WO1995033822A1

WO1995033822A1 - Growth medium for conifer embryogenic tissue

Info

Publication number: WO1995033822A1
Application number: PCT/NZ1994/000051
Authority: WO
Inventors: Dale Raymond Smith
Original assignee: New Zealand Forest Research Institute Limited
Priority date: 1993-04-23
Filing date: 1994-06-02
Publication date: 1995-12-14
Also published as: BR9302220A; AU6859894A; ZA934807B

Abstract

This invention relates to growth media for capturing and sustaining the growth of conifer embryogenic tissue and conifer embryos. The media have concentrations of the inorganic ions in the ranges shown in table (1). Using media of the invention allows good growth of embryogenic tissue and conifer embryos which can subsequently be germinated to obtain plants. With these media there is no need to use plant regulator/hormones such as auxins and/or cytokinins for embryogenic tissue capture and maintenance.

Description

Growth medium for conifer embryogenic tissue

This invention relates to growth media for growing embryogenic tissue and

embryos.

BACKGROUND TO THE INVENTION

A basic plant medium commonly used for capturing and sustaining the growth of plant embryogenic tissue is known as the Murashige Skoog (MS) medium. This medium is based on the ratios and concentrations of minerals found in tobacco leaves. It has been possible to successfully grow embryogenic tissue from many plant species in media which have slight variations from the concentrations/ratio of the minerals in the MS medium. Unfortunately, the MS medium or variations thereof are not ideal for growing coniferous tissue.

One growth medium known as the Weyerhaeuser medium (Gupta and Pullman 1990, 1991a, 1991b) has been developed particularly for coniferous embryogenic tissue, based on chemical analysis of the composition of pine seeds. This medium is significantly different from the MS medium in both its ratios and concentrations. Unfortunately the Weyerhaeuser medium only works in relation to a few conifer genotypes and therefore is too

specific for general conifer propagation. There are other problems associated with the Weyerhaeuser medium and the media referred to above which will become apparent from the

following; discussions. With embryogenesis, an aim is to obtain as many embryos as possible from a single seed. The natural growth of a seed proceeds in six main stages:

a) The first stage has an embryo consisting of between one to three cells attached to the archegonium and positioned within the corrosion cavity

of the seed.

b) The second stage has a number of embryos multiplying and developing with each embryo having less than 64 cells. It is usually at this

growth stage (zygotic polyembryogenesis) that embryos are placed onto or into the growth medium.

c) The third stage is growth of the embryo away from the archegonium and towards the end of the corrosion cavity. The long axis of the embryo develops and assumes a cylindrical shape with a complex of elongated cells, the suspensor, at one end, and with a rounded head at the other end where the apical meristem will eventually develop. This is known as a bullet stage.

d) The fourth stage is the development of cotyledonary tissue at the shoot apex of the embryo, at the root end of which is a group of cells known as the suspensor zone. e) The fifth stage is the further development and maturation of embryos, with the formation and greening of cotyledons, formation of an epicotyl or shoot apex, and formation of a hypocotyl. This stage of development ends with emergence of a root (radicle), that is, the process of germination.

f) The sixth stage is the establishment of the germinated embryo as a plant capable of growing in soil.

Prior growth media do not encourage the natural zygotic polyembryogenic state which occurs in the seeds. Instead, with the previous media it has been necessary to dissect out embryos at the cotyledonary stage. Plant growth regulators (hormones) are then applied to the cells of the body of the embryo or at the point of attachment of the suspensor to encourage the cells to differentiate back to nonspecialized cells which can then be multiplied. This process, often referred to in the literature as somatic embryogenesis, has been widely reported for Picea (Spruce) species, and to a lesser degree for other conifers.

One problem with the above process is that there is in effect double handling involving first the development of specialised tissue, reversion of same to basic cell types and then re-growth of the tissue to form mature embryos. Another problem is that the growth hormones used (such as 2,4-D) may induce somaclonal variation. That is, the ideal genotype which is being cloned may be corrupted by the growth hormones and the resultant embryo may not be true to type. It is an object of the present invention to address the above problems, or at least to provide the public with a useful choice.

Further objects and advantages of the present invention will become apparent from the following description.

SUMMARY OF THE INVENTION

According to one aspect the present invention comprises growth media effective for capturing and sustaining the growth of embryogenic tissue and for subsequent development, maturation and germination of conifer embryos, including inorganic ions within the concentration ranges given in Table 1:

TABLE 1

ION CONCENTRATION RANGE mmoles/1

N0₃ 8-27

NH₄ 0.95-3

Ca 0.08-0.25

Fe 0.05-0.15

Na 1.9-5.75

Zn 0.045-0.135

Cu 4.5xl0-³-1.5xl0^"2 ION CONCENTRATION RANGE mmoles/1

Mg 0.8-2.5

It should be appreciated that the growth medium may be prepared in liquid or solid form.

Reference throughout this specification will be made to the use of the present invention with respect to embryogenic tissue from conifers such as Pinus radiata, Pinus taeda, Pinus elliotii, and Pseudotsuga menziesii, however it should be appreciated that the present invention may be able to be used with embryogenic tissue coming from other conifers.

A medium produced in accordance with the present invention provides an environment for the embryos to grow in and the applicant has found that a number of distinctive advantages have arisen.

The first advantage is that the present invention provides for sustained zygotic polyembryogeny in-vitro. Unlike the situation with the previous media, it is generally no longer necessary to dissect embryos at the late pre-cotyledonary stage out of seed nor is it necessary to apply growth hormones. When using a medium of the present invention, the embryogenic tissue grows out of the seed naturally, as illustrated in the examples herein. Another advantage of the present invention is that the medium is suitable for growing a high percentage of genotypes.

According to another aspect the invention provides a method of growing embryogenic tissue comprising the step of growing the tissue on a growth medium as

described above.

The medium and method of the invention are particularly suitable for capturing and sustaining the growth in-vitro of embryogenic tissue of conifers in particular, including Pinus radiata, Pinus taeda, Pinus elliotii and Pseudotsuga menziesii, for example.

