JPH10136815A - Tissue culture method for production cotton plant capable of making in-vitro growth of large amount - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the tissue culture method capable of regenerating cotton plants which may be grown in vitro in a large amt. and have a fertilization capability from the specific tissues of the cotton plants. SOLUTION: The seeds of the cotton plants are treated to get rid of bacteria- molds and these seeds are cultured for three days to several weeks in the presence of light in a bright period of 16 hours at 20 to 40 deg.C in a first medium consisting of salts, vitamins, carbon sources, gelatinizing agents. Cotlyledon nodes partly with perfect cotlyledons or without the cotlyledons are cut from the resulted nursery stock and these cotlyledon nodes are kept cultured for at least 4 weeks until many shoots are formed in the presence of the light of the bright period of 16 hours at 20 to 40 deg.C in a second medium consisting of salts, vitamins, carbon sources, gelatinizing agents and plant hormones for the purpose of propagation of the shoots. The formed shoots are collected. These shoots are transferred to a root inductive medium consisting of the salts, vitamins, carbon sources, gelatinizing agents and plant hormones and are cultured until the roots grow in the presence of the light of the bright period of 16 hours at 20 to 40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a tissue culture method for producing a large amount of viable cotton plants in vitro from specific tissues of cotton plants. The method of the present invention provides genotype-independent, direct multiple shoot propagation, and uses modern methods of agricultural biotechnology and genetic engineering to enable micropropagation, potential mutant selection, and genetic Open the possibility of producing improved cotton plants. This method provides an important step in the success of cotton improvement programs using tissue culture.

[0002]

BACKGROUND OF THE INVENTION Cotton is an important crop worldwide, cultivated primarily for fiber purposes. The seeds are an important source of livestock feed. Cotton has influenced the economic development of countries around the world. Therefore, modern agricultural biotechnology methods for cotton improvement are of great interest worldwide. This has increased the importance of developing tissue culture methods to facilitate the application of modern methods of cotton plant genetic engineering.

Several reports have been published on cotton tissue culture. They relate to direct shoot differentiation and somatic embryogenesis using suspension and callus cultures. The list is shown below for reference.

[0004] Organ formation and regeneration linked to micropropagation by tissue culture have been successful in several plant species, such as the kidney bean (Phaseolus) species (Rubluo, A and Carta, Rubulo A & Kartha). KK), 1985, In vitro culture of apical meristems of various kidney bean (Phaseolus) species and varieties, J. of Plant physiol 119: 425-43
3). Glycine max (Shetti,
K, Asano, K and Osawa, K (Shetty
K., Asano Y and Oosawa K) 1992, Glycine max Merr with allantoin and amide
ill) stimulation of in vitro shoot organogenesis. Plant Sei.
81: 245-252), Cajanus caja
n) (Shiv Prakash N, Pental D
& Bhalla-Sarin N) 1994, Regeneration of pigeonpea (Cajanus cajan) from cotyledons through multiple shoot formation, Plant Cell Rep. 13: 6
23-622), carnations (Claire-Annev, A, Jancheba, S, and Dons, H. (Claire-Annev)
A. Yancheva S and Dons H) 1995, cells having nodal regions of carnation shoots show high potential for adventitious shoot formation, Plant Cell tiss. Org Cult. 40: 154-157).

[0005] Regeneration of plants by the tissue culture method is well established. Organogenesis or somatic embryogenesis has led to the successful regeneration of a wide variety of plant species in vitro. Organogenesis results in the formation of an organ, the shoot (or root), which can be isolated and induced to grow the root (or shoot) to produce the entire plant, while somatic embryogenesis results in the formation of shoots (or shoots). A somatic embryo (both embryo that grows without genetic fertilization) that has both the root and the root grows to become the whole plant. The ability of each plant part or cell to regenerate into a complete plant (totipotent (totipotenc
y)) is a well-known phenomenon, but it is necessary to study each plant or plant part in order to invent conditions that allow such regeneration. Several factors governing the growth and differentiation of such cultures have been determined. Establishing interactions between different groups of plant hormones, growth regulators alone or in combination, is involved in several relationships that exist between cells, tissues and organs. Thus, the success of differentiation depends on the type of explant, the physiological conditions of the explant and the physical and chemical environment of the explant in culture. For this reason, the science of tissue culture has been directed to the optimization of the physiological conditions of the source plant, the type of explant, the culture conditions, and the plant hormones used to initiate tissue culture. This demonstrates that developing a new method for growing plants by tissue culture is by no means trivial.

A major aspect to be considered on a case-by-case basis is the type of plant growth regulator and the amount of plant growth regulator that induce regeneration. In addition, the chemical composition of the medium, growth temperature, and other culture conditions play important roles in inducing organogenesis and somatic embryogenesis, and in the development of shoot and root maturation and healthy fertilizing plants. Fulfill. Responses to media, hormones and growth conditions vary from plant species to plant species and to varieties. Therefore, inventing conditions for efficient plant regeneration, organogenesis, and somatic embryogenesis requires the development of expertise for specific plants.

Another major area of need for innovation in tissue culture is to identify those parts of the plant that are regenerated abundantly in response to culture conditions. Not all parts of some plants are regenerated efficiently. Successful tissue culture of plants includes the parts of the plant that have been identified for totipotency (called explants), the physiological status of the explants,
And a complex combination of growth conditions, especially growth regulators that determine the success of a plant in tissue culture. Different explants of a plant usually show very different responses to conditions due to growth. General principles cannot be applied for reproduction. In each case, identification of explants and identification of culture conditions are innovative steps for the development of a tissue culture method for regenerating one plant into many plants or somatic embryos.

[0008] Currently, there is no detailed literature on the formation of multiple shoots from tissue explants of cultured varieties of cotton. The present invention describes for the first time a detailed method of organ formation from small parts of cotton seedlings (called explants) to obtain multiple shoots of cotton plants by tissue culture. The method shows wide application to all the varieties tested by the present inventors and is able to efficiently mature shoots, which makes it very useful in agricultural biotechnology for micropropagation and genetic transformation. is there. This method has great potential in modern agricultural biotechnology legislation cotton improvement programs.

Several reports dealing with tissue culture conditions that cause somatic embryogenesis (the structure that produces normal plants without fertilization) (Davidonis GH and Hamilton, and Davidonis GH and Hamilton).
ton RH) Gossyp 1983, Gossyp
ium hirsutum L) Plant regeneration from callus tissue, Plant
Sci. Lett. 32: 89-93; Shoemaker RC, Couche LJ & Galbrait
h DW) 1986, cotton (Gossipium hirsutumu L)
ypium hirsutum L), characterization of somatic embryogenesis and plant regeneration, Plant Cell Rep. 3: 178-181; Trollinder, N.L. and G.J., J.R. (Trolinder)
NL and Goodin JR) 1987; Cotton Gossypium Hirsutum
Somatic embryogenesis and plant regeneration of L (Gossypium hirsutum L), Plant Cell Rep. 6: 231-234, Finer J. 1988. Gossyp
hirsutum), plant regeneration from somatic embryogenic suspension cultures, Plant Cell Rep. 7: 399-402) has been reported.
Several months are required for the onset and maturation of somatic embryogenesis.
The method is highly genotype dependent (Trolinder NLand Xhix
ian C.) 1989, genotype specificity of the somatic embryogenic response of cotton, Plant Cell Rep. 8: 133-136), and thus applies to a limited group of varieties. Initiation and maturation of somatic embryogenesis requires several months, and plants regenerated through somatic embryogenesis are often reported to be cytologically and morphologically abnormal ( Sterry DM, Altman DW, Kohel Rz, Rangan TS, & c. St. DM, Altman, DW, Kochel, Earl Zett, Langan, TS, and Komesky, Y.
ommeskey E) 1989, Cytological abnormalities of cotton body cell clones from callus cultures, Genome 32: 762-770). Plants that grew through somatic embryogenesis were often reported to be nonfertile (Trolinder NL and Goodin JR).
87, Gossypium hirs
utum L) somatic embryogenesis and plant regeneration, Plant Cell Re
p. 6: 231-234).

