CN104488711A - Method for obtaining intervarietal hybrids of eustoma grandiflorum rapidly - Google Patents
Method for obtaining intervarietal hybrids of eustoma grandiflorum rapidly Download PDFInfo
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Abstract
The invention discloses a method obtaining intervarietal hybrids of eustoma grandiflorum rapidly. The method comprises the following steps of: hybridizing male and female parents of different varieties of eustoma grandiflorum, and taking embryos of seeds, wherein the embryos of seeds serve as explants; putting the embryos into an inoculation culture medium, culturing, and inoculating each bottle with 10-15 embryos; transferring the embryos to a subculture medium in 35 days, and subculturing to obtain seedlings; transferring the seedlings to a differential medium, and propagating to obtain more seedlings; transferring the seedlings to a rooting medium, rooting, and screening to obtain single plants; transplanting the single plants, and managing the living plants of eustoma grandiflorum normally to obtain the first-generation novel hybrids of eustoma grandiflorum. By adopting the embryo culture technique and the tissue culture method, excellent hybrids of eustoma grandiflorum can be obtained in only 0.5-1 year, and the breeding time can be shortened greatly.
Description
Technical field
The invention belongs to technical field of tissue culture, relate to cross breeding method, be specifically related to a kind of method of quick acquisition Lisianthus variety difference.
Background technology
Lisianthus, strain state is merrily and lightheartedly stagnant spills, and pattern elegance is lucid and lively, and flower shape uniqueness is lovely, is all unanimously one of potted flower all the fashion in the world and cut-flower kind.
Lisianthus originates in United States Nebraska and Texas, and property happiness is warm, moistening and sunny.More cold-resistant, not water-fast wet.Introduce a fine variety European and Japanese by crossbreeding and improvement, now become seductively charming moving, abnormal novel flowers.Wherein Dutch Ke Saxinzhadeng (K.SahinZaden) company and Ji Futebuluomaizhadeng (KieftBloemzaden) company are that the breeding of Lisianthus and the popularization of new varieties are done a lot of work.The florist's shop of European Countries and the windowsill of family are found everywhere.In Japan, sakata company of world-renowned flower seed company achieves preeminent accomplishment in Lisianthus breeding.
At present, intervarietal cross breeding is still one of main method of the seed selection of Flower New Variety kind.The selection cross of external Lisianthus has been carried out more than 100 year; the large quantities of new varieties of seed selection; and be domesticly also in the stage of introducing a fine variety now; manyly obtain crossbreed technology by hybridization means and also fail to break through; as, there is after fertilization obstacle in the intervarietal cross of Lisianthus part, causes hybridization to can not get seed; have a strong impact on the recombinant of the favorable genes such as balloonflower root pattern, leaf look, therefore solved this difficult problem and balloonflower root breeding of new variety is had great importance.
And utilize embryo culture technique to overcome after fertilization obstacle between Lisianthus kind, directly can obtain hybrid new breed.Production is applied to acceleration Lisianthus special something lost resource technical support is provided.
Lisianthus cottage propagation coefficients comparison is low, and in addition because of the many employings of seminal propagation is F1 generation seed, and offspring's segregation ratio is comparatively large, makes variet complexity quite serious, therefore advocates and adopt tissue culture propagation method, make the proterties that offspring keeps good.Therefore utilize tissue-culturing quick-propagation, batch production high quality seedling has important practical value.
Many scientific research institutions and production unit adopt the stem section of Lisianthus and blade to carry out the Fast-propagation of seedling as explant tissue culture propagation method.Disclose in the literary composition of " tissue-culturing rapid propagation of Lisianthus " (Zhou Junying, agricultural science and technology communication, 2004,5) and Lisianthus seed is carried out technology numerous soon as explant to it; " effect of plant hormone in Lisianthus tissue-culturing rapid propagation " (Zhan Gesheng, Agriculture of Anhui is circulated a notice of, AnhuiAgri.Sci.Bull.2004,10 (6): 52-53,59) what choose in a literary composition be Lisianthus young leaflet tablet is explant, adopt different hormones to process it and study the effect of hormone in Lisianthus tissue-culturing rapid propagation, visible, the stem section of Lisianthus, blade and seed can, as the explant in tissue culture procedures, also not utilize balloonflower root embryo to carry out tissue cultures to obtain the research of variety difference as explant to it at present.