The medium and method of the invention are also particularly suitable for the development, maturation and germination of somatic embryos of conifers in particular including Pinus radiata, Pinus taeda, Pinus elliotii and Pseudotsuga menziesii, for example.

DETAILED DESCRIPTION OF THE INVENTION

The medium of the invention comprises ions within the concentration ranges of Table 1 above, which are given again in column A of Table 2 below while column B gives preferred ranges for the ion concentrations and column C gives ion concentrations of one particularly preferred medium. Ion concentrations within 5% of those of column C are also highly preferred. TABLE 2 MEDIA ION CONCENTRATIONS (mmoles/1) A B C

ION

NO₃ 8-27 13-23 17.8

NH₄ 0.95-3 1.5-2.5 1.96

Ca 0.08-0.25 0.12-0.21 0.17

Fe 0.05-0.15 0.07-0.13 0.10

Na 1.9-5.75 2.9-4.9 3.85

Zn 0.045-0.135 0.06-0.12 0.09

Cu 4.5xl0-³-1.5xl0^"2 7xl0-³-1.2xl0^"2 9.cixl0^"3

Mg 0.8-2.5 1.2-2.0 1.62

Table 3 provides a comparison between the ion concentrations of one preferred medium of the invention with the Weyerhaeuser medium, the Murashige Skoog medium and a general tissue culture medium for radiata pine improved at New Zealand Forest Research Institute Limited, Rotorua, New Zealand and referred to as FRI-LP. It can be seen again that there are distinct differences between the concentration of ions in the preferred medium and in the previous media.

Previously it was thought that a relatively high level of calcium was required in growth media, however the level of calcium in the preferred medium in Table 3 has been effective for many coniferous genotypes. Another noticeable difference between the preferred medium and the prior art media is the higher level of sodium, copper and zinc in the preferred medium.

TABLE 3

MEDIA ION CONCENTRATION COMPARISON

(mmoles/1)

ION PREFERRED WEYERHAEUSER MURASHIGE FRI-LP

MEDIUM MEDIUM SKOOG MEDIUM

MEDIUM

N03 17.80 20.54 39.40 32.96

NH4 1.96 7.54 20.61 5.00

TOTAL 19.76 28.09 60.02 37.96

N

P 1.96 1.00 1.25 1.98

K 14.16 10.02 20.05 19.79

Ca 0.17 1.00 2.99 5.08

Mg 1.62 2.50 1.50 1.46

Cl 3.42X10^"1 1.00 5.99 2.10x10^

Fe 0.10 0.02 0.10 0.11 ION PREFERRED WEYERHAEUSER MURASHIGE FRI-LP MEDIUM MEDIUM SKOOG MEDIUM

MEDIUM

S 1.83 1.14 1.73 1.69

Na 3.85 0.06 0.20 0.22

B 0.13 0.25 0.10 0.10

Mn 1.62xl0^"2 6.21xl0^"2 l.OOxlO^"1 8.97xl0^"2

Zn 0.09 0.05 0.03 0.03

Cu 9.61xl0-³ 5.01x10^ l.OOxlO^"4 l.OOxlO^'3

Mo 8.27x10^ 5.17X10^"4 1.03xl0^"3 1.03xl0-³

Co 8.41X10^"4 5.25x10"* 1.05X10-⁴ 1.05x1a⁴

I 6.02xl0^"3 2.50xl0^"2 5.00xl0^"3 4.82x10^

It should be appreciated that the composition of the preferred medium above is given by way of example only and that other ratios and concentrations may be used, within the ranges of Table 1 above.

It is the concentrations of the inorganic ions listed in Table 1 which distinguish media of the invention from other media such that the medium of the invention is able to sustain the growth and proliferation of embryogenic tissue of a wide range of conifer types. Potassium, chloride, phosphate, manganese, borate, sulphate, iodide, molybdenum and cobalt ions are preferably included in the medium. The media of the invention preferably also contain other nutrients generally used in conifer embryogenesis such as a carbon source. In the preferred media vitamins especially thiamine, nicotinic acid, and pyridoxine are preferably also included. Inositol, sucrose, agar and glutamine and other amino acids (particularly asparagine, arginine, citrulline, oriώhine, lysine, alanine and proline) are also preferably present in preferred media. The concentrations of some of the other components can usefully be varied according to the stage of the embryogenesis. For example it is preferred to include glutamine and other amino acids in relatively high concentrations for the later stages of embryogenesis. Particularly preferred media include those of the solutions of Tables A5 to A9 at the end of this disclosure. Also preferred are media of the invention having concentrations of the nondistinguishing components at levels between 50% and 150% of those found in Tables A5 to A9. Highly preferred are media with concentrations of the non-distinguishing components at 75 % to 125 % of those found in the solutions of Tables A5 to A9. The preferred pH for media of the invention is in the range 5.5-5.9. Agar may be included in preferred media at 0.5-1.5g/l (w/v). Sucrose may be included in preferred media

at 5-50g/l (w/v).

According to another aspect of the invention there is provided a process for conifer embryogenic tissue capture and embryogenic tissue maintenance. In embryogenic tissue capture it is preferred that the seeds are surface sterilised and entire megagametophytes containing the immature zygotic embryos are dissected and placed directly onto a medium of the invention. Media with concentrations of each ion in the lower half of the ranges shown in Table 1 are preferred for this step. The medium shown in Table A5 is particularly preferred for this. Culture is carried out at an appropriate light intensity. Ambient photo period is preferably used and the temperature is preferably about 24°C. It is preferred that plant growth regulators such as auxins and cytokinins are not present. Preferred species for the practice of the invention include Pseudotsuga menziesii, Pinus taeda, Pinus elliotii and

Pinus radiata.

Embryogenic tissue will grow for up to a month on the capture medium but for sustained maintenance it is recommended that conifer embryogenic tissue is maintained on standard embryogenesis medium (Table A6). Again for the maintenance of conifer embryogenic tissue it is preferred not to use auxins or cytokinins.