[0010] These regeneration methods have been successfully used in Agrobacterium-mediated genetic transfer of cotton (Umbeck P, Johnso, U.S.A.).
n G, Barton K. Swain W) 1987, genetically transformed cotton Gossypium hirsutum.
L), Plant Bio./Technology 5: 263-266; Philosopher, E, Devoir, L.D., Merlot, J. D., Hulk, L.E., Amerson, N.
El, Rashka, Kee and Murray, Eee (Firoozabady E., DeBoer LD, Merlo JD, Halk LE,
Amerson, NL, Rashka KE and Murrey EE) 1987, Agrobacterium tumefaciens
faciens) Gossipium hirsutumu L (Gos
transformation of S. sypium hirsutum L and regeneration of transgenic plants, Plant Mol. Biol. 10: 105116), and particle bombardment (Finer, J-J and McMullen M) 1990, particle bombardment Gossypium hirsutum el via Gossyp
hirsutum L), Plant Cel-I Rep. 8: 586
-589). Some bacterial genetics, such as genes encoding herbicide resistance (Baylay, C., trolinder, N.L., R.C., Morgan, M., Xenberry,
Jay E and Orderable DW (Bayley C, Trolinder NL, Ray C, Morgan M, Quisen
berry JE and OW DW) 1992, imparting 2,4-D resistance to cotton, Theo, Appl. Genet. 83: 45-649) and the Bacillus thuringiensis endotoxin gene (Parrack, FJ, Deeton, Earl W, Armstrong, Tea
A, Fuchs, R.L., Sims, S.R., Green Plate, J.T., and Fishhoff, D.A. (Perlak FJ, Deaton RW, Arms
trong TA, Fuchs RL, Sims SR, Greenplate JT & Fisch
hoff DA) 1990, insect-resistant cotton, plants. Bio / Technology
8: 939-943) has been successfully expressed in transgenic cotton plants. However, other breeds did not respond to the tissue culture, i.e., somatic embryogenesis,
These advances are currently limited only to cotton Coker varieties.

A method has been described for growing a single shoot in vitro from a shoot apical tissue of a cotton plant under sterile tissue culture conditions (Gould, Jay, Bannister,
Es, Hasegawa, O, Fahima, M, Smith, Earl H (Gould J, Banister S, Hassegawa O, Fahi
ma M., Smith RH) 1991, Regeneration of Gossypium hirsutum and G. barbadense from shoot apical tissue for transformation,
Plant Cell Rep. 10: 12-16). Apical meristems and shoot tips of cotton plants have been used in cultures to obtain adventitious buds and multiple shoots (Bajaj, YPS and ManjeetGill 1986). Gossypium by meristem culture
) Species preservation, Indian J of Exp. Biol. 24: 581-583.
). In these reports, the response of the culture was genotype-dependent and rooting was rare. Because of this problem, the reatomicity of these methods is not very good.

Recently, McCabeDE and Martinelli (McCabeDE and Martinelli)
BJ) 1993, Transformation of excellent cotton varieties by meristem particle bombardment, Bio / Technology 11: 596-598; Clan, C.A., Lin, J., Cary, J.W., and Cleveland, T.E. Chlan CA, L
in J, Cary JW & Cleveland TE) 1995, Impact Transformation and Regeneration of Transgenic Cotton from Meristems, Plan
t Mol. Bio. Rep. 13: 31-37) Cotton hypocotyls were used to introduce genes by the impact transformation method. In these reports, one apical meristem grew to one shoot. Next, the regenerated shoots were rooted with a suitable medium to obtain mature cotton plants. In all such reports, 1
Transformation and regeneration were inefficient because one apical meristem became one mature plant and many transformants were chimeric with respect to the expression of the transforming gene. Such a method is greatly improved by developing a method in which many plants grow from one explant as disclosed in the present invention.

Table 1 summarizes the state of the art of tissue culture methods related to cotton plants covered by patents or described in the literature. Next, we describe our method in comparison with the known state of the art.

The abbreviations used in the text of the plant growth regulator (hormone) used in the medium are as follows: B
AP (6-benzylaminopurine or 6-benzyladenine), 2iP (γ, γ-dimethylallylaminopurine), Kin (kinetin), 1AA (indoleacetic acid), NAA (naphthaleneacetic acid), 1BA (indolebutyric acid), TDZ ( 1-phenyl-3,1,2,3 thidiazol-5-yl urea), DU (diphenyl urea), P
U (U-1-phenylN4pyridyl) urea) and 2,
4-D (2,4-diphenoxyacetic acid).

[0015]

[Table 1] Table 1 Report Mode of regeneration Plant hormone Explant Remarks ────────────────────────────────── ── 1. Davidonis GH and Hamilton RH 1983, Plant regeneration from callus tissue of G. hirsutum L, Plant Sci. Lett. 32: 89- 93 Somatic embryogenesis NAA and Kine cotyledons Two year old G. hirsutum L Coker 310 grown in LS medium containing 30 gm / l in the absence of NAA and kin Callus was used. 30% of the cultures gave somatic embryogenesis. 2. Davidnis, G.H., Mumma, R.O., and Hamilton, R.H. (1987), Regulated Regeneration of Cotton Plants from Tissue Culture, U.S. Pat. No. 4,672,035.

[0016] 3. Somatic embryogenesis and planting of shoemakers RC, Couche, L.S. and Galbraith, Shoemaker RC, Couche LS & Galbraith DW 1986, cotton (Gossypium hirsutum L) Characterization of Product Regeneration, Plant Cell Rep. 3: 178-181 Somatic Embryogenesis NAA, Kine Hypocotyls Seventeen varieties of cotton Gossypium hirsutum L were evaluated for somatic embryogenesis. After a series of callus morphologies in media containing MS salts, NAA and kin, a few weeks later, the calli were observed for the presence of somatic embryogenesis: varieties coker 210 and coker 315 Embryos were isolated and grown into plants 4. Trollinder, N.L. and Gougin, J.R. nd Goodin JR) 1987; Somatic embryogenesis and plant regeneration of Gossypium hirsutum L, Plant Cell Rep. 6: 231-234 2,4-D, Kin hypocotyl 6-week old callus Spherical embryos were observed in the culture, at which stage the calli were sub-cultured in growth media free medium, and after 3-4 weeks the suspension was sieved to collect the spherical, heart-type embryos. The collected embryos were allowed to grow to maturity on a solidifying medium.The mature embryos were germinated into plants, and many plants grown in this manner were non-fertile (only 15% were fertile). 5. Trollinder NL and Xhixi an C. 1989, Genotype specificity of the somatic embryogenic response of cotton, Plant Cell Rep. 8: 12,4- D, Kin Hypocotyls 38 Gossypium cultivar strains and strains And breeds (race) were screened for somatic embryogenesis in the manner developed for Coker 312. The screen showed that there was a genotypic change in embryogenesis. Only a few genotypes can be applied to the developed model.

[0017] 6. Sterry DM, Altman SW, Kohel RZ, Rangan TS, & Commeskey E 1989, Cotton, Kochel, Earl Zett, Langan, TS, Komski, Y Cytologic abnormalities in culture, Genome 32: 762-770 High frequency chromosomal abnormalities were observed in plants regenerated by somatic embryogenesis, and many plants regenerated by somatic embryogenesis were unable to reproduce . 7. Finer J. 1988. Plant regeneration from somatic embryogenic suspension culture of Gossypium hirsutum, Plant Cell Rep. 7:39 9-402 Somatic embryogenesis NAA, Kin, Picloram, 2,4- D Cotyledons A sustainable embryogenic suspension culture was developed. Embryogenic tissues were transferred to auxin-free medium and embryos were observed. The resulting plants were not fertile. 8. Finer, Jay J, 1990, Efficient method for regenerating cotton from cultured cells, Patent No. ZA / Z8808599.

