Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Native Hawaiian Ferns: The Stage IV acclimation of two native Hawaiian ferns, palapalai (Microlepia strigosa and hapu'u (Cibotium chamissoi) were satisfactorily met. In addition a culturally important Hawaiian fern, ho'i'o (Diplaziium sandwichianum) was successfully propagated and acclimated. Paphiopedilum orchids: A protocol for culture of shoot explant in vitro was successfully developed for 5 species and 8 hybrids as well as for a species of genus Phragmipedium. Lycopediella: A protocol for in vitro clonal propagation of Lycopodiella cernua was developed. Psilotum: The same protocol as developed for Lycopodiella was also successful with Psilotum nudum. Huperzia: For Huperzia phyllantha growth was obtained only when inorganic salt formulation of MS was diluted to 0.1X concentration in nutrient medium. Orchid: Germplasm of Phalaenopsis amabilis variety rosenstromii was successfully increased in vitro from seed and established ex vitro. Information on Native Hawaiian Ferns were disseminated by presentation of workshops for faculty, graduate students, and conservation groups. Information on paphiopedilum was presented at seminar for the Orchid Growers of Hawaii. This represented the first successful protocol for the initiation of in vitro culture of this genus in the over 50 years since in vitro culture has been applied for micropropagation of orchids. PARTICIPANTS: Training was extended to two 10 month terms for postgraduate student. TARGET AUDIENCES: Target audiences were for graduate students in conservation, horticulturists interested in expansion of marketable goods. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The successful rapid micropropagation of native Hawaiian ferns provides the industry of marketable plants as well as another product for use in landscape construction. Success in culture of hapu'u should enable initiation of studies on the time required for the restoration of tree fern forests. Clonal plants of Psilotum and other related plants should enable further studies of plant extracts, e.g. floral preservatives as detected in use with live flowers. Success in rapid and reliable clonal propagation of paphiopedilum orchids foretell the addition of this orchid to the current expanding orchid cut flower and potted plant industry.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: An acclimation protocol initiated in late 2010 has improved the survival rate for micropropagatied palapalai (Microlepia strigosa). It consists of prehardening plantlets in the laboratory, by holding them in open flasks of water (50 ml) for one month, before potting. "Hydroculture" does not seem to work with hapu'u (Hawaiian tree fern) sporelings, and other Stage IV trials were begun in 2011. In these trials, the sporelings, are placed in various containers with peat-perlite medium moistened with either MS salts (l/2 or 1/4 strength) or Miracle Gro. The nutrient mediua for growing explants of Huperzia phyllantha and Lycopodium venustulum have been established. A protocol for establishment of aseptic cultures of Paphiopedilum species and hybrids from selected plants grown in the greenhouse has been established. PARTICIPANTS: Two non-compensated researchers and emeritus researcher are currently involved with this project. TARGET AUDIENCES: Commercial growers, landscapers,and foresters interested in diversifying their inventory should be interested in this project. Project also has solved a problem in initiation of clonal propagation of select species and hybrids of orchid group which has escaped solution for over 60 plus years. PROJECT MODIFICATIONS: None
Impacts
Data for 2011, compared to previous years of this project, show that hydroculture has improved the survival rate of palapalai from 54 percent to 82 percent. It's too soon to know the outcome of the new trials with hapu'u, but the low survival rate is expected to improve. Project results to date were publicly reported through two poster presentations in 2011. One in six native Hawaiian vascular plants is a fern, yet they are seldom available. Having protocols for two important species already is increasing the availability. The procedures used may offer models for making additional Hawaiian fern species available for use in forest restoration, landscaping and Hawaiian cultural practices. Explants of two more paphiopedilum hybrids are now growing in culture, proving thus far that our test nutrient medium is suitable for initiating cultures.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The Stage IV acclimation method increases the survival rate for palapalai fern, production timetable and reduces labor. It increases the availability of true Hawaiian palapalai fern by industry. Timetable for hapu'u production justifies cost of these plants. Few plants given to local botanical gardens survive well. Hawaiian Fern Project has been described in "GREEN: Hawaii's Sustainable Living Magazine in Fall 2010. Of 11 selections of paphiopedilum, 9 clones have been successfully initiated. Explants are most successful when stock plants are grown in well drained medium. Several paphiopedilum clones have proliferated well and removed ex vitro. PARTICIPANTS: Two non-compensated cooperators and a Visiting Colleague are currently involved with this project. TARGET AUDIENCES: Commercial growers of ferns especially for landscaping have realized and accepted use of plants produced by in vitro techniques owing to the uniformity of the product. Success in micropropagation of selected paphiopedilum plants should effect a change in the inventory of quality plants for the industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A few of the hapu'u ferns established ex vitro have been distributed to local botanical gardens and have been successfully established. Potential for establishment of regeneration time for this fern is excellent. Demand and acceptance by commercial growers for palapalai fern produced through micropropagation high. Description of micropropagation of paphiopedilum for industry in preparation.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Ferns: A small number of spores sown in vitro of hapu'u (Cibotium chamissoi), an endemic Hawaiian tree fern, has produced plants. The endemic fern kilau (Dropteris glabra) was propagated in vitro. Orchids: Paphiopedilum seeds sown in vitro on sterile solid agar medium germinated very well. Liquid medium was not suitable. Applicability of propagation of paphiopedilum in vitro of more selected horticulturaly useful plants appear feasible. Lycopodium: Production of plantlets in vitro was successful in all in vitro steps but transfer to in vivo conditions has not been successful. PARTICIPANTS: Two non-compensated cooperators and a Visiting Colleague are currently involved with this project. TARGET AUDIENCES: Commercial growers interested in diversifying their inventory should be interested in this project. Professional and commercial companies who propagate orchids should find information new and relevant. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Ferns: Plants of hapu'u derived from spores grown in vitro have been distributed to three botanical gardens and to the public through the gardens' plant sales. Orchids: Paphiopedilum seeds germinated best on low nutrient salt medium. Upon germination of paphiopedilum seeds on solid agar medium, numerous multiple shoots were formed. In liquid medium paphiopedilum seeds did not germinate.
