Method for large scale generation of artificial seeds comprising symbiota

Abstract

The present invention relates to new methods of selecting and breeding organisms, in particular organisms which exhibit symbiotic behavior with symbionts such as fungal endophytes or epiphytes or bacterial microbiome in plants, and to new organisms and symbiota developed thereby. More particularly, the present invention provides artificial seeds comprising symbiota, and methods for preparing and using such artificial seeds, as well as plants, plant seeds and other plant parts derived from artificial seeds or symbiont-containing plants of the present invention.

Claims

1. An artificial seed, comprising: a plant embryo inoculated with one or more symbionts and subsequently coated with a first coating to encapsulate the embryo.

2. The artificial seed according to claim 1, wherein said artificial seed is further coated with a second coating layer.

3. The artificial seed according to claim 2, wherein said second coating layer includes added nutrients.

4. The artificial seed according to claim 2, wherein said second coating layer is a nutrient deprived layer.

5. The artificial seed according to claim 1, wherein the embryo is from a plant selected from the group consisting of grasses and legumes.

6. The artificial seed according to claim 1, wherein the symbiont is a fungal endophyte.

7. The artificial seed according to claim 1, wherein the embryo is treated to create one or more points of entry for the symbiont.

8. The artificial seed according to claim 1, wherein the first coating comprises calcium alginate.

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

(1) In the figures:

(2) FIG. 1 shows artificial seeds generated through Ca-alginate coating of perennial ryegrass embryos using a coating with Ca-alginate matrix without added nutrients.

(3) FIG. 2 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with coloured Ca-alginate matrix. Artificial seeds of perennial ryegrass coloured with Queen Green (90610); a) air-dried artificial seeds; b) artificial seeds plated on germination medium. Artificial seeds of perennial ryegrass coloured with Queen Pink (92330); c) air-dried artificial seeds; d) artificial seeds plated on germination medium.

(4) FIG. 3 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with multiple Ca-alginate matrix layers. a) Artificial seeds of perennial ryegrass coated with first coating (non-coloured) Ca-alginate layer (layer A) with added nutrients. b) Artificial seeds of perennial ryegrass coated with two (first layer A; non-coloured plus second layer B; Queen Green-coloured) Ca-alginate layers with added nutrients; c) double-coated artificial seeds placed on germination medium.

(5) FIG. 4 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with multiple Ca-alginate matrix layers. a)-c) Cross-sections of artificial seeds of perennial ryegrass coated with first coating (non-coloured) Ca-alginate layer (layer A) and second coating with Queen-Pink or Queen-Green coloured Ca-alginate layer (layer B). d)-e) Cross-sections of artificial seeds of perennial ryegrass coated with first coating (non-coloured) Ca-alginate layer (layer A) and second coating with Queen-Green coloured Ca-alginate layer (layer B).

(6) FIG. 5 shows germination of seeds, embryos and artificial seeds of perennial ryegrass cv. Bronsyn E (endophyte free, 2668 seed batch). a) Original seeds: 1% germination frequency on filter paper; b) Surface-sterilized seeds: 10% germination frequency on filter paper; c) Isolated embryos: 48% germination frequency on germination medium; d) Artificial seeds (with germination medium): 40% germination frequency on MS medium.

(7) FIG. 6 shows germination of seeds, embryos and artificial seeds of perennial ryegrass cv. Bronsyn E+ (endophyte plus, 2667 seed batch). a) Original seeds: 10% germination frequency on filter paper; b) Surface-sterilized seeds: 30% germination frequency on filter paper; c) Isolated embryos: 90% germination frequency on germination medium; d) Artificial seeds (with germination medium): 81% germination frequency on MS medium.

(8) FIG. 7 shows germination of artificial seeds and development of artificial-seed derived seedlings in perennial ryegrass.

(9) FIG. 8 shows freshly isolated seed-derived embryos of perennial ryegrass individually placed in wells of a) 96-well and b) endophyte mycelium suspension added to individual wells and allowed to partly air-dry under laminar flow prior to c) production of artificial seeds coated with Ca-alginate layer.

