Paula M. Pijut

Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications

Hardwood forests and plantations are an important economic resource for the forest products industry worldwide and to the international trade of lumber and logs. Hardwood trees are also planted for ecological reasons, for example, wildlife habitat, native woodland restoration, and riparian buffers. The demand for quality hardwood from tree plantations will continue to rise as the worldwide consumption of forest products increases. Tree improvement of temperate hardwoods has lagged behind that of coniferous species and hardwoods of the genera Populus and Eucalyptus. The development of marker systems has become an almost necessary complement to the classical breeding and improvement of hardwood tree populations for superior growth, form, and timber characteristics. Molecular markers are especially valuable for determining the reproductive biology and population structure of natural forests and plantations, and the identity of genes affecting quantitative traits. Clonal reproduction of commercially important hardwood tree species provides improved planting stock for use in progeny testing and production forestry. Development of in vitro and conventional vegetative propagation methods allows mass production of clones of mature, elite genotypes or genetically improved genotypes. Genetic modification of hardwood tree species could potentially produce trees with herbicide tolerance, disease and pest resistance, improved wood quality, and reproductive manipulations for commercial plantations. This review concentrates on recent advances in conventional breeding and selection, molecular marker application, in vitro culture, and genetic transformation, and discusses the future challenges and opportunities for valuable temperate (or “fine”) hardwood tree improvement.

Candidate gene association mapping for winter survival and spring regrowth in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) is a widely cultivated cool-season grass species because of its high quality for forage and turf. Susceptibility to freezing damage limits its further use in temperate zones. The objective of this study was to identify candidate genes significantly associated with winter survival and spring regrowth in a global collection of 192 perennial ryegrass accessions. Significant differences in winter survival (WS), percentage of canopy green cover (CGC), chlorophyll index (Chl), and normalized difference vegetation index (NDVI) were found among accessions. After controlling population structure, LpLEA3 encoding a late embryogenesis abundant group 3 protein and LpCAT encoding a catalase were associated with CGC and Chl, while LpMnSOD encoding a magnesium superoxide dismutase and LpChl Cu-ZnSOD encoding a chlorophyll copper-zinc superoxide dismutase were associated with NDVI or Chl. Significant association was also discovered between C-repeat binding factor LpCBF1b and WS. Three sequence variations identified in LpCAT, LpMnSOD, and LpChl Cu-ZnSOD were synonymous substitutions, whereas one pair of adjacent single nucleotide polymorphisms (SNPs) in LpLEA3 and one SNP in LpCBF1b resulted in amino acid change. The results demonstrated that allelic variation in LpLEA3 and LpCBF1b was closely related to winter survival and spring regrowth in perennial ryegrass.

Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda.

AbstractKey messageThis transformation and regeneration protocol provides an integral framework for the genetic improvement ofFraxinus profunda(pumpkin ash) for future development of plants resistant to the emerald ash borer.AbstractUsing mature hypocotyls as the initial explants, an Agrobacteriumtumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7xa0days on Murashige and Skoog (MS) medium with 22.2xa0μM 6-benzyladenine (BA), 4.5xa0μM thidiazuron (TDZ), 50xa0mgxa0L−1 adenine hemisulfate (AS), and 10xa0% coconut water (CW) prior to transformation. Hypocotyls were transformed using 90xa0s sonication plus 10xa0min vacuum infiltration after Agrobacterium was exposed to 100xa0μM acetosyringone for 1xa0h. Adventitious shoots were regenerated on MS medium with 22.2xa0μM BA, 4.5xa0μM TDZ, 50xa0mgxa0L−1 AS, 10xa0% CW, 400xa0mgxa0L−1 timentin, and 20xa0mgxa0L−1 kanamycin. Timentin at 400 and 20xa0mgxa0L−1 kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (β-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through nfluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB.

Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls

An Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for white ash (Fraxinus americana) using hypocotyls as the initial explants. Hypocotyls isolated from mature embryos germinated on Murashige and Skoog (MS) medium supplemented with 22.2xa0µM 6-benzyladenine (BA) and 0.5xa0µM thidiazuron (TDZ) were transformed using A. tumefaciens strain EHA105 harboring the binary vector pq35GR containing a fusion gene between neomycin phosphotransferase (nptII) and gusA, as well as an enhanced green fluorescent protein (EGFP). Explants were transformed in a bacterial suspension with 100xa0µM acetosyringone using 90xa0s sonication and 10xa0min vacuum infiltration. Putative transformed shoots representing seven independent lines were selectively regenerated on MS medium with 22.2xa0µM BA, 0.5xa0µM TDZ, 50xa0mgxa0L−1 adenine sulfate, 10xa0% coconut water, 30xa0mg L−1 kanamycin, and 500xa0mgxa0L−1 timentin. Timentin at 500xa0mgxa0L−1 was optimal for controlling excess bacterial growth, and transformed shoots were selected using 30xa0mgxa0L−1 kanamycin. The presence of GUS (β-glucuronidase), nptII, and EGFP in transformed plants was confirmed by polymerase chain reaction (PCR). Reverse transcription-PCR and fluorescence microscopy confirmed the expression of EGFP. Transgenic microshoots were rooted (80xa0%) on woody plant medium supplemented with 4.9xa0µM indole-3-butyric acid, 2.9xa0µM indole-3-acetic acid, and 500xa0mg L−1 timentin, and subsequently acclimatized to the culture room. This transformation protocol provides the framework for future genetic modification of white ash to produce plant material resistant to the emerald ash borer.

Improvement of Agrobacterium-mediated transformation and rooting of black cherry

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105 harboring a PsAGAMOUS RNAi plasmid (pART27-PsAGRNAi). The effects of Agrobacterium culture density and cocultivation duration on transformation efficiency were examined using EHA105 harboring either pBI121-MDL4 or pBI121-PsTFL1. In addition, the effect of the binary vector on transformation efficiency was also studied. Fifteen-minute vacuum infiltration without sonication produced the highest transformation efficiency (21.7%) in experiments using pART27-PsAGRNAi. OD600 values of 1.0 and 1.5 resulted in a transformation efficiency of 5% when pBI121-PsTFL1 was used for transformation. Transformation efficiency of 5% was also obtained from 3-d cocultivation using construct pBI121-MDL4 whereas no shoots regenerated after 4-d cocultivation. The binary vectors used also impacted transformation efficiency. PCR and quantitative-PCR analyses were used to confirm the integration of transgenes and determine the copy number of the selectable marker gene, neomycin phosphotransferase II, in 18 putative transgenic lines. Rooting of transgenic black cherry shoots was achieved at a frequency of 30% using half-strength Murashige and Skoog medium supplemented with 2% sucrose, 5xa0μM naphthaleneacetic acid, 0.01xa0μM kinetin, and 0.793xa0mM phloroglucinol, and the resulting transgenic plants were successfully acclimatized.

Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra

Black ash (Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloem-feeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators to induce callus and adventitious shoot bud formation. Significant effects of explant, and plant growth regulator interactions were found. The frequency of callus formation ranged from 77.8 to 94.4% and 88.9–100% from single leaflets and intact compound leaves, respectively, with no significant difference between treatments. For adventitious shoot bud induction, however, 22.2xa0µM 6-benzylaminopurine (BA) combined with 31.8xa0µM thidiazuron (TDZ) was the best treatment regardless of the initial leaf explant type, showing 21.1 and 28.8% shoot bud induction, with 1.5 and 1.9 adventitious shoots per explant, from single leaflets and intact compound leaves, respectively. The regenerated shoot buds were elongated on MS medium supplemented with Gamborg B5 vitamins plus 2xa0mgxa0L−1 glycine (MSB5G), 13.3xa0µM BA, 1xa0µM indole-3-butyric acid (IBA), and 0.29xa0µM gibberellic acid. The elongated shoots were continuously micropropagated through nodal stem sectioning until used for rooting. An average of 85.2% of the microshoots were successfully rooted in woody plant medium containing 5.7xa0µM indole-3-acetic acid plus 4.9xa0µM IBA with a 10-day initial dark culture, followed by culture under a 16-h photoperiod. Rooted plantlets were acclimatized to the greenhouse and showed normal plant growth and development with 100% survival. This regeneration protocol would be useful for mass propagation for conservation of F. nigra and for use in genetic transformation for EAB resistance.

Agrobacterium-mediated transformation of black cherry for flowering control and insect resistance

