Appendices

Propagation Guide Supplement

Appendix A Tetrazolium (TZ) Test for Seed Viability

Appendix B Maintenance Routines and Seasonal Considerations

Appendix CTransportation Considerations and Planting Stress

CENTER FOR PLANT RESTORATION & COASTAL PLANT RESEARCH

1 General Method The TZ test is a biochemical test for seed viability. The test distinguishes between living and dead tissues within a single seed. Viable seeds appear pink to red in color when they are soaked in TZ solution; non-living tissue remains uncolored. The reaction takes place equally well in dormant and non-dormant seeds. Results can be obtained within 24 hours and often less. The TZ chemical, 2,3,5-triphenyltetrazolium chloride, is soluble in water making a colorless solution, a 1% solution is commonly used. Although the solution deteriorates with exposure to light, it will remain in good condition for several months if stored in the refrigerator in an aluminum foil wrapped bottle. The solution should be discarded if it becomes yellowish. A predetermined weight or number of seeds can be placed in a petri dish. Add enough TZ solution to completely cover the seeds. Place a piece of aluminum foil over the petri dish to block out the light. For seeds that require more than 24 hours, check the level daily and add more water as needed; if the seeds dry out the TZ test will not be successful. After the biochemical reaction has occurred, the number of viable (red-stained) seeds can be counted, and an estimate of the viability of a whole collection of seeds can be made. Not all viable seeds in the collection may germinate and this needs to be taken into consideration if the seeds are to be used for direct sowing.

2 Special Considerations Some types of seeds may need additional preparation for the TZ test to enhance absorption of the dye and yield a positive result:

a) Any hard covering such as an endocarp (pit or stone inside a fruit) or scale must be removed.

b) Seeds must be made wet to activate the enzymes that will cause the color reaction.

c) Seeds may need to be cut longitudinally to expose the embryo in large seeded grasses, or cut transversely 1/4 to 1/3 away from the end with the radicle (embryonic root) in small seeded grasses.

d) Seeds are soaked in TZ solution for 24 hours in the dark. Cut seeds require a shorter time; intact seeds require 24 hours or more.

e) Interpretation of results depends upon the kind of seed. In grasses completely colored embryos indicate good seed.

Distichlis spicata PHOTO CPR

Individual seeds before and after soaking in TZ solutionPHOTO CPR

Petri dishes of seeds before and after soaking in TZ solutionPHOTO CPR

Appendix ATetrazolium (TZ) Test for Seed Viability

1 Watering Routine The amount of watering needed will depend on the season. More water is required during hotter summer months. If a tray or holding pond appears to be losing more water than normal try to find a leak and repair or replace.

Summer - Inside Greenhouse: Marsh plants being grown in trays should be monitored daily for water uptake. Generally the plants growing in containers require the holding trays to be filled daily. In addition a morning and afternoon mist is also beneficial.

Summer - Outside: Marsh plants being grown in holding ponds should be monitored daily for water uptake. Generally the plants growing in containers require the shallow holding ponds to be filled three times per week. In addition running a sprinkler in the morning and afternoon is also beneficial.

Winter - Inside Greenhouse: Marsh plants being grown in trays should be monitored every other day for water uptake. This may vary depending on the temperature setting of the heating system. Generally the plants growing in containers require the holding trays to be filled every other day. In addition a morning or afternoon mist is also beneficial.

Winter - Outside: Marsh plants being grown in shallow holding ponds should be covered with clear plastic to trap the heat inside. These structures should be monitored once a week for water uptake. Generally the plants growing in containers under these structures require the holding ponds to be filled every two weeks.

2 Fertilizing, Weeding and Other Maintenance It is beneficial to inspect individual plants every other week. This is a time consuming process but should be done for a successful yield. Familiarity with the morphology of the young plants being propagated is important. Plants are fertilized with a full strength (per manufacturer directions) water soluble 20-20-20 general purpose fertilizer monthly, poured directly onto the soil. Remove any weeds that maybe growing in the container by hand. It is important to remove all of the weed, including the roots, to prevent re-growth. Undesirable seeds can be introduced into the containers from the soil and from the wind. This problem can be reduced by using sterile soil. Plants being grown outside are more susceptible to wind blown seeds than plants grown in the greenhouse. 3 Growing Plants Outdoors Plants can be placed outdoors once they are replanted in 4 or 6" pots. This helps to free up space in the greenhouse for more seedlings. Place the pots in 4 or 6" deep holding ponds (5 x 10 ft) constructed with a wood frame using 2 x 6" treated lumber and lined with heavy duty black plastic sheeting (6 mil). These ponds will hold 200 six inch pots or 450 four inch pots and will retain a large amount of water, reducing the need to add water as frequently. Plants being grown outside during the summer will require daily monitoring of the water levels in the holding ponds. During the winter months, plants being grown outdoors will need to be covered with heavy duty clear plastic sheeting (6 mil) supported by a frame constructed with cinder blocks and wood to trap the heat inside like a mini greenhouse.

Outdoor holding pondsPHOTO CPR

Watering in the greenhousePHOTO CPR

Appendix B Maintenance Routines and Seasonal Considerations

4 Growing Plants Year Round Plants being grown for longer than 12 months should be repotted into larger containers to prevent them from stunting and becoming root-bound. Older plants may require the dead leaves and stems to be trimmed periodically to avoid excessive shading of the new developing shoots.