(10) FIG. 9 shows artificial seeds produced by method 1.

(11) FIG. 10 shows germinating artificial seeds produced by method 1.

(12) FIG. 11 shows artificial seeds produced by method 2.

(13) FIG. 12 shows artificial seeds produced by method 2 with endophyte outgrowth.

(14) FIG. 13 shows artificial seeds produced by method 3.

(15) FIG. 14 shows artificial seeds produced by method 3 with endophyte outgrowth.

(16) FIG. 15 shows germinating artificial seeds produced by method 3.

(17) FIG. 16 shows endophyte suspensions at different dilution rates.

EXAMPLE 1METHOD FOR LARGE-SCALE GENERATION OF GRASS-ENDOPHYTE SYMBIOTA (ARTIFICIAL SEEDS)

(18) The objective of the work was to develop an efficient, robust and low-cost method for large-scale production of grass endophyte symbiota. The method should be: a) applicable to inoculation of 10s-100s of endophyte in 100s-1000s of grass genotypes; b) applicable to perennial ryegrass, tall fescue and Brachiaria; and c) applicable to inoculation of novel and designer endophytes with de novo generated genetic variation [i.e. induced mutagenesis (ionizing radiation, colchicine), targeted mutagenesis, transgenesis, cisgenesis, intragenesis, etc.].

(19) The method should further enable next-generation ab initio molecular breeding, selection and evaluation of grass-endophyte symbiota [rather than breeding and selection of grass host followed by endophyte inoculation and symbiota evaluation only].

(20) The experimental strategiesand corresponding experimental stepsimplemented include:

(21) 1. Large-Scale Perennial Ryegrass Seed-Derived Embryo Isolation and Artificial Seed Production

(22) A. Develop an efficient, low-cost, large-scale seed surface-sterilization method;

(23) B. Develop an efficient, low-cost, large-scale seed-derived embryo isolation method;

(24) C. Develop an efficient, low-cost, large-scale artificial seed production method;

(25) D. Test germination frequency and germination stages of artificial seeds;

(26) E. Assess endophyte presence in seedlings derived from artificial seeds generated with embryos isolated from endophyte-plus seeds;

(27) 2. Large-Scale Endophyte Inoculation into Perennial Ryegrass Artificial Seeds

(28) F. Develop an efficient, low-cost, large-scale endophyte inoculation method for artificial seeds [based on seed-derived embryo inoculation with endophyte mycelium followed by artificial seed production including double/multiple coating (inner layer plus endophyte, outer layer as pseudo-aleurone/endosperm) of artificial seeds]; and

(29) G. Assess endophyte presence in seedlings derived from artificial seeds generated with embryos isolated from endophyte-minus seeds inoculated with novel endophytes.

(30) Large-Scale Perennial Ryegrass Seed-Derived Embryo Isolation and Artificial Seed Production

(31) A) Seed Surface Sterilization Method

(32) The seed surface sterilization method implemented includes the following steps:

(33) Day 1: seeds were soaked in 10% sulphuric acid overnight.

(34) Day 2: treated with 10% Domestos for 20 min and stored at 24 C after wash with distilled sterile water.

(35) Day 3: treated with 10% Domestos for 20 min and stored at 24 C after wash with distilled sterile water, followed by embryo isolation [see B) below].

(36) Four independent experiments were conducted with 200 seeds each.

(37) No bacterial or fungal contamination was observed.

(38) B) Embryo Isolation Method

(39) Based on the successful surface-seed sterilization method [see A) above], 1,000 ryegrass seed-derived embryos can be isolated by one person within 4 hours.