[0018] 9. Langan, TS 1993, Cotton Regeneration, Patent No. 5244802 Somatic Embryogenesis NAA, Kin Hypocotyl, Cotyledon, Immature Embryo Callus is initiated in MS medium containing NAA and Kin And subcultured every 3 weeks for growth. Somatic embryos were seen 4-6 weeks after placing the tissue in callus initiation medium. A number of variants have been found to be embryogenic in vitro. SJ2, SJ4, SJ5, SJ2C, GC510, B1644, B2710. 10. Gawel NJ and Robacker CD 1990, Somatic embryogenesis of two Gossypium hirsutum genotypes on semisolid versus liquid growth medium, Plant Cell Tissue Organ Culture 23: 201-204 Somatic Embryogenesis 2,4D, Kin Petiole from mature flowering plant A comparative test was carried out on somatic embryogenesis in liquid versus solid media, and Coker 312 Culture in liquid medium was more embryogenic in both named T-25. 11. Bajaj, YPS and Gil M. 1986; Preservation of Gossypium species by shoot tip and meristem culture, Indian J. of Exp. Biol. 24: 581- 583 Callus cultures, adventitious bud cultures, and multiple shoots NAA, IAA, Kin shoot tips The response of the culture is genotype dependent, with only a few cultures giving multiple shoots and very rooted. It was rare.

[0019] 12. Gould, J, Banister, S, Hasegawa, O, Fahima, M, Smith, Gh J, Banister S, Hassegawa O, Fahima M., S mith RH 1991, from shoot apical tissue for transformation Regeneration of Gossypium hirsutum and G. barbadense in Japan, Plant Cell Rep. 10: 12-16 Organogenesis from preexisting meristems None Apical shoot meristems Using this method G. barbadense Pima S-6 and G. hirsutum 19 varieties with normal fertility were regenerated, but one explant Only one shoot was formed from one piece and rooting could not be optimized. 13. Umback P, Johnson G, Barton K. Swain W 1987, genetically transformed cotton Gossypium hirsutum L, Plant Bio./Technology 5: 263-266 14. Umbak, P. 1991, discloses a method for producing cotton cells transformed by tissue culture, patent number INA 168950, Transformation of cotton and regeneration of plants 2,4-D, Kin hypocotyl Genetic transformation of cotton. Cotton immature tissues were transformed in vitro by Agrobacterium tumefaciens-mediated genetic transformation. The resulting cotton tissue was selected for drug and screened for transformation. The transformed culture was then induced to obtain somatic embryos. Somatic embryos were grown into mature plants. Coker 310, 312, and 5110 were transformed in this manner.

[15] 15. Firoozabady E., DeBoer LD, Merlo, Jay Dee, Hulk, L.E., Amerson, N.L., Rashka, K.E., and Murray, E.E. Transformation of transgenic plants and transformation of cotton Gossypium hirsutum L with Agrobacterium tumefaciens by Merlo JD, Halk LE, Amerson, NL, Rashka KE and Murrey EE) 1987. 10: 105-116 Cotton Transformation and Plant Regeneration 2iP, NAA Cotyledons Cotyledon explants were transformed from 12-day-old seedlings to regenerate plants. For regeneration, explants treated with Agrobacterium tumefaciens were transferred to a medium containing bacteriostatic and selective agents so that only the transformed cells produced callus. Callus was subcultured in hormone-free medium to give transgenic plants. Using this method, G. hirsutum Coker 201 was transformed. 16. Perlack FJ, Deaton RW, Armstrong, Armstrong, T.A., Fuchs, R.L., Sims, S.R., Greenplate, and Fishhoff, D.A. TA, Fuchs RK, Sims SR, Greenplate & Fischhoff DA) 1990, insect-resistant cotton, plants. Bio / Technology 8: 939-943 2,4-D, Kin Hypocotyl Bacillus thuringiensis Kurstaki ) The insect control protein gene of the variant HD-1 [cry 1A (b) and HD73 Cry1A (c)] was truncated and transformed into the cotton hypocotyl via Agrobacterium tumefaciens. Then, a somatic embryo was obtained from the transformed cells, and finally, an insect-resistant cotton plant of G. hirsutum Coker 312 was obtained. .

17. Bayley, C, Trolinder, NL, Ray, C, Morgan, M, Xenenberry, Jay E, and O'Daw Dee Brew 1992, Impartation of 2,4-D resistant cotton, Theo, Appl. Genet. 83: 645-649 2,4-D, Kin Hypocotyl 2,4-D monooxygenase from Alcaligenus eutrophus The gene tfdA was isolated, modified, expressed in tobacco, and transformed into cotton plants with Agrobacterium tumefaciens to obtain Coker strain 312, a 2,4-D resistant plant. 18. Langan T, Rajsekran, Hudspeth and Yenofsky (1989) Cotton regeneration and transformation, Patent No. EP344302 Transformation and regeneration of transgenic plants (via somatic embryogenesis) NAA, Kin hypocotyl, cotyledon, immature embryo cotton (SJ2, SJ4, SJ5, SJ2-1, GC510, B1644, B27110, B1810, Picker Variety of Siokra and stripper FC2017) Regeneration and transformation. Transformation was performed with Agrobacterium tumefaciens somatic embryos obtained from calli germinated in Beasley and Ting's medium.

[0022] 19. Finer JJ and McMullen 1990, Transformation of Gossypium hi rsutum L via particle bombardment, Plant Cel-I Rep. 8: 586-589 Trans. Transformation and regeneration of transgenic plants (via somatic embryogenesis) NAA, Kin, 2,4-D, picloram Discs of cotyledon leaves High-density particles containing plasmid DNA are transformed into embryogenic plant cells An accelerated particle bombardment method was performed on embryogenic suspension cultures of cotton. These cells were then subjected to somatic embryo development in the presence of a selection material (Hygromycium). Coker 310 variants were transformed in this manner. 20. McCabe DE and Martinelli BJ 1993, Transformation of excellent cotton varieties by meristem particle bombardment, Bio / Technology 11: 596-598 from pre-existing meristems Organogenesis BA (short treatment for 15 hours) Hypocotyl This method excises hypocotyls from germinating seeds and sprays particles containing foreign genes onto hypocotyls. Plants grew from the treated hypocotyls and the transformed plants were screened. Only one plant grew from one axis. The following cotton varieties were transformed using this method. Pima, sea island cotton, new world cotton (upland) varieties. 21. McCabe DE and Martinelli BJ 1992, Particle-mediated transformation of cotton, patent PCT / US90 / 0172, Same as above.

[0022] 22. Clan, C.A., Lin., J., Carey, J.W., and Cleveland, T.E. (Chlan CA, Lin J., Cary JW & Cleveland TE) 1995, Impact Transformation of Transgenic Cotton from Meristems Regeneration method, Plant Mol. Bio. Rep. 13: 31-37 Organ formation from pre-existing meristems BA, IAA Hypocotyls Hypocotyls are aseptically removed and burst pressure of DNA coated 1.6A ° gold particles Impact was performed at 90 to 110 kg / cm ² . After impact, these tissues grew to whole plants in the presence of kanamycin selection. One axis gave only one plant. ────────────────────────────────────

[0024]

SUMMARY OF THE INVENTION The present invention provides, for the first time, an efficient method of growing a new meristem zone with apical explants from cotton seedlings and providing large numbers of shoots. In addition, the explants form organ-forming masses at the base, which are divided into smaller parts to maintain a cycle in which regeneration of multiple shoots is repeated with good reproducibility. The method of the invention is very simple and applicable to a wide variety of Gossypium species and species. The method is largely genotype independent, rapid, and convenient for micropropagation and genetic transformation applications.

[0025]

The method of the present invention uses a different starting material (explant) from previous reports and gives the same as the original plant because it is based on direct organogenesis; Gives simple plants that are simple and capable of large amounts of fertilization. The reports and patents in Table 1 vary greatly depending on the variety (genotype). The method described by the applicant is applicable to many variants of different species. Reports 20 and 22 in Table 1
The patented claim 21 of Table 1 uses hypocotyls as explants, and in this method, as described by the applicant,
One shoot is formed instead of several shoots. While reports 4 and 6 in Table 1 give non-fertile plants, the method of the present invention gives fertilizing tendon lake plants.

The success or failure of the method of the present invention depends on the age of the seedlings and the method by which the explants are removed from the seedlings for culturing according to the method of the present invention. In the method of the present invention, cotyledonous nodes (also called cotyledon axils or apical meristems of seedlings) are
Take from ~ 20 days old seedlings. When explants obtained from such seedlings are cultured, various numbers of shoots are obtained depending on the variety. Apical meristems without cotyledons or with cotyledons one or part and with 0.5 cm hypocotyls gave multiple shoots. This method provided a large amount of shoots in one step from apical meristems taken without cotyledons. Growth regulator requirements varied depending on the explant. When the apical meristem was taken without cotyledons, both BAP and kinetin were required for multiple shoot growth. On the other hand, when one or a part of one cotyledon was included, regeneration of multiple shoots required only BAP. The hormone combinations and explants we used are different from those reported in any of the reports listed in Table 1. Regeneration of multiple shoots in our method was successful at a range of hormone levels. For example, BAP is 1
It works efficiently at concentrations of 0-100 μM and gives shoots from the apical axilla in the presence of one or some cotyledons. For cotyledon axils only, BAP 4- with kinetin 2-20 μM
Works efficiently at 20 μM. As described in Table 1, the growth regulator, or BAP, along with kinetin has not previously been used for the development of methods for multiple shoot regeneration of cotton. In reports 11 and 12 of Table 1, the method gives poor roots, while the method of the present invention is superior in this respect.

The method of inducing the growth of multiple shoots involves cells other than the original meristem, which naturally grow into one plant. We have identified explants (also known as cotyledons or axils) that give rise to new meristems that result in multiple shoots when cultured in media supplemented with appropriate concentrations of commonly used growth regulators. This method works for any variant of cotton,
It can be applied to cultivar, strain or race, so this method is a new method, has no genotype restriction and shortens the time required for regeneration which is a serious problem in the prior art Is done.

The present invention provides for the first time an efficient method of growing a new meristem zone with apical explants from cotton seedlings and providing several shoots. In addition, the explants form organ-forming masses at the base, which are divided into smaller parts to maintain a cycle in which regeneration of multiple shoots is repeated with good reproducibility.

Earlier patents in the area relating to cotton include US Pat.
A4672035 and U.S. Pat. No. 5,244,802 (where we present Gosipium spp. Via somatic embryogenesis).
Gossypium hirsutum L Coker disclosed a method of regenerating plants from 310 varieties of cotton callus; US-A 86937384 and CA
A1, 1335799 (here disclosed a method of cotton regeneration and transformation involving somatic embryogenesis based on a regeneration method)
And WOA1 9215675 (where the inventor
Discloses a method of impact-mediated transformation of cotton embryos, from which transgenic plants were grown.

[0030] The method described in the present invention is not intended for somatic embryogenesis. The method of the present invention is very simple to employ commercially and has application to a wide variety of Gossypium varieties and species. It is largely genotype independent and is useful for micropropagation and genetic transformation applications. Unlike current methods, the method of the present invention does not form plants with morphological and cytogenetic abnormalities.

The method of the present invention uses a different starting material (explant) from that used in previous reports and is based on direct organogenesis, so that it is short, convenient and fertile. Give a normal plant.

OBJECTS OF THE INVENTION It is a primary object of the present invention to provide an improved method for efficient organ formation of cotton varieties for the purpose of maintaining and regenerating large amounts of plants from specific plant tissues.

Another object of the present invention is to provide a method of regenerating shoots of cotton plants, such that the shoots multiply and easily and efficiently produce roots to form mature fertile cotton plants. That is.

Yet another method of the present invention is to provide a cotton plant applicable to DNA-coated microprojectiles for genetic transformation using Agrobacterium or for delivery of DNA to plant cells. It is an object of the present invention to provide an improved method for organ-forming tissue culture.

Yet another method of the present invention is to provide a method for growing disease-free cotton plants for the replacement and preservation of disease-free germplasm.

To achieve the above object, the present applicant has
A method for regenerating a large number of viable fertilizable cotton plants by tissue culture starting from the cotyledon node (also called cotyledon axilla or cotyledon axis) with or without one or a part of the cotyledon of the cotton seedling. The method comprising the steps of: i) treating cotton plant seeds in a conventional manner to remove bacteria / mold (contaminants) in a conventional manner; ii) treating the seeds treated in step (i) in a first medium Wherein the first medium comprises: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, and d. A medium comprising a gelling agent and having a pH in the range of 5.4 to 6.2,
Autoclaved and cultured for 20-4
3 days to several weeks at a temperature of 0 ° C. in the presence of 16 hours light (photoperiod) light (at least 40 μmol / m ² s), iii. Cutting cotyledon nodes (explants) with or without partial or complete cotyledons from the seedlings obtained in step (ii), iv. The explant obtained in step (iii) is cultured in a second medium for shoot growth, wherein the second medium comprises: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, d. Plant hormone (plant growth regulator) alone, 0.1 ~
100 μM 6-benzylaminopurine, 6-benzyladenine, γ, γ-dimethylallylaminopurine, kinetin, 1-phenyl-3,1,2,3thidiazole-5
Plant hormones in combination with cytokinins such as ilurea, e. A medium comprising a gelling agent and having a pH in the range of 5.4 to 6.2, which has been sterilized by an autoclave and cultured for 20 minutes.
16 hours of light (40 μmol / m ²⁾ at a temperature of ４０40 ° C.
(v) culturing is continued for at least 4 weeks until many shoots are formed; (vi) collecting the formed shoots; (vii) transferring the collected shoots to a root-inducing medium (third medium). Transfer, where the third medium is: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, d. A plant hormone (a plant growth regulator) of 50 μM or less consisting of an auxin selected from indoleacetic acid, indolebutyric acid and naphthaleneacetic acid; e. A medium consisting of a gelling agent and having a pH in the range of 5.4 to 6.2, which is sterilized in an autoclave, and (viii) a light of 16 hours at a temperature of 20 to 40 ° C. ( 4
0 μmol / m ² s) until the roots are formed.

A preferred feature of the present invention is that the salt of MS medium, B
Use first, second and third media consisting of 5 media vitamins, a carbon source and a gelling agent. However, the second and third media may further contain a plant hormone (a plant growth regulator) and a gelling agent. The most preferred salts of MS media used for the first, second, and third media are
It has the following composition: Ingredient Concentration (mg / L) Salt of MS Medium: NH ₄ NO ₃ 1650 KNO ₃ 1900 MgSO ₄ .7H ₂ O 370 MnSO ₄ H ₂ O 169 ZnSO ₄ .7H ₂ O 8.6 CuSO _{4 · 5H 2 O 0.025 CaCl 2 ·} 2H 2 O 440 KI 83 CoCl 2 · 2H 2 O 0.025 KH 2 PO 4 170 H 3 BO 3 6.2 Na 2 MoO 4 · 2H 2 O 0.25 FeSO 4・ 7H ₂ O 27.85 Na ₂ EDTA 37.3 Myo-inositol 100

Suitable vitamins for use in the first, second and third media are selected from the following: Ingredient Concentration (mg / L) Nicotinic Acid 1.0 Pyridoxine Hydrochloride 1.0 Thiamine Hydrochloride 10 .0

The carbon source used in the first, second and third media is selected from sucrose or glucose, and such a carbon source used ranges from 1 to 6% w / v. .