In one embodiment of the present invention a growth medium may be used having the same ratios of ions as shown for the preferred medium in Table 3 but with a different overall ion concentration. For instance, at the zygotic polyembryogenesis stage of the embryo development (stage b above) a medium may be used which has only one half of the concentration of each of the ions listed for the preferred medium in Table 3 (eg see Table A5). The tissue may be grown on this medium until a sustained extragametophytic growth of tissue has been established and can be relocated on full strength medium such as that of Table A6. Again it is noted that the level of calcium is an important factor with respect to a growth medium suitable for growing embryogenic tissue either at stage b or at later stages.

The growth medium may also contain 4-9 gm/1 of gellan gum. Media of this embodiment have been found to be particularly useful for conifer embryo maturation. The gellan gum marketed under the name Gelrite ™ has been found to be particularly useful. Transfer from embryo development medium with conventional Gelrite content of about 3 gm/1 to an embryo maturation medium with 5-7 gm/1 (preferably 6 gm/1) Gelrite followed by subsequent transfer to another embryo maturation medium differing in that the Gelrite concentration was 4-5 gm/1 (preferably 4.5 gm/1 Gelrite) gave surprisingly good yields of embryos. It appears that transient use of very high Gelrite levels (eg 6 gm/1) stimulates the maturation of somatic embryos.

Water vapour permeable films or filters may be used in conjunction with the medium of the invention. Use of such films or filters in embryogenesis has been described in co-pending application Ser No 991,994 filed December 17, 1992. Using water vapour permeable films or filters allows the avoidance of buildup of condensation. Thus, problems such as those relating to the presence of freely available liquid on the surface of solid media are avoided. Also the films or filters allow a controlled rate of water loss. This is preferably in the range of 90-150 gm/sq meter per day. A rate particularly suitable for Pinus radiata embryos is 118 gm/sq meter per day. Many types of films may be used provided they have the desired qualities of being able to seal against microbial infection and are sufficiently permeable to water vapour. For instance, the film may be made from plastics material such as polyvinyl chloride (PVC). Among those films suitable are those sold under the trade mark VITAFILM by the Goodyear Tyre & Rubber Co (Australia) Ltd. In this range the OMNI, VW, MWT and F10 V/S are all useful with the medium of the invention for conifer embryogenesis. When the somatic embryos maturing on the medium of the invention are in vessels covered with a water permeable film, evaporation of liquid from the medium makes it less available to the embryos, mimicking the changes in matric potential that occur during natural embryo development. As a consequence of the loss of water, the concentration of ions within the medium increases, however without taking it outside the range useful for conifer embryogenesis. I have been unable to duplicate the beneficial effect of the water permeable film simply by transferring embryos to medium with higher concentrations of medium components equivalent to that attained when water vapour is lost through a water permeable film. I have concluded therefore that the beneficial effect of the use of water permeable film is not due to an increase in osmotic potential of the medium, or to the increase in concentration of substrates such as sucrose. I believe rather that the effect of the film is to decrease the availability of water to the developing embryo at a precise and critical stage of maturation.

According to another aspect of the invention there is provided a process for harvest of mature embryos with developed cotyledons, and subsequently treating them to allow a high percentage of germination, such that the conversion of cotyledonary stage embryos to plants growing in soil averages or exceeds 50% . Conversion of somatic embryos to plants at an efficiency approaching that necessary for the commercial application of somatic embryogenesis has not been previously reported for conifers other than spruces. Germination is preferably carried out on media with inorganic ions at concentrations of 50 % to 100% of those listed in Table 2, column C and Table 3. Particularly preferred for this is the medium of Table A10. EXAMPLES

The following Examples further illustrate the invention. The Examples illustrate the use of the medium in embryogenic tissue capture and in embryogenic tissue maintenance for capture and maintenance of embryogenic tissue of Pseudotsuga menziesii,

Pinus taeda, Pinus elliotii and Pinus radiata and for subsequent embryo development and maturation, and subsequent germination and conversion to plants in soil.

Standard procedures for the preparation of plant tissue culture media are followed. Media as described below are sterilised by autoclaving for 20 minutes at 121°C. Organic components are filter sterilised, and are added to the medium after autoclaving. Culture is carried out in 90 mm petri dishes each containing 22 ml of medium.

EXAMPLE 1 - EMBRYOGENIC TISSUE CAPTURE

Seeds were removed from cones of Pinus radiata, Pinus taeda, Pinus elliotii, and Pseudotsuga menziesii at an appropriate stage of development of the zygotic embryos (for instance for Pinus radiata in New Zealand, from early December to early January), and the seeds were surface sterilised and entire megagametophytes containing the immature zygotic embryos were dissected and placed directly onto Standard Embryogenic Tissue Capture Medium (Table A 5). Dishes were cultured at low light intensity (5 microEinsteins m"² sec"¹) under ambient photoperiod. The temperature was maintained constant throughout at 24°C +/- 1°C. As a variation, zygotic embryos at any stage of development up to the formation of cotyledonary primordium formation may be dissected and placed directly onto Standard Embryogenic Tissue Capture Medium (Table A5), or onto Standard Embryogenesis Medium (Table A6) or Embryo Development Medium (Table A7).

Embryogenic tissue grew out onto the medium over the next three months, and tissue pieces greater than 2 millimetres across were transferred to Standard Embryogenesis Medium.

This protocol differs from that described by other authors, for example in the patents granted to Gupta et. al (1990, 1991a, 1991b ), and in the references cited therein. The major difference is that the mineral salt composition differs markedly from that used by the above named authors, and in the references which they cite (see Table 3).