(40) Artificial Seed Production Method

(41) Ca-Alginate Coating of Perennial Ryegrass Embryos into Artificial Seeds

(42) i) Coating with Ca-Alginate Matrix without Added Nutrients

(43) For the Ca-alginate coating of perennial ryegrass embryos into artificial seeds using a coating with Ca-alginate matrix without added nutrients, the following steps were undertaken: Embryos were freshly isolated and mixed with 3% sodium alginate solution. Alginate drops were placed into 50 mM calcium chloride solution while stirring at 60 rpm. Each drop contains one embryo. Artificial seeds were collected after 15 min stirring and washed with sufficient distilled sterile water.

(44) Artificial seeds were placed on germination medium MS or MS+1 mg/L BAP.

(45) FIG. 1 shows artificial seeds generated through Ca-alginate coating of perennial ryegrass embryos using a coating with Ca-alginate matrix without added nutrients.

(46) ii) Coating with Ca-Alginate Matrix with Added Nutrients

(47) For the Ca-alginate coating of perennial ryegrass embryos into artificial seeds using a coating with Ca-alginate matrix with added nutrients, the following steps were undertaken: Embryos were freshly isolated and mixed with 3% sodium alginate in modified MS medium consisting of MS (without CaCl2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g/L MES. Alginate drops (containing individual embryos) were placed in 50 mM calcium chloride solution while stirring at 60 rpm. Each drop contains a single seed-derived isolated embryo. Artificial seeds were collected after 15 min stirring and thoroughly washed with distilled sterile water. Artificial seeds were placed on MS medium plates for germination.
iii) Coating with Coloured Ca-Alginate Matrix

(48) For the Ca-alginate coating of perennial ryegrass embryos into artificial seeds using a coating with coloured Ca-alginate matrix with added nutrients, the following steps were undertaken:

(49) Embryos were freshly isolated and mixed with 3% sodium alginate in modified MS medium consisting of MS (without CaCl2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g/L MES.

(50) Different food dyes [i.e. 10 L/ml Queen Green (90610) or Queen Pink (92330)] were added to the sodium alginate coating solution to colour coating matrix thus establishing basis to demonstrate potential for multi-layer coating.

(51) Alginate drops (containing individual embryos) were placed in 50 mM calcium chloride solution while stirring at 60 rpm.

(52) Each drop, contains a single seed-derived isolated embryo.

(53) Artificial seeds were collected after 15 min stirring and thoroughly washed with distilled sterile water.

(54) Artificial seeds were placed on MS medium plates for germination.

(55) FIG. 2 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with coloured Ca-alginate matrix.

(56) iv) Coating with Multiple Ca-Alginate Matrix Layers

(57) For the Ca-alginate coating of perennial ryegrass embryos into artificial seeds using a coating with multiple Ca-alginate matrix layers, the following steps were undertaken: Embryos were freshly isolated and mixed with 3% sodium alginate in modified MS medium [consisting of MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g/L MES] as the first coating layer (layer A) to make artificial seeds. Alginate drops (containing individual embryos) were placed in 50 mM calcium chloride solution while stirring at 60 rpm. Each drop contains a single seed-derived isolated embryo. Artificial seeds coated with layer A were collected after 15 min stirring and thoroughly washed with distilled sterile water. The average diameter of the artificial seed freshly coated with layer A is 4 mm. Artificial seeds coated with layer A were placed in Petri dish and allowed to air-dry for 1-2 hours in a laminar flow cabinet. The diameter of the air-dried artificial seed coated with layer A is 2 mm. Air-dried artificial seeds coated with layer A were mixed with 3% sodium alginate in modified MS medium [consisting of MS (without CaCl2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g/L MES] coloured with food dye [i.e. 10 L/ml Queen Green (90610)] as the second coating layer (layer B) to make double-coated artificial seeds; following the same procedure.

(58) FIG. 3 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with multiple Ca-alginate matrix layers.

(59) FIG. 4 shows Ca-alginate coating of perennial ryegrass embryos into artificial seeds using coating with multiple Ca-alginate matrix layers.

