Comparative Anatomy And Molecular Studies Of In Vitro And In Vivo Laticifers And Cell Marker Assisted Breeding In The Rubber Tree

The rubber tree(Hevea brasiliensis Muell.Arg.)is a perennial crop and the most economically important latex producing plant.Conventional breeding cycle spans approximately 30 years until the final choice of clones,which has led breeders to develop suitable methods for early selection of clones with high yield potential.This research aims to develop new method to shorten the breeding cycle based on relationships between lacifer frequencies and natural rubber yields.Laticifer cells are highly specialized cells that synthesize and store natural rubber.Morphological and functional differences of different laticifers are largely unknown,but such information is important for breeding and cultivation practices.The laticifers not only present in the trunk but are also found in leaves,petioles,young shoots,and roots.This research has systematically studied anatomical differences between two in vivo laticifer types and the callus laticifers,the transcriptome differences between the primary and secondary laticifers,as well as the laticifer frequencies in the trunk and calli of different cultivars and investigated their relationships to rubber yields.The main results are as follows: 1.Callus induction and plant regeneration in the rubber treeCallus were induced from many tissue types of the rubber tree such as anther,young shoot stem,and young petiole,and plantlets were regenerated from anther callus via somatic embrogenesis.The frequencies of both callus induction and embryogenesis were all genotype-dependent.The callus of cultivar Reyan 7-33-97 that has high yield potential,grew faster than the callus of cultivars with lower rubber yield.Cultivars with strong somatic embryogenesis and plantlet regeneration also had higher rubber yield.These results suggested that callus cultures may serve as a model to to study latex yield potential of the rubber tree genotypes.2.Comparative morphology of in vivo and in vitro laticifer cells in rubber treeMorphology of laticifer cells in callus induced from anther,young shoots,and petiole and primary and secondary laticifers in the trunk of adult trees was compared.Results indicate that the primary laticifers are unbranched,non-articulated,and anastomosing,and have bumpy lateral walls when matured.The secondary laticifers have smooth lateral walls and perforated end walls and belong to articulated anastomosing laticifers.The laticifer cells in the callus cultures show a novel morphology different from the morphology of laticifer cells in planta,excluding their origin from explants.They are morphologically similar to the secondary laticifers with smooth lateral walls and end walls,and similar relative cell wall thickness.3.Genome-wide identification,characterization of the MADS-box gene family in the rubber tree and their involvement in laticifer development97 MADS-box genes were identified in the rubber tree through transcriptomes and genome mining.93.8% of the genes were mapped onto the genome scaffolds in correspondence to the coverage(93.8%)of current version of sequenced genome.Phylogenetic analysis indicates that type II MADS-box genes have been more actively duplicated than their orthologous genes in Arabidopsis and rice,so that most(70,72.2%)of the MADS-box genes in the rubber tree belong to type II subfamily.This is a high percentage compared to that in Arabidopsis(43.7%)and rice(56.8%).Moreover,69 out of 70 type II genes in the rubber tree are transcribed,and they are mostly predominantly expressed in flowers,and some genes are predominantly expressed in laticifers,suggesting their roles in both flower and laticifer development.The number of type I genes in the rubber tree is only 27(27.8%),a much smaller number compared to their orthologous genes in Arabidopsis and rice,and most of the type I genes(55.6%,15)are silent and are most probably pseudogenes.The high birth rate and low death rate of type II genes and low birth rate and high death rate of type I genes may corresponds to the special developmental requirement of the rubber tree,e.g.the development of laticifer system for biosynthesis of cis-polyisoprene,the rubber.Moreover,atypical MIKC* factors(e.g.HbMADS1 in S-clade,and HbMADS20 in P-clade)are identified.These genes are diverged to typical MIKC* genes in sequences and facilitate functions required in laticifer development and rubber biosynthesis,which is not necessary in Arabidopsis and rice.4.Accelerating rubber tree breeding by cell marker assisted selection: comparative analysis of in vivo and in vitro laticiferous cells and their correlation with rubber yieldThe investigation was performed on relationships between laticifer cells in callus induced from young shoot stem,the secondary laticifers in the trunk of adult trees,and rubber yields,in order to develop suitable methods for the early selection of rubber tree clones with high yields.The results show that the laticifer density in callus and the laticifer ring number in bark are both genotype-dependent,and they are both highly correlated with the rubber yields with correlation coefficients R=0.846(P<0.002)and R=0.899(P<0.002)for the callus and bark,respectively.Since callus can be induced from tissues of young seedlings in cross-pollinated populations,the laticifer density in callus can serve as an early selection marker of genotypes in cross-pollinated populations for high rubber yields,and thus shorten the breeding cycle.