1. Ohelo seed (left) and germinating seedlings (right)
In Vitro Storage and Cryopreservation of ‘Ōhelo Seed and Tissue Cultures
Barbara M. Reed1, Sugae Wada2, Esther E. Uchendu2,3 and Francis Zee4 1USDA-ARS National Clonal Germplasm Repository, Peoria Road, Corvallis, OR ; 2Dept. of Horticulture, Oregon State University, Corvallis, OR 97331; 3University of Guelph, Dept. of Plant Agriculture, Ontario Agriculture College, Guelph, Ontario, Canada N1G 2W1; and 4USDA-ARS Pacific Basin Agricultural Research Center (PBARC) P.O. Box 4487, Hilo, Hawaii
‘Ōhelo plant
Abstract.
‘Ōhelo (Vaccinium reticulatum Smith) is a native shrub of Hawaii and Maui commonly found in disturbed, open sites at 640 to 3700 meter elevation. Three cultivars: Kilauea, Red Button, and Nene were selected for ornamental use due to their compact form and bright red and waxy new growth. This study tested in vitro cold storage and the cryopreservation of shoot tips of these cultivars and of seed for germplasm preservation. Shoot cultures in tissue-culture bags were maintained in good condition in 4 ºC cold storage for 16 months before requiring repropagation. Shoot tips were moderately desiccation sensitive, but could be cryopreserved by any of three protocols. Regrowth following controlled rate cooling was 42% to 50%; encapsulation dehydration 60% to 75%; and PVS2 vitrification 45% to 70%. Wild collected seed was tested for germination after 3 month at room temperature or -20°C storage and after cryopreservation. Room-temperature stored seed displayed dormancy that was broken by cryopreservation. High levels of germination were noted for the -20°C stored seed and the cryopreserved seed. These studies show that ‘ōhelo germplasm can be successfully cold stored as tissue cultures or cryopreserved as shoot tips or seeds.
Cold Acclimation (CA).
Shoot cultures of all cultivars were incubated at 22 °C with 8 h low light (10 μE•m-2) and -1 °C with 16 h dark CA conditions for two weeks prior to each cryopreservation technique (Reed, 1990). Shoots for cold storage were CA for one week.
Cold Acclimation (CA). Shoot cultures of all cultivars were incubated at 22 °C with 8 h low light (10 μE•m-2) and -1 °C with 16 h dark CA conditions for two weeks prior to each cryopreservation technique (Reed, 1990). Shoots for cold storage were CA for one week.
Red Button
Kilauea
Nene
Shoot cultures of ‘‘ōhelo cultivars stored at 4˚C for 12 to 16 months in semipermeable tissue culture bags on WPM without plant growth regulators. Shoot cultures were planted and sealed in the bags, grown for 1 wk in the growth room and 1 week in cold acclimation before storage in the cold room with 12 hr of low light. These shoots would be rated 3 (good) or 4 (very good).
Cold Storage of Shoot cultures.
The shoot cultures (3 cm) were placed on 10 ml WPM with 7 g•L-1 agar without plant growth regulators (PGR) in semi-permeable plastic tissue-culture bags (Star Pac Bags, Garner Enterprises, Conroe, TX). One shoot was placed in each section of 5-section bags and three bags of each cultivar were stored. Shoots in the storage containers were placed in the growth room for one week and then CA as above for one week before being placed in storage at 4 °C with a 12-h photoperiod under low light (7 μmol •m-2•s-1). Shoots were evaluated at 4 month intervals.
Shoot Tip Cryopreservation.
Shoot tips were plunged into liquid nitrogen
(LN -196°C) for cryopreservation following encapsulation dehydration (ED), PVS2 vitrification (VIT) and controlled rate cooling (CC). Vials with cryopreserved samples were rapidly rewarmed in 45 °C water for 1 min and in 25 °C water for one min before the samples were rinsed and plated on WPM medium in 24-well culture plates (Costar, Cambridge, MA).
Objectives.
Develop alternative storage techniques for ‘ōhelo cultivars and germplasm.
Commercial micropropagation of ‘ōhelo.
In vitro storage of shoot cultures
Cryopreserved storage of shoot tips
Cryopreserved storage of seed
Response of three ‘ōhelo (Vaccinium reticulatum) cultivars to in vitro cold storage. The cultures were subjectively rated (5 high-0 low) at 4 month intervals and any cultures rated <2 were removed for repropagation.
Micropropagation. V. reticulatum cultivars Kilauea [VAC 1817 (No 6-7)], Red button [VAC 1818 (No 6-9)] and Nene [VAC 1819 (No 6-6)] were multiplied on woody plant medium (WPM) (Lloyd and McCown, 1980), with 5 mg•L-1 2ip, 30 g•L-1 sucrose, 6 g•L-1 agar (Difco, Detroit, MI), at pH 5.2 (Zee et al., 2008). Standard growth conditions were at 25 °C with a 16 h light and at 22 °C with 8 h dark photoperiod (40 μE•m-2•s-1). Shoots were acclimated in a tube structure with mist before moving to the greenhouse.
Ohelo seed (left) and germinating seedlings (right)
Response of three ‘ōhelo (Vaccinium reticulatum) cultivars to cryopreservation with three commonly used techniques. All shoot cultures were cold acclimated for 2 wks before cryopreservation. Regrowth > 40% is acceptable for long-term storage in liquid nitrogen.
Seed Germination.
Seeds were equilibrated for 24 h in a desiccator over Drierite and divided into two sets. Set A was stored at -20˚C for 3 months; Set B was stored at room temperature (~25˚C) for 3 months. Blue germination blotters were fully soaked with DI water and fifty seeds were placed on a blotter. Additional DI water (5 ml per dish) was added and seeds were incubated at 25 ˚C with 8 h light /
15 ˚C 16 h dark for 5 months.
Seed Cryopreservation.
Duplicate vials of 50 seeds from Sets A and B were stored in liquid nitrogen for 1, 6 and 12 months. Two vials removed at each time period were germinated as above (n=100).
Control seeds were germinated on the initial liquid nitrogen storage date (n=200).
Germination of control ‘ohelo seed lots held at room temperature (RT) or at -20˚C for 3 months and the same seed lots following cryopreservation for 12 months.
Conclusions
Micropropagation: ‘Ōhelo shoot cultures can be commercially micropropagated, acclimated and grown into potted plants.
Medium-term storage: Shoot cultures can be cold stored in vitro for more than 16 months without a loss of viability.
Long-term storage: Shoot tips can be successfully cryopreserved with any of the three protocols.
Germination: ‘Ōhelo seed become dormant when stored at room temperature.
Seed storage: ‘Ōhelo seed stored at -20 C or in liquid nitrogen germinates within two months at high rates.
‘Ohelo liner plants from in vitro cultures showing foliage and flower color.
This project was supported by the Specialty Crops Research Initiative Grant
and the U.S. Department of Agriculture, Agricultural Research Service CRIS project ( D).