AU676016B2 - Growth medium - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): NEW ZEALAND FOREST RESEARCH INSTITUTE LIMITED Invention Title: GROWTH MEDIUM **e The following statement is a full description of this invention, including the best method of performing it known to me/us: GROWTH MEDIUM 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, 199 la, 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.

*4*I

A

I

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.

*e *e 2 f) Te 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 Pice (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 spccialised 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 soniaclonal 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.

SUMMvARY OF 1118 INVENTIQN According to one aspect the present invention comprises growth media effective for capturing and sustaning 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 I CONCENTRAITON RANGE mmoles/l

NO

3 8-27 N'H4 0.95-3 Ca 0.08-0.25 Pe 0,05-0.15 Na 1.9-5.75 Zn 0.045-0.135 C11 4,5x10' 3 -1.x~Y 2 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 thc present invention with respect to embryogenic tissue from conifers such as Pinus radiala, Pinus taeda, pi'nus elliotii, and Pseudotsuga nwnziesil, however it should be appreciated that the present invention may be able to be used with embryogenic tissue coming from other conifers.

4 S. S

S

*5 S S *5 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 polyombryogeny 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, Pintu elliotil and Pseudotsuga menzlesil, 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 ellioti and Pseudotsuga menziesil, for *p example.

DETAILED DUCRIPTI()N Qri Ttffi MENIM The medium of the invention comprises ions within the concentration ranges of Table I 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 (2 are also highly preferred, TABLE 2 MEDIA ION CONCE3NTRATIONS (m moles/l)

ION

NO

3

NH

4 Ca Fe Na ZnI Cu Mg 8-27 0.95-3 0.08-0.25 0,05-0.15 1.9-5.75 0.045-0.135 4.5x10'-t.5x 10.2 0.8-2.5 (3-23 1.5-2.5 0.12-0.21 0,07-0.13 2.9-4.9 0.06-0.12 7xl 0*3-1 .2x10, 2 1.2-2.0 17,8 1.96 0.17 0.10o 3.85 0.09 9.61x10 3 1.62

S

Table 3 provIdes; a comparison between the ion concentrations of one preferred medium of the invention with the Weyerhaeu~te medium, the Murashige Skoog medium and a general tissue culture medium for radlata pine Improved at Now Zealand Forest Research 6

S

S S

S.

S. S

S

S.

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 MURASIGOE FRI-LP MEDIUM MEDIUM SKOOG MEDIUM

MEDIUM

N03 17.80 20.54 39.40 32.96 NH14 1.96 7.54 20.61 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 ge *4ee 0* 4*40 Good *4 4* 4** gf• 4 4 *4 4 4* 4.

SO S eS 0* 0 00*0 Cl 3.42x10-l 1.00 5.99 2.10X10' Fe 0.10 0,02 0.10 0.11 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 1.62x10" 6,21x10' 1.0O0xlO. 8,97x100 Zn 0.09 0.05 0.03 0.03 Cu 9.61x0* 5.01x104 1.00oox10 .1 Ox10 3 Mo 8.27x104 5,17x00" 1.03x10' 1,03xlO' Co 8.41x04 5.25x10 4 1,05x104 1.05x10 4 I 6.02x1O 2.50x10" 5.Ox10 3 4,82x10A 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, lodide, molybdenum and cobait ions are preferably Included in the medium. The media of the invention preferably also contain other nutrients generally used in conifer embryogenesis such as 8 000* 0 00 0 *s 0 00 00 0 *r 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, ornithine, lysine, alanine and proline) are also preferably present in preferred media. The concentrations of some of the other components can usefully be varied accordin~g 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 omibryogenesis. Particularly preferred media include those of the solutions of Tables AS to A9 at thie end of this disclosurc, Also preferred are media of the invention having concentrations of the nondistinguishing components at levels between 50% and 150% of those found iii Tables A5 to A9, H-ighly preferred aro media with concentrations of the non-distinguishing components at to 125% of those found in the solutions of Tables AS to A9, The preferred pH for niedia of the Invention is in the range 5.5-59 Aea ma eicuenpeerremdi *at O,5-1.5g/1 Sucrose may be included in preferred media at 5-50g/l (Wv).

