WO2005030988A1

WO2005030988A1 - Method of predicting and/or modifying conversion potential of embryogenic tissues of plants

Info

Publication number: WO2005030988A1
Application number: PCT/CA2004/001743
Authority: WO
Inventors: Vindhya Amarasinghe; Dilsiri Palitha Dharmawardhana; William Alexander Lane; Wolfgang Schuch
Original assignee: Cellfor Inc.
Priority date: 2003-09-26
Filing date: 2004-09-24
Publication date: 2005-04-07
Also published as: UY28540A1; AR045838A1

Abstract

The predictability of conversion of an embryogenic tissue to somatic embryos can be predicted by means of a genetic marker. The genetic marker may be the level of expression of any gene that varies in amount in a way that correlates to the observed actual conversion rate to embryos. By measuring the level of expression of such a marker gene in embryogenic tissue, a prediction can be made about the potential of that tissue to convert to embryos. A particular gene that can be used in this way is a gene that has been identified as a glycine-rich protein that includes a marker sequence as shown in SEQ ID NO:1. The level of expression of this gene can be measured by quantitative real-time PCR or alternative method, e.g. an immunological method.

Description

METHOD OF PREDICTING AND/OR MODIFYING CONVERSION POTENTIAL OF EMBRYOGENIC TISSUES OF PLANTS

TECHNICAL FIELD

This invention relates to the conversion of embryogenic tissues to somatic embryos during the reproduction of plants by plant tissue culture methods. More particularly, the invention relates to ways of predicting or assessing whether a particular line or batch of embryogenic tissue will convert to somatic embryos at a rate suitable for commercial acceptability, and then proceeding with conversion to embryos and eventually to seedlings based on such prediction or assessment. The invention relates particularly, but not exclusively, to conifer species.

BACKGROUND ART

Methods of plant tissue culture have been used for many years for the reproduction of plant species. Such methods are particularly suited for producing gymnosperm species, especially conifers for generating large numbers of clones of superior trees useful for reforestation. Normally, a suitable explant or donor tissue is taken from a part of a desirable plant and is placed on at least one culture medium to produce a cell mass (biomass) capable of further differentiation and development. The cell mass is sometimes referred to as callus or embryonal- suspensor mass. The cell mass can be maintained and proliferated in the undifferentiated state indefinitely, and can if desired be frozen at cryogenic temperatures or dried for long term storage. When suitable, the cell mass can be manipulated (e.g. transferred to an induction medium) to stimulate or induce differentiation into immature somatic embryo structures, which can then be cultured further into mature embryos. The mature embryos may subsequently be germinated and grown into conifer seedlings ready for commercial planting. The seedlings produced in this way are genetically identical and may all exhibit superior qualities. Protocols for somatic embryogenesis typically involve several stages from initiation of embryogenic tissue through to somatic embryo maturation and germination.

For initiation of conifer embryogenic cell lines whole gametophytes containing immature fertilised embryos or dissected immature fertilised embryos are used as explants. The explants are placed on one or more "initiation media", which may include growth regulators, to induce the formation of embryogenic tissue. For maturation of conifer somatic embryos, initiated embryogenic cell lines are then placed onto several further media to maintain and develop the embryogenic tissue and multiply the number of embryo initials. Embryogenic tissue is then generally placed on a "maturation medium" to encourage the tissue to form mature embryos.

Patents providing background as to the use of embryogenesis to create somatic embryos include U.S. Pat. No. 5036007, U.S. Pat. No. 5,034,326, U.S. Pat. No. 5,041,382, U.S. Pat. No. 4,957,866, and U.S. Pat. No. 5,294,549. The disclosures of these patents are incorporated herein by reference.

A particular problem that is encountered with this procedure is that the stimulation ofthe cell mass to differentiate into somatic embryos is not always as successful as would be desired and sometimes only low yields of somatic embryos are produced. Since the conversion rate may not be apparent for a number of months, a considerable amount of time and resources is often invested in unproductive tissue samples. This can significantly reduce the productivity and the economic success ofthe entire tissue culture procedure.

There has been no explanation as to why some tissues samples are highly receptive to the stimulation of differentiation and others are not. Not only can there be enormous variations between tissues obtained from different plants (different genotypes), but also between different samples or batches of tissue from the same original plant or tissue line. It is therefore difficult to predict which tissue mass samples will be productive in the conversion to embryos and which will not.

DISCLOSURE OF THE INVENTION

It is therefore an object ofthe present invention, at least in its preferred forms, to provide a method of predicting the conversion potential of embryogenic tissue of plants, especially conifers, so that such prediction can be used to improve the reproduction rate of such plants by tissue culture techniques. Such a method can be used to avoid attempted stimulation of unproductive tissue, thus improving the yield and productivity an overall tissue culture program.

Another object ofthe invention, at least in preferred forms, is to modify (e.g. increase) the conversion rate of cell lines that otherwise have low rates of conversion.

According to one aspect ofthe invention, there is provided a method of predicting embryo conversion potential of embryogenic tissue of plants, which comprises: identifying a gene of a plant, the level of expression of which gene in lines or batches of embryogenic tissue ofthe plant correlates to an observed conversion rate ofthe embryogenic tissue to somatic embryos, thus establishing a correlation between the level of expression ofthe gene in embryogenic tissue and rates of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level ofthe gene while maintaining said tissue prior to conversion, and from said established correlation, predicting the potential conversion rate ofthe embryogenic tissue ofthe line or batch.

