CN107155866A - The method that perennial forage maize is cultivated using non-multiple corn allopolyploid - Google Patents
The method that perennial forage maize is cultivated using non-multiple corn allopolyploid Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses the method that perennial forage maize is cultivated using non-multiple corn allopolyploid, belong to forage maize breeding method field.The present invention is using the non-multiple corn allopolyploid MTP94 containing corn, friction standing grain and teosinte chromosome as female parent, directly selected as paternal hybrid, then in first-filial generation using corn, friction standing grain or tetraperennial teosinte and cultivate perennial forage maize.Non- multiple corn allopolyploid is used to cultivate perennial forage maize first by the inventive method, is that perennial forage maize cultivation opens new way；Secondly, the inventive method can make the intermolecular hybrid of different Related species materials, and Germplasm Bases are widened significantly, and the cross combination between the different plant species for adapting to different ecological environment can be designed as needed；In addition, the inventive method breeding cycle is short, only need first-filial generation directly to select kind, and by vegetative propagation hybrid vigor fixing, be not required to the production of hybrid seeds every year and sowing, breeding and production cost are low.
Description
Technical field
The invention belongs to perennial forage maize breeding method field, and in particular to utilize non-multiple corn allopolyploid
The method for cultivating perennial forage maize.
Background technology
Forage grass is the basis of careless animal husbandry development, is related to the quality and safety of livestock products.Low quality forage grass increase is fed into
This, and harm animal health.China frequently occurs in recent years milk, meat products quality security problem are all close with domestic forage grass shortage
Cut is closed.Grain is hidden in grass, grain, soil conservation, Xing Mu, rich people can be saved, with output efficiently, it is product safety, resources conservation, environment-friendly
The advantages of.At present, forage grass already turns into most potential industry in feed industry.
Corn (Zea mays L.) is because with higher biomass and nutritive value, being described as " king of feed ".Feeding jade
Rice is used as harvest object not for the purpose of harvesting seed using aerial part (stem, leaf and fruit).By the end of, state in 2016
Family's authorization fodder maize kind totally 22, wherein Zea 20, only 2 kinds of class Sorghum, including annual teosinte and
Beautiful grass 1.Zea veriety is cenospecies with beautiful grass 1, and annual teosinte is conventional variety.Except careless 1 extra of jade,
It is annual.Fodder maize based on cenospecies needs the annual production of hybrid seeds, annual to plant, and not only cultivation management is bothered, cost
Height, and seed selection cycle length, the slowly effect of self-mating system, and height relies on the seed selection experience of breeder, it is difficult to cultivate real work
For the kind of the quality and yield requirement of feed.
Compared with annual fodder maize, perennial forage grass has root system deeper, and carbon sequestration and water holding capacity are stronger, and needs
Fertilizer, water are less；Being planted in marginal land, black fallow need not cultivate, the advantages of plantation was benefited for many years in 1 year.But current rural areas of south China
Tall and big perennial grass lack, and single varieties are needed badly to cultivate and meet many of specific ecological environment and market development needs
Year raw forage grass new varieties.
In maize race, the affiliation of friction standing grain category and Zea is nearest.Corn (n=10) and friction standing grain dyeing
Body radix (n=18) is different, hybridizes not affinity, hybridizes extremely difficult success.Gama grass (finger-like friction standing grain is also known as,
Tripsacum dactyloids L., 2n=72) it is a kind of perennial warm season bunch type dogstail.Due to its well developed root system, for many years
Life, breeding cycle length, to hold green phase length, palatability good, in North America frequently as a kind of high quality forage (.Maize such as Eubanks
Genetics Cooperation
Newsletter.1993.67:41.).Further, since power of regeneration is strong, wide adaptability, can in acid, alkalescence and
Grown under wetland environmental condition, but also as a kind of excellent soil improvement and soil-and-water conservation effect (Jack R etc.
