WO1986007378A1

WO1986007378A1 - Method for clonal propagation of coniferous trees

Info

Publication number: WO1986007378A1
Application number: PCT/US1986/001231
Authority: WO
Inventors: David F. Karnosky; Alexander M. Diner
Original assignee: Michigan Technological University; University of Michigan Ann Arbor
Current assignee: Michigan Technological University; University of Michigan Ann Arbor
Priority date: 1985-06-06
Filing date: 1986-06-06
Publication date: 1986-12-18
Anticipated expiration: 1987-12-06
Also published as: EP0222904A1; AU5965586A

Abstract

Coniferous trees, such as European larch and jack pine, are clonally micropropagated by germinating surface-sterilized seeds on translucent, gel-solidified water, removing the seed coat from the germinated seeds, separating cotyledons and hypocotyls from the embryonic tissue and incubating same on a gel-solidified bud initiation medium containing about 5 to about 15 mg/l of a cytokinin, such as benzylaminopurine, and 0 to about 0.01 mg/l of an auxin, such as naphthalene acetic acid, to promote the formation of adventitious buds, transferring the budded tissue to a bud development medium free of plant growth regulators and containing activated carbon to promote the growth of the adventitious buds, transferring the budded tissue to a shoot elongation medium free of plant growth regulators and activated carbon to promote growth of shoots to a length suitable for initiation of root growth, and separating shoots and introducing individual shoots into a nutrient medium for initiating root growth. In one embodiment, needles and stems from plantlets grown from cotyledons and hypocotyls separated from embryonic tissue are recycled for use as source tissue which is incubated in the bud initiation medium to produce subsequent and successive generations of plantlets.

Description

METHOD FOR CLONAL PROPAGATION OF CONIFEROUS TREES

BACKGROUND OF THE INVENTION

This invention relates to methods for colonal propagation of coniferous trees and, more particularly, to a tissue culture method for accomplishing same.

European larch (La ix decidua) and jack pine (Pinus banksiana) are highly variable species with excellent potential for forest regeneration in northern regions of the United States. In comparative growth trials of European larch and otner conifers, European larch has consistently outgrown native conifers in both height and diameter. Pulping studies indicate that 18-23 year old European larch can produce higner Kraft yields than 50-60 year old jac< pine and with pulp strength properties similar to those of jack pine.

The larches are notoriously unreliable in seed production and seed quality. Consequently, the capability of obtaining viable seed from superior European larches is currently the primary limiting factor to increased use of this species for reforestation. Jack pine, the major coniferous pulp species in the Great Lakes states and a native to regions extending from the lower Rocky Mountains in Canada to northern New England, is one of the least site-demanding tree species. Thus, it is particularly useful for reforesting sandy soil types with low fertility. For the foregoing reasons, there is a need for methods capable of reliable and consistent clonal propagation of superior seed sources of European larch, jack pine and other coniferous trees. Representative prior methods for clonal propagation of coniferous trees are disclosed in United States Patent Nos. 4,417,417 (Mehra-Pelta) , 4,337,921 (Mehra-Pelta et al.), 4,354,327 (Semitzer et al.), 4,353,184 (Abo El-Nil), and 4,217,730 (Abo El-Nil) , and literature references Campbell et al._. Can. J. Bot., 53 (16): 1652 (1975), Mehra-Palta et al, Tappi, 61(1) :37 (1978) and Bonga, Can. J. Bot. 60:1357 (1982).

Other plant propagation techniques are disclosed in United States Patent Nos. 4,463,522

(Lindemann) , 4,361,984 (Dunstan) , 4,301,619 (Janick et al.), and 4,204,366 (Janick et al.).

SUMMARY OF THE INVENTION

An object of the invention is to provide a tissue culture method for clonal micropropagation of larch species, particularly European larch, and other coniferous trees, such as jack pine.