Publications
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: A procedure for the micropropagation of indigenous Hawaiian fern palapalai (Micropepis strigosa) has been refined. Plantlets were distributed to Army Ecosystem Restoration Program. Several hybrids of paphiopedilum (orchid) have been successfully initiated in aseptic culture. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Army Ecosystem Restoration Program on Oahu has reported that some of the first indigenous palapalai fern produced by project and outplanted in 2006 are thriving in the wild and cannot be distinguished from natural populations if it were not for the markers. Hawaiian tree fern spores have been germinated and limited number of plantlets derived. This may enable, some day, to initiate a project on the rate of restoration of Hawaiian tree fern forests. Information derived on explanting of paphiopedilum orchid hybrids is still too preliminary for dissemination.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Since the demand for best quality Hawaiian palapalai (palapalai from the Hawaiian gene pool) continues, further laboratory experiments have been initiated. Another indigenous fern, neke, is also available for freshwater wetland restoration. Stage IV research with hapu'u utilizes a mist bench. Another species of hapu'u is being tried. Aseptic cultures of 8 Paphiopedilum accessions were initiated. One explant each from 2 accessions has already grown into rooted plantlet. Preliminary results indicate that coconut water is required in the nutrient medium for successful culture initiation. Through invitro culture of young embryos, a new interspecific hybrid between Brassavola (Rhyncolaelia) glauca and Brassavola nodosa has been produced.
PARTICIPANTS: Yoneo Sagawa, PhD. Professor of Horticulture John T. Kunisaki, M.S. Assistant Horticulturist, Retired Kay Lynch, B.S.
TARGET AUDIENCES: Target audience is conservationists, nurseries, hybridizers.
PROJECT MODIFICATIONS: Success in explanting sterile cultures depend not only the conditions in external environment when going from in vitro to ex vitro but also on the stage of growth within the in vitro environment.
Impacts
Further manipulation of in vitro cultures of palapalai is essential for more efficient rapid micropropation. Neke, an indigenous fern, is now available for freshwater wetland restoration projects. Growth stage for explanting of sterile cultures is important for survival. Coconut water is required in nutrient medium for initiation of paphiopedilum cultures. Embryo culture in vitro is essential for production of unique hybrids.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
The efficiency of micropropagation for the production of Hawaiian palapalai fern for native forest restoration was demonstrated. Paphiopedilum orchid explants are being grown into plantlet stage in vitro for clonal increase. No further progress on lycopodium and Hupererzia. Incompatibility between two genera and two species of orchids has been overcome by use of in vitro technique.
Impacts
Development of commercially viable protocol for clonal increase of paphiopedilum should assist orchid industry to meet the increasing demand commercially for these orchids. Hawaiian palapalai fern is a tool available for restoration projects and for use in ecological research. Overcoming species and genera incompatilities of orchids should provide a broader spectrum of unique hybrids and germplasm for future breeding.
Publications
- Kim, Mi-Seon, C. W. Morden, Y. Sagawa, J. Kim 2003. The Phylogeny of Phalaenopsis Species. Proc. of Nagoya International Orchid Conf. 2003, 41-43.
- Sagawa, Y. and W. Rehrig 2005. Observations on Phalaenopsis Research: Past, Present and Future. Taiwan International Orchid Symposium 2005, 364-376.
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Progress 10/01/04 to 09/30/05
Outputs
Our surface sterilization and explanting procedure for aseptic culture initiation was successfully tested on a lycopodium, Hupererzia phallantha and a culture-recalcitrant orchid genus, Paphiopedilum. Three new fern species - pala'a (Spenomeris chinensis), neke (Cyclosorus interruptus) and laukahi (Pneumatopteris hudsoniana) were successfully placed into aseptic culture. Paphiopedilum XSusan Tucker which was micropropagated from plant ex vitro was successfully flowered in the greenhouse.
Impacts
Success in development of surface sterilization and explanting procedure will be useful for other species. More native Hawaiian ferns can be placed into culture. Flowering of paphiopedilum explanted from ex vitro plant will enable commercial production of clonal plants since paphs are destined to become the second orchid genus destined for mass production.