.Proceedings of the National Academy of Sciences. 1977.74(8):3494-3497).However,
Due to its there are problems that low setting percentage, easy shattering, make seeling industry not enough, significantly limit friction standing grain
Popularization and application.Diploid friction standing grain is zoogamy, and polyploid is facultative apomixis, mainly diploid sporogony,
I.e. intraovular archespore or megasporocyte meiosis form unreduced gamete body after being obstructed, and then direct development is formed
Embryo, then endosperm is formed through pseudogamy, finally develop into the seed (.Crop such as Kindiger Science.1996. 36 (2):250-
255.).The characteristic of this facultative apomixis also has after a small number of (1%~6%) sexual hybridizations in addition to maternal instinct offspring is produced
Generation, and part sexual progeny can still carry out the apomixia (.Apomixis such as Grimanelli Newsletter.1995.8:
35-37.), this just provided for the improvement of friction standing grain herbage variety and realization that its good characteristic is transferred into cultivated maize can
Energy.Its perennial root, apomixia and the degeneration-resistant, Resistant Gene that carry are the excellent kinds for cultivating perennial forage maize kind
Matter resource.Abundant leaf amount, a small amount of stalk are conducive to it to be processed into grass product, referred to as " king of dogstail " and " ice cream grass ".But
Its blade edge has sharp sawtooth, is unfavorable for artificial harvest；Early growth speed is slow, and grass yield is low.
Teosinte (Teosinte) is the general designation of all wild species in addition to cultivated maize kind in Zea, is and corn parent
The nearest class plant of edge relation, the offspring of fertility is resulted in corn hybridization.Annual teosinte tillering ability is strong, branch
Leaf is in great numbers, reproducibility is strong, can repeatedly cradle, and is the forage plant of high-quality.Tetraperennial teosinte (Z.perennis, 2n
=40) and Zea diploperennis (Z. diploperennis, 2n=20) has that perennial, tillering ability is strong, trophosome
In great numbers the features such as.Tetraperennial teosinte is the unique Tetraploid of Zea (2n=4x=40), with prosperity
Rhizome, the frost of resistance to severe and waterlogging, can as the Cold resistant genes for cultivating perennial forage maize excellent donor.
Corn growth gesture is strong, and friction standing grain has perennial characteristic, and tetraperennial teosinte has perennial and cold-resistant
Property, the characteristics of reproducibility is strong.How by the good characteristic of three species condense together cultivation perennial forage maize be always
The focus and difficult point of corn breeding concern.Tang of Sichuan Agricultural University is prayed woods and is equal to what is introduced with Beijing City Agriculture and Forestry Institute within 2006
Come from hybrid F of the tetraploid corn (corn is doubled) in the U.S. between gama grass1(2n=56) and tetraploid are for many years
Give birth to class corn hybridization and obtain ternary hybrid, have one plant of vigorous, tillering ability of growth strong, perennial in the ternary hybrid of gained
Ternary hybrid, is named as MTF-1 (Tripsazea creammaize T., 2n=76).MTF-1 chromosome number is 76
(wherein maize chromosome 20, tetraperennial teosinte chromosome 20, gama grass chromosome 36), is one
The individual allohexaploid containing corn, tetraperennial teosinte and a full set of genome of friction standing grain；MTF-1 not only overcomes and rubbed
Wiping standing grain setting percentage is low, percentage of seedgermination is low and the very thin shortcoming easily lodged of tetraperennial teosinte stalk, while also retaining
The wildnesses such as perennial, tiller many, vegetative growth phase length, show powerful hybrid in plant strain growth gesture and biological yield
Advantage (the expensive Sichuan Agricultural Universities Master's thesis of Su Yue, 2009).But the ternary kind power of regeneration is relatively weak, when surviving the winter
Temperature is easily dead less than -4 DEG C, influences the regeneration of Second Year, is unfavorable for utilizing it directly as perennial forage grass.Patent《Profit
The method that the perennial forage grass kind of aneuploid is cultivated with corn allopolyploid》(CN103548674A) disclose and utilize MTF-
1 method for cultivating perennial forage maize kind.