If a suitable gene is already known, the method may consist merely ofthe measurement ofthe expression level ofthe gene and then to make a prediction based on the measured expression level. The method ofthe invention is preferably used with embryogenic tissue of a coniferous plant, e.g. all Pinus spp, Picea spp and Pseudotsuga spp., and particularly loblolly pine, radiata pine, Douglas fir and other commercial conifer species. 5 For conifers, a conversion rate of at least 200 germinants per gram of tissue is deemed to be acceptable and less than 50 is considered to be unacceptable for commercial tissue culture. Of course, the cut-off between acceptable and unacceptable is always a matter of choice and tissue in the marginal range of 50 to

10 200 germinants per gram may be accepted depending on other circumstances. It can therefore be said that conifer tissues having a predicted conversion rate of less than 50 germinants per gram should normally be discarded and tissues with higher predicted conversion rates may be accepted, particularly those with conversion rates of 200 germinants per gram of tissue and above. These figures may also

15 vary from species to species and even from tissue culture to tissue culture (as some may be more valuable than others and thus more acceptable at low conversion rates). It is believed that, for any particular procedure, an appropriate cut-off point will be apparent to person skilled in the art and may be higher or lower than the values given above.

20 Preferably, the expression level ofthe gene in a line or batch ofthe tissue is measured by means of quantitative real time polymerase chain reaction, although other suitable techniques may be used if capable of detecting levels of gene expression in embryogenic tissues.

25 A suitable gene that has been found to correlate with embryo conversion is one which contains the DNA sequence of SEQ ID NO: 1 shown below, or a gene having at least 40% identity thereto at the nucleotide level (or, more preferably,45,50,55,60, 65, 70, 75, 80, 85, 90, 95 or 99% identity) and at least

30 60% at the amino acid level (more preferably at least 65, 70, 75, 80, 85, 90 or 95% identity) throughout the sequence. The sequence comparisons are carried out using ClustalW™ programme at the European Bioinformatics Institute using the default settings. The identity values given are for the full length ofthe available coding sequences ofthe respective sequences.

SEQ ID NO:l - SEQUENCE OF AMPLIFIED REGION

>118b

GTGCGGAGAGTCTGGACACTTTGCTAGAGACTGCACTAACGGTGGCGGTGCTGGT GGTGGAGGCGGCGGTGGCGGGGGCAGCTGTTATCAGTGCGGTGACTTCGGTCATT TTGCCCGAGATTGCCCTAACCGTA

According to another aspect ofthe invention, there is provided a method of selecting a line or batch of embryogenic tissue of a coniferous plant that has a high predicted rate of conversion to embryos, which method comprises: identifying a gene of a coniferous plant, the expression of which gene in lines or batches ofthe tissue correlates to the observed conversion ofthe tissue to somatic embryos, thus establishing a correlation between expression ofthe gene in embryogenic tissue and levels of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level of said gene while maintaining said tissue prior to conversion, from said established correlation, predicting the conversion potential ofthe embryogenic tissue ofthe line or batch; and selecting said line or batch for subsequent conversion to somatic embryos only if said predicted conversion potential ofthe line or batch exceeds a predetermined level.

According to yet another aspect ofthe invention, there is provided a method of converting embryogenic tissue of a coniferous plant, which method comprises: identifying a gene of a coniferous plant, the expression of which gene in lines or batches ofthe tissue correlates to the observed conversion ofthe tissue to somatic embryos, thus establishing a correlation between expression ofthe gene in embryogenic tissue and levels of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level of said gene while maintaining said tissue prior to conversion, from said established correlation, predicting the conversion potential ofthe embryogenic tissue ofthe line or batch; selecting said line or batch for subsequent conversion to somatic embryos only if said predicted conversion potential ofthe line or batch exceeds a predetermined level; and converting the embryogenic tissue to somatic embryos by culturing the tissue on one or more conversion media.

According to yet another aspect ofthe invention, there is provided a method of producing somatic embryos of a coniferous plant, which method comprises: obtaining a batch of embryogenic tissue from a coniferous plant and maintaining said tissue on a tissue maintenance medium, measuring a level of expression of a gene of said tissue maintained on said maintenance medium, said gene being a gene whose expression correlates to an observed conversion ofthe tissue to somatic embryos according to an established correlation; if said level of expression correlates to a level of somatic embryo conversion at or higher than a predetermined value, inducing said embryogenic tissue to convert to somatic embryos; if said level of expression correlates to a level of somatic embryo conversion lower than said predetermined value, discarding said batch and obtaining another batch ofthe same line or another line of said coniferous plant, repeating said procedure until a batch exhibits a level of expression at or higher than said predetermined value is obtained; and inducing conversion of said tissue of said batch having said level of expression at or higher than said predetermined value to somatic embryos.