5 Diseases, Insects and Pests Diseases on marsh and dune plants are generally not a problem. However, with the propagation of large numbers of the same species in the greenhouse some problems might be encountered. Aphids, and powdery leaf mildew may become problematic in the humid summer conditions, while rodents and snakes may become a problem during cooler winter months as they seek heat inside. Aphids can be eliminated by using a commercial mild liquid dish soap diluted with water. This can be applied repeatedly by using a spray bottle. Also, the infected plants can be isolated or destroyed. Often placing the plants outdoors will reduce the severity of the problem as birds and insects eat the aphids. Powdery leaf mildew can be reduced by removing infected plants outdoors, where improved air circulation and reduced humidity will generally kill the organism. Rodents can be discouraged by using mouse traps baited with peanut butter. Alternatively, filling holes and covering openings with a wire mesh can prevent the animals from entering the greenhouse.

Covered raceway pools to grow aquatic plants PHOTO CPR

Eastern Lubber Grasshopper (Romalea guttata)PHOTO CPR

Appendix B Maintenance Routines and Seasonal Considerations

CENTER FOR PLANT RESTORATION & COASTAL PLANT RESEARCH

Beware of alligators in and around marsh and dune vegetationPHOTO CPR

1 Plant Preparation for Transportation Preparing the plants for transportation to the restoration site should be started at least 1 month in advance. Plants should be weeded. If the restoration site is exposed to high salinity, plants can be salt hardened by gradually adding salt to the water over a period of about one month to result in a final concentration of 10 ppt (parts per thousand). A full strength water soluble fertilizer (20-20-20) should be applied two weeks before transplanting. Finally, the containers should be checked to see if the plant roots have grown together. If the roots are intertwined, cut them to separate the pots for transportation.

2 Maintaining Good Health Before Transplanting Generally the plant roots should not be out of water for longer than 24 hours. The plants should be watered twice a day during the summer and at least once a day in the winter to keep the exposed roots wet during transportation. While awaiting planting, pots can be placed in shallow water at the transplant site provided there is a protected location with no waves or strong currents that could damage the plants.

3 Planting Methods and Considerations Observe the restoration site before moving the plants to the site. Most restoration sites consist of dredge or fill material, which may take time to settle. Soil erosion may also be a factor at the restoration site. Coconut fiber material (coir) can be used to reduce the energy of the water and maintain soil moisture, until the plants can become established. To increase the probability of success at a restoration site you should consider the requirements of each plant species that is to be transplanted, such as elevation of the site, correct planting depth, spacing of transplants, etc. Planting methods will vary from species to species. It is important that the roots get access to water or the plants will die. Application of slow-release pellet fertilizer may also be useful at the time of planting, especially in sandy substrates.

4 Post-Planting Maintenance and Evaluation Monitoring of the restoration site should be done at the site 1, 3, 6, 12, and 24 months after transplanting to evaluate the success. Observations to record include: number of surviving plants, damage from storm debris or soil erosion, and how quickly bare areas coalescence (fill in) between transplants. Problem areas at the restoration site may require supplemental replanting.

Preparing Ruppia maritima for transplanting PHOTO CPR

Vallisneria americana in tubs for eventual transportation and transplanting PHOTO CPR

CENTER FOR PLANT RESTORATION & COASTAL PLANT RESEARCH

Appendix C Transportation Considerations and Planting Stress

CPR Propagation Guide TeamDr. Patrick Biber J.D. Caldwell Scott R. Caldwell Matthew Marenberg

Further InformationUSDA PLANTS profile: http://plants.usda.gov/java/

Center for Plant Restoration and Coastal Plant Research:http://sites.google.com/site/coastalplantrestoration/home

Field guide and images of Coastal Mississippi Wetland plant species: http://jcho.masgc.org/

NRCS Jamie L. Whitten Plant Materials Center:Plant Guide for Establishing Coastal Vegetation on the Mississippi Gulf Coasthttp://www.plant-materials.nrcs.usda.gov/pubs/mspmspu7271.pdf

This brochure is funded in part with qualified outer continental shelf oil and gas revenues by the Coastal Impact Assistance Program, Bureau of Ocean Energy Management, Regulation and Enforcement, U.S. Department of the Interior through a grant award to the Mississippi Department of Marine Resources. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the opinions or policies of the U.S. Government. Mention of trade names or commercial products does not constitute their endorsement by the U.S. Government.

Dr. Patrick BiberEmailbotanyGCRL@gmail.com Phone 228 872 4200 Fax 228 872 4204 Mail703 East Beach Drive Ocean Springs, MS 39564

Appendices

CPR Brochures

GrassesSaltgrass Distichlis spicataBitter Panicum Panicum amarum Smooth Cordgrass Spartina alternifloraBig Cordgrass Spartina cynosuroidesSaltmeadow Cordgrass Spartina patensGulf Cordgrass Spartina spartinaeSeaoats Uniola paniculata

SedgesSturdy Bulrush Schoenoplectus robustusChairmaker's Bulrush Schoenoplectus americanus

RushesBlack Needlerush Juncus roemerianus

Beach and InlandSea-Oxeye Daisy Borrichia frutescensCrimsoneyed Rosemallow Hibiscus moscheutosBeach Morning-Glory Ipomoea imperati

Submerged AquaticsShoalgrass Halodule wrightiiWidgeongrass Ruppia maritimaTurtlegrass Thalassia testudinum American Eelgrass Vallisneria americana