Publications
- Liang, H., Sagawa, Y. and Li, Q. 2005. Effects of rutin on vegetative growth of mung bean (Vigna radiata) seedlings and its interaction with indoleacetic acid. Jour. Plant Physio. and Mol. Biol. 31(4):361-368.
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Progress 10/01/03 to 09/30/04
Outputs
Tissue cultured awa 'Mo'i' plantlets were successfully established in greenhouse. With this, work on awa has been completed. Of 85 surviving plants of spathoglottis upon treatment with colchicine, 16 likely and 21 possibly converted plants have been identified via guard cell measurements. Chromosome counts are needed to verify conversion. Comparison of propagation of palapalai via in vitro culture and spore culture in the nursery indicates that in vitro tissue culture is by far more efficient for production of pure and quality plants for commercial production.
Impacts
Development of in vitro micropropagation procedure provides Pacific Island countries the possibility of transfer of sterile germplasm. Identification of tetraploid spathoglottis provide germplasm for production of triploids for release in nature. Comparison of in vitro and ex vitro micropropagation of native forms shows that in vitro system results in pure and quality plants for commercial production.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Micropropagation work on awa varieties, SIG and Mahakea, has been completed. Lycopodium plantlets are being rooted in mixture of perlite/black cinder medium. Psilotum propagules are now at clonal increase stage. Spathoglottis orchid seedlings treated with colchine for various time intervals are large enough ex vitro for analysis. Preliminary work in measurement of guard cells indicate some effects from treatments. A donation of approximately 100 select genotypes of paphiopedilum orchids has been received. Major effort for palapalai fern has been towards production from initiation in vitro through transfer ex vitro and use as landscape plant material.
Impacts
A one week workshop on micropropagation of awa was presented for SPC Plant Protection Service in Suva, Fiji for the benefit of Pacific Island Countries. A one week workshop on orchid culture was presented sponsored by IRETA with representatives from 13 Pacific countries in attendance.
Publications
- Kunisaki, J.T., Araki, A. and Sagawa, Y. 2003. Micropropagation of 'awa. CTAHR Extension Circular. Biotechnology. 4:11 pp.
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Progress 10/01/01 to 09/30/02
Outputs
Orchids: Doritis (an orchid) plantlets derived in vitro from sterile leaf explants have been increased and are ready for transplant ex vitro. Upon establishment ex vitro, fidelity of products will be investigated. Awa: All cultivars are in the final stage of micropropagation, establishment under natural conditions. A laboratory manual for awa micropropagation has been prepared and is awaiting funding for preparation of color brochure by PIO. Lycopodium and Psilotum: Rooting of derived shoots is being attempted in perlite and chopped sphagnum moss under aseptic conditions.
Impacts
Upon establishment of fidelity of leaf micropropagation protocol, a more rapid method will be available to the industry. Success in awa micropropagation makes preservation of Hawaiian awa germplasm feasible. Preservation and exchange of Pacific Island germplasm are also possible.
Publications
- No publications reported this period
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Progress 10/01/00 to 09/30/01
Outputs
Orchids: a protocol for micropropagation of Doritis, an orchid, has been established by use of explants from leaves of various maturity. Awa: Following the procedure developed for the initial established cultivar, three more clones have been increased aseptically and are being rooted and will be transferred to the greenhouse. Psilotum: Other rooting media besides gel are being tested for successful transfer of derived shoots to greenhouse conditions.
Impacts
If leaf explants are successful for production of uniform non-mutant mature plants, there should be an increase in efficiency and speed of micropropagation of orchids. If the procedure for awa is applicable to many cultivars, preservation and exchange of germplasm should succeed.
Publications
- No publications reported this period
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Progress 10/01/99 to 09/30/00
Outputs
Awa: First established cultivar has been rooted and transferred to greenhouse. Other cultivars have been initiated and established. Lycopodium: Shoots are being rooted for transfer to greenhouse conditions. Protea: Attempts to establish cultures of P. cynaroides have not been successful. Psilotum: Established cultures are being clonally increased. Spathoglottis: S. kimballiana seeds have been treated with various concentrations of colchicine and various time periods in vitro; plants have been established in greenhouse. Variants will be tested for ploidy.
Impacts
The first non-proprietary protocol for propagation of awa through tissue culture should be available for dissemination to growers in Hawaii as well as other interested parties especially in the Pacific countries.
Publications
- No publications reported this period
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Progress 10/01/98 to 09/30/99
Outputs
No progress to report. This project was initiated on 10/01/1999.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 10/01/97 to 09/30/98
Outputs
Proteas: Proliferating bodies have emerged on explants of newly selected leucospermum, Hybrid #83, and are being increased clonally. No proliferation has emerged on cultured explants of Hybrid #74 and #93 as yet. Lycopodium: Problem of disinfestation of explants has been overcome. With procedure as develped, at least 6 clones have been initiated into culture. Piper methysticum: Although aseptic culture have been initiated, the disinfestation procedure needs improvements because success rate is very low. Orchids: Since a clone of paphiopedilum has been established, experiments are currently in progress to determine the best way for root induction. Clonal propagation of phalaenopsis is being realigned from explanting shoot tips and axillary buds from vegetative plants to shoots derived from node culture in vitro of inflorescences. Native Hawaiian Plants: Wikstroemia uva-ursi (Akia) has been successfully cultured in vitro and multiplied to commercially useful levels.
