CN102657093A - Somatic embryogenesis method of phyllostachys heterocycla var. pubescens - Google Patents

Abstract

The invention discloses a somatic embryogenesis method of phyllostachys heterocycla var. pubescens, which includes the following steps that an explant of phyllostachys heterocycla var. pubescens is appropriately disinfected and then is induced on an inductive medium to produce a callus tissue; after being processed with multiplication culturing, the callus tissue firstly forms an embryoid on a differential medium and then the embryoid bourgeons and forms a regenerated plant on a germination medium; finally the regenerated plant is processed with the cultivations of seedling toughening up and seedling exercising and then is transplanted to survive. The invention solves the technical difficulty in somatic embryogenesis of phyllostachys heterocycla var. pubescens and provides a technology platform for the technical breeding of scattered bamboo plants.

Description

The method that a kind of mao bamboon somatic embryo takes place

Technical field

The present invention relates to forest biotechnology and field of tissue culture, specially refer to the mao bamboon somatic embryo and take place.

Background technology

Twentieth century along with the fast development of Plant Biotechnology in the world, has also increased the bamboo biotechnology research since the eighties both at home and abroad gradually, and the report of at present relevant with this patent in the world bamboo regenerating system aspect mainly contains:

Nineteen eighty-two Metha etc. has reported that India's thorn bamboo (Bambusa arundinacea) mature embryo induces callus and regeneration plant (list of references 1).

Nineteen eighty-three Huang and Murashige belong to from Phyllostachys (Phyllostachy), indocalamus that (blade of Sasa) 、 le Sinobambusa (Bambusa) and tender stem apex induce callus (list of references 2).

Rao in 1985 etc. have induced callus with male bamboo (Dendrocalamus.strictus) mature seed, and then regeneration plant (list of references 3).

Yeh in 1986 and Chang with the inflorescence of green bamboo (B.oldhamii), Folium Bambusae beecheyanae (B.beecheyana) with somatic embryo occurring mode regeneration plant (list of references 4,5).

Hassan in 1987 etc. have reported the plant regeneration (list of references 6) of firm bamboo (Ph.viridis) blade callus.

Huang in 1989 etc. have reported that the tender stem apex with green bamboo (B.oldhamii), fishpole bamboo (Ph.aurea) and indocalamus (S.pygmaea) carries out callus induction, but formation regeneration plant (list of references 7) takes place the callus organ of only being induced by BA and NAA.

Nineteen ninety Tsay report utilizes sinocalamus latiflorus (Sinocalamus latiflora) flower pesticide to induce callus and realize plant regeneration (list of references 8).

Usefulness Dendrocalamus hamiltonii (D.hamiltonii) stalk buds such as Godbole induced callus and realized plant regeneration (list of references 9) in 2002.

Ogita utilized phyllostachys nigra var. henonis (Ph.nigra var.henosis) bamboo shoots to induce callus in 2005, but the easy brown stain of callus, not regeneration (list of references 10).

Utilizations such as Gillis in 2007 crust bitter bamboo (B.balcooa) small ear induces callus and has set up high-efficiency regeneration system (list of references 11).

Cheah in 2011 etc. utilize little buddha bamboo (B.ventricosa) stalk bud to induce callus and realize plant regeneration (list of references 12).

China mainland scholar starts late in the research aspect the bamboo biotechnology.

Fault state rather waited the cultivation of having reported bambusa textile (D.membranceus) and India thorn bamboo (B.arundinacea) (list of references 13-14) in 1991; Wu Tao in 2008 etc. have reported the cultivation (list of references 15) to spun gold cizu (B.affinis ' Viridiflavus '); All belong to the research through the strain of callus regeneration bamboo, its explant is tender shoots.The Yuan Jin tinkling of pieces of jade in 2009 etc. are that explant induction goes out callus and realizes plant regeneration with show filal obedience bamboo (B.multiplex) embryo and small ear, with the regeneration efficiency of kind of embryo explants higher (list of references 16).Qiao Gui honor in 2010 etc. are that explant induction goes out callus and obtains regeneration plant (list of references 17) with sinocalamus latiflorus flower pesticide, and the same year, Zhang et al. was that explant induction goes out callus and realizes plant regeneration (list of references 18) with the embryo of Dendrocalamus hamiltonii.2011 Pei petrels etc. are explant with thunder bamboo (Ph.violascens) embryo, and dedifferentiation produces callus, callus regeneration sprout (list of references 19).

