Full Text: 1 Introduction Genus Phaius was established by Jao de Loureiro 1790 in Flora Cochinchinensis and comprises more than 30 species around tropical parts of Old world from Asia, Australia, Africa and various islands of Pacific and Indian oceans (Loureiro, 1790; Lucksom, 2007; Chowdhery & Agrawala, 2013). Phaius tankervilleae was introduced in England in 1778 from China by John Fothergill. With course of time, these species were naturalized by introduction in South America, Caribban Islands; Central America (Panama) and was listed in catalogue of plant species at Royal Botanical Gardens, Kew (Desmond, 1995) and The European Garden Flora (Cullen et al., 2011). In India, few species are naturally available in North-Eastern states and legally categorized as endangered; and it was protected under Schedule-I of Indian Wild Life (Protection) Act, 1972 and has serious trade restrictions on international commercial export selling’s under Appendix-II of international conventions (Bhuyan, 2010; CITES, 2016). Efforts were also initiated for conservation through long-term seed storage (Hirano et al., 2009) and mass multiplication (Malemnganba et al., 1994). Both in-situ and ex-situ conservation programmes of P. tankervilleae were initiated in India (Kanwal, 2014). Presence of P. tankervilleae from other Indian states like Uttarakhand from Corbett National Park, North-Western Himalaya (Naithani et al., 2009) and Southern India (George & Mathew, 2016) indicates spatial distribution in India. P. flavus (Blume) Lindl., geographically located in Assam, Eastern Himalayas and P. mishmensis (Lindl. & Paxton) Rchb., and P. nanus Hook.f., are the other common Phaius species available in India (Pearce & Cribb, 2002). Phaius orchids are considered as highly decorative and have great ornamental value with long inflorescences, bigger size flowers, more florets & different colours with evergreen long broad elliptic pleated leaves suitable indoor cultivation; otherwise often treated as invasive orchids (Ackerman, 2012). Even it has medicinal application in traditional medicine and used in natural dye indigo preparations in Arunachal Pradesh (Kanwal, 2014). The predominant brownish-blue hue of flowers especially when old or damaged may indicate the presence of rich chemicals (Pearce & Cribb, 2002). Generally, P. tankervilleae is very commonly cultivated than other species (Schuiteman & de Vogel, 2000). However, the commercial varieties of Phaius are unavailable for domestic cultivation and not registered under ‘Protection of Plant Varieties and Farmers Rights Act, 2002’ for Plant Breeder’s rights in India. At international level, various experiments were conducted at large scale to develop novel Grex/hybrids (https://www.orchids.org/grexes/phaius-magniflorus); but only few hybrids are available within closed circuits by amateur growers, which are not yet legally protected nor notified by Plant Variety Protection (PVP) of TRIPS agreement or similar governing laws enacted under Patent Cooperation Treaty (http://www.aos.org/orchids/orchids-a-to-z/letter-p/phaius.aspx). Further, the occurrence of natural hybrids involving wild species was not reported so far in genus Phaius. Nototribic mode of pollination assisted by insects was found in Phaius tankervilleae (Banks ex L’Herit) Bl (Buragohain et al., 2016). However, auto self pollination was absent due to obstructive floral structures, although genetic barriers were never reported (Bhuyan, 2010). Royal Horticultural Society (RHS) data base reveals only one hybrid ‘Ashworthianus’ obtained from P. flavus (seed parent) and P. wallichii (male parent) in 1893 by Sanders (St Albans). So far, no hybrid registered with P. flavus and P. tankervilleae as parents for both direct and reciprocal crosses (http://apps.rhs.org.uk/horticulturaldatabase/orchidregister/orchidregister.asp). Development of hybrids using rare and endangered species for commercial sales will reduce the threatening pressure on their wild species (Kishor & Sharma, 2009). At gobal level, there are no trade restrictions on hybrids derived from Orchid species for import and exports as per ‘Convention on International Trade in Endangered of Wild Flora and Fauna’ (CITES, 2004; Devadas et al., 2010). Hence, in present study an attempt has been made to develop new interspecific hybrid using native indigenous species available and novel progeny generated characterized at flowering stage to assess the taxonomic and breeding value for further genetic improvement. 2 Materials and methods 2.1 Mother plants Phaius species viz., P. tankervilleae (Banks ex l’Heritier) Bl., and P. flavus (Bl.) Lindl. were used as parent material in the present studies, these were catalogued species accessions from the ‘National Active Germplasm Site’ (NAGS) collections at ICAR-NRC on Orchids, Pakyong, Sikkim. 