2 HARC Board of Directors 2003

2 HARC Officers 2003

3 Director's Letter

5 100 Years Ago: 1903

6 Article

9 Sugarcane Research

14 Tropical Fruit Research

21 Coffee Research

25 Forestry Research

26 Miscellaneous Crops

30 Services

32 Personnel

33 Sugar Production 2001-2003

33 Publications and Presentations

T A B L E O F C O N T E N T S

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On the Inside

Front cover images, clockwise, from top right corner: (1) Leaf print of transgenic papaya, showing the expression of reporter protein

(2a) Tobacco plant inoculated with pPVX204 plasmid DNA under natural light at 9 DPI(2b) Tobacco plant inoculated with pPVX204 plasmid DNA under UV at 9 DPI(3) Apple snail eggs(4) Papaya flowers(5) Drs. Qingyi Yu and Ray Ming examining BAC Southern film(6) Taro shoots

Background Image: Cultivars of Hawaiian sugarcane (Saccharum spp.) - photo courtesy of Maui Nui Botanical Gardens, Kahului, Maui

Editorial team: Susan Schenck and Blake Vance

Photo credits: Ricelle Agbayani, Jim Carr, Jason Fujii, Mel Jackson, Heather McCafferty, Ray Ming, Chifumi Nagai, Blake Vance and Judy Zhu

Design and layout: Cyndi Flugum, Design Mill, Ltd.

Mr. G. S. Holaday, Co-ChairmanGeneral ManagerHawaiian Commercial & Sugar Company

Mr. E. A. Kennett, Co-ChairmanPresident and ManagerGay & Robinson, Inc.

Mr. J. Hoxie Vice President, Agricultural GroupHawaiian Commercial & Sugar Company(Retired 3/11/03)

Mr. F. KigerVice President, Agricultural GroupHawaiian Commercial & Sugar Company(Elected 6/3/03)

Mr. C. OkamotoTreasurerGay & Robinson, Inc.

Dr. L. IngamellsHawaiian Commercial & Sugar Company

O F F I C E R S / B O A R D

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Ms. Stephanie A. Whalen (President)Director, Experiment StationHawaii Agriculture Research Center

Dr. Robert V. Osgood (Vice-President)Hawaii Agriculture Research CenterResigned 9/30/03)

Dr. Kuo-Kao Wu (Vice-President)Hawaii Agriculture Research CenterElected 9/30/03)

Mr. Anthony L. Lannutti (Secretary-Treasurer)Hawaii Agriculture Research CenterElected 2/4/03)

HARC Officers — 2003

HARC Board of Directors — 2003

he year 2003 was the 109th year ofcontinuous research and serviceperformed by HARC for the agricul-tural sector in Hawaii. The sugar-

cane industry continues to lead Hawaii'sagriculture in demonstrating its foresight insponsoring research activities that contributeto its sustainability.

Harvested acres of 19,851 for 2003 pro-duced 261,009 tons of raw sugar. The aver-age tons sugar per acre was 13.15, a 3.79%increase from the 12.67 in 2002. By compar-ison, the average tons sugar per acre 10 yearsago was 10.47 from the harvest of 64,705acres. The 25.6% gain in yield for the indus-try ref lects not only an increase in theremaining companies' performance but thefavorable physical environment and infra-structure the remaining companies have ascompared to those that dropped out of thebusiness.

At one time, the Hawaii sugar industry operated in 12 physically different definable environ-ments. Several of the plantations dealt with more than one environment requiring differentsugarcane varieties and cultural practices. This was the challenge for the Experiment Station'sbreeding and selection program as well as its agronomists. While the many micro-environ-ments frustrated the century-old search for a single alternative crop for the thousands of acresavailable, they offered a wealth of opportunities for agricultural diversification. This organiza-tion makes full use of its breadth of experience in these micro-environments and its results-ori-ented culture to develop new agricultural businesses and improve existing agricultural prod-ucts.

This annual report continues its reflection of highlights from 100 years ago followed by a fea-ture article on sustainability and sugarcane's exemplary lead in this area. You will read howpursuing the latest technology is still a priority for us, although now it is applied not only tosugarcane but to many of the other major tropical crops in Hawaii. Cutting edge technologiesdeveloped in the private sector are first and foremost applied to the major crops of the world.By definition, Hawaii's crops are minor crops. HARC in collaboration with the University ofHawaii College of Tropical Agriculture and Human Resources and the USDA-AgriculturalResearch Service works to apply these technologies to Hawaii's crops as reflected in the arti-cles which follow.

Developing resistance to yellowleaf virus, evaluating the genetic diversity of smut pathovars,evaluating transgene silencing mechanisms and producing a biologically active pharmaceuticalprotein along with continuing breeding and selection, highlight our sugarcane program.

D I R E C T O R ’ S L E T T E R

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Director's Letter

Stephanie A. Whalen, President and Director since 1994

Major efforts are focused on the molecular biology of papaya including the cloning and char-acterization of flower development, investigating systemic acquired resistance and disease andpest resistance through gene insertion, and discovering a primitive Y chromosome.

For coffee, a genetic map was constructed, transgenic nematode-resistant plants are beingdeveloped and improved quality is targeted both in the breeding and selection work as well asin shading effects.

A vascular wilt disease was identified as the cause of dieoff of koa trees. This could be of verysignificant concern as it has been found on all the islands and at many elevations. Our priorfield trials with koa suggest that there is some resistance within the population. Before initiat-ing future field trials, seedlings will be screened for resistance to support our goal to select thebest seed sources for the expansion of this uniquely high-value Hawaiian crop.

The agronomic work in taro in the past years was followed this year with the first report of suc-cessfully inserting a useful gene into taro demonstrating that this technology can improve taro'sresistance to diseases. Also in taro, a botanical extract was found to successfully control anextremely economically damaging pest, the apple snail.

Contract services have become a small but important part of our goal to develop new agricul-tural businesses or bring them to the state. We are proud that this year the potato certifica-tion business we have been building for 5 years was successful and passed on to farmers mak-ing room for us to develop and nurture another.

In this day and age when few people know where their food comes from, outreach is taken seri-ously by all the staff. They participate through assisting in the State Science Fair, the islandfarm fairs, visitor presentation and facility tours and schools and business meeting presenta-tions. I am deeply appreciative of their volunteered time in this area and in their flexibility inthe continuing transition both internally to this organization and externally in the agriculturalcommunity.

I also want to thank the sugarcane industry for its continuing support and commitment toresearch, to other commodity groups we are working with and to our clients on the mainlandand other parts of the world.

Respectfully,

Stephanie A. WhalenPresident and Director

D I R E C T O R ’ S L E T T E R

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A L O O K B A C K

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he Hawaii Planters’ Monthlyreported that: “By the end of 1903 we find thatthe station had increased its staff,

which now included the director, Mr.Eckart, four chemists, Messrs. Peck,Werthmueller, Jordan, and Thompson, andwith the field work still in the hands of Mr.Clarke. There had also been an addition tothe laboratory buildings and theExperiment Station Committee for 1903lament, 'It is unfortunate that the area ofthe Station grounds (Makiki) is so small, asthe field experiments have to be restrictedmuch more than is desirable, especially atsuch times when the necessity arises for fal-lowing portions of the land.'”

By 1903, the sugarcane leafhopper hadspread to all the islands and caused suchserious damage that the entire sugar indus-try was threatened with extinction.Parasites from Ohio were being propagat-ed, but tangible results remained elusive.

Of the 55 plantations listed in thePlantation Directory (The Planters'Monthly, vol. XXII, 49 (Jan. 1903)), onlyHawaiian Commercial & Sugar Co. andGay & Robinson are still in operation.

Results of an irrigation experiment usingsalt water revealed that the yield of caneand sugar were reduced by half as thegrains of salt per gallon was increased from50 to 200. Other 1903 events included:The 6-mile Waimea (Kikiaola) Ditch onKauai and the Opaeula Ditch on Oahuwere completed; the 12.5-mile HonokahauDitch on Maui was under construction.These ditches were fundamental to irrigat-ing sugarcane grown on the leeward sidesof these islands.

On January 13th, 102 Koreans arrived inHawaii to work at the Waialua Plantation.

T100 Years Ago: 1903

They were followed by nearly 500 moreKoreans by that July who worked on BigIsland, Kauai and Oahu plantations.

Fuel oil was first used for pumping at Kihei,Hawaiian Commercial & Sugar Co., Paiaand Haiku plantations.

The first message was sent by telegraphcable from Hawaii to the mainland (SanFrancisco). This Pacific cable linked SanFrancisco, Hawaii, Guam and thePhilippines.

Alexander Young Hotel opened in Honoluluon July 31.

The E. K. Fernandez Shows began entertain-ing the families of Hawaii with what was tobecome circus shows.

- Blake Vance

E. D. Tenny, elected president of the Hawaiian Sugar Planters'Association on November 23

he Hawaiian sugarcane industry has a long history of sustainable agronomic, diseaseand pest control practices. As one of the first agriculture industries to adopt thepractice of production and release of parasitoid insects to control sugarcane pestinsects, it has not resorted to the use of insecticidal chemicals on Hawaiian planta-

tions. Although numerous severe diseases have appeared over the years, control measures havefollowed Integrated Pest Management (IPM) practices by using good cultural practices, seedfield disease surveys, breeding for resistance, quarantine regulations, and hot water seed treat-ment. Only a single, low toxicity fungicide, Tilt, is in use as a seed dip treatment to controlCeratocystis on planting material. Water use practices are carefully monitored and drip irriga-tion was installed to avoid overuse of water resources and to prevent runoff. Millwater fromfactory operations is recycled to fields. Mulching of fields helps cut down on weed problems,but herbicides are still necessary to a limited extent. Because of its sustainability, the Hawaiiansugarcane industry is still in business on the same fields after more than one hundred years.

The Hawaii Agriculture Research Center also supports sustainable agriculture for all Hawaiiancrops. Small farmers growing many different local crops need to learn IPM and sustainablepractices in order to reduce the use of pesticides and bare-ground fallow. HARC is engaged inseveral projects to attain this goal. Sustainable agriculture is more complex than conventionalfarming and requires greater levels of effort and skills. The challenge now is to demonstrate itspotential profitability, advantages to the environment and to increase its use in Hawaiian agri-culture. HARC has carried out several research projects designed to increase and improve uponHawaii agriculture's sustainability.

Adaptation of asparagus production to Hawaii's environment. Asparagus was evaluated for its pro-duction sustainability and marketability in Hawaii (HARC Annual Report 1999). California cul-tivars proved to have the highest yields. The crop can be adapted to continual year-round pro-duction by cutting the water supply periodically to allow the ferns to die back and restart newspears production. In temperate climates the dormant period always occurs over the wintermonths, but in Hawaii it can be made to occur at any time of year. Thus farms can stagger pro-duction in field sections to supply the market all year. Fertilizer rates and disease and pest con-trol can also be managed in a sustainable production system. After the plants have establisheda good root mass, there is no need to replant for at least 15 years. Since the project wasinstalled, acreage in asparagus has increased.

Use of molasses amendments to overcome nematode damage to papaya. The benefits of amendingsoil with molasses were recognized years ago in the sugarcane industry. During the process ofdecomposition, molasses appeared to reduce damage to roots caused by root parasites.Finding new uses for this sugar production by-product as a soil amendment would benefit sus-tainability of other crops as well as the sugar industry. It was applied to papaya trees that hadceased to be productive because they were heavily infested with reniform nematodes (HARCVegetable Report 3, Oct. 2001). The molasses was injected into the irrigation tubing at a dilu-tion rate of 1:20 once a week for 12 weeks. Counts of nematodes decreased somewhat. Treesregained their green color, leaves began to grow again and trees began to produce marketablefruits. The effect was probably due to a number of soil factors other than nematodes.Molasses also had slight, but positive effects on Maui onions and Chinese cabbage.

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TSustainable Agriculture in HawaiiS. Schenck

A R T I C L E

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Wood ear mushroom production in forest understory. Timber production is a possible sustainableagricultural industry in Hawaii. Koa in particular would help to preserve Hawaii's naturalecosystem while producing high quality timber products. For generating additional revenuewhile the trees mature, production of edible wood ears (pepeiao) mushrooms was investigated(HARC Annual Report 1997). A simple production method was devised that takes advantageof fallen or cut tree branches. The mushroom spawn can be produced in bags of bagasse, asugarcane milling byproduct, and inoculated into pieces of wood using a hand drill. The inoc-ulated wood pieces are left on the forest floor and the harvestable wood ear fruiting bodiesstart to appear within about three months. Harvesting can continue for three months. Thewood ears are then simply dried and sold for cooking in soups and sauces.

Natural farming methods in vegetable production. A project was installed to demonstrate a farm-ing system using crop rotation, cover crops and green manures to maintain soil fertility and tex-ture and manage diseases and pests. It was designed to demonstrate the sustainability of thismethod under Hawaiian growing conditions and to inform farmers and gardeners of its prac-tical value. The field site was located at Waialua High School on Oahu and was supervised byS. Schenck and N. Kawachi, a high school teacher with a background in agriculture. Most ofthe hands-on work was performed by the high school students. A crop rotation schedule wasplanned and carried out for the duration of the three-year project. The produce was sold bythe students to local markets or used to supply high school agricultural field days and sales.The principles of soil nutrient conservation and replenishment with alternating crops and inter-crop covercrops are applicable all or in part to various small farms and gardening situationsand, as it was designed to do, this project informed students and others about these methods.

Liquid Compost Factor (LCF) to improve tomato plant vigor and increase tolerance to nematodes. LiquidCompost Factor is a product made by ABR, Maui from molasses, pineapple mill waste and othernatural products in which an edible fungus is cultured. The resultant liquid acts to increasegrowth rate, vigor and plant tolerance to various stress factors. Since it is composed of edible,non-toxic substances, it is usable in organic farming production and any other farming or gar-dening practice where pesticides are not warranted or desired. We tested the product in severaldifferent tomato trials, arugula, and turfgrass. Further trials are continuing. In field plots oftomato that were heavily infested with root-knot nematodes there was an increase in fruit num-ber production when LCF was applied as a soil drench pre- and post-planting.

Evaluation of Paecilomyces lilacinus as a biocontrol agent of nematodes in tomato and cucumber. Thenematode parasitic soil fungus Paecilomyces lilacinus has shown promise in controlling nema-todes on a number of vegetable and fruit crops and one strain is certified for commercial salein Europe. We are currently testing this soil fungus applied as a soil drench on tomato andcucumber in field plots infested with root-knot and reniform nematodes.

Cover crops to reduce the practice of bare-ground fallow. Certain cover crops such as Sunn hemp(Crotalaria juncea), are known to reduce levels of parasitic nematodes, keep weeds under con-trol, reduce erosion between crop cycles, and add nutrients to soil. Several projects have beenundertaken to quantify these effects and develop IPM systems using cover crops. Managementof root-knot and reniform nematodes in tomato using Sunn hemp was successful (HARCAnnual Reports 1998 and 1999). Sunn hemp proved less effective in controlling nematodes inginger, although a growth response was observed, probably due to increased soil nitrogen(HARC Annual Report 2000). During 2003, a large cover crop project was initiated in severalOahu farms to demonstrate to farmers the advantages of cover cropping as compared to the

practice of bare-ground fallowing. Bare-ground fallow on Oahu's North Shore has resulted inmuch soil run-off into the ocean and threatens the offshore ecosystem with silting. The covercrops in the trial include Sunn hemp, oats, lana vetch, winter wheat and barley. The responseof farmers and their willingness to participate in the project has been encouraging. The proj-ect is scheduled to continue through 2004.

Additional Information

Farming Organically. Ever Green, by Lois Taylor. Honolulu Star Bulletin, July 3, 1998.

Hawaii Organic Farmers Association webpage, http://www.hawaiiorganicfarmers.org/

Hue, N.V. and J.A. Silva. 2000. Organic soil amendments for sustainable agriculture: organicsources of nitrogen, phosphorus, and potassium. pp 133-144 In Plant Nutrient Managementin Hawaii's Soils. J.A. Silva and R.S. Uchida, Eds. CTAHR, University of Hawaii.

Managing Cover Crops Profitably, 2nd Edition 1998. Sustainable Agriculture Network.Beltsville, MD.

McSorley, R. and D.W. Dickson. 1995. Effect of tropical rotation crops on Meloidogyne incog-nita and other plant-parasitic nematodes. Supplement to the Journal of Nematology27(45):535-544.

Ochse, J.J. and W.S. Brewton. 1954. Preliminary report on Crotalaria versus nematodes.Proceeding Florida State Horticulture Society 67:218-219.

Organic: A Growing Market. Honolulu Advertiser, January 21, 1998.

Practicing MOA Nature Farming in Hawaii. 1998. Pan American MOA Foundation, Publisher:Miami, FL

Thurston, H.D. 1984. page 19 in Tropical Plant Diseases. The American PhytopathologicalSociety, St. Paul.

A R T I C L E

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R E S E A R C H R E P O R T S

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ugarcane yellowleaf syndrome iscaused by a luteovirus, sugarcaneyellowleaf virus (ScYLV). There arereports of yield losses due to ScYLV

infection. Yellow leaf virus infections resultedin yield losses in Hawaii in the 1990s whenwhole fields turned yellow. Virus infection isstill present and widespread in all susceptiblecultivars and has been shown to be spread bycertain common aphid species. Infectionwithin the plant spreads first to the juvenile,growing plant parts and young leaves and iseventually found throughout the entireplant. The virus also spreads by plantinginfected seed pieces. In the currently selectedHawaiian commercial cultivars, ScYLV leafyellowing symptoms usually appear undercertain environmental stress conditions,especially cool temperatures and drought.However, preliminary tests in Hawaii andLouisiana indicated that the infection isharmful to the plants’ performance evenwhen the plants are symptomless. The reduc-tion in growth rate, tillering and sucker for-mation appears to be greater in youngplants. Since all plants of the susceptible cul-tivars in plantation fields contain the virus,no clear-cut comparison of infected andvirus-free plants of the same cultivar on thesame location has so far been possible.

This project will undertake to cre-ate ScYLV-resistant sugarcanethrough transformation. Genesilencing seems to be a universalmechanism for plant resistance toviral infection and posttranscrip-tional gene silencing has alreadybeen achieved in sugarcane bytransformation with an untrans-latable piece of Sorghum MosaicVirus. A similar approach wasattempted in this project withScYLV. The ScYLV genome andthe functions of the viral proteinsare known (F. Moonan, J. Molina

SSugarcane ResearchDeveloping Transgenic Sugarcane for ScYLV Resistance

and T. E. Mirkov (2000) Virology 269:156-171). Therefore, the strategy for productionof a ScYLV-resistant cane can be straightfor-ward. A H62-4671 variety has been trans-formed with a non-translatable DNAsequence piece of the viral coat protein,which will lead to gene silencing of viral RNAupon infection. Sugarcane seems to have avery powerful gene silencing system, whichpromises success of the project.

About twelve independent transgenic linesderived from H62-4671 were produced andPCR analysis has been carried out to verifythe presence of coat protein gene and selec-table marker gene, NPTII. A virus challengeassay to test the resistance level has been car-ried out on the greenhouse-grown transgeniclines. About 30 plants were inoculated withaphids feeding on the virus-infected cane,H73-6110 and plants were left in the green-house until new leaves were developed.Tissue blot was used to detect the virus in theinoculated plants. We are in the process ofdata collection. Southern blot analysis toverify the genome copy and insertion is also inprocess.

– Y. J. Zhu, G. Osterman, C. Moritomo, H.McCafferty, R. Agabayani, A. Lehrer, S. Schenckand P. Moore (USDA/ARS)

Greg Osterman is inoculating sugarcane with virus transmis-sion vector aphids

ene silencing, including post-transcriptional gene silencing(PTGS), is now recognized as anessential gene regulation mech-

anism which exists in plants, animals andall higher organisms. This process isimportant for regulation of endogenousgenes, and in plants, PTGS is an importantdefense mechanism against viral infection.Plants engineered for virus resistance usePTGS of a virus gene, inserted in the plantgenome, to protect against that virus.Although there are many useful applica-tions of PTGS, this process can create amajor problem when trying to engineerplants for high level expression of a trans-gene. We are studying the mechanisms ofPTGS with one goal being to control PTGS.We are taking two general approaches: oneis to control the number and pattern oftransgene integrations so as to avoid con-figurations like inverted repeats which cantrigger PTGS. The other approach is toidentify viral genes that act to suppressPTGS, and determine their mode of action.

In the first approach, we have used threemethods. The first of these involves bom-barding plant cells with linear DNA con-taining only the gene expressioncassette(s), instead of using entire circularvector plasmids, as is done conventionally.This method has been used in rice to pro-duce “predominantly single- or low” trans-gene copy lines. These low copy rice linesunderwent transgene silencing at a muchlower frequency than did lines produced byconventional bombardment. In sugarcane,we found this method did not produce achange in transgene copy number, withmost lines still containing many copies.Even in those few lines recovered with lowcopy number, transgene expression wasnot enhanced.

The second method utilizes the Cre-loxrecombination system to resolve multiple

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Mechanisms of Transgene Silencing

G transgene copies to a single copy. Thismethod did produce a high percentage oflow copy lines; however, these lines did notshow elevated gene expression.

The third method utilizes the Ac-Ds trans-poson system to insert transgenes by trans-position. This method has been used inbarley to produce single copy lines whichunderwent transgene silencing at a muchlower frequency than did conventionalmulti-copy lines. This system can producesingle copy insertions, but may also directtransgene insertions to actively transcribedregions of plant chromosomes, whichwould be an additional advantage. Overfifty transgenic sugarcane lines have beenproduced with this method. Transgenecopy number and expression analysis ofthese plants is underway.

Our study of viral suppressors of PTGS iscurrently focused on the P0 gene of ScYLV.Transient expression experiments indicatethis gene can suppress silencing in cornleaves. Experiments to determine whichspecific parts of the P0 gene product havethe suppressor activity, and other experi-ments to determine at what step of thePTGS process P0 acts, are underway.

If either of these approaches to understand-ing PTGS leads to methods of controllingthe process, this will provide a big boost toour efforts to use sugarcane as a biofactoryto produce high-value proteins.

– T. Mangwende, M-L. Wang, S. Ancheta, J.Clayton, C. Goldstein, J. Carr (USDA/ARS) andH. Albert (USDA/ARS)

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new sugarcane smut pathovarthat infected the previouslyresistant cultivar H78-7750appeared in Hawaii in 2001.

Field trials subsequently showed that manyof the Hawaii sugarcane cultivars differedin susceptibility to the old and new Ustilagoscitaminea isolates (HARC Annual Report2001-2002). Molecular diversity of U. scit-aminea and other fungal species was ana-lyzed by amplified fragment length poly-morphism (AFLP) markers. Thirty-seven U.scitaminea isolates and five isolates ofother fungal species were f ingerprintedwith 310 AFLP markers.

Sugarcane smut fungal isolates were col-lected from Maui, Oahu and Kauai. Inaddition, U. hordei from barley, two iso-lates of U. maydis from corn, and two unre-lated sugarcane pathogens were includedin the trial. Fungal DNA was isolated by amethod using EB buffer (0.1 M Tris, 0.05M EDTA, 0.5 M NaCl, 1 g kg-1 NaHSO3, 1g kg-1 sodium dodecyl sulfate, 2 g kg-1CTAB) and agitation with glass beads.DNA digestion followed the protocol ofVos et al. (1995). Selective amplificationwas performed using labeled EcoR I andPst I primers and followed the protocol ofQiagen (Valencia, CA). Eleven pairs ofAFLP primers were used to fingerprint thegenomic DNA. Polymorphic AFLP markersgenerated by each primer combinationranged from 21 to 27, producing an accu-mulated total of 310 markers.

The genetic similarity of the 37 U. scita-minea samples ranged from 0.85 to 1.0and averaged 0.95. This suggested littlegenetic variation among the Hawaiian iso-lates or between the old and new patho-types. However, there were dramatic dif-ferences in genomic composition betweenthe three Ustilago species. Genomic DNAsequences are constantly evolving as shownby the polymorphisms of AFLP markers

A detected among isolates. The manner offungal transmission by airborne teliospores,the ease of cross-fusing between haploidsporidia from different isolates, and theapparent mutation and recombination dur-ing dikaryon formation allow for continualchange and recombination within the rela-tively small and isolated population of U.scitaminea in Hawaii. This may account forthe appearance of new pathotypes and fortheir subsequent disappearance throughhybridization.

A slight genetic mutation that is notdetectable by AFLP analysis can result in U.scitaminea overcoming host resistance.Nonetheless, selection for resistance in thebreeding program is still necessary in orderto avoid releasing highly susceptible culti-vars. But hot water treatment of plantingmaterial should not be neglected, even forresistant cultivars, because of the possibilityof the appearance of new pathogen strains.

- S. Schenck, H. M. Pearl, Z. Liu, P. H. Moore(USDA/ARS) and R. Ming

Reference:

Vos, P., R. Hogers, M. Bleeker, M. Reijans, T.van de Lee, M. Hornes, A. Frijters, J. Pot, J.Peleman, M. Kuiper, and M. Zabeau, 1995.AFLP: a new technique for DNA fingerprint-ing. Nucleic Acids Res. 23:4407-4414.

Evaluation of Genetic Diversity of Ustilago scitaminea Pathotypes

reviously, we reported the produc-tion of transgenic sugarcane andrice plants expressing a high-valuepharmaceutical protein, granulo-

cyte macrophage-colony stimulating factor(GM-CSF, HARC Annual Report 2001-2002). In field trials, some sugarcane linesproduced up to 0.03% of the total solubleprotein (tsp) as GM-CSF. The transgeniclines differed for gene promoter, sub-cellu-lar localization signals, andpresence/absence of a peptide tag foraffinity purification. All of the lines in thehighest expression group (>0.01% tsp) con-tained expression constructs directing theprotein to remain in the endoplasmic retic-ulum (ER). Protein processing and foldingis carried on in the ER; these experimentsshow that extended time in the ER is essen-tial for stable production of GM-CSF insugarcane.

In July of 2002, approximately 50 selectedtransgenic sugarcane plants were trans-planted to an experimental field at theKunia substation for evaluation. Theexpression levels remained stable for atleast 10 months after transplanting. Wealso observed relatively uniform expressionthroughout the plants. Within a singleplant, small variations (less than 3X) weredetected between leaves and internodes ofdifferent ages. Furthermore, addition of acostly cocktail of proteinase inhibitors andantioxidants was found to be essential forpreventing degradation of GM-CSF whenextracting the protein from sugarcaneplants.

The GM-CSF produced in the sugarcaneand rice showed native biological activity ina human cell proliferation assay. Extractsfrom sugarcane calli, sugarcane leaves, andrice grains stimulated the division of bonemarrow (TF-1) cells in a manner essentiallyidentical to purified, commercially pro-

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PProduction of a Biologically Active Pharmaceutical Protein in Sugarcane and Rice

duced GM-CSF, indicating the plant-pro-duced GM-CSF had close to 100% nativebiological activity. In GM-CSF extractedfrom rice grains which had been stored atroom temperature for over 12 months, con-centrations remained similar to freshly har-vested grain, and activity remained at thesame high level, indicating that the GM-CSFis very stable when expressed in rice seeds.This stability provides storage and shippingoptions which would not be available for aprotein expressed throughout the plantbody.

Developing a crop plant as a biofactory toproduce high-value proteins involves twomajor steps: engineering the plant to pro-duce the desired protein with the correctfolding and processing so the protein hasbiological activity, and engineering the plantto produce the protein at a level highenough to make extraction and purificationeconomically feasible. The first of thesehurdles has now been cleared for both sug-arcane and rice. Current efforts are focusedon achieving higher levels of accumulationfor our model protein.

– M-L. Wang, S. Ancheta, J. Clayton, C.Goldstein and H. Albert (USDA/ARS)

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ugarcane breeding season beganon November 21, 2002 and wascompleted in the f irst week ofJanuary, 2003. Six biparental

crosses were made between LCP85-384 asthe sole pollen donor for six Hawaiian com-mercial cultivars. Polycrosses (732) weremade from 2,500 tassels of 346 breedingclones. In 2003, we evaluated 464 seedlingclones harvested from 23 yield tests (FT7s),of which 22% were selected for further yieldtrails. During the year, 23 FT7 tests wereinstalled from seedling clones selected inand before 2002.

50,544 seedlings raised from true sugar-cane seeds were transplanted in FT1 trial;53,520 FT1 plants planted in 2002 wereratooned; 2,532 clones were planted inFT4; and 577 selected clones from FT4(installed in 2002) will be advanced to FT5in 2004. The top 4 commercial varietiesranked by occupied acreage at the end of2003 were H78-7750, H77-4643, H65-7052 and H78-3567.

H78-7750 continued to be the leading vari-ety for the fourth year. It occupied a totalof 21,964 acres or 48.3% of total cane area.H77-4643, ranked number two since 2001,occupied 9,377 acres or 20.6% of totalcane area. It has been the leading varietyon Kauai since 1996. H65-7052 moved upto the third ranked variety, this year occu-pying 6,955 acres or 15.3% of total canearea. H78-3567 declined in acreage to thefourth position. It occupied 4,277 acres orabout 9.4% of the sugarcane area.

New clones with commercial potential areH91-4392, H93-4068 and H93-4398 forMakaweli soil on Kauai; H90-5555, H90-7453 and H95-5783 for rocky and dryareas on Maui; H88-6401, H95-0181 andH95-1446 for sandy areas on Maui; H86-3792, H87-4394 and H90-0598 for wind-ward Maui. H83-7061 and H87-5794 had

S the top FT7 records in the leeward regionand H87-4319 is the best clone for millwater irrigated fields.

Sugarcane breeding continues to be one ofHARC's most important activities.Changing plantation practices and newindustry prerogatives make the developmentof new cultivars a critical concern.Changing weather patterns and the appear-ance of new pathogen strains require theplantations to have a number of differentcultivars on hand. The Genetics andPathology Department has always been ableto take advantage of its international con-nections with other sugarcane breeders toimport new breeding stock and to sendHawaiian clones to other locations for dis-ease resistance screening for diseases notpresent in Hawaii.

– K. K. Wu

Breeding and Selection

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ematode damage and preco-cious flowering are two seriousproblems faced by the Hawaiipineapple industry. Since

pineapple does not have natural resistanceto nematodes, the growers have been rely-ing on nematicides to reduce damage.Besides adding to the production cost,nematicides may not be available in thefuture due to environmental concerns.Spontaneous flowering of pineapple caus-es the fruit to ripen outside of the normalharvest period. This also adds to the pro-duction cost for the growers. In collabora-tion with the pineapple industry, Universityof Hawaii, and Leeds University (UK), aproject is in progress to genetically engineerpineapple plants for nematode resistanceand flowering control. Our strategy is touse a rice gene encoding a proteinaseinhibitor, cystatin, to combat the nema-todes. The anti-sense version of a pineap-ple gene encoding aminocyclopropane-1-carboxylic acid (ACC) synthase, a keyenzyme in ethylene biosynthesis, is used tosuppress the spontaneous flowering.

In early 2003, we established a fast andreliable method for screening the putativeplants for the presence of transgene usingpolymerase chain reaction (PCR).However, we did not find any positiveplants from the transformations per-

NTropical Fruit ResearchGenetic Transformation of Pineapple with Nematode Resistance and Flowering Control

formed before 2003. Since then, we adopt-ed an improved plant selection proceduredeveloped by Eden Perez (UH), in whichtransformed tissues were isolated by repeat-ed cutting and regeneration of the plantmaterials with increasing levels of selection.A transgenic line (#51) carrying the anti-sense ACC synthase gene for flowering con-trol was obtained using this method. Line51 repeatedly tested positive using PCR andproduced roots rapidly in media with a highlevel of selection (25 mg L-1 of hygromycin).Approximately 500 plantlets of Line 51 and200 control plants are at the rooting stage inpreparation for field tests.

From August to December 2003, we havetransformed 9400 leaf bases usingAgrobacterium carrying either pGU13 orpKLD121. Cystatin gene is under the controlof a constitutive promoter in pGU13 or apineapple root-specif ic promoter inpKLD121. We also infected 5400 leaf baseswith Agrobacterium carrying pGU6, con-taining anti-sense ACC synthase under thecontrol of a constitutive promoter. We arein the process of subjecting the plantletsregenerated from these transformations tothe new selection procedure.

– M-L Wang, G. Uruu (UH), R. Paull (UH), J.Buenafe and C. Nagai

Discovery of a Primitive Y Chromosome in Papaya

apaya, a polygamous angiospermwith male, female, and hermaphro-dite forms, offers several advan-tages for genetic and evolutionary

studies. These include a small genome, ashort generation time, numerous flowertypes, a unique evolutionary process infemale flowers, an intriguing sex determina-tion system, and an established transforma-

P tion system. Papaya sex determination hasbeen a frequent subject of genetic analysesbecause it is directly related to efficient com-mercial fruit production.

Mechanisms of sex determination are diverseand have evolved independently in manygroups of animals and plants. The mostfamiliar system involves the structurally dis-

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15

tinct (heteromorphic) sex chromosomes (Xand Y, or Z and W) that are homozygous inone sex and heterozygous in the other. Maleheterogamety has evolved more often thanfemale heterogamety. Heteromorphic sexchromosomes are believed to have evolvedfrom a homologous pair of autosomes.However, empirical data to support thishypothesis has been lacking. Sex chromo-some evolution is associated with suppres-sion of recombination by chromosomalrearrangement around the sex determina-tion gene, and subsequent degeneration ofthe Y chromosome. In the most advancedsystems, the sex chromosomes are morpho-logically distinctive, and there is no geneticexchange between them for part orall of their length. In humans, 95%of the Y chromosome is suppressedfor recombination.

High-density genetic mapping, finemapping, and physical mapping ofthe sex determination gene led tothe discovery of a papaya primitiveY chromosome with a small male-specific region, the MSY, that isabout 10% of the chromosomeshowing severe suppression ofrecombination and degenerationof DNA sequences. The MSY con-sists of a mosaic of conserved, X-degenerat-ed, and ampliconic sequences. High fre-quencies of sequence duplications andtransposable element insertions con-tributed to the degeneration of the MSY

– Z. Liu, P. H. Moore (USDA/ARS), H. Ma, C.M. Ackerman, M. Ragiba, Q. Yu, H. M. Pearl, M.S. Kim, J. W. Charlton, J. I. Stiles (UH), F. T. Zee(USDA/ARS), A. H. Paterson (Univ. GA) and R.Ming

High-Density Linkage Mapping Revealed Suppression ofRecombination at the Sex Determination Locus in Papaya

apaya (Carica papaya L.) is a fruitcrop cultivated worldwide in tropi-cal and subtropical regions. It isbelieved to be native to tropical

America where it has undergone a long peri-od of selection. Papaya is polygamous withthree basic sex types: female, male, and her-maphrodite. Hermaphrodite trees producea pyriform-shaped fruit that is preferred in

P

Papaya male flowers

resulting in low-gene density. One uniquefeature of the incipient Y chromosome inpapaya is the small physical size of the MSYregion. The small size and the mosaic struc-ture of sequence degradation in the MSYregion suggest a recent origin of the papayasex chromosomes. Hermaphrodite and maleplants of papaya share identical DNAsequences in most parts of the MSY region,suggesting that divergence between male andhermaphrodite is a second step of sex chro-mosome evolution after recessive mutationsresulted in producing the female sex. Thisfinding provides direct evidence for the originof sex chromosomes from autosomes.

the market. However, seeds from hermaph-rodite trees always segregate into hermaph-rodites and females at the ratio of 2:1 andthe sex types of the plants can be determinedonly by inspection of the flowers. Therefore,it is a general practice for farmers to plantthree to five seedlings in one hill, allowingthem to grow for four to six months until thesex types are identified, followed by removal

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of the undesired plants leaving only her-maphrodite plants. Genetic mapping is thefirst step towards cloning the sex determina-tion gene and ultimately to development oftrue hermaphrodite papaya vari-eties.

A high-density genetic map ofpapaya was constructed using 54F2 plants derived from cultivarsKapoho and SunUp with 1501markers, including 1498 amplifiedfragment length polymorphism(AFLP) markers, the papayaringspot virus coat protein marker,morphological sex type, and fruitflesh color. These markers weremapped into 12 linkage groupscovering a total length of 3294.2cM, with an average distance of2.2 cM between adjacent markers.This map revealed severe suppres-sion of recombination around the sex deter-mination locus with a total of 225 markersco-segregating with sex types. This high-den-sity genetic map is essential for cloning of

Segregating papaya F2 population used for genetic mapping

specific genes of interest such as the sexdetermination gene and for the integration ofgenetic and physical maps of papaya.

Cloning and Characterization of Flower Development Genes in Papaya

nstability of papaya f lowers,revealed by environmentally influ-enced sex reversal and stamencarpellody, results in fruit malfor-

mation making it unmarketable.Hermaphrodite f lowers may changetoward maleness by carpel abortion ortoward femaleness by stamen carpellody,(i.e., transforming stamens into carpels),whereas male and female f lowers mayrevert toward hermaphrodism. Incompletesex reversal results in a continuous gradedseries of flower types. For example, undernatural conditions in Hawaii, hermaphro-dite flowers can have from 0 to 10 stamensand 1 to 10 carpels, whereas the truefemale flowers have 0 stamens with 5 vas-cular traces and male flowers have 10 sta-mens and an aborted pistil. Although

I female flowers are fairly stable, stamens infemale flowers were observed by Hofmeyr in1939 and recently by Manshardt. Sex rever-sal in papaya flowers is controlled by genet-ic and environmental factors, including tem-perature, nutritional status and moisture.

Papaya is a good model system to studyflower development in perfect flowers anddioecious flowers. Based on knowledge offlower development in the model plantsAntirrhirum and Arabidopsis, we are cloningand characterizing homologous genes asso-ciated with carpel development in papaya.The Arabidopsis class C organ identity geneAGAMOUS has a papaya homolog namedPAG that shares 85% identity withArabidopsis AGAMOUS. Genomic Southernanalysis showed that papaya has only one

– H. Ma, P. H. Moore (USDA/ARS), Z. Liu, M. S.Kim, Q. Yu, M. M. M. Fitch (USDA/ARS), T.Sekioka (UH), A. H. Paterson (Univ. GA) and R.Ming

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copy of PAG that is expressed at a high levelin carpels. The Arabidopsis gene LFY, apositive regulator of AGAMOUS, has apapaya homolog PFL that shares 65% iden-tity with LFY. PFL encodes a protein shar-ing 71% identity with the LFY homologs ofthe tree species California sycamore(Platanus racemosa) andblack cottonwood (Populustrichocarpal). Despite exten-sive sequence similarity in twoconserved regions, the proline-rich and acidic motifs differbetween PFL and its LFY coun-terparts from other plants.This difference may not affectthe gene function as demon-strated by research on thePinus radiata LFY homologNeedly. Genomic and BACSouthern analyses indicatethat like PAG, PFL exists as asingle copy in the papayagenome. In situ hybridizationresult showed that PFL isexpressed at a relatively low level in theshoot apical meristem of very youngseedlings but it is expressed at a high levelin the floral meristem. Hua1, another regu-

lator of stamen and carpel identities, has apapaya homolog named Phua1 that sharesabout 82% identity with the ArabidopsisHua1 gene. Understanding the papaya flo-ral development process could lead tostrategies for controlling these problems offruit production.

Papaya male flowers

– Q. Yu, P. H. Moore (USDA/ARS), C.Ackerman, H. H. Albert (USDA/ARS), R. E.Paull (UH) and R. Ming

lants have natural defense respons-es against pathogens which can beinduced by avirulent pathogens. Anattack by one avirulent pathogen at

one point on the plant can trigger enhancedresistance against a broad spectrum ofpathogens throughout the plant body; thisresponse is termed systemic acquired resist-ance (SAR). SAR has been studied exten-sively in model plant systems like arabidop-sis, and it has been shown that SAR can beinduced by application of salicylic acid orstructurally similar chemicals like benzothia-diazole (BTH).

We have initiated a study of SAR in papayato build the base for development ofimproved papaya cultivars or protective

PSystemic Acquired Resistance in Papaya

treatments. Root drench of papayaseedlings with BTH resulted in increasedtolerance to the virulent pathogenPhytophthora palmivora, increased ß-1,3-glucanase and chitinase activities (both aredefense related enzymes), and increasedaccumulation of a PR1 (a gene widely usedas a marker for SAR) mRNA. All these indi-cate that papaya has a SAR response whichcan be induced by BTH, making this chemi-cal a valuable research tool and possiblefuture field treatment.

Four members of the papaya PR-1 genefamily were cloned. BTH reduced mRNAaccumulation for two of them andincreased it in the other two. One of these,PR-1d, was induced over twentyfold higher

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apaya, one of the most importantfruit crops in the tropics, is suscepti-ble to a variety of pathogens includ-ing fungi, bacteria and viruses(Nishijima, 1994) that reduce yields

and marketability of fruit. Simultaneous con-trol of both Papaya Ringspot Virus (PRV) andfungal diseases would decrease dependenceon fungicides and significantly improve pre-and post-harvest fruit quality to increase pro-ductivity.

Phytoalexins have been shown to be impor-tant natural components in the defense ofplants against fungal infection. Several fruitcrops, including grapevine and peanut syn-thesize the stilbene-type phytoalexin, resvera-trol, (trans-3,4’,5-trihydroxy-stilbene) whenattacked by pathogens. Under a researchmaterial transfer agreement, we haveobtained a transformation construct fromBayer AG that contains the stilbene synthasegene (Vst1) from grapevine under control ofits own inducible promoter and ahygromycin-resistance gene under the controlof a CaMV35S-promoter (Hain et al., 1993).The beauty of this construct is that stilbenebiosynthesis specif ically depends on theproduct of the stilbene synthase gene sincethe precursor molecules for the formation ofhydroxy-stilbenes, malonyl-CoA and p-coumaroyl-CoA, are both commonly presentin plants. The end product of the action of

than its normal level. Accumulation of themRNA for this gene increased for at least14 days after BTH treatment. In contrast,both chitinase and ß-1,3-glucanase activi-ties peaked after 1 to 2 days then returnedto base levels at approx. 10 days.

The arabidopsis NPR1 gene plays an essen-tial regulatory role in SAR. An NPR1 genewas cloned from papaya; it contains struc-tural domains similar to the arabidopsisgene. These domains are involved in pro-tein-protein interactions and nuclear local-

P

ization, which are essential for function inSAR of arabidopsis. The papaya NPR1 geneis expressed constitutively and is slightlyinduced by BTH treatment. Overall, thesefindings indicate that the basic elements ofpapaya SAR resemble the pathway asdescribed in arabidopsis so the knowledgeand resources available for this model sys-tem can be widely applied in our study ofpapaya.

- Y. J. Zhu, X. Qiu (UH), M-L. Wang, P. Moore(USDA/ARS) and H. Albert (USDA/ARS)

Improved Disease Resistance in Transgenic Papaya with a Grapevine Vst1 Gene

the enzyme on these precursors is stilbene, anatural compound present in several con-sumed fruits and vegetables, which should beacceptable in papaya fruit. Furthermore,using a gene with a pathogen-inducible pro-moter means that stilbene synthase will beexpressed only at a low basal level in trans-genic plants unless there is a pathogen attack.Following a transitory rise in expression, theexpression is expected to return to a low levelwhen the pathogen fails to establish.

A stilbene synthase gene (Vst1) has been iso-lated from grapevine (Vitis vinifera L.) andtransformed into a range of species toincrease resistance of host to pathogens towhich they were originally susceptible. Sinceresveratrol at 1.0 mM inhibited in vitro myceli-um growth of P. palmivora, we hypothesizedthat papaya resistance to this pathogen mightbe increased by transformation with thegrapevine stilbene synthase construct pVst1,containing the Vst1 gene and its pathogeninducible promoter. Multiple transformedlines were produced, clonally propagated,and evaluated with a leaf-disk bioassay andwhole plant response to inoculation with P.palmivora. We found RNA transcripts of stil-bene synthase were induced in plant linestransformed with the grapevine pVst1 con-struct shortly after pathogen inoculation andthat the transformed papaya lines exhibitedincreased resistance to P. palmivora. Five

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independent transgenic lines, Vst7, Vst8,Vst11, Vst12 and Vst14, along with non-transformed control plants were selected forleaf-disk assay to assess tolerance of plantsto P. palmivora. Twenty microliters of sporesuspension (1 x 106 spores mL-1) were pipet-ted onto the center of leaf-disks cultured onMS medium in the Petri plate. Plates wereplaced in the growth chamber maintained at24 oC with 12 h light and 100% relativehumidity. Water soaked spots were observedon the non-transformed controls within 24hrs; necrotic lesions appeared later and pro-ceeded to expand. Necrotic lesions wereobserved on all treated leaves except those

inoculated with a water extract from culturemedium without the fungus. Diameters oflesions measured 3 days after inoculationwere significantly smaller (P<0.05) in all fiveVst1-transformed lines than in the non-trans-formed controls. On the average, the lesionsof the transgenic plants were reduced about25 to 30% in diameter and about 40 to 50% ininfection area. The immature transformedplants appeared normal and will be advancedto field trials to evaluate their utility.

– Y. J. Zhu, R. Agabayani, M. Jackson, C. S. Tang(UH) and P. Moore (USDA/ARS)

n traditional selection systems, themost widely used selectable mark-ers are the antibiotic resistancegenes such as neomycin phospho-

transferase gene. Although proven successfulfor the production of transgenic plants, thepresence of such genes may be undesirable.Because of this, the use of mannose, whichcannot be metabolized by many plantspecies, was developed as a new selectionstrategy.

In this study, mannose was used as a selec-table agent and the phosphomannose iso-merase (PMI) gene as the selectable marker.Transformed cells are able toutilize mannose as a carbonsource and grow in theabsence of other carbonsources such as glucose andsucrose. Cells geneticallytransformed to express PMIacquire a growth advantage(positive selection) on man-nose-containing media,which makes mannose a use-ful selection agent for thegeneration of transgenicplants.

We evaluated the use of PMI as a selectablemarker for the recovery of transgenic papayaplants following biolistic bombardment of

Phosphomannose-Isomerase as an Efficient Selectable Marker in Genetically Engineered Papaya

I papaya embryogenic callus. In this study, weconcluded that papaya embryogenic calluscannot utilize mannose as the sole carbonsource and PMI can be used as a selectablemarker for transformation of papaya.Transformation efficiency was higher thanwith an antibiotic selectable marker.Transformed papaya embryogenic calli withPMI can utilize mannose as efficiently assucrose. A new technology was developedusing alternative selectable marker genes forgenetic engineering which should improveconsumer acceptance of “genetically modi-fied” agricultural products.

– R. Agbayani, H. Albert (USDA/ARS), P.Moore (USDA/ARS) and Y. J. Zhu

Transgenic shoot regenerated fromMannose-resistance calli

Transgenic plant about 4weeks after transplanting

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he productivity of papaya (Caricapapaya L.) is often limited by itssusceptibility to a number of natu-ral enemies. Papaya pests include

nematodes, aphids, leafhoppers and mites.

The aim of this study is to generate papayaplants with improved pest resistance. Forplant transformation, a biolistic gene gunprocedure was used. A gene encoding a pro-tein with known entomotoxic properties wasintroduced into a commercial papaya culti-var. The protein selected was a lectin, termedGNA, from the snowdrop lily,Galanthus nivalis.

Lectins are naturally-occur-ring proteins which possess atleast one non-catalyticdomain that binds reversiblyto a specif ic mono- oroligosaccharide. They areubiquitous not only in plantsbut also in animals andmicroorganisms. In animalsand microorganisms, theirmain function is cell recogni-tion. In plants, their role is stillsomething of an enigma.Some have been shown to betoxic to insects. Feeding stud-ies have shown that the GNAprotein has an insecticidalnature but is non-toxic tohigher animals.

It is hoped that papaya plantsexpressing GNA may have improved resist-ance to defoliating pests. Also, plants mayhave some resistance to nematodes whichcan be detrimental to the growth and devel-

Engineering Papaya for Improved Pest Resistance

T opment of papaya. GNA was recently shownto have potential as an anti root-knot nema-tode protein when expressed in Arabidopsisthaliana plants.

We have generated a number of independentpapaya lines expressing the GNA protein.Lines were first identified by molecular analy-sis. Protein expression was then examined. Ithas been determined that the recombinantGNA protein is biologically active. Futureexperiments will investigate the resistance ofthe transgenic papaya plants to pest attack.

– H. McCafferty, P. Moore (USDA/ARS) and Y.J. Zhu

Two common pests of papaya:Above: Stevens leafhopper(Empoasca stevensii)

Right: Carmine spider mite(Tetranychus cinnabarinus)

ur coffee breeding and selectionprogram, to develop a uniquelyHawaiian coffee, is in its 7thyear and over 100 hybrid fami-

lies have been produced. The program'sobjectives are to produce coffee cultivarsadapted to Hawaii's specific growing envi-ronments with desirable characteristics,such as, enhanced flavor, excellent cuppingquality, large bean size, increased yield anddisease resistance.

In 1999, a large scale coffee crossing pro-gram was undertaken (HARC AnnualReport 1999, p. 19) using potentially elite

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rabica coffee is traditionallyseed propagated, since themajority of arabica cultivars areinbred and growers can easily

obtain enough seedlings of their cultivars.However, hybrid arabica coffee cultivarsneed to be cloned (vegetatively propagat-ed) in order to preserve their superior traitsfor commercial cultivation. A protocol wasdeveloped at HARC (HARC Annual Report1997, p. 22) to vegetatively propagate ara-bica coffee by rooted cuttings. Efficiencyof rooting was 28% and varied among cul-tivars tested. The protocol is useful only forsmall-scale propagation. Cloning viasomatic embryos is the method of choicefor production of large quantities ofpropagules (HSPA Annual Report 1991, p.51-52). Induction of coffee somaticembryos takes 6-8 months from initiation.

Experiments were conducted for in vitropropagation of arabica coffee using in vitroshoot multiplication for an alternative tocuttings and somatic embryo derivedpropagules. Vertical shoots of maturedcatuai and typica trees were used as

A

O

explants. Surface sterilization of shoot tipswas optimized using various combinationsof sodium hypochlorite concentrations (0.6-1.8%) and treatment durations (10-30min). Fifty percent of shoots treated with1.2% hypochlorite for 20 min kept tips greenduring the first 28 days of culture, whereasno significant difference was found amongtreatments to establish clean shoot cultures.Effects of sucrose (0-40%), cytokininsincluding BA (6-benzylaminopurine, 0-10mg L-1), kinetin (1.5 mg L-1) and 2iP (2.5mg L-1) were also tested on new shootgrowth in culture. Addition of NAA (0.02mg L-1) did not help increase initial shoottip growth. In 43 days, we obtained 18 invitro propagules (37.5%). All the propaguleshad 1-2 shoots. We were not successful atroot induction from these shoots usinggrowth regulators such as IBA (indole-3-butyric acid). These preliminary results indi-cated that in vitro shoot tip culture is not asefficient a propagation method as cloningvia somatic embryos.

– L. Fournier (E.S.I.T.P.A. Rouen, France), J.Clayton and C. Nagai

Coffee ResearchCloning of Arabica Coffee

Progress of Coffee Breeding and Selection Program

trees with desirable characteristics selectedfrom five coffee growing areas in Hawaii. Bymid-2000, approximately 1500 progenytrees from 165 crosses were under carefulfield cultivation at Kunia (HARC AnnualReport 2000, p. 21). Tree morphology,yield, cherry/bean characteristics and cup-ping quality of the original selected treeswere evaluated by the fall of 2001 and fruit(cherry)/seed (bean) characteristics wereevaluated in the fall of 2002.

Twelve superior hybrid families were selectedfrom the original 120 crosses made in 1999.Selection was based on tree morphology

and height, cherry sizeand yield potential.These promising cof-fees included larger-bean Mokka hybridsand higher yieldingMargogipe (averagetaste, large-bean, low-yield) hybrids. InDecember 2003, acupping test of 2 newhybrids of Margogipe(H99-34: YellowCatuai x Margogipe,H99-36: MA1-12-mokka x Margogipe)was conducted atCathy Cavaletto's lab-oratory at UH. H97-1700 (KA17C-YellowCatuai) was used as a control. A seven-judge panel found no significant differencein cupping including dry and wet aroma,acidity, flavor and body. This preliminarycupping test indicated that size and yield ofcoffee were modified without changingcupping quality. Seeds of these trees weregrown in the HARC Maunawili greenhouse

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Coffee hybrid families (H99- series) at Kunia Substation

- C. Nagai, R. V. Osgood, J. Clayton, C.Cavaletto (UH) and S. Bittenbender (UH)

Genetic Transformation of Coffee for Nematode Resistance Using Cysteine and Serine Proteinase Inhibitors

oot-knot nematodes, Meloidogynespp., adversely affect coffee pro-duction in many coffee-growingregions. Meloidogyne konaensis

causes severe damage to Coffea arabica cv.Typica (‘Guatemala’) grown on the Island ofHawaii. A weakened root system and overalldecline of the tree occur when a nematodeinfestation is severe. Grafting Typica on C.liberica rootstocks is currently providinggrowers with partial resistance to M. konaen-sis. In an effort to increase resistance levels,genetic engineering a Typica rootstock isbeing explored.

Cystatin, a cysteine proteinase inhibitor fromrice, and a dual construct, cystatin with acowpea trypsin inhibitor were chosen for their

R success in reducing nematode levels in othercrops. The cystatin gene was driven by theCaMV35S promoter or tubulin (root specific)promoter with the NPTII gene for selection.The dual construct was driven by the 35S pro-moter (HARC Annual Report 2001-2002, p.26-27). Transformation was performed usingboth Agrobacterium tumefaciens and particlebombardment.

Plants were produced from selected somaticembryos (SE) in culture media with an antibi-otic, G418. Plants were regenerated from 29of the 1,100 selected somatic embryo linesinoculated with A. tumefaciens. Fifteen plantlines were produced from the 1,200 selectedSE lines using the particle bombardmentmethod.

and then shipped to Kauai in October forfield evaluation at Kauai Coffee Co. Thefirst field evaluation at a commercial coffeefield will be initiated in early 2004.

he coffee breeding and selectionprogram initiated in 1997 has asits objective the development ofnew cultivars adapted to

Hawaiian growing conditions and havingdesirable characteristics. It is made moredifficult by the limited genetic diversitydetected among arabica coffee cultivars.This lack of genetic diversity in the genepool of arabica coffee limits the potentialfor germplasm improvement. The occa-sionally occurring spontaneous interspecif-ic hybrids have been widely used forimproving disease and pest resistance inarabica coffee. One extensively used sourceis the Timor hybrid (2n = 44), discoveredon the island of Timor, that was derivedfrom a spontaneous interspecif ic crossbetween C. arabica and C. canephora.One example of the use of the Timorhybrid in coffee breeding programs is thedevelopment of the coffee leaf rust-resist-ant Catimor cultivar. This was derived froma cross between the Caturra cultivar andthe Timor hybrid. Another spontaneousinterspecific hybridization between C. ara-bica and C. liberica increased the geneticdiversity among C. liberica introgressed

PCR analysis confirmed the pres-ence of the cystatin gene in 12lines of plants (Figure). Multipleplants were obtained in severalSE lines. Eleven PCR-positiveplants were obtained from theSE line B17-3 using two differentprimer sets. These multipleplants will be used for the nema-tode bioassay in the University ofHawaii greenhouse next year.Further molecular analyses,Western and Southern blottingswill also be performed.

– R. Myers-Cabos (UH), C. Nagai,B. Sipes (UH), D. Schmitt (UH), H.Atkinson (Leeds Univ.)

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T

Transgenic coffee with nematode resistance genes, cystatin and trypsininhibitors

Construction of a Genetic Map for Arabica Coffee

lines and was used as the main source ofrust resistance in coffee breeding programsin India.

Molecular marker linkage maps are beingused successfully in many crop species fordirected germplasm improvement. Markerassisted selection allows for screening oflarge numbers of trees for a gene of interestat an early stage of growth and reduces thenumber of backcrosses required to obtainquality traits. As reported in the HARC2001-2002 Annual Report, we used ampli-f ied fragment length polymorphisms(AFLPs) to construct a genetic linkage mapon a pseudo F2 population of arabica coffee(Coffea arabica L.) derived from a crossbetween the cultivars Mokka hybrid andCatimor. Sixty trees from this populationwere selected on the basis of plant heightdistribution to construct a linkage map. Atotal of 456 dominant markers and eight co-dominant markers were generated from 288AFLP primer combinations.

The results to date determined that, of thetotal number of markers generated, 68%were from Catimor, 30% from Mokka hybrid

raditionally, coffee was grown asa shade crop and this is still thecase in some parts of the coffee-growing world. Shade-grown

coffee has often been associated with high-er quality at cupping. Therefore, a collab-orative effort is underway between HARCand the University of Hawaii to determinewhat level of shade might be suitable forgrowing high quality coffee in Hawaii.Trials are going to be undertaken at a num-ber of sites around the state, includingHARC's Kunia substation, where extensivecoffee plantings have been made for breed-ing purposes and are available for this kindof study. Different kinds of shading will bestudied, including spraying a kaolinite mix-ture directly onto the leaves of the plant.HARC's Analytical Chemistry Laboratorywill be undertaking analyses of a number ofcoffee components in green and roastedcoffee harvested from this study.

and 2% co-dominant. This dis-tribution suggests that the het-erozygosity within the Catimorsub-genomes was twice thatwithin the Mokka hybrid sub-genomes. Linkage groups wereconstructed resulting in 16major linkage groups contain-ing 4 to 21 markers, and 15small linkage groups consistingof 2 to 3 linked markers each.The total length of the mapwas 1802.8 cM with an averagedistance of 10.2 cM betweenadjacent markers. This geneticmap will serve as the frame-work for mapping quantitativetrait loci (QTL) controllingsource-sink traits in the samepopulation.

– H. M. Pearl, C. Nagai, P. H. Moore(USDA/ARS), D. L. Steiger, R. V. Osgood and R.Ming

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T

Jamie Clayton and Heather Pearl collecting leaf samples in coffeefield.

Quality Aspects of Shade-Grown Coffee

Currently, the project is in its early stages,and pruning of coffee trees to be included inthe study has just been completed. A grad-uate student will soon be working at HARCto develop methods for roasted coffeeanalysis. Green coffee methods were devel-oped in a previous study. When the studytrees are ready for harvest, all analyticalwork associated with this project will beundertaken at HARC.

– M. C. Jackson, T. Idol (UH), S. Steiman (UH)and H. C. Bittenbender (UH)

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oa (Acacia koa) is the dominanttree species in much of theremaining native Hawaiianforests and provides important

habitat for Hawaiian plants and animals,many of which are endangered. NativeHawaiian culture places a high importanceon koa for uses such as canoe building andcarving. Koa is the basis of an estimated$50 million hardwood furniture and craftsindustry in Hawaii yet little is known aboutthe suitability of diverse island seed sourcesfor sustainable planting at commercialsites. Information is also lacking on themanagement of koa for commercial, sus-tainable operations on former agriculture,ranch and degraded native forest lands. Atpresent, koa is an endemic tree of greatvalue but little is known about manage-ment. Protecting and restoring koa forests,both natural and plantation, is therefore ahigh priority among land managers andcommercial foresters in the state.

The vascular wilt disease Fusarium oxyspo-rum f.sp. koae, commonly known as koawilt, was recently identified as a diseasecausing dieoff of koa trees in Hawaii. Littleis known of its ecology or origin, but it isfound on all of the main islands of theHawaiian chain. It infects trees through

KForestry ResearchKoa Research at HARC

their roots and attacks the vascular systemcausing eventual death. Koa plantings atlower elevation have especially been impact-ed by this pathogen, limiting managers'abilities to use koa in forestry and restora-tion projects. At Maunawili, Oahu, Acaciakoa families’ survival percent ranged from alow of 4.0% to a high of 91.6%. The averagefamily survival percent was 35.4%. However,the two best families had survival percent-ages of 91.6% and 75%, respectively. Koa wiltin natural forests at high elevations has alsobeen found on the islands of Maui andHawaii. On Maui, 4200 acres are affectedby the disease in Haleakala National Park.Koa survival was reported to be 50% in areasaffected by koa wilt on Maui. Recently,there have been outbreaks of koa wiltreported on the island of Hawaii in VolcanoNational Park.

HARC’s forestry program is primarily aimedat selecting the best suited seed sources ofkoa for specific sites in Hawaii. Koa familyvariation in HARC field trials strongly sug-gests the presence of resistance to koa wiltdisease. Work continued on screening ofkoa seedling families for genetic resistanceto koa wilt (Table 1.)

– N. Dudley

Table 1. HARC Koa Seedling Families Trial Network

Site Island

Maunawili (HARC) OahuKapa’a (Midler Trust) KauaiHonolua Plantation (Maui Pineapple Company) MauiKula (L. Dorcay Trust) MauiHumu’ula (Parker Ranch/DHHL) Big Island

aro had a market value of 2.7 mil-lion dollars in Hawaii in 2003(Hawaii Agricultural StatisticsServices) and is an important seg-

ment of Hawaii's agriculture. However,due to its minor crop status, there are fewpesticides registered for this crop and nonefor fungal pests commonly occurring ontaro (Ooka, 1994). Thus, there is a need todevelop biologically-based managementstrategies for economically signif icantpest/disease problems on Hawaii’s impor-tant crops. Genetic transformation of cropplants can result in increased resistance topests. Such transgenic crops provide bio-logically based pest management with low-ered production costs and a cleaner envi-ronment.

HARC, UH and USDA are collaborating todevelop a taro transformation system. Thespecific objectives are: a) to develop aregeneration system for transformed taroto produce fertile plants; b) to develop atransformation system for taro, using selec-tion genes, a reporter gene and the putativefungal resistance gene, rice chitinase gene(CHI111); and c) to test for increased fun-gal resistance of transgenic taro.

We have established tissue cultures of tarousing meristem tip culture. Shoot tips oftwo commercial taro cultivars, ‘Bun Long’and ‘Maui Lehua’, were explanted using themethod of Hartman (1974) to avoiddasheen mosaic virus. An ELISA test(Agdia Kit for Dasheen Mosaic virus ofPathoScreen) was performed on tissue-cul-tured taro to confirm the absence of virus.

More than 20 tissue culture media withvarying phytohormones were tested for cal-lus and shoot initiation for two taro culti-vars. Only two media were found toinduce embryogenic calli, with most of themedia inducing shoot growth.

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TMiscellaneous CropsDevelopment of a Transformation and Regeneration System for Taro

To select transformed tissue in an antibioticmedium, the appropriate level of an antibi-otic needs to be established. Kill curves fortaro tissue culture were carried out in theMS+BA (4.0 mg L-1) media with varyingconcentrations of the antibiotic, G418. Aconcentration of 50 mg G418 L-1 inhibitedgrowth of taro and will be used in futureselection media.

Five plasmids (pHP12679, pHP5897,pBI121, pBISN1 and pAHC27), all contain-ing the ß-glucoronidase (GUS) reportergene, were tested for GUS transient expres-sion after being delivered by biolistic bom-bardment to taro shoots. Two plasmids(PHP12679 and PHI 121) that gave thehighest expression will be used for latertransformations of taro.

A rice chitinase gene has been successfullyinserted into taro using particle bombard-ment with 1% transformation efficiency.The transgenic plantlets have been multi-plied for further fungal resistance tests. Wealso have successfully transformed a ricechitinase gene into taro calli using an effi-cient Agrobacterium-mediated transforma-tion method. This is the first report oftransformation of taro with a useful geneand showed that the genetic transformationtechnique can be successfully applied to theimprovement of taro resistance to diseases.

– J. Zhu, M. M. M. Fitch (USDA/ARS), P. H.Moore (USDA/ARS), L. He (UH), S. C.Miyasaka (UH), M. Tanabe (UH) and J. Cho(UH)

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awaiian `awa, although origi-nating in the South Pacific, nowcomprises approximately 13 cul-tivars that are morphologically

and chemically distinct from their SouthPacific counterparts. Because of 'awa'spropensity to spontaneously mutate, gen-erations of astute growers were able tocarefully select mutant plants with themost pleasing and useful psychoactiveeffects. The result is that Hawaiian `awatoday represents some of the best to befound anywhere.

With the gradual diversification of agricul-ture in Hawaii, the Hawaii AgricultureResearch Center's Analytical ChemistryLaboratory has moved to develop researchprojects that service the needs of this fastgrowing agricultural sector. HARC nowanalyzes the bulk of the `awa commercial-ly grown and sold throughout theHawaiian islands, and also services theneeds of growers and distributors in otherregions. Clients need accurate, dependableinformation on overall kavalactone con-tent and the relative concentrations ofeach of the six major kavalactones, theactive ingredients.

Traditional cultural practices indicate that`awa should be grown in partial shade.No clear practices for fertilization rate anddegree of pruning have ever been estab-lished. Therefore, HARC, together withcollaborators at the University of Hawaii,College of Tropical Agriculture and HumanResources (Dr. H. C. Bittenbender, Dr. C.S. Tang) are evaluating the effects ofparameters such as light, pruning and fer-tilization rate on overall kavalactone con-tent in the root. The work began inSeptember 2000 and is still continuing.The project is based at the UH Magoonhorticultural facility and utilizes a novelbasket cultivation system based upon

HDetermination of Environmental Factors for Increased Kavalactones in 'Awa

experimentation by Big Island `awa grow-ers. This system allows for the close moni-toring of irrigation and fertilization rates,and also the amount of sunlight. Its great-est advantage is that it facilitates the sam-pling of root pieces. The basket is simplyopened, root pieces excised and then thebasket re-sealed. In addition, soil-bornepests such as the root knot nematode areeliminated. By the end of the project, theimportance of light, fertilization and prun-ing will be established and cultural practicesoptimized so that kavalactone content canbe maximized. HARC is providing analyticalsupport to this effort.

Recently, there has been evidence suggestingthat solvent extracts of `awa are responsiblefor liver disease in a relatively small numberof people regularly taking this form of sup-plement. In collaboration with Dr. C. S.Tang's group at the University of Hawaii,HARC is currently working on def iningmethodology and plant physiologicalprocesses involved in natural production ofalkaloids found in `awa stems that havebeen shown to be toxic to human liver cellsgrown in culture.

In addition, a HARC/UH collaborationresulted in the development of a very rapidand inexpensive method for determiningindividual kavalactone content using nearinfrared reflectance spectroscopy (NIRS).

– M. C. Jackson, H. C. Bittenbender (UH) and C.S. Tang (UH)

his project is aimed at developinga system to transiently express ahigh level of foreign protein inplants without tissue-culture pro-

cedures. Plants offer several advantages overother, more traditional expression systems,for the production of high-value products. Ithas been demonstrated that plants canexpress, fold, assemble and process complexforeign proteins. There may be significanteconomic benefits in the production of bulkquantities of valuable pharmaceutical prod-ucts in plants compared with animal celllines or transgenic animals, and there may befewer safety concerns associated with use ofplant expression systems.

Several studies have demonstrated thatplant viruses can be used as vehicles to intro-duce and express foreign proteins in plants.Many plant viruses multiply to high levels inplants, leading to concomitantly high levelsof foreign protein expression when a foreignprotein gene is incorporated in the viral con-struct. Virus delivery does not lead to per-manent incorporation of the transgene intoplants. Nevertheless, depending on whichvirus is used, virus multiplication and geneexpression can continue for long periods(weeks or months). Plant virus expressionvectors have several other potential advan-tages over the more commonly used trans-genic plant technology. In this study, weused two model genes to evaluate expressionand function of foreign genes in plants. Oneof the genes coded for human granulocyte-macrophage colony-stimulating factor (GM-CSF) and the other for Citrus AP24(CsAP24), an antifungal protein.

GM-CSF has important clinical applicationsin the treatment of neutropenia and aplasticanemia and reduction of infections associat-ed with bone-marrow transplants. In thisstudy, potato virus X (PVX) viral vector sys-tem was evaluated for efficient introduction

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TDeveloping Highly Efficient Transient Expression System with Plant Viral Vector

and expression of the gene for GM-CSF pro-tein. Nicotiana benthamiana plants wereinoculated with the plasmid DNA of PVX vec-tor containing GM-CSF gene driven byCaMV35S promoter. The expression level andsize of recombinant GM-CSF protein weredetermined with ELISA and Western blotanalysis. The results showed that leaf age sig-nificantly affected GM-CSF protein produc-tivity with younger leaves expressing a higherlevel of recombinant protein. Protein expres-sion levels declined slightly over several daysfollowing induction. The two leaves abovethe inoculated leaves also produced signifi-cant amounts of systemic GM-CSF. Proteinextracts of the Nicotiana tobacco leaves con-tained up to 2% recombinant GM-CSF pro-tein. The protein extract actively stimulatedthe growth of human TF-1 cells, suggestingthat GM-CSF expressed via the PVX viral vec-tor was biologically active.

PVX viral vector was also used to identify thefunctional role of AP24 proteins in plant dis-ease resistance. PVX viral vectors were con-structed for CsAP24 and for CsAP24 genewithout a start codon. PVX vector containingthe CsAP24 gene without the start codoncaused virus disease symptoms in the inocu-lated plants. The reason may be due to sur-plus or untranslatable CsAP24 RNA interfer-ence. When Phytophthora parasitica wasplated on V8 agar medium containingCsAP24 protein, fungal spore germinationwas inhibited, but not mycelial growth.Nicotiana benthamiana plants infected withPVX vector without the CsAP24 gene devel-oped severe disease symptoms when chal-lenged with the fungus. The plants inoculat-ed with PVX viral vectors containing theCsAP24 gene showed more resistance to P.parasitica. It is proposed that endogenousCsAP24 protein promoted fungal resistancein N. benthamiana plants.

– F. Zhou, P. Moore (USDA/ARS) and Y. J. Zhu

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he Golden Apple Snail (Pomaceacanaliculata) is a major pest inwetland taro patches in Hawaii,causing major crop losses. Hawaii

taro production in 2001 was estimated at6.4 million pounds, with a sales value ofabout $3.4 million, a 9% decrease from 2000(State of Hawaii, Dept. of AgricultureAgricultural Statistics Service Report, March22, 2002). This was in large part due to theeffects of the Golden Apple snail. GoldenApple snail was introduced into Hawaii,Japan and many other countries in SoutheastAsia from South America as a source of foodin the early 1980s. However, after its com-mercial markets had failed, discarded andescaped snails invaded taro and rice ecosys-tems and have been causing significant eco-nomic damage. In Hawaii, these snails werealso purposely introduced into taro paddies(lo`i), the reasoning being that they could beharvested for food. However, the conse-quences of this action were not fully under-stood at the time. The snails are voracious,fast growing and with a huge reproductivepotential. A single female can produce asmany as 15,000 offspring per year, and canthrive in water at a density of 1,000 snails persquare meter. They mature within 60 to 85days and spawn at weekly intervals and havebeen described as the most damaging pestever to hit neotrop-ical areas. Thesnails very quicklyspread throughouttaro lo`i, via theextensive and inter-connected irriga-tion system. In1996, the HawaiiState Departmentof Agriculture sta-tistical servicesrecorded that in1992, approxi-mately 60,000 lb.of fresh taro wasmarketed from the islands of Oahu, Molokaiand Maui, yet in 1996, only approximately

TSnails

10,000 lb. was marketed; an 84% decline,largely due to apple snails. This picture ofrapid and overwhelming infestation is reflect-ed elsewhere in the world. For example, $1million has been spent annually to control thesnails in rice paddies in Taiwan since 1982. Inaddition, an estimated 20% of farm incomewas spent on apple snail control in thePhilippines in 1993. Since taro grows in waterwhere there are aquatic animals sharing thesame ecosystems, chemicals are not allowedfor pest control.

In 2003, HARC conducted limited field stud-ies of two botanical extracts. Extracts weremade from plants that had shown promise inprior toxicology studies. The field trial was ini-tiated in December 2003 and is scheduled tobe completed in May of 2004. Of the twoextracts tested, application of one of theextracts has resulted in an average snail mor-tality rate of greater than 90% compared withthe untreated control plots, 66 days after theinitiation of the trial. The majority of the snaildeaths were observed within 15 days after ini-tiation of the test.

HARC will use the data gained from the trial toto move toward an EPA registration of theextract for use as a molluscicide in wetlandtaro.

– M. C. Jackson

Apple snail setupApple snail eggs on taro leaf stems

ince the mid-1950s, agricultureand the military have employedmany compounds for pest controland other uses. Some of these are

extremely long lived, including DDT, hep-tachlor, PCP, PCBs and PAHs. There are avariety of means by which soils could betreated to remove persistent chemicals, forinstance, removal of the contaminated soiland incineration or landfill. Consideringthe extremely large quantities of soilinvolved and the lack of a suitable landfill ora facility large enough to incinerate the vol-umes that would be required, these alterna-tives are not suitable.

The research undertaken in this projectsought to address the problem in a differentmanner; by isolating strains of saprophytic,wood-rotting fungi present in Hawaii thatcan efficiently degrade chemicals such asthose described above, in soils, in situ.Several wood-rotting basidiomycetes havebeen reported to be extremely activedegraders. The results from this project canbe used as the basis for further studies offield delivery systems and evaluation ofeffectiveness, ultimately resulting in an effi-cient, environmentally friendly means ofbioremediating contaminated soils.

Certain saprophytic fungi have already beenshown to efficiently degrade persistent con-taminants in soils. However, the reportedstrains were isolated in regions other thanHawaii and consequently they may be poor-ly adapted to Hawaii’s environment. Inaddition, there is difficulty in legally import-ing these microorganisms. There is there-fore a need to find effective fungal speciesgrowing within the state. The varied micro-climates and ecosystems within the islandsprovide a rich repository of fungi that mayhave unique properties with respect to theirability to degrade persistent compounds.The U.S. military in Hawaii and other

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SDegradation of Chlorinated Organic Compounds Using Saprophytic Fungi from Hawaii's Forests

Pacific basin regions will benefit from a tech-nology developed in an area and climate thatis similar to theirs.

Wood-degrading fungi growing in forests onOahu and Hawaii were collected, isolated inpure culture in vitro and identified. Fungi weregrown in liquid culture and test compoundsadded at 1 µg mL-1. After seven days, certainspecies of fungi were able to significantlydegrade some chlorinated pesticides andPCBs and PAHs. Radio-labeled chlorinatedtest compounds, used in some experiments,demonstrated that the radio-label used wasreleased as a gas, probably CO2. The extensivedegradation indicated little possibility of theformation of potentially toxic intermediates insignificant quantities. Expanding the work bymixing selected fungi with soils proved diffi-cult. Some results were encouraging but wereinconclusive that degradation was occurring.

– M. C. Jackson, J. Pitz, S. Schenck and D. Hemmes(UH)

n keeping abreast of the latestenvironmental regulatory develop-ments, HARC assists Hawaii'sfarmers in correctly applying the

rules to everyday farming activities. HARCcontinues to provide training for membersand others who need to know and under-stand these specialized and often highlytechnical requirements. HARC worked thisyear with the Hawaii state legislature, regu-latory agencies, and various stakeholdergroups to help develop appropriate envi-ronmental policies relating to nonpointsource pollution, surface and coastal waterquality, agricultural air quality issues, cropprotection chemicals, and endangeredspecies habitat, among others.

– J. Ashman

IServicesEnvironment

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Quality Assurance Unit

Computer System Administration

ARC continued to participate in thepesticide registration process underthe Environmental ProtectionAgency's Federal Insecticide

Fungicide, and Rodenticide Act. An independ-ent Quality Assurance Unit (QAU) is main-tained to inspect and audit related studies.The QAU participated in a sugarcane magni-tude of residue study. HARC's facilities wereinspected for compliance with the EPA'sGood Laboratory Practice standards by oneof our sponsors. As a member of the HawaiiPesticide Advisory Committee, input from thesugarcane industry was provided to the StateDepartment of Agriculture.

— B. Vance

HLaboratory ServicesAnalytical Chemistry Services

ur contract laboratory provided ana-lytical chemistry services to Hawaii’sagricultural community, industries,government agencies and to U.S.

mainland and international companies. Weanalyzed for natural and synthetic compoundsin plants, soil, water and air using gas chro-matography and high-performance liquid chro-matography. Our analytical residue determina-tions ranged from quick screens for generalinformation to detailed research studies includ-ing pesticide registration projects for the UnitedStates Environmental Protection Agency(USEPA).

Pesticide registration for use of imidacloprid inpapaya was completed this year (ref., FederalRegister, 68 (114) June 13, 2003). HARC'swork began in 1998 and provided field phaseand analytical residue phase data for imidaclo-prid in papaya. Funding was provided byHawaii's Department of Agriculture and thePapaya Action Committee, PAC. Minor CropPest Management, IR-4, a national agriculturalprogram to clear pest control agents for minorcrops, prepared the final imidacloprid registra-tion report for USEPA. This registration of imi-dacloprid not only allows use on papaya, butalso on a crop grouping including star apple,black sapote, sapodilla, mango, canistel andmamey sapote. The tolerance for imidaclopridis 1 ppm. Labels have been updated forProvado® and Admire®. Imidacloprid pro-vides protection against sucking insects such asleafhoppers and aphids on many crops.Without treatment, heavy infestations ofleafhoppers have destroyed papaya fields.

Agricultural commodities and products thatare exported from the United States and othercountries must meet the pesticide residue toler-ance standards of the import country.Analytical chemistry determinations were per-formed on pineapple, papaya, Kona coffee, ste-via, and wood products to show that theseproducts comply with Japan's import toleranceregulations.

– J. Pitz

O

ew users to the local area network(LAN) were provided access toHARC's LAN, an e-mail account ifappropriate and an introduction to

the network and use policies. HARC's com-puter inventory grew by 5 computers, 1 print-er, 1 hub and 1 switch. The LAN at theExperiment Station is still tightly integratedwith the USDA/ARS and their users are pro-vided technical support. Continued assistancewas provided to users of Sun Microsystem'soffice software suite, StarOffice, which under-went 2 upgrades. Limited testing of the Linux(vs. Windows) operating system on the desk-top was begun. To complement our disasterrecovery plan, Standard OperatingProcedures for the major procedures werewritten or revised. Users were reminded of thenecessity to keep their PCs updated with thelatest antivirus signatures and security patch-es. Anti-SPAM software at the user level waspromoted. The firewall's software and hard-ware was upgraded and maintained. The fileserver was replaced as was its network operat-ing system and relocated to a more securelocation; RAID was deployed. The dial-up e-mail system was replaced by a fasterPOP3/SMTP system that further allowedusers to access their e-mail off-site. Lowerrates were secured for our telephone system.

– B. Vance

N

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Henry Cortes, ExperimentalistAmy Dela Cruz, Laboratory Technician

Peizhu Guan, Research AssistantPeggy Hiraki, Laboratory TechnicianTara Katayama, Research Assistant

Walter Kitagawa, Laboratory AssistantTerryl Leong, Special Projects Assistant

John David (JD) McEnerney, Research AssistantRhonda O'Kane, Research Assistant

Heather Pearl, Research AssistantXiaohui Qiu, Research Assistant

Lal Ratnapala, Research AssistantMeghan Riley, Research Assistant

Josienellie Rosete, Laboratory AssistantJacy Suenaga, Laboratory Assistant

Sasa Tom, Research AssistantErin Yafuso, Laboratory Assistant

George Yamamoto, Special Projects Assistant

Kunia and Maunawili SubstationsRudy Dizor, Mechanical Operator

Angel Galvez, Mechanical OperatorRogelio Fernandez, ExperimentalistRoland Fernandez, ExperimentalistErnest Gamatero, Experimentalist

Richard Kinoshita, Breeding Station SuperintendentRogelio Pascua, Experimentalist

Leslie Poland, Kunia Farm ManagerJohn Rockie, Experimentalist

Roger Styan, Experimentalist, Supervisor

Maui SubstationAlbert Arcinas, Maui Farm Manager

Artemio Bacay, Field WorkerTeodoro Bonilla, Field WorkerRomeo Cachola, Field Worker

Luis Dela Cruz, Weighing Machine OperatorWilson Galiza, ForemanGael Ito, Experimentalist

Pacifico Padilla, Senior Field WorkerDomingo Vallecera, Field Worker

Kauai SubstationFernando Garcia, Field WorkerNarciso Garcia, Field Worker

StudentsLinda He, University of HawaiiHao Ma, University of Hawaii

Roxana Myers, University of HawaiiShawn Steiman, University of Hawaii

Xiaohui Qiu, University of Hawaii

Administration and Support StaffStephanie Whalen, President and Director

Robert Osgood, Vice President and Assistant DirectorJanet Ashman, Environmental Specialist

Tracy Bacnis, SecretaryFlorida Chow, Human Resources

Becky Clark, Computer Operations ClerkElon Clark, Buildings and Grounds Superintendent

Ladislao Gonzalez, Watchman, MaintenanceAnthony Lannutti, Controller

Ann Marsteller, LibrarianCharlene Onishi, Accounts PayableCynthia Pinick, Executive Secretary

Blake Vance, LAN Administrator and Quality Assurance

StaffHenrik Albert, Molecular Biologist, USDA, ARS

Jim Carr, Biological Laboratory Technician, USDA, ARSNicklos Dudley, Forester

Maureen Fitch, Plant Physiologist, USDA, ARSMel Jackson, Chemist

Ray Ming, Plant Molecular GeneticistPaul Moore, Plant Physiologist, USDA, ARS

Chifumi Nagai, Plant Breeder, BiotechnologistJerry Pitz, Chemist

Lance Santo, AgronomistSusan Schenck, Plant Pathologist

Hugh Smith, EntomologistBen Somera, Sugar TechnologistKuo Kao Wu, Sugarcane BreederAileen Yeh, Hawaii Coordinator

Judy Zhu, Biochemist

Research AssociatesCindy Goldstein

Zhiyong LiuRagiba Makandar

Tichaona MangwendeHeather McCafferty

Kaloian NickolovMing-Li Wang

Qingyi YuFengyong Zhou

Laboratory Research Assistants andExperimentalists

Christine Ackerman, Research AssistantRicelle Agbayani, Research Assistant

Susan Ancheta, Laboratory TechnicianJosephine Buenafe, ExperimentalistJoseph Charlton, Research AssistantJamie Clayton, Research Assistant

S U G A R P R O D U C T I O N , E T C .

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Publications and Presentations

Albert, H.H., X. Qiu, M-L. Wang and P.H. Moore (2003) Suppression subtractive hybridizationanalysis of BTH-induced genes in papaya. West Sect ASPB Oct. 10-11. Plant Genomics 2003:p. 21.

Albert, H.H., M-L. Wang, P.G. Lemaux and P.H. Moore (2003) Toward single-copy transgeneintroduction for sugarcane. 4th ISSCT Molecular Biol. Workshop. p. 32.

Albert, H.H., M-L. Wang, P.G. Lemaux and P.H. Moore (2003) The Ac-Ds transposon system -a tool for engineering and research in sugarcane. Plant, Animal & Microbe Genomes XI. W191.p. 43.

Albert, H.H. and H. Wei (2003) Promoter of the sugarcane ubi4 gene. US Patent No.6,638,766.

Bowers, J.E., C. Abbey, S. Anderson, C. Chang, X. Draye, A.H. Lattu, R. Jessup, C. Lemke, J.Lennington, Z. Li, Y-R. Lin, S-C. Liu, L. Luo, B.S. Marler, R. Ming, S.E. Mitchell, D. Qiang, K.Reischmann, S.R. Schulze, D.N. Skinner, Y-W. Wang, S. Kresovich, K.F. Schertz and A.H.Paterson (2003) A high-density genetic recombination map of sequence-tagged sites forSorghum, as a framework for comparative structural and evolutionary genomics of tropicalgrains and grasses. Genetics, 165:367-386.

Sugar Production2001ACRES TONS RAW TONS SUGAR

COMPANY HARVESTED SUGAR (96•) PER ACRE

Gay & Robinson, Inc. 4,193 54,691 13.04*Hawaiian Commercial & Sugar Co. 15,101 191,512 12.68Totals & average 19,294 246,203 12.76**

2002Gay & Robinson, Inc. 4,754 54,196 11.40*Hawaiian Commercial & Sugar Co. 16,557 215,888 13.04Totals & average 21,311 270,084 12.67**

2003Gay & Robinson, Inc. 4,191 55,267 13.19Hawaiian Commercial & Sugar Co. 15,660 205,742 13.14Totals & average 19,851 261,009 13.15**

* Includes Kekaha salvage cane** Weighted average

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Dudley, N. (2003) Evaluating Hawaiian “Awa” (Piper methysticum) varieties for growth per-formance and kavalactone concentration in a Koa Agroforestry system. International Union ofForest Research Organization - All Division 5 Conference, Forest Products Research, March 11-15, Rotorua, New Zealand.

Fitch, M., T. Leong, N. Saito, G. Yamamoto, A. Dela Cruz, A. Yeh, S. White, S. Maeda, S.Ferreira and P. Moore (2003) Photoautotrophic Rooting and Growth of Papayas In Vitro. Am.Soc. Plant Physiol. (Abstr. P572) p.131.

Fitch, M.M.M., T. Leong, N. Saito, G. Yamamoto, A. Dela Cruz, A. Yeh, S. White, S.H. Maeda,S. Ferreira and P.H. Moore (2003) Control of bacterial contamination in large scale papayamicropropagation. In Vitro Cell Develop Biology – Plants 39 (Abstr.) 19A.

He, X., S.C. Miyasaka, M.M. Fitch, Y.J. Zhu and P.H. Moore (2003) Development of a genetictransformation and regeneration system for taro. 15th Ann. CTAHR Student Research Symp.(Abstr.) p. 21. and Am. Soc. Plant Physio. (Abstr. P647) p. 144.

Lehrer, A.T. and S. Schenck (2003) Purification of sugarcane yellow leaf virus and antiserumproduction (Abstr.) VII ISSCT Sugarcane Pathology Workshop, May 11-16, Baton Rouge, LA.

Liu, A., P.H. Moore, H. Ma, M.S. Kim, R. Makandar, H. Pearl, J. Charlton, Q. Yu, J.I. Stiles,M.M. Fitch, A.H. Paterson and R. Ming (2003) The genetic basis of sex determination inpapaya. Am. Soc. Plant Physiol. (Abstr. P501) p. 118.

Liu, Z., P.H. Moore, C.A. Ackerman, Q. Yu, A.H. Paterson and R. Ming (2003) A primitivechromosome in papaya controls sex determination. Plant Genetics 2003: Mechanism ofGenetic Variation. Oct. 22-26. Snowbird, Utah.

Liu, Z., P.H. Moore, H. Ma, M.S. Kim, Q. Yu, J.I. Stiles, M.M.M. Fitch, A.H. Paterson and R.Ming (2003) Physical mapping of sex determination gene in papaya. Plant, Animal & MicrobeGenomes XI. P541. p. 210. and International Plant and Animal Genome IX Conference. Jan.11-15, San Diego, CA.

Liu, Z., P.H. Moore, H. Ma, M.S. Kim, R. Makandar, H. Pearl, J. Charlton, Q. Yu, J.I. Stiles,M.M.M. Fitch, A.H. Paterson and R. Ming (2003) The genetic basis of sex determination inpapaya. Plant Biology 2003. July 27-30, Honolulu, HI.

Ma, H., P.H. Moore, Z. Liu, M.S. Kim, M.M.M. Fitch, T. Sekioka, A.H. Paterson and R. Ming(2003) Preliminary molecular characterization of the sex determination locus in papaya. Plant,Animal & Microbe Genomes XI. P542. p. 210. and International Plant and Animal Genome IXConference. Jan. 11-15, San Diego, CA.

Manshardt, R.M. and P.H. Moore (2003) Natural history of papaya and the Caricaceae. Am.Soc. Plant Physiol. (Abstr. P842) p. 177.

McCafferty, H., P. Moore and J. Zhu (2003) Towards improved insect resistance in papaya.Am. Soc. Plant Physiol. (Abstr. P731) p. 158.

Ming, R., P.H. Moore, Z. Liu, H. Ma, Q. Yu, M.S. Kim, M.M.M. Fitch and T. Sekioka (2003)Papaya genomics. Am. Soc. Plant Physiol. (Abstr. P999) p. 203.

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Ming, R., P.H. Moore, Z. Liu, H. Ma, Q. Yu, M.S. Kim, M.M.M. Fitch and T. Sekioka (2003)Papaya genomics. Am. Soc. Plant Biol. Annual Meeting July 27-30, Honolulu, HI.

Miyasaka, S.C., M.M. Fitch, Y-J. Zhu and P.H. Moore (2003) Development of a genetic trans-formation and regeneration system for taro. Am. Soc. Plant Physiol. (Abstr.).

Osgood, R. V. (2003) Cane Planter, Sugarcane Yield and Record Yield. Sugar Journal 66 (7):6.

Pearl, H., C. Nagai, P.H. Moore, D.L. Steiger, R.V. Osgood and R. Ming (2003) Construction ofa genetic map for Arabica coffee using AFLP markers. Plant, Animal & Microbe Genomes XI.P551. p. 212 and International Plant and Animal genome IX conference. Jan. 11-15, San Diego,CA.

Qiu, X., M-L. Wang, Y.J. Zhu, P.H. Moore and H.H. Albert (2003) Global expression profilingof pathogen-defense related genes from papaya. Am. Soc. Plant Physiol. (Abstr. P792) p. 168.

Qiu, X., M. Wang, Y.J. Zhu, W.B. Borth, J. Hu, P.H. Moore and H.H. Albert (2003) Isolationand expression profiling of genes in papaya systemic acquired resistance. 15th Ann. CTAHRStudent Research Symp. (Abstr.) p. 26.

Qiu, X., M. Wang, Y.J. Zhu, P.H. Moore and H.H. Albert (2003) Global expression profiling ofpathogen-defense related genes in papaya. Am. Soc. Plant Biol. Annual Meeting. July 25-30,Honolulu, HI.

Qiu, X., Y.J. Zhu, M-L. Wang, R. Ming, P.H. Moore, D. Gonsalves and H.H. Albert (2003)Isolation and characterization of genes involved in papaya (Carica papaya L.) systemic acquiredresistance. Proc. International Congress Plant Pathology (2): P12.2 p. 169.

Schenck, S. (2003) Differentiation of Ustilago scitaminea races in Hawaii. VII ISSCT SugarcanePathology Workshop, May 11-16, Baton Rouge, LA and (with R. Ming) Am. Phytopath. Soc.Pac. Div. Meeting, June 22-24, Kailua-Kona, HI.

Schenck, S., M.W. Crepeau, K.K. Wu, P.H. Moore, Q. Yu and R. Ming (2003) Native HawaiianSaccharum officinarum sugarcanes: Genetic diversity and relationship to modern commercialSaccharum hybrid cultivars. 4th ISSCT Molecular Biol. Workshop. p. 29.

Steiger, D.L., P.H. Moore, F. Zee, Z. Liu and R. Ming (2003) Genetic relationships ofmacadamia cultivars and species revealed by AFLP markers. Euphytica. 132:269-277.

Wang, M., C.S. Goldstein, P.H. Moore and H.H. Albert (2003) Towards introduction of single-copy transgenes in sugarcane. American Society of Plant Biologist Annual Meeting. July 25-30,Honolulu, HI.

Wei, H., M-L. Wang, P.H. Moore and H.H. Albert (2003) Comparative expression analysis oftwo sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J. PlantPhysiol. 160: 1241-1251.

Wu, K.K. and S. Schenck (2003) A brief history of the Hawaii sugar industry. Sugar Journal 66(7): 14.

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Yu, Q., A.M. Alvarez, P.H. Moore, F. Zee, M.S. Kim, A. de Silva, P.R. Hepperly and R. Ming(2003) Molecular Diversity of Ralstonia solanacearum isolated from ginger in Hawaii.Phytopathology 93:1124-1130.

Yu, Q., P.H. Moore, H.H. Albert, R.E. Paull and R. Ming (2003) Cloning and characterizationof flower development genes in papaya. Am. Soc. Plant Physiol. (Abstr. P508) p. 120. and Am.Soc. Plant Biol. Annual Meeting, July 25-30, Honolulu, HI, and 15th Ann. CTAHR StudentResearch Symp. (Abstr.) p. 30.

Zhou, L., C.C. Chen, R. Ming, D.A. Christopher and R.E. Paull (2003) Apoplastic invertase andits enhanced expression and post-translation control during fruit maturation and ripening. J.Amer. Soc. Hort. Sci. 128:628-635.

Zhou, F., M-L. Wang, H. Albert, P.H. Moore and Y.J. Zhu (2003) Production of GM-CSF pro-tein using PVX vector on N. benthamiana plants. Am. Soc. Plant Physiol. (Abstr. P810) p. 17.

Zhu, Y.J., R. Agbayani and P.H. Moore (2003) Green fluorescent protein as a sole selectablemarker facilitates genetic transformation of papaya (Carica papaya L.). 7th InternationalCongress of Plant Molecular Biology, Barcelona, Spain. (Abstr. S26-63) p. 388.

Zhu, Y.J., C.S. Tang and P.H. Moore (2003) Metabolic engineering phytoalexins from theVitaceae improves antifungal activity in tropical plant. (Abstr. P1020) In Vitro Cell Devel. Biol.39:24.

Zhu, Y J., C.S. Tang, S.E. Ferreira, M.M.M. Fitch, D. Gonsalves and P.H. Moore (2003)Developing transgenic resistance to fungal diseases in papaya (Carica papaya L.). Proc.International Congress Plant Pathology (2): P12.3 p. 169.