AN IN VITRO RESCUE METHOD FOR IMMATURE EMBRYO OF AVOCADO

Abstract

Provided is an in vitro rescue method for immature embryo of avocado, and the method includes: collecting immature avocado hybrid fruits, stripping immature zygotic embryos from the sterilized fruits; inducing maturation of zygotic embryos in different mediums selected according to the maturities of zygotic embryos; and avocado seed embryos growing to normal plants under light conditions after germination. Provided is a technology for the in vitro culture rescue of immature embryo of avocado, wherein the zygotic embryos at different developmental stages are induced to maturation under in vitro conditions, and after maturation, the seed embryos are induced to germinate and form healthy plants. The technology of the present invention can effectively improve the survival rate of hybrid avocado zygotic embryo, increase the success rate of hybrid avocado, rescue the hybrid offspring, reduce the loss of hybrid offspring, ensure the genetic integrity of hybrid group, and provide the key technology for hybrid breeding and genetic research of avocado, etc.

Claims

1. An in vitro rescue method for immature embryo of avocado, characterized in that the said method comprises: (1) pre-treatment of explants collecting immature fallen fruits of avocado, stripping immature zygotic embryos from the sterilized fallen fruits; selecting the zygotic embryos with maturity below 80%, increasing the maturity to 80-100% by accompanying culture, and then performing a germination culture. (2) preparing a basal medium, accompanying mediums and a germination medium according to the dispensations, wherein the dispensation of the basal medium is: TABLE-US-00006 Concentrations Ingredients of basal medium (mg/L) (mg/L) Macroelements: Potassium nitrate KNO.sub.3 1600-2200 Ammonium nitrate NH.sub.4NO.sub.3 1500-1800 Potassium dihydrogen phosphate KH.sub.2PO.sub.4 150-180 Magnesium sulfate MgSO.sub.4•7H.sub.2O 350-400 Calcium chloride CaCl.sub.2•2H.sub.2O 420-460 Trace elements: Potassium iodide KI 0.60-0.9  Boric acid H.sub.3BO.sub.3 2.0-4.0 Manganese sulfate MnSO.sub.4•4H.sub.2O  8-12 Zinc sulfate ZnSO.sub.4•7H.sub.2O 1.5-2.5 Sodium molybdate Na.sub.2MoO.sub.4•2H.sub.2O 0.20-0.30 Cobalt chloride CoCl.sub.2•6H.sub.2O 0.020-0.30  Cupric sulfate CuSO.sub.4•5H.sub.2O 0.020-0.30  Iron salt: Ethylenediaminetetraacetic disodium 35-40 salt acid Na.sub.2•EDTA Ferrous sulfate FeSO.sub.4•7H.sub.2O 25-30 Organic Inositol  80-120 composition: Nicotinic acid VB5 0.8-1.2 Pyridoxine hydrochloride VB6 0.8-1.2 Thiamine hydrochloride VB1 0.8-1.2 the dispensations of accompanying mediums a, b, c, d, e and f are as follows a. Half-strength basal medium 300 to 500 mg/L casein hydrolysate 8-12% coconut water 8 to 12% sucrose 1 to 3 g/L agar powder; and b. Half-strength basal medium 300˜500 mg/L glutamine 8-12% coconut water 8 to 12% sucrose 1-3 g/L agar powder c. Half-strength basal medium 300-500 mg/L casein hydrolysate 8-12% coconut water 4-6% sucrose 2-4 g/L agar powder; and d. Half-strength basal medium 300-500 mg/L glutamine 8-12% coconut water 4-6% sucrose 2-4 g/L agar powder. e. Basal medium 300-500 mg/L casein hydrolysate 8-12% coconut water 2-4% sucrose 4-6 g/L agar powder; and f. Basal medium 300-500 mg/L glutamine 8-12% coconut water 4-6% sucrose 4-6 g/L agar powder; adding each component other than coconut water to the vessel respectively, stirring, and then adjusting the pH value of each medium to 5.5-6.5; after sterilizing and cooling the mediums, adding the filtered and sterilized coconut water to the cooled and sterilized mediums to obtain the accompanying mediums a, b, c, d, e, and f. The dispensation of the germination medium is Basal medium 2.0-4.0 μM 6-benzylaminopurine 4-8 g/L agar powder 2.5-3.5% sucrose. (3) selection of initial accompanying mediums selecting the appropriate dispensations of the accompanying mediums according to the different maturities of the zygotic embryos; for seed embryos within 100 days after pollination, selecting either of the accompanying medium a or b as the initial accompanying medium; for seed embryos between 100 and 200 days after pollination, selecting either of accompanying medium c or d as the initial accompanying medium; for seed embryos after 200 days after pollination, either of accompanying medium e or f is selected as the initial accompanying medium; (4) accompanying culture of zygotic embryos and selection and replacement of the accompanying mediums; during the pre-heart-shape stage of the zygotic embryos, the accompanying medium a orb is used for accompanying culture; when the zygotic embryos are cultured into the torpedo stage, the accompanying medium is replaced by the accompanying medium c or d for accompanying culture. When the zygotic embryo is cultured into the maturation stage, the accompanying medium is replaced by the accompanying medium e or f for accompanying culture; after the zygotic embryos are cultured to maturation stage, they are cultured in the germination medium for germination culture; The accompanying culture is carried out under dark conditions; (5) Germination culture; the seed embryos obtained from the accompanying culture are put into the germination medium and germination culture is carried out at 25° C.±1° C.; light culture is carried out during the germination culture until the seed embryos are cultured into normal rooted seedlings.