Summary of the invention
For present situation; the present invention will provide a kind of method embryo through fertilization being obtained fast balloonflower root variety difference as explant by tissue culture technique; provide not only a kind of method of acquisition Lisianthus new varieties completely newly; the problem of its squamous subculture variation can also be solved; survival rate is higher; not by the impact in season, significantly reduce propagation cost, meet large-scale production needs.
In order to solve the problem, the invention provides a technical scheme as follows:
A method for quick acquisition Lisianthus variety difference, comprises the following steps:
(1) father, female parent are carried out bagging isolation, the next morning in one day before flowering, get fresh paternal pollen and female parent is pollinated, after pollination, carry out bagging isolation;
(2) after winning pollination, achene is pollinated, superclean bench takes out ovary, after by ovary running water 5 ~ 10min, after using the ethanol disinfection 30 ~ 50s of 70 ~ 75% again, be placed in the mercuric chloride solution 4 ~ 5min of 0.1%, then use aseptic water washing 5-6 time, after flushing, scratch seed coat with aseptic blade, get its embryo as explant;
(3) explant is placed in inoculation medium to cultivate, every bottle graft kind embryo 10-15; Proceed to after 35d in subculture medium and carry out squamous subculture, forward to after obtaining seedling in differential medium expand numerous, expand numerous obtain seedling after, transferred to by seedling in root media and carry out culture of rootage, screening obtains monomer plant;
(4) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 4d, healthy and strong and when having 5-7 sheet true leaf, 6-8cm height until Lisianthus seedling, then untie sealed membrane, hardening 6d, be transplanted in the matrix of sterilization, temperature controls at 24-26 DEG C, front 7d humid control, about 70%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions;
(5) field management: by the Lisianthus seedling normal management of transplant survival, obtain first-filial generation Lisianthus new varieties.
In above-mentioned steps two, get the time of ovary for 8 ~ 12d after pollination.
In described step 3, inoculation medium is: MS+2,4-D 2mg/L+6-BA 1.5mg/L;
The medium of above-mentioned squamous subculture is MS+6-BA 1.0mg/L+NAA 0.1mg/L;
Above-mentioned differential medium consists of MS+6-BA 0.2mg/L;
Above-mentioned root media consists of 1/2MS+NAA0.1 ~ 0.2mg/L+IBA0.1 ~ 0.2mg/L+ sucrose or white granulated sugar 20 ~ 30g/L+ agar 7 ~ 8g/L.
The condition of culture of above-mentioned inoculated and cultured is temperature 24-26 DEG C, pH is 6.3, and the photoperiod is that 14h illumination/10h is dark, and light intensity is 1200Lx;
The condition of culture that above-mentioned squamous subculture and differentiation are cultivated is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx;
The condition of culture of above-mentioned culture of rootage is cultivation temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6,14-18h/d, intensity of illumination 1400Lx.
Beneficial effect of the present invention:
Utilize embryo culture technique to overcome after fertilization obstacle between Lisianthus kind, directly can obtain hybrid new breed, production is applied to the special something lost resource of acceleration Lisianthus and provides technical support.
The tradition plantation needs time of about 1 year could harvest to some extent, and utilizes tissue culture technique that the time of Lisianthus about half a year can be made just to gather in the crops, and substantially reduces breeding cycle, utilizes continuous print shoot proliferation, have effect fast and effectively.Traditional breeding way wants to obtain excellent Hybrid, need through continuous 4 ~ 5 generations hybridization, about needs time of 3 ~ 5 years could the stable crossbreed of acquired character, and the present invention directly obtains hybrid new breed by embryo culture technique, substantially reduces breeding time.
The present invention, while acquisition crossbreed, can also obtain the plant of detoxification, have good resistance, is not easily infected by the virus, and reduce extermination of disease and insect pest spent cost, the survival rate of seedling is also very high, therefore plays the effect of killing two birds with one stone.