The plant hormone used in the second medium is
It was selected from cytokinin, cytokinin active urea or a combination thereof. Preferably, the plant hormone used in the second medium is a cytokinin such as 6-benzylaminopurine, 6-benzyladenine, zeatin, (γ, γ dimethylallylaminopurine), kinetin; 1-phenyl-3 -1,2,3 thidiazol-5-yl urea and diphenyl urea, 4-1-phenyl N (pyridyl)
It is selected from cytokinin-active ureas such as urea. The growth regulator used in the third medium is an auxin such as indoleacetic acid, naphthaleneacetic acid and indolebutyric acid.

Cotyledonous nodes (explants) are isolated from germinated seedlings from 2 days to several weeks old, and the explants used are taken from cotton plants grown in the field or grown in tissue culture. The basal regenerated tissue (remaining after collecting the shoots in the first cycle) is removed from steps (iv)-
Repeat the culture in (viii). The salt concentration of the MS medium used is the full or half level already described on a weight / volume basis. The gelling agent used is 0.2-
It is selected from 1.2% w / v agar, gelrite (phytagel) or any gelling agent.

Further, shoots regenerated by the tissue culture method of the present invention can be used for micropropagation of cotton plants. Further, the cotyledon node or basal regenerated tissue produced by the method of the present invention may contain Agrobacterium (Agrobacterium).
ium) or can be used for genetic transformation via bombardment of DNA-coated particles. In addition, shoots regenerated by the tissue culture method can be used for the production of disease-free cotton plants, or such shoots can be used for the replacement and preservation of disease-free cotton germplasm. .

The most preferred methods of the present invention include: (i) treating cotton plant seeds to remove contaminants such as bacteria / mold; (ii) treating seeds for germination in Table 2. Culture in the indicated medium, where the medium is

[0044]

Table 2 Component concentration (mg / L) Salts of MS medium: NH ₄ NO ₃ 1650 KNO ₃ 1900 MgSO ₄ .7H ₂ O 370 MnSO ₄ H ₂ O 169 ZnSO ₄ .7H ₂ O 8.6 CuSO ₄ .5H ₂ O 0.025 CaCl ₂ .2H ₂ O 440 KI 83 CoCl ₂ .2H ₂ O 0.025 KH ₂ PO ₄ 170 H ₃ BO ₃ 6.2 Na ₂ MoO ₄ .2H ₂ O 0.25 FeSO ₄ .7H ₂ O 27.85 Na ₂ EDTA 37.3 Myo-inositol 100 B. B5 medium vitamin nicotinic acid 1.0 pyridoxine hydrochloride 1.0 thiamine hydrochloride 10.0 C.I. Carbon source: sucrose / glucose 30000.0 Gelling agent 0.2-1.2% w / v

A medium having a pH in the range of 5.4 to 6.2, which has been sterilized by an autoclave; (iii) cutting off the cotyledon node together with the cotyledon axilla obtained in the step (ii); The explant obtained in step (iii) is cultured in a medium shown in Table 3, wherein the medium is

[0046]

Table 3 Component concentration (mg / L) Salts of MS medium: NH ₄ NO ₃ 1650 KNO ₃ 1900 MgSO ₄ .7H ₂ O 370 MnSO ₄ H ₂ O 169 ZnSO ₄ .7H ₂ O 8.6 CuSO ₄ .5H ₂ O 0.025 CaCl ₂ .2H ₂ O 440 KI 83 CoCl ₂ .2H ₂ O 0.025 KH ₂ PO ₄ 170 H ₃ BO ₃ 6.2 Na ₂ MoO ₄ .2H ₂ O 0.25 FeSO ₄ .7H ₂ O 27.85 Na ₂ EDTA 37.3 Myo-inositol 100 B. B5 medium vitamin nicotinic acid 1.0 pyridoxine hydrochloride 1.0 thiamine hydrochloride 10.0 C.I. Carbon source: sucrose / glucose 30000.0 Hormone (growth regulator) Cytokinin 0.1-100 μM Gelling agent 0.2-1.2% w / v

A medium having a pH in the range of 5.4 to 6.2, which is sterilized by an autoclave, and (v) a light (40 μm) of 16 hours at a temperature of 20 to 40 ° C. Mol / m ² s) for 4-6 weeks or until many shoots are formed, (vi) collecting the formed shoots; (vii) transferring the collected shoots to a root-forming medium shown in Table 4. , Where this medium is

[0048]

Table 4 Component concentration (mg / L) Salts of MS medium: NH ₄ NO ₃ 1650 KNO ₃ 1900 MgSO ₄ .7H ₂ O 370 MnSO ₄ H ₂ O 169 ZnSO ₄ .7H ₂ O 8.6 CuSO ₄ .5H ₂ O 0.025 CaCl ₂ .2H ₂ O 440 KI 83 CoCl ₂ .2H ₂ O 0.025 KH ₂ PO ₄ 170 H ₃ BO ₃ 6.2 Na ₂ MoO ₄ .2H ₂ O 0.25 FeSO ₄ .7H ₂ O 27.85 Na ₂ EDTA 37.3 Myo-inositol 100 B. B5 medium vitamin nicotinic acid 1.0 pyridoxine hydrochloride 1.0 thiamine hydrochloride 10.0 C.I. Carbon source: sucrose / glucose 30000.0 Hormone (growth regulator) Auxin 50 μM or less Gelling agent 0.2-1.2

A medium having a pH in the range of 5.4 to 6.2, which is sterilized by an autoclave; and (viii) culturing the shoots at a temperature of 20 to 40 ° C. until roots are formed. It comes from things.

The seedlings thus formed can be transferred to soil to grow cotton plants, if necessary.

In the present invention, the success or failure of the present invention depends on the age of the seedling and the method of removing explants from the seedling for culturing by the method of the present invention. For optimal results, cotyledons may be taken along with axils from 2-20 day old seedlings. However, depending on the combination of varieties and plant hormones used, the largest number of shoots can be obtained from explants taken from seedlings of a particular age.

In yet another embodiment of the method of the present invention,
It can be repeated using the basal mass remaining after collecting the shoots as described in step (vi). The basal mass is cut into 2-6 pieces, cultured as in step (iv) and the remaining steps up to step (viii) are continued to obtain a second cycle of shoot growth.

The details of the method of the present invention are as follows:
These are surface sterilized prior to use to produce seeds free of mold contaminants. Many methods of sterilization are known for surface sterilization. Such methods include at least one
There is a method of soaking the seed in a solution containing two sterilants. Such sterilants include sodium hypochlorite, calcium hypochlorite, mercuric chloride, alcohol, sulfuric acid, and the like. For surface sterilization of seeds, 0.05-0.5% w
/ V mercuric chloride aqueous solution with continued stirring for 1 to 15 minutes, then washed thoroughly (3 to 10 times) with deionized sterile water, and then seeded with ethanol or rectified alcohol (50 to 50).
100% w / v) for 30 seconds to 10 minutes.

Surface-sterilized seeds are used to germinate
On water containing a gelling agent such as 0.6-1.2% w / v agar in germination bottles, on damp paper towels, or on Murashige and Skoog.
B.) Salt or any other conventional medium, vitamins or other vitamin mixtures of B5 medium, and any carbon source such as 1-6% w / v sucrose or glucose, and 0.6-1.2 % W / v agar or 0.2-0.5%
w / v gelrite (phytagel)
el)), place on a culture medium containing a gelling agent, adjust the pH of the culture medium to 5.6 to 6.2, and adjust
Autoclave at lb / cm for 20 minutes.

Placing 1-2 seeds in a 50 ml glass culture tube containing 10-15 ml of germination medium or in a large (250 ml) plastic or glass jar containing 50 ml of medium Can be. For germination, seeds may be incubated at a temperature of 20-40 ° C. with 16 hours of light (at least 40 μmol / m ² s). The incubation is continued until the seeds germinate and the roots and sprouts emerge and give cotyledons that are sufficiently widespread.

Cotyledonous nodes (explants) are preferably obtained from 2 to 20 day old seedlings after germination in a contaminant-free environment known in the art, ie, in a laminar flow with a sterile sharp scalpel or knife. Obtained by cutting. The cotyledon nodes along the axils are cut out so as not to damage the node tissue.