2. The method according to the claim 1, characterized in that in the step (1), the surface of the fallen fruits and the seeds are sterilized.

3. The method according to the claim 2, characterized in that the said sterilization treatment comprises: soaking in a 0.5 v/v % sodium hypochlorite solution followed by rinsing in sterile distilled water for three times; the said 0.5 v/v % sodium hypochlorite solution further containing 10 drops/L of Tween 20.

4. The method according to the claim 1, characterized in that, in the step (2), the said dispensation of the germination medium is formulated as: basal medium: 2.22 μM 6-BA; 6 g/L agar powder; 2.5-3.5% sucrose; wherein the dispensation of said basal medium is formulated as: TABLE-US-00007 concentrations Ingredients of basal medium (mg/L) (mg/L) Macroelements: Potassium nitrate KNO.sub.3 1900 Ammonium nitrate NH4NO.sub.3 1650 Potassium dihydrogen phosphate KH.sub.2PO.sub.4 170 Magnesium sulfate MgSO.sub.4•7H.sub.2O 370 Calcium chloride CaCl.sub.2•2H.sub.2O 440 Trace elements: Potassium iodide KI 0.75 Boric acid H.sub.3BO.sub.3 3.0 Manganese sulfate MnSO.sub.4•4H.sub.2O 10 Zinc sulfate ZnSO.sub.4•7H.sub.2O 2.0 Sodium molybdate Na.sub.2MoO.sub.4•2H.sub.2O 0.25 Cobalt chloride CoCl.sub.2•6H.sub.2O 0.025 Cupric sulfate CuSO.sub.4•5H.sub.2O 0.025 Iron salt: Ethylenediaminetetraacetic acid disodium 37.3 salt Na.sub.2•EDTA Ferrous sulfate FeSO.sub.4•7H.sub.2O 27.8 Organic Inositol 100 composition: Nicotinic acid VB5 1.0 Pyridoxine hydrochloride VB6 1.0 Thiamine hydrochloride VB1 10

5. The method according to claim 4, characterized in that in the step (2), the dispensations of the accompanying mediums a, b, c, d, e, f are as follows. a. Half-strength basal medium 400 mg/L casein hydrolysate 10% coconut water 10% sucrose 2 g/L agar powder; and b. Half-strength basal medium 400 mg/L glutamine 10% coconut water 10% sucrose 2 g/L agar powder; c. Half-strength basal medium 400 mg/L casein hydrolysate 10% coconut water 5% sucrose 3 g/L agar powder; and d. Half-strength basal medium 400 mg/L glutamine 10% coconut water 5% sucrose 3 g/L agar powder. e. Basal medium 400 mg/L casein hydrolysate 10% coconut water 3% sucrose 5 g/L agar powder; and f. Basal medium 400 mg/L glutamine 10% coconut water 3% sucrose 5 g/L agar powder;

6. The method according to the claim 1, characterized in that, in the step (3), for the seed embryos within 100 days after pollination with a poor effect of the initial accompanying culture selected or a poor development, after stripping the seeds of their seed coats and sterilizing them, keeping the seeds growing at a temperature of 25° C.±1° C. in a pre-culture medium, with 16 hours of light irradiation during the growth, and continuing the rescue of immature embryos after the seeds return to normal growth. wherein the dispensation of the pre-culture medium is formulated as: half-strength basal medium: 2.0-2.5 μM 6-benzylaminopurine; 4-8 g/L agar powder; 2.5-3.5% sucrose;

7. The method according to the claim 1, characterized in that in the step (3), the pH value of the medium is adjusted to 5.74 using an acid reagent; preferably, the said acid reagent is hydrochloric acid.

8. The method according to the claim 1, characterized in that, in the step (3), the said sterilization condition is autoclaving at 121° C. and 0.1 MPa for 15-20 minutes.