Scaevola coriacea has also been successfully multiplied in vitro and removed to in vitro in the nursery.
Impacts
(N/A)
Publications
- WEI, W.S., HWANG, W.-I., KIM , S.Y. and SAGAWA, Y. 1997. Induction of Somatic Embryogenesis in Azadirachta indica. Plant Cell, Tissue & Organ Culture. 50:91-95.
- LYNCH, K. 1997. Toward a Protocol for in vitro Propagation of Scaevola coriacea (Goodeniaceae). Newsletter of Hawn.Bot.Soc. 36:64-65.
- TANIWIDJAJA, C., WEBB, D.T. and SAGAWA, Y. 1998. Micropropagation of Akia (Wikstroemia uva-ursi A.Gray). Plant Cell, Tissue & Organ Culture. 53:85-90.
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Progress 10/01/96 to 09/30/97
Outputs
Orchids: With success in clonal multiplication of paphiopedilum, shoots are now being utilized to determine the best way for rooting. Native Hawaiian Plants: Wikstroemia uva-ursi (Akia) has been successfully cultured in vitro and multiplied to commercially useful levels. Scaevola coriacea has also been successfully mutliplied in vitro and experiments are in progress for removal to in vivo in the nursery. Proteas: Culture of recently selected pin cushion, Leucospermum 'Hybrid 36' x reflexum, was initiated to induce proliferation which will be later subjected to colchicine treatments in attempt to develop a tetraploid of superior horticultural qualities. Piper methysticum: Preliminary work showed that culture initiation of this plant will be difficult due to contamination problems, unless changes to the usual disinfestation process are made. Lycopodium: Work in improving disinfestation of explants and rooting of derived plants is still being continued.
Impacts
(N/A)
Publications
- TENIWIDJAJA,C.,WEBB,D.T. and SAGAWA,Y. 1997. Micropropagation of Akia (Wikstroemia uva-ursi A. Gray). Pl. Cell, Tissue & Organ Culture. (In Press).
- SU, W.W., HUANG, W.I., KIM, S.Y. and SAGAWA, Y. 1997. Induction of somatic embryogenesis in Azadirachta indica A. Juss. Pl., Cell, Tissue & Organ Culture. (In Press).
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Progress 10/01/94 to 09/30/95
Outputs
Lycopodiums: Initial step of disinfestation of explants still needs to be improved. However, enough plantlets have been obtained through the micropropagation process and are being transplanted to pot culture to determine their transferability to natural conditions. Proteas: More Leucospermum cultivars have been placed in culture. Somatic embryos of Protea 'Mayday' have extended shoot growth but have not rooted although treated with NAA. IBA will be tested to promote rooting. Plantlets derived from both friable callus and protoplasts from friable callus of Phalaenopsis #150 when removed to greenhouse are highly variable and difficult to grow. On the other hand, plantlets from friable callus of Phalaenopsis #2423 when removed from invitro to the greenhouse are vigorous growers.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
Analysis of growth response of friable Phalaenopsis orchid callus showed that interms of fresh weight it grows relatively slowly. Addition of IAA or BA to culture medium did not promote growth suggesting that the callus is autotrophic. Growth was also not affected by change of sucrose levels from 2.5 to 30 gms/liter. Upon transfer of callus to medium without sucrose, somatic embryogenesis was induced. Supplements of a wide range of plant growth regulators or nitrogen sources combined with various sucrose levels showed that the dominant factor for somatic embryogenesis was sucrose level. Proteas: Leucospermum 'Pohaku La Hawaii', L. 'Hoku Hawaii', and L. 'Red Sunset' have developed proliferating bodies which are now being increased clonally. Protea 'Mayday' has developed somatic embryos which are being stimulated to grow into plantlets.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
The growth and regenerative capacity of friable callus from phalaenopsis (orchid) appears unaffected after many years of subculture. The plants regenerated from protoplast culture are still being maintained in vitro and are difficult to remove to in vivo conditions. A second callus of phalaenopsis has been initiated and is in multiplication. Tissue culture of a famous named cattleya clone which is positive for CYMV & ORV has been established and is undergoing multiplication. When adequate number of propagules is obtained those will be used for experiments for elimination of virus. Protea: Promising cultivars, Leucaspermum hybrids athryn' and achel' have been cultured and now are being clonally propagated. As yet, cultivars of Protea have not been successfully grown in culture.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
Anthuriums: Problem of establishing in vitro culture of Ozaki, the cultivar grown in greatest number for cut flower production, has been overcome. In comparison to all other successfully cultured cultivars, Ozaki must be established in medium of much lower nutrient salt concentration. Proteas: Medium formulation used successfully for clonal increase of Leucospermum cultivars has been found to be unsuitable for Protea cultivars. Protea cultivars may require lower salt concentrations in the medium because, in test media of salt dilutions up to 0.1X, explants survived for longer periods. Greater dilutions than 0.1X is now being tested. Orchids: Preliminary studies of plants derived from protoplasts indicate presence of chromosomal disturbances as well as retardation in growth. Regenerative capacity of friable callus after numerous passages in culture is also being tested. Success of micropropagation should be based on examination of final products.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
Proteas -- Axilllary bud explants of another genus Protea neriifolia 'Pink Mink'has been successfully established in culture. When explants attain sufficient size, clonal increase will be initiated. Orchids -- A truly friable callus has been isolated for the first time ever from an orchid, Phalaenopsis sp. Manipulation of callus via alteration of sucrose concentration resulted in callus growth at high levels and embryogenesis at low levels. Embryogenic callus has produced embryoids which have differentiated into plants which are beginning to flower in the greenhouse. Fidelity of derived plants has not been established. Protoplasts have been successfully isolated from friable callus which in turn have differentiated plants in vitro -- a first for orchid tissue culture. Upon transfer to greenhouse, fidelity of derived plants will be determined.