Cotyledon nodes (explants) were obtained at the concentrations shown in Table 2 on a weight / volume basis with Murashige and Skoog.
Skoog) salt or half its concentration, vitamin or any other vitamin composition in B5 medium, 1-6% w / v of sucrose or any carbon source of glucose, commonly used growth regulators, e.g. Quinine-BAP (6-benzylaminopurine), 6-benzyladenine, kinetin, zeatin, and 2iP (γ, γ-dimethylallylaminopurine), and 0.6 to 1.2% of a gelling agent w / v agar or 0.2-0.5%
w / v gelrite (phytagel)
el)). PH of the medium is 5.4 ~
After adjusting to 6.2, autoclave at 121 ° C., 15 pounds / cm for 20 minutes. The composition of the medium for shoot propagation is shown in Table 3.

The culture was subjected to a 16 hour light (40-100 μmol / m ² s) white fluorescent lamp at a temperature of 20-40 ° C.
Incubate until multiple shoots are formed. This is achieved by bulging the basal part of the explant. At this stage the shoots are collected and on a weight / volume basis the concentrations of Murashige and Skoog salt or half of the concentrations shown in Table 3, vitamins in B5 medium or any other vitamin composition known in the art, Any carbon source of 1-6% w / v sucrose or glucose, auxin-type growth regulators for root induction, such as indoleacetic acid, naphthaleneacetic acid, indolebutyric acid (50 μM or less), gelling agent. 0.6-1.2% w / v agar or 0.2-0.5% w / v gelrite
After adjusting the pH of the medium to 5.6 to 6.2, the mixture is autoclaved at 121 ° C. and 15 lb / cm for 20 minutes. The composition of this medium for inducing roots is shown in Table 4. Culture until roots develop and shoots grow to give 2-4 vortex leaves.

The remaining basal tissue (after collecting the shoots)
It is divided into several parts and cultured again in the medium shown in Table 3 to allow a second cycle of shoot growth.

The culture is continued until a plurality of shoots are formed. At this stage, the shoots are collected again and transferred to the medium for root formation shown in Table 4. The remaining basal mass is again cultured for the next cycle of shoot growth. Thus, starting from one explant, up to 30 shoots can be collected in three cycles of culture.

Such shoots with well-developed roots are removed from the culture and washed with sterile deionized water to remove trace nutrients present on the root surface, and then the seedlings are vegetatively grown. Nutrient solutions, such as Hoagla
nd) Maintain in solution (without sugar). Once vegetative growth has begun, they are transferred to vermiculite and finally to soil for growing mature plants with seeds.

The method of the present invention for inducing a high frequency of shoots allows whole plants to grow. The basis of multiplication to obtain multiple shoots from cotyledons involves cells other than the original meristem, which multiply and grow naturally into one plant. We have identified explants that give new meristems that become multiple shoots when cultured in media supplemented with appropriate concentrations of appropriate growth regulators. The method of the present invention can be applied to any variety of cotton. This method is a new approach, not limited to genotype, and reduces the time required for shoot regeneration, a serious problem in the prior art.

The method of the present invention relates to the differentiation of shoots from cells other than preexisting meristems of germinated seeds. Therefore, culturing the explant in the presence of an appropriate concentration of a growth regulator would likely alter the cell programming to produce multiple shoots. New meristems produced by the method according to the invention produce some shoots.

At present, there is no detailed method for forming multiple shoots from explants of any tissue of any cultivar of cotton. Reports dealing with tissue culture methods leading to micropropagation have been limited to plant regeneration via somatic embryogenesis. Only a few varieties,
That is, the Coker strain responds well to the already used methods of somatic embryogenesis. The growth process of plant regeneration through somatic embryogenesis takes several months.
Moreover, plants obtained by somatic embryogenesis are morphologically and cytogenically abnormal and infertile. The present invention provides, for the first time, a detailed method of organogenesis of explants that provide multiple shoots with cotton. According to the method of the present invention, explants are repeatedly sub-cultured in a medium supplemented with a suitable concentration of a growth controlling substance, and a range of 3 to 30 (the number is important)
You can regenerate multiple shoots. This has not been suggested in previous publications and is very important for growing multiple seedlings from one explant.

The following examples are intended to illustrate the invention.
It should in no way be considered as limiting the scope of the invention.

[0066]

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Sources of explants and genotypes Seeds of cotton (G. hirsutum variety Khandwa-2) treated with 0.1% mercuric chloride for 7 minutes Sterilized. The seeds are then washed five times with sterile distilled water and alcohol (90% v /
v) Burned for 30 seconds. This sterilized seed,
Murashige and Skoog salt, B5
Of vitamins, 3% w / v sucrose and 0.8% w / v agar as a gelling agent, and the pH of the medium was adjusted to 5.8 before autoclaving.
The two seeds were placed in 50 ml glass tubes each containing 15 ml of medium. Seeds were placed in a microbe-free environment (ie, laminar flow) with sterile forceps. The seeds on the medium were incubated at 25 ± 2 ° C. with a white fluorescent light (60 μmol / m ² s) for 16 hours light until the seeds germinated and roots and sprouts were formed with well-extended cotyledons. .

Explants (small pieces of shoots used for regeneration experiments as starting material) were cut out using 6-day-old seedlings. Explants of different types (eg, hypocotyl sections, discs from cotyledons, mature embryos isolated from seeds, immature isolated from immature seeds, with or without one or part of the cotyledons) Embryos, cotyledons (axilla), and hypocotyls of about 0.5 cm) were cut out.

These different explants were combined with Murashige and Skoog salts, B5 vitamins,
% W / v glucose, 22.19 μM 6-benzyladenine and 0.6% w / v gelling agent agar, and the pH of the medium before autoclaving.
Was adjusted to 5.8. Culture was continued until multiple shoots were regenerated. It was observed that the apical meristem together with both cotyledons failed to produce multiple shoots. Hypocotyl sections, cotyledons and mature embryos as well as immature embryos failed to produce multiple shoots. Cotyledon nodes taken with 0.5 cm hypocotyls and one cotyledon or part of cotyledon give rise to multiple shoots in the medium. Cotyledons taken apart from cotyledons also produced a large number of shoots in media containing 8.87 μM 6-benzyl-adenine and 9.29 μM kinetin.

Identification of the types of cotyledonous explants and the combinations of growth regulators to which they respond is an important aspect of the present invention.

Example 2 Media-Dependent Response of Explants Treated with 0.1% w / v mercuric chloride for 7 minutes, washed 6 times with sterile distilled water, and then seeded with alcohol (90% w / v)
v) for 30 seconds and burned, cotton (G. hirsutum L. Khandwa-2) seeds were removed. This sterilized seed is used for Murashige and Skoog (Mu
rashige and Skoog) salt, B5 vitamin, 3% w / v
Of sucrose and 0.8% w / v agar (before autoclaving, adjust the pH of the medium to 5.
8). For germination, 2 seeds of 15 ml each
And stored in glass tubes containing medium. Seeds on the medium were incubated at 25 ± 2 ° C. with a white fluorescent light (60 μmolm ⁻² s ⁻¹ ) for a 16 h light period. Seeds were sown on the medium with sterile forceps in a contaminant-free environment (laminar flow). The culture was continued until the seeds germinated and roots and sprouts were formed with well-extended cotyledons.

Axils (explants) of seeds having cotyledons were cut out using 6-day-old seedlings. Explants were cut with a sharp scalpel. This explant is mixed with Murashige and Skoog salt, B5 vitamins, 3% w
/ V glucose and / or sucrose, 22.19μ
Plated on medium containing M 6-benzyladenine and 0.6% w / v gelling agent (medium pH adjusted to 5.8 before autoclaving).