According to another aspect of the invention there is ptovided a process for conifer enibryogenic, tissue capture and embryogenie tissue maintenance, In embryogenic tissue capture it is prefcrred that the seeds are surface sterihised and entire megagarnetophytes containing the Immature zygotic embryos are dissected and placed directly onto a medium of the invention. Media with concentrations of each ion in tho lower half of :ranges s.howni In Table 1, are preferred for this step. The medium shown In Table AS is particularly preferred for this, Culture Is carried out at an appropriate light intensity, Ambient pho.o period Is preferably used and the temperature is preferably about 24'C, it is pref~erred that plant growth regulators such as awrdiis and cytokinins are flt present, Prcferred species for the practice of the invention include Pseudo tsu~qa inemlexii, PN1111 ioxd(h, Pflus elild and PiniUs radlata.

timbryogenic tiss~ue wilt 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). A\gain 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 mediumi in Table 3 but with a difforent overall ion concentration, For Instance, at the tygotic polyembryogenesis stage of the embryo dtveloprp-nt (stage b above) a medium may be used which has only one half of the concentration of each of the ions listed for the preferrtd medium i 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/I of gellan gum, Media of this embodiment have been found to be particularly useful for conifer embryo maturation.

The gcllan, gum marketed under the name Geirite T" has been found to be particularly :useful. Transfer from embryo development medium with conventicnal Geirite content about 3 gmI to an embryo maturation medium with 5-7 gm/I (prefetably 6 gm/I) Gelcite followed by subsequent transfer to another embr-yo maturatidon medium differing In that the Geirite concentration was 4-5 gm/i (preftrably 4.5 gmn/I Gclritc) gave surprisingly good yields of embryos, it appears that ttanslent use of very high G3eirite levels (eg 6 gm/I) 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 copending 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 t.in 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 osmotle 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 matutation.

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 growipe, in soil averages or exceeds 2~.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 to 100% of those listed in Table 2, column C and Table 3. Particularly preferred *0***for this Is the medium of Table

EXAMPUIS

The following Examples further illustrate the invention, The E~xamples Illustrate the use of the medium in embryogenic tissue Capture and in embryogenic tissue maintenance for capture and maintenance of embryogento tissue of Pseudotsuga innziesil, Pinus toeda, Pin us elliolil 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 tollowed. Media as described below are sterilised by autoclaving for 20 minutes at 1211 0 C, Organic components are filter sterilised, and are added to the medium after autoclaving. Culture is carried out In 90 mmn petri dishes each containing 22 ml of mediu~m.

I

EXAMPLE 1 EMBRYOGENIC TISSUE CAPIURE Sccds were removed from cones of Pin us radiara, Pinus taeda, Pinus elliofli, and Pseudotsuga ineni~esil at an appropriate stage of development of the zygotic embryos (for instance for Pinus radiala 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 Thnbryogenic Tissue Capture Medium (Table A Dishes were cultured at low light intensity micro~insteins rn- 2 swc 4 under ambient photoperiod. The temperature was maintained constant throughout at 24 0 C Ve...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 }3mbryogenic Tissue Capture Medium (Table A5), or onto Standard Embryogenesis Medium (Table A6) or Elmbryo Development Medium (T'able A7), Embryogenic tissue grew out onto the medium over the next three months, anid tissue pieces greater than 2 millimetre-s across were transferred to Standard Enibryogenesis Medium, This protocol differs from that described by other authors, for example in the patents granted to Gupta ct. al (1990, 1991a) 1991 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, anid in the references which they cite (see Table 3).

I

The mineral salt formula of this invention allows the capture of embryogenic tissue without the need Jo resort to the use of plant growth regulators such as auxins (eg.