Impacts
(N/A)
Publications
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Progress 10/01/88 to 09/30/89
Outputs
Proteas--Better in vitro rooting of Leucospermum microcuttings was obtained in perlite than in agar medium. Roots in perlite possessed root hairs and branched. Another cultivar, Leucospermum 'Hybrid 24' was increased in vitro and established successfully under natural conditions. Orchids--Friable calli have been induced and isolated from Phalaenopsis tissue cultures. Multiplication and differentiation is regulated by manipulation of sucrose in the medium. With supplemental sucrose calli multiply and are orange-yellow in color. Without supplemental sucrose calli turn green and differentiate embryoids and plantlets. This may be the first report of induction, isolation and manipulation of friable calli of orchids. Others--Aseptic microcuttings of Grevillea 'Robyn Gordon' have been successfully rooted in sterile perlite and 1/2 liquid MS medium after pretreatment with IBA. A few plants have been flowered. Review of "Micropropagation of Floriculture Crops" and future areas
for orchid research are reviewed in 2 separate chapters in forthcoming volume 5 of "Handbook of Plant Cell Culture".
Impacts
(N/A)
Publications
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Progress 10/01/87 to 09/30/88
Outputs
Proteas--Plantlets from in vitro culture of Leucospermum hybrid 'Hawaii Gold' were established under natural conditions. Modifications are being made to the final stage of in vitro culture to reduce mortality of plantlets transferred to natural conditions. Another cultivar, Protea hybrid 'Mayday', has increased rapidly and is now being rooted. Australian Plant Introductions: Clonal increase of all introductions was terminated since sufficient number of plantlets were produced. Orchids--There is, as yet, no commercially feasible method for rapid clonal propagation of two commercially important genera, Phalaenopsis and Paphiopedilum. Response of phalaenopsis to in vitro culture is different from other orchids. Instead of adventitious shoots, callus has been induced, multiplied and induced to undergo embryogenesis. Plantlets are being grown to flowering for analysis of stability. Application of suspension and protoplast culture for rapid clonal propagation has been
initiated.
Impacts
(N/A)
Publications
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Progress 10/01/86 to 09/30/87
Outputs
Transfer of rooted plantlets of Leucospermum hybrid 'Hawaii Gold' to natural conditions has not been successful. Even under aseptic conditions plantlets have not been established in vermiculite, perlite, or peat moss or mixtures of these. With procedure developed for 'Hawaii Gold', plantlets have been obtained from propagules of Protea hybrid 'Mayday' and propagules have developed on explants of Leucospermum lineare x L. glabrum. AUSTRALIAN PLANT INTRODUCTIONS: Cultures of Anigozanthus gabrielae, A. humilis, A. manglesii 'Red Emperor', and Macropedia fulginosa have been terminated after sufficient quantities of each were transferred to Maui. Clonal increase is being continued for Actinodium cunninghamii, Lechenaultia hybrid, Verticordia grandis and Chamelaucium megapetalum. To obtain rapid and uniform clonal propagation of horticultural crops, callus of phalaenopsis orchid, which to date has been relatively recalcitrant, was induced from axillary buds on
inflorescences. Basal medium with sucrose produces chlorophyll-less callus whereas deletion of sucrose produces green callus. Embryogenesis was induced and a substantial number of plantlets were obtained. These plantlets when established in the greenhouse will be tested initially for cytological uniformity and finally for morphological uniformity especially when in flower. Protoplast cultures were also obtained from callus cells.
