Functional implications of the specialized staminal appendages in alpine ginger (Roscoea spp.: Zingiberaceae).

Floral organs are widely believed to enhance the pollination and reproductive success of angiosperms. However, the functional implication of some floral structures is still unknown. In this study, we explored the functional role of staminal appendages on male and female reproductive success of Himalayan Roscoea spp. and tested if their function differed between species with biotic pollination and autonomous selfing. Phenotypic manipulation is a powerful approach to test the functional effect of a particular trait on plant fitness. We compared various proxies of pollination success between intact flowers and flowers with manually excised staminal appendages. We found that the rate of visitation did not differ between intact and manipulated flowers. Our results revealed that in outcrossing Roscoea spp., the staminal appendages act as triggering devices to facilitate pollen release and deposition and also to manipulate the foraging position of pollinators to ensure both male and female reproductive success. In contrast, in autonomously selfing Roscoea spp., the removal of staminal appendages did not affect any aspect of pollination processes. Our results suggest that the staminal appendages are an integral component of outcrossing in Roscoea spp. and are maintained by selection pressure through both male and female reproductive success. This study provides important insights on how variation in breeding systems can provoke changes in the structure and function of floral organs among congeners.

Water mediates fertilization in a terrestrial flowering plant.

Water-mediated fertilization is ubiquitous in early land plants. This ancestral mode of fertilization has, however, generally been considered to have been lost during the evolutionary history of terrestrial flowering plants. We investigated reproductive mechanisms in the subtropical ginger Cautleya gracilis (Zingiberaceae), which has two pollen conditions - granular and filiform masses - depending on external conditions. We tested whether rain transformed granular pollen into filiform masses and whether this then promoted pollen-tube growth and fertilization of ovules. Using experimental manipulations in the field we investigated the contribution of water-mediated fertilization to seed production. Rain caused granular pollen to form filiform masses of germinating pollen tubes, which transported sperm to ovules, resulting in fertilization and seed set. Flowers exposed to rain produced significantly more seeds than those protected from the rain, which retained granular pollen. Insect pollination made only a limited contribution to seed set because rainy conditions limited pollinator service. Our results reveal a previously undescribed fertilization mechanism in flowering plants involving water-mediated fertilization stimulated by rain. Water-mediated fertilization is likely to be adaptive in the subtropical monsoon environments in which C. gracilis occurs by ensuring reproductive assurance when persistent rain prevents insect-mediated pollination.

Reproductive isolation in alpine gingers: How do coexisting Roscoea (R. purpurea and R. tumjensis) conserve species integrity?

Multiple barriers may contribute to reproductive isolation between closely related species. Understanding the relative strength of these barriers can illuminate the ecological factors that currently maintain species integrity and how these factors originally promoted speciation. Two Himalayan alpine gingers, Roscoea purpurea and R. tumjensis, occur sympatrically in central Nepal and have such similar morphology that it is not clear whether or how they maintain a distinct identity. Our quantitative measurements of the components of reproductive isolation show that they are, in fact, completely isolated by a combination of phenological displacement of flowering, earlier for R. tumjensis and later for R. purpurea, and complete fidelity of visitation by different pollinator species, bumblebees for R. tumjensis and a long-tongued fly for R. purpurea. Furthermore, the nectar of R. tumjensis flowers is available to the shorter tongued bumblebees while R. purpurea nectar is less accessible, requiring deep probing from long-tongued flies. Although flowering phenology is a strong current barrier that seemingly obviates any need for pollinator discrimination, this current pattern need not reflect selective forces occurring at the initial divergence of R. tumjensis. There has been considerable pollinator switching during the radiation of the Himalayan Roscoea, and the association of flowering time with type of pollinator in these sympatric species may have originated among the earliest or latest flowering individuals or populations of an ancestor to exploit either bumblebee activity early in the breeding season or long-tongued fly abundance later in the season. These two sympatric Roscoea species add to accumulating evidence of the primacy of prezygotic pollination traits in speciation among angiosperms even in the absence of postzygotic incompatibility.