2,4-Dichiorophenoxy acetic acid, Indole acetic acid, I-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 Embry~ogenic 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 inenziesit, inus toeda, Pinus MIMIi, and Pinus radlara without need to alter the protocol an any way for each of the above named species. 155r00 explants from different control pollinated cone parents of Pinus radlata were put into culture on the Standard Embryogenic Tissue Capture Medium and after adjustment of results for contamination, up to 100% of the whole megagarnetophyto explants gave rise to embryogenic tissue, when the tissue placed into culture was developmentally competent to form embryogenic tissue. The mean response of the best result for each cone parent was 32.9 for lus radiata. For Pin us taeda, Pin us elliotdi and Pseudotsuga menzlesi about 30% of megagametophyte explants at an appropriate stage of development gave :rise to embryogenic tissue, EXAMPLE 2 EMBRYOGEIRIC 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 suapcnsor 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 maitenance of conifer embryogenic tissue differs from that described by other authors, for example in the patents granted to Gupta at, al (1990, 1991a, 1991b and In the references cited therein. ilie major differen~ce Is that the mineral Wat composition differs markedly from that used by the above named authors, i: ~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-Dichlorophcnoxy acetic acid, Indole. acetic acid, t-Napthylacetic acid, or Indole-butyric-acid) and/or cytokinins (eg. 6-Benzylamino Purine, Zeatin, or N6-(2 Isopentonylladenine).

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, EXAMPLE 3 EMBRYO DEVELOPMENT 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 I 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 Embryogcnic 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" S. 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" appcart.ce. 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.

0 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 I (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/I, Abscisic acid in the range 5 mg/I to 25 mg/i, and Gclrite at a concentration between 4.5 gm/I and 6 gmi/i. A concentration of 6 gm/I, is the preferred level of Geirite for the first transfer from Embryo Development Medium to Embryo Maturation Medium (refer Table A8).

The usefulness of manipulating the Geirite, levels in this manner is illustrated by the following eikperiments.

Embryogenic tissue of a competent plant-forming clone of Pinus radilaa was transferred from Embryo Development Medium onto Embryo Maturation Medium #1 gelled with 8 grams/litre Difco Bacto agar, or 3 grams/litre gellari gum (Geirite or 6 grams/litre gellam gum. Each treatment had eight replicate dishes. The yield of harvestable somatic embryos, capable of' germination and conversion to plants was assesse when embryo production ceased after six weeks. Results are shown in Table 4.

TABLE 4

S

*5 S S

*S

S* S S *5 Treatment Total number of embryos Average yield per dish 8 g/l Bacto agar 0 0.0 3 gI Geldtel~ 16 I6 gI Ge rite'm 1 41 17,6J As a refinement to this protocol, I have found that it is usefuil to further enhance embryo yield by removing the tissue from Embryo Maturation Medium #1 with 6 grams/litre Geirite after thrce weeks, and placing subdivided tissue pieces onto Embryo Maturation, Medium #2 with 4.5 grams/litre (3elrite. Figure I illustrates a maturation protocol. taking advantage of these effects and those obtained from the use of water vapour permeable films.

EXAMPLE 5 TRE EFFECT OF USE OF WATER VAPOUR-PERM-EABLE 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 raiata embryogenic cell line was distributed in equal amounts at random over a number of petri dishes containing the BMM#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. Tile use of Vitafilm Omnniproduced the greatest yield of somatic embryos for this particular cell line in tis experiment.

TABLE 5 The effect of permeable films on thle yield of somatic embryos, Film/Closure Total number of Average somatic somatic embryos per dish embryos Plastic petri dish lid/cling film 28 Vitafilm Flo V/S 5 1 12.7 Vtailim. VW 62 15.5 Vitatilm Omni-film 97 24,2 Vitafilm MW F 50 12.5 EXAMPLE 6 rThE EVFECT OF FILMS ON WATER LOSS FROM GROWTH MEDIUM IN PETI DISHES.

ease.

*6* so Petri dishes containing solid somatic embryo development medium were covered with plastic lids, scated oik with clling film, or with one of four different gas Permeable plastkc 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 241C Incubator under dhe same conditions ais used for somatic embryo development.

The meanl watcr loss from dishes after 9 days when covercd with lids of different films was determined. Water loss was correlated with the somatic embryo counits from identical dishecs of medium cultured under the same conditions. A correlaton between embryo formation and water loss of 0.994 was determined statistically for thle film.

covered dishes. Bxperimental results are shown in Table 6.

TABLE 6 Embryo formation and water loss from media Lid/Filn 'Number of Embryos Water Loss (gm/9 days) Lid 28 0 Vitaiirn Flo V/S 51 4.88 Vitatjim. VW 62 5.14 Vitailm Omni-flim 97 6.73 Vitaflm MWT 50 4.60 The water loss from dishes giving tile highest yield of somatic embryos, that Is those covered with Vitafilm Omni-film, was determined to be 118 g/r dish area-/day.

set* *Goo a. a 6* 9a a a. S 9 *5 a. 0 a a a .a In another experiment, following approxilmately three weeks of culture on EMM//1, tissue of four plant-form'ing Pinus radkzta 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 (Jeirite at 4.5gm/I as described In Tablo A9, Some of the dishes retained plastic lids which were scaled at thle margis with Impermeable domestic cling film. Other dishes were Instead covered with orw )f two different plastic, films, As an. additional treatment, lidded, sealed dishes contained an ethylene absorbing agent (potassium permanganate on a matrix of .,-Inum oxide). After a period of eight days, cotyledonary stage somatic embryos wer, :stcd. 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.

6 Cell Line Treatment Mean embryos per 5% LSD test dish 122 Standard dish 14,5 b Vitafilm Omnfilm 78.0 a Vitafilm MWT 11010 a 380 Ethylene absorber 26.5 b 125 Standard dish 4.0 b VitLfIm OmnifiIm 17.0 a Vitaflim MWT 14.5 a 380 Etthylnce absorbcr 5.0 b 6*r *6 *6 6e A13 Standard dish 1.0 b Vitatlm Omnifllm 6.5 ab Vitafilm MWT 9,5 a 380 Ethylene absorber 2.0 ab A17 Standard dish 1,5 -i Yitafilm Ornirim l o a vitafihin MW 0.5 a 380 Ethylene absorber M ILI a,b 'Difference in results of treatments bearing the sMe leftr were not statisticilly significant, @6 *6@ 6 @6 6 *66 6 6 66*6 *666 6 *66 66 6 The results in Table 7 indicate that the use of water-pernieable films In tho XUMM/t2 stage enhaoos the production of cotyledonary stage somatic cmbryos, and also that there is probably an optimal rate of water loss appropriate to each plant cell line (clone) when grown on EMM#2. Thc- results also show that up to more than 100 riabryos at the cotyledonary stage may be obtained from a single petri dish for Pinus radlata. With Pin uta wed the results of this step are similar with as imany as 10-20 embryos at the cotyledonary 4atgo being produced per dish.

EXAMPLE 7 RECOVE3RY OF PLANTS 14ROM SOMATIC EMBRYOS Wolf developed somatic embryos were transferred to NZ FRI Embryo Germination Medium (Table A10). Dishes were seated with clIngfilm, and maintained under shade cloth i standard incubator conditions, Embryos were Incubated at 24 degree Celsius, under a light intensity of approximately 40 micro Trinste~ns mrisec 1 and a 6 hour photoperiod, For somatic embryos from now cell lines, roots appeared as early as 10 dayi after transfer to Germination Mediu!".. cell lines age, roots took longer to emerge but usually appeared within 12 weeks, *t New cell, lines produced many somatic embryos \4f high quality. Generally, these wcre V896* dark green, had long cotyledons, and normally formed a deinite epicotyl whilo adli in sterile otlture, Fimbryos of this typti tend to form roots quickly, and a high conversion efflcle, cy (perccntgge of harvested mature cotyledonary stage embryos converting to 4:00plants In soil, greater than 50%) is observed. I have obserw 'i that tie growth and of aquality of plants Is directly related to the quality of the epicotyl at time of pricking-Out 0""04of embryos into propagation medium. Somatic embryos with very short epicotyls do not perform as well as those which have well developed snoots 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 opicotyl for carbon fixation In-vivo, Setting of Germinated 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: parts fine peat parts perlite 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 a of Pinus radlata, Pinus taeda, Pinls elliotii, and Pseudotsuga menziesli using the Standard Embryogenic Tissue Capture Medium described in Table A5S This medium has 0 proved to be satisfactory for all the conifer species named above without alteration.

