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Somatic embryogenesis and subsequent plant regeneration from zygotic embryo derived callus of Rubber (hevea Brasiliensis muell. arg).

Md. Mahbubur Rahman
Bangladesh Forest Research Institute, Chittagong-4000, Bangladesh

Maziah Mahmood
Institute of Bio Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

Norhani Abdullah
Institute of Tropical Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

Noor Azmi Shaharuddin
Bangladesh Forest Research Institute, Chittagong-4000, Bangladesh

Waheeda Parvin
Faculty of Agriculture, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia

Abstract/ Executive Summary:

A protocol has been developed for induction, maturation and germination of the zygotic embryo derived callus of the rubber tree (Hevea brasiliensis Muell. Arg.). The influence of plant growth regulators (PGRs) including 2,4-D, α-NAA, picloram, GA₃ and TDZ on MS and MMS were studied. Optimum calli were induced on MS supplemented with 2.0 mg/l 2, 4-D. The best callus growth and proliferation was recorded on MS fortified with 2.0 mg/l 2, 4-D + 2.0 mg/l BAP + 0.5 mg/l NAA. The maximum embryonic calli were induced on MS + 2.0 mg/l 2, 4-D + 2.0 mg/l Kn medium. Embryo induction, differentiation and maturation were obtained on MMS (MS +Vit B5). The rooted plantlets were produced on half strength MS without any supplements. The novelty of this study is the induction of embryos and plant regeneration from zygotic embryo explants of Hevea for the first time. The protocol developed in this study will facilitate mass propagation of high yielding rubber clones as well as to develop transgenic rubber plants with desired genes through genetic transformation.

Keywords: Hevea brasiliensis, Zygotic embryo, Somatic embryogenesis, Regeneration

Location: Malaysian Rubber Board, Malaysia

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Rubber wood

Objectives:

To develop a unique plant regeneration system that could be used in mass propagation as well as genetic modification of H. brasiliensis.

Methodology:

Hevea (Clone RRIM 901) seeds were obtained from the Malaysian Rubber Board, Malaysia. After removing the seed coat, the surface sterilization of endosperm was done for 15 min with 15% Clorox. There after, one drop of Tween 20 was added which followed by washing with sterile water for 5 - 6 times. The embryos were excised from con the callus induction medium. MS basal medium with B5 vitamins and different auxins including 2, 4-D, NAA, picloram, and Dicamba combination with cytokinins BAP and Kn at different concentrations were used for callus induction and growth. The concentrations of auxin varied from 0 - 5.0 mg/l and that of cytokinins 0 - 5.0 mg/l. The media pH was adjusted to 5.8 with 3% sucrose and 2.75 g/l gelrite followed by 20 min of autoclaving at 121ºC. The growth room condition was maintained at 25 ± 2ºC in the darkness. Each culture flask contained 30 ml medium with three embryos. Callus induction frequency was recorded after 4 weeks of culture initiation.The calli were sub-cultured every 2 weeks to optimize the callus growth and proliferation. MS supplemented with different concentrations of 2, 4-D, BAP and Kn in presence of NAA was tried. The callus diagonal length, fresh and dry weight/culture, and morphologies were recorded every week in order to optimize the growth and to obtain callus maintenance medium. The calli (3 weeks old) were transferred to the embryo induction medium. The MS was supplemented with B5 vitamins, amino acid glutamine (100 mg/l), 10% coconut water as organic supplements and 200 mg/l casein hydrolysate with 5% sucrose. The phytohormones added was within the range of 0.5 - 3.0 mg/l Kn, 0 - 3.0 mg/l GA₃, 0.1 mg/l NAA and 0.5 mg/l 2, 4-D. The cultures were kept at 25 ± 2ºC in darkness for 4 weeks. Apex induction and maturation of the embryos were examined on the modified MS medium supplemented with phytohormones BAP, GA₃ and TDZ. Plant regeneration from induced embryos was achieved in full or half strength hormone free MS medium. Visual observations were made to record the embryo induction percentage, maturation and germination. Completely randomized design (CRD) was used to carry out the experiments. Data were analyzed using SAS and means were statistically compared using LSD test at p < 0.05 significance level.

Source of Information: Plant Tissue Cult. & Biotech. 27(1): 51?61, 2017 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It was observed that the combination of BAP and GA₃ was required for embryo induction, maturation and germination. After 3 - 4 weeks of growth, embryos started to become greenish, and showed bipolar differentiation. The combination of an auxin and BAP was used to induce somatic embryos from immature and mature zygotic embryos of Quercus sp. (Wilhelm 2000). Embryo germination was initiated by the bipolar differentiation of (root and shoot primordial) the mature embryos. The embryos developed plantlets after 4 to 5 weeks of cultureon half strength MS medium. Embryo germinated in hormone free as well as on medium fortified with phytohormone. Embryo germination was enhanced by the addition of GA₃ on culture medium. The plantlets which grown in the medium containing GA₃were more vigorous than in hormone free medium. Maximum plant conversion was observed in 0.3 mg/l GA₃ and 0.2 mg/l IBA supplemented medium. Addition of IBA (0.1 mg/l 0.5 mg/l) in culture medium also favoured root elongation. About 80% of somatic embryos initiated germination and produced rooted plantlets. The plant regeneration protocol developed from zygotic embryo derived callus of H. brasiliensis (Clone RRIM 901) could be used for other rubber genotypes to produce mass scale planting materials. The regenerative embryogenic tissues established in this study could also be used to develop transgenic rubber plants with desirable genes.

Knowledge Management: Journal