New Approaches to Seaweed Aquaculture: Developing a Biosecure and Reliable Seed Stock for the Emergent Northeast Edible Seaweed Industry

Research goal: To develop long-term maintenance technologies for current (sugar kelp - Saccharina latissima) and emerging seaweeds (Winged Kelp – Alaria esculenta and Reds – Dulse) in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.

Our research formally began in the Fall of 2021 for this project and as part of this project we established a cryogenic center with an auto-fill liquid nitrogen dewar for the storage of cryogenically preserved seaweed seed/spores.

During the fall of 2021 we targeted two phenotypes of sugar kelp (Saccharina latissima), which included skinny and wide blade phenotypes.  Sporulation and cryogenic freezing activities were conducted in October and November 2021 for each phenotype, respectively.  For each sporulation of seed the following methodology was applied:

Natural sources of sugar kelp (Saccharina latissima) reproductive tissue were obtained by the co-PIs from of mature sporophytes collected from two locations along the midcoast, Maine and key morphological features were recorded (stipe length, blade length and width). For each phenotype the surface of the reproductive tissue was soaked in sterile seawater for 5–10 min, cleaned with a damp cloth, scraped with a clean razor blade then stored on paper towels in the dark for 24 hours at 10°C. The following day the meiospores were harvested following the procedures of Flavin et al 2013 and Redmond et al 2014.  Attempts were made in the fall of 2022 to obtain more natural sources of reproductive tissue for additional cryogenic tests but natural skinny kelp beds had been overharvested and were not available.  We will be working with two farmers in 2023 (see Cooperators) to obtain all reproductive sorous tissue to complete this project and to provide cryogenic seed stock training.

Cyrogenic Freezing Procedure:

For the “skinny kelp” phenotype two cryoprotectants mixed 1:1 in 2 mL cryovials were used and the meiospores were frozen using a ThermoFisher CryoMed controlled rate freezer (CRF) at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen vapor. For the “wide blade” phenotype three cryoprotectants were used and mixed 1:1 in 2 mL cryovials and frozen via CRF.  For each phenotype the following number of vials were collected and stored:

Skinny blade Kelp: cryoprotectant 1 = 97 vials, cryoprotectant 2 = 96 vials

Wide blade Kelp: cryoprotectant 1 = 75, cryoprotectant 2 = 76, & cryoprotectant 3 = 55.

The number of vials depended on the meisopore density for each of the sporulation event. To minimize the genetic variation between different kelp blades, the meiospores from several fronds were combined for each sporulation event and downstream cryogenic freezing. There are advantages and disadvantages to doing this, as each kelp frond with have different meiospore vitality (as per previous research), but in order to test a broad range of cryoprotectants we chose to combine meiospores from different blades in order to generate many vials for downstream testing and differentiation into sporophytes.

Thaw Procedure:

For cyrogenic revival of meiospores both the freezing process and thawing process are important aspects of meiospore survival and differentiation.  It is vitally important to remove / dilute the cryoprotectants as soon as possible. Two thawing techniques were examined for each cryoprotectant used for our two Saccharina latissima phenotypes (skinny and wide blade), 1) rapid thaw in water bath and 2) CryoThaw: a simple tube adapter to expedite and automate thawing.

• Rapid thaw in water bath: Each frozen meiospore vial is held in a 20 C water bath until a pea sized pellet appears and then the vial content is placed in 9 mL of chilled (4 C) Prov50/2 media, and inverted 5 times the evaluated for vitality using Fluorescein Diacetate (FDA) and observed for motility.
• CryoThaw (Beddall et. al, 2016 DOI: 1016/j.jim.2016.08.009): Using a small tube adaptor (commercially available) the frozen uncapped meiospore cryovial is placed at the entrance of a 15 mL centrifuge tube containing 9 mL Prov50/2 media (chilled to 4 C), the centrifuge is programmed to 22 C, 1500 RPM, for 5 min. Once the vial thaws the meiospore pellet is in the media within minutes and are diluted rapidly and within a controlled environment. The 15 mL vial is removed and inverted 5 times prior to analysis for vitality (as in step 1). The goal of this method is to provide a more controlled environment for thawing and diluting meiospores with the goal to remove the cryoprotectant as soon as possible.

Vitality Testing:

After thawing, 1 mL of each thawed solution (from different cryoprotectants) is stained with 10 uL of FDA working stock and incubated at 10 C for 20 mins prior to flow cytometric analysis on a BioRad ZE5 Cell Analyzer.  All vital meiospores will fluorescence green with blue laser excitation. The total number of meiospores is determined using red fluorescence (as an indicator of chlorophyll) as well as the number of vital cells.  The percent vital cells is determined and recorded for each vial and cryoprotectant.

Motility Testing:

After thawing, 50 uL of each thawed solution is observed under the microscope using a hemocytometer to determine if any of the thawed cells are motile – an indication of vitality.

Meiospore Differentiation examination:

After thawing, X mL of each thawed solution is combined with 4 mL of Prov50/2 media and placed into a well of a 6-well tissue culture plate and incubated in 12/12 light/dark cycle to monitor for meiospore differentiation and eventual sporophyte formation.

Other Kelp species examined to date:  In fall of 2022 a protocol for releasing Winged Kelp (Alaria esculenta) spores from reproductive fronds was developed in order to test spore cryopreservation techniques (TBD in 2023).  The sporophylls were collected from natural sources (Pemaquid Point, ME), and returned to the lab on ice for processing. The sporophylls were removed from the fronds and scraped with a razor blade to remove mucus and contaminates from the surface.  Afterwards the sporophylls were divided into two treatments to induce spore release.  1) Betadine dip: sporophylls were dipped in a Betadine solution (500 uL of Betadine to 500 mL of DIW) for 2 mins then removed into a tray of filtered seawater (FSW) and scraped with a razor blade again, sandwiched between paper towels, and incubated overnight in the dark at 12 C. 2) FSW treatment: sporophylls were dipped in FSW and scraped with a razorblade to remove mucus and surface contaminates, afterwards all scraped sporophylls were dipped in FSW for 30 seconds then removed and scraped again with a razor blade. All sporophylls were sandwiches between paper towels and incubated overnight at 12 C.  Of the two treatments the one with the most success was the Betadine dip.  Unfortunately, the season and all the reproductive tissue had been harvested the next time we attempted a sporulation for cryopreservation.  We will plan to begin this process in Spring/Summer 2023 – using reproductive tissue provided by seaweed nurseries (see cooperators).