Embryogenic tissue of Pinus radlata, Pinus taeda, Pinus elllotil, and Pseudotsuga rnenziesil 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, Pirnta taeda, Pilus ellio11l, and Pvelldotsilga menzlesil 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 medim 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 Len harvested from these media and transferred via standard germination and nursery ptocedures to soil, Over 4000 plants from 50 clones of Pin us radiata have been grown in soil in the greenhouse, nursery bed, or In field trials. By way of exam~ple, for one collection of Pinzs radlata, 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, The best conversion of mature somatic embryos of** to plants in soil was 73% (1001 plants from 1367 somatic embryos). Plants from somatic embryos of Pinus toeda have also been transferred to soil by the same process, .:..EXAMPLE 8 EFFECTIVE RAN g OF CONCENTRATIONS OF PREFERRED MEDIUM MMNRAL 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 concentrationsn of components other thanfinorganic tons 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 Stage of Developmemt medium mineral elements

C@

C C

C..

ETC ETP EDM EM#1 EM#2 EG (6) 1/8 trength* 1/4 strength 1/2 strength Full strength x strength 2x strength ETG Embryogenic Tissue Capture ETP Embryogenic Tissue Proliferation on Standard Embryogenesis Medium EDM Embryogenic Development on Development Medium EM#I Embryo Maturation #1 EM#2 Embryo Maturation #2 EG Embryo Germination C. C C C *r C C* C C

C+

does not gel optimal growth useful growth slower growth not effective This table represents the effective range of concentration of preferred medium mineral elements for Pinus radlata. 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 nienziesii for the first 4 stages.

MEDIA FOR EMBRYOGENIC TISSUE CAPTURE, MAINTENANCE, DBVELOPMENT, AND MATURATION TABLE Al Major Ion Stock: 6* ooe oo.

09*0 5 I o "Z oo 4 Se *r Compound

KNO

3 MgSO 4 .7H 2 0 CaCI 2 ,2HO0 NaNO 3

NH

4

H

2

PO

4 make up to 400 ml Weight gm 14,31 4.00 0.25 3.10 2,25

-I,

TABLE A2 Minor Ion Stock Compound MnSO 4 .41120

HI

3

E

3 Zn$0 4 .7H 2 0

KI

CuSO 4 .51120 Na 2 MoO 4 ,.2H 2 0 CoC1 2 .611 2 make up to 200 ml Weight mg 36,0 80.0 250.0 10.0 24.0 es

L*S*

9*t* S S TABLE A3 Iron stock -to make 1 litre FeSO 4 .711 2 0 1.5 gm Na 2 EDTA 2.0 gm, TABLE A4 Vitamin stock to make I litre, Thiamine HCd 0.5 gm Nicotinic acid 0,s gm, Pyridoxine HC0 0.05 gm,

S

S S

S*

OS

5@ TABLE A5 Standard Embryogenic Tissue Capture Medium per litre of medium major ion stock minor ions stock Iron chelate stock Vitamin stock Inositol Sucrose charcoal (Merck, activated) Difco Bacto agar 20 nil 10 ml 10 ml 5 mi 0.5 gm, 10.0 gm 2.0 gm.

8,0 gm 9*

S.

pH adjust to 5.6-5.8 before addition of agar and autoclaving Table A6. Standard Embryogenesis Medium (embryogenie tissue, maintenance medium)

S

S. S

S

SO S Major ion stock Minor ion stock Iron chelate stock Vitamin stock Inositol Sucrose Difco Bacto agar per litre of medium.

40 ml 20 ml, 20 ml.

