Marc-Philippe Carron

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

SummaryA culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N6-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.

Influence of atmospheric gases, particularly ethylene, on somatic embryogenesis of Hevea brasiliensis

The atmosphere of the culture vessel is an important factor for successful somatic embryogenesis in Hevea brasiliensis (Müll. Arg.). Considerable release of carbon dioxide and ethylene occurred during the development of calli. By avoiding the accumulation of gas, unconfined conditions were the most favourable for inducing somatic embryogenesis. Trapping of ethylene was as favourable for calli development and for somatic embryogenesis as unconfined conditions. Inhibition of ethylene synthesis by the adding of aminooxyacetic acid to the medium, also favoured the embryogenic process, and inhibition of ethylene action by the adding of silver nitrate to the medium enhanced significantly embryo production.

Histology of early somatic embryogenesis inHevea brasiliensis: The importance of the timing of subculturing

Somatic embryos ofHevea brasiliensis can be obtained by culturing thin sections of inner tegument of seed on two successive different media, MH1 and MH3. Histological study showed that in calli cultured on non-renewed medium MH1 for 40 days, the embryogenesis process initiated on the 20th day did not produce results owing to early degeneration of the cells involved in the embryogenic pathway. However, typical embryogenic cells formed when medium MH1 was renewed once during the first phase of culture (between day 20 and day 30). Proembryos developed when the calli were subcultured on medium MH3 10–15 days later. Embryogenic cells did not form when there was frequent renewal of medium MH1 or early subculturing on MH3 after less than 40 days of culture on MH1. Methodical histological monitoring of the development of embryogenic quality of calli thus made it possible to define the optimum culture sequences for the embryogenesis process and which are favourable for regular obtaining of proembryos.

Somatic embryogenesis in rubber ( Hevea brasiliensis Müll. Arg.)

Natural rubber, produced by Hevea brasiliensis (Mull. Arg.) has been known since ancient times and the Mayas and Aztecs used it in their daily life and religious ceremonies (Serier, 1993). In forests, this tropical tree can reach 40 m in height and 5 m in circumference. It is much smaller in plantations, where twice-weekly tapping of the tree bark considerably reduces its growth. Paradoxically, cultivation of this tree is very recent, beginning in Southeast Asia at the turn of the century, i.e., outside its original zone in the Amazonian basin, where “South American Leaf Blight”, caused by the fungus Microcyclus ulei is rife. The normal cultivation zone lies between 20° N and 20° S, apart from plantations in China and northern India (Compagnon, 1986). The areas planted cover around 7 million hectares, primarily in Thailand, Indonesia and Malaysia, but also in China, India, Africa and Latin America.

Callus friability and somatic embryogenesis in Hevea brasiliensis

The influence of plant growth regulators, sucrose, calcium and various macronutrient media on callus friability and somatic embryogenesis was investigated inHevea brasiliensis Müll. Arg. Friable and embryogenic calli were spontaneously formed in two rubber tree clones (PR 107 and RRIM 600) on the Medium for Hevea (MH), with 3,4-dichlorophenoxyacetic acid (3,4-d), kinetin and sucrose, while compact embryogenic calli were enhanced in three other clones (PB 260, PB 235 and GT1). Callus friability was enhanced in clone PB 260 when the concentration of one growth factor (3,4-d or kinetin) was reduced from 4.5 μLM to 0.45 μM during the first culture, or when high sucrose or calcium levels 351 mM and 12 mM, respectively) were maintained during subcultures. The different macronutrient media did not alter callus texture but only use of MH and Murashige and Skoog (MS) media led to somatic embryogenesis. Friable calli obtained by modifying the auxin/cytokinin balance lost their embryogenic potential. In contrast, those obtained on media with high sucrose or calcium concentrations were mainly composed of embryogenic cells embedded in a mucilaginous matrix. Such calli could be of potential interest for establishing embryogenic cell suspension cultures.