The mineral salt formula of this invention allows the capture of embryogenic tissue without the need to resort to the use of plant growth regulators such as auxins (eg. 2,4-Dichlorophenoxy acetic acid, Indole -3- acetic acid, 1-Napthylacetic acid, or Indole-butyric-acid) and/or cytokinins ( eg. 6-Benzylamino Purine, Zeatin, or N6-[2 Isopentenyljadenine). While plant growth regulators such as auxins or cytokinins may on occasions enhance the growth of tissue on the Standard Embryogenic Tissue Capture Medium, the Standard Embryogenesis Medium or the Embryo Development Medium, their use is not essential when explants are put into culture at the appropriate stage of development of the immature zygotic embryo. The simple protocol described has been successfully used to capture embryogenic tissue of Pseudotsuga menziesii, Pinus taeda, Pinus elliotii, and Pinus radiata without need to alter the protocol an any way for each of the above named species. 15500 explants from 10 different control pollinated cone parents of Pinus radiata were put into culture on the Standard Embryogenic Tissue Capture Medium and after adjustment of results for contamination, up to 100% of the whole megagametophyte explants gave rise to embryogenic tissue, when the tissue placed into culture was developmen tally competent to form embryogenic tissue. The mean response of the best result for each cone parent was

32.9% for Pinus radiata. For Pinus taeda, Pinus elliotii and Pseudotsuga menziesii about 30% of megagametophyte explants at an appropriate stage of development gave rise to embryogenic tissue.

EXAMPLE 2 - EMBRYOGENIC TISSUE MAINTENANCE

Embryogenic tissue has been found to continue to grow for up to a month on the Standard Embryogenic Tissue Capture Medium, however this medium is unsuitable for sustained tissue maintenance. The conifer embryogenic tissue was more effectively maintained on Standard Embryogenesis Medium (Table A6).

Tissue development was maintained in a primitive state on Standard Embryogenesis Medium, ideally with embryos never developing past the eight-celled stage before dissociating into simple embryonic initials. These embryos may have a single suspensor cell attached to the embryo initial - further development of suspensors is not encouraged on this medium, and the tissue does not become "bulky". 2.1 Plant growth regulators

This protocol for the maintenance of conifer embryogenic tissue differs from that described by other authors, for example in the patents granted to Gupta et. al (1990, 1991a, 1991b ), and in the references cited therein. The major difference is that the mineral salt composition differs markedly from that used by the above named authors, and in the references which they cite.

The ion composition of the medium that is described here allows the maintenance of embryogenic tissue of conifers without the need to resort to the use of plant growth regulators such as auxins (eg. 2,4-Dichlorophenoxy acetic acid, Indole -3- acetic acid, 1-Napthylacetic acid, or Indole-butyric-acid) and/or cytokinins (eg. 6-Benzylamino Purine, Zeatin, or N6-[2 Isopentenyljadenine).

Plant growth regulators such as 2,4-D and BAP may stimulate apparent growth of embryogenic tissue, in part due to formation of suspensor cells, but the use of plant growth regulators is not necessary, and confers no benefits. While we have recovered sound, mature somatic embryos from some cell lines maintained on medium with 2,4-D and BAP, my experience has been that these cell lines lose their plant-forming potential much sooner than the same cell lines which have been maintained on the Standard Embryogenesis Medium without plant growth regulators. It is usually the experience of practitioners of the art of somatic embryogenesis of conifers that cell lines quickly lose the ability to form mature embryos, and subsequently

plants.

The ability to retain the plant-forming potential of conifer cell lines for periods of in excess of one year is a benefit of the use of the unique mineral salt composition which is described here. This benefit is possibly due to the fact that plant growth regulators are not required for embryogenic tissue capture and maintenance in the medium described herein. Most cell-lines grown on the preferred medium showed ill-thrift or died when placed on the Weyerhaeuser medium.

EXAMPLE3 -EMBRYODEVELOPMENT

Embryo development was encouraged by transfer of tissue to an Embryo Development Medium (Table A7). Embryogenic tissue from the maintenance medium was suspended in liquid medium in a sterile McCartney bottle. Embryogenic tissue was used at the rate of 1 gm per 4 ml of Cell Suspension Medium (Standard Embryogenesis Medium with the agar omitted). When the suspension was finely divided, it was dispensed as 0.25 ml aliquots, three per 90 mm. petri dish containing Embryo Development Medium which consisted generally of Standard Embryogenic Tissue Maintenance Medium to which was added amino acids including glutamine at 550 mg/1, and which was gelled with 3.0 gm/1 Gelrite rather than with Difco Bacto agar (see Table A7). The first step in somatic embryo development is marked by the continued multiplication of the cells in an individual embryo to form a compact mass, often referred to in the literature as the "proembryo" or "proembryonic mass". It is roughly equivalent to the "globular" stage of development in dicotyledonous angiosperms. As this embryo mass forms, the single suspensor cell develops into a multi- stranded structure, also called the suspensor. The embryo head continues to develop, and assumes a cylindrical shape with a rounded head. This stage is sometimes referred to in the literature as the "bullet" stage.

Culture dishes were incubated under the same conditions as the maintenance stage. Over approximately 2 to 3 weeks the tissue rapidly increased in bulk, and took on a "spiky" appearance. Over the next 2 to 3 weeks, "bullet" stage embryos with well defined suspensors became readily visible to the unaided eye. At this point, the tissue masses were subdivided, and are transferred to Embryo Maturation Medium.

EXAMPLE 4 - EMBRYO MATURATION

Each "spot" of tissue on the Embryo Development Medium which was judged to be at a suitable stage of development was subdivided and the pieces transferred to Embryo Maturation Medium # 1 (Table A8). Embryo Maturation Medium # 1 is similar to Embryo Development Medium, but contains higher concentrations of amino acid, such as glutamine at 5 - 10 gm/1, Abscisic acid in the range 5 mg/1 to 25 mg/1, and Gelrite at a concentration between 4.5 gm/1 and 6 gm/1. A concentration of 6 gm/1, is the preferred level of Gelrite for the first transfer from Embryo Development Medium to Embryo Maturation Medium (refer Table A8). The usefulness of manipulating the Gelrite levels in this manner is illustrated by the following experiments.

Embryogenic tissue of a competent plant-forming clone of Pinus radiata was transferred from Embryo Development Medium onto Embryo Maturation Medium ffl gelled with 8 grams/litre Difco Bacto agar, or 3 grams/litre gellan gum (Gelrite ™) or 6 grams/litre gellan gum. Each treatment had eight replicate dishes. The yield of harvestable somatic embryos, capable of germination and conversion to plants was assessed when embryo production ceased after six weeks. Results are shown in Table 4.