Ginger and the beetle: Evidence of primitive pollination system in a Himalayan endemic alpine ginger (Roscoea alpina, Zingiberaceae).

The Himalayan endemic alpine genus Roscoea, like other members of ginger family, exhibits the combination of floral traits that would fit pollination by long distant foragers such as bees, birds or flies. We studied the pollination biology of Roscoea alpina, observed potential floral visitors and determined their foraging behaviour, visitation frequency and pollination efficiency, to seek evidence in support of the pollination syndrome hypothesis. We also measured the floral spectra of R. alpina flowers to evaluate if signals fit with the currently known framework for observed floral visitors. We found that R. alpina have autonomous selfing and pollinator-mediated crossing, but lack apomixis. We observed that a beetle (Mylabris sp.), and a moth (Macroglossum nycteris) visit the flowers of R. alpina for pollen and nectar feeding respectively. Our field observations, the stigmatic pollen count and fruit set data indicated that the visit by the beetle was legitimate, while that of the moth was illegitimate. Emasculated flowers visited by beetles set as many fruits and seeds/fruit as auto-selfed and naturally pollinated flowers, while emasculated flowers excluded from beetle visits did not set fruit and seed; indicating that a single visit of a beetle to the flowers of R. alpina can facilitate pollination. We found that flower spectral signal of R. alpina does not fit typical spectra previously reported for beetle or bee-visited flowers. Our results suggest that, to ensure reproductive success in alpine habitat, R. alpina has evolved autonomous selfing as a predominant mode of reproduction, while beetle pollination would promote genetic diversity of this plant species. The visitation of beetles to the flowers of R. alpina, despite floral signal mismatch with the classically associated beetle vision, suggests that a different visual processing may operate in this plant-pollinator interaction at high altitudes.

Leaf structure affects a plant's appearance: combined multiple-mechanisms intensify remarkable foliar variegation.

The presence of foliar variegation challenges perceptions of leaf form and functioning. But variegation is often incorrectly identified and misinterpreted. The striking variegation found in juvenile Blastus cochinchinensis (Melastomataceae) provides an instructive case study of mechanisms and their ecophysiological implications. Variegated (white and green areas, vw and vg) and non-variegated leaves (normal green leaves, ng) of seedlings of Blastus were compared structurally with microtechniques, and characterized for chlorophyll content and fluorescence. More limited study of Sonerila heterostemon (Melastomataceae) and Kaempferia pulchra (Zingiberaceae) tested the generality of the findings. Variegation in Blastus combines five mechanisms: epidermal, air space, upper mesophyll, chloroplast and crystal, the latter two being new mechanisms. All mesophyll cells (vw, vg, ng) have functional chloroplasts with dense thylakoids. The vw areas are distinguished by flatter adaxial epidermal cells and central trichomes containing crystals, the presence of air spaces between the adaxial epidermis and a colorless spongy-like upper mesophyll containing smaller and fewer chloroplasts. The vw area is further distinguished by having the largest spongy-tissue chloroplasts and fewer stomata. Both leaf types have similar total chlorophyll content and similar  F v/F m (maximum quantum yield of PSII), but vg has significantly higher F v/F m than ng. Variegation in Sonerila and Kaempferia is also caused by combined mechanisms, including the crystal type in Kaempferia. This finding of combined mechanisms in three different species suggests that combined mechanisms may occur more commonly in nature than current understanding. The combined mechanisms in Blastus variegated leaves represent intricate structural modifications that may compensate for and minimize photosynthetic loss, and reflect changing plant needs.

The comparative reproductive biology of a tetraploid species, Hedychium villosum, and its diploid progenitor H. tenuiflorum (Zingiberaceae).