(60) Freshly isolated seed-derived embryos of perennial ryegrass are individually placed in wells of a) 96-well or b) 384-well plates. With the aid of a disposable syringe sodium alginate solution is added to the individual wells and single embryos in alginate solutions are loaded in the syringe. With the aid of the syringe individual embryos coated with alginate solution are dropped into polymerising CaCl2 solution under agitation for production of artificial seeds. The use of 96-well plate is preferred over the 384 well plate for production of artificial seeds of perennial ryegrass.

(61) Assessing Germination Frequency of Artificial Seeds

(62) In order to assess germination frequency of artificial seeds, the following steps were undertaken:

(63) Germination of Seeds, Embryos and Artificial Seeds of Perennial Ryegrass Cv. Bronsyn E.sup. (Endophyte Free, 2668 Seed Batch)

(64) Seed germination frequency was comparatively assessed for (FIG. 5):

(65) a) Original seeds: 1% germination frequency on filter paper;

(66) b) Surface-sterilized seeds: 10% germination frequency on filter paper;

(67) c) Isolated embryos: 48% germination frequency on germination medium;

(68) d) Artificial seeds (with germination medium): 40% germination frequency on MS medium.

(69) Germination of Seeds, Embryos and Artificial Seeds of Perennial Ryegrass Cv. Bronsyn E+(Endophyte Plus, 2667 Seed Batch)

(70) Seed germination frequency was comparatively assessed for (FIG. 6):

(71) a) Original seeds: 10% germination frequency on filter paper;

(72) b) Surface-sterilized seeds: 30% germination frequency on filter paper;

(73) c) Isolated embryos: 90% germination frequency on germination medium;

(74) d) Artificial seeds (with germination medium): 81% germination frequency on MS medium.

(75) FIG. 7 shows germination of artificial seeds and development of artificial-seed derived seedlings in perennial ryegrass.

(76) Assessing Endophyte Presence in Seedlings Derived from Artificial Seeds

(77) In order to assess endophyte presence in seedlings derived from artificial seeds, the following experiments were undertaken:

(78) Endophyte Presence in Seedlings Derived from Seeds and Artificial Seeds of Perennial Ryegrass Seed Cv. Bronsyn E+(Endophyte Plus, 2667 Seed Batch)

(79) Twenty seedlings of Bronsyn E+(2667) seeds germinated on filter paper were transferred to soil.

(80) Twenty five seedlings from germinated artificial seeds generated with Bronsyn E plus (2667) seed-derived embryos were transferred to soil. The embryos in artificial seeds were sterilized using 10% H.sub.2SO.sub.4 overnight treatment.

(81) Following 6 week grow-out of seedlings derived from seeds and artificial seeds, endophyte presence was assessed based on endophyte-specific SSR test.

(82) Twenty seedlings of Bronsyn E plus (2667; containing ST endophyte) seeds germinated on filter paper were transferred to soil, leading to 13 of 19 seedlings (68%) testing positive for ST endophyte presence in the endophyte-specific SSR test.

(83) Twenty five seedlings from germinated artificial seeds generated with Bronsyn E plus (2667) seed-derived embryos were transferred to soil. The embryos in artificial seeds were sterilized using 10% H.sub.2SO.sub.4 overnight treatment, leading to 19 of 23 seedlings (83%) testing positive for ST endophyte in the endophyte-specific SSR test, clearly indicating that the methods for seed surface sterilization, large-scale embryo isolation, and artificial seed production with Ca-alginate coating do not negatively affect viability of a resident endophyte.

(84) Large-Scale Inoculation of Endophytes in Perennial Ryegrass Artificial Seeds

(85) Different methods for the large-scale inoculation of endophytes in perennial ryegrass artificial seeds were developed, with examples of methods 1 to 3 described below:

(86) Inoculation of Isolated Seed-Derived Embryos with Endophyte Mycelium and Production of Endophyte-Infected Artificial Seeds in Perennial Ryegrass

(87) Freshly isolated seed-derived embryos of perennial ryegrass are individually placed in wells of a) 96-well and b) endophyte mycelium suspension added to individual wells and allowed to partly air-dry under laminar flow prior to c) production of artificial seeds coated with Ca-alginate layer (FIG. 8).