10 ml 1.0 gm 30.0 gm 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 glutamine asparagine arginine minor amino acids stock milligram per litre 110 105 2 mal per litre 9* 0 *999 0 9 9 Table A6 b Minor amino acid stock amino acid *9*9 9* 9. 9*0* 9 9* 9 9* *9 *0 9 99 citrulline ornithiae lysine alanine proline 1.58 1152 1.10 018 0.7 Make up to 800 ml with double distilled water.

Dispense into 40 ml aliquots.

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

Adjust PH to 5.6-5.8 and filter sterilise before use.

TCable A7 Embryo Development Medium Major ion st,,ck Minor ion stock Iron chelate stock Vitamin stock Inositol.

Sucrose Kelco, Geirite per litre of medium 40 nit 20 ml 20 mil 10 mil 1,0 gm 30.0 gm 3.0 gm set.

PH1 adjust to 5.6-5.8 before addition of agar and autoclaving.

Add the following filter sterilised amino acids after autoclaving, major amino acids glutamine asparagine arginino minor amino acids stock (as per Table A6b) milligram per litre 550 510 175 10 ml per 111tre *9 Table A8 Embryo Maturation medium #11 (EMM//1) To make one litre of medium Step I Major Ion stock 40 ml Minor !on stock 20 MIl Iron chelate stock 20 ml, Vitamin stock 10 ml Inositol, 1 gm Sucrose 30 gm *0Dissolve In double distilled water, and adjust volume to allow for addition of filter stedilised components.

Adjust pH to 5.7 Add (Jelrite 3 gm per 500 mi flask (6 gmn per litre) then add pHf adjusted liquid.

Autoclave, Step 2 Dissolve with heating to give final volume of 50 inl Minor amino acid stock 40 ml Glutainine 7.3 8m Asparagine 2.1 gm Arginine 0,7 gm Abscislc Acid 15 mg (dissolve in IN NaCUI) Filter sterilise and add to autoclaved medium TABLE A9 Embryo Maturation Medium (EM1#2) Prepare as for BMM#1.

Substitute Getrit 2.25 gmn per 500 ml flask (4,5 gmn per ltre) 9* a.

a. a a TABLE AIO Embryo Germination Medium (NZRI EGM) Por litre of medium Major ion stock 24 mld Minor ion stock 12 ml Iron chelate stock 12 nil Vitamin stock 6 ml Inositol 0.6 gm.

Glucose 30 gin Adjust to PH 5.70 and autoclave Add flter sterilised amino acids in aqueous solution adjusted ta pH1 5.70 Arginlne 0,26 gfln Glutam-ine 0.40 gm P~Iine0.02 gm Aspects of the present invention havt.. been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing fromn the scope of tho invention.

00 off* 4000

S

*4 55 S S *5 S. S S S

S.

L Referen=e Cited: Oupa, Pullman 1990; Methodforo reproelucing conferottplants by soinlle emrbryogenesix. United Statess Patent number 4,957866, September 18, 0990.

Oupta, ullman 1991a., Mthodfor reprodurcing contreous plats by ilng somatlic embry2ogenesis usrinR bscisic acll nd osinwcpotenial varlallon. United States Patent Numbr 5,03,007, Jly 30 1991.

Ouplal PXK, Pullmnan 1991b: R18gh concentlration enrich e t qor~Uterryon cels. Unlet States Patent number 5,01,382, Augulst 20 1991, ago foot Soso No to 0:64 00 of oas so*$ too.

