IN VITRO CULTURE METHOD TO INCREASE THE BIOMASS AND INCREASE THE NUMBER OF VARIETY PLANTS AND PRUNUS SPP INJERT HOLDER, PARTICULARLY PRUNUS AVIUM

Abstract

The present invention is an in vitro culture method to increase both the biomass and the number of seedlings in a number varieties and rootstock types of Prunus spp., particularly Prunus avium, and the use of said culture method, which is a useful means of micropropagating in vitro any variety and rootstock type of Prunus spp., in particular Prunus avium.

Claims

1. An in vitro culture method to increase the biomass and the number of seedlings of different varieties and rootstocks of Prunus spp. CHARACTERIZED by including the following stages: a) Collecting shoots from the donor plant, b) Washing the shoots with water for 10 minutes and disinfecting them with a 50% commercial bleach solution at for 20-40 minutes, c) Submerging the shoots in a pesticide for 20-40 minutes, d) Submerging the shoots in an indolebutyric acid solution (IBA) for 20-40 minutes, e) Collecting the buds and introducing them into a temporary immersion system made up of media A and B, wherein immersions are performed 2 and 8 times per day with a duration of 1 to 6 minutes for the duration of the 10-20 days culture period. f) The in vitro seedlings are obtained after the require culture period.

2. In vitro culture method to increase the biomass and the number of seedlings of different varieties and rootstocks of Prunus spp. Pursuant to claim No. 1 CHARACTERIZED because on stage e), the temporary immersion system contains a medium B, which consists of a modified DKW base medium with a concentration of 0.001 to 0.1 mg/L of indole butyric acid (IBA), 0.1 to 3 mg/L of 6-benzylaminopurine (BAP), 25 gr/L of sucrose, 0.5 gr/L of ascorbic acid and 0.1 gr/L of inositol.

3. In vitro culture method to increase the biomass and the number of different varieties and rootstocks of Prunus spp Pursuant to claim No. 1 CHARACTERIZED because on stage e), the temporary immersion system contains a medium A, which consists of a modified DKW base medium with 0.001 to 0.01 mg/L of indole butyric acid (IBA), 0.1 mg/L of 6-benzylaminopurine (BAP), 25 gr/L of sucrose, 0.5 gr/L of ascorbic acid and 0.1 gr/L of inositol.

4. In vitro culture method to increase the biomass and the number of seedlings of different varieties and rootstocks of Prunus spp. Pursuant to claim No. 1 CHARACTERIZED because on stage e), the temporary immersion system contains a medium A, which consists of a modified DKW base medium with 0.001 to 0.01 mg/L of indole butyric acid (IBA), 0.6 mg/L of 6-benzylaminopurine (BAP), 25 gr/L of sucrose, 0.5 gr/L of ascorbic acid and 0.1 gr/L of inositol.

5. Use of the in vitro culture method to increase the biomass and the number of seedlings of different varieties and rootstocks of Prunus spp. Pursuant to claim No. 1 CHARACTERIZED by its usefulness to micropropagate in vitro any variety and rootstock of Prunus spp.

6. Use of the in vitro culture method to increase the biomass and the number of seedlings of different varieties and rootstocks of Prunus spp. Pursuant to claim No. 1 CHARACTERIZED by its usefulness to micropropagate in vitro Prunus avium.

Description

DESCRIPTION OF FIGURES

[0026] FIG. 1. Schematic figure of the immersion system. Immersion system scheme for the in vitro culture of different varieties of Prunus spp buds in 150 mL flasks containing 40 mL of propagation medium.

[0027] FIG. 2. Pictures of the initial material of segmented explants and effect on their growth when included in a temporal immersion system. Panel A shows the initial material for each Prunus spp variety; panel B shows the segmented explants from the initial material to be included in the temporary immersion system; panel C shows the explants after their exposure for 14 days in the temporary immersion system.

[0028] FIG. 3. Chart for multilevel parameters analysis immersion number (N), immersion time (T), and culture medium (M). Panel A shows the multilevel analysis for the different varieties considering the biomass production (Qx) as a parameter in relation to the number of immersions. Panel B shows the multilevel analysis for the different varieties considering the number of plants produced (Px) as a parameter in relation to the number of immersions.