Another object of the invention is to provide such a method which is capable of germinating seeds to produce larger and healthier seedlings for use as a starting material for clonal propagation.

A further object of the invention is to provide such a method which is capable of producing shoots of suitable length for root initiation within reduced time periods. A still further object of the invention is to provide a method for initiating root growth and development from plantlets located either jji vitro in agar or the like or in vivo in a soilless mix. A yet further object of the invention is to provide a method capable of producing several successive generations of plantlets from the embryonic tissue from one seed source.

Other objects, aspects and advantages of the invention will become apparent to those skilled in the art upon reviewing the following detailed description and the appended claims.

The invention provides a method of clonal propagation of coniferous trees, particularly various larch species and jack pine and more particularly European larch. The method includes the steps of germinating surface-sterilized seeds, removing the seed coat and separating cotyledons and hypocotyls from the remaining embryonic tissue, incubating the separated cotyledons and hypocotyls on a gel-solidified bud initiation medium containing about 5 to about 15 mg/1 of a cytokinin and 0 to about 0.1 mg/1 of an auxin to promote formation of adventitious buds, transferring the budded tissue to a gel-solidified bud development medium free of plant growth regulators and containing activated carbon to promote the growth of adventitious buds to shoot length, transferring the resulting tissue to a gel-solidified shoot elongation medium free of plant growth regulators and activated carbon to promote growth of individual adventitious buds to a length suitable for initiation of root growth, and separating shoots from the plantlets and treating the separated shoots to initiate root growth. In one embodiment, the seeds are germinated on translucent gel-solidified water. In one embodiment, root growth is initiated by introducing individual shoots either into a nutrient medium for n vitro rooting or into a soilless mix for _i_n vivo rooting.

The invention also provides a remultipliσation technique whereby the needles and stems from plantlets grown from cotyledons and hypocotyls separated from the embryonic tissue are recycled for use as source tissue wnich is incubated on the bud initiation medium to produce subsequent and successive generations of plantlets. The recycled needles and stems can be used as a source for at least 4 successive generations of plantlets from one seed source.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The process of the invention can be used with a variety coniferous trees, including pines such as jack pine (Pinus banksiana) . It is well suited for use with European larch (Larix decidua) , tamarack (Larix larcina) , western larch (Larch occidentalis) , Japanese larch (Larix leptolepis) and larch hybrids. At the present time it is believed to be most particularly suited for use with European larch.

Select seeds from superior trees are first sterilized. Since seeds lots are invariably contaminated with spore-laden debris, the surfaces preferably are sterilized in a two-step process. In one preferred process, seeds are immersed in aqueous hydrogen peroxide, preferably about 30%, for about 5 to about 30 minutes, preferably about 10 minutes, while being agitated in covered petri dishes or trie like. These seeds are then rinsed one or more times with a sterile, demineralized water, preferably three times-- a quick rinse, 5 minute soak and a quick rinse. The moist seeds are then retained in covered petri dishes or the like at room temperature for about 4 to about 8 hours, preferably about 6 hours. During this period, any residual viable spores will germinate and can be destroyed by repeating the above immersion steps, which are followed by rinsing. The surface-sterilized seeds are germinated in. suitable manner, such as by placing on firmly-gelled, sterile, demineralized water in petri dishes or the like. The germination medium preferably is solidified with a translucent gelling agent, such as with about 2% agar. The dishes are sealed with a parafilm or the like and the seeds are incubated therein at room temperatures and under ambient illumination for about 5 to about 7 days.

The use of a translucent, firm water gel for germination provides several advantages. It provides a solid substrate from which the seeds can i bide necessary moisture. Also, individual seeds can be distinctly separated so that they can be conveniently monitored (on the clear gel) for visible growth from contaminating spores which can be selectively excised and discarded. This germination procedure has been found to yield larger, healthier seedlings than those produced by germinating in hydrogen peroxide.