Impacts
(N/A)
Publications
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Progress 10/01/85 to 09/30/86
Outputs
Banana: Approx. 100 derived plantlets of dwarf Brazilian banana were transplanted to the field and are growing uniformly with inflorescence now emerging. Two other varieties from New Zealand are being clonally increased in vitro for subsequent field testing. This work is in cooperation with the staff fruit specialist. Protea: Propagules of Leucospermum hybrid 'Hawaii Gold' have been increased, differentiated into shoots and then rooted. However, problems of necrosis of propagules during differentiation and low rooting percentage of shoots have been encountered. Axillary bud explants of another plant, Protea hybrid 'Mayday', have developed propagules which are now being clonally increased. Australian Plant Introductions: Cultures of the following Australian plants are being clonally increased for subsequent field testing on Maui: Anigozanthus gabrielae, A. humulis, A. manglesii 'Red Emperor', Macropedia fulginosa, Actinodium cunninghamii, Lechenaultia hybrid,
Verticordia grandis, and Chamelaucium megalopetalum. This work is in cooperation with the staff horticulturist on Maui. Inflorescence explants of Spathiphyllum, Agapanthus, Xanthosoma, Alpina and Phalaenopsis have yielded plantlets and circumented major disinfestation problems when using other explants. Reproductive plantlet capacity is not affected by orientation of spadix segments in vitro in the case of Spathiphyllum floribundum. Although successful, the efficiency of root explants for micropropagation is limited.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 09/30/85
Outputs
BANANA: Clonal increase of dwarf Brazilian banana through tissue culture was successful on agar medium of 1/2 X MS nutrient, 20 g/liter sucrose and 5 mg/liter BA. Higher BA concentrations (10 and 15 mg/liter) produced more shoots but these remained dormant for a long period before sprouting with plantlets. Derived plantlets were successfully transferred to natural conditions and will be grown to maturity to determine whether genetic stability was maintained during clonal increase. PROTEA: Growing of differentiated shoots from protea callus into plantlets has not been successful. Shoots become chlorotic and rapidly die even with addition of activated charcoal and/or increasing calcium level in the media and frequent transfers to fresh medium. Increasing phosphorus and boron levels in the media is now being investigated. PANCRATIUM: Callus has been induced from explants of Pancratium littorale which has a new phenanthridone, pancratistatin, which has promising
anti-leukemic activity. INFLORESCENCE CULTURE: Problem of low success in tissue culture due to difficulty in disinfestation of explants has been solved with tropical ornamentals such as Spathiphyllum, Agapanthus, Xanthosoma, and Maranta by in vitro culture of inflorescence in MS nutrients, 20 g/1 sucrose and 1 mg/liter BA. In fact flowers need to be present in cultures although plantlets arise from base of petiole. Detailed studies of plantlet origin and development merit study.
Impacts
(N/A)
Publications
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Progress 01/01/84 to 12/30/84
Outputs
PROTEA: Axillary shoot explants have proliferated into callus type growth with many shoots in liquid 1/2 MS nutrient medium supplemented with 0.2-0.5 mg/liter BA. Higher levels of BA was inhibitory. Attempts to elongate these proliferated shoots into plantlets have not been successful. Initial axillary bud explants must be cultured in liquid medium; no explant on agar has as yet survived. ANTHURIUM, STRELITZIA, PLUMERIA: Work on recalcitrant anthurium cultivars and strelitzia has been terminated since no promising results have been obtained. Plumeria ovary explants form callus on media of 1.0-3.0 mg/liter NAA and 1.0-3.0 mg/liter BA more readily than leaf explants but are more difficult to disinfest. BANANA: Disinfested shoot explants have proliferated into multiple shoots on media of 5.0, 10.0, 15.0 mg/liter BA. At 5.0 mg/liter BA, shoots appear normal while at higher BA rates the shoots were stunted. GERMPLASM STORAGE: After storage (dark, 26C) of
Cattleytonia Rosy Jewel tissue cultures for 41 weeks in liquid modified Vacin and Went medium with coconut water (15% by volume), 90% recovered and differentiated plantlets when subcultured to solid modified Vacin and Went medium and placed in continous light at 26C.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
Germplasm storage: When tissue cultures of Cattleytonia Rosy Jewel were stored (dark, 4-5 C) on modified Vacin and Went solid or liquid with or without coconut water (15% by volume), cultures in liquid medium with coconut water survived twice as long (10 wks) as those on solid medium with coconut water (4.5 wks). Tissues stored (dark, 4-5 C) inmodified Vacin and Went medium + CW (15% by volume) + sucrose (10%) survived 15 weeks' storage. Protea: Disinfestation sequence of 20 min. soak in 10% clorox followed by 20 min in 5% clorox yielded approx. 60% viable auxillary explants of protea but was toxic to apical shoot explants and insufficent for flower bud disinfestation. Axillary shoots in liquid MS medium with 0.2 mg/1 BA have turned green but have not elongated or proliferated. Anthurim, Strelitzia, Plumeria: More axillary shoot explants of recalcitrant Anthurium andreanum and inflorescence explants of strelitzia were cultured in BA but multiple shoots have not been
obtained. Plumeria shoot and inflorescence explants have been difficult to disinfest due to latex exudation. However, callus can be readily induced on leaf explants but attempts to differentiate the callus into shoots have not been successful.
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Progress 01/01/82 to 12/30/82
Outputs
Anthurium: 10,960 A. andreanum plantlets (8,100 UH383, Paradise Pink; 2,860 UH721) were shipped to Waiakea Branch Station for nursery culture and eventual dissemination to industry. Three hundred sixty plantlets (UH731) were transferred for disease research (Plant Pathology) and 360 (UH721) to nurseryman R. Komori for trial. Two thousand eight hundred plantlets of A. scherzerianum (1,300 A318-7 and 1,500 A363-42) were distributed through Big Island Assn. of Nurserymen. Dendrobium: Propagation of 4 clones--'K339-19', 'K384-308', 'K318-11' and 'K350-3' was completed and transferred to greenhouse. Plumeria: Callus are readily indiced on leaf section but differentiation has been unsuccessful when treated with different growth regulators at various concentrations. Strelitzia reginae: Very promising results have been obtained; inflorescence node sections have produced multiple shoots. Attempts are being made to determine if response is related to inflorescence age and/or
cultivar differences. Preservation: Tissue cultures of Cattleytonia Rosy Jewel stored (dark, 4-5 degrees C) in liquid modified Vacin and Went medium with 15% (v/v) coconut water and sucrose concentrations (0 to 10%) were tested for survival by subculture to solid modifed VW + 15% CW medium at 25-27 degrees C.
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Progress 01/01/81 to 12/30/81
Outputs
Anthurium: 600 plantlets of A. schezerianum clone A318-7 and 400 of clone A363-42 and 1,100 plantlets of A. andreanum clone 517 were transferred to the Waiakea Branch Station for eventual dissemination to industry. A. andreanum plantlets (775) of UH517 derived through callus cultures have also been sent to Waiakea Branch Station for evaluation of genetic stability. Dendrobium: 4 clones, 'K339-19', 'K384-308', 'K318-11', 'K350-3' are being clonally increased for advanced testing. Plumeria: Callus has been induced leaf explant but will not survive subculture without leaf. Preservation: Adequate stock of tissue culture of Cattleytonia Rosy Jewel has been established to initiate storage experiments at room temperature (26 degrees C) and 4.5 degrees C both in the dark and with various concentrations (0-4%) of sugar.
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Progress 01/01/80 to 12/30/80
Outputs
Anthurium schezerianum cultivars, 'A363-42' and 'A318-7' and A. andraenum 'UH383' are now being clonally increased in vitro. Plantlets of these cultivars will be released to industry during this coming year. Culture of A. andraenum cultivars 'UH517', 'UH711', 'UH712', and 'UH721' have been initiated but still have not attained sufficient growth for clonal increase. Plantlets have been obtained from calli induced on A. andraenum 'Mauna Kea' leaves. These plantlets will be grown in Waiakea and will be later utilized in studies of genetic stability of plants obtained from tissue culture. Preliminary work on clonal increase of Strelitzia reginae and Plumeria acuminata has been initiated to determine the most suitable surface disinfestation procedure and the most suitable plant part to utilize.
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Progress 01/01/79 to 12/30/79
Outputs
Over 25,000 plantlets of Anthurium andraeanum 'Marian Seefurth' have been transferred to Dept. of Research and Development, County of Hawaii for distribution to growers. Cultures of 'Marian Seefurth', 'Calypso', and 'Mauna Kea' were transferred to local commercial tissue culture firms for continued increase. Cultivar response to nutrients seem to differ because 'Anuenue' and 'Trinidad', which grew poorly on nutrient formulation used for other cultivars, are now growing vigorously after the concentration of inorganic nutrients was reduced by half. Plantlets of A. schezerianum will attain sufficient size in about 2 months, after which clonal increase can be initiated. Dormancy problem was encountered when shoot tips of Aglaonema pseudobracteatum were cultured. Cultured shoots enlarged but did not sprout into plantlets even after 2-l/2 months. Tests with different growth regulators will be made for possible release of dormancy.
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Progress 01/01/78 to 12/30/78
Outputs
Rapid clonal propagation through tissue culture of Dracaena marginata cv. Tricolor has been accomplished through plants derived from callus induced from stem explants. However, plantlets obtained look more like D. marginata than D. marginata cv. Tricolor. This strongly suggests that D. marginata cv. Tricolor is a sport of D. marginata and is a periclinal chimera. Further evidence is being accumulated to substantiate this hypothesis. Plantlets of Anthurium andraenum cv. Marian Seefurth have overgrown in culture awaiting proper procedure for transferring plantlets to industry and would require re-culturing to ensure good establishment after transplanting to natural conditions. Cultivars, Calypso, Anuenue, and Mauna Kea are being increased clonally. Cultures of A. schezerianum have been initiated since several selected plants may have potential as potted plants.
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Progress 01/01/77 to 12/30/77
Outputs
Because of high contamination, initiation of aseptic cultures of Anthurium andreanum was a major problem. With improved surface-sterilization technique, the yield of sterile explants has been increased to 50% or better from previous 10% or less. With more initial explants, clonal increase of a cultivar can proceed much more rapidly. Plantlets of 'Marian Seefurth' and 'Calypso' are being transplanted to natural conditions for flowering to determine the genetic stability of this propagation method. Clonal propagation of and pathogen elimination from ginger (Zingiber officinale Roscoe), a speciality crop in Hawaii, was achieved by in vitro culture of vegetative buds from rhizomes. Plants derived have been distributed to commercial growers for establishment of mother blocks. In-vitro culture of embryos from palm seeds of Pritchardia kaalae and Veitchia joannis resulted in faster and more reliable germination. Species which are rare or have germination problems should
be tested.