[0029] FIG. 4. Chart for biomass production for the variety based on the immersion parameters applied. The chart shows the biomass production results for each variety (Maxma 14, Colt, Van and Rainier) in relation to the different immersion parameters, where T is the immersion time, N the number of immersions and m the type of culture medium, which could be culture medium A and culture medium B.

[0030] FIG. 5. Chart for number of seedlings produced for the variety based on the immersion parameters applied. The chart shows the seedlings production number results for each variety (Maxma 14, Colt, Van and Rainier) as per the different immersion parameters, where T is the immersion time, N the number of immersion and m the type of culture medium, which could be culture medium A and culture medium B.

[0031] FIG. 6. Chart of sucrose consumption for the variety based on the immersion parameters applied. The chart shows the sucrose consumption level results for each variety (Maxma 14, Colt, Van and Rainier) as per the different immersion parameters, where T is the immersion time, N the number of immersion and m the type of culture, medium which could be culture medium A and culture medium B.

DESCRIPTION OF THE INVENTION

[0032] The current invention refers to an in vitro culture method to increase both the biomass and the number of seedlings of different Prunus spp varieties and rootstocks, particularly Prunus avium.

[0033] The in vitro culture method involves the following stages: [0034] a) Collecting shoots from the donor plant, [0035] b) Washing the shoots with water for 10 minutes and disinfecting them with a 50% commercial bleach solution for 20-40 minutes, [0036] c) Submerging the shoots in a pesticide for 20-40 minutes, [0037] d) Submerging the shoots in an indolebutyric acid solution (IBA) for 20-40 minutes, [0038] e) Collecting the buds and introducing them into a temporary immersion system made up of media A and B, wherein immersions are performed 2 and 8 times per day with a duration of 1 to 6 minutes for the duration of the 10-20 days culture period. [0039] f) The in vitro seedlings are obtained after the required culture period.

[0040] In this invention, whenever reference is made to the varieties and species of the Prunus genus, they correspond, but are not limited to: Prunus accumulans, Prunus africana, Prunus amplifolia, Prunus amigdaloides, Prunus amygdalus, Prunus annularis, Prunus argentea, Prunus besseyi, Prunus brachybotrya, Prunus brachypetala, Prunus brasiliensis, Prunus brittoniana, Prunus buxifolia, Prunus capollin, Prunus cerasifera, Prunus cercocarpifolia, Prunus chamissoana, Prunus cornifolia, Prunus debilis, Prunus detrita, Prunus divaricata, Prunus douglasii, Prunus emarginata, Prunus erythroxylon, Prunus espinozana, Prunus fasciculata, Prunusferganica, Prunus ferruginea, Prunus fortunensis, Prunus gentryi, Prunus glandulosa, Prunus guanaiensis, Prunus hainanensis, Prunus havardii, Prunus herthae, Prunus hintonii, Prunus huantensis, Prunus integrifolia, Prunus lanata, Prunus laurocerasus, Prunus leiocarpa, Prunus lichoana, Prunus ligus trina, Prunus lundelliana, Prunus mexicana, Prunus microphylla, Prunus moritziana, Prunus myrtifolia, Prunus nachichevanica, Prunus oblonga, Prunus oleifolia, Prunus omissa, Prunus opaca, Prunus ovalis, Prunus padifolia, Prunus pleiantha, Prunus prunifolia, Prunus pumila, Prunus punctata, Prunus ravenii, Prunus rhamnoides, Prunus rotunda, Prunus rufa, Prunus ruiziana, Prunus samydoides, Prunus sellowii, Prunus serotina, Prunus simonii, Prunus skutchii, Prunus spinosa, Prunus stipulata, Prunus subcoriacea, Prunus subcorymbosa, Prunus tetradenia, Prunus trichopetala, Prunus tucumanensis, Prunus ulei, Prunus urotaenia, Prunus ussuriensis, Prunus valida, Prunus virens, Prunus wurdackii.

[0041] In particular, it refers to Prunus avium, Prunus armeniaca, Prunus persica, Prunus mahaleb, Prunus insititia, Prunus dulcis, Prunus cerasus Prunus salicina and Prunus domstica.