Black cherry is one of the most valuable hardwood species for cabinetry, furniture, and veneer. The goal of this study was to develop transgenic black cherry plants with reproductive sterility and enhanced insect resistance. Black cherry TERMINAL FLOWER 1 (PsTFL1) was overexpressed under the control of the CaMV 35S promoter in black cherry via Agrobacterium-mediated transformation, as a strategy for gene containment. PsTFL1 is a homolog of ArabidopsisTFL1 which is known to play a key role in regulating flowering time by counteracting with FLOWERING LOCUS T and repressing the transcription of the floral-related genes. The elevated expression level of PsTFL1 was proven to be able to significantly delay flowering and cause abnormal floral structure which led to sterility in Arabidopsis. Therefore, the overexpression of PsTFL1 was expected to induce the similar phenotype in black cherry to achieve reproductive sterility. To enhance insect resistance in black cherry, the black cherry endogenous genes encoding prunasin hydrolase isoform 3 (PH3) and mandelonitrile lyase isoform 4 (MDL4) were inserted into black cherry under the control of the phloem-specific promoter rolC or the CaMV 35S promoter. The two enzymes catalyze the hydrolysis of prunasin to mandelonitrile and the dissociation of mandelonitrile to hydrogen cyanide (HCN), respectively, and the overexpression of PH3 or MDL4 might accelerate the release of toxic HCN and lead to an effective protection from cambial-mining insects. Three independent transgenic lines of 35S::PsTFL1, three of rolC::MDL4-FLAG, two of rolC::PH3-FLAG, and eight of 35S::MDL4-FLAG were obtained. The integration of transgenes and the copy number of neomycin phosphotransferase were examined by polymerase chain reaction (PCR) and quantitative PCR (qPCR) analysis. The mRNA levels of PsTFL1, MDL4, and PH3 were examined by real-time qPCR and were compared to the wild-type. The expression level of PsTFL1 in the three 35S::PsTFL1 lines were 3.7–5.8-times higher than that of the wild-type. However, the mRNA level of MDL4 in the 35S::MDL4-FLAG lines and rolC::MDL4-FLAG lines, and the mRNA level of PH3 in the rolC::PH3-FLAG lines showed no significant change indicating that transgene silencing was induced. Western blot analysis was carried out using anti-FLAG antibody to detect the FLAG-tagged PH3 in the transgenic line that had slightly increased mRNA level of PH3, but no signal was detected.

Genetic fidelity assessment of in vitro-regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2u2009±u20093.6%) was obtained on MS medium with 10.8xa0μM NAA and 4.4xa0μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2xa0μM BA and 4.6xa0μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3u2009±u20096.3%). Maximum rooting of shoots (73.3u2009±u20095%) was achieved using half-strength MS medium with 4.9xa0μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77u2009×u2009105 protoplasts (Pp) per gram fresh weight with 94u2009% viability; after 60xa0min pre-plasmolysis with 0.7xa0M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7xa0M mannitol, 1.5u2009% cellulase Onozuka R10, and 1u2009% pectolyase Y-23 for 6xa0h. Liquid Kao and Michayluk medium containing 2.7xa0μM α-naphthaleneacetic acid (NAA) and 2.2xa0μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5u2009×u2009105xa0Ppxa0ml−1. Protoplast-derived microcalli became visible after 3–4xa0weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4xa0μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2xa0μM BA plus 4.6xa0μM zeatin after 2–3xa0weeks, with an overall shoot organogenesis efficiency of 78–93u2009%. Rooting of microshoots on half-strength MS medium containing 4.9xa0µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62u2009%. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

Origin of adventitious roots in black walnut (Juglans nigra) softwood cuttings rooted under optimized conditions in a fog chamber

AbstractnHigh-quality black walnut (Juglans nigra L.) logs are of great economic value and are used in the manufacture of high-end products. Indigenous to the central hardwood region, black walnut has been commercially cultivated for many years, and genetic improvement and selections have resulted in superior timber genotypes. The recalcitrance of black walnut cuttings to form adventitious roots is the greatest hurdle for mass propagation of improved material. The goal of this research was to improve the frequency of adventitious root formation in black walnut cuttings, and investigate anatomical changes during root development. Softwood cuttings (15–20xa0cm) were collected from juvenile and mature sources of elite genotypes, dipped for 60xa0s in 31.1, 62.2, or 93.2xa0mM indole-3-butyric acid-potassium salt (K-IBA), or 36.9, 73.8, or 110.7xa0mM indole-3-butyric acid (IBA), and then inserted into a moist medium consisting of 3 perlite: 1 coarse vermiculite (v/v). Cuttings were placed in bench-top fog chambers or a mist bench for 5xa0weeks. To visualize anatomical changes during root formation, stems were fixed in formaldehyde, embedded in paraffin, serially sectioned, and stained on sequential days throughout root development. Rooting was greatest (72%) for cuttings exposed to 93.2xa0mMxa0K-IBA and placed in the fog chamber, while cuttings treated with IBA rooted at lower frequencies (16–22.2%). Cuttings in the mist bench often deteriorated and rooted at lower frequencies independent of the auxin type. Anatomical analysis revealed adventitious root initials by day 16 and root primordia formation by day 18. Rooted cuttings survived acclimatization to the greenhouse.