Micrografting Technique of Hevea brasiliensis In Vitro Plantlets

Materials and reagents

Biological materials

Here, 2.5-months-old vitroplants were used, after germination of the somatic embryos, having reached between 1 and 2 leaf stages with root development equivalent in size to the one of the aerial part.

1. Hevea in vitro plantlets of interest for scion sampling. Plantlets should have the same diameter as the rootstock to make it easier to cut the scion

2. Hevea in vitro–rooted plantlets of interest to be grafted (serving as a rootstock)

Materials

1. Sterile round filter paper 85 mm diameter as culture support (Whatman, catalog number: 1001085)

Reagents

1. Agar agar (Sigma, catalog number: A1296)

2. NH₄NO₃ (Labover, catalog number: A0501-1000)

3. KNO₃ (Sigma, catalog number: 1050611000)

4. NaH₂PO₄·H₂O (Labover, catalog number: 13429448)

5. CaCl₂·2H₂O (Sigma, catalog number: 1420021000)

6. MgSO₄·7H₂O (Sigma, catalog number: 23039)

7. H₃BO₃ (Sigma, catalog number: B6768)

8. MnSO₄·H₂O (Sigma, catalog number: M7899)

9. ZnSO₄·7H₂O (Sigma, catalog number: Z0251)

10. CuSO₄·5H₂O (Sigma, catalog number: C8027)

11. NaMoO₄·2H₂O (Labover, catalog number: S0525-25)

12. KI (Sigma, catalog number: P8166)

13. CoCl₂·6H₂O (Sigma, catalog number: C8661)

14. Myo inositol (Sigma, catalog number: I7508)

15. Nicotinic acid (Labover, catalog number: N0611.0100)

16. Pyridoxin HCl (Labover, catalog number: P0612.0050)

17. Thiamin HCl (Labover, catalog number: T0614.0025)

18. D(+) Biotin (Labover, catalog number: B0603.1000)

19. Calcium pantothenate (Sigma, catalog number: C0400000)

20. Ascorbic acid (Labover, catalog number: A0602.0100)

21. Choline chloride (Sigma, catalog number: C1879)

22. Cysteine HCl (Labover, catalog number: C0706.0100)

23. Glycine (Labover, catalog number: G0709.1000)

24. Riboflavin (Labover, catalog number: R0613.0025)

25. FeSO₄·7H₂O (Sigma, catalog number: 1039630001)

26. Na₂EDTA·2H₂O (Sigma, catalog number: E6635)

27. Sucrose (Labover, catalog number: S0809.9025)

28. Osmosis water

Solutions

1. MACRO MS 10× stock solution (see Recipes)

2. MICRO MH 100× stock solution (see Recipes)

3. Vitamins 1,000× stock solution (see Recipes)

4. Fe-EDTA 100× stock solution (see Recipes)

5. Germination culture (GER) medium (see Recipes)

Recipes

For each stock solution, mix the components, in the order listed, with osmosis water. These stock solutions can be kept for one month in the refrigerator or for up to one year in the freezer at -20 °C.

1. MACRO MS 10× stock solution

2. MICRO MH 100× stock solution

3. Vitamins 1,000× stock solution

4. Fe-EDTA 100× stock solution

5. Germination culture (GER) medium

Mix all medium components in osmosis water. Adjust pH to 5.8. Autoclave for 20 min at 120 °C. Dispense 5 mL of liquid medium into sterile tubes and the semi-solid medium in Petri dishes. Media can be stored for 1–2 weeks at room temperature in the dark.

Equipment

1. Laminar flow hood

2. Magnifying glass

3. Sterile fines clamp

4. Sterile large clamp (for plantlet manipulation)

5. Sterile scalpel

6. Sterile razor blade

7. Sterile blade holder

8. Food-grade plastic film

Software and datasets

1. XLSTAT software (for data analysis) (Addinsoft, version 2023.1.1., Lumivero [13])

Procedure

The entire protocol, except for media preparation, must be carried out under sterile conditions using sterile equipment.

A. Material preparation

1. Prepare GER medium (see Recipes).

2. Sterilize a circle of filter paper 85 mm in diameter in a capped glass tube (used as support for the grafted plant) as described in Figure 1.

3. After autoclaving GER medium, pour it into the glass tube.

Figure 1. Folding the filter paper into the glass tube. A. Place the paper disc on the lid of a glass tube. B. Fold the paper disc over the lid. C. Pierce the center of the folded disk with a pointed object. This will allow the taproot to pass through. D. Insert the folded disc into the tube that will contain the grafted seedling.

B. Rootstock preparation (under a magnifying glass)

1. Carefully remove the in vitro plantlets out of the glass tube (Figure 2A).

Figure 2. Plant material preparation. A. In vitro plantlet at the right stage of development. B. Rootstock after pruning of cotyledons and taproot. C. Grafting window preparation. D. Scion at the correct size equivalent to the diameter of the rootstock.

2. Prune cotyledons and stem from in vitro plantlets used as rootstock (Figure 2B).

3. Prune taproot at 6 cm below the collar (Figure 2B).

4. Prepare the grafting window by cutting a slightly oblique slit in the stem at least 2 cm above the collar (Figure 2C). Take care not to weaken or break the rootstock stem.