The seed coats of the germinated seeds are then removed and discarded and tne resulting tissues temporarily collected in sterile. demineralized water. The embryos and the enclosing gameophyte tissue are then surface-sterilized in a suitable manner. For example, these tissues can be surface-sterilized by immersing them for about 5 minutes in a 15% aqueous solution of a commercial bleach, such as Chlorox, and rinsed three times in sterile, demineralized water as described above.

The intact embryo is first aseptically removed from the enclosing gametophytes tissue and the latter tissue discarded. Cotylodons are then cut from the embryo at a point at or near the hyocotyl-radicle junction and the radicle discarded. Hypocotyl and separated cotyledons are placed horizontally and partially inserted into a gel-solidified bud initiation medium containing a cytokinin and incubated thereon for a time sufficient to induce formation of adventitious buds. The bud initiation medium may also contain an auxin and contains conventional nutrients required for gymnosperm tissue culture.

While various cytokinins known to function as plant growth regulators can be used in the bud initiation medium, benzylaminopurine (BAP) is presently preferred. The amount of cytokinin varies depending primarily on the species being propagated. For most species and particularly European larch, the amount of cytokinin used can be about 5 to about 15 mg/1. preferably about 10 mg/1.

Various auxins known to function as plant growth regulators can be used in the bud initiation medium, including indole-3-buryric acid, idole-3-butyric acid, indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid and naphthalene acetic acid and mixtures thereof. If used, the presently preferred auxin is napthalene acetic aicd (NAA) , particularly for European larch. The amount auxin of used can be 0 to about 0.1 mg/1, preferably no more than about 0.01 mg/1. A preferred bud initiation medium is set forth in Table 1. This medium is modified from the medium of Brown and Lawrence, Forest Science, 14:62-68 (1968) and has a pH of 5.5. Other bud initiation media containing the same or similar nutrients required for gymnosperm tissue with cytokinin and with or without an auxin can be used.

TABLE I Bud Initiation Medium

Component Concentration (mg/1)

sucrose 10,000 agar 10,000

1-glutamine 1,460

KN0₃ 740

CaCl₂.2H₂0 440

MgS0₄.7H₂0 370

KH₂P0₄ 170

KC1 100 myo-inositol 50

Na₄EDTA..2H₂0 42

FeS0₄.7H₂0 28 nS0₄.H₂0 17

Benzlyaminopurine (BAP) 10

ZnS0₄.7H₂0 8.6

H₃B0₃ 6.2

KI 0.83 nicotinic acid 0.50

NaMo0₄.2H₂0 0.25 pyridoxine hydrochloride 0.10 thiamine hydrochloride 0.10

CoCl₂.6H₂0 0.025

naphthalene acetic acid (NAA) 0.01 Petri dishes or the like containing the bud initiation medium and tissue are sealed with parafilm or the like and the tissue incubated for a time sufficient to induce formation of adventitious buds. This incubation period typically is about

1 week to about 4 weeks, preferably about 2 to about 3 weeks. While the environmental conditions for this incubation generally are not particularly critical, temperatures in the range of about 18 to about C are preferred. For example, the tissue can be incubated at room temperature, preferably under constant illumination at about 1,000 to about 5,000 lux, either mixed flourescent (e.g., 70%) and incandescent (e.g., 30%) or 100% cool white flourescent.

The incubated tissue is then aseptically transferred to a gel-solidified bud development medium, free from plant growth regulators and containing activated carbon, to promote growth of the adventitious buds. A preferred bud development medium is set forth in Table II. This medium is a half strength GDI medium, modified from the medium of Mott and Amerson, North Carolina Agricultural Research Service Technical Bulletin No. 271 (1982) and has a pH of 5.5. Other bud development medium free from plant growth regulators and containing activated carbon and conventional nutrients required for gymnosperm can be used.