2.2 Hybridization programme A series of crossing programme was carried out with Phaius species and other orchid species from 2005 to 2014 to assess the cross compatibility nature, with long term objective to generate novel inter-specific and inter-generic hybrids for Orchid genetic improvement. Based on flowering synchrony, Phaius species were crossed with other species and hybrids from other genera for several years viz., Paplionanthe, Calanthe, Dendrobium, Dienia, Coelogyne, Phalaenopsis, Lycaste, Thunia, Cymbidium, Arundina, Paphiopedilum, Vanda, Coelogyne and Vanda group (Table 1). The hybridization was done by emasculation of female flowers by removing anther cap and pollinia. Fresh pollinia were collected from male parent and attached to stigma surface of the column for pollination. Both direct and reciprocal crosses were made using P. tankervilleae (Banks ex l’Heritier) Bl., and P. flavus (Bl.) Lindl. at ICAR-NRC Orchids, Sikkim. One to two flowers were crossed for each combination due to limitation of flowering plants availability. Generally the stigmatic surface of female plants was highly receptive and sticky; however the pollen bags were used to cover the inflorescence to avoid cross pollination with insects. Once the pollination succeeded, flower colour changed in to dark and lip dries after 3-4 days and success of pod set was presented in Table 1. Few crosses were made by selfing using pollen of the same flower on stigma to understand self compatibility among Phaius species. 2.3 Progeny development Mature ellipsoid capsules were harvested after 10-11 months from pod formation after crossing and seedlings were raised using in-vitro techniques. The response of in-vitro studies will be discussed in separate paper. Among the capsules obtained from crosses, seedlings were obtained from selfed progenies and crossed progenies from PBX-11-21 (direct cross) and PBX-11-25 (reciprocal cross) series. Progenies of PBX-11-21 and PBX-11-25 flowered in early 2017 and mid 2018 after four and five years respectively after shifting seedlings for growing conditions under normal greenhouses. All the standard package practices for cultivation of Phaius progenies for growth were followed using pots (Devadas et al., 2008). Morphological characters are recorded at the full bloom stages and colour of the flowers was recorded with the help of ‘Royal Horticultural Society’ (RHS) colour chart (Simpson, 2011). The general descriptions for the morphological characters were based on quantitative data and expressivity of qualitative characters like flower colour at Pakyong, Sikkim (Altitude 1,300 MSL) location are recorded. 3 Results & Discussion 3.1 Success of hybridization A total of 91 handmade pollinations were made that includes self pollinations to study both cross compatibility and self incompatibility nature in Phaius species. All 12 crosses attempted for selfing in both species resulted 91.67% pod setting. The status of pod setting with other species and genera are presented in Table 1. Success of crossing at species and genera level was recorded 13.18% pod formation, with success rate of in-vitro seed germination at 14.28%. Crosses viz., PBX-05-290, PBX-06-78, PBX-06-308, PBX-09-03, PBX-14-265, PBX-14-274 resulted pod set, when P. tankervilleae was used as one of the parent in hybridization programme; however failed to achieve germination of seedlings. Number of pedigree crosses resulted viable seedlings, excluding selfed crosses is limited to 2.19% among crosses performed. Results indicate narrow genetic behaviour of Phaius with limited scope for response to genetic improvement through conventional hybridization programmes. 3.2 Incompatibility behaviour of Phaius Sympodial orchids like Calanthe masuca, Lycaste crueata, Paphiopedilum villosum showed only capsule development, when used as female parents in the present study. Similarly, capsule development was recorded in P. tankervilleae, when Calanthe triplicata, Paplionanthe teres and Coelogyne flaccida var. cristata were used as male parents. It indicates the possibility of pollen influence due to presence of hormones on flower colour change as an indication of pollination with capsule formation, without producing viable seeds (Burg & Dijkman, 1967; Arditti et al., 1971). High level of incompatibility was observed Phaius with other Orchid genera, like sympodial orchid viz., Calanthe, Coelogyne, Phalaenopsis, Lycaste, Dienia, Cymbidium, Thunia, Paphiopedilum, Coelogyne, Eria and monopodial orchids viz., Paplionanthe, Dendrobium, Arundina and Vanda (Table 1). Both the pre and post-zygotic barriers could be the reasons with Phaius for reproductive isolation between species and the genera for unsuccessful hybridizations (Edmands, 2002). The long journey of pollen grains that usually stimulate ovule formation after pollination, which is distinct in orchid reproductive system (Arditti, 1992) and hurdles in pollen tube elongation after pollen germination might operate among other incompatible crosses. 3.3 Self and cross compatibility in Phaius In the present study the crosses viz., PBX-11-21 to 23 using P. tankervilleae as female parent and P. flavus as male parent (direct crosses) along with their reciprocal crosses (PBX-11-24 to 26) yielded capsule/ pod formation with viable progeny development. Similarly, except P. flavus the attempted self pollinations in P. tankervilleae formed capsule development along with successful progeny development. It is indicates the cross compatibility nature of species among Phaius and self compatible nature within species. The true hybridity of crosses was confirmed with F1 progeny testing and selfed progenies resulted female type of plants only. 3.4 Description of novel of Phaius cross (PBX-11-22) The data was collected on mature flowering plants (F1) of Phaius cross (PBX-11-22) based on ‘Common Descriptors of Orchids’ essentially required by Plant breeders (Table 2). The comparative descriptions of hybrid with parental species for demarcation of floral traits are shown in Table 3. The new hybrid plant (Figure 1), inflorescence (Figure 2) and as well as flower configuration along with lip differences was depicted in Figure 3 for identification of specific characters. The floral configuration of novel Phaius hybrid progeny (PBX-11-22) is compared on both front and ventral view to assess the exact shape, size and colour of parts (Figure 4). The floral parts of P. tankervilleae, which was used as one of the parent depicted separately for trait comparison (Figure 5). Other parent, P. flavus (#Old NOAC 659) was lost during course of time from the active collections, due to disease infection and limited population; but morphological characters were compared from previous year’s data. The reciprocal combination of same cross, where P. flavus and P. tankervilleae were used as female and male parents (PBX-11-25/01) flowered during July-Aug, 2018. The morphological features of plants derived from reciprocal cross are similar to direct cross, except smaller size of flowers and light tinge of pale green colour on tips of sepals and petals (Figure 6). Phaius flowers produced from both direct (PBX-11-22) and reciprocal (PBX-11-25) crosses are found to be similar in flower traits and colour. The characterization of progenies at F1 stage, indicate the novel features with combined features of floral characters from both parents. The flower colour found to be more attractive due to rich yellow-orange colour, than the yellow coloured P. flavus. The size of sepals and petals were found be intermediate; but shape was similar to P. tankervilleae with acute tip. Characters of lip found to be grand with distinct and dark crimson colour with thick stripes starting adjacent to three obscure calli extending up to tip of terminal lobe and spreading all over anterior lobe on yellow-orange back ground. Lateral lobes of lip were erect and semi-open covering above column, similar to P. flavus and lip colour sometimes extended towards the lower margin of lateral lobes. In case of P. tankervilleae the lateral lobes were overlapped, which was not found in any Phaius F1 progenies. A similar cross was noticed as ‘Joan Hart’ by G. Coram from web reference (http://orchids.wikia.com/wiki/Phaius_Joan_Hart; Orchidwiz, 2013) with similar features, but morphological description of plant was not available. In several other countries, Phaius species ranked as endangered species (Harrison et al., 2004; Simmons et al., 2017) and efforts were lacking to develop hybrids through systematic breeding approaches. Hence, the present novel Phaius breeding lines developed can serve as model to develop new varieties. Conclusion This scientific study could be useful for botanists and taxonomist to reassess genetic diversity among species of Phaius and associated gene flow. High incompatibility with other orchid genera indicates narrow genetic base, but found to be compatible within genera and also self compatible. The new primary hybrid from indigenous species developed can serve as an ideal reference material for taxonomical studies and can useful as genetic stock for cultivation as terrestrial plants with good adaptability in sub-tropical conditions. Population development at F2 generation may unravel the genetic control of various traits and assist in construction of classical genetic map in Phaius. Further improvement can be done through markers, mutation breeding and hybridization with its near relatives like Calanthe etc. Acknowledgement Authors thank the Dr. R. P. Medhi, Former Director of the institute and acknowledge the contribution of orchid germplasm collections of genus Phaius during 1999-2004 under NATP project by (late) Dr. V. Nagaraju, then Acting Director and Principal Scientist (Horticulture). Authors also thank skilled supporting staff Shri. Dawa Bhutia and Shri. Arjun Gurung for proper field maintenance of Phaius crossed progenies. Conflict of interest Authors hereby declare that there is no conflict of interest that could possibly arise.