(88) Method 1: Direct Inoculation of Isolated Embryos with Endophyte Suspension Prior to Ca-Alginate Coating

(89) Method 1, inoculation of isolated seed-derived embryos with endophyte mycelium and production of endophyte-infected artificial seeds in perennial ryegrass, is based on direct inoculation of isolated embryos with endophyte suspension prior to Ca-alginate coating as follows:

(90) Freshly isolated embryos of perennial ryegrass are incubated with endophyte suspension (1/16 dilution) for 30 mins at RT in individual wells of 96-well plates.

(91) Inoculation suspension is removed from well and inoculated embryos are allowed to partly air-dry on filter paper disks.

(92) Artificial seeds are produced (FIG. 9) with endophyte-inoculated embryos with 3% sodium alginate-containing modified MS growth medium [MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP].

(93) Artificial seeds are allowed to germinate on MS medium for germination.

(94) Freshly isolated embryos of perennial ryegrass are directly inoculated with endophyte suspension (1/8 dilution), partly air-dried and then coated with Ca-m alginate in individual wells of 96-well plates.

(95) Artificial seeds from perennial ryegrass directly inoculated with endophyte and then coated with Ca-alginate layer are able to germinate on MS germination medium (FIG. 10).

(96) Method 2: Direct Coating of Isolated Embryos with Endophyte-Containing Ca-Alginate Layer

(97) Method 2, inoculation of isolated seed-derived embryos with endophyte mycelium and production of endophyte-infected artificial seeds in perennial ryegrass, is based on direct coating of isolated embryos with endophyte-containing Ca-alginate layer as follows:

(98) Embryos of perennial ryegrass are freshly isolated in endophyte suspension (1/16 dilution) in individual wells of 96-well plates.

(99) Two-fold concentration sodium alginate (6%) modified MS medium [MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP] is added to the individual wells to coat embryos with an endophyte-containing alginate layer.

(100) Artificial seeds are produced with endophyte-layer coated embryos (FIG. 11).

(101) Artificial seeds are allowed to germinate on MS medium for germination.

(102) Embryos of perennial ryegrass are freshly isolated and coated with endophyte suspension (1/8 or 1/16 dilutions) with Ca-alginate then added to generate an endophyte-containing alginate layer coating the embryos in individual wells of 96-well plates.

(103) Following culture, endophyte out-growth is observed from the endophyte-containing alginate layer used to coat the isolated embryos of perennial ryegrass (irrespectively of endophyte suspension dilution rate used; FIG. 12) demonstrating viability of the endophyte included in the Ca-alginate coating layer.

(104) Method 3: Double-Coating of Artificial Seeds Generated from Endophyte Inoculated Isolated Embryos

(105) Method 3, inoculation of isolated seed-derived embryos with endophyte mycelium and production of endophyte-infected artificial seeds in perennial ryegrass, is based on double-coating of artificial seeds generated from endophyte-inoculated isolated embryos as follows:

(106) Freshly isolated embryos of perennial ryegrass are coated with an endophyte suspension (1/16 dilution), mixed with alginate [6% Ca-alginate in modified MS medium (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP] to generate a first coating layer containing endophytes in individual wells of 96-well plates.

(107) Artificial seeds with a first endophyte-containing alginate layer coating freshly isolated embryos of perennial ryegrass are blot-dried on filter paper in laminar air flow for 30 mins and then coated with a second alginate layer of 3% Ca-alginate without any nutrients,

(108) Double-coated artificial seeds with endophyte-containing layer coated embryos of perennial ryegrass are then germinated on MS medium.

(109) Second coating with nutrient deprived medium of endophyte-inoculated artificial seeds aims to reduce endophyte out-growth during germination and restrict endophyte growth in close proximity to isolated perennial ryegrass embryo (FIG. 13).