Plant growth regulators, while important stimuli for bud initiation, inhibit the growth and development of the adventitious bud primordia. Consequently, if plant growth regulators remain in contact with the tissue, suitable growth of the adventitious buds will not take place within a reasonable period of time. The activated charcoal in the bud development medium absorbs, and thus TABLE II Bud Development Medium

Component Concentration (mg/1)

sucrose 10,000 activated charcoal 10,000 agar 10,000

KN0₃ 500

KC1 150

MgS0₄.7H₂0 125

( H₄)₂S0₄ 100

CaCl₂.2H₂0 75

NaH₂P0₄.H₂0 45

Na₄EDTA.2H₂0 42

Na₄HP0₄ 15

FeS0₄.7H₂0 13.

myo-inositol 5

KI 0.38

Na₂Mo0₄.2H₂0 0.13

CoCl₂.6H₂0 0.13 thiamine hydrochloride 0.1 nicotinic acid 0.1 pyridoxine-hydrochloride 0.1 eliminates from tne bud development medium, any residual plant growth regulator inadvertently carried with the tissue from the bud initiation medium. The amount of activated charcoal used in the bud medium usually is about 1%.

Petri dishes or the like containing the bud development medium and tissue are sealed with parafilm or the like and the tissue is incubated for a time sufficient for the adventitious buds to grow to identifiable individual shoots. This incubation period typically is about 4 weens to about 6 weeks. The temperature and illumination conditions can be the same as those for the incubation on the bud initiation medium. The incubated tissue is transferred to a gel-solidified shoot elongation medium, free from plant growth regulators and containing nutrients required by gymnosperms, and incubated thereon to elongate shoots. A preferred shoot elongation medium is set forth in Table III. This medium is modified from the medium of Litvay et al.. Institute of Paper Chemistry Technical Paper Series No. 115 (1981) and has a pH of^" 5.5. Other shoot elongation media free of plant growth regulators and containing conventional nutrients required for gymnosperm in known concentrations can be used. For example, the medium of Gresshof and Doy, Planta 107:161 (1972) at half or full strength can be used as a shoot elongation medium. However, it has been found that the shoot elongation medium set forth in Table III TABLE III Shoot Elongation Medium

Component Concentration (mg/1)

sucrose 10,000 agar 10,000

MgS0₄ 950

KN0₃ 740

KCI 100 myo-inositol 50

ZnS0₄.7H₂0 43

Na₄EDTA.2H₂0 42

FeS0₄.7H₂0 28

CaCl₂.2H₂0 22

MnS0₄.H₂0 21 nicotinic acid 5

KI 4

Na₂Mo0₄.2H₂0 1. 25 pyridoxine hydrochloride 1.00 thia ine hydrochloride 1.00 CuS0₄.5H₂0 0.50 CoCl₂.6H₂0 0.13

more rapid growth for European larch and, therefore, is more preferred at the present time.

After the shoots have grown to a length of at least about 2 mm, preferably at least about 5 mm, on the shoot elongation medium, they are cut from the basal matrix of embryonic tissues and the bases of individual excised shoots are inserted into freshly prepared shoot elongation medium and these shoots are incubated thereon until they reach a rootable length. Generally, the excised shoots will be maintained on the shoot elongation medium until they reach a length of at least about 5mm, preferably at least about 10 mm. The shoots usually grow at different rates and the faster growing ones can be cut from the embryonic tissue upon reaching the desired length.

In a preferred method, the shoots are incubated on freshly prepared shoot elongation medium for successive 2- to 4-week periods at room temperature and under about 1,000 to about 2,000 lux cool white fluorescent illumination, or until the snoots reach the desired minimum length for rooting.