[0042] The scope of this invention considers furthermore the varieties of Prunus, which could be, but are not limited to: Early burlat, Brooks, 210, Moreau, Tulare, Lapins, Rainer, 719, Garnet, Sweet Georgia, Black republican, Van, SHG, Sommerset, Vanda, Kordia, Stella, Hartland, Bing 260, Bing wab 13, Ruby, Van compact, Sunset bing, Black Tartarian, Sylvia, Sweet heart, NY 7690, Techlova, Karina, Summit, Regina, Celeste, Compact Stella, Napoleon, New star, Zirat 900, Sam, Hedelfingen, Sunburst, Lambert, Schmidt or Schneider.

[0043] Particularly, in stage c) of this method, the pesticide to use can be any pesticide capable of eliminating bacterial and fungal endogenous pathogens present in the explants; the preferred pesticide is benomile.

[0044] In stage 3) of the proposed method, the buds are collected and introduced in a temporary immersion system made up of the medium A and B, more details are included below:

TABLE-US-00001 Medium A Medium B DKW base medium modified with DKW base medium modified with 0.1-0.001 mg/L of indole 0.10 mg/L of indole butyric acid butyric acid (IBA), 0.1-3 mg/L (IBA), 0.6 mg/L of 6- of 6-benzylaminopurine (BAP), benzylaminopurine (BAP), 25 gr/L 25 gr/L of sucrose, 0.5 gr/L of of sucrose, 0.5 gr/L of ascorbic acid and 0.1 gr/L of ascorbic acid and 0.1 gr/L of inositol inositol

[0045] The immersion system includes the culture media A and B, whichon the basis of a temporal immersion protocol consisting in 2 to 8 immersions per day lasting 1 to 6 minutes during the 10 to 20 culture days periodempower the growth of different Prunus genus varieties and rootstocks. The system activates in an automatic and controlled manner the output of the culture medium for the number of times and durations already indicated. We considered a total volume between 50 mL and 2 L for each culture medium.

[0046] After the bud incubation of 10 to 20 [days] in the temporal immersion system, it is possible to obtain in vitro seedlings.

[0047] To determine the seedlings' growth, it is suggested to evaluate the sucrose consumption, biomass production, and number of generated seedlings.

Examples of the Application

Example 1: In Vitro Culture Method to Increase the Biomass and Increase the Number of Seedlings from Prunus avium Genotypes in Comparison with the Micropropagation Protocol in Solid Medium

[0048] In this example, the best parameters and conditions to use in the temporal immersion bioreactor to obtain the greater growth or biomass increase in the following Prunus genotypes are presented: MAXMA-14 and Colt rootstock, and the Van and Rainier varieties. Concurrently with this, we compared the performance from different immersion protocols in relation to a micro propagation protocol from the varieties in a solid culture medium.

[0049] First, tissue portions from the genotypes submitted were collected, which were then washed with water for 10 minutes. Subsequently, the shoots were disinfected with 50% commercial bleach for 20 minutes, they were immersed in a 2% benomil solution for 20 minutes, and then stored at a temperature of 4 C. for 900 hours. Before planting the plant tissue, it was submerged in a solution with 1000 ppm of indolebutyric acid (IBA) for 20 minutes, and then planted in chambers at a temperature of 24 C. From their growth, the buds were collected and introduced in the in vitro culture using 150 mL flasks containing 40 mL of propagation medium. The temporal immersion system consisted of independent explants organized in a system of 10 bioreactors in a parallel setting.

[0050] The experiment was performed using a set of 100 seedlings of each genotype as the supply of explants: For this we evaluated the following immersion parameters: [0051] Immersion time (T): 1 and 3 minutes. [0052] Number of immersions (N): protocols of 2 and 4 immersions. [0053] Culture medium (m): Culture medium A (0.01 mg/L IBA, 0.1 mg/L de BAP) and B (0.01 mg/L IBA, 0.6 mg/L BAP).

[0054] The explants used correspond to segmented explants from the initial material (FIG. 2, panels a and b). The explants in the temporary immersion system were withdrawn after 14 days of exposure, and it was observed that they had achieved a significant growth (FIG. 2, panel c).