TABLE 4

Treatment Total number of embryos Average yield per dish

8 g/1 Bacto agar 0 0.0

3 g/1 Gelrite™ 16 2.0

6 g/1 Gelrite™ 141 17.6

As a refinement to this protocol, I have found that it is useful to further enhance embryo yield by removing the tissue from Embryo Maturation Medium ffl with 6 grams/litre Gelrite after three weeks, and placing subdivided tissue pieces onto Embryo Maturation Medium #2 with 4.5 grams/litre Gelrite. Figure 1 illustrates a maturation protocol taking advantage of these effects and those obtained from the use of water vapour

permeable films. EXAMPLE 5 - THE EFFECT OF USE OF WATER VAPOUR-PERMEABLE FILMS

ON EMBRYO MATURATION

This example illustrates the use of water vapour permeable films on yield of embryos obtained from culture on the medium of the invention. Tissue of a plant-forming Pinus radiata embryogenic cell line was distributed in equal amounts at random over a number of petri dishes containing the EMM#1 medium as described in Table A8. Some of the dishes retained plastic lids which were sealed at the margins with impermeable domestic cling film. Other dishes were instead covered with one of four different plastic films. In this experiment, over a period of ten days cotyledonary stage somatic embryos were harvested directly from EMM#1 (Table A8). The results are shown in Table 5 below. Each treatment had four dishes of embryo-forming tissue. The use of Vitafilm Omni-film produced the greatest yield of somatic embryos for this particular cell line in this experiment.

TABLE 5 The Effect of Permeable Films on the Yield of Somatic Embryos

Film/Closure Total number of somatic Average somatic embryos embryos per dish

Plastic petri dish lid/cling film 28 - 7.0

Vitafilm F10 V/S 51 12.7

Vitafilm VW 62 15.5

Vitafilm Omni-film 97 24.2

Vitafilm MWT 50 12.5 EXAMPLE 6 - THE EFFECT OF FILMS ON WATER LOSS FROM GROWTH MEDIUM IN PETRI DISHES.

Petri dishes containing solid somatic embryo development medium were covered with plastic lids, sealed on with cling film, or with one of four different gas permeable plastic films. Initial weights of the dishes were recorded, and weight loss noted at intervals of 2-3 days. There were four replicates of each treatment, and dishes were maintained in a 24°C incubator under the same conditions as used for somatic embryo development.

The mean water loss from dishes after 9 days when covered with lids of different films was determined. Water loss was correlated with the somatic embryo counts from identical dishes of medium cultured under the same conditions. A correlation between embryo formation and water loss of 0.994 was determined statistically for the film covered dishes. Experimental results are shown in Table 6.

TABLE 6 - Embryo formation and water loss from media

Lid/Film Number of Embryos Water Loss (gm/9 days)

Lid 28 0

Vitafilm F10 V/S 51 4.88

Vitafilm VW 62 5.14

Vitafilm Omni-film 97 6.73 Lid/Film Number of Embryos Water Loss (gm/9 days)

Vitafilm MWT 50 4.60

The water loss from dishes giving the highest yield of somatic embryos, that is those covered with Vitafilm Omni-film, was determined to be 118 g/m² dish area/day.

In another experiment, following approximately three weeks of culture on EMM#1, tissue of four plant-forming Pinus radiata embryogenic cell lines was subdivided and distributed in equal amounts at random over a number of petri dishes containing the EMM#2 medium which had Gelrite at 4.5gm/l as described in Table A9. Some of the dishes retained plastic lids which were sealed at the margins with impermeable domestic cling film. Other dishes were instead covered with one of two different plastic films. As an additional treatment, lidded, sealed dishes contained an ethylene absorbing agent (potassium permanganate on a matrix of aluminum oxide). After a period of eight days, cotyledonary stage somatic embryos were harvested. The results are shown in Table 7 below. Each treatment had four dishes of embryo-forming tissue.

TABLE 7 The effect of permeable films on yield of cotyledonary stage somatic embryos.

Cell Line Treatment Mean embrvos per 5 % LSD test dish 122 Standard dish 14.5 b

Vitafilm Omnifilm 78.0 a

Vitafilm MWT 380 110.0 a

Ethylene absorber 26.5 b

125 Standard dish 4.0 b

Vitafilm Omnifilm 17.0 a

Vitafilm MWT 380 14.5 a

Ethylene absorber 5.0 b

A13 Standard dish 1.0 b

Vitafilm Omnifilm 6.5 ab

Vitafilm MWT 380 9.5 a

Ethylene absorber 2.0 ab

A17 Standard dish 1.5 a

Vitafilm Omnifilm 1.0 a Vitafilm MWT 380 0.5 a

Ethylene absorber 0.5 a

a,b Difference in results of treatments bearing the same letter were not statistically significant.

The results in Table 7 indicate that the use of water-permeable films in the EMM#2 stage enhances the production of cotyledonary stage somatic embryos, and also that there is probably an optimal rate of water loss appropriate to each plant cell line (clone) when grown on EMM#2. These results also show that up to more than 100 embryos at the cotyledonary stage may be obtained from a single petri dish for Pinus radiata. With Pinus taeda the results of this step are similar with as many as 10-20 embryos at the cotyledonary stage being produced per dish.

EXAMPLE 7 - RECOVERY OF PLANTS FROM SOMATIC EMBRYOS

Well developed somatic embryos were transferred to NZ FRI Embryo Germination Medium (Table A10). Dishes were sealed vith clingfilm and maintained under 50% shade cloth in standard incubator conditions. Embryos were incubated at 24+/-1 degree Celsius, under a light intensity of approximately 40 micro Einsteins m^"2sec¹, and a 16-hour photoperiod. For somatic embryos from new cell lines, roots appeared as early as 10 days after transfer to Germination Medium. As cell lines age, roots took longer to emerge but usually appeared within 12 weeks.

New cell lines produced many somatic embryos of high quality. Generally, these were dark green, had long cotyledons, and normally formed a definite epicotyl while still in sterile culture. Embryos of this type tend to form roots quickly, and a high conversion efficiency (percentage of harvested mature cotyledonary stage embryos converting to plants in soil, greater than 50%) is observed. I have observed that the growth and quality of plants is directly related to the quality of the epicotyl at time of pricking-out of embryos into propagation medium. Somatic embryos with very short epicotyls do not perform as well as those which have well developed shoots of 5 mm or longer. It is likely that the cotyledons of somatic embryos do not function as photosynthetic organs, and that the embryos rely on the epicotyl for carbon fixation in-vivo.

Setting of Geπninated Embryos

Germinated somatic embryos were set under ambient glasshouse conditions into 350 mm x 295 mm x 55 mm nursery flats containing the following propagation medium:

1.5 parts fine peat

1.0 parts perlite

0.5 parts fine pumice

Diazinon, 1/2 teaspoon per tray Magnesium ammonium phosphate 30 gm per tray

Normal nursery procedures for the exflasking of tissue cultured plant material are followed subsequently.

The protocols described above, using the unique mineral salt medium described in Table 3 have been used to capture embryogenic tissue from whole megagametophyte explants of Pinus radiata, Pinus taeda, Pinus elliotii, and Pseudotsuga menziesii using the Standard Embryogenic Tissue Capture Medium described in Table A5. This medium has proved to be satisfactory for all the conifer species named above without alteration. Embryogenic tissue of Pinus radiata, Pinus taeda, Pinus elliotii, and Pseudotsuga menziesii has been proliferated and maintained, often for periods of several years, by regular 14 day transfers to fresh dishes of Standard Embryogenesis Medium as described in Table A6. This medium has proved to be of use for these four species without alteration.

Embryogenic tissue of Pinus radiata, Pinus taeda, Pinus elliotii, and Pseudotsuga menziesii has been proliferated and the formation of "bullet" stage somatic embryos has been observed upon transfers to Embryo Development Medium as described in Table A7. This medium has proved to be of use for these four species without alteration.

Mature somatic embryos have been observed on EMM#1 and EMM#2. Several thousand mature cotyledonary stage somatic embryos of over 50 clones of Pinus radiata have been harvested from these media and transferred via standard germination and nursery procedures to soil. Over 4000 plants from 50 clones of Pinus radiata have been grown in soil in the greenhouse, nursery bed, or in field trials. By way of example, for one collection of Pinus radiata, 28 different cell lines (clones) produced 6066 mature

cotyledonary stage embryos. Of these 2980 plants were successfully established in soil, giving an average conversion of 49.1 % . The best conversion of mature somatic embryos to plants in soil was 73% (1001 plants from 1367 somatic embryos). Plants from somatic embryos of Pinus taeda have also been transferred to soil by the same process. EXAMPLE 8 EFFECTIVE RANGE OF CONCENTRATIONS OF PREFERRED

MEDIUM MINERAL ELEMENTS

Similar experiments to Examples 1-4 and 7 were carried out using ion concentrations, 0.125, 0.25, 0.5, 1.0, 1.5 and 2.0 times those of the preferred medium of Table 3 at each developmental stage and also for embryo germination. The results are presented in Table 8. The concentrations of components other than inorganic ions is the same for each of the six different strength solutions and appropriate for the step being investigated. The concentration of the other components are those used in Examples 1-4 and 7 for the particular step being investigated. TABLE 8 Effective range of concentration of preferred medium mineral elements

Stage of Development

ETC ETP EDM EM#1 EM#2 EG

(1) (2) (3) (4) (5) (6)

1/8 - - - - - -

strength* ETC ETP EDM EM#1 EM#2 EG

(1) (2) (3) (4) (5) (6)

1/4 - - - - - -

strength

1/2 + + + + + + + + + + + + strength

Full + + + + + + + + + + + + + + + strength

1.5 x - + + + ^' + + strength

2 x — — - - - - strength

ETG = Embryogenic Tissue Capture

ETP = Embryogenic Tissue Proliferation on Standard Embryogenesis Medium

EDM = Embryogenic Development on Development Medium

EM#1 = Embryo Maturation ffl

EM#2 = Embryo Maturation #2

EG = Embryo Germination

* does not gel + + + optimal growth

+ + useful growth

4- slower growth

not effective

This table represents the effective range of concentration of preferred medium mineral elements for Pinus radiata. The concentrations giving the best result (+ + +) for Pinus radiata also gave good results for Pinus taeda at every stage, for Pinus elliotii for the first 3 stages, and for Pseudotsuga menziesii for the first 4 stages.

MEDIA FOR EMBRYOGENIC TISSUE CAPTURE, MAINTENANCE, DEVELOPMENT, AND MATURATION TABLE Al - Major Ion Stock:

Compound Weight gm

KN0₃ 14.31

MgS0₄.7H₂0 4.00

CaCl₂.2H₂0 0.25

NaN0₃ 3.10

NH₄H₂P0₄ 2.25

make up to 400 ml TABLE A2 - Minor Ion Stock

Compound Weight mg

MnS0₄.4H₂O 36.0

H₃BO₃ 80.0

ZnS0₄.7H₂O 250.0

Kl 10.0

CuS0₄.5H₂0 24.0

Na₂Mo0₄.2H₂O 2.0

CoCl_2.6H₂O 2.0

make up to 200 ml

TABLE A3 - Iron stock - to make 1 litre

FeS0₄.7H₂0 1.5 gm

Na₂EDTA 2.0 gm

TABLE A4 - Vitamin stock - to make 1 litre

Thiamine HCl 0.5 gm

Nicotinic acid 0.5 gm

Pyridoxine HCl 0.05 gm

TABLE A5 - Standard Embryogenic Tissue Capture Medium per litre of medium major ion stock 20 ml minor ions stock 10 ml

Iron chelate stock 10 ml

Vitamin stock 5 ml

Inositol 0.5 gm

Sucrose 10.0 gm

Charcoal (Merck, activated) 2.0 gm

Difco Bacto agar 8.0 gm

pH adjust to 5.6-5.8 before addition of agar and autoclaving

Table A6. Standard Embryogenesis Medium (embryogenic tissue maintenance medium) per litre of medium