9. The method according to the claim 1, characterized in that in the step (5), the conditions of the said light culture are: irradiation by Grolux lamps to ensure plant growth; the irradiation intensity is 40 mmol.Math.m.sup.−2.Math.s.sup.−1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0095] FIG. 1 is a schematic view of the flow of one embodiment of the method of the present invention.

[0096] FIG. 2 is a schematic view of the process of another embodiment of the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention is further described below by specific embodiments.

[0098] Provided is a rescue method for immature embryo of avocado, and the said method comprising the steps as following:

(1) Pre-Treatment of Explants

[0099] For materials of physiological fruit drop, materials of other shedding and the materials that need embryo rescue, preferably performing the embryo rescue within 48 hours of fruit drop or fruitage falling, which helps increase the success rate of rescue.

[0100] After harvest, the fruits were sterilized on the surface by immersion in the 0.5% (v/v) sodium hypochlorite solution containing 10 drops/L of Tween 20 for 10 min, followed by rinsing in the sterile distilled water for three times. By sterilizing the fallen fruit and the seed embryos, to ensure a sterile state of the seed embryos, and ascertain the successful rescue of the seed embryos and correct results.

[0101] The sterilized fruits were cut longitudinally under aseptic conditions; the seeds were taken out and the fruits were sterilized on the surface by immersion in 0.5% (v/v) sodium hypochlorite solution for 10 minutes, followed by rinsing in sterile distilled water for three times. The sterilized seeds were carefully stripped of the immature zygotic embryos.

(2) Embryo Basic Maturation Culture Method

[0102] The maturity of the zygotic embryos is judged from practical experiences. Direct germination culture is performed on the zygotic embryos with 80%-100% embryo maturity. Put the zygotic embryos with 80%-100% embryo maturity into the germination medium for germination culture, and choose the intensity of germination medium according to the development status of avocado and embryo maturity; the medium intensity is chosen according to the different development status of avocado and embryo maturities; a liquid medium is chosen if the maturity is low, and a semi-solid medium is chosen if the maturity is moderate, meanwhile, according to the embryo development status, the culture medium is gradually changed from the liquid medium to the semi-solid culture medium and finally to solid culture medium.

[0103] Zygotic embryos with embryo maturity of 80% or less are the objects to be rescued.

[0104] Zygotic embryos with 80% or less embryo maturity were selected and cultured to 80-100% maturity, and the germination culture is performed next.

[0105] The mediums to be used for accompanying culture to germination culture include: (1) basal medium, (2) germination medium and (3) accompanying medium.

[0106] The dispensations of the three mediums listed above and the methods of preparation are described below.

[0107] The dispensation of the germination mediums is:

Basal Medium:

[0108] 2.22 μM 6-BA;

[0109] 6 g/L agar powder;

[0110] 3% sucrose;

[0111] wherein the dispensation of the basal medium is

TABLE-US-00003 concentration Ingredients of basal medium (mg/L) (mg/L) Macroelements: Potassium nitrate KNO.sub.3 1900 Ammonium nitrate NH.sub.4NO.sub.3 1650 Potassium dihydrogen phosphate KH.sub.2PO.sub.4 170 Magnesium sulfate MgSO.sub.4•7H.sub.2O 370 Calcium chloride CaCl.sub.2•2H.sub.2O 440 Trace elements: Potassium iodide KI 0.75 Boric acid H.sub.3BO.sub.3 3.0 Manganese sulfate MnSO.sub.4•H.sub.2O 10 Zinc sulfate ZnSO.sub.4•7H.sub.2O 2.0 Sodium molybdate Na.sub.2MoO.sub.4•2H.sub.2O 0.25 Cobalt chloride CoCl.sub.2•6H.sub.2O 0.025 Cupric sulfate CuSO.sub.4•5H.sub.2O 0.025 Iron salt: Ethylenediaminetetraacetic acid disodium 37.3 salt Na.sub.2•EDTA Ferrous sulfate FeSO.sub.4•7H.sub.2O 27.8 Organic Inositol 100 composition: Nicotinic acid VB5 1.0 Pyridoxine hydrochloride VB6 1.0 Thiamine hydrochloride VB1 10

[0112] Note: In the above table, iron salts were obtained in tissue culture by firstly dissolving disodium ethylenediaminetetraacetate (Na 2-EDTA) and ferrous sulfate FeSO.sub.4-7H.sub.2O respectively, and then mixing with each other in the preparation.

[0113] {circle around (1)}Choosing the accompanying medium according to different embryo maturations a total of 120 seed embryos within 100 days after pollination, 100 d-200 d after pollination and 200 d after pollination were selected from the fallen seeds of avocado, and the following mediums were respectively used for culture.

[0114] The dispensations of the accompanying mediums a, b, c, d, e, and f and the principles of selection are as follows.