According to still another aspect ofthe invention, there is provided a method of producing somatic embryos of a coniferous plant, which method comprises: obtaining a batch of embryogenic tissue from a coniferous plant and maintaining said tissue on a tissue maintenance medium, measuring a level of expression of a gene of said tissue maintained on said maintenance medium, said gene being a gene identified by containing a DNA sequence according to SEQ ID NO: 1 or a sequence having at least 40% identity thereto; if said level of expression correlates to a level of somatic embryo conversion at or higher than a predetermined value, inducing said embryogenic tissue to convert to somatic embryos; if said level of expression correlates to a level of somatic embryo conversion lower than said predetermined value, discarding said batch and obtaining another batch ofthe same line or another line of said coniferous plant, repeating said procedure until a batch exhibits a level of expression at or higher than said predetermined value is obtained; and inducing conversion of said tissue of said batch having said level of expression at or higher than said predetermined value to somatic embryos.

According to yet another aspect ofthe invention, there is provided a method of identifying a plant family or genotype having good potential for conversion to embryos at a high rate during tissue culture, which method comprises: identifying a gene of a plant, the level of expression of which gene to a high conversion rate of plant tissue to somatic embryos, thus establishing a correlation between the level of expression ofthe gene in embryogenic tissue and rates of subsequent conversion ofthe tissue to embryos; testing at least one plant of a family or genotype to measure the level of expression ofthe gene in embryogenic tissue of the plant, and predicting the potential for conversion ofthe plant or genotype from the level of expression ofthe gene.

Furthermore, the invention relates to a polypeptide having the sequence of SEQ

ID NO:5 below or at least 60% similarity thereto.

Still further aspects ofthe invention relate to the identification of families or genotypes of plants having a disposition to high conversion rates and to such families or genotypes thus identified. Also, the invention relates to cell lines identified as having high conversion potential.

BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1, 2 and 3 are schematic diagrams illustrating steps in procedures carried out according to preferred embodiments ofthe present invention; Figs.4 to 6 are graphs of fluorescence versus PCR cycle number used for comparison of levels of expression of a certain gene in lines or batches of embryogenic tissue of loblolly pine, as explained in the Examples below; Fig. 7 is a graph correlating gene expression to production of plantable germinants as explained in the Examples below; and Figs. 8, 9 and 10 show similarities between gene 118 ofthe present invention and similar genes of other plants.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is based, at least in part, on the concept ofthe inventors that different undifferentiated cell mass samples (embryogenic tissue) exhibit different rates of conversion to healthy mature somatic embryos when subjected to the same environmental conditions for reasons relating to (or causing) differences in expression levels of one or more genes ofthe embryogenic tissue. That is to say, the expression of one or more genes is required for high rates of conversion, or alternatively the expression of one or more genes may be a consequence ofthe presence or occurrence of other (unknown) factors that lead to high conversion rates.

The inventors observed that different samples or batches of embryogenic tissue produced from the same plant may exhibit different degrees of embryo conversion under the same conditions, and theorized that mere differences of genetic make-up (differences of DNA coding of genes among various genotypes) may not be the main or only reason for the observed differences in rates of embryo conversion. Instead, the inventors postulated that differences of expression of one or more genes may be the cause, or a least a symptom, ofthe different performances during cell differentiation. That is to say, poor expression (or perhaps, in some cases, over-expression) of one or more genes in a particular sample causes or is a symptom of poor conversion ofthe biomass to healthy somatic embryos. Such differences of expression may be a result of differences of treatment or exposure of a sample cell mass to environmental conditions or methods of handling. Accordingly, in the present invention, differences in gene expression between tissue samples are correlated with the performance of those samples during differentiation treatments to produce somatic embryos. Such differences of gene expression that correspond well (directly or inversely) with embryo conversion performance are regarded as markers of good or poor conversion performance. Consequently, once a marker gene has been identified in this way, a particular tissue sample can be tested for levels of expression ofthe marker gene and the results of such tests used to predict the likelihood that a particular sample will give good or poor conversion results. Samples likely to give poor results may then be discarded before differentiation has commenced or progressed to a significant extent.

The method is illustrated in simplified form in Fig. 1 ofthe accompanying drawings. A batch of embryogenic tissue is first maintained (reference numeral 1) in normal culture maintenance conditions and a sample is then tested for expression ofthe gene already determined to be a marker (reference numeral 2). If the sample is found to have a high level of expression (arrow 3), the batch is converted to embryos (reference numeral 4) and then the embryos are subjected to the steps required to cause them to mature and germinate (reference numeral 5) so that they develop into healthy seedlings 6. If the expression ofthe gene is indeed a reliable indicator of conversion potential, a large percentage ofthe embryogenic cells will convert to health embryos. If the sample is found to have a low level of expression (arrow 7), the batch is discarded (reference numeral 8) in order to avoid wasting resources on a batch with low conversion potential.

To confirm this approach, the inventors screened the expression of nearly 150 genes of embryogenic tissue of loblolly pine (Pinus taeda) at the culture maintenance stage using quantitative real-time PCR and correlated the measured expression with subsequent embryo productivity. Quantitative real-time (kinetic) PCR (polymerase chain reaction) was used to measure gene expression. This is a widely-used method of detecting and quantifying very small amounts of DNA or RNA, e.g. the amounts associated with active expression of a gene. Specifically, masses of embryogenic cells of different genotypes from different culturing stages (TX, PI and P2) were frozen in liquid nitrogen and the total RNA ofthe tissue was isolated under RNAase-free conditions with RNA Easy kits (Qiagen). The isolated RNA was quantified and equivalent quantities of each sample were used as a template for reverse transcription (Invitrogen Superscript III Reverse transcriptase kit, Cat#l 8080-44). The resulting cDNA was used as a template for amplifying the sequence of interest (marker gene) using primers, and hot start thermal polymerase and sybregreen (QuantitectSybre™ Green PCR Kit: Qiagen Cat#204143) and real time thermocycle (Roche Light Cycler) according to the manufacturer's protocol (Meuer, S., Wittwer, C, and Nakagawara, K. 2001 Rapid Cycle Real-Time PCR: Methods and Applications, Springer Verlag, Berlin).