When the shoots have reached the desired length for rooting, e.g., 10-15 mm, they are removed from the shoot elongation medium and treated for initiation of roots, such as dipping the shoot bases into a commercial rooting powder and then placing these shoots in a soilless mix (e.g., a mixture of peat, perlite and vermiσulite at a ratio of 1:2:1) under an intermittent mist for ijn vivo rooting or by transferring to a root initiation medium containing one or more auxins for In vitro rooting. For example, the bases of the shoots can be inserted into a half strength GD medium modified by the addition of about 0.5 mg/1 of naphthalene acetic acid. Petri dishes or the like containing the root initiation medium and shoots are sealed with parafilm or the like and incubated under temperature and illumination conditions similar to that for shoot elongation for about 1 to 2 weeks to initiate formation of adventitious roots on the shoots. Since the auxin (naphthalene acetic acid) inhibits root development, the shoots must be removed from the root initiation medium after this time period in order to prevent degeneration or deterioration of the adventitious roots. The shoots are transferred from the root initiation medium to a root development medium free from plant growtn regulators, for. example, a half strength GD medium (free from plant growth regulators) and incubated tnereon until the root length for each plantlet reaches at least 10 mm, preferably at least 15 mm. This usually takes up to about 4 weeks.

Once the roots have reached .the desired length, the shoots can be transferred to a soilless mix like that mentioned above, preferably in a mist chamber in a greenhouse at a temperature of about 18 to about 24° C and under natural illumination supplemented with about 1,000 to about 2,000 lux mixed incandescent and fluorescent illumination. For example, the supplemental illumination can be 1500 lux mixed cool-white and Agro-Lite (General Electric Corporation) fluorescent illumination on a 16-hour daylight cycle. The rooted shoots are allowed to grow in the soilless mix until they reach a size of 3 to 4 inches which usually takes about 1 to about 3 months. The above procedure and media for i_n vitro clonal micropropagation of coniferous trees using embryonic tissues can be used indefinitely to increase clone size by using shoot tissues from successive generations of tissue culture plantlets. During shoot elongation, primary needles can be extracted from the plantlets and they, along with residual stems stripped of needles, can be recycled for multiplication. That is, they are introduced into the bud initiation medium and subsequently treated as described above (i.e., successively incuDated in bud development, shoot elongation and root initiation media) to produce plantlets.

Successive generations of plantlets can be produced by this multiplication process, selecting tissues from the parent plantlets or clones shown to have rapid growth and/or large numbers. European larch plantlets through four generations have been produced, one generation from seed embryo and three successive generations from plantlet tissues, with an average of 5 plantlets for every plantlet generation used as a source of tissue for adventitious bud initiation. Such a multiplication scheme equates to a clone of 3,125 plantlets from a single unrooted plantlet within 5 cycles of micropropagation.

Without further elaboration, it is believed that one skilled in the art, using the preceding description, can utilize the present invention to its fullest extent. The following examples are presented to exemplify an embodiment of the invention and should not be construed as limitations thereof. EXAMPLE 1

European larch and jack pine seeds were surface sterilized with 30% hydrogen peroxide for 4 minutes and germinated for 5 to 7 days on moist filter paper in sterile petri plates placed under fluorescent illumination on a 14-hour photoperiod. The seed coats were removed and the remaining tissue was placed in a 10% aqueous solution of Chlorox for 5 minutes. After 3 rinses in sterile, distilled water, the gametophyte tissues were removed and the cotyledons were excised and placed on a modified GD medium containing 5 ppra benzylaminopurine (BAP) , 1% Difco Bacto agar and 1% sucrose. The medium pH was adjusted to 5.5 before autoclaving. Sealed petri plates were incubated for 1 to 2 weeks at 20° C under constant 1,000 lux mixed -incandescent and cool-white fluorescent illumination to initiate formation of adventitious buds.

The cotyledons were then transferred to a half strength modified GD medium with 1% charcoal and incubated thereon for 2 weeKs to promote growth of the adventitious buds. The tissue was then transferred to a half strength GD medium without charcoal and was incubated to promote shoot elongation until shoots of 2-5 mm in length were formed. These shoots were excised from the cotylodens and regularly subcultured in a freshly prepared half strength GD medium.

