Puna- ja valkoherukoiden solukkolisäys ja kryosäilytys

Cryopreservation of red and white curran

Alkamispäivä

01.04.2016

Päättymispäivä

31.03.2017

Tiivistelmä

1) Tausta ja tavoitteet


Background
The currant cultivars belonging to the Finnish mandate variety list of long term preservation were confirmed in the RIBESCO project (Core collection of Northern European gene pool of Ribes – RIBESCO AGRI GEN RES action 071, 2007-2011). The aim of the RIBESCO project was to establish cooperation between national collections in different countries in order to find the most valuable part of each collection and organise a decentralised core collection for the safe and recognised conservation of edible currants and gooseberry. As continuation of this work, in a two-part project led by Marjatta Uosukainen (Agrifood Research Finland) and financed by the Ministry of Agriculture and Forestry of Finland, the in vitro culture and cryopreservation methods of currants were studied 2009-2011 and 2011-2014 black currant mandate varieties were given a heat treatment and in vitro cultures checked to be free of blackcurrant reversion virus (BRV) were obtained through meristem culture. These cultures were multiplied for about six months and cold-acclimatised, after which shoot tips were excised for long-term cryopreservation using droplet-vitrification-method.
By 2014, the cryopreservation work was successfully carried out with in vitro cultured BRV-free materials of all the Finnish black currant mandate accessions except one, Melalahti, that fell behind other accessions because the plants for heat treatment were only sent to Laukaa one year later and they were heat treated and found BRV free in 2014. During the in vitro culture phase we noticed that Melalahti needs special care compared to the routine methods used, different type of growth media and long day conditions, as some other Northern accessions. As the project funding had already ended, the cryopreservation of this black currant was carried out in the beginning of 2015 under the working budget of the Finnish National Programme for Plant Genetic Resources. The controls thawed from cryopreservation showed good immediate survival after cryopreservation but poor regrowth of new in vitro cultures. The routine methods used had given at least 40 % regrowth after cryopreservation with all other black currants, but with Melalahti we first had regrowth as with other accessions, but only 2 % started multiplying and most only browned soon. We tried 24 hour light treatment and the same special growth media that was earlier used for multiplication of this accession, but even this did not solve the problem. During 2015 we studied and found a growth media suitable for this tricky Melalahti accession with financing from the Finnish National PGR programme and we are at the moment preparing also this accession for final long-term preservation. So the case of black currants will be closed.
Research scientist Saija Rantala is doing her PhD thesis work on cryopreservation of currants. Also her work has shown that in vitro culture of black currants is in general easier than in vitro culture of red currants, which also affects their cryoability. For this reason, the long term cryopreservation was started with black currants (using freezing of both in vitro cultured shoot tips and dormant buds) and in vitro culture of red and white currants was studied further. There are clear differences between cultivars in in vitro culture success. In 2014-2015 the in vitro culture of red and white currants was one of the work packages in a project led by Pauliina Palonen (University of Helsinki) and financed by the Ministry of Agriculture and Forestry of Finland, carried out at Luke with red currant cultivars ‘Punainen hollantilainen’, ‘Katri’, ‘Punahilkka’ and white currant cultivar ‘Lepaan Valkea’. These results are still preliminary, but can be used to choose applicable growth media for starting the cryopreservation studies on these cultivars.


Aim
The aim of this project is to find suitable cryopreservation method for several red currant varieties. The testing of the cryopreservation methods for red and white currants has only been made in very small scale until now, and this is an obligatory next step in order to continue the long-term cryopreservation of the mandate variety collections with red and white currants.

2) Toteutus

Materials
Punaherukalla (Ribes Rubrum group) on ollut käynnissä solukkolisäystutkimusta lajikkeilla ’Punainen hollantilainen’, ´Katri’ and ‘Punahilkka’ sekä valkoherukalla
‘Lepaan Valkea’ (Ribes Rubrum group), joka on osoittautunut helpoksi kasvattaa eri solukkolisäysalustoilla ja saattaa siksi olla hyvä lajike kryosäilytyamenetelmän testaamiseen.

Red currant (Ribes Rubrum group) cultivars ’Punainen hollantilainen’, Katri’ and ‘Punahilkka’ have been included to the on-going in vitro culture study at Luke, so that the in vitro cultures and also some data about the in vitro growth media suitable for those cultivars are available and the cultures can be multiplied for this project to study the cryopreservation on these cultivars. ‘Punainen Hollantilainen’ is a synonyme to ‘Red Dutch’, ‘Prinz Albert’ and ‘Rotes Wunder’ so although an old European variety, it also is a Finnish mandate list cultivar and can be treated as some kind of international standard among the more local cultivars. ‘Katri’ (or ‘Rotes Wunder Katri’) is also synonyme to ‘Red Dutch’, ‘Prinz Albert’ and ‘Punainen hollantilainen’, it is a strain selected in Finland with high vitamin C content. ‘Punahilkka’ however is a Finnish variety.
White currant cultivar ‘Lepaan Valkea’ (Ribes Rubrum group) is also included in the on-going in vitro culture study at Luke and has shown to be easy to multiply on several growth media types and this can be an easy cultivar, to try the cryopreservation with it.

Methods

Pisara-vitrifikaatio-kryomenetelmän modifikaatiota testataan.
Hyödynnetään vähän myös aiemmin kryotankkiin pakastettuja in vitro -viljelmistä eristettyjä ja lepotilaisten silmujen materiaaleja.

The droplet-vitrification cryopreservation method has been successfully applied at Luke for long-term cryopreservation of in vitro grown shoot tips of black currants, but some modifications will need to be tested for success with red and white currants. One general variable to be tested is the duration of the plant vitrification solution 2 pretreatment. The in vitro growth media before shoot tip eradication, the sugar pretreatment before freezing and the growth media during regrowth are also important factors affecting the cryopreservation and may need to be tested. For these tests fresh in vitro grown shoot tips can be grown and cold acclimated or materials already frozen into the cryotank ( either in vitro -grown or dormant bud materials) can be used for testing the regrowth media after cryopreservation.


3) Tuotokset


As the main outcome of this Project, cryopreservation protocol for red and white currants will be established. The applicable results will be reported to the Finnish National Genetic Resources Programme and NordGen, and published as part of a scientific paper.


4) Vaikuttavuus ja käytäntöön vienti


The national plant genetic resources working group for fruits and berries has discussed the continuation of the red and white currant work in its meetings. The long-term preservation of these cultivars is the aim, for which we apply for resources to fulfill this work. Later also methods for gooseberry cryopreservation would be needed and the success with in vitro culture and cryopreservation methods of red and white currants will help also with this species.

5) Asiakkaat
The Project will benefit The Finnish National Plant Genetic Resources Pogramme and the NordGen organisation.