| Sweet glycosides produced per mu V Total amount (g) | 3708.9 |
Example 7
The other conditions and procedure were the same as in example 1 except that in the preliminary culture after sterilization of the original lo han guo seeds and the preliminary culture after sterilization of the triploid lo han guo seeds, the preliminary culture conditions were changed to dark conditions at 25-27 ℃ for 7 days. The field test was carried out under the same conditions, and the results were as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.56 |
| Average weight (g) of 100 fruits | 67.48 |
| Mu number yield (one) | 9638 |
| Sweet glycosides produced per mu V Total amount (g) | 3642.1 |
Example 8
The other conditions and procedures were the same as in example 1 except that when tetraploid Lo Han Guo and hexaploid Lo Han Guo were crossed, the male flowers of the hexaploid were pollinated by the female flowers of the tetraploid. The field test was carried out under the same conditions, and the results were as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.57 |
| Average weight (g) of 100 fruits | 68.83 |
| Mu number yield (one) | 9815 |
| Sweet glycosides produced per mu V Total amount (g) | 3850.7 |
Comparative example 1
The other conditions and procedures were the same as in example 1, except that a Lo Han Guo variety with a large fruit size (70-80 g of a single fruit) and a glycoside V content of about 0.46% was selected, and the double induction treatment was directly performed without pre-culture after the sterilization treatment. The field test was carried out under the same conditions, and the results were as follows:
comparative example 2
The other conditions and procedures were the same as in example 1 except that after the sterilization of triploid momordica grosvenori seeds, the double induction treatment was directly performed without pre-culture. The field test was carried out under the same conditions, and the results were as follows:
| pentaploid seedless momordica grosvenori | |
| Sweet glycoside V content (%) | 0.55 |
| Average weight (g) of 100 fruits | 67.32 |
| Mu number yield (one) | 9630 |
| Sweet glycosides produced per mu V Total amount (g) | 3565.6 |
Note: each of the examples and comparative examples described above used the same diploid Lo Han Guo batch as a control.
Claims (7)
1. A cultivation method of a pentaploid seedless momordica grosvenori comprises the following steps: the diploid momordica grosvenori is subjected to double induction treatment to obtain tetraploid momordica grosvenori, and the tetraploid momordica grosvenori is hybridized with the triploid momordica grosvenori subjected to double induction treatment to obtain the pentaploid momordica grosvenori; the method comprises the following steps of (1) taking diploid momordica grosvenori seeds for pre-culture; hybridizing the diploid momordica grosvenori and the tetraploid momordica grosvenori to obtain triploid momordica grosvenori, and pre-culturing seeds of the triploid momordica grosvenori; after pre-culture, carrying out double induction treatment; the pre-culture is to culture the disinfected fructus momordicae seeds for 1 to 2 days at 36 to 38 ℃ under the sterile condition in the dark, and then culture the fructus momordicae seeds for 4 to 5 days at 25 to 26 ℃; the double induction treatment is to insert momordica grosvenori seeds into a colchicine MS liquid culture medium for treatment, wherein the concentration of colchicine is 3-5 mg/L; the induction treatment is carried out under the conditions of 25-28 ℃, 16-20 hours of illumination per day, 2000-3000lux of illumination, and 4-8 hours of dark culture.
2. The cultivation method as claimed in claim 1, wherein the MS liquid medium is MS medium +25-35g/L sucrose and has a pH value of 5.5-6.
3. The method of claim 1, wherein the hybridization to obtain the pentaploid Lo Han Guo is pollination of hexaploid female flowers with tetraploid male flowers.
4. The method of claim 1, wherein crossing diploid Lo Han Guo and tetraploid Lo Han Guo is performed by pollinating tetraploid female flowers with diploid male flowers.
5. A cultivation method of a pentaploid seedless momordica grosvenori comprises the following steps:
(1) peeling, cleaning and disinfecting momordica grosvenori seeds, pre-culturing, performing multiple induction treatment and culture, and performing chromosome multiple identification after the momordica grosvenori seeds germinate and grow;
(2) carrying out tissue culture propagation culture and rooting culture on identified tetraploid momordica grosvenori seedlings;
(3) hardening the seedling of the tetraploid momordica grosvenori after rooting and planting the diploid momordica grosvenori together, hybridizing the diploid momordica grosvenori and the tetraploid momordica grosvenori, and obtaining triploid momordica grosvenori as the fruit;
(4) peeling the seeds of the fruits obtained in the step (3), cleaning, disinfecting and pre-culturing; performing multiple induction treatment and culture, and performing chromosome multiple identification after the fructus momordicae seeds germinate and grow;
(5) placing the identified six-ploid Grosvenor momordica fruit seedlings in an MS proliferation culture medium for tissue culture propagation culture and rooting culture;
(6) transplanting the tetraploid momordica grosvenori seedlings in the step (2) and the hexaploid momordica grosvenori seedlings in the step (5) into a field for hybridization, and obtaining the pentaploid momordica grosvenori fruits; taking seeds in the fruit of the polyploid momordica grosvenori to germinate and then cultivating the seeds in a field, remaining female plants after blooming, removing male plants, pollinating the polyploid momordica grosvenori by diploid pollen, and obtaining the polyploid seedless momordica grosvenori after fruiting; the pre-culture in the step (1) and the step (4) is to activate the disinfected fructus momordicae seeds for 1 to 2 days at 36 to 38 ℃ under the dark condition and culture the fructus momordicae seeds for 4 to 5 days at 25 to 26 ℃ under the aseptic condition;
in the step (1) and the step (4), the multiple induction treatment refers to that the momordica grosvenori seeds are inserted into an MS liquid culture medium of colchicine for treatment, and the concentration of the colchicine is 3-5 mg/L.
6. The cultivation method as claimed in claim 5, wherein the conditions of the induction treatment are 25-28 ℃, 16-20 hours per day under light, 2000-3000lux of illumination, 4-8 hours of dark cultivation; the MS liquid culture medium is MS culture medium plus 25-35g/L sucrose, and the pH value is 5.5-6.
7. The method of claim 5, wherein the crossing of diploid Lo Han Guo and tetraploid Lo Han Guo in step (3) is performed by pollinating tetraploid female flowers with diploid male flowers; in the step (6), the tetraploid momordica grosvenori and the hexaploid momordica grosvenori are hybridized to pollinate the hexaploid female flowers by the tetraploid male flowers.
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