Anthurium anther culture was successfully established using half-anthers as explants. Explants were cultured on Winarto-Teixeira basal medium (WT-1) containing 0.01 mg/l a-naphthalene acetic acid (NAA), 0.5 mg/l thidiazuron (TDZ), and 1.0 mg/l 6-benzylaminopurine (BAP), or on New Winarto-Teixeira basal medium (NWT-3) supplemented with 0.02 mg/l NAA, 1.5 mg/l TDZ, and 0.75 mg/l BAP for callus initiation. Regenerated calli produced multiple shoots on WT-1, which were then rooted in NWT-3 supplemented with 1% activated charcoal. Plantlets were acclimatized ex vitro using a mixture of burned rice husk, rice husk, and bamboo peat (1:1:1, v/v/v) as the potting medium. There was considerable morphological and cytological diversity of regenerants derived from anther culture, which are described in detail in this study. The callus cluster color ranged from green to light green and had a high regeneration capacity (7.3 and 4.8 shoots/callus cluster), light reddish-yellow callus showed moderate regeneration (2.6 shoots/callus cluster), while reddish-yellow callus had the lowest regeneration capacity (1.5 shoots/callus cluster). Morphological variations clearly observed in regenerants derived from this technique included alterations in plant size, peduncle length, spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also cytological variations in both in vitro and ex vitro regenerants of anther culture with 23-29% haploids, 5-10% aneuploids, 56-69% diploids, and 3-4% triploids. The results strengthen other studies in which the development of anther cultures, especially via callus formation, resulted in morphological and cytological alterations. These variations have been discussed to great length in this paper.

Background: Variation in shape and size of many floral organs is related to pollinators. Evolution of such organs is driven by duplication and modification of MADS-box and MYB transcription factors. We applied a combination of micro-morphological (SEM and micro 3D-CT scanning) and molecular techniques (transcriptome and RT-PCR analysis) to understand the evolution and development of the callus, stelidia and mentum, three highly specialized floral structures of orchids involved in pollination. Early stage and mature tissues were collected from flowers of the beepollinated Phalaenopsis equestris and Phalaenopsis pulcherrima, two species that differ in floral morphology: P. equestris has a large callus but short stelidia and no mentum, whereas P. pulcherrima has a small callus, but long stelidia and a pronounced mentum. Results: Our results show the stelidia develop from early primordial stages, whereas the callus and mentum develop later. In combination, the micro 3D-CT scan analysis and gene expression analyses show that the callus is of mixed petaloid-staminodial origin, the stelidia of staminodial origin, and the mentum of mixed sepaloid-petaloid-staminodial origin. SEP clade 1 copies are expressed in the larger callus of P. equestris, whereas AP3 clade 1 and AGL6 clade 1 copies are expressed in the pronounced mentum and long stelidia of P. pulcherrima. AP3 clade 4, PI-, AGL6 clade 2 and PCF clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a tradeoff between DIV clade 2 expression with SEP clade 1 expression in the callus, on the one hand, and with AP3 clade 1 and AGL6 clade 1 expression in the stelidia and mentum on the other. Conclusions: We detected differential growth and expression of MADS box AP3/PI-like, AGL6-like and SEP-like, and MYB DIV-like gene copies in the callus, stelidia and mentum of two species of Phalaenopsis, of which these floral structures are very differently shaped and sized. Our study provides a first glimpse of the evolutionary developmental mechanisms driving adaptation of Phalaenopsis flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid-petaloid-staminodial origin of the orchid mentum.

Konservasi anggrek spesies alam merupakan langkah penting untuk menghindari kepunahan akibat rusaknya habitat alamnya. Tujuan penelitian ialah mengetahui kemampuan tumbuh biji anggrek spesies alam pada media Vacin dan Went (VW) dan menentukan jenis anggrek spesies yang telah berhasil dikonservasi melalui biji. Anggrek spesies alam yang digunakan sebanyak 46 spesies yang berasal dari 18 genus anggrek, yakni Phalaenopsis, Dendrobium, Vanda, Ascocentrum, Paphiopedilum, Rhyncostilis, Neofinetia, Epidendrum, Arachnis, Dimorphosis, Phaius, Spathogottis, Trichoglottis, Arundina, Cymbidium, Renanthera, Armodorum, dan Gramathophylum. Polinasi bunga anggrek dilakukan dengan metode selfing maupun sibling. Buah dipanen saat warna buah berubah menjadi kuning kehijauan dan/atau tekstur buah menjadi lebih lunak. Selanjutnya, sebelum kultur biji, buah disterilkan dan biji disebar pada media VW. Hasil yang diperoleh menunjukkan bahwa buah anggrek hasil polinasi mengalami kemasakan antara 34-280 hari setelah polinasi. Biji hasil selfing dari 41 anggrek spesies (dari 46) dapat berkecambah pada media VW dengan umur berkecambah berkisar antara 10-69 hari setelah sebar. Sebanyak 19 anggrek spesies alam mampu membentuk protokorm di atas 70% dan semua protokorm mampu membentuk planlet. Selanjutnya, 19 spesies dapat diaklimatisasi dengan kisaran waktu antara 272-552 hari setelah terbentuk protokorm, Sebanyak 16 spesies belum dapat diaklimatisasi karena planlet yang masih kecil, sedang 6 spesies tidak tumbuh. Arundina graminifolia merupakan anggrek yang paling cepat membentuk protokorm dan Grammatophylum sp. merupakan anggrek yang paling lama membentuk protokorm. Biji Ascocentrum aurantiacum, Aerides odorata, Phalaenopsis luddemanniana, P. violacea, dan Cymbidium finlaysonianum tidak mampu membentuk protokorm. Dari penelitian ini diketahui bahwa media VW dapat digunakan untuk konservasi anggrek spesies alam melalui perbanyakan dengan menggunakan biji.

Deceptive Ceropegia pitfall flowers are an outstanding example of synorganized morphological complexity. Floral organs functionally synergise to trap fly-pollinators inside the fused corolla. Successful pollination requires precise positioning of flies headfirst into cavities at the gynostegium. These cavities are formed by the corona, a specialized organ of corolline and/or staminal origin. The interplay of floral organs to achieve pollination is well studied but their evolutionary origin is still unclear. We aimed to obtain more insight in the homology of the corona and therefore investigated floral anatomy, ontogeny, vascularization, and differential MADS-box gene expression in Ceropegia sandersonii using X-ray microtomography, Light and Scanning Electronic Microscopy, and RT-PCR. During 10 defined developmental phases, the corona appears in phase 7 at the base of the stamens and was not found to be vascularized. A floral reference transcriptome was generated and 14 MADS-box gene homologs, representing all major MADS-box gene classes, were identified. B- and C-class gene expression was found in mature coronas. Our results indicate staminal origin of the corona, and we propose a first ABCDE-model for floral organ identity in Ceropegia to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.