Endogenous cytokinins as biochemical markers of rubber-tree (Hevea brasiliensis) clone rejuvenation

The endogenous levels of isopentenyladenine, isopentenyladenosine, zeatin and zeatin riboside and the ability forin vitro axillary shoot organogenesis and rhizogenesis were compared between mature and rejuvenated clones ofHevea brasiliensis (Müll. Arg.). Enhancement of thein vitro organogenesis ability of rubber-tree clones following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by an increase of zeatin riboside levels in shoots used as starting material forin vitro micropropagation. Furthermore, the zeatin level, inin vitro shoots of clones treated byin vitro micrografting, and consequently capable of axillary shoot and root organogenesis, was higher than inin vitro shoots of non treated mature material incapable of in vitro organogenesis. We conclude that the endogenous zeatin-like cytokinin level (free and ribosylated forms) can be considered as a reliable marker for the recovery ofin vitro shoot and root organogenesis after rejuvenating treatments in rubber-tree clones.

Identification of differentially expressed cDNA sequences and histological characteristics of #Hevea brasiliensis# calli in relation to their embryogenic and regenerative capacities

Different friable Hevea callus lines from the same genotype can display different embryogenic and regenerative potentials. These lines can not be distinguished on the basis of macroscopic criteria. A histological and molecular study was undertaken to characterize the differences existing between five callus lines with different potentials. The genes differentially expressed during induction were analyzed using the differential display (DD-RT) technique. Twenty-eight cDNAs were found to be differentially expressed during induction in the embryogenic regenerating line (ER). Embryogenic nodules were formed earlier in the ER lines than in the embryogenic non-regenerating lines and were completely absent in the non-embryogenic line. Of these 28 cDNAs, five could be used to distinguish between calli prior to induction, thereby enabling an early diagnosis of friable Hevea callus embryogenic potential.

Rubber (Hevea brasiliensis Müll. Arg.)

Hevea brasiliensis (Fig. 1) belongs to the family Euphorbiaceae. It is the highest rubber-yielding among the nine species of the genus Hevea (H. brasiliensis, H. camporum, H. guianensis, H. microphylla, H. nitida, H. pauciflora, H. rigidifolia, H. spruceana) (Compagnon 1986).

Influences of aging and cloning methods on the capacity for somatic embryogenesis of a mature Hevea brasiliensis genotype.

We compared embryogenic capacities of integument explants excised from three sources of the Hevea brasiliensis (Müll. Arg.) mature genotype PB 260. The three sources were 17-year-old (BT 86) and 7-year-old (BT 96) budded trees and 7-year-old emblings (EM 96). The highest proportions of embryogenic calluses obtained from the total number of integument explants initially used were from trees of EM 96 origin, followed by BT 96 trees, with explants from BT 86 trees producing the lowest number of embryogenic calluses. Further initiation of embryogenic callus lines from the primary somatic embryos derived from the three sources was successful only for EM 96. Somatic embryo cultures from BT 86 and BT 96 sources produced only friable calluses that could not be further amplified. Overall, somatic embryo explants derived from EM 96 responded over a wider range of 3,4-dichlorophenoxyacetic acid and kinetin concentrations than the somatic embryo explants from BT 86 and BT 96 origins. The effects of chronologic, ontogenetic and physiologic aging on explant capacity for somatic embryogenesis and on the overall efficiency of the process in H. brasiliensis are discussed.

Comparison of endogenous ABA and IAA contents in somatic and zygotic embryos of Hevea brasiliensis (Müll. Arg.) during ontogenesis

Abstract IAA and ABA contents were measured in somatic embryos and in zygotic embryos and the surrounding endosperm in Hevea brasiliensis . Two successive phases development and maturation, were defined during ontogenesis using morphological phological growth and water status criteria. Endosperm contained high amounts of IAA (10–25 nmol/g dry wt.) throughout development and high ABA (40 nmol/g dry wt.) at the end of the development phase. Zygotic embryos contained high IAA concentrations at the start of the development phase; the maximum was observed in early torpedo stage (when length was 5 mm) and the level then decreased progressively until the end of the maturation phase. ABA accumulated (26 nmol/g dry wt.) in zygotic embryos at the end of growth and then its concentration dropped during maturation. It would therefore appear firstly that the IAA and ABA contents were always higher in the endosperm than in the embryo and secondly that the very low IAA and ABA contents characterize maturity in these two types of tissue. The results support the hypothesis of a major role of IAA in embryo development and of ABA in causing and controlling maturation. In comparison, IAA and ABA contents of somatic embryos were low (0.6–2 nmol/g dry wt.) and varied little during ontogenesis. Their variation was very different to that observed in ovulo and might be related to the abnormal development and low germination percentages observed.