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Progress 01/01/76 to 12/30/76
Outputs
An efficient system of propagation of some aroids and monocots has been established by induction of multiple shoots on stem segments with nodes culturedin vitro in a basal medium supplemented with benzyl amino purine (BA). Propagation by node culture has been successful with Scindapsis aureus, Philodendron oxycardium, Philodendron lacerum, Spathiphyllum 'Clevelandii', Alocasia cucullata, Zingiber officianle, and Neomarica coerulea. Polyploidy wasnot found in plantlets obtained by node culture. Anatomical study of node cultures suggests that several epidermal cells and cells of some subepidermal layers are involved in forming buds. Histochemical and microauto-radiographic studies showed increases in DNA, RNA, proteins, reducing sugars, activities of respiration enzymes, and activity of peroxidase prior to bud formation. Two Anthurium andreanum cultivars, 'Marian Seefurth' and 'Calyso', are now being rapidly increased clonally by culturing stem explants on basal medium
with BA. Other cultivars have been placed in culture but explants have not grown sufficiently for the rapid clonal increase.
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Progress 01/01/75 to 12/30/75
Outputs
When techniques for clonal propagation of dendrobiums through shoot-tip culture were published (Sagawa & Shoji, 1967, Kim, Kunisaki & Sagawa, 1970) for Dendrobium phalaenopsis, Den. J. Thomas, and other evergreen types, the authors were not aware that a commercially feasible technique for rapid clonal propagation of Den. nobile types was not available. Through experimentation, a commercially acceptable technique utilizing a modified Vacin and Went medium hasbeen established for Den. nobile types which, in recent years, upon introductionfrom nurseries in Japan has commanded premium prices in Hawaii as potted plants and as potential cut flowers. Propagation through embryo culture of palms such as Pritchardia & Veitchia with small embryos has been found to be feasible. Future studies will concentrate on palms of commercial importance for rapid clonal increase or on palms which are known to have problems in germination fromseed. Investigation on the clonal propagation of
Dracaena marginata through tissue culture is still being continued to obtain more consistent and rapid induction of multiple shoots for the technique to be considered commercially feasible. Although anthurium cultivars are now being propagated through the technique developed by our laboratory, the initial sterilization process is being improved to obtain greater numbers of explants so that propagation can proceed faster. Ti tissue culture has been terminated and a paper has been submitted for publication.
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Progress 01/01/74 to 12/30/74
Outputs
Phalaenopsis orchids which are among the commercially important orchids as pot plants and wedding bouquets have been limited in production owing to the unavailability of large numbers of selected clones. Phalaenopsis have previously responded poorly to rapid clonal increase by tissue culture. Spontaneous plantlet formation has been found to occur on the tip and nodes of the inflorescence and roots. However, these yield only limited numbers and in limited number of clones. A relatively successful method for clonal increase ofPhalaenopsis has been developed by induction of plantlets in vitro on nodes frominflorescences and sub-culturing shoot tips from them. Ample numbers of plantlets have been produced of several types of Phalaenopsis and these have been transferred to the greenhouse for flowering. With other horticulturally useful plants, ti and anthurium have been successfully propagated clonally through tissue culture. Application of BA to the medium has induced
multiple shoots on explants and the plantlets, after being rooted, have been successfullygrown under natural conditions. Selected anthurium clones are now being cultured in this manner for release to growers.
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Progress 01/01/73 to 12/30/73
Outputs
For establishing productive acreages of ornamental and fruit crops which are highly heterozygous and axesually propagated, methods for rapid clonal increase of selected clones are highly desirable. For some orchids, tissue culture of shoot-tips and young inflorescences have been demonstrated in our laboratory to be highly efficient sources for clonal propagation. In addition, the roots of Dendrobium Lady Hay and Dendrobium J. Thomas have responded to 2,4-D at 0.5-1.0 ppm by forming tumorous growths which through subsequent sub-culture can either be induced to differentiate plantlets or more tumorous growth. The tumor is theresult of mitotic activity at the periphery of the vascular cylinder. This is the first time that orchid roots have ever been successfully used for clonal propagation; other investigators have published unsuccessful attempts. In clonal propagation of other horticulturally useful plants, callus cultures have been obtained for anthurium, dracaena
and ti. For the latter two plants, roots have been readily induced from calli. Problems of inducing plantlet formation more readily from these calli are currently being investigated.
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Progress 01/01/72 to 12/30/72
Outputs
In the case of ornamental and fruit crops which are highly heterozygous, productive acreages of asexually propagated crops require rapid increase of selected clones. For rapid asexual propagation of orchids, vegetative shoot tips have generally been the source of explants. Although removal of the shoot tips from sympodial orchids may not be considered too drastic, removal of shoot tips from monopodial orchids frequently results in death of the plant. This problem has been overcome by development of a rapid propagation method by utilizing the inflorescence of sarcanthine orchids. Methods for clonal propagation of other horticulturally useful plants such as aroids, dracaenas, gingers, and foliage plants are currently in progress.
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