When the shoots reached a length of 5 to 10 mm, they were placed in a half strength GD medium containing 0.5 ppm naphthalene acetic acid and incubated tnereon for 2 weeks to initiate root formation. The shoots were then transferred back to a half strength GD medium free of plant growth regulators to promote root growth. Plantlets having a total root length of at least 1 cm were transferred to a soilless mixture of peat, perlite and vermiculite (at a ratio of 1:2:1) in a greenhouse mist chamber and grown therein for approximately 1 month prior to placement on greenhouse benches.

Results of shoot and root initiation from these tests are set forth below in Table IV.

TABLE IV

Shoot Initiation European larch Jack Pine

No. Seed Sources Tested 17 4

Total No. Seeds Plated 388 416

No. Seeds with Multiple Shoots 369 167 Percent Seeds with Multiple Shoots 95.1% 40.1% Average No. Shoots Per Clone 24.6 3.6 Maximum No. Shoots Per Clone 348 28

Root Initiation

No. Clones Tested 36 Average No. Ramets Per Clone 4.3

Percent Rooting (All Clones) 47.7%

EXAMPLE 2-

A series of tests were run on European larch embryonic tissue and recycled plantlet tissue (multiplication technique) to evaluate bud and root initiation. In these tests, European larch seeds were surface sterilized twice for 10 minutes each (separated by 6 hours) in 30% hydrogen peroxide and rinsed 3 times in sterile, demineralized water after each sterilization treatment. The seeds were then germinated for 5-7 days on 2% agar-solidified water in sterile petri plates under fluorescent illumination on a 14-hour photoperiod. After the seed coats were removed, the gametophytes with embryos were surface sterilized for 5 minutes in a

15% aqueous Chlorox solution and then rinsed 3 times in sterile, demineralized water. Cotyledons and hypocotyls were excised and separately placed on bud initiation medium in petri plates having the composition set forth in Table I. The plates were sealed with parafilm and incubated at 20-22° C under constant 1,000 lux mixed fluorescent (70%) and incandescent (30%) illumination for 1-4 weeks.

The tissues were transferred from the oud initiation medium to a bud development medium having the composition set forth in Table II and incubated thereon for 2 weeks under the same temperature and illumination conditions as during bud intiation. The cotyledons were transferred from the bud development medium to a freshly prepared shoot elongation medium having the composition set forth in Table III for successive 2-week periods of incubation until shoots were at least 10 mm in length.

The base of these shoots were inserted into a root initiation medium having the composition set forth in Table II, except that it did not contain charcoal and was supplemented with 0.5 mg/1 naphthalene acetic acid. The shoots were incubated on the root initiation medium for 2 weeks under the above temperature and illumination conditions. The shoots were then transferred to freshly prepared root development medium having the composition set forth in Table II, except that it did not contain activated charcoal. The shoots were subcultured on this root development medium every two weeks until the total root length per plantlet reached or exceeded 1 cm. These plantlets were then transferred to a soilless mixture in the manner described in Example 1. Needles were stripped from some unrooted plantlets and these needles and the stripped stems were recylced to freshly prepared bud initiation medium to produce new generations of plantlets. The results from these tests are set forth below in Table V.

TABLE V

A. Bud Initiation of European Larch Embryonic Tissue No. seed sources tested 18 No. genotype (embryos), plated 377-0

No. genotypes initiating buds 3318

Percent genotypes initiating buds 88

Average no. shoots/clone 22.6

Maximum no. shoots/clone 348 B. Bud Initiation of European Larch Plantlet Tissues No. plantlets plated 208

No. plantlets showing multiple shoots 131

Percent plantlets showing multiple shoots 63

Average no. shoots per plantlet per generation 5 Maximum no. shoots per plantlet per generation 34 C. Root Initiation of European Larch Plantlets No. genotypes treated for initiation of roots 80 No. plantlets treated for initiation of roots 280 No. treated plantlets showing initiated roots 139 Percent treated plantlets showing initiated roots 49.6 EXAMPLE 3