The inventors found (in test no. 118) that the expression of a particular sequence (part of a specific gene) at a high level correlated well with a high embryo conversion rate. The specific sequence identified in this way is SEQ ID NO: 1.

Higher levels of expression ofthe gene containing this sequence correlated with higher embryo productivity, medium levels correlated with medium conversion and low levels correlated with low embryo conversion. The level of expression of this gene in embryogenic tissue maintained on standard maintenance medium is therefore considered to be a marker that is predictive ofthe level of conversion of the embryogenic tissue to somatic embryos.

The gene in question (containing the particular sequence mentioned above and sometimes referred to herein as the "118 gene" because ofthe test number referred to above) is a gene described as a "glycine rich protein (contig7202)". The partial cDNA sequences were assembled and deposited at the University of Minnesota, Center for Computational Genomics and Bioinformatics TIGR database under ID numbers Dirl 3-7202, TC22024 and TC22025, respectively. The EST sequences and Genbahk ID numbers that were used to assemble these sequences are shown in Table 1 below. TABLE 1

Contg# EST ID# GB# TC22025 NXPV_06₂_E12 BQ697927 NXPV_015_D12 BG318580 NXSI_041_E10 BF609204 XSI_112_C07 BG040533 NXSI_148_A11 BG275803 NXCI_053_A08 BE643974 NXSI_090_E08 CD025901 NXSI_108_E05 BG040463 NXSI_065_A12 BQ701488 NXSI_038_D03 BF518349 NXSI_076_F08 BF777863 NXCI_054_B06 BE644065 NXNV027A12 CD027113 NXNV027A12 AW290452 NXPV_009_A10 BG318219 NXSI_001_A09 BF516618 NXRV100_D07 BQ655877 NXCI_069_H06 BE758675 NXSI_059_D06 BF610503 NXSI_043_E10 BF609519 NXCI_131_E12 BF186193 NXCI_130_E12 BF186085 NXNV 081 F08 A 736926

TC22024 NXSI_110_D10 BG040271 NXNV009E05 CD02667₃ NXNV009E05 A ₂8999₂ PP023A10 BX249354 NXRV_0 _B06 CD0₂452₃ NXPV_027_E08 BG₃19399 PP098A02 BX254₃87 PP065E07 BX252494 PP098C07 BX₂5441Q NX V_008_E11 BM1₃₃551 hl_PLlCSUH AI92016₂ NXSI_125_F04 BQ70₂160 NXCI_076_F06 BE76₂071 NXCI_056_G01 BE656772 PP023C02 BX249₃69 NXSI_041_E06 BF609₂00 NXPV_062_G06 BQ69794₂ NXPV_052_D12 BQ697754 NXPV_101_H05 BI₃97606 XPV_003^11 CD02₂Q72 NXCI_101_H06 BE9968₃0 NXPV_016_D10 BG3187₃5 NXRV056_D04 CD024598 NXPV 033 D06 CD02₂₂68 The assembled sequence of this gene is shown below as SEQ ID NO:4: t

SEQ ID NO: 4 - NUCLEOTIDE SEQUENCE

>ContigId: 7202 TCGAGAGGGTTTGGAGACGAGGGTTTTTTGTTCTGTGTTGGTTGTTCTTT GAGGGATTCGAACCATGTCTGAGGGGAAGAGATCCGGAAAGGTTAAGTGG TTCGATTCGACTAAGGGTTATGGTTTCATAACCCCTGATGATGGCAGCGA GGACCTGTTTGTGCATCAGACTTCGATTCACTCCAATGGGTTTCGATCGC TGGCCGAGGGAGAGGCCGTCGAGTACACGGTCGAGATTGAGAATGGAGGA AGAACCAAGGCCCTAGATGTTACAGGGCCCGATGGAGCCTTTGTCCAGGG TAACAGTGGAGGAGGCGGCGGTGGAGGCCGAGGAGGCGGTAGAGGCGGAG GTGGTGGTGGCTACGGCGGTGGTGGTTATGGCGGCGGCGGTGGTGGTTAC GGCGGCGGTGGGTATGGCGGTGGTGGTGGATGGAACGGAGGTGGCGGTGG CGGCCGAGGTGGTGGTCGTGGAGGAGGTAGGGGAGCCGGTGGCGGCGGCG GTGGCGGCGGTGGAAGCTGTTACAAGTGCGGAGAGTCTGGACACTTTGCT AGAGACTGCACTAACGGTGGCGGTGCTGGTGGTGGTGGCGGCGGTGGCGG GGGCAGCTGTTATCAGTGCGGTGACTTCGGTCATTTTGCCCGAGATTGCC CTAACCGTGAGGGTTAGGCATATCTGAACCGTGCCCTTTCTATCTTCTCG TTCTACTTCAGCGTTTTGAATGTTGTGCCCGCTGCCGCTACCCACGCCTT CCCCAAGATTTCGTGGTTTGTGGCTTTTGTTGTTGTAAGCCTGCTCCGGT TTTGGGTATGTCTGGCTTAAATATTTTACGTTGTTTTCAAATTTGCAGGC TTAGTTATGTTTGTAAGGGTTCTCTACTGTAATGTAAGGTACTGAATGAA CTATTTATCCATAGACTCTCTAGGAGTGGAGCCGGGCCATAATTTGGCGG TAGAAGATCTATTTTTGCTACTATTGGCTAGGGATATACACTCTTCTTTT TTAATTGTG