C. Scion preparation (under a magnifying glass)

1. Cut a beveled graft from the apex or another bud of the selected in vitro plant. Its size should match the diameter of the rootstock stem (Figure 2D).

2. While waiting to hold the rootstock under the magnifying glass, leave the scion on GER semi-solid medium for a few seconds to avoid drying and oxidation, without closing the Petri dish with its lid (Figure 2D).

D. Grafting (under a magnifying glass)

1. Place the scion in the oblique slit and surround it with a piece of semi-solid medium (grafting steps, Figure 3A).

2. Transfer the grafted material into a glass tube containing liquid GER medium; stabilize the grafted plant by placing it in the middle of the folded filter paper (Grafting steps, Figure 3B).

3. Hermetically seal the capped culture tubes with food-grade plastic foil. No aeration is required until bud break and graft development.

Figure 3. Grafting steps in optimal conditions. A. Placement of the graft in the slit and protection with a piece of solid medium. B. Correct positioning of the grafted plant into the filter paper base.

E. Graft re-growth

1. Grow plantlets for one month at 27 °C, under a light intensity of 60 μmol m^-2·s^-1 with a 12/12 h light/dark photoperiod.

2. Monitor and suppress the restart of cotyledon buds twice a week. Under sterile conditions, partially remove the plantlet from the disinfected tube and remove the bud with a sterile blade.

3. When the grafted bud breaks and regenerates a growth unit (Figure 4A), transfer the plant to the greenhouse for acclimatization when the leaves are fully expanded (Figure 4B). Use a 2 L pot for full development for two months. During this step, use a transparent plastic cup for 2 weeks to maintain humidity around the young grafted plant (Figure 4C). Then, transfer plantlets into 7 L pots for up to one year (Figure 4D). The soil used here was GO M2 (Jiffy), pH 5, with the following growing conditions: average temperature of 28–32 °C, humidity 50%–70%, manual watering 2–3 times a week, and natural lighting.

Figure 4. Graft re-growth, acclimatization, and plant development in the greenhouse. A. Graft re-growth. B. Grafted plantlet with fully developed leaves. C. Acclimatization step in the greenhouse. D. Development of the grafted plant after one year.

Data analysis

To establish a robust protocol in rubber trees, several parameters were tested on enough plants to allow statistical analysis using XLSTAT software. These parameters were tested on in vitro plants of clone PB 260 regenerated by somatic embryogenesis. In fact, two types of media (liquid or semi-solid), as well as a junction protection treatment (none, liquid, or semi-solid) and various times of rootstock bud pruning (day of grafting, 3 weeks after grafting) were tested. Statistical analysis was carried out by parametric k-proportions comparison including khi², Montecarlo, and Marascuilo tests.

Validation of protocol

Throughout the process, adverse effects such as root damage and bud break on the rootstocks were monitored (Figure 5). Root damage that occurred in the semi-solid medium was avoided in the liquid medium. Bud break on the rootstock was avoided by pruning all the cotyledonary buds on the rootstock on the day of grafting. Grafted buds survived thanks to the use of a drop of semi-solid medium at the junction. The two best treatments, consisting of a treatment at the scion-rootstock junction with semi-solid medium and a pruning of the rootstock buds, resulted in a grafted bud break rate ranging from 63% to 73% (Figure 5).

Figure 5. Effect of rootstock culture medium, the treatment at the junction, and the pruning of the rootstock buds on the development of the grafted buds. A. Histograms showing the effectiveness of treatments on root damage, graft survival, bud breaking, and acclimatization in the greenhouse. B. Details of treatments applied and number of grafted plants used. Statistical analyses were carried out using a parametric k-proportions comparison. Values with the same letter are not significantly different at the 0.05 probability level.

General notes and troubleshooting

Preliminary trials revealed several problems, including root damage due to the resistance of the semi-solid medium, bud break on the rootstock competing with the grafted bud, and, most importantly, drying of the grafted buds. According to the literature, stem and root pruning reduced early bud break on rootstock in citrus [14]. These authors also showed that root damage could be reduced using liquid medium. Interestingly, a drop of medium at the junction prevents the drying of grafted buds in pear and apple shoot tip grafting [15]. The first critical step is pruning the cotyledons, which consists of removing the cotyledons and underlying buds to avoid competition with the graft, as soon as the rootstock is prepared and without damaging the stem (see section B). Grafting is, therefore, done on a long internode without any developed buds. Monitoring the buds is the second critical step; they are removed carefully once or twice a week (see section E). This successful micrografting method developed on rubber clone PB 260 should then be tested on several other commercial clones. This technique will speed up the production of plant material for rootstock–scion interaction studies for commercial rubber clones as well as transgenic material for various agronomic traits. This technique may also facilitate rubber tree rootstock breeding.

Acknowledgments

The authors would like to thank Dr. Pablo Aleza (IVIA) and Dr. Mirea Bordas (Agromillora) for fruitful discussions and advice.

Competing interests

The authors declare that they have no financial and non-financial competing interests.

References

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