Micropropagation of sweet chestnut (Castanea sativa Mill.)

Abstract

Pitomi kesten je u Hrvatskoj devastirana vrsta zbog raka kestenove kore i truleži korijena, a posljednjih godina i novih štetočina. Sadnja novih stabla otpornijih genotipova bila bi važan pothvat. Nove sadnice moguće je proizvesti generativno, ali zbog stranooplodnje, sadni materijal u tom slučaju nije uniforman. Mikropropagacija kao metoda vegetativnog razmnožavanja nameće se kao rješenje. Cilj ovog istraživanja bio je razviti protokol za mikropropagaciju pitomog kestena (Castanea sativa Mill.). Vegetacijski vršci izolirani su pod stereomikroskopom iz aksilarnih pupova izdanaka korijena i postavljeni na uspostavni medij. U pokusima su kao eksplantati korišteni mikroizdanci (0,5 – 1 cm) ili nodalni segmenti. Ispitan je utjecaj citokinina 6-benzilaminopurina i zeatina na učinkovitost mikropropagacije. Također su ispitani učinci različitih vrsta agara i različitih koncentracija giberelinske kiseline. Nodalni segmenti dali su signifikantno veći broj izdanaka u odnosu na mikroizdanke. Međutim, dužina najdužeg izdanka i prosječna dužina izdanaka bila je signifikantno veća na mikroizdancima. Eksplantati su se značajno bolje razvili na mediju MS s koncentracijom 1,5 mg/L 6-benzilaminopurina (BAP), u odnosu na medij MS s 1,5 mg/L zeatina. Najveći broj izdanaka po eksplantatu (2,7) dobiven je iz nodalnih segmenata na MS mediju s 1,5 mg/L BAP-a. Najveća dužina najdužeg izdanka (9,5 mm) i najveća prosječna dužina izdanaka (8,9 mm) ostvarena je s mikroizdancima na mediju MS s 1,5 mg/L BAP-a.Sweet chestnut is a devastated species in Croatia, since it has been plagued by chestnut blight and ink disease, and in recent years new pests. Planting new trees of more resistant genotypes would be an important undertake. New seedlings can be produced generatively, but because of allogamy, the planting material in this case is not uniform. Micropropagation as a vegetative propagation method implies a solution. The objective of this study was to establish the protocol for micropropagation of sweet chestnut (Castanea sativa Mill.). Shoot tips from axillary buds of young root shoots were isolated under stereomicroscope and placed on establishing medium. As explants either microshoots (0.5 – 1 cm) or nodal segments were used. The influence of cytokinins 6-benzylaminopurine and zeatin on microprogation efficacy was tested. The effects of different agars and different concentrations of gibberellic acid were also tested. The nodal segments had significantly higher number of shoots per explant than the microshoots. However, the length of the longest shoot and the average length of shoots was significantly higher on the microshoots. Explants developed significantly much better when propagated on MS medium 1.5 mg/L 6-benzylaminopurine (BAP) then on MS medium with 1.5 mg/L zeatin. The highest number of shoots per explant (2.7) was obtained from nodal segments on the MS medium with 1.5 mg/L BAP. The largest length of the longest shoot (9.5 mm) and the largest average shoots length (8.9 mm) were obtained on the microshoots on MS medium with 1.5 mg/L BAP