MTP94 (Tripsazea creammaize T., 2n=94) is that Sichuan Agricultural University utilizes MTF-1
The imperfect 2n gametes (2n=74) that (Tripsazea creammaize T., 2n=76) is produced and the perennial class of tetraploid are beautiful
The chromosome that n gametes (n=20) hybridization that rice (Z.perennis, 2n=40) 9475 is produced is synthesized by 2n+n mode of reproduction
Number is the perennial corn allopolyploid of non-multiple of 94, and its chromosome is configured to maize chromosome 20, rub standing grain dyeing
Body 34 and tetraperennial teosinte chromosome 40 (Dong Xingchun, Sichuan Agricultural University's Master's thesis, 2015).
MTP94 has the advantages that perennial and cold resistance is strong, but the shortcomings of there is low biological yield and poor regenerability, it is difficult to directly
It is used as forage maize application.
From the foregoing, have been disclosed in the prior art and cultivate perennial by bridge of the corn allopolyploid of ortholoidy
The method of forage maize kind, but do not find to cultivate perennial forage maize using the corn allopolyploid of aneuploidy
Method.
The content of the invention
The present inventor has been surprisingly found that non-multiple corn allopolyploid MTP94 has female part can in research process
The characteristic educated；Further research also found, with corn, tetraperennial teosinte or friction standing grain as male parent to MTP94
Pollination, can all obtain solid offspring, the present invention is exactly to be completed on the basis of having been surprisingly found that herein.Present invention aims at offer
A kind of method that perennial forage maize is cultivated using non-multiple corn allopolyploid.
The present invention is achieved through the following technical solutions：
The method that the present invention cultivates perennial forage maize using non-multiple corn allopolyploid, comprises the following steps：
(1), when spring ground temperature rises to more than 10 DEG C, to non-multiple corn allopolyploid (Tripsazea
Creammaize T.) carry out the breeding of point root and stem of certain plants, or by vegetative propagation sides such as stalk cuttage upper one year, press strip, underground layering or tissue cultures
The seedling that formula is obtained, is that 2m × 2m carries out single-strain planting by seeding row spacing；Meanwhile, plant male parent in batches around it；
(2), before spinning, its filigree is cut short to 1 by non-multiple corn allopolyploid female fringe bagging, and before pollination
~3cm, is pollinated with paternal pollen to non-multiple corn allopolyploid；Or under the conditions of isolation, make male parent beautiful to non-multiple
Rice allopolyploid free pollination；After harvesting, drying after seed maturity, -20 DEG C of refrigerators are stored in；
(3) seed obtained by step (2), is tedded into 3~4 hours under the sun, then planted in phjytotron
In nutritive cube, temperature be 25 DEG C~30 DEG C, humidity be to carry out vernalization under the conditions of 60%~80%；Treat seedling length to the heart of 3 leaf 1
When, the tip of a root is taken, is then that 2m × 2m carries out Plant colonization in crop field according to seeding row spacing；In Adult plant, selection plant height >=200cm,
Grass length >=250cm, base portion girth >=35cm, tiller number >=20, sugared brix >=10%, blade surface are without obvious scab, stalk
The upright plant without the individual plant green forage yield >=20kg that lodges, cradles；The individual plant of middle choosing is expanded numerous into nothing using stalk cottage method
Property system；
(4), carry out chromosome number identification to the clonal tip of a root selected by step (3), and using in situ hybridization (GISH) and
FISH (FISH) is constituted to clonal chromosome and identified, each 30~50 cells of clonal verification；Choosing
Total chromosome number is selected for 60~72, wherein maize chromosome number is 8~12, friction standing grain chromosome number is 14~18, four
Times perennial class maize chromosome number of body is the clone of 38~42；
(5) clone of wintering rate >=100% in the clone selected by step (4), is entered into variety comparative test；Seedling is stem
Stalk cuttage seeding or point root and stem of certain plants seedling, are had a competition using the product that the perennial forage maize kind of authorization or its parent are carried out at least 2 years as control
Test；Cradled respectively at taking out male phase beginning, selection green forage yield is higher than check variety 15%, and crude protein content >=9.0%DM, phase
To feeding value (RFV) >=80 strain, as new perennial forage maize kind.
Non- multiple corn allopolyploid described in above method step (1) or (2) refer to containing corn, friction standing grain and
The aneuploid ternary hybrid of tetraperennial teosinte chromosome.