The culture was continued until a plurality of shoots were formed. Multiple shoots were observed in the presence of 30 gl ^-1 sucrose or glucose. However, based on comparative studies using glucose, sucrose and glucose + sucrose for differentiation of multiple shoots, glucose was selected as the preferred carbon source in the medium. In the medium containing sucrose alone or in combination with glucose, the phenols leached in large quantities and the medium turned brown. Leaching of phenols was minimal when glucose was provided as the sole carbon source. When the medium was rich in phenols, shoot growth was reduced and explants eventually died. The use of glucose (rather than sucrose) in cotton is an important aspect of the present invention. This prevents leaching of phenols. This protocol does not require frequent subculture of growing explants. In previous reports, this was an important issue in cotton tissue culture.

Cotyledon nodes (explants) were also cultured. Murashige and Skoog salt, B5 vitamins, 3% w / v glucose, different concentrations of growth regulators [eg 2.22 μM to 44.38 μM BAP
(6-benzyladenine), 2.32 μM-44.47
μM KIN (Kinetin) and 2.46 μM-49.
21 μM 2iP (γ, γ dimethylallylaminopurine) and a combination of 6-benzyladenine with dimethylallylaminopurine and kinetin], and 0.6
Cotyledon nodes (explants) were also cultured in a medium containing% w / v gelling agent agar (pH of the medium was adjusted to 5.8 before autoclaving).

The culture is grown until a plurality of shoots are formed.
With a white fluorescent lamp (60 μmolm ^-2 s ^-1 ), 2
Incubated at 5 ± 2 ° C. The results obtained are summarized in Tables 5 and 6.

[0075]

Table 5 Effect of cytokinin on shoot development from explants (cotyledons with one cotyledon) of cultivar "Khandwa-2" in the first cycle of regeneration Explant-forming properties Per explant Notes Concentration (μM) Shoots (%) Average number of shoots formed, ± SE (first cycle) ^* BAP (2.22) 66 0.8 ± 0.17 (a) BAP (4.44) 73 1.23 ± 0.27 (a) BAP (8.87) 70 2.90 ± 0.57 (b) BAP (13.31) 66 3.28 ± 0.60 (b) BAP (22.19) 80 4.46 ± 0.68 (b) BAP (33.09) 66 3.07 ± 0.65 (b ) Less shoot growth BAP (44.38) 60 3.33 ± 0.49 (b) Less shoot growth KIN (2.32) 26 0.8 ± 0.2 (a) KIN (4.65) 33 0.8 ± 0.2 (a) KIN (9.29) 26 2.00 ± 0.54 (b) ) KIN (13.94) 53 2.20 ± 0.41 (b) KIN (23.23) 60 3.10 ± 0.43 (b) Root formation KIN (34.85) 60 2.00 ± 0. 31 (b) Root formation KIN (46.47) 60 2.80 ± 0.48 (b) Root formation 2iP (2.46) 20 0.6 ± 0.24 (a) 2iP (4.92) 26 0.4 ± 0.16 (a) 2iP (9.84) 40 0.46 ± 0.16 ( a) 2iP (14.76) 53 0.73 ± 0.52 (a) 2iP (24.61) 60 1.261 ± 0.53 (a) Callus formation 2iP (36.91) 30 1.25 ± 0.41 (a) Callus formation 2iP (49.21) 30 1.09 ± 0.42 (a) Callus formation Note: Data shown are means ± SE (standard error) calculated from the results of three independent experiments. When the treatments classified as a and b were analyzed by the Student's t test, there was a significant difference between treatments at P> 0.05, but no significant difference within treatments. BAP (6-benzyladenine), Kin (kinetin) and 2iP (γ, γ dimethylallylaminopurine).

[0076]

Table 6 Combined effect of cytokinin on the first cycle shoot development from explants (cotyledons with one cotyledon) of modified medium variety "Khandwa-2" Different cytokinin concentrations Explants per explant (μM) Formability Shoots formed Shoots (%) Average number ± SE ^* BAP KIN 2iP 4.44 4.65-50 0.75 ± 0.21 (a) 8.87 9.29- 33 1.00 ± 0.36 (a) 13.31 13.94-66 2.13 ± 0.19 (b) 22.19 23.23-86 3.86 ± 0.48 (c) 4.44 4.65 2.46 60 0.73 ± 0.18 (a) 4.44 9.29 4.92 40 1.19 ± 0.34 (a) 4.44 13.94 9.84 50 1.28 ± 0.38 (a) 4.44 23.23 24.61 66 2.20 ± 0.45 (b) 4.44 46.47 49.21 66 2.16 ± 0.27 (b) Note: Data shown are mean ± calculated from the results of three independent experiments SE (standard error). When the treatments classified as a, b, and c are analyzed by the Student's t test, there is a significant difference between the treatments at P> 0.05, but there is no difference within the treatments. BAP (6-benzyladenine), Kin (kinetin) and 2iP (γ, γ dimethylallylaminopurine).

It is clear from the results that changes in the concentration of the growth regulator give different responses with respect to the development of multiple shoots. This can be used to develop optimal conditions for the regeneration of the varieties.

Example 3 Cultivar / Species-Dependent Response Gossypium hirsutum
L) and 13 of C. arboreum
Seeds of different varieties were treated with 01% w / v mercuric chloride for 7 minutes, washed six times with sterile distilled water, and then burned by soaking in alcohol (90% v / v) for 30 seconds.

Sterilized seeds of different varieties were sown on a medium containing Murashige and Skoog salts, vitamins of B5, 3% w / v sucrose and 0.8% w / v agar. (The pH of the medium was adjusted to 5.8 before autoclaving). For germination, the two seeds were stored in glass tubes each containing 15 ml of medium.
Seeds on media, the white fluorescent lamp (60μmolm ^-2 s ^-1) 1
Incubated at 25 ± 2 ° C. with a 6 hour light period. Seeds were sown on the medium with sterile forceps in a contaminant-free environment (laminar flow). The culture was continued until the seeds germinated and roots and sprouts were formed with well-extended cotyledons.

Cotyledon nodes (explants) using 6-day-old seedlings
Was cut out. Explants were cut with a sharp scalpel.
Explants are Murashige and Skoog
) Salt, B5 vitamin, 3% w / v glucose, 2
2.19 μM 6-benzyladenine and 0.6% w
/ V gel medium agar (121 ° C).
The pH of the medium was adjusted to 5.8 before autoclaving at 15 pounds / cm ² for 20 minutes). Cultures are grown at 25 ± 2 ° C. with 16 hours of light using white fluorescent light (60 μmolm ⁻² s ⁻¹ ).
Incubated. Culture was continued until multiple shoots were formed.

In this step, the shoots are collected and Murashige and Skoog salt, B5 vitamin, 3% w / v sucrose, 2.69 μM concentration of a growth control substance (naphthalene acetic acid) NAA6- Transferred to medium containing benzyladenine and 0.8% w / v gelling agent agar (pH of the medium was 5.3 before autoclaving).
Adjusted). The results obtained are summarized in Table 7.

[0082]

Table 7 22. Organogenesis capacity of cotton varieties with modified medium containing 19 μM 6-benzyladenine Varieties Explant formation Primary per explant Secondary shoots per explant (%) Number of shoots (first cycle) Number of shoots (second cycle) ± SE ± SE Gossypium 80 4.46 ± 0.70 (a) 5.80 ± 0.91 (a) hirsutum cv. Khandwa-2 G.hirsutum 66 2.10 ± 0.56 (b) 7.08 ± 0.13 (a) cv. PKV081 G.hirsutum 73 1.90 ± 0.44 (b) 4.66 ± 0.86 (a) cv. RS810 G.hirsutum 90 2.70 ± 0.42 (b) 4.30 ± 0.73 (a) cv. Pusa37 G.hirsutum 80 1.60 ± 0.45 (b) 3.10 ± 0.70 (b) cv. Pusa G.hirsutum 70 1.40 ± 0.40 (b) 2.80 ± 0.57 (b) cv. Stoneville G.hirsutum 70 2.40 ± 0.63 (b) 3.50 ± 0.87 (a) G .hirsutum 60 2.10 ± 0.56 (b) 2.50 ± 0.63 (b) cv. CA-1193 G.hirsutum 70 1.50 ± 0.44 (b) ND ³ cv. Jurhat G.hirsutum 90 1.90 ± 0.44 (b) ND cv. Sima G .hirsutum 87 2.37 ± 0.37 (b) ND cv. LH1343 G.arboreum 50 1.00 ± 0.39 ( ) 2.20 ± 1.00 (b) cv. Shyamly G.arboreum 40 0.50 ± 0.16 (c) 0.90 ± 0.34 (a) cv. Lohit 1. Data were collected for each of the 10 methods as described in Methods.
Mean ± SE (standard error) calculated from the results of three independent experiments repeated 15 times. 2. When the processes classified as a, b and c are analyzed by a statistical test, there is a significant difference between the processes at P> 0.05, but there is no difference within the processes. 3. Not implemented.