(110) Artificial seeds with a first endophyte-containing alginate layer coating freshly isolated embryos of perennial ryegrass are blot-dried on filter paper in laminar air flow for 30 mins and then coated with a second alginate layer of 3% Ca-alginate without any nutrients.

(111) Endophyte growth is mainly restricted to inner alginate coating layer for a period of up to 3 weeks (FIG. 14).

(112) Embryos of perennial ryegrass are freshly isolated directly in endophyte suspension (1/8 dilution), then partly air-died and coated with a first alginate layer [3% Ca-alginate in modified MS medium (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP] in individual wells of 96-well plates.

(113) Artificial seeds with directly endophyte-inoculated embryos of perennial ryegrass are stored at 4 C overnight and then coated with a second alginate layer of 3% Ca-alginate without any nutrients.

(114) Double-coated artificial seeds with directly endophyte-inoculated embryos of perennial ryegrass are then germinated on MS medium.

(115) Double-coated artificial seeds with directly endophyte-inoculated embryos of perennial ryegrass germinated on MS medium show germination rates comparable to the original seed batch used for embryo isolation (FIG. 15).

(116) Assessing Endophyte Presence in Seedlings Derived from Artificial Seeds with Seed-Derived Embryos Inoculated with Novel Endophytes

(117) In order to assess endophyte presence in seedlings derived from artificial seeds with seed-derived embryos inoculated with novel endophytes (e.g. NEA11) using Method 1, the following experiment was undertaken:

(118) Endophyte Presence in Seedlings Derived from Artificial Seeds Produced with Embryos from Perennial Ryegrass Seed Cv. Bronsyn E (Endophyte Minus, 2668 Seed Batch) Inoculated with Novel Endophyte NEA11

(119) Following 6 week grow-out of seedlings derived from artificial seeds, endophyte presence was assessed based on endophyte-specific SSR test.

(120) Twenty-three seedlings from germinated artificial seeds generated with Bronsyn E minus (2668) seed-derived embryos inoculated with NEA11 using Method 1 were transferred to soil. 6 of 23 seedlings (i.e. 26%) tested positive for NEA11 endophyte presence in the endophyte-specific SSR test demonstrating the establishment of symbiota (Table 1). Endophyte presence in symbiota established from germinated artificial seeds generated with perennial ryegrass seed-derived embryos inoculated with novel endophyte NEA11 using Method 1 was confirmed following 3 months after transfer to soil.

(121) TABLE-US-00001 TABLE 1 Assessing Endophyte Presence in Seedlings Derived from Artificial Seeds with Seed-Derived Embryos Inoculated with Novel Endophytes SSR Marker NLESTA1QA09 NLESTA1NG03 NLESTA1CC05 Endophyte Seedling Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 detected 2668_4 153 184 226 167 NEA11 2668_15 153 184 226 167 NEA11 2668_1 153 184 226 167 NEA11 2_2 153 184 226 167 NEA11 2668 Bb1 153 184 226 167 NEA11 2668_13 153 184 226 167 NEA11
Large-Scale Inoculation of Designer Endophytes in Perennial Ryegrass Artificial Seeds

(122) Large-scale inoculation of designer endophytes derived from induced mutagenesis through colchicine-treatment (e.g. NEA12dh17) or derived from X-ray mutagenesis (e.g. IRM1-35) in perennial ryegrass artificial seeds is carried out using methods 1 to 3 described above.

(123) Freshly isolated embryos of perennial ryegrass are incubated with designer endophyte (e.g. NEA12dh17, IRM1-35) suspension (1/16 dilution) for 30 mins at RT in individual wells of 96-well plates.

(124) Inoculation suspension is removed from well and inoculated embryos are allowed to partly air-dry on filter paper disks.

(125) Artificial seeds are produced with designer endophyte-inoculated embryos with 3% sodium alginate-containing modified MS growth medium [MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP].

(126) Artificial seeds are allowed to germinate on MS medium for germination.

(127) Freshly isolated embryos of perennial ryegrass are directly inoculated with designer endophyte (e.g. NEA12dh17, IRM1-35) suspension (1/8 dilution), partly air-dried and then coated with Ca-alginate in individual wells of 96-well plates.