Described non-multiple corn allopolyploid refer to MTP94 (Tripsazea creammaize T.2n=94) or
Person MTP94 Derivative line.
The total chromosome number of the MTP94 is 94, and its chromosome is configured to：Maize chromosome 20, friction standing grain chromosome
34 and tetraperennial teosinte chromosome 40.
Described MTP94 Derivative line refers to the maize chromosome contained 20, the friction standing grain being bred as by parent of MTP94
The aneuploid ternary hybrid of chromosome 34 and tetraperennial teosinte chromosome 40.
MTP94 source：Sichuan Agricultural University is produced with MTF-1 (Tripsazea creammaize T., 2n=76)
The reproduction that passes through 2n+n of the n gametes that produce of imperfect 2n gametes (2n=74) and tetraperennial teosinte (2n=40)
Mode is synthesized.Compared with MTF-1, MTP94 genome turns into maize chromosome 20, friction standing grain chromosome 34 and four times
The perennial class maize chromosome of body 40；2 friction standing grain chromosomes are wherein reduced, the perennial class of 20 tetraploids are added beautiful
Rice chromosome.
The public can obtain MTP94 from Sichuan Agricultural University.
Male parent described in above method step (1) or (2) refers to corn (Zea mays L.), friction standing grain
One kind of the material such as (Tripsacum L.) or teosinte (Teosinte).
Described corn (Zea mays L.) refers to one of corn inbred line, cenospecies, collective species or farm variety etc.
Kind.
Described teosinte refers to Diploid Perennial teosinte (Z.diploperennis, 2n=20), tetraploid for many years
Raw class corn (Z.perennis, 2n=40), maize seed in great numbers (Z.luxurians, 2n=20), Nicaragua's maize seed
(Z.nicaraguensis, 2n=20), zea mexicana subspecies (ssp.mexicana, 2n=20), Wei Weitenange subspecies
One of (ssp.huehuetenangensis, 2n=20) or small clever corn subspecies (ssp. parviglumis, 2n=20) etc.
Kind.
Described tetraperennial teosinte can be tetraperennial teosinte (Z.perennis,
2n=40) 9475.
Described friction standing grain (Tripsacum L.) refers to gama grass (Tripsacum dactyloides, 2n
=4x=72) or other friction standing grain species.
Described teosinte and friction standing grain material can be obtained from Mexico's wheat and corn improvement center (CIMMYT) is open.
A point root and stem of certain plants breeding described in above method step (1) or (5) refers to the seedling that turns green using plant base portion as planting stock
The method for carrying out vegetative propagation.
Cutting propagation described in above method step (1), (3) or (5) refers to carry out using the stipes of cut-out as skewer bar
The method of vegetative propagation.
Press strip described in above method step (1) refers to will after ripe tiller (stipes carries axillary bud) blade is rejected
Whole piece stem is buried in the earth or is only buried in the earth stipes, treats that eustipes part new talent was just cut off with maternal plant into the heart stage of three leaf one, with this
The method for carrying out vegetative propagation.
Underground layering described in above method step (1) refers to ripe tiller (stipes carries axillary bud) to be cut from maternal plant root
It is disconnected, whole tiller is buried in the earth, treats eustipes part new talent to be unearthed into the heart stage of three leaf one and cuts off, carried out with this asexual with female branch
The method of breeding.
Tissue cultures described in above method step (1) refer to have cell complete with the tip of a root, stem end, stipes, leaf, embryo etc.
The mitogenetic method for carrying out tissue culture propagating of tissue of energy property.
Grass length in above method step (3) refers to stretch plant tillering branches and leaves piece perpendicular to ground, and measurement ground is arrived
The vertical height of blade tip.
Base portion girth in above method step (3) is the length measured that made a circle with tape measure close proximity to ground outside all tillers
Degree.
Sugared brix in above method step (3) is contained in juice with oozing out after being fractureed in the middle part of sugared brix instrument measurement stalk
Soluble solid percentage.
