Efficient Micropropagation of Chestnut Hybrids(Castanea spp.)Using Modified Woody Plant Medium and Zeatin Riboside

The chestnuts genus(Castanea spp.)is comprised of economically important trees native to the Northern hemisphere that are used as food and hardwood timber.Here,a very efficient method for micropropagation of European×Japanese chestnut hybrids(Castanea sativa×C.crenata)is described.Woody Plant Medium was used as the basal medium.In vitro shoots of four rootstock cultivars were micropropagated without shoot-tip necrosis on multiplication medium containing 5.7 or 11.4μmol·L^(−1)zeatin riboside,and were rooted on rooting medium containing 2.46μmol·L^(−1)indolebutyric acid.Monthly shootmultiplication rates for each cultivarwere 2–5 folds.In vitro rooting percentages for four cultivars were 87%for‘Maraval’,67%for‘Marigoule’,93%for‘Marsol’,and 97%for‘Précoce Migoule’.Within a 5 week period,80%–95%of rooted shoots were successfully acclimated under high humidity conditions after they were planted in either soil or rockwool.

Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants

Flowering locus T(FT)is a primary integrator in the regulation of plant flowering.Overexpressing a blueberry(Vaccinium corymbosum L.)FT gene(VcFT)(herein VcFT-OX)resulted in early flowering and dwarfing in‘Aurora’plants(herein‘VcFT-Aurora’).In this study,we found that VcFT-OX reduced shoot regeneration from leaf explants.To investigate the potential roles of the phytohormone pathway genes associated with VcFT-OX,differentially expressed(DE)genes in leaf tissues of‘VcFT-Aurora’plants were annotated and analyzed using non-transgenic‘Aurora’plants as a control.Three DE floral genes,including the blueberry SUPPRESSOR of Overexpression of constans 1(VcSOC1)(gibberellin related),Abscisic acid responsive elements-binding factor 2(VcABF2)and protein related to ABI3/VP1(VcABI3/VP1)(ethylene-related),are present under both the phytohormone-responsive and the dwarfing-related Gene Ontology terms.The gene networks of the DE genes overall showed the molecular basis of the multifunctional aspects of VcFT overexpression beyond flowering promotion and suggested that phytohormone changes could be signaling molecules with important roles in the phenotypic changes driven by VcFT-OX.

VcFT-induced mobile florigenic signals in transgenic and transgrafted blueberries

FLOWERING LOCUS T(FT)can promote early flowering in annual species,but such role has not been well demonstrated in woody species.We produced self and reciprocal grafts involving non-transgenic blueberry(NT)and transgenic blueberry(T)carrying a 35S-driven blueberry FT(VcFT-OX).We demonstrated that the transgenic VcFT-OX rootstock promoted flowering of non-transgenic blueberry scions in the NT(scion):T(rootstock)grafts.We further analyzed RNASeq profiles and six groups of phytohormones in both NT:T and NT:NT plants.We observed content changes of several hormone metabolites,in a descending order,in the transgenic NT:T,non-transgenic NT:T,and non-transgenic NT:NT leaves.By comparing differential expression transcripts(DETs)of these tissues in relative to their control,we found that the non-transgenic NT:T leaves had many DETs shared with the transgenic NT:T leaves,but very few with the transgenic NT:T roots.Interestingly,a number of these shared DETs belong to hormone pathway genes,concurring with the content changes of hormone metabolites in both transgenic and non-transgenic leaves of the NT:T plants.These results suggest that phytohormones induced by VcFT-OX in the transgenic leaves might serve as part of the signals that resulted in early flowering in both transgenic plants and the non-transgenic NT:T scions.

Agrobacterium tumefaciens-Mediated Transformation of Wild Tobacco Species Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa

Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg·L-1 kanamycin and 250 mg·L-1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg·L-1 kanamycin and 250 mg·L-1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T0 plants and T1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii.

VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant

The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants.Mu-legacy is a valuable blueberry mutant,in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene(VcRR2).Mu-legacy plants,compared with nontransgenic‘Legacy’plants,show dwarfing,promotion of flower bud formation,and can flower under nonchilling conditions.We conducted transcriptomic comparisons in leaves,chilled and nonchilled flowering buds,and late-pink buds,and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and‘Legacy’plants.These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENTCOPALYL DIPHOSPHATE SYNTHETASE 1(VcGA1),which induces new homeostasis of hormones,including increased gibberellin 4(GA4)levels in Mu-legacy leaves.Consequently,increased expression of VcRR2 and VcGA1,which function in cytokinin responses and gibberellin synthesis,respectively,initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches.In nonchilled flower buds,29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and‘Legacy’plants,of which 22 were chilling responsive.Thus,these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions.We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.