橡胶树体外植物幼苗的微接技术

Florence Dessailly; Pascal Montoro; Sémi Melliti; Julie Leclercq

doi:10.21769/BioProtoc.5215

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Peer-reviewed

Micrografting Technique of Hevea brasiliensis In Vitro Plantlets

橡胶树体外植物幼苗的微接技术

FD Florence Dessailly email

PM Pascal Montoro

SM Sémi Melliti

JL Julie Leclercq

发布: 2025年02月20日第15卷第4期 DOI: 10.21769/BioProtoc.5215 浏览次数: 848

评审: Samik BhattacharyaPreeti SinghAnonymous reviewer(s)

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Abstract

To prepare Hevea brasiliensis plantations, selected planting material is propagated by grafting using illegitimate seedlings as rootstocks, whose paternal genotype is unknown. Recent advances in rubber tree in vitro cloning propagation open the possibility of using these techniques to supply new planting material. Micrografting is a promising technique to speed up the preparation of plant material for rootstock–scion interaction studies. This article describes the implementation of an efficient micrografting technique from Hevea in vitro plants from clone PB 260. The procedure combines several conditions to preserve the root system and the grafted scion and to prevent any breakage of rootstock buds. This technique paves the way for clonal propagation and holds potential for further development on other rubber clones for further studies on the interaction between rootstock and scion.

Key features

• This protocol requires rejuvenated in vitro plantlets.

• Requires between 40 and 60 days before scion bud break.

• Sterile working conditions.

Keywords: Budding (芽接)

Somatic embryogenesis (体细胞胚胎发生)

Background

Hevea brasiliensis is the main source of natural rubber, which is harvested by regular tapping of the trunk, and accounts for 42% of world rubber consumption. Like many perennial species, the rubber tree is an allogamous species that requires grafting to clone selected material. In contrast to fruit tree species in which the scion and the rootstock are the selected planting material, rubber tree propagation has been carried out by grafting, using scion clonal material and illegitimate seedlings as rootstocks, whose paternal genotypes are unknown. This planting material has resulted in heterogeneity in rubber plantations due to the non-clonal rootstock component. Microcutting and somatic embryogenesis have been introduced to overcome this heterogeneity problem [1,2]. The use of clonal rootstock material could be an alternative to improve the productivity of rubber plantations [3]. In fruit trees like chestnut [4], micrografting is also used to obtain disease-free material for early detection of scion/rootstock incompatibility. A first attempt at micrografting has been made in Hevea to rejuvenate clonal lines for micropropagation [5]. Perrin and collaborators micrografted rubber bud tips onto one-month-old sterile seedlings used as rootstocks. Microcutting lines were then established from this micrografted material. Because of the low multiplication rate and the time required for rejuvenation in the microcutting procedure, somatic embryogenesis emerged as an alternative. Over more than a decade, somatic embryogenesis has evolved into a short-term process using compact callus and a long-term propagation method using friable embryogenic callus lines [2,6]. The latter has been widely and successfully used for the implementation of genetic modifications [7–9] and functional characterization of genes of interest [10–12]. However, even though these techniques are robust and well-established, they are still restricted to some rubber clones. On the other hand, mastery of micrografting is still insufficient to support studies such as the analysis of rootstock/scion interactions using rejuvenated and clonal material. The aim of this research is to propose an efficient micrografting procedure in rubber using in vitro–rejuvenated plantlets from somatic embryogenesis.

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

Reagent	Final concentration
NH₄NO₃	400 μM
KNO₃	400 μM
NaH₂PO₄·H₂O	40 μM
CaCl₂·2H₂O	30 μM
MgSO₄·7H₂O	60 μM

2. MICRO MH 100× stock solution

Reagent	Final concentration
H₃BO₃	15,008 μM
MnSO₄·H₂O	10,000 μM
ZnSO₄·7H₂O	4,000 μM
CuSO₄·5H₂O	148 μM
NaMoO₄·2H₂O	99 μM
KI	500 μM
CoCl₂·6H₂O	101 μM

3. Vitamins 1,000× stock solution

Reagent	Final concentration
Myo inositol	300 μM
Nicotinic acid	20 μM
Pyridoxin HCl	3 μM
Thiamin HCl	2 μM
Biotin	0.2 μM
Calcium pantothenate	1 μM
Ascorbic acid	1 μM
Choline chloride	1 μM
Cysteine HCl	60 μM
Glycine	5 μM
Riboflavin	1 μM

4. Fe-EDTA 100× stock solution

Reagent	Final concentration
FeSO₄·7H₂O	10 μM
Na₂EDTA·2H₂O	10 μM

5. Germination culture (GER) medium

Reagent	Final concentration
Macro MS 10×	0.5×
Micro MH 100×	1×
Fe-EDTA 100×	1×
Vitamins 1,000×	1×
Sucrose	234 mM
Agar	7 g/L

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

English

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文章信息

稿件历史记录

提交日期: Sep 5, 2024

接收日期: Dec 23, 2024

在线发布日期: Feb 9, 2025

出版日期: Feb 20, 2025

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如何引用

Dessailly, F., Montoro, P., Melliti, S. and Leclercq, J. (2025). Micrografting Technique of Hevea brasiliensis In Vitro Plantlets. Bio-protocol 15(4): e5215. DOI: 10.21769/BioProtoc.5215.