[0055] To determine the performance of the explants exposure from the different varieties, we determined the parameters for sucrose consumption, the final biomass by weighing the material produced, and the number of resulting plants based on a recount. For the specific case of the sucrose consumption of the explants, we used the Kit Sucrose Assay (Sigma-Aldrich) following the manufacturer's instructions.

[0056] For the analysis of results, we applied multilevel factorial analysis, by means of which the best responses regarding the number of plants (PX) and the biomass (Qx) were determined.

[0057] Results of the Performance of the Immersion Parameters Evaluated

[0058] When the biomass (Qx) production was studied, the most important variable turned out to be the number of immersions.

[0059] When applying a total of 4 times of immersion for 1 minute and using the medium B as propagation medium, the Maxma-14 genotype was the one with the greater biomass production with 23.851 g (FIG. 3a). Regarding the number of seedlings produced, we observed that the number of seedlings produced depended on the medium, the number of immersions, and the genotype used. Through a protocol comprising 3 or 4 immersions, for 1 or 3 minutes, and with medium B, we obtained the best results in explants from the MAXMA-14 genotype (11710 explants) (FIG. 3b).

[0060] In the case of micropropagation of the MAXMA-14 variety, the immersion system made up by the medium B, with N=3 or 4 and T=1 or 3 min (FIG. 4; bars B,3,3 and B,1,4 in axis X) produced the highest biomass (23.851 g). For Colt we obtained the highest values using the medium A at N=4 and T=1 min (bar A.4.1) and medium B at N=3 and T=3 min (bar B.3.3); in both cases the production reached 17.31 g.

[0061] In the case of the Van variates, better performance of biomass production was obtained when using medium A, N=3 and 4, and T=1 and 3 min (bars A,3,3 and A,1,4) producing 26.21 g. In the case of Rainier, the greater biomass was produced when using medium A under the conditions of N=3 and 4 and T=1 and 3 min (bars A,3,3 and A,1,4), producing its best performance of 18.1 g of biomass.

[0062] Regarding the number of seedlings produced (FIG. 5), we observed that variety MAXMA-14 showed the greatest number of seedlings under all the conditions whenever medium B was used. Additionally, we observed a good performance when the later was micro-propagated in medium A under the conditions of N=4 and T=3 min, reaching a maximum production of 11710 y 11510 seedling, respectively. The Colt explants had their greater number of seedlings produced with the use of medium B under conditions of N=2 for T=3 min (7910 explants) and N=3 for T=1 min (7110).

[0063] Micropropagation Performance Comparison Between Temporary Immersion Systems and Micropropagation in a Solid Medium.

[0064] At the same time, we compared the micropropagation performance by using the temporary immersion bioreactor system and by carrying out micropropagation of the same explants in a solid culture medium.

[0065] The solid medium used contains a DKW medium (Driver and Kuniyuki, 1984) supplemented with 0.01 mg/L IBA, 500 mg/L ascorbic acid, 100 mg/L of inositol and 7 g/L of Phytagel.

[0066] Again, the micropropagation performance was determined based on final biomass, number of seedlings produced, and sucrose consumption.

[0067] Biomass Production and Number of Seedlings

[0068] It was observed that the biomass production for genotypes MAXMA-14 and Colt rootstock is greater using the immersion system in comparison to the observations made when the micropropagation is performed in the semisolid medium. (FIG. 4).

[0069] In the case of the Van variety, most of the cultures in the temporary immersion system showed, except for the protocol with the medium A, N=3 and T=1 min, a higher production performance in comparison to the culture in solid (FIG. 5).

[0070] With respect to the culture of the Rainer genotype, it did not show significant differences between micropropagation in the immersion system and culture in a solid medium.

[0071] Sucrose Consumption

[0072] The sucrose consumption in the culture of the MAXMA-14 variety, in general, showed differences between culture in the immersion system and that in a solid medium (FIG. 6). Similarly, the Colt explants consume different amounts of sucrose in the immersion systems evaluated in comparison to the semisolid medium.

[0073] The behavior of the varieties also showed the same tendency observed in the rootstock, that is, different consumptions in comparison with the micropropagation in a semisolid medium.

[0074] Finally, the Van explants showed the same consumption of sucrose in the semisolid medium that in the 4 conditions evaluated in the immersion system.