The calculation formula of wintering rate (%) described in above method step (5) is：Seedling number/first year of turning green in spring cradles
When tiller number × 100.
Compared with prior art, the present invention has the advantage that and had the beneficial effect that：(1) the inventive method is beautiful using non-multiple
Non- multiple corn allopolyploid, is used to cultivate perennial forage grass jade first by the rice fertile characteristic of allopolyploid female part
Rice, the not cultivation of only perennial forage maize opens a kind of new approach, and also has on genetics research great meaning
Justice.(2) non-multiple corn allopolyploid MTP94 used in the inventive method, has polymerize corn, gama grass and many
The genome of year raw tetraploid class corn, it not only has the perennial character of rub standing grain and tetraperennial teosinte,
And the Apomixis of heredity friction standing grain, and the characteristic such as cold-resistant, tiller, with extensive hereditary basis, not only
The difficulty of corn and its sibling species inter-genera distant hybridization can be overcome as bridge material, and has widened cultivation forage maize significantly
Hereditary basis, can according to the genetic make up designed the need for different ecological environment and variety type between different species, from
And cultivate the perennial forage maize for adapting to different ecological condition, meeting different needs；In addition, be alternatively arranged as bridge material to
The transfer of common cultivation corn moves the excellent degeneration-resistant, disease and insect resistance having in Wild related germplasm or Quality Gene resource.(3) it is of the invention
Method breeding cycle is very short, direct to first-filial generation need to only select, you can be bred as perennial forage maize kind；And mesh
The preceding conventional fodder maize based on cenospecies, the breeding cycle length of its self-mating system, waste time and energy, breeding cost it is high and high
Degree relies on the experience in breeding of breeder.(4) the inventive method is different from traditional forage maize breeding method, by distant hybridization,
Polyploid breeding and Clone Breeding combine together seed selection perennial forage maize.The variety type of forage maize is also from comprehensive product
Kind, conventional variety, cenospecies are extended to Clonal Cultivar, and variety type is more diversified.(5) the inventive method passes through trophosome
Hybrid vigor fixing, and bred by asexual manners such as point root and stem of certain plants, cuttages, the good characteristic of kind can be not only kept, is prevented
The only degeneration of hybrid vigour, and without the production of hybrid seeds every year as existing fodder maize, without planting every year, once plant
Plant, can for many years use, greatly save labour, and farming cost, production cost is substantially reduced.(6) the inventive method is utilized
The perennial forage maize variety yield of incubation is high, and selected strain First Year green forage yield is up to 80t/hm2, grass 1 beautifulr than control
Number high by 16.08%, Second Year fresh grass is up to 60t/hm2More than, higher by 47.10% than compareing, fresh grass total amount reaches 149t/hm within 2 years2With
On, it is higher by 32.24% than compareing；High disease resistance, whole breeding time has no obvious scab, and cold resistance is good, frost can be restrained oneself, four
River basin and all areas of basin being capable of overwinterings.Stalk is rich in soluble sugar, and palatability is good, domestic animal eating；Forage quality is good,
Tasseling stage cradles CP up to 9.0%DM, RFV more than 90%.
Brief description of the drawings
Fig. 1 .MTP94 plant photos.
The solid seed photo of Fig. 2 .MTP94 filial generations.
The dyeing material body GISH+FISH of Fig. 3 numberings 9910 identifies microphoto.
The dyeing material body GISH+FISH of Fig. 4 numberings 9919 identifies microphoto.
The material plant photo of Fig. 5 numberings 9919.
Embodiment
With embodiment, the present invention is described further below, but the scope of the present invention is not construed as limiting.Such as nothing
Method therefor is all conventional method in special instruction, example below.
The method that embodiment 1 cultivates perennial forage maize using non-multiple corn allopolyploid
Carry out as follows：
(1), the spring in 2013, in Chongzhou City of Sichuan Agricultural University during more than 10 DEG C of base ground temperature, by MTP94 (Tripsazea
T.2n=94, the public can obtain MTP94 biomaterials to creammaize from Sichuan Agricultural University) carry out dividing root and stem of certain plants breeding to be planted
Seedling (see Fig. 1), and single-strain planting is carried out according to seeding row spacing 2m × 2m, meanwhile, tetraploid is planted in batches for many years in 2~4m around
Raw class corn (Zea perennis, 2n=40) 9475 is (public quoted from international corn wheat improvement center (CIMMYT) of Mexico
Crowd can obtain " 9475 " biomaterial from CIMMYT).