The corresponding translated peptide sequence is shown below as SEQ ID NO:5:

SEQ ID NO: 5 - AMINO ACID SEQUENCE

>7202

MSEGKRSGKVKWFDSTKGYGFITPDDGSEDLFVHQTSIHSNGFRSLAEGEAVEYT VEIENGGRTKALDVTGPDGAFVQGNSGGGGGGGRGGGRGGGGGGYGGGGYGGGGG GYGGGGYGGGGGWNGGGGGGRGGGRGGGRGAGGGGGGGGGSCYKCGESGHFARDC TNGGGAGGGGGGGGGSCYQCGDFGHFARDCPNREG

The primers used for detecting the sequence of SEQ ID NO: 1 in the quantitative real-time PCR procedure carried out by the inventors are shown below.

Forward Primer 118f [SEQ ID NO:2]: TACAAGTGCGGAGAGTCTGG

Reverse Primer 118r [SEQ ID NO:3]:

GATAGAAAGGGCACGGTTCA

The experimental detail showing the correlation between high levels of expression of this gene and embryo productivity is shown in Example 1 below.

Samples of tissue cultures showing expression of this gene at a high level may be taken as an indication of high probability of embryogenic productivity. The term "high", as well as terms "medium" and "low", are assigned somewhat subjectively with reference to the amplification ofthe ribosomal sequences, but for the tests using the above primers having a ribosomal copy number of 10 - 10 , the term "high" was considered to be sequence copies in the range of 10⁷ - 10⁸, medium was 10⁵ - 10⁶, and low was 10² - 10³. Persons skilled in the art will, from experience, be able to determine levels of expression of a gene that are considered to be high, medium or low.

While it is particularly convenient to employ quantitative real-time PCR as indicated above to measure levels of expression of a particular gene in embryogenic tissue, other methods may also be employed, such as immunological methods. For example, an antibody to the expressed protein or peptide may be raised, and the antibody used in a Western blot or ELISA-type test to determine the presence and relative amount ofthe expressed protein . It is believed that the protein sequence of SEQ ID NO:5, and homologous sequences having at least 60% similarity (preferably 65, 70, 75, 80, 85, 90, 95 or 99% similarity), is novel and is useful for such testing purposes, so it is considered to form part ofthe present invention.

While the present invention has been described as a method of predicting the likely conversion rate of specific samples of embryogenic tissue, it can also be used to identify families and genotypes of plants that consistently have good potential to produce effective embryogenic lines. That is to say, plant families or genotypes that consistently show high expression ofthe marker gene in their embryogenic tissue can be identified as having good conversion potential. A screening test may thus be devised in which different families or genotypes of plants are used to produce embryogenic tissue from their explants, and the tissue tested as above. The resulting plants families found to have a high propensity for embryo conversion may then be selected for further investigation and commercialization.

The inventors carried out a sequence similarity searches on publicly available databases and found that there is a region (RNA binding domain) ofthe gene under consideration that has high similarity to a cold-inducible RNA binding protein in wheat. This domain corresponds to amino acid residues 19 to 38 of protein sequence SEQ ID NO:5. The amino acid alignment for the gene product is shown in Fig. 8. In the figure, SEQ ID NO: 5 (118) is aligned with amino acid sequences of Nicotiana sylvestris (SEQ ID NO:6), Triticum sativum (SEQ ID NO:7) and two strains of Arabidopsis thaliana (SEQ ID NO:8 and SEQ ID NO:9).

This indicates to the inventors that this sequence is likely coding a RNA binding protein in addition to or instead of a structural gene (see, for reference, Karlson et al., (2002) "A Cold Regulated Nucleic Acid Binding Protein of Wheat Shares a Domain with Bacterial Cold Shock Proteins.", The Journal of Biological Chemistry, Vol. 277:35248-35256). The functioning of this gene as a cold- inducible gene regulator leads the inventors to predict that embryogenic tissue samples may be made more productive in their conversion to embryos by subjecting the tissue sample to a cold treatment before stimulation to produce embryos. The cold treatment should be carried out at a sufficiently low temperature and for a sufficient duration to cause increased expression ofthe gene. Such a treatment may be, for example, treatment carried out at a temperature below about 8°C for a period of time from half an hour up to several hours, e.g. treatment in the range of 2 to 6°C for a period of at least half an hour (e.g. Vi to 1 hour). Of course, different temperatures and durations may be required to optimize the cold treatment for different genotypes, and such details will be apparent from simple trial and experimentation for each genotype employed. A cold-treatment of this kind before commencing induction of embryo development will help to assure a high rate of embryo productivity, at least for plants that otherwise express the protein of SEQ ID NO: 5, or its homologs, only at low or intermediate levels.