A series of comparative tests were run using two different shoot elongation medium, one having the composition set forth in Table III and the other being a half strength GD medium. A comparison of the length of the adventitious shoots after a 4-week incubation period were as follows:

Shoot Length, mm

Test No. Shoot Elongation Media

Modified Litvag

GD 1/2 (Table III)

1 4.0 7.8

2 2.5 6.8

3 4.1 6.3 4 1.9 4.4

5 2.7 5.5"

Average 2.9 6.9

Note: (1) Average length of 10 tallest plantlets per petri dish of 30 plantlets.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of the invention and, without departing from the spirit and scope thereof, make various changes and modifications to adapt it to various usages. For instance, while the composition and concentration of preferred media and preferred incubation conditions for European larcn have been described in some detail, those skilled in the art will readily recognize necessary modifications to those compositions, concentrations, and incubation conditions to obtain optimum results, particularly with other coniferous species.

Claims

1. A tissue culture method for clonal micropropagation of coniferous trees including the steps of : (a) germinating surface-sterilized seeds of a coniferous tree;

(b) removing the seed coating from the germinated seed;

(c) separating cotyledons and hypocotyls from the embryonic tissue;

(d) incubating the separated cotyledons and hypocotyls on a gel-solidified bud initiation medium containing about 5 to about 15 mg/1 of a cytokinin and 0 to about 0.1.mg/1 of an auxin to promote formation o'f adventitious buds:

(e) transferring the budded tissue to a gel-solidified bud development medium free of plant growth regulators and containing activated carbon to promote the growth of the adventitious buds to shoot length;

(f) transferring the resulting tissue to a gel-solidified shoot elongation medium free of plant growth regulators and activated carbon to promote growth of individual adventitious buds to a length suitable for initiation of root growth; and

(g) separating snoots from the plantlets and treating the separated snoots to initiate root growth.

2. A tissue culture method according to Claim 1 wherein step (g) is carried out by introducing tne separated shoots either into a nutrient medium for n vitro rooting or into a soilless mix in a greenhouse for _ir vivo rooting.

3. The tissue culture method according to Claim 1 wherein the seeds are from European larch, tamarack. Western larch, larch hybrids, and jack pine.

4. The tissue culture method according to Claim 3 wherein the cytokinin is benzylaminopurine.

5. The tissue culture method according to Claim 4 wherein the auxin is naphthalene acetic - acid and the amount thereof is 0 to about 0.01 mg/1.

6. The tissue culture method according to Claim 3 wherein step (a) is carried out by incubating the seeds on translucent, gel-solidified sterile water.

7. The tissue culture method according to Claim 3 wherein the seeds are sur ace-sterilized by immersing in aqueous hydrogen peroxide for about 5 to about 30 minutes, rinsing with sterile water, allowing the moist seeds to stand for about 4 to about 8 hours, again immersing the seeds in aqueous hydrogen peroxide for about 5 to about 30 minutes, and then rinsing with sterile water.

8. The tissue culture method according to Claim 3 wherein the separated individual shoots are subcultured for about 2 week to about 4 weeks on freshly prepared shoot elongation medium prior to step (g) .

9. The tissue culture method according to Claim 8 wherein step (g) is carried out by inserting the bases of individual shoots into a root initiation medium containing gymnosperm nutrient medium containing an auxin.

10. The tissue culture method according to Claim 8 wherein the shoot initiation medium contains about 0.5 mg/1 naphthalene acetic acid.

11. The tissue culture method according to Claim 3 wherein needles and stems from plantlets grown from cotyledons and hypocotyls separated from the embryonic tissue in step (c)^' are recycled for use as source tissue which is incubated on the bud initiation medium to produce subsequent and successive generations of plantlets.

12. The tissue culture method according to Claim 11 wherein the recycled needles and stems are used as source tissue for at least four successive generations of plantlets.