With regard to mao bamboon, Zhou Hong (2005), Lu Juanjuan (2006) utilize the moso bamboo shoot bud to induce callus respectively, but do not obtain regeneration plant (list of references 20-21).Li Nan utilizes the mao bamboon seed to induce callus, but growth is slow, quality is loose, and water stainization is serious, fails to obtain regeneration plant (list of references 22).Yue Jinjun etc. have studied the influence to mao bamboo seed embryo callus induction rate and quality of different placements and cutting mode, do not see and realize plant regeneration (list of references 23).In sum, the plant regeneration system of scattered bamboo through callus is not perfect, and the research of mao bamboon only is in the callus induction stage, does not all see the acquisition regeneration plant.The mao bamboon somatic embryo generation system of stability and high efficiency has become the key technology obstacle of restriction mao bamboon biotechnology breeding.

The invention provides the method that the mao bamboon somatic embryo takes place, will technology platform be provided for cell engineering, gene engineering and the molecular biological research of mao bamboon.

The main reference document

1.Mehta?UI，Rao?VR，Ram?HYM.Somatic?embryogenesis?in?bamboo[C].In?Proceeding?5th?Interenational?Cogress?of?Plant?tissue?culture&cell?Culture，Tokyo，Japan，1982，109-110

2.Huang?LC，Murashige?T.?Tissue?culture?investigations?of?bamboo?I.Callus?cultures?of?Bambusa，Phyllostachys?and?Sasa[J].Bot?Bull?Acad?Sin，1983，24，31-52

3.Rao?IU，Bamanuja?Rao?IV，Narang?V.Somatic?embryogenesis?and?regeneration?of?plants?in?the?bamboo?Dendrocalamus?strictus.Plant?Cell?Reports，1985，4：191-194

4.Yeh，ML?and?WC?Chang.Somatic?embryogenesis?and?subsequent?plant?regeneration?from?inflorescence?of?Bambusa?beecheyana?Munro?var.beecheyana[J].Plant?Cell?Reports，1986，5：409-411

5.Yeh，ML?and?WC?Chang.Plant?regeneration?through?somatic?embryogenesis?in?callus?culture?of?green?bamboo(Bambusa?oldhamii?Munro)[J].Theoretical?and?Applied?Genetics，1986，73：161-163

6.HASSAN?AAE，DEBERGH?P.Embryogenesis?and?plantlet?development?in?the?bamboo?Phyllostachys?viridis(Young)McClure[J].Plant?Cell，Tissue?and?Organ?Culture，1987(10)：73-77

7.Huang?LC，Huang?BL?and?Chen?WL.Tissue?culture?investigations?of?bamboo?IV.Organogenesis?leading?to?adventitious?shoots?and?plants?in?excised?shoot?apices[J].Environ?Exp?Bot，1989，29，307-315

8.Tsay?HS，Yeh?CC，Hsu?JY.Embryogenesis?and?plant?regeneration?from?anther?culture?of?bamboo[Sinocalamus?latiflora(Munro)McClure][J].Plant?cell?Reports，1990，9：349-351

9.Godbole?S，Sood?A，Thakur?R，et?al.Somatic?embryogenesis?and?its?conversion?into?plantlets?in?a?multipurpose?bamboo，Dendrocalamus?hamiltonii?Nees?et?Arn.Ex?Munro.Current?Science?2002，83：885-889

10.Shinjiro?Ogita.Callus?and?cell?suspension?culture?of?bamboo?plant，Phyllostachys?nigra.Plant?Biotechnology，2005，22(2)，119-125

11.Gillis?K，Gielis?J，Peeters?H，et?al.Somatic?embryogenesis?from?mature?Bambusa?balcooa?Roxburgh?as?basis?for?mass?production?of?elite?forestry?bamboos[J].Plant?Cell，Tissue?and?Organ?Culture，2007(91)：115-123

12.Cheah?KT，Chaille?LC.Somatic?Embryogenesis?From?Mature?Bambusa?ventricosa[J].Biotechnology，2011，1-5

13. fault state is peaceful, Zhuge Qiang. bamboo callus culture and plant regeneration [J]. bamboo research transactions, 1991,10 (4): 4

14. fault state is peaceful, Zhuge Qiang. the bambusa textile cell suspension cultures is separated [J] with protoplast. forestry scientific research, 1994,7 (1): 44-47

15. Wu Tao, Lu Juanjuan, Ding Yulong etc. spun gold cizu callus culture and plant regeneration research [J]. forestry science and technology exploitation, 2008,22 (2): 19-22

16. the Yuan Jin tinkling of pieces of jade turns round and look at little flat Lee Lu Bin Yue Jin army Guo Yao Na Guangping. show filal obedience bamboo callus induction and plant regeneration [J]. forest-science, 2009,45 (3): 35-39,172.

17. the flourish Li Hai grandson Jiang Jing ancestor of battalion of Qiao Gui repaiies Zhuo Renying. the acquisition of sinocalamus latiflorus anther culture and regeneration plant [J]. Botany Gazette, 2010 (1): 88-90.