Major ion stock 40 ml

Minor ion stock 20 ml

Iron chelate stock 20 ml

Vitamin stock 10 ml

Inositol 1.0 gm

Sucrose 30.0 gm

Difco Bacto agar 8.0 gm

pH adjust to 5.6-5.8 before addition of agar and autoclaving add the following filter sterilised amino acids after autoclaving:

major amino acids milligram per litre glutamine 110 asparagine 105 arginine 35

minor amino acids stock 2 ml per litre

Table A6 b Minor amino acid stock amino acid gm citrulline 1.58 ornithine 1.52 lysine 1.10 alanine 0.8 proline 0.7

3.1 Make up to 800 ml with double distilled water.

3.2 Dispense into 40 ml aliquots.

3.3 Freeze immediately, store frozen, and thaw only on day of use.

3.4 Adjust pH to 5.6-5.8 and filter sterilise before use. Table A7 - Embryo Development Medium per litre of medium Major ion stock 40 ml

Minor ion stock 20 ml

Iron chelate stock 20 ml

Vitamin stock 10 ml

Inositol 1.0 gm

Sucrose 30.0 gm

Kelco Gelrite 3.0 gm

pH adjust to 5.6-5.8 before addition of agar and autoclaving.

Add the following filter sterilised amino acids after autoclaving.

major amino acids milligram per litre glutamine 550 asparagine 510 arginine 175

minor amino acids stock 10 ml per litre

(as per Table A6b) Table A8 - Embryo Maturation medium #1 (EMM#1)

To make one litre of medium Step 1

Major ion stock 40 ml

Minor ion stock 20 ml

Iron chelate stock 20 ml

Vitamin stock 10 ml

Inositol 1 gm

Sucrose 30 gm

Dissolve in double distilled water, and adjust volume to allow for addition of filter sterilised components.

Adjust pH to 5.7

Add Gelrite 3 gm per 500 ml flask (6 gm per litre) then add pH adjusted liquid. Autoclave.

Step 2

Dissolve with heating to give final volume of 50 ml

Minor amino acid stock 40 ml

Glutamine 7.3 gm

Asparagine 2.1 gm

Arginine 0.7 gm

Abscisic Acid 15 mg (dissolve in IN NaOH)

Filter sterilise and add to autoclaved medium

TABLE A9 - Embryo Maturation Medium ffl (EMM#2)

Prepare as for EMM#1

Substitute Gelrite 2.25 gm per 500 ml flask (4.5 gm per litre)

TABLE AlO - Embryo Germination Medium (NZRI EGM)

Per litre of medium

Major ion stock 24 ml

Minor ion stock 12 ml

Iron chelate stock 12 ml

Vitamin stock 6 ml

Inositol 0.6 gm

Glucose 30 gm

Gelrite 5.0 gm

Adjust to pH 5.70 and autoclave

Add filter sterilised amino acids in aqueous solution adjusted to pH 5.70 Arginine 0.26 gm

Glutamine 0.40 gm

Proline 0.02 gm

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the invention.

References Cited:

Gupta, P.K., Pullman G.S., 1990: Method for reproducing coniferous plants by somatic embryogenesis. United States Patent number 4,957,866, September 18, 1990.

Gupta, P.K. , Pullman G.S., 1991a: Method for reproducing coniferous plants by using somatic embryogenesis using abscisic acid and osmotic potential variation. United States Patent Number 5,036,007, July 30 1991.

Gupta, P.K., Pullman G.S. , 1991b: High concentration enrichment of conifer embryonal cells. United States Patent number 5,041,382, August 20 1991.

Claims

1. A growth medium effective for maintaining conifer embryogenic tissue or for subsequent development, maturation or germination of conifer embryos including inorganic ions in the following concentrations:

ION CONCENTRATION RANGE

(mmoles/1)

N0₃ 8-27

NH₄ 0.95-3

Ca 0.08-0.25

Fe 0.05-0.15

Na 1.9-5.75

Zn 0.045-0.135

Cu 4.5xl0^"3-1.5xl0^"2

Mg 0.8-2.5

2. A growth medium as claimed in claim 1 including inorganic ions in the concentration

ranges: ION CONCENTRATION RANGE (mmoles/1)

NO₃ 13-23

NH₄ 1.5-2.5

Ca 0.12-0.21

Fe 0.07-0.13

Na 2.9-4.9

Zn 0.06-0.12

Cu 7xl0^"3-1.2xiσ²

Mg 1.2-2.0

3. A growth medium as claimed in claim 1 which is free of or does not depend on the use of plant growth regulators (hormones, phytohormones) of the auxin and/or cytokinin type.

4. A growth medium effective for sustaining the growth of conifer embryogenic tissue or for the development or maturation of conifer embryos including inorganic ions in the concentrations: ION CONCENTRATION (mmoles/1)

N0₃ about 17.8

NH₄ about 1.96

Ca about 0.17

Fe about 0.10

Na about 3.85

Zn about 0.09

Cu about 9.6xl0^"3

Mg about 1.62

5. A growth medium as claimed in claim 4 and which is free of or does not depend on the use of plant growth regulators (hormones, phytohormones) of the auxin and/or cytokinin type.

6. A growth medium as claimed in claim 5 including inorganic ions in the concentrations:

ION CONCENTRATION (mmoles/1)

N03 17.80 ION CONCENTRATION (mmoles/1)

NH4 1.96

TOTAL 19.76

N

P 1.96

K 14.16

Ca 0.17

Mg 1.62

Cl 3.42X10^"1

Fe 0.10

S 1.83

Na 3.85

B 0.13

Mn 1.62xl0^"2

Zn 0.09

Cu 9.61xl0^"3

Mo 8.27x10^

Co 8.41x10^ ION CONCENTRATION (mmoles/1)

I 6.02xl0^"3

7. A medium of claim 1 including 5g/l-50g/l (w/v) sucrose.

8. A medium of claim 1 including 0.5g/l-1.5g/l (w/v)agar.

9. An embryo maturation medium of claim 1 further including 4 to 9 grams gellan gum per litre.

10. An embryo maturation medium of claim 1 further including 5 to 7 grams per litre gellan gum.

11. A medium of claim 1 including glutamine and at least one other amino acid selected from asparagine, arginine, citrulline, ornithine, lysine, alanine and proline.