Embodiment
Embodiment 1 one kinds obtains the method for Lisianthus variety difference fast, comprises the following steps:
(1) select cultivar " Lu Xita " respectively as male parent A, tree peony type great Hua purple kind " mythology " as maternal B, growth selection is vigorous, the plant that petal is large, before flowering
Within one day, carry out bagging isolation, the next morning, get fresh paternal pollen and female parent is pollinated, after pollination, carry out bagging isolation;
(after 2 pollinations, 12d wins the rear achene of pollination, superclean bench takes out ovary, after ovary is used running water 10min, after using the ethanol disinfection 50s of 70% again, be placed in the mercuric chloride solution 4 ~ 5min of 0.1%, then use aseptic water washing 5-6 time, after flushing, scratch seed coat with aseptic blade, get its embryo as explant;
(3) explant is placed in inoculation medium to cultivate, every bottle graft kind embryo 10; Inoculation medium is: MS+2,4-D 2mg/L+, 6-BA 1.5mg/L; Condition of culture is temperature 24-26 DEG C, pH is 6.3, and the photoperiod is that 14h illumination/10h is dark, and light intensity is 1200Lx;
(4), after cultivating 35d in inoculation medium, proceed in subculture medium and carry out squamous subculture.Medium is MS+6-BA 1.0mg/L+NAA 0.1mg/L; Condition of culture is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx;
(5) differentiation cultivate: forward to after obtaining seedling after inoculation in differential medium expand numerous.Differential medium consists of MS+6-BA 0.2mg/L; Condition of culture is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx;
(6) culture of rootage: expand numerous obtain seedling after, transferred to by seedling in root media and carry out culture of rootage, screening obtains monomer plant; Root media consists of 1/2MS+NAA0.1mg/L+IBA0.2mg/L+ white granulated sugar 30g/L+ agar 7g/L.Cultivation temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6,14-18h/d, intensity of illumination 1400Lx.
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 4d, healthy and strong and when having 5-7 sheet true leaf, 6-8cm height until Lisianthus seedling, then untie sealed membrane, hardening 6d, be transplanted in the matrix of sterilization, temperature controls at 24-26 DEG C, front 7d humid control, about 70%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions.
(8) field management: after the Lisianthus seedling field planting 15d of transplant survival, seedling-slowing stage terminates, and now removes sunshade net, in conjunction with watering, executes thin fertile 1 time, because Lisianthus prefers the environment of warm and moist, therefore sprays water 2 times with mist watering can blade face every day.
Rich water quality management: every 7d executes ammonium fertilizer 1 time, every 667m
2fertilizing amount is 4 ~ 5kg.At the beginning of 5 months, intensity of illumination strengthens gradually, covers the sunshade net of 50% outward to reduce illumination burning to seedling, will strengthen ventilating management, be beneficial to the elongation growth of seedling stem in greenhouse at greenhouse film.When seedling grows to 20cm, the rhombus net weaved into nylon rope carries out drawing in the net process, prevents flower seedling from lodging, draws 1 layer of net later, draw 3 layers altogether every 20cm.
Blooming period management: from April 15, each kind is buddingged successively, Lisianthus enter nourish and grow and reproductive growth go forward side by side the phase.The Lisianthus in this period, except nitrogen fertilizer application, should increase the amount of application of phosphorus potash fertilizer, weekly the KH of foliage-spray 1 ‰
2pO
41 time, reduce blade face water spray number of times gradually, wet between dry between water management, not ponding in furrow.When there being bud open, stopping blade face water spray, preventing ponding fickle in love from rotting.
The control of damage by disease and insect: Lisianthus resistant to diseases and insects is comparatively strong, and in whole vegetative period, part kind has graywall to occur, and can spray carbendazol wettable powder 800 times of liquid and mancozeb 1000 times of liquid of 50%, every 3d sprays 1 time, it is better that 2 kinds of medicaments are used alternatingly effect.After 2 weeks, graywall is controlled, and after 20d, disease disappears completely.
In addition, the primary pest of harm Lisianthus is Liriomyza and black cutworm, Liriomyza bores food Lisianthus blade, black cutworm stings the root of food Lisianthus, control Liriomyza can the clean emulsion of fly mite of foliage-spray 500 ~ 800 times, poisoning black cutworm can with the phoxim baythion breast liquid irrigating root of 800 ~ 1000 times, and 1 time weekly, insecticidal effect is more remarkable.
