Identification of key genes responsible for green and white colored spathes in Anthurium andraeanum (Hort.).

Modern anthuriums, Anthurium andraeanum (Hort.) are among the most popular flowering plants and widely used for interior decoration. Their popularity is largely attributed to the exotic spathes with different colors. Previous studies have reported color development in red spathe cultivars, but limited information is available on key genes regulating white and green colored spathes. This study analyzed anthocyanin, chlorophyll, and carotenoid contents as well as transcript differences in spathes of eight cultivars that differed in spathe colors ranging from red to white and green. Results showed that increased expression of a transcription factor AaMYB2 was associated with elevated levels of anthocyanin in spathes, but decreased expression of AaMYB2 and increased expression of AaLAR (leucoanthocyanidin reductase) and AaANR (anthocyanidin reductase) were accompanied with the accumulation of colorless proanthocyanidin, thus the white spathe. As to the green colored spathe, chlorophyll content in the green spathe cultivar was substantially higher than the other cultivars. Correspondingly, transcripts of chlorophyll biosynthesis-related genes AaHemB (porphobilinogen synthase) and AaPor (protochlorophyllide oxidoreductase) were highly upregulated but almost undetectable in white and red spathes. The increased expression of AaHemB and AaPor was correlated with the expression of transcription factor AaMYB124. Subsequently, qRT-PCR analysis confirmed their expression levels in nine additional cultivars with red, white, and green spathes. A working model for the formation of white and green spathes was proposed. White colored spathes are likely due to the decreased expression of AaMYB2 which results in increased expression of AaLAR and AaANR, and the green spathes are attributed to AaMYB124 enhanced expression of AaHemB and AaPor. Further research is warranted to test this working model.

Triploid cultivars of Cymbidium act as a bridge in the formation of polyploid plants.

Triploid is considered a reproductive barrier and also a bridge in the formation of polyploids. However, few reports are available in Cymbidium. In this study, diploid 'Xiaofeng', sexual triploid 'Yuchan' and 'Huanghe' of Cymbidium were used to evaluate hybridization compatibility of the triploids. Results showed that the sexual triploids were fertile whether they were used as male or female parents. 'Yuchan' produced male gametes of 1x, 1x~2x, 2x, 2x~3x, and 3x at frequencies of 8.89%, 77.78%, 6.67%, 3.33%, and 3.33%, respectively; while 'Huanghe' produced 3.33% 1x, 80.00% 1x~2x, 8.89% 2x, 5.56% 2x~3x, and 2.22% 3x male gametes. The cross of 'Xiaofeng' with 'Yuchan' produced progenies with a wide range of ploidy levels, including one diploid, 34 2×~3× aneuploids, 12 triploids, and one tetraploid, indicating that male gametes produced by sexual triploid were fertile and could be transmitted and fused with egg cells. On the other hand, 10 progenies obtained from the cross of 'Yuchan' × 'Xiaofeng' were all aneuploids. The cross of 'Yuchan' with 'Huanghe' produced 40 progenies including three 2×~3× aneuploids, nine 3×~4× aneuploids, 21 tetraploids, six 4×~5× aneuploids, and one pentaploid, suggesting that 2x gametes, instead of the unreduced ones played a more important role in the formation of tetraploids. The survival rates of the hybrids were all above 80.00%, with the tetraploids at 96.67%. Cytological analysis revealed that during meiosis of sexual polyploids, two chromosome sets of the 2n gamete were inclined to enter into the same daughter cell, resulting in the production of 2x gametes. Our results indicate that the triploid cymbidiums are not reproductive barrier but serve as a bridge in the formation of polyploid plants.

Unreduced Male Gamete Formation in Cymbidium and Its Use for Developing Sexual Polyploid Cultivars.

Polyploidy plays an important role in crop improvement. Polyploid plants, particularly those produced through unreduced gametes (2n gametes), show increased organ size, improved buffering capacity for deleterious mutations, and enhanced heterozygosity and heterosis. Induced polyploidy has been widely used for improving floriculture crops, however, there are few reported sexual polyploid plants in the floriculture industry. This study evaluated nine cultivars of Cymbidium Swartz and discovered that 2n male gametes occurred in this important orchid. Depending on cultivars, 2n male gamete formation frequencies varied from 0.15 to 4.03%. Interspecific hybrids generally produced more 2n male gametes than traditional cultivars. To generate sexual polyploid plants, seven pairs of crosses were made, which produced five triploid and two tetraploid hybrids. Two triploid hybrids were evaluated for in vitro regeneration and growth characteristics. Compared to the diploid parents, the triploids were more easily regenerated through rhizomes or protocorms, and regenerated plants had improved survival rates after transplanting to the greenhouse. Furthermore, the sexual polyploid plants had more compact growth style, produced fragrant flowers, and demonstrated heterosis in plant growth. Through this study, a reliable protocol for selection of appropriate parents for 2n gamete production, ploidy level evaluation, in vitro culture of polyploid progenies, and development of new polyploid cultivars was established. Our study with Cymbidium suggests that the use of 2n gametes is a viable approach for improving floriculture crops.

Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium.

Cymbidium, one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium 'Xiaofeng' (CXF), which can be efficiently micropropagated, and terrestrial Cymbidium sinense 'Qijianbaimo' (CSQ), which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs) were identified between CXF and CSQ during shoot induction. Gene ontology (GO) enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid) metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium.