Musa sp. (aka Bana)
Partial Automation of Banana Micropropagation
Hawaii is the only significant commercial producer of banana (Musa sp.) in the US. Banana production in Hawaiʻi totaled 9,250 tons in 2009. However, the Hawaiian banana industry suffers serious losses from Banana bunchy top virus (BBTV). Production of micropropagules is an important method for production of disease-free planting material. Uniformly sized micropropagated plants also have an advantage for new plantings. During the past ten years, HARC produced over 250,000 banana micropropagules using stationary liquid micropropagation protocol for all three commercial types of banana including Williams, Apple, and Saba cooking banana. Cost of production, however, is high and many small farmers cannot afford to plant micropropagules. We developed a more efficient protocol of banana micropropagation using partial automation in a large culture container system, Rita® (VITROPIC, Saint-Mathieu-Treviers, France). This method uses filtered air to push liquid medium every 4 - 6 hours into an upper chamber where propagules are placed. The HARC protocol was established by modifying the protocols used in Costa Rica and France. In vitro culture of banana was established using HARC’s stationary liquid culture system (Annual Report 1997 p.25). When the propagules started multiplying (2-3 months from culture initiation), they were placed in the Rita containers for partial immersion. We successfully established shoot multiplication in Rita® for three varieties: Saba, Apple, and William. Comparison of propagation efficiency was conducted for all three varieties. Total fresh weight and propagule numbers produced in various culture media with BA 0-10 mg/L clearly showed that the Rita system resulted more propagules within six weeks. PM2 and PM4 media produced the most efficient propagule production. The Rita system produced 30-40% higher number of shoots than those in the stationary liquid Magenta boxes. Twice as much root mass per plant was obtained in root induction medium from Rita-derived propagules as compared to those from the stationary liquid method. Comparisons of Rita and liquid Magenta cultures clearly showed that the Rita system produced higher numbers of propagules and greater fresh weight in both Apple and Williams varieties. We planted propagules of both Apple and Williams in the nursery of a commercial operation. No difference was found in survival rate (range: Apple 89%-100%, Williams 100%) or plant growth between the stationary liquid Magenta box and the Rita methods for either variety. No variant mutants were observed among the propagules multiplied in various culture media in the Rita system. The Rita method successfully lowered production cost estimates, since most of the cost of propagule production is the labor. Cost of production by the Rita system was about 30-40% less than the earlier method. Although careful handling of medium changes is required for Rita, the overall labor requirement is significantly reduced. Rita culture system however has its limitation in size. We tested 20-60 propagules per Rita container (200 ml capacity) as starting propagules. When higher numbers of propagules were placed in the container, lower multiplication and more contamination was observed. However, once scaling up of the proven Rita banana culture system is achieved, higher multiplication and overall more efficient propagule production is expected with further cost reduction.
Hawaii is the only significant commercial producer of banana (Musa sp.) in the US. Banana production in Hawaiʻi totaled 9,250 tons in 2009. However, the Hawaiian banana industry suffers serious losses from Banana bunchy top virus (BBTV). Production of micropropagules is an important method for production of disease-free planting material. Uniformly sized micropropagated plants also have an advantage for new plantings. During the past ten years, HARC produced over 250,000 banana micropropagules using stationary liquid micropropagation protocol for all three commercial types of banana including Williams, Apple, and Saba cooking banana. Cost of production, however, is high and many small farmers cannot afford to plant micropropagules. We developed a more efficient protocol of banana micropropagation using partial automation in a large culture container system, Rita® (VITROPIC, Saint-Mathieu-Treviers, France). This method uses filtered air to push liquid medium every 4 - 6 hours into an upper chamber where propagules are placed. The HARC protocol was established by modifying the protocols used in Costa Rica and France. In vitro culture of banana was established using HARC’s stationary liquid culture system (Annual Report 1997 p.25). When the propagules started multiplying (2-3 months from culture initiation), they were placed in the Rita containers for partial immersion. We successfully established shoot multiplication in Rita® for three varieties: Saba, Apple, and William. Comparison of propagation efficiency was conducted for all three varieties. Total fresh weight and propagule numbers produced in various culture media with BA 0-10 mg/L clearly showed that the Rita system resulted more propagules within six weeks. PM2 and PM4 media produced the most efficient propagule production. The Rita system produced 30-40% higher number of shoots than those in the stationary liquid Magenta boxes. Twice as much root mass per plant was obtained in root induction medium from Rita-derived propagules as compared to those from the stationary liquid method. Comparisons of Rita and liquid Magenta cultures clearly showed that the Rita system produced higher numbers of propagules and greater fresh weight in both Apple and Williams varieties. We planted propagules of both Apple and Williams in the nursery of a commercial operation. No difference was found in survival rate (range: Apple 89%-100%, Williams 100%) or plant growth between the stationary liquid Magenta box and the Rita methods for either variety. No variant mutants were observed among the propagules multiplied in various culture media in the Rita system. The Rita method successfully lowered production cost estimates, since most of the cost of propagule production is the labor. Cost of production by the Rita system was about 30-40% less than the earlier method. Although careful handling of medium changes is required for Rita, the overall labor requirement is significantly reduced. Rita culture system however has its limitation in size. We tested 20-60 propagules per Rita container (200 ml capacity) as starting propagules. When higher numbers of propagules were placed in the container, lower multiplication and more contamination was observed. However, once scaling up of the proven Rita banana culture system is achieved, higher multiplication and overall more efficient propagule production is expected with further cost reduction.
Anthuriums
Micropropagation
Commercial production of anthuriums would benefit from an ample supply of planting materials for new and re-planted greenhouses and shadehouses. A project was initiated to determine whether the rate of micropropagation and the number of plants that could be produced within a certain time interval could be increased. Our existing tissue culture stocks of control plants of Marian Seefurth were used as starting material. About 240 plates of Marian Seefurth were inventoried and served as stocks from which small shoot tip cuttings were used to produce new plants. Within six months, we produced about 5000 shoots from the plant clusters. Four to five months after shoot tip cuttings were prepared, about 900 of the shoots were ready to pot in the greenhouse. Losses as a result of contamination were approximately 20%, but we were able to decrease contamination by replenishing them more frequently. In early April 2008, we shipped the first samples to commercial growers to determine if the plants were suitable for planting. Without increasing the original number of stock plates, at least 10,000 shoots could be generated in one year. Increasing the stock plate number should result in even larger yields of shoots. In 2010, we shipped 4300 plants of a second cultivar provided as a micropropagated culture in 2009. In mid-2011 we shipped 3500 plants, 2800 from new callus cultures that we had initiated in 2008 to 2009. Thus, micropropagation can be carried out either with pre-existing plant cultures or by initiating callus cultures and regenerating plants. Callus and shoot cultures from about 20 different cultivars were developed and all except one were micropropagated. The exception is a cultivar obtained in 2009 that until today has not yet generated calli but we are still trying. Different growth media formulations are being evaluated to determine whether rooted shoots can be developed in less than four to five months.
Transformation for Increased Disease Resistance
Anthurium andraeanum Hort. cultivars for the cut flower market are highly susceptible to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae. They are also susceptible to damage from the burrowing nematode, Radopholus similis, and the root-knot nematode, Meloidogyne javanica. About 700+ independently transformed lines of two anthurium cultivars, Marian Seefurth and Midori, were obtained by our group. About 400 of the transgenic lines were shipped under USDA permit to Hilo where the tissue cultured plants were acclimatized to greenhouse conditions for screening for resistance to the pests. X. a. pv dieffenbachiae testing commenced in late March 2008 starting with 15 Marian Seefurth clones containing genes for an Arabidopsis nonpathogenesis-related protein (NPR1). NPR1 controls the cascade of gene activation events in response to pathogen attack. Clones also contained the gene attacin, a lytic peptide from the giant luna moth Hyalophora cecropia and an attacin + T4 lysozyme gene. T4 lysozyme is a lytic protein from the T4 phage virus. Three control Marian Seefurth clones were also tested. Some of the lines were tolerant to bacterial blight in green house experiments. Nematode resistance tests will be carried out on potted plants and in cinder beds infested with nematodes.
A sample of 22 transgenic lines with the NPR1 gene and six transgenic lines with the attacin gene all showed positive PCR results using primers specific for the nptII selection gene. These results show that the selection protocol is stringent. Four lines tested were all PCR positive for the attacin gene . A single band was found in lines NPR1-29-1, NPR1-31-1, NPR1-49-3, and NPR1-49-3B, indicating a single-copy transgene insertion of NPR1 into the anthurium genome . This was the first clear Southern blot result for transgenic anthurium lines produced in our lab. High quality genomic DNA extracts are critical for good PCR and Southern blot results.
Of the 700+ lines we obtained from transformation of anthurium, about three quarters contained a gene for bacterial resistance while about one quarter contained one or two nematode resistance genes (rice cystatin and/or cowpea trypsin inhibitor genes).
A different study undertaken in 2008 sought nematode resistance using RNA interference (RNAi) in which genes important to nematode health and fertility are transformed into a plant. When the pest ingests the plant substances, the transgene products stimulate nematode genes to destroy the pest's health and fertility RNAs, resulting in decreased growth, fertility, and/or death. Constructs were created by a team at Kansas State University and shipped to HARC. We have obtained about 20 transgenic lines. PCR determinations for selection and transgenes in the first putative line are to be completed shortly.
Transformation for Novel Flower Color
Growth in the floriculture industry is enhanced by the creation of new shapes, colors, and fragrances in traditional blossoms. The introduction of a blue rose by Suntory company of Japan inspired many to dream of other blue flowers. Why not anthurium? We are collaborating with a researcher in Britain and with University of Hawaii researchers to create such an anthurium. We wish to produce a purple or blue, large, heart-shaped anthurium for the local industry. A set of genes in the anthocyanin biosynthetic pathway will be used. We will observe transient expression of the genes by bombarding them into anthurium spathes of different colors and will also stably transform cultivars expressing a range of spathe colors. The time span from inserting the genes into anthurium to first bloom is approximately 28 months.
Commercial production of anthuriums would benefit from an ample supply of planting materials for new and re-planted greenhouses and shadehouses. A project was initiated to determine whether the rate of micropropagation and the number of plants that could be produced within a certain time interval could be increased. Our existing tissue culture stocks of control plants of Marian Seefurth were used as starting material. About 240 plates of Marian Seefurth were inventoried and served as stocks from which small shoot tip cuttings were used to produce new plants. Within six months, we produced about 5000 shoots from the plant clusters. Four to five months after shoot tip cuttings were prepared, about 900 of the shoots were ready to pot in the greenhouse. Losses as a result of contamination were approximately 20%, but we were able to decrease contamination by replenishing them more frequently. In early April 2008, we shipped the first samples to commercial growers to determine if the plants were suitable for planting. Without increasing the original number of stock plates, at least 10,000 shoots could be generated in one year. Increasing the stock plate number should result in even larger yields of shoots. In 2010, we shipped 4300 plants of a second cultivar provided as a micropropagated culture in 2009. In mid-2011 we shipped 3500 plants, 2800 from new callus cultures that we had initiated in 2008 to 2009. Thus, micropropagation can be carried out either with pre-existing plant cultures or by initiating callus cultures and regenerating plants. Callus and shoot cultures from about 20 different cultivars were developed and all except one were micropropagated. The exception is a cultivar obtained in 2009 that until today has not yet generated calli but we are still trying. Different growth media formulations are being evaluated to determine whether rooted shoots can be developed in less than four to five months.
Transformation for Increased Disease Resistance
Anthurium andraeanum Hort. cultivars for the cut flower market are highly susceptible to bacterial blight caused by Xanthomonas axonopodis pv. dieffenbachiae. They are also susceptible to damage from the burrowing nematode, Radopholus similis, and the root-knot nematode, Meloidogyne javanica. About 700+ independently transformed lines of two anthurium cultivars, Marian Seefurth and Midori, were obtained by our group. About 400 of the transgenic lines were shipped under USDA permit to Hilo where the tissue cultured plants were acclimatized to greenhouse conditions for screening for resistance to the pests. X. a. pv dieffenbachiae testing commenced in late March 2008 starting with 15 Marian Seefurth clones containing genes for an Arabidopsis nonpathogenesis-related protein (NPR1). NPR1 controls the cascade of gene activation events in response to pathogen attack. Clones also contained the gene attacin, a lytic peptide from the giant luna moth Hyalophora cecropia and an attacin + T4 lysozyme gene. T4 lysozyme is a lytic protein from the T4 phage virus. Three control Marian Seefurth clones were also tested. Some of the lines were tolerant to bacterial blight in green house experiments. Nematode resistance tests will be carried out on potted plants and in cinder beds infested with nematodes.
A sample of 22 transgenic lines with the NPR1 gene and six transgenic lines with the attacin gene all showed positive PCR results using primers specific for the nptII selection gene. These results show that the selection protocol is stringent. Four lines tested were all PCR positive for the attacin gene . A single band was found in lines NPR1-29-1, NPR1-31-1, NPR1-49-3, and NPR1-49-3B, indicating a single-copy transgene insertion of NPR1 into the anthurium genome . This was the first clear Southern blot result for transgenic anthurium lines produced in our lab. High quality genomic DNA extracts are critical for good PCR and Southern blot results.
Of the 700+ lines we obtained from transformation of anthurium, about three quarters contained a gene for bacterial resistance while about one quarter contained one or two nematode resistance genes (rice cystatin and/or cowpea trypsin inhibitor genes).
A different study undertaken in 2008 sought nematode resistance using RNA interference (RNAi) in which genes important to nematode health and fertility are transformed into a plant. When the pest ingests the plant substances, the transgene products stimulate nematode genes to destroy the pest's health and fertility RNAs, resulting in decreased growth, fertility, and/or death. Constructs were created by a team at Kansas State University and shipped to HARC. We have obtained about 20 transgenic lines. PCR determinations for selection and transgenes in the first putative line are to be completed shortly.
Transformation for Novel Flower Color
Growth in the floriculture industry is enhanced by the creation of new shapes, colors, and fragrances in traditional blossoms. The introduction of a blue rose by Suntory company of Japan inspired many to dream of other blue flowers. Why not anthurium? We are collaborating with a researcher in Britain and with University of Hawaii researchers to create such an anthurium. We wish to produce a purple or blue, large, heart-shaped anthurium for the local industry. A set of genes in the anthocyanin biosynthetic pathway will be used. We will observe transient expression of the genes by bombarding them into anthurium spathes of different colors and will also stably transform cultivars expressing a range of spathe colors. The time span from inserting the genes into anthurium to first bloom is approximately 28 months.