(9) the Lisianthus new varieties obtained and parent's cultivated character are compared in table 1.
Table 1
As can be seen from the comparison result, the crossbreed that the present invention obtains, pattern is beautiful, Hua Jing great, and ornamental value is high, and number of on average blooming is many, and seedling stage, growing way was vigorous, showed except hybrid vigour.Compare with conventional breeding methods, substantially reduce breeding time.
Embodiment 2 one kinds obtains the method for Lisianthus variety difference fast, comprises the following steps:
(1) select cultivar " mini " as male parent A respectively, " A Linna 3 ", as maternal B, growth selection is vigorous, the plant that petal is large, within one day, carry out bagging isolation before flowering, the next morning, get fresh paternal pollen and female parent is pollinated, after pollination, carry out bagging isolation;
(after 2 pollinations, 10d wins the rear achene of pollination, superclean bench takes out ovary, after ovary is used running water 10min, after using the ethanol disinfection 50s of 75% again, be placed in the mercuric chloride solution 4 ~ 5min of 0.1%, then use aseptic water washing 5-6 time, after flushing, scratch seed coat with aseptic blade, get its embryo as explant;
(3) explant is placed in inoculation medium to cultivate, every bottle graft kind embryo 15; Inoculation medium is: MS+2,4-D 2mg/L+6-BA 1.5mg/L; Condition of culture is temperature 24-26 DEG C, pH is 6.3, and the photoperiod is that 14h illumination/10h is dark, and light intensity is 1200Lx;
(4), after cultivating 35d in inoculation medium, proceed in subculture medium and carry out squamous subculture.Medium is MS+6-BA 1.0mg/L+NAA 0.1mg/L; Condition of culture is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx;
(5) differentiation cultivate: forward to after obtaining seedling after inoculation in differential medium expand numerous.Differential medium consists of MS+6-BA 0.2mg/L; Condition of culture is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx;
(6) culture of rootage: expand numerous obtain seedling after, transferred to by seedling in root media and carry out culture of rootage, screening obtains monomer plant; Root media consists of 1/2MS+NAA0.1mg/L+IBA0.2mg/L+ white granulated sugar 30g/L+ agar 7g/L.Cultivation temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6,14-18h/d, intensity of illumination 1400Lx.
(7) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 4d, healthy and strong and when having 5-7 sheet true leaf, 6-8cm height until Lisianthus seedling, then untie sealed membrane, hardening 6d, be transplanted in the matrix of sterilization, temperature controls at 24-26 DEG C, front 7d humid control, about 70%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions.
(8) field management: after the Lisianthus seedling field planting 15d of transplant survival, seedling-slowing stage terminates, and now removes sunshade net, in conjunction with watering, executes thin fertile 1 time, because Lisianthus prefers the environment of warm and moist, therefore sprays water 2 times with mist watering can blade face every day.
Rich water quality management: every 7d executes ammonium fertilizer 1 time, every 667m
2fertilizing amount is 4 ~ 5kg.At the beginning of 5 months, intensity of illumination strengthens gradually, covers the sunshade net of 50% outward to reduce illumination burning to seedling, will strengthen ventilating management, be beneficial to the elongation growth of seedling stem in greenhouse at greenhouse film.When seedling grows to 20cm, the rhombus net weaved into nylon rope carries out drawing in the net process, prevents flower seedling from lodging, draws 1 layer of net later, draw 3 layers altogether every 20cm.
Blooming period management: from April 12, each kind is buddingged successively, Lisianthus enter nourish and grow and reproductive growth go forward side by side the phase.The Lisianthus in this period, except nitrogen fertilizer application, should increase the amount of application of phosphorus potash fertilizer, weekly the KH of foliage-spray 1 ‰
2pO
41 time, reduce blade face water spray number of times gradually, wet between dry between water management, not ponding in furrow.When there being bud open, stopping blade face water spray, preventing ponding fickle in love from rotting.
(9) the Lisianthus new varieties obtained and parent's cultivated character are compared in table 2.