(2), the autumn in 2013, the bagging before MTP94 spinnings, and cut MTP94 filigrees to 1~3cm before pollination, to improve
Setting percentage.Male parent is done in the loose powder phase with 9475 to pollinate to MTP94.After harvesting (see Fig. 2), drying after seed maturity, -20 are stored in
DEG C refrigerator.
(3) in, the spring in 2014, gained seed in (2) is tedded into 3~4h under the sun, to improve seed enzyme activity, promotes seed
Sprout.Then sow in nutritive cube in the controlled environment chamber, in 28 DEG C ± 2 DEG C of temperature, vernalization under the conditions of humidity 70% ± 5%,
Success vernalization 30, random number is 991~9930.When seedling length to 31 heart of leaf, Sichuan is colonized in by seeding row spacing 2m × 2m
Chongzhou City of agriculture university base, cultivates and looks after meticulously.
(4) in, the autumn in 2014, individual plant primary election, selection plant height >=200cm, grass length >=250cm, base portion week are carried out in tasseling stage
Length >=35cm, tiller number >=20, sugared brix >=10%, blade surface are without obvious scab, stalk without lodging, cradle individual plant fresh grass
Yield >=20kg individual plant, has 12 individual plants and is selected in.And the strain of middle menu is expanded numerous into clone using stalk cuttage mode.
(5) selected clonal total chromosome number in tip of a root tabletting identification (4), is passed through；And use genomic in situ hybridization
(GISH) selected clonal chromosome is constituted and identified with FISH (FISH).Selective staining body sum is 60
~72, wherein maize chromosome number 8~12, friction standing grain chromosome number 14~18, tetraperennial teosinte dyeing
Body number is the strain of 38~42.As a result it is 993,995,9910 (see Fig. 3), 9911,9919 (to be shown in Table 1) therefrom to select numbering
(see Fig. 4), 9921,9923 totally 7 chromosome numbers and constitute and meet the strain of target call.
The Chromosome Identification result of the selected perennial forage maize strain of the present invention of table 1.
(6), variety test.Spring in 2015, it is control to examine the beautiful grass of kind 1 and parent 9475 with state, carries out kind
Comparative test.Experimental design is plot area 4m × 4.5m, and seeding row spacing 1.5m × 2m, 3 repetitions, surrounding sets protection row, in
The variety comparative test for carrying out continuous 2 years in 2015~2016 years.Investigate wintering rate by the end of March spring in the 2016th, and in taking out male phase beginning
Each clone is cradled, and crude protein content is detected, selection wintering rate >=100%, crude protein content >=9.0%
DM, relative feeding value (RFV) >=80, and green forage yield are higher than the strain of check variety more than 15%.As a result (2 Hes are shown in Table
Table 3) select numbering be 9910,9911,9919 (see Fig. 5) totally 3 meet target call perennial forage maize kind.
The perennial forage maize strain correlated traits comparative test result of the present invention of table 2.