The manner in which the cold treatment can be employed is illustrated by way of example in Fig. 2 ofthe accompanying drawings. This illustration shows a test for expression being carried out after the cold treatment and a decision to discard or continue being based on the resulting expression detected. According to the illustrated procedure, a batch of embryogenic tissue is first maintained (reference numeral 10) and is then subjected to a cold treatment (reference numeral 11), e.g. the batch is held at a temperature of 2 to 6°C for about half an hour), a sample is then tested for expression ofthe gene determined to be a marker (reference numeral 12). If the sample is found to have a high level of expression (arrow 13), the batch is converted to embryos (reference numeral 14) and then the embryos are subjected to the steps required to cause them to mature and germinate (reference numeral 25) so that they develop into seedlings 16. If the expression of the gene is a reliable indicator of conversion potential, a large percentage ofthe embryogenic cells will convert to health embryos. If the sample is found to have a low level of expression (arrow 17), the batch is discarded (reference numeral 18) in order to avoid wasting resources on a batch with low conversion potential.

The expression testing step may, however, be discarded on the assumption that the cold treatment will improve the expression level ofthe gene (and therefore improve the rate of conversion) of all cell lines. The resulting procedure will then be as shown in Fig. 3. According to this procedure, a batch of embryogenic tissue is first maintained (reference numeral 20) in culture and then the entire batch of embryos is subjected to a cold treatment (reference numeral 21), e.g. the batch is held at a temperature of 2 to 6°C for about half an hour. The batch is then converted to embryos (reference numeral 24) and then the embryos are subjected to the normal steps required to cause them to mature and germinate (reference numeral 25) so that they develop into seedlings 26. The cold treatment enhances the expression ofthe gene and thus increases the rate of conversion to healthy 5 embryos.

In view ofthe homology ofthe 118 gene to the gene found in wheat, it is believed that the invention, in all of its aspects, is suitable for use with wheat and other crop plants, such as oats, barley, legumes, canola, and the like.

10 The 118 gene has also been compared with genes in species of red runner bean and spruce, leading to the conclusion that the invention is effective for all angiosperms and gymnosperms, although the invention is particularly suitable for conifers, as already noted above. The comparisons are shown in Fig. 9 and Fig.

15 10, respectively. In the case of red runner bean as shown in Fig. 9, two EST sequences (SEQ ID NO:10 and SEQ ID NO:l 1) derived from Phaseolus embryos were found to be highly homologous to pine 118 sequence (SEQ ID NO:4) (63 and 72% identity, respectively). In the case of spruce as shown in Fig. 10, it was found that several EST sequences (SEQ ID NO:12, SEQ ID NO:13, SEQ ID

20 NO: 14 and SEQ ID NO: 15) have high homology to the 118 gene (SEQ ID NO:4) (65-75% identity).

EXAMPLE 1

25 Tests were carried out on four culture lines of loblolly pine embryogenic tissue. One ofthe lines (Q3802) was known to convert to embryos at a high rate of production, two (03971 and 03621) were known to convert to embryos at only a very low rate, and the final line (L3514) was an intermediate producer of embryos.

30 The tissue samples were obtained from cryogenic storage and were placed on a maintenance media (TXS) to produce and maintain embryogenic tissue mass. The tissue samples were screened by means of quantitative real time PCR for expression of nearly 150 genes using forward and reverse primer sequences (including those of SEQ ID NOs:2 and 3) produced for this purpose. The employed technique allowed for the detection not only ofthe expression ofthe genes being tested for, but also ofthe level of expression.

The embryogenic tissue was then converted by transferring the tissue first to an induction medium PI and then to an induction medium P2 and the number of embryos thereby formed was measured. The results are shown in Table 2 below.

The embryo conversion results were correlated with the gene activity to assess whether the activity of any gene corresponded to the productivity of embryo production. That is to say, a comparison was made to see if any gene expressed poorly when productivity was low, and highly when productivity was high, etc. Primer pair 118f and 118r (SEQ ID NO:2 and SEQ ID NO:3) specific to a glycine rich cell wall protein were found to identify a gene that had the required correlation and could thus be used as a marker for prediction of conversion productivity.

Accompanying Figs. 4 to 6 illustrate the expression of this gene in the various cell lines of Table 2. It will be seen that the gene is expressed highly in line Q3802 (TXS, PI), but only at a low level in lines 03971 (Fig. 4) and 03621 (Fig. 5). In the case of L3514, the expression ofthe gene was at an intermediate level (Fig. 6), and so was the embryo productivity (Table 2).