From the results in this table, it is very clear that this method works well for all 13 cotton varieties. However, when cultured in a medium containing 22.19 μM 6-benzyladenine, G. hirsutum (G.
sutum varieties were more responsive, while G. arboreum was less responsive and species-related differences in organogenesis were observed.

Further, in the first cycle of regeneration, shoots were removed for root development, and the basal mass of the remaining explants was cultivated again in medium for the next cycle of regeneration. The regenerated material was collected again, transferred to medium containing the appropriate concentration of growth regulator for root development, and the remaining basal mass re-cultured in the medium for the next cycle of regeneration. Thus, in three cycles of culture, up to 30 shoots could be recovered starting from a single explant.

[0085]

According to various aspects of the present invention, an easy, efficient and reproducible method for frequently inducing direct differentiation of regenerated products and plant regeneration from several varieties of cotton. Is provided. The method of the present invention provides for direct differentiation of shoots and provides many of the advantages described above. The rapid and high frequency of direct shoot morphogenesis routinely obtainable by cultivation by this method is attributed to the genetic and / or impact transformation of cotton varieties by Agrobacterium tumefaciens. It is expected to facilitate the law.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Aroku Kumar Srivastava India 226001 U. P. Looknow, Lana Pratap Marg, National Botanical Research Institute (72) Inventor Shiv Kumar Gupta India 226015 U. P. Looknow, Central Institute of Medicinal and Aromatic Plants, P.K. Oh. Simap

Claims (19)

[Claims] 1. A fertilizing cotton plant capable of growing in large quantities by tissue culture starting from a cotyledon node (also called cotyledon axis or cotyledon axil) with or without one or a part of the cotyledon of the cotton seedling (I) treating cotton plant seeds to remove bacteria / mold (contaminants) by a conventional method, and (ii) treating the seeds treated in step (i) in a first medium. Wherein the first medium comprises: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, and d. A medium comprising a gelling agent and having a pH in the range of 5.4 to 6.2,
Autoclaved and cultured for 20-4
3 days to several weeks in the presence of 16 hours of light (photoperiod) light (at least 40 μmol / m ² s) at a temperature of 0 ° C. for 16 hours;
(Iii) cutting cotyledon nodes (explants) with or without partial or complete cotyledons from the seedlings obtained in step (ii),
(Iv) culturing the explant obtained in step (iii) in a second medium for shoot growth, wherein the second medium comprises: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, d. 0.1 as plant hormone (plant growth regulator)
Any combination of cytokinins such as １００100 μM 6-benzylaminopurine, 6-benzyladenine, γ, γ-dimethylallylaminopurine, kinetin, and cytokinin-active urea derivatives; e. A medium comprising a gelling agent and having a pH in the range of 5.4 to 6.2,
Autoclaved and cultured for 20-4
Light for 16 hours at a temperature of 0 ° C. (40 μmol / m ² /
performed in s ^2); (v) number of shoots continued for at least 4 weeks culture until form; (vi) were collected form the shoots; (vii) Harvested shoots roots inductive medium (third medium) Where the third medium is: a. Salts of any conventional media, b. Vitamins in any conventional media, c. A carbon source, d. A plant hormone (a plant growth regulator) of 50 μM or less consisting of an auxin selected from indoleacetic acid, indolebutyric acid and naphthaleneacetic acid; e. A medium comprising a gelling agent and having a pH in the range of 5.4 to 6.2,
Autoclaved and (viii)
Culturing at a temperature of 20-40 ° C. for 16 hours with light (40 μmol / m ² s) in the light phase until roots are formed. 2. The method according to claim 1, wherein the first, second and third mediums comprise salts of MS medium, vitamins of B5 medium, a carbon source, and a gelling agent. 3. The method according to claim 1, wherein the second and third mediums comprise salts of MS medium, vitamins of B5 medium, carbon sources, plant hormones (plant growth regulators) and gelling agents. 4. The method of claim 1, wherein the first, second, and third media comprise the following salts of MS medium: Component Concentration (mg / L) Salt of MS Medium: NH ₄ NO ₃ 1650 KNO ₃ 1900 MgSO ₄ .7H ₂ O 370 MnSO ₄ H ₂ O 169 ZnSO ₄ .7H ₂ O 8.6 CuSO ₄ .5H ₂ O 0.025 CaCl ₂ .2H ₂ O 440 KI 83 CoCl ₂ .2H _{2 O 0.025 KH 2 PO 4 170} H 3 BO 3 6.2 Na 2 MoO 4 · 2H 2 O 0.25 FeSO 4 · 7H 2 O 27.85 Na 2 EDTA 37.3 myoinositol 100 5. The method according to claim 1, wherein the vitamins of the first, second and third media are selected from the following: Component Concentration (mg / L) Nicotinic acid 1.0 Pyridoxine hydrochloride 1 0.0 Thiamine hydrochloride 10.0 6. The carbon source used in the first, second and third media is selected from sucrose or glucose.
The method of claim 1. 7. The method of claim 1, wherein the carbon source used ranges from 1 to 6% w / v. 8. The plant hormone used in the second medium is selected from any of cytokinins or cytokinin-active ureas used alone or in combination, depending on the explant used. The method of claim 1. 9. The plant hormone used in the second medium is 6-benzylaminopurine, 6-benzyladenine, zeatin, (γ, γ-dimethylallylaminopurine), cytokinin such as kinetin, 1-phenyl. −
9. The method of claim 8, wherein the method is selected from cytokinin active ureas, such as 3-1, 2,3 thidiazol-5-yl urea, phenyl urea and 4-1-phenyl N (4 pyridyl) urea. 10. The method of claim 1, wherein the cotyledonous nodes (explants) are isolated from germinated seedlings that are 2 days to several weeks old. 11. The method according to claim 1, wherein the explant used is taken from a cotton plant seedling grown in the field or grown in tissue culture. 12. The regenerated basal tissue (remaining after collecting the shoots in the first cycle) is repeatedly cultured in steps (iv) to (viii) of claim 1 to maintain the reproduction of the seedlings. The method of claim 1. 13. The method according to claim 1, wherein the concentration of the salts of the MS medium used is at the level of the whole or half of the first, second or third medium on a weight / volume basis. And the method according to 12. 14. The gelling agent used is 0.2 to 1.
14. The method according to claims 1, 4, 5, 11 to 13 selected from agar, gelrite (phytagel) or any gelling agent at a concentration of 2% w / v. 15. The method according to claim 1, wherein the phytohormone (a growth regulator) used in the third medium is selected from auxins such as indoleacetic acid, indolebutyric acid and naphthaleneacetic acid. 16. The shoots regenerated by a tissue culture method can be used for micropropagation of cotton plants.
The method described in. 17. The cotyledon node (sapling axillary) or basal regenerative tissue can be used for genetic transformation based on Agrobacterium-based or bombardment of DNA-coated particles. The method of claim 1. 18. A shoot regenerated by a tissue culture method,
The method according to claim 1, which is used for producing disease-free cotton plants. 19. A shoot regenerated by a tissue culture method,
2. The method according to claim 1, which can be used for the replacement and preservation of disease-free cotton germplasm.