The perennial forage maize strain green forage yield comparative test result of the present invention of table 3
Claims (10)
1. the method for perennial forage maize is cultivated using non-multiple corn allopolyploid, it is characterised in that including following step
Suddenly：
(1), when spring ground temperature rises to more than 10 DEG C, to non-multiple corn allopolyploid (Tripsazea creammaize
T. a point root and stem of certain plants breeding, or the kind that the vegetative manners such as stalk cuttage upper one year, press strip, underground layering or tissue cultures are obtained) are carried out
Seedling, is that 2m × 2m carries out single-strain planting by seeding row spacing；Meanwhile, plant male parent in batches around it；
(2), before spinning, by non-multiple corn allopolyploid female fringe bagging, and before pollination cut short its filigree to 1~
3cm, is pollinated with paternal pollen to non-multiple corn allopolyploid；Or under the conditions of isolation, male parent is allowed to non-multiple corn
Allopolyploid free pollination；After harvesting, drying after seed maturity, -20 DEG C of refrigerators are stored in；
(3) seed obtained by step (2), is tedded into 3~4 hours under the sun, the nutrition in phjytotron is then planted
In alms bowl, temperature be 25 DEG C~30 DEG C, humidity be to carry out vernalization under the conditions of 60%~80%；When seedling length to 31 heart of leaf, take
The tip of a root, is then that 2m × 2m carries out Plant colonization in crop field according to seeding row spacing；In Adult plant, selection plant height >=200cm, grass be long >=
250cm, base portion girth >=35cm, tiller number >=20, sugared brix >=10%, blade surface are without obvious scab, the upright nothing of stalk
Lodge, cradle individual plant green forage yield >=20kg plant；The individual plant of middle choosing is expanded numerous into clone using stalk cottage method；
(4) chromosome number identification, is carried out to the clonal tip of a root selected by step (3), and utilizes in situ hybridization (GISH) and fluorescence
In situ hybridization (FISH) is constituted to clonal chromosome and identified, each 30~50 cells of clonal verification；Selection dye
Colour solid sum is 60~72, and wherein maize chromosome number is 8~12, and friction standing grain chromosome number is 14~18, tetraploid
Perennial class maize chromosome number is the clone of 38~42；
(5) clone of wintering rate >=100% in the clone selected by step (4), is entered into variety comparative test；Seedling is stalk skewer
Seedlings plugging or point root and stem of certain plants seedling, using the perennial forage maize kind of authorization or its parent as the variety comparative test of control progress at least 2 years；Point
Do not cradle in taking out male phase beginning, selection green forage yield is higher than check variety 15%, and crude protein content >=9.0%DM, relative raise
With the strain of value (RFV) >=80, as new perennial forage maize.
2. according to the method described in claim 1, it is characterised in that the non-multiple corn described in its step (1) or (2) is heterologous
Polyploid refers to MTP94 (Tripsazea creammaize are T.2n=94) or MTP94 Derivative line.
3. according to the method described in claim 1, it is characterised in that the male parent described in its step (1) or (2) refers to corn
One kind of the material such as (Zea mays L.), friction standing grain (Tripsacum L.) or teosinte (Teosinte).
4. method according to claim 3, it is characterised in that described corn (Zea mays L.) refers to corn selfing
One kind of system, cenospecies, collective species or farm variety etc..
5. method according to claim 3, it is characterised in that described teosinte refers to Diploid Perennial teosinte
(Z.diploperennis, 2n=20), tetraperennial teosinte (Z.perennis, 2n=40), maize seed in great numbers
(Z.luxurians, 2n=20), Nicaragua's maize seed (Z.nicaraguensis, 2n=20), zea mexicana subspecies
(ssp.mexicana, 2n=20), Wei Weitenange subspecies (ssp.huehuetenangensis, 2n=20) or small clever corn
One kind of subspecies (ssp.parviglumis, 2n=20) etc..
6. method according to claim 5, it is characterised in that described tetraperennial teosinte be tetraploid for many years
Raw class corn (Z.perennis, 2n=40) 9475.
7. method according to claim 3, it is characterised in that described friction standing grain (Tripsacum L.) refers to orchardgrass shape
Rub standing grain (Tripsacum dactyloides, 2n=4x=72) or other friction standing grain species.
8. according to the method described in claim 1, it is characterised in that point root and stem of certain plants breeding described in its step (1) or (5) refer to
The method that the seedling that turns green of plant base portion carries out vegetative propagation for planting stock.
9. according to the method described in claim 1, it is characterised in that the cutting propagation described in its step (1), (3) or (5) is
Refer to the method that vegetative propagation is carried out using the stipes cut off as skewer bar.
10. according to the method described in claim 1, it is characterised in that the tissue cultures described in its step (1) refer to the tip of a root,
Stem end, stipes, leaf, embryo etc. have the mitogenetic method for carrying out tissue culture propagating of the tissue of cellular omnipotency.
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