EXAMPLE 2

The 118 gene was tested as a marker for predicting the success of production of mature embryos. Twenty nine sample callus tissues were tested for expression of the gene and subsequent conversion to plantable germinants. The results are shown in Table 3 below and are illustrated in Fig. 7 ofthe accompanying drawings. Plantable germinants differ from mature embryos in that they have undergone germination. Germination is itself a major developmental step which is controlled by factors different from those that control embryo maturation. This is believed to account for some discrepancies seen in the results (e.g. sample 21 - Q3802 D pull), as low results may be an indication of poor germination rather than poor embryo production. TABLE 3

Notations:

¹ Sample identity number for me PCR reaction

² Gene expression level in a single sample

³ Average of three replications

⁴ Experiment identity for the cryo experiment

⁵ Loblolly pine genotype

⁶ Descriptions from sample tubes

⁷ Culture medium identity

⁸ Number of plantable germinants

⁹ Standard deviation

Different aspects ofthe invention are defined in the following claims. However, these claims should not be regarding as limiting the scope of this invention and instead are merely representations of specific aspects or embodiments. It will be apparent to persons skilled in the art from the present description that other embodiments or aspects may be novel and inventive, and it is the intention ofthe inventors to protect all forms ofthe invention disclosed herein.

Sequence Free Text

In SEQ ID NO: 12 ofthe accompanying Sequence Listing, the free text under item <223> states that "n represents any nucleotide" and "n is a, c, g or t".

Claims

CLAIMS:

1. A method of predicting embryo conversion potential of embryogenic tissue of plants, which comprises: identifying a gene of a plant, the level of expression of which gene in lines or batches of embryogenic tissue ofthe plant correlates to an observed conversion rate ofthe embryogenic tissue to somatic embryos, thus establishing a correlation between the level of expression ofthe gene in embryogenic tissue and rates of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level of said gene while maintaining said tissue prior to conversion, and from said established correlation, predicting the potential conversion rate ofthe embryogenic tissue ofthe line or batch.

2. The method of claim 1 , wherein the expression level of the gene in a line or batch of the tissue is measured by means of quantitative real time polymerase chain reaction.

3. The method of claim 1 , wherein the expression of the gene in a line or batch of the tissue is measured by means of an immunological technique.

4. The method of claim 1 , wherein the gene contains the sequence of SEQ ID NO:4 or a sequence having at least 40% identity to SEQ ID NO:4.

5. The method of claim 1 , wherein the plant is a conifer.

6. The method of claim 5, wherein conifer is selected from the group consisting of loblolly pine, radiata pine and Douglas fir.

7. The method of claim 1 , wherein the plant is an angiosperm.

8. Embryos of a plant converted from embryogenic tissue predicted to have a high rate of conversion according to the method of claim 1.

9. A method of selecting a line or batch of embryogenic tissue of a coniferous plant that has a high predicted rate of conversion to embryos, which method comprises: identifying a gene of a coniferous plant, the expression of which gene in lines or batches ofthe tissue correlates to the observed conversion ofthe tissue to somatic embryos, thus establishing a correlation between expression ofthe gene in embryogenic tissue and levels of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level of said gene while maintaining said tissue prior to conversion, from said established correlation, predicting the conversion potential ofthe embryogenic tissue ofthe line or batch; and selecting said line or batch for subsequent conversion to somatic embryos only if said predicted conversion potential ofthe line or batch exceeds a predetermined level.

10. The method of claim 9, wherein the expression level ofthe gene in a line or batch of the tissue is measured by means of quantitative real time polymerase chain reaction.

11. The method of claim 9, wherein the gene contains a the DNA sequence of SEQ ID NO:4 or at least 40% identity to SEQ ID NO:4.

12. The method of claim 9, wherein the plant is a conifer selected from the group consisting of loblolly pine, radiata pine and Douglas fir.

13. The method of claim 9, wherein said line or batch is selected only if said predicted conversion potential exceeds 50 germinants per gram of tissue.

14. A method of converting embryogenic tissue of a coniferous plant, which method comprises: identifying a gene of a coniferous plant, the expression of which gene in lines or batches ofthe tissue correlates to the observed conversion ofthe tissue to somatic embryos, thus establishing a correlation between expression ofthe gene in embryogenic tissue and levels of subsequent conversion ofthe tissue to embryos; for a particular line or batch of said tissue, measuring the expression level of said gene while maintaining said tissue prior to conversion, from said established correlation, predicting the conversion potential ofthe embryogenic tissue ofthe line or batch; selecting said line or batch for subsequent conversion to somatic embryos only if said predicted conversion potential ofthe line or batch exceeds a predetermined level; and converting the embryogenic tissue to somatic embryos by culturing the tissue on one or more conversion media.

15. The method of claim 14, wherein the expression level ofthe gene in a line or batch ofthe tissue is measured by means of quantitative real time polymerase chain reaction or equivalent method.

16. The method of claim 14, wherein the gene contains a the DNA sequence of SEQ ID NO:4 or at least 40% identity to SEQ ID NO:4.

17. The method of claim 14, wherein the plant is selected from the group consisting of loblolly pine, radiata pine and Douglas fir.

18. The method of claim 14, wherein said line or batch is selected only if said predicted conversion potential exceeds 50 germinants per gram of tissue.