4 Ito

Claims (16)

1.9-5.75 Za 0,045-0.135 Cu45x10i x0 Mg 082 0* 0 0 0 0 0

2. A growth medium ranwges: as climed In claim I including inorganic ions In the concentration ION CO1."N9N1TRArON RANGE NO 3

13-23 Ca 0,12-0.21 PCe 0.07-0.13 Na 2.9-4.9 Z11 0.06-0.12 CU 7x1O0-1 ,2:x1' 2 Mg 1 .2-2.0 3. A growth medium as Claimed in claim .1 which is NA~ Of or does not depen(' -1 the use of plant growth regulators (hormones, phytohormone.a) of' the auxin and/A cytokinin type. 4. A growth medium dffective, for sustaining the growth of conifer enibryogenlo tissue or for the deveoporint or maturation of conifer embryos icluding inorganic ionis In the concentrations: C C. 9C*C C. C. C C S S. LON CONCENTRAITON (mnioles/1) N03 about 17.8 NI-14 about 1,916 Ca about 0,.17 Fe about 0, Na about 3,85 Zn about 0409 Cu about 9.6xlo0 3 Mg about 1,62 A growth medium as claimed in claim 4 and which 19. free of or does not depend on the use of plant growth regulators (hormone~s, phytohormones) of the auxin and/or cytokinin type, so*. 6. A growth medium as claimed in claim including inorganic ions in the concentrations: 5.55 5S S S. S Gee* a. a a a a 7. A medium of claim 1 including 5g/l-50g/1 sucrose. 8. A medium of claim 1 including O.5gli-1,5g/1 (w/v)agar. 9, An embryo maturation medium of claim 1, further including 4 to 9 grams gellan gum per litre. An embryo maturation medium of claim 1 further including 5 to 7 grains per litre gellan gum, 11. A medium of claim 1 including glutamine and at least one other amino acid selected from asparagine, arginine, olitrulline, ornithine, lysine, alanine and proilne. 12, A medium of claim I including sucrose, gellan gum, glutainine and at least one amino acid chosen from asparagine, arginine, citrulline, ornithine, lysine, alanine and got. pro0ene a....pole 13. A mecdium of claim 12 Including 5 milligrams per litre to 25 milligrams per litre 4-6 of abscislc acid. to14. A method of growing conifer embryogenic tissue including the stop of growing the tissue on a growth medhim Including inorganic ions in the concentrution ranges: ION CONCENTRATON RANGE (mrnoles/l) NO 3 8-27 N1-14 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 .5X1O 3 -1.SX0 2 a a

15. A method of growing conifer em-bryogenic tissue including the step of placing whole megagarnetophytes containing embryos at the potyembryogencais stage onto a growth mediumn including inorganic ions in the concentration ranges: I p. a p 0* ION CONCENTRATION RANGE (mmoleS/l) N0 3 8.9-17,8 N14 4 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 4,8x103-9.6ltxl0' Mg0.116

16. A method of capturing conifer embryogcnic tissue at the zygotic polyembryogenesls stage including the step of placing whole megagometophytcS on a medium including ions at concentrations shown below: ION CONCENTRATION (mrnoleS/1) NO 3 about 8.9 NH 4 about 0.98 Ca about 0.085 Fe about 0.05 N a about 1.925 Znt about 0.045 CU about 4.8X10, 3 Mg about 0.81

17. A method as claimed in claim 14, wherein die ernbryogenic tissue is derived from Pinus rodiata, Pinus taeda, Pimis MlIMtI or Pseudotsuga tnenziesii.

18. Pinus A method as claimed in claim 16 wherein the embryogenic tissue is derived from radiata, Pinus taeda, Pinus eliotii or Pseiidotsiga, mnenzesii.

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

20. A nmcthod 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: 4 I.e. 4 *1 C C C C ION CONCE3NTRATION (mmoles/1) N03 17.80 NI{4 1.96 TOTAL 19.76 N P 1.96 K 14.16 Ca 0.17 Mg 1.62 CI 3,42x10 1 l Fe 0.10 s 1.53 Na 3.85 B 0.13 MAn I .62x10' 2 Zn 0.09 Cu 9.61x10' Mo 8.27x10, 4 e Co 8.41x10 4 I 6.02x10 3 22, A method as claimed in claim 20 wherein said conifer is Pinus radiata.