Table 2
As can be seen from the comparison result, the crossbreed that same the present invention obtains, pattern is beautiful, Hua Jing great, and ornamental value is high, and number of on average blooming is many, and seedling stage, growing way was vigorous, showed hybrid vigour.
Conventional seed breeding method approximately needs the time of 3 ~ 5 years, and the present invention adopts embryo culture technique conjunctive tissue cultural method, only needs the time of 0.5 ~ 1 year just can obtain excellent crossbreed, substantially reduces breeding time.
Claims (4)
1. obtain a method for Lisianthus variety difference fast, it is characterized in that, comprise the following steps:
(1) father, female parent are carried out bagging isolation, the next morning in one day before flowering, get fresh paternal pollen and female parent is pollinated, after pollination, carry out bagging isolation;
(2) after winning pollination, achene is pollinated, superclean bench takes out ovary, after by ovary running water 5 ~ 10min, after using the ethanol disinfection 30 ~ 50s of 70 ~ 75% again, be placed in the mercuric chloride solution 4 ~ 5min of 0.1%, then use aseptic water washing 5-6 time, after flushing, scratch seed coat with aseptic blade, get its embryo as explant;
(3) explant is placed in inoculation medium to cultivate, every bottle graft kind embryo 10-15; Proceed to after 35d in subculture medium and carry out squamous subculture, forward to after obtaining seedling in differential medium expand numerous, expand numerous obtain seedling after, transferred to by seedling in root media and carry out culture of rootage, screening obtains monomer plant;
(4) acclimatization and transplants: first blake bottle is moved to greenhouse, natural conditions lower refining seedling 4d, healthy and strong and when having 5-7 sheet true leaf, 6-8cm height until Lisianthus seedling, then untie sealed membrane, hardening 6d, be transplanted in the matrix of sterilization, temperature controls at 24-26 DEG C, front 7d humid control, about 70%, reduces humidity later gradually, until under transplanted seedling is placed in natural conditions;
(5) field management: by the Lisianthus seedling normal management of transplant survival, obtain first-filial generation Lisianthus new varieties.
2. the method for a kind of quick acquisition Lisianthus variety difference according to claim 1, is characterized in that, in described step 2, gets the time of ovary for 8 ~ 12d after pollination.
3. the method for quick acquisition Lisianthus variety difference according to claim 1 and 2, it is characterized in that, in described step 3, inoculation medium is: MS+2,4-D 2mg/L+6-BA 1.5mg/L; The medium of described squamous subculture is MS+6-BA 1.0mg/L+NAA0.1mg/L; Described differential medium consists of MS+6-BA 0.2mg/L; Described root media consists of 1/2MS+NAA0.1 ~ 0.2mg/L+IBA0.1 ~ 0.2mg/L+ sucrose or white granulated sugar 20 ~ 30g/L+ agar 7 ~ 8g/L.
4. the method for quick acquisition Lisianthus variety difference according to claim 1 and 2, is characterized in that, the condition of culture of described inoculated and cultured is temperature 24-26 DEG C, pH is 6.3, and the photoperiod is that 14h illumination/10h is dark, and light intensity is 1200Lx; The condition of culture that squamous subculture and differentiation are cultivated is temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6.3,12h/d, intensity of illumination 1600Lx; The condition of culture of culture of rootage is cultivation temperature is 23 ± 1 DEG C, and pH is the photoperiod of 6,14-18h/d, intensity of illumination 1400Lx.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105265313A (en) * | 2015-10-13 | 2016-01-27 | 湖北大学 | Eustoma russellianum seedling cultivation method |
| CN110338052A (en) * | 2019-05-31 | 2019-10-18 | 张掖市绿之源农业发展有限公司 | A kind of Lisianthus cross-breeding method |
| CN113508694A (en) * | 2020-04-10 | 2021-10-19 | 辽宁省农业科学院 | Cuttage seedling raising method for tender branches with leaf frame net container |
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| CN110338052A (en) * | 2019-05-31 | 2019-10-18 | 张掖市绿之源农业发展有限公司 | A kind of Lisianthus cross-breeding method |
| CN113508694A (en) * | 2020-04-10 | 2021-10-19 | 辽宁省农业科学院 | Cuttage seedling raising method for tender branches with leaf frame net container |
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Application publication date: 20150408 |