19. A method of producing somatic embryos of a coniferous plant, which method comprises: obtaining a batch of embryogenic tissue from a coniferous plant and maintaining said tissue on a tissue maintenance medium, measuring a level of expression of a gene of said tissue maintained on said maintenance medium, said gene being a gene whose expression correlates to an observed conversion ofthe tissue to somatic embryos according to an established correlation; if said level of expression correlates to a level of somatic embryo conversion at or higher than a predetermined value, inducing said embryogenic tissue to convert to somatic embryos; if said level of expression correlates to a level of somatic embryo conversion lower than said predetermined value, discarding said batch and obtaining another batch ofthe same line or another line of said coniferous plant, repeating said procedure until a batch exhibits a level of expression at or higher than said predetermined value is obtained; and inducing conversion of said tissue of said batch having said level of expression at or higher than said predetermined value to somatic embryos.

20. The method of claim 19, wherein the expression level ofthe gene in a line or batch ofthe tissue is measured by means of quantitative real time polymerase chain reaction or equivalent method.

21. The method of claim 19, wherein the gene contains a the DNA sequence of SEQ ID NO:4 or at least 40% identity to SEQ ID NO:4.

22. The method of claim 19, wherein the plant is selected from the group consisting of loblolly pine, radiata pine and Douglas fir.

23. The method of claim 19, wherein said line or batch is selected only if said predicted conversion potential exceeds 50 germinants per gram of tissue.

24. A method of producing somatic embryos of a coniferous plant, which method comprises: obtaining a batch of embryogenic tissue from a coniferous plant and maintaining said tissue on a tissue maintenance medium, measuring a level of expression of a gene of said tissue maintained on said maintenance medium, said gene being a gene identified by containing a DNA sequence according to SEQ ID NO: 4 or a sequence having at least 40% identity thereto; if said level of expression correlates to a level of somatic embryo conversion at or higher than a predetermined value, inducing said embryogenic tissue to convert to somatic embryos; if said level of expression correlates to a level of somatic embryo conversion lower than said predetermined value, discarding said batch and obtaining another batch ofthe same line or another line of said coniferous plant, repeating said procedure until a batch exhibits a level of expression at or higher than said predetermined value is obtained; and inducing conversion of said tissue of said batch having said level of expression at or higher than said predetermined value to somatic embryos.

25. The method of claim 24, wherein said coniferous plant is loblolly pine.

26. A method of identifying a plant family or genotype having good potential for conversion to embryos at a high rate during tissue culture, which method comprises: identifying a gene of a plant, the level of expression of which gene to a high conversion rate of plant tissue to somatic embryos, thus establishing a correlation between the level of expression ofthe gene in embryogenic tissue and rates of subsequent conversion ofthe tissue to embryos; testing at least one plant of a family or genotype to measure the level of expression ofthe gene in embryogenic tissue ofthe plant, and predicting the potential for conversion ofthe plant or genotype from the level of expression ofthe gene.

27. A polypeptide having the sequence of SEQ ID NO: 5 or a sequence having at least 60% similarity to SEQ ID NO:5.

28. A method of improving the rate of conversion of an embryonic tissue of a plant to somatic plant embryos, which comprises: establishing a mass of embryogenic tissue suitable for conversion to embryos; subjecting the mass of tissue to a cold treatment; and subsequently inducing the tissue to convert to somatic embryos.

29. The method of claim 28, wherein the cold treatment comprises cooling the mass of tissue to a temperature below about 8°C.

30. The method of claim 28, wherein the cold treatment comprises cooling the mass of tissue to a temperature in the range of 2 to 6°C for a period of time of at least about half an hour.

31. The method of claim 28, wherein said tissue exhibits a level of expression of a protein of SEQ ID NO: 5 prior to said cold treatment, said level being lower than a maximum level of expression detectable in other samples of embryogenic tissue ofthe same genotype.

32. The method of claim 28, wherein the plant is a conifer.

33. A method of identifying genotypes of plants having a predisposition to high conversion rates when embryogenic tissue of said plants is induced to convert to embryos during tissue culture, which method comprises: producing embryogenic tissue of said plants and maintaining said tissue on a maintenance medium, measuring the expression level of a gene of said plant that correlates to the rate of conversion of embryogenic tissue ofthe plant to somatic embryos; and identifying plants that have a disposition to high conversion rates based on said measurement ofthe expression level of said gene.

34. Plants of a genotype having a predisposition to high conversion rates when embryogenic tissue of said plants is induced to convert to embryos during tissue culture, said plants having been identified by a the method of claim 33.

35. A method of identifying a cell line of a plant having a predisposition to high conversion rates when induced to convert to embryos during tissue culture, which method comprises: maintaining said cell line on a maintenance medium, measuring an expression level of a gene of said plant that correlates to the rate of conversion of embryogenic tissue ofthe plant to somatic embryos; and identifying cell lines that have a disposition to high conversion rates based on said measurement ofthe expression level of said gene.

36. A cell line of a plant having a predisposition to high conversion rates when induced to convert to embryos during tissue culture, said cell line having been identified by a method according to claim 35.

37. A method of predicting embryo conversion potential of embryogenic tissue of plants, which comprises: for a particular line or batch of said tissue, measuring the expression product of a I gene while maintaining said tissue prior to conversion, said gene having an expression level in embryogenic tissue that has an established correlation to the subsequent conversion rate ofthe embryogenic tissue to somatic embryos, and from said established correlation, predicting the potential conversion rate ofthe embryogenic tissue ofthe line or batch.

38. The method of claim 37, wherein said expression product is a protein of SEQ ID NO: 5 or a protein having at least 60% homology to SEQ ID NO: 5.