The evolutionary advantages of polyploidy may result from a number of changes in floral traits and breeding system, which may enable polyploids to exploit new habitats and become widespread. In this study, we comparatively investigated the floral biology of the tetraploid species Hedychium villosum and its diploid progenitor H. tenuiflorum, to assess reproductive divergence between the two species. The results showed that flowers of the tetraploid species last longer and produce more nectar than did diploid species. The flowering times of the two species did not overlap at all. Observations of floral visitors in natural populations demonstrated that butterflies and hawkmoths were effective pollinators of both species, but there was a significant difference in butterfly and hawkmoth assemblages between the two species. The hand-pollination experiments and pollen tube growth experiments suggested that diploid H. tenuiflorum was self-incompatible, while tetraploid H. villosum was completely self-compatible. H. villosum has a much wider distribution range and occupies more diverse habitats than H. tenuiflorum. Polyploidisation may enable tetraploid H. villosum to exploit new habitats previously unavailable to diploid H. tenuiflorum.

Stigmatic fluid aids self-pollination in Roscoea debilis (Zingiberaceae): a new delayed selfing mechanism.

BACKGROUND AND AIMS: Delayed selfing is the predominant mode of autonomous self-pollination in flowering plants. However, few delayed selfing mechanisms have been documented. This research aims to explore a new delayed selfing mechanism induced by stigmatic fluid in Roscoea debilis, a small perennial ginger. METHODS: Floral biology and flower visitors were surveyed. The capacity of autonomous selfing was evaluated by pollinator exclusion. The timing of autonomous selfing was estimated by emasculation at different flowering stages. The number of seeds produced from insect-pollination was assessed by emasculation and exposure to pollinators in the natural population. The breeding system was also tested by pollination manipulations. KEY RESULTS: Autonomous self-pollination occurred after flowers wilted. The stigmatic fluid formed a globule on the stigma on the third day of flowering. The enlarged globule seeped into the nearby pollen grains on the fourth flowering day, thus inducing pollen germination. Pollen tubes then elongated and penetrated the stigma. Hand-selfed flowers produced as many seeds as hand-crossed flowers. There was no significant difference in seed production between pollinator-excluded flowers and hand-selfed flowers. When emasculated flowers were exposed to pollinators, they produced significantly fewer seeds than intact flowers. Visits by effective pollinators were rare. CONCLUSIONS: This study describes a new form of delayed autonomous self-pollination. As the predominant mechanism of sexual reproduction in R. debilis, delayed self-pollination ensures reproduction when pollinators are scarce.

Reproductive biology of two Himalayan alpine gingers (Roscoea spp., Zingiberaceae) in China: pollination syndrome and compensatory floral mechanisms.

According to the concept of pollination syndromes, floral traits reflect specialisation to a particular pollinator or set of pollinators. However, the reproductive biology of endemic, and often specialised, plants may require increased attention as climate change accelerates worldwide. Species of Roscoea endemic to the Himalayan region have striking orchid-like flowers with long corolla tubes, suggesting pollination by long-tongued insects. Until now, the reproductive biology of species of Roscoea has been poorly documented. We investigated the floral biology, breeding system and pollination ecology of R. cautleoides and R. humeana, from Hengduan Mountains, a global biodiversity hotspot in southwest China. We also tested whether floral longevity increases pollination success. Pollination experiments showed that the two species were self-compatible and depended on insects for fruit production. Over several flowering seasons we did not observe any potential pollinators with long tongues that matched the corolla tube visiting flowers in centres of distribution. The principal pollinators observed were pollen-collecting generalist bees, with low visitation frequencies. In general, members of the ginger family are characterised by short-lived (usually 1 day) flowers, but flowers of R. cautleoides and R. humeana last 8 and 6 days, respectively. Removing stigmas decreased fruit set in both study populations. Our results suggest that the original pollinators may have been long-tongued insects that are now absent from the Chinese Himalayas because habitats have responded to climate change. However, long-lived and self-compatible flowers, coupled with the presence of generalist pollinators, are traits that have allowed these gingers to reproduce and continue to persist in the alpine habitats.

Long-term conservation through cryopreservation of immature seed of Mantisia spathulata and Mantisia wengeri; two endangered plants of north-east India.

A successful protocol for long-term conservation of two endangered plants viz. Mantisia spathulata and M. wengeri has been devised through cryopreservation of immature seeds. Immature seeds of both the species were precultured in 0.6 M Sucrose and 2 M Glycerol for 3 h at 24 ± 2 degree C. Precultured seeds were then desiccated under the airflow of 27 ± 3 m min -1 velocity inside laminar air flow cabinet for different time periods. The seeds were then cryostored in liquid nitrogen for an hour. A maximum of 40 percent and 36.6 percent seed germination was recorded after cryostorage at moisture contents of 26.0 percent and 16.2 percent for M. spathulata and M. wengeri respectively. To protect these rare plants against loss due to disease, insect damage, or natural disaster a back up collection has been established using the protocol and applied to a large number of immature seeds that were obtained from the ex situ plants growing in the experimental garden of the North-eastern Hill University, Shillong.

Rapid multiplication and in vitro production of leaf biomass in Kaempferia galanga through tissue culture

An efficient protocol has been established for rapid multiplication and in vitro production of leaf biomass in Kaempferia galanga L, a rare medicinal plant. Different plant growth regulators like Benzyladenine (BA), Indoleacetic acid (IAA), Indolebutyric acid (IBA), Napthaleneacetic acid (NAA) and adenine sulphates (Ads) have been tried for induction of multiple shoots using lateral bud of rhizome as explants. The highest rate of shoot multiplication (11.5 +/- 0.6) shoot/explant as well as leaf biomass production (7.4 +/- 0.3) gram/explant was observed on Murashige and Skoog medium supplemented with Benzyladenine (1 mg/l) and Indoleacetic acid (0.5 mg/l). Data of shoot multiplication and leaf biomass production were statistically analysed. Upon excission of leaves after 2 months of culture under sterile condition, the base of each plantlet was transferred to fresh media which could produce the same leaf biomass within another 2 months in a 50 ml culture tube containing 20 ml and 250 ml conical flasks containing 30 ml Murashige and Skoog medium. The rate of multiplication and leaf biomass production remained unchanged in subsequent subcultures. The regenerated plantlets were acclimatized in greenhouse and subsequently transferred to the field. Survival rate of the plantlets under ex vitro condition was 95 percent. Genetic fidelity of the regenerants was confirmed using random amplified polymorphic DNA (RAPD) marker. The protocol could be commercially utilized for large scale production of true-to-type plantlets and as an alternative method of leaf biomass production in Kaempferia galanga.

Caracterização anatômica e fitoquímica de folhas e rizomas de Hedychium coronarium J. Kõnig (Zingiberaceae) / Anatomical and phytochemical characterization of leaves and rhizomes from Hedychium coronarium J. Kõnig (Zingiberaceae)

Devido a grande potencialidade na utilização de Hedychium coronarium, na medicina popular e também como biorremediadora no tratamento de efluentes, objetivou-se uma diagnose dos órgãos, folha e rizoma, para elucidar resultados estruturais e fitoquímicos. A folha é anfiestomática, com predominância de estômatos na face abaxial. Em ambas as superfícies foliares há projeções de cera epicuticular sobre as paredes anticlinais das células epidérmicas. O mesofilo dorsiventral apresenta hipoderme multisseriada (3 camadas) em ambos os lados. O parênquima clorofiliano é diferenciado em paliçádico (1-2 camadas) e lacunoso (4-5 camadas) com muitos espaços intercelulares e ocorrência de idioblastos cristalíferos. Na nervura central, o aerênquima ocorre em único arco na região abaxial. Os feixes vasculares distribuem-se aleatoriamente e são de diferentes tamanhos, pequenos, médios e grandes, envolvidos por fibras. Os feixes menores localizam-se no lado abaxial da nervura. A triagem fitoquímica das folhas mostrou a presença de saponinas e ausência de taninos, antraquinonas, alcalóides e flavonóides. Por meio de Cromatografia em Camada Delgada foram identificadas as presenças de cariofileno e mirceno no óleo essencial bruto obtido a partir das folhas de H. coronarium.

Due to the great potentiality regarding the use of Hedychium coronarium in folk medicine and also as a bioremediator in effluent treatment, this study aimed to diagnose leaf and rhizome in order to elucidate structural and phytochemical results. Hedychium coronarium leaf is amphistomatal, with predominance of stomata on the abaxial surface. On both leaf surfaces, there are epicuticular wax projections over the anticlinal walls from epidermal cells. The dorsiventral mesophyll presents multiseriate (3 layers) hypoderm on both sides. The chlorophyllian parenchyma is differentiated into palisade (1-2 layers) and spongy (4-5 layers) with many intercellular spaces and some crystalliferous idioblasts. In the midrib, the aerenchyma occurs in a single arc on the abaxial surface. The vascular bundles are randomly distributed and present different sizes: small, medium and large, involved by fibers. The smallest bundles are found on the abaxial rib surface. The leaf phytochemical analysis showed the presence of saponins and lack of tannins, anthraquinones, alkaloids and flavonoids. Using thin-layer chromatography, the presence of caryophyllene and myrcene was detected in the crude essential oil obtained from H. coronarium leaves.

High frequency plantlet regeneration from rhizomatous buds in Mantisia spathulata Schult. and Mantisia wengeri Fischer and analysis of genetic uniformity using RAPD markers.

A protocol has been devised for enhanced in vitro regeneration of critically endangered Mantisia spathulata Schult. and Mantisia wengeri Fischer. Highest Bud Forming Capacity (BFC) of 6.10 +/- 0.55 with an average of 19.93 +/- 3.19 roots was obtained for M. spathulata within 5-6 weeks in Murashige and Skoogs (MS) medium supplemented with a combination of 10.0 microM of N6-benzyladenine (BA) and 2.5 microM of alpha-naphtalene acetic acid (NAA). For M. wengeri, BFC of 7.82 +/- 0.73 and 20.86 +/- 1.65 roots was achieved in MS media supplemented with a combination of 5.0 microM BA and 2.5 microM of NAA RAPD markers were used to evaluate the genetic stability of in vitro raised hardened plantlets. Similarity coefficient among the regenerated plants ranged between 0.85-0.98 for M. spathulata and 0.83-0.98 for M. wengeri. Maximum of 88 and 90% genetic similarity were obtained between in vitro raised hardened plantlets and mother stock of M. spathulata and M. wengeri, respectively through RAPD analysis. The hardened plantlets after RAPD analysis on being transferred to soil of experimental garden showed no marked phenotypic variations in vegetative or floral characteristics.

Autonomous selfing provides reproductive assurance in an alpine ginger Roscoea schneideriana (Zingiberaceae).

BACKGROUND AND AIMS: Reproductive assurance, the ability to produce seeds when pollinators or mates are scarce, is thought to be the major advantage of selfing in flowering plants. However, few studies have performed a direct cost-benefit analysis of the selective advantage of selfing, particularly given a long-term perspective among populations or across several flowering seasons within population. This study examined the fertility consequences of autonomous selfing in Roscoea schneideriana (Zingiberaceae), a small perennial Himalayan ginger typically found in habitats at around 3000 m a.s.l. METHODS: The floral biology of R. schneideriana was studied in natural populations; the capacity for autonomous selfing was estimated using pollinator exclusion experiments; the timing of selfing was quantified by anther removal at different times during flowering; whether autonomous selfing increases seed production was tested by emasculating flowers; and the magnitude of inbreeding depression was estimated by comparing relative performance of progeny from self- and cross-pollinations. Pollinator observations were also conducted in the natural populations. KEY RESULTS: The hooked stigmas of most flowers curl towards the anther and can contact pollen grains at an early stage of anthesis. Flowers with potential pollinators excluded set of as many seeds per fruit as hand-selfed and opened flowers. Autonomous selfing mostly occurs within 2 d of anthesis and can increase seed production by an average of 84 % in four populations during the flowering seasons of 2005-2007. Visits by effective pollinators were extremely rare. The cumulative inbreeding depression of R. schneideriana was 0.226. CONCLUSIONS: Autonomous selfing in R. schneideriana is achieved by stigmas curling towards the anthers early in flowering. It is suggested that under the poor pollination conditions, autonomous selfing has been selected for in this alpine ginger because it provides substantial reproductive assurance with very low costs.