[0115] For the seed embryos within 100 days after pollination, either of the accompanying medium a or b is selected for the accompanying culture.

a. Half-Strength Basal Medium

[0116] 400 mg/L casein hydrolysate

[0117] 10% coconut water

[0118] 10% sucrose

[0119] 2 g/L agar powder; and

b. Half-Strength Basal Medium

[0120] 400 mg/L glutamine

[0121] 10% coconut water

[0122] 10% sucrose

[0123] 2 g/L agar powder

[0124] For seed embryos at 100 to 200 days after pollination, either of the accompanying medium c or d is selected for accompanying culture.

c. Half-Strength Basal Medium

[0125] 400 mg/L casein hydrolysate

[0126] 10% coconut water

[0127] 5% sucrose

[0128] 3 g/L agar powder; and

d. Half-Strength Basal Medium

[0129] 400 mg/L glutamine

[0130] 10% coconut water

[0131] 5% sucrose

[0132] 3 g/L agar powder.

[0133] For seed embryos after 200 days after pollination, either of the accompanying medium e or f is selected for accompanying culture.

e. Basal Medium

[0134] 400 mg/L casein hydrolysate

[0135] 10% coconut water

[0136] 3% sucrose

[0137] 5 g/L agar powder; and

f. Basal Medium

[0138] 400 mg/L glutamine

[0139] 10% coconut water

[0140] 3% sucrose

[0141] 5 g/L agar powder.

[0142] The distributions of the dispensations for the 120 seed embryos and their accompanying mediums are shown in Table 1 below.

TABLE-US-00004 TABLE 1 Dispensation of Number of accompanying Embryo maturity embryos/granule medium within 100 days after 20 a pollination 20 b between 100 d-200 d after 20 c pollination 20 c after 200 d after 20 e pollination 20 f

(3) Preparation of Culture Solution

[0143] Adjusting the pH value of all the above accompanying mediums to 5.5-6.5 with hydrochloric acid, autoclaving the accompanying mediums at 121° C. and 0.1 MPa for 15-20 min; adding coconut water to the cooled and sterilized accompanying medium after cooling the accompanying mediums.

(4) Selection and Replacement of the Accompanying Mediums

[0144] In the pre-heart-shape stage of embryo culture, choosing medium a or b, after the embryos were cultured into the torpedo stage, choosing medium c or d, and after maturity stage choosing medium e or f.

[0145] After 2-3 weeks of using the corresponding accompanying medium for each maturity, replacing the medium with a new one, or the accompanying medium being replaced to a accompanying medium for the next maturity according to the maturity of the seed embryos until the seed embryos mature.

[0146] The accompanying culture should be performed at 25° C.±1° C. with darkness or low light.

(5) Germination Culture

[0147] When the seed embryos in the accompanying culture reached the germination period, the seed embryos were placed in the germination medium for cultivation. Meanwhile, cotyledons on the accompanying medium were partially removed to induce the germination of the embryos, and the medium was replaced at an interval of 2-3 weeks.

[0148] In the germination culture, the seed embryos were kept growing at a temperature of 25° C.±1° C. Germination culture was carried out under light conditions with a 16 h photoperiodic incubation during the germination culture. The specific operations were: irradiation by Grolux lamp with an irradiation intensity of 40 mmol.Math.m.sup.−2.Math.s.sup.−1; the irradiation intensity is 1 KLux=18 μmol.Math.m.sup.−2.Math.s.sup.−1 under solar light source during the daytime, to promote the growth of the plants until they were cultured into normal rooted seedlings.

TABLE-US-00005 TABLE 2 Summary of seed embryo rescue results of avocado Dispensation of Number of accompanying Embryo maturity embryos/granule medium rescue results within 100 days after 20 a 75% pollination 20 b 76% between 100 d-200 d after 20 c 90% pollination 20 c 90% after 200 d after 20 e 95% pollination 20 f 96%

[0149] As is seen from Table 2, the success rate of rescue of the seed embryos with lower maturity (e.g., within 100 days after pollination) is lower than that of the seed embryos with higher maturity. For the seed embryos pollinated for more than 200 days with higher maturity, the success rate of rescue could exceed 95%, and the rescue and recovery of most seed embryos are achieved.

[0150] Through the method of the present invention, the fruit fallen in the process of hybridization culture of avocado can be rescued, and the recovery and effective use of the fallen seeds of avocado are realized, and the loss of hybridization results are reduced. The technology of the present invention can effectively improve the survival rate of zygotic embryos of hybrid avocado, increase the success rate of avocado hybridization, rescue the hybrid offspring, reduce the loss of hybrid offspring, ensure the genetic integrity of the hybrid group, and provide key technology for avocado hybrid breeding and genetic research, etc.