18.Zhang?N，Fang?W，Shi?Y，et?al.Somatic?embryogenesis?and?organogenesis?in?Dendrocalamus?hamiltonii[J].Plant?Cell，Tissue?and?Organ?Culture，2010(103)：325-332

19. Pei petrel,, woods the new year, Fang Wei etc. the Primary Study [J] of thunder bamboo somatic embryo inducement. Botany Gazette, 2011,46 (2): 170-178

20. week is grand, He Gang. mao bamboon callus culture research [J]. Hunan forestry science and technology, 2005,32 (4): 41-42.

21. reed is beautiful. the tissue culture of several kinds of economic bamboo kinds. Nanjing Forestry University's master thesis [D] .2006,33

22. Li Nan, golden heroes, Peng Huazheng etc. mao bamboon seed sprouting characteristic and callus induction ability pre-test [J]. Zhejiang forestry science and technology, 2009,29 (3): 73-76

23. Yue Jin army, Guo Guangping, Yuan Jinling, etc. the Primary Study of mao bamboo seed embryo callus induction and differentiation [J]. Molecular Plant Breeding (network edition), 2011,9:1425-1430

Characteristics of the present invention

Make a general survey of domestic and international present Research to the bamboo plant regenerating system, what build up regenerating system so far mainly is the bamboo kind of growing thickly, less about the report of scattered bamboo, and efficient is low, poor repeatability, does not see the report of most important scattered bamboo kind-mao bamboon regenerating system.Scattered bamboo kind is because physiological habit is different with the bamboo that grows thickly, and the cultivation difficulty is big, although existing how tame unit has carried out research, rarely has progress.Process is carried out big quantity research and test, and important improvement and innovation have been carried out in similar research, and the inventor has grasped callus induction of mao bamboon and the key technology that somatic embryo takes place, and discloses the method that the mao bamboon somatic embryo takes place in the world first.

Summary of the invention

The method that a kind of mao bamboon (Phyllostachys heterocycla var.pubescens) somatic embryo takes place; Its characteristic comprises: explant sterilization, callus induction and propagation, embryoid induction, sprouting and plant regeneration; And strong sprout, refining seedling and transplanting process, concrete steps are following:

(1) explant sterilization: select healthy full kind real, earlier with the Ethanol Treatment of 75% (v/v) 1 minute; Adding 0.2% clorox that 5-6 drips Tween-80 with every liter again handled 15-20 minute; Then for several times with aseptic water washing; Inoculate after blotting surface moisture with aseptic paper at last;

(2) callus induction and propagation: the explant after will sterilizing is inoculated on the inducing culture; The dark 20-30 of cultivation angel callus forms; Callus inducing medium is to be the basis with the MS medium; Add 2 of 1-8mg/L, the proline of the ZT of 4-D, 0.1-5mg/L, the carragheen of 10g/L, 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L; Then the callus of faint yellow densification is separated; Be inoculated on the subculture medium enrichment culture 70-90 days; Proliferated culture medium is to be the basis with the MS medium; Add 2 of 1-8mg/L, the glutamine of the carragheen of 4-D, 10g/L, the proline of 500mg/L, 500mg/L, the caseinhydrolysate of 300mg/L;

(3) embryoid induction, sprouting and plant regeneration: the callus behind the enrichment culture is inoculated on the embryoid induction medium; The dark cultivation 20-40 days; Embryoid is formed; The embryoid induction medium is to be the basis with the MS medium, adds the ZT of 1-8mg/L, the BA of 1-3mg/L, the KT of 1-5mg/L, the maltose of 30g/L, the carragheen of 10g/L; Then embryoid is inoculated on the differential medium, illumination cultivation 30-50 days, embryoid was sprouted the formation regeneration plant gradually; Differentiation culture is to be the basis with the MS medium, adds the proline of 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L, the carragheen of 10g/L; Light application time 12 hours/day, intensity of illumination 800-2000lux;

(4) strong sprout, refining seedling and transplanting: the plantlet of somatic embryo regeneration is inoculated on the strong seedling culture base cultivated 50-60 days; Uncork refining seedling 5-7 days; Take out the medium that seedling cleans root, clean root with 0.1% liquor potassic permanganate again, then seedling is planted in the medium of sterilizing; The scattered light that shelters from heat or light was cultivated after one month, and seedling becomes to live; Described strong seedling culture base is to be the basis with the MS medium, adds the NAA of 1-4mg/L, the TDZ of 0.2-0.5mg/L, the carragheen of 10g/L.

Embodiment

Below in conjunction with mao bamboon (Phyllostachys heterocycla var.pubescens) instance the present invention is elaborated.