Botany: a new self-pollination mechanism.

Pollen grains from most flowering plants are transported by wind or animals and deposited on the receptive surface of the stigma of a different individual, but self-pollination is also common. We have discovered a new process for self-pollination in the laterally orientated flowers of a Chinese herb, in which a film of pollen is transported from the anther (pollen sacs) by an oily emulsion that slides sideways along the flower's style and into the individual's own stigma. This mode of self-pollination is a new addition to the broad range of genetic and morphological mechanisms that have evolved in flowering plants (angiosperms), and may be common in species growing in shady, windless and insect-poor habitats.

In vitro multiplication in Kaempferia galanga linn.

Kaempferia galanga is an important medicinal plant that is facing threat of extinction owing to indiscriminate and unsustainable harvesting in the wild. Conventional breeding is difficult in this plant, and in vitro multiplication is important to conservation and propagation. Leaf and rhizome explants of Kaempferia were aseptically cultured on MS medium with various combinations of indole-3-acetic acid (IAA), benzyl amino purine (BAP), napthalene acetic acid (NAA), 2-4-dichlorophenoxy acetic acid (2,4-D) and kinetin at concentrations ranging from 0.5 to 2.5 mg/L. High-frequency organogenesis and multiple shoot regeneration was induced from rhizome explants on MS medium supplemented with 0.5 mg/L of IAA and 2.5 mg/L of BAP. Rooting was induced in MS medium with 0.5 mg/L of IAA and 2 mg/L of BAP.

Pollen histochemistry and pollen : ovule ratios in Zingiberaceae.

BACKGROUND AND AIMS: Pollen grains of 37 species from 11 genera in the family Zingiberaceae were examined to assess qualitatively starch or lipid contents; the pollen grain and ovule numbers per flower and pollen : ovule ratios were also counted and calculated. Pollen : ovule ratios were studied to reveal patterns of variation in the Zingiberaceae. METHODS: Freshly open flowers with dehiscing anthers were collected at random from plants growing in natural habitats or in botanical gardens. Presence of lipids or starch in pollen grains was tested by Sudan solution and IKI solution, respectively, and examined under a microscope. To estimate the pollen and ovule numbers per flower, one anther from each bud was carefully dissected and all pollen grains were counted; ovaries were carefully dissected out of each flower and counted. Whenever possible, at least 10-15 buds were used in the determination. KEY RESULTS: Thirty-three of all the 37 species examined had starchy pollen. Starch was not found in only four species and lipid was not found in only one species; among the four tribes in subfamily Zingiberoideae, all species of Zingibereae and Globbeae had pollen with no starch, Alpineae and Hedychieae had pollen with and without starch, whereas, all species of subfamily Costoideae had starchy pollen with abundant lipids. The mean pollen : ovule ratios in the members of the Zingiberaceae investigated range from 3.25 +/- 1.56 to 616.52 +/- 117.83. CONCLUSIONS: The pollen nutrition types seemed not related to mating systems. The pollen : ovule ratios in members of the Zingiberaceae with the same breeding system are noticeably lower than those recorded by previous authors. The lower pollen : ovule ratios in this family are presumed to be related to the highly efficient pollination systems, mediated by pollen which can be quite glutinous and the relatively large stigma area. In most of the Alpinia species the anaflexistylous flowers have much larger numbers of pollen grains and higher pollen : ovule ratios than the cataflexistylous flowers. There are significant differences in mean pollen grain numbers and pollen : ovule ratios between different life forms but ovule numbers are approximately the same.