Factors affecting in vitro regeneration in the model tree Populus trichocarpa: II. Heritability estimates, correlations among explant types, and genetic interactions with treatments among wild genotypes

Abstract

To enhance the sensitivity of an ongoing Genome Wide Association Study (GWAS) for in vitro shoot regeneration and genetic transformation, a wide range of factors that can affect regeneration rate and, therefore, expression of genetic diversity were studied. Included were explant types and sources; direct versus indirect regeneration; nitrogen salts and micronutrient levels in basal medium; sucrose, auxin, and cytokinin types and levels; light intensity and quality; melatonin and serotonin; antibiotics (to exogenously control contaminants); antioxidants; and the ethylene-mitigating agent silver nitrate. Genotype was a statistically significant source of variance in response to nearly every treatment and for every measure of regeneration noted in shoots and roots, and initial generation of callus. Nonetheless, broad-sense heritability estimates varied widely as a result of variable degrees of environmental modification and statistical interactions with genotype. Compared to leaves, stem and petiole explants were highly correlated in their expression of genetic variation in response to treatments, a likely result of their functional similarity. Based on literature review, this study appears to be the most intensive analysis of plant natural genetic variation in response to in vitro manipulations published. It should help to guide development of improved and highly heritable treatments in other plant species that employ organogenic regeneration systems.

Factors affecting in vitro regeneration in the model tree Populus trichocarpa I. Medium, environment, and hormone controls on organogenesis

“…Indirect Regeneration The first experiment sought to assess the rates of shoot regeneration under an indirect versus a direct regeneration system, using hormones commonly employed in the Strauss laboratory for this purpose in poplars. As detailed in a companion paper (Table S1 in Ma et al 2022), treatment effects were statistically significant for both callus and shoot regeneration, as well as for treatment x explant interaction. When averaged over all 20 genotypes, the rate of callus formation was, as expected, much higher with the indirect system (on average, greater than 80% of explants with callus) compared to the direct system (less than 20% of explants with callus) (Figure S1 E1, Table S3), and both leaf and petiole explants behaved similarly.…”

“…These boxplots present the effect of different treatments on the growth of different explant types averaged over genotypes as biological replicates. The variation among genotypes was graphed and statistically analyzed using ANOVA, and presented in a companion paper (Ma et al 2022). Scatter plots of these average traits are presented to show the correlation between different traits as well as between different explant types.…”

“…To gain insight into the replicability of regeneration phenotypes across experiments that were conducted at widely different times, subsets of data that utilized the same genotype/ treatment combinations were extracted for analysis; ANOVA and graphical results are presented in Ma et al 2022.…”

“…Treatments that substantially elevate the rates of regeneration are also identified. Analyses of genetic control, H 2 , and genetic interactions with treatment factors are presented in a companion paper (Ma et al 2022). To avoid contamination, in vitro explants were often used in early experiments; however, because the planned GWAS experiments employed in vivo-derived plants (because of the scale of the work), in vitro studies were often repeated with in vivo explants, and later experiments focused predominantly on in vivo explants.…”

“…To avoid contamination, in vitro explants were often used in early experiments; however, because the planned GWAS experiments employed in vivo-derived plants (because of the scale of the work), in vitro studies were often repeated with in vivo explants, and later experiments focused predominantly on in vivo explants. Despite the recalcitrance of this species to common methods of regeneration and genetic transformation, treatments that enabled high levels of successful regeneration in many genotypes were identified, and as shown in a companion paper (Ma et al 2022), these regeneration responses were highly heritable.…”