23. taeda, A method as claimed in claim 20 wherein said conifer is Pinus radiata, Pinus Pinus elliotil or Pseudotsuga menziesii. 0 0 0 0 24, A method according to claim 14 wherein said tissue is cultured in a vessel covered with a water vapour permeable film. 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: TON CONCENTRATION (mmoles/1) NO 3 about 8.9 NI, about 0.98 #0.0 0 *0 0 0* 0S *0 0 0@ *0 Ca about 0,085 Fe about 0.05 N'a about 1.925 Zn about 0.045 Cu. about 4.8x10 3 l Mg9 about 0.81

28. A conifr embryo germination medium according to claim I including inorganic ions in the following concentrations: *000 *800 0 0000 4000 S 0**0 *000 0* 00 0000 0*00 0000 S. S 50 0* 00 S S S. ION CONCENTRATION RANGE (MmnIClS/I) N0 3 8.9-17,8 NIL. 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. 8404Y-9. 61M 0 Mg 0.81-1.62

29. A mediumq as claimed in claim 28, which is the medium of Table A method for growing conifer plants including: dissecting out megagametaphytes from seeds of cones at the appropriate stage of development of the zygotic embryos; placing maid megagametophytes on a medium as claimed in claim 27; growing embryogenic tissue for up to a monthi on said medium; transferring the embryogenic tissue to a second growth medium including inorganic ions in the concentrations: see* 000. ION CONCENTRATION (mmoles/l) N03 17.80 NH14 1.96 TOTAL 19,76 N P t.96 K 14.16 ca 0 117 Mg 1.62 C1 3.42XI1Y 1 1'e 0.10 OS.. S.

55.5 S 55 S5*5 S S55* 5* 4 45 SS *5 5 S S S. S 1,83 Na 3.85 B 0.13 Mn 1. 62x10- 2 Zn 0.09 CU 9.6t1401 Mo 80.27xl104 CO 8.41 x IO4 I 6.02x10- 3 transferring die embryogenic tissue to a third medium Including Inorganic ions In (lie concentrations of the second medium and further Including about 550 mg/i glutamine and one or more of asparagine, arginine, citrulino, ornithine, lysinc, alanine, and peoline, said third medium being gelled with gellan gum; 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/i glutarnine, one or more of asparagine, arginino, citrullinc, ornithine, lysine, alanine, and :prollne, 5-25 mg/I abscisic acid and 4-9 gm/i gellan gum; harvesting mature cotyledonary stage embryos gerinating said cotyledonary stage embryos; and transferring to soil, so 31. A conifer plant derived from embryogenic tissue grown on a medium of claim 1. 32. A method of growing conifer plants of claim wherein said conifer is Pinus radlata or Pinus taeda. 33. A growth medium of claim 1 for growing conifer enmbryogenic tissue selected from the media of Tables AS, A6, A7, A8 and A9. 34. A process for producing a conifer plant including the steps of growing conifer embryogenic tissue by the method of claim 14, obtaining mature cotyledonary stage embryos, and germinating said cotyledonary stage embryos. A method of claim 34, in which said conifer is Pinus xadiata or Pinus taeda. DATED THIS 12TH DAY OF DE~CEMBER 1996 15 NEW ZEALAND FOREST RESEARCH INSTITUTE LIMITED *0e* By Its Patent Attorneys: GRIFFITH HACK Fellows Institute of Patent Attorneys of Australia stA11rcfk**0pott0100NZ *Q1t S.. 00 e00 13ST.RACT This invention relates to growth media for capturing and sustaining the growth of coifir embryogenic tissue and conifer embryos, The media have concentrations of the following inorganic Ions in the ranges shown below. TAB3LE I ION CONCENTRATION RANGE (m moles/i) NO 8-27 NH 4 0.95-3 Ca 0,08-0,25 Na 1,9-$,75 Z 0,0$0.135 Cu 4,SX1O-4.5x'o- MA 0,8-2,5 Using media of the invention ws good growth of emrbryogenic tissue and conifCe embryos which can subsequently be germinated to obtain plants. With these mnedia there is no need to use plant regulator/hormons such as auxins and/or cytokinios for embryogenti tissue capture and maintnance *000 0S00 0S S 0 0005 SS 0