12. A medium of claim 1 including sucrose, gellan gum, glutamine and at least one amino acid chosen from asparagine, arginine, citrulline, ornithine, lysine, alanine and proline.

13. A medium of claim 12 including 5 milligrams per litre to 25 milligrams per litre of

abscisic acid.

14. A method of growing conifer embryogenic tissue including the step of growing the tissue on a growth medium including inorganic ions in the concentration ranges:

ION CONCENTRATION RANGE (mmoles/1)

NO₃ 8-27

NH₄ 0.95-3

Ca 0.08-0.25

Fe 0.05-0.15

Na 1.9-5.75

^" Zn 0.045-0.135

Cu 4.5xl0^"3-1.5xl0^"2

Mg 0.8-2.5

15. A method of growing conifer embryogenic tissue including the step of placing whole megagametophytes containing embryos at the polyembryόgenesis stage onto a growth medium including inorganic ions in the concentration ranges:

ION CONCENTRATION RANGE

(mmo'~ ^,;)

NO₃ 8.9-17.8 ION CONCENTRATION RANGE (mmoles/1)

NH₄ 0.98-1.96

Ca 0.085-0.17

Fe 0.05-0.10

Na 1.925-3.85

Zn 0.045-0.09

Cu 4.8xl0^"3-9.61xl0^"3

Mg 0.81-1.62

16. A method of capturing conifer embryogenic tissue at the zygotic polyembryogenesis stage including the step of placing whole megagometophytes on a medium including ions at concentrations shown below:

ION ^" CONCENTRATION (mmoles/1)

N0₃ about 8.9

NH₄ about 0.98

Ca about 0.085 ION CONCENTRATION

(mmoles/1)

Fe about 0.05

Na about 1.925

Zn about 0.045

Cu about 4.8xl0^"3

Mg about 0.81

17. A method as claimed in claim 14, wherein the embryogenic tissue is derived from Pinus radiata, Pinus taeda, Pinus elliotii or Pseudotsuga menziesii.

18. A method as claimed in claim 16 wherein the embryogenic tissue is derived from Pinus radiata, Pinus taeda, Pinus elliotii or Pseudotsuga menziesii.

19. A method as claimed in claim 16 wherein the embryogenic tissue is derived from Pinus radiata.

20. A method according to claim 14 including promoting/allowing development of cotyledonary tissue at the shoot apex of the embryo on said medium.

21. A method for maintaining embryogenic tissue, or for development or maturation of conifer somatic embryos including the step of growing the tissue on a growth medium including ions in the concentrations as shown below:

ION CONCENTRATION (mmoles/1)

N03 17.80

NH4 1.96

TOTAL 19.76

N

P 1.96

K 14.16

Ca 0.17

Mg 1.62

Cl 3.42x10 '

Fe 0.10

S 1.83

Na 3.85

B 0.13 ION CONCENTRATION (mmoles/1)

Mn 1.62xl0^"2

Zn 0.09

Cu 9.61xl0^"3

Mo 8.27x10^

Co 8.41x10^

I 6.02xl0^"3

22. A method as claimed in claim 20 wherein said conifer is Pinus radiata.

23. A method as claimed in claim 20 wherein said conifer is Pinus radiata, Pinus taeda, Pinus elliotii or Pseudotsuga menziesii.

24. A method according to claim 14 wherein said tissue is cultured in a vessel covered with a water vapour permeable film.

25. A method according to claim 21 wherein said tissue is cultured in a vessel covered with a water vapour permeable film.

26. A method as claimed in claim 24 wherein the film allows the transmission of water vapour at the rate of between 90-150 gm/sq metre per day.

27. A growth medium for capturing conifer embryogenic tissue at the zygotic polyembryogenesis stage including inorganic ions in the following concentrations:

ION CONCENTRATION (mmoles/1)

N0₃ about 8.9

NH₄ about 0.98

Ca about 0.085

Fe about 0.05

Na about 1.925

Zn about 0.045

Cu about 4.8xl0^"3

Mg about 0.81

28. A conifer embryo germination medium according to claim 1 including inorganic ions in the following concentrations: ION CONCENTRATION RANGE (mmoles/1)

NO₃ 8.9-17.8

NH₄ 0.98-1.96

Ca 0.085-0.17

Fe 0.05-0.10

Na 1.925-3.85

Zn 0.045-0.09

Cu 4.8xl0^"3-9.61xl0^"3

Mg 0.81-1.62

29. A medium as claimed in claim 28, which is the medium of Table AlO.

30. A method for growing conifer plants including:

(a) dissecting out megagametaphytes from seeds of cones at the appropriate stage of development of the zygotic embryos;

(b) placing said megagametophytes on a medium as claimed in claim 27;

(c) growing embryogenic tissue for up to a month on said medium;

(d) transferring the embryogenic tissue to a second growth medium including inorganic ions in the concentrations:

ION CONCENTRATION (mmoles/1)

Co 8.41x10^

I 6.02xl0^"3

(e) transferring the embryogenic tissue to a third medium including inorganic ions in the concentrations of the second medium and further including about 550 mg/1 glutamine and one or more of asparagine, arginine, citrulline, ornithine, lysine, alanine, and proline, said third medium being gelled with gellan gum;

(f) transferring the embryogenic tissue to a fourth medium which is an embryo maturation medium including inorganic ions in the concentrations of the second medium and further including 5-10 gm/1 glutamine, one or more of asparagine, arginine, citrulline, ornithine, lysine, alanine, and proline, 5-25 mg/1 abscisic acid and 4-9 gm/1 gellan gum;

(g) harvesting mature cotyledonary stage embryos (h) germinating said cotyledonary stage embryos; and (i) transferring to soil.

31. A conifer plant derived from embryogenic tissue grown on a medium of claim 1.

32. A conifer plant of claim 30 wherein said conifer is Pinus radiata or Pinus taeda.

33. A growth medium of claim 1 for growing conifer embryogenic tissue selected from the media of Tables A5, A6, A7, A8 and A9.