(1) select full healthy plant real, earlier with 75% (v/v) Ethanol Treatment 1 minute; Adding 0.2% the clorox that 5-6 drips Tween-80 with every liter again handled 15-20 minute; Then for several times with aseptic water washing; Wait to inoculate after blotting surface moisture with aseptic paper at last;

(2) explant after will sterilizing is inoculated on the callus inducing medium; Inducing culture is to be the basis with the MS medium; Add 2 of 4mg/L; The proline of the ZT of 4-D, 0.1mg/L, the carragheen of 10g/L, 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L, 26 ℃ of dark cultivations 20-30 days, explant forms the callus of grain of rice size; The callus of faint yellow densification is separated; Be inoculated on the shoot proliferation medium; Proliferated culture medium is to be the basis with the MS medium; Add 2 of 2mg/L, the glutamine of the carragheen of 4-D, 10g/L, the proline of 500mg/L, 500mg/L, the caseinhydrolysate of 300mg/L are secretly cultivated and can be bred more than one times in 30 days;

(3) embryo callus with shoot proliferation is inoculated on the embryoid induction medium; The embryoid induction medium is to be the basis with the MS medium; Add the ZT of 4mg/L, the BA of 1.5mg/L, the KT of 2.5mg/L, the maltose of 30g/L, the carragheen of 10g/L; 26 ℃ of dark cultivations 20-40 days, most callus form embryoid; Then embryoid is inoculated on the differential medium; Differentiation culture is to be the basis with the MS medium; Add the proline of 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L, the carragheen of 10g/L, illumination cultivation 30-50 days, light application time 12 hours/day; Intensity of illumination 1200-2000lux, embryoid sprout the formation regeneration plant gradually;

(4) strong sprout, refining seedling and transplanting: the plantlet of somatic embryo regeneration is inoculated in the strong seedling culture base; The strong seedling culture base is to be the basis with the MS medium, adds the NAA of 4mg/L, the TDZ of 0.2mg/L, the carragheen of 10g/L, cultivates 50-60 days; Uncork refining seedling 5-7 days; Take out the medium that seedling cleans root, clean root with the low concentration liquor potassic permanganate again, medium (the mountain region yellow soil: vermiculite: peat soil=1: 1: 1) of then seedling being planted to and sterilizing; The scattered light that shelters from heat or light was cultivated after one month, and seedling becomes to live.

Claims (1)

1. the method that takes place of a mao bamboon (Phyllostachys heterocycla var.pubescens) somatic embryo; Its characteristic comprises: the explant sterilization; Callus induction and propagation; Embryoid induction, sprouting and plant regeneration, and strong sprout, refining seedling and transplanting process, concrete steps are following:

(1) explant sterilization: select healthy full kind real, earlier with the Ethanol Treatment of 75% (v/v) 1 minute; Adding 0.2% clorox that 5-6 drips Tween-80 with every liter again handled 15-20 minute; Then for several times with aseptic water washing; Inoculate after blotting surface moisture with aseptic paper at last;

(2) callus induction and propagation: the explant after will sterilizing is inoculated on the inducing culture; The dark 20-30 of cultivation angel callus forms; Callus inducing medium is to be the basis with the MS medium; Add 2 of 1-8mg/L, the proline of the ZT of 4-D, 0.1-5mg/L, the carragheen of 10g/L, 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L; Then the callus of faint yellow densification is separated; Be inoculated on the subculture medium enrichment culture 70-90 days; Proliferated culture medium is to be the basis with the MS medium; Add 2 of 1-8mg/L, the glutamine of the carragheen of 4-D, 10g/L, the proline of 500mg/L, 500mg/L, the caseinhydrolysate of 300mg/L;

(3) embryoid induction, sprouting and plant regeneration: the callus behind the enrichment culture is inoculated on the embryoid induction medium; The dark cultivation 20-40 days; Embryoid is formed; The embryoid induction medium is to be the basis with the MS medium, adds the ZT of 1-8mg/L, the BA of 1-3mg/L, the KT of 1-5mg/L, the maltose of 30g/L, the carragheen of 10g/L; Then embryoid is inoculated on the differential medium, illumination cultivation 30-50 days, embryoid was sprouted the formation regeneration plant gradually; Differentiation culture is to be the basis with the MS medium, adds the proline of 500mg/L, the glutamine of 500mg/L, the caseinhydrolysate of 300mg/L, the carragheen of 10g/L; Light application time 12 hours/day, intensity of illumination 800-2000lux;

(4) strong sprout, refining seedling and transplanting: the plantlet of somatic embryo regeneration is inoculated on the strong seedling culture base cultivated 50-60 days; Uncork refining seedling 5-7 days; Take out the medium that seedling cleans root, clean root with 0.1% liquor potassic permanganate again, then seedling is planted in the medium of sterilizing; The scattered light that shelters from heat or light was cultivated after one month, and seedling becomes to live; Described strong seedling culture base is to be the basis with the MS medium, adds the NAA of 1-4mg/L, the TDZ of 0.2-0.5mg/L, the carragheen of 10g/L.