(128) Artificial seeds from perennial ryegrass directly inoculated with designer endophytes (e.g. NEA12dh17, IRM1-35) and then coated with Ca-alginate layer are able to germinate on MS germination medium leading to the establishment of symbiota. Designer endophyte presence and identity in the symbiota generated following large-scale inoculation of designer endophytes derived from induced mutagenesis through colchicine-treatment (e.g. NEA12dh17) or derived from X-ray mutagenesis (e.g. IRM1-35) in perennial ryegrass artificial seeds is demonstrated using an endophyte-specific SSR test.

(129) Large-Scale Inoculation of Transgenic Endophytes in Perennial Ryegrass Artificial Seeds

(130) Large-scale inoculation of transgenic endophytes derived from genetic transformation of NEA12 endophyte with plasmid containing a chimeric gene for expression of the DsRed fluorescent marker gene (e.g. NEA12-DsRed) in perennial ryegrass artificial seeds is carried out using method 1 described above.

(131) Freshly isolated embryos of perennial ryegrass are incubated with transgenic endophyte (e.g. NEA12-DsRed) suspension (1/16 dilution) for 30 mins at RT in individual wells of 96-well plates.

(132) Inoculation suspension is removed from well and inoculated embryos are allowed to partly air-dry on filter paper disks.

(133) Artificial seeds are produced with transgenic endophyte-inoculated embryos with 3% sodium alginate-containing modified MS growth medium [MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP].

(134) Artificial seeds are allowed to germinate on MS medium for germination.

(135) Freshly isolated embryos of perennial ryegrass are directly inoculated with transgenic endophyte (e.g. NEA12-DsRed) suspension (1/8 dilution), partly air-dried and then coated with Ca-alginate in individual wells of 96-well plates.

(136) Artificial seeds from perennial ryegrass directly inoculated with transgenic endophyte (e.g. NEA12-DsRed) and then coated with Ca-alginate layer are able to germinate on MS germination medium leading to the establishment of symbiota with transgenic endophytes. Transgenic endophyte presence and identity in the symbiota generated following large-scale inoculation of transgenic endophyte (e.g. NEA12-DsRed) in perennial ryegrass artificial seeds is demonstrated using an endophyte-specific SSR and transgene-specific PCR test.

(137) Large-Scale Inoculation of Novel Endophytes in Tall Fescue Artificial Seeds

(138) Large-scale inoculation of novel endophytes from tall fescue (e.g. NEA17, NEA19, NEA20) in tall fescue artificial seeds is carried out using method 1 described above.

(139) Freshly isolated embryos of tall fescue are incubated with novel fescue endophytes (e.g. NEA17, NEA19, NEA20) suspension (1/16 dilution) for 30 mins at RT in individual wells of 96-well plates.

(140) Inoculation suspension is removed from well and inoculated embryos are allowed to partly air-dry on filter paper disks.

(141) Artificial seeds are produced with novel endophyte-inoculated embryos with 3% sodium alginate-containing modified MS growth medium [MS (without CaCl.sub.2)+750 mg/L glutamine+5 M CuSO.sub.4+1.95 g MES+1 mg/l BAP].

(142) Artificial seeds are allowed to germinate on MS medium for germination.

(143) Freshly isolated embryos of tall fescue are directly inoculated with novel fescue endophytes (e.g. NEA17, NEA19, NEA20) suspension (1/8 dilution), partly air-dried and then coated with Ca-alginate in individual wells of 96-well plates.

(144) Artificial seeds from tall fescue directly inoculated with novel fescue endophytes (e.g. NEA17, NEA19, NEA20) and then coated with Ca-alginate layer are able to germinate on MS germination medium leading to the establishment of symbiota. Novel endophyte presence and identity in the symbiota generated following large-scale inoculation of novel fescue endophytes (e.g. NEA17, NEA19, NEA20) in tall fescue artificial seeds are demonstrated using an endophyte-specific SSR test.

