Induction of Axillary Bud Swelling of Hevea brasiliensis to Regenerate Plants through Somatic Embryogenesis and Analysis of Genetic Stability.

To overcome rubber tree (RT) tissue culture explant source limitations, the current study aimed to establish a new Hevea brasiliensis somatic embryogenesis (SE) system, laying the technical foundation for the establishment of an axillary-bud-based seedling regeneration system. In this study, in vitro plantlets of Hevea brasiliensis Chinese Academy of Tropical Agricultural Sciences 917 (CATAS 917) were used as the experimental materials. Firstly, the optimum conditions for axillary bud swelling were studied; then, the effects of phenology, the swelling time of axillary buds (ABs), and medium of embryogenic callus induction were studied. Plantlets were obtained through somatic embryogenesis. Flow cytometry, inter-simple sequence repeat (ISSR molecular marker) and chromosome karyotype analysis were used to study the genetic stability of regenerated plants along with budding seedlings (BSs) and secondary somatic embryo seedlings (SSESs) as the control. The results show that the rubber tree's phenology period was mature, and the axillary bud induction rate was the highest in the 2 mg/L 6-benzyladenine (6-BA) medium (up to 85.83%). Later, 3-day-old swelling axillary buds were used as explants for callogenesis and somatic embryogenesis. The callus induction rate was optimum in MH (Medium in Hevea) + 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) + 1.5 mg/L 1-naphthalene acetic acid (NAA) + 1.5 mg/L Kinetin (KT) + 70 g/L sucrose (56.55%). The regenerated plants were obtained after the 175-day culture of explants through callus induction, embryogenic callus induction, somatic embryo development, and plant regeneration. Compared with the secondary somatic embryo seedling control, axillary bud regeneration plants (ABRPs) were normal diploid plants at the cellular and molecular level, with a variation rate of 7.74%.

Optimization of the Transformation Protocol for Increased Efficiency of Genetic Transformation in Hevea brasiliensis.

The recurring growth of bacterium in newly developed resistant cells and a minimal level of bacterial infection rate are the main limiting factors of Agrobacterium-mediated transformation experiments in Hevea brasiliensis. The current study aimed to optimize crucial factors of the transformation protocol in order to obtain an efficient transformation experimental model for Hevea using cotyledonary somatic embryos as explants. Transformation conditions such as antibiotic concentration, preculture duration, Agrobacterium concentration, sonication and cocultivation conditions were analyzed using the binary vector pCAMBIA2301. Transient transformation was confirmed by GUS histochemical staining. The best transformation efficiency was observed when the explants were not cultured on a preculture medium that contained acetosyringone at a level of 100 µM. The best results were obtained using a bacterial density of 0.45 at OD 600 nm, 50 s of sonication of explants in a bacterial liquid culture and a total incubation time of 18 min in the same bacterial suspension. Transmission electron microscopical analysis confirmed the impacts of sonication on bacterial infection efficiency. Cocultivation conditions of 22 °C and 84 h of darkness were optimal for the transfer of T-DNA. Agrobacterium was eliminated with 500 mg/L of timentin, and the selection of transformants was performed using 100 mg/L of kanamycin in the selection medium. The presence of transgene was confirmed in the resistant embryos by polymerase chain reaction (PCR). The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest into the Hevea genome for the breeding of this economically important plant species in the future.

Comparative analysis of latex transcriptomes reveals the potential mechanisms underlying rubber molecular weight variations between the Hevea brasiliensis clones RRIM600 and Reyan7-33-97.

BACKGROUND: The processabilities and mechanical properties of natural rubber depend greatly on its molecular weight (MW) and molecular weight distribution (MWD). However, the mechanisms underlying the regulation of molecular weight during rubber biosynthesis remain unclear. RESULTS: In the present study, we determined the MW and particle size of latex from 1-year-old virgin trees and 30-year-old regularly tapped trees of the Hevea clones Reyan7-33-97 and RRIM600. The results showed that both the MW and the particle size of latex varied between these two clones and increased with tree age. Latex from RRIM600 trees had a smaller average particle size than that from Reyan7-33-97 trees of the same age. In 1-year-old trees, the Reyan7-33-97 latex displayed a slightly higher MW than that of RRIM600, whereas in 30-year-old trees, the RRIM600 latex had a significantly higher MW than the Reyan7-33-97 latex. Comparative analysis of the transcriptome profiles indicated that the average rubber particle size is negatively correlated with the expression levels of rubber particle associated proteins, and that the high-MW traits of latex are closely correlated with the enhanced expression of isopentenyl pyrophosphate (IPP) monomer-generating pathway genes and downstream allylic diphosphate (APP) initiator-consuming non-rubber pathways. By bioinformatics analysis, we further identified a group of transcription factors that potentially regulate the biosynthesis of IPP. CONCLUSIONS: Altogether, our results revealed the potential regulatory mechanisms involving gene expression variations in IPP-generating pathways and the non-rubber isoprenoid pathways, which affect the ratios and contents of IPP and APP initiators, resulting in significant rubber MW variations among same-aged trees of the Hevea clones Reyan7-33-97 and RRIM600. Our findings provide a better understanding of rubber biosynthesis and lay the foundation for genetic improvement of rubber quality in H. brasiliensis.

A missense mutation in the VHYNP motif of a DELLA protein causes a semi-dwarf mutant phenotype in Brassica napus.

Although dwarf genes have been widely used to improve lodging resistance and enhance harvest index in cereal crops, lodging is still a serious problem in rapeseed (Brassica napus) production. A semi-dwarf B. napus mutant, ds-1, was identified through EMS mutagenesis of a microspore-cultured DH line. The mutant had a significant reduction in height due to a lower first branch position and shorter internodes when compared with wild-type cultivars. This dwarfism was inherited as a single semi-dominant gene, ds-1. DS-1 locus was mapped to chromosome A6, and co-segregated with a microsatellite marker BnEMS1125 derived from the gene BnRGA. BnRGA encodes a DELLA protein that functions as a GA signaling repressor. The expression of a mutant BnRGA allele from ds-1, Bnrga-ds, caused dwarf phenotypes in Arabidopsis. Comparative sequencing of RGA open-reading frames (ORFs) of ds-1 and wild-type cultivars revealed a single proline (P)-to-leucine (L) substitution that may lead to a gain-of-function mutation in GA signaling. The expression of the Arabidopsis homolog, Atrga-ds, bearing this site-directed mutation also rendered dwarf phenotypes in Arabidopsis, which demonstrated that the P-to-L mutation in the VHYNP motif of Bnrga-ds is responsible for the dwarfism. A yeast two-hybrid assay confirmed that this mutation inhibited the interaction between Bnrga-ds/Atrga-ds and the GA receptor, AtGID1A, in the presence of GA(3), suggesting that the conserved proline residue in the VHYNP motif of DELLA protein directly participates in DELLA-GID1 interaction. Identification and characterization of the dwarf gene ds-1 will facilitate its utilization in improving lodging resistance in Brassica breeding.