EXAMPLE 2ENDOPHYTE INOCULATION METHOD IN PERENNIAL RYEGRASS

(145) This example describes enhancement of endophyte inoculation frequency following puncturing isolated embryos of perennial ryegrass with an hypodermic needle prior to inoculation using method 1 (direct inoculation) or method 2 (coating with endophyte containing Ca-alginate layer).

(146) Embryos isolated from perennial ryegrass seeds were inoculated with endophyte NEA11 using either methods 1 or 2, with endophyte suspensions at different dilution rates (1/4, 1/8, 1/16; see FIG. 16) subjected, with and without wounding of embryos with a hypodermic needle. Puncturing of embryos prior to inoculation greatly enhanced inoculation efficiency, demonstrated by SSR-based endophyte detection in 6 week old symbiota recovered from artificial seeds derived from inoculated embryos (see Table 2).

(147) TABLE-US-00002 TABLE 2 Number and frequency of endophyte-inoculated perennial ryegrass plants recovered following different endophyte inoculation treatment methods PDB Endophyte Detected Treatment Method conc Wounding NEA11 E- Inoculation % A 1 1/16 No 0 42 0 B 1 1/8 No 0 42 0 C 1 1/8 Puncture 11 29 27.5 D 2 1/16 No 0 42 0 E 2 1/8 No 0 42 0 F 2 1/8 Puncture 9 9 50 Method 1: Direct Inoculation of Isolated Embryos with Endophyte Suspension Prior to Ca-Alginate Coating Method 2: Direct Coating of Isolated Embryos with Endophyte-Containing Ca-Alginate Layer

EXAMPLE 3ENDOPHYTE INOCULATION METHOD IN PERENNIAL RYEGRASS AND TALL FESCUE

(148) This example describes enhancement of endophyte inoculation frequency following puncturing isolated embryos of perennial ryegrass (L. perenne) and tall fescue (F. arundinacea) with an hypodermic needle prior to inoculation using method 1 (direct inoculation) or method 2 (coating with endophyte containing Ca-alginate layer).

(149) Method 1: Direct Inoculation of Isolated Embryos with Endophyte Suspension Prior to Ca-Alginate Coating

(150) Method 2: Direct Coating of Isolated Embryos with Endophyte-Containing Ca-Alginate Layer

(151) Embryos isolated from seeds from different varieties were inoculated with different endophytes (NEA11 and NEA17) using either methods 1 or 2, with and without wounding of embryos with hypodermic needle. Puncturing of embryos prior to inoculation greatly enhanced inoculation efficiency, demonstrated by SSR-based endophyte detection in 6 week-old symbiota recovered from artificial seeds derived from inoculated embryos (see Table 3).

(152) TABLE-US-00003 TABLE 3 Number and frequency of endophyte-inoculated perennial ryegrass and tall fescue plants recovered following different endophyte inoculation treatment methods No. of artificial seeds No. of artificial seeds Method 1 Method 1 plus wounding Species Variety Experiment Endophyte Total Negative Positive Total Negative Positive L. perenne Alto 1 NEA11 (LpTG-2) 42 42 0 20 16 4 2 NEA11 (LpTG-2) 21 21 0 21 20 1 3 NEA11 (LpTG-2) 84 84 0 40 29 11 F. arundinacea Dovey 1 NEA11 (LpTG-2) 42 42 0 40 39 1 L. perenne Alto 1 NEA17 (FaTG-2).sup. 42 42 0 42 42 0 F. arundinacea Dovey 1 NEA17 (FaTG-2).sup. 70 70 0 35 35 0 Finesse 2 NEA17 (FaTG-2).sup. 42 42 0 70 70 0 No. of artificial seeds No. of artificial seeds Method 2 Method 2 plus wounding Species Variety Experiment Endophyte Total Negative Positive Total Negative Positive L. perenne Alto 1 NEA11 (LpTG-2) 84 84 0 18 9 9