In Vitro Culture of Young Embryo in Polyembryonic Citrus (I)

Abstract

Polyembryonic satsuma mandarin (Citrus unshiu Marc.) was crosspollinated with trifoliate orange (Poncirus trifoliata L.).
Embryos were isolated from immature seeds 90, 120, and 140 days after pollination.
Isolated embryos were divided into the following five stages:
Stage I: Globular-shaped embryo (less than 0.1mm in dia.)
Stage II: Early heart-shaped embryo (0.1mm-0.3mm in dia.)
Stage III: Middle _??_ (0.3mm-0.4mm _??_)
Stage IV: Late _??_ (0.4mm-0.5mm _??_)
Stage V: Torpedo-shaped embryo (greater than 0.5mm in dia.)
These embryos were cultured on Murashige and Skoog (MS) medium and MS medium supplemented with 20% non-autoclaved cucumber juice (C. J.) under aseptic conditions.
The growth of embryos and the number of differentiated cotyledons were greater when 90 and 120 day embryos except Stage V were cultured on the medium with cucumber juice whereas no such effects were observed on 140 day embryos.
On the contrary, root differentiation and rooting were reduced by the addition of cucumber juice.
These findings indicate that there would be two factors or complexes present in cucumber juice; (1) a factor causing both growth and differentiation of cotyledon, (2) a factor causing inhibition of root growth.
For the successful rearing of heart-shaped embryos isolated 90 and 120 days after pollination, it appears that the following series of subcultures at one month intervals would be recommended:
(1) Initial culture of heart-shaped embryos on MS medium with 20% C. J. and 3% sucrose.
(2) Subculture of those embryos on MS medium with 3% sucrose without C. J.
(3) Subculture of them on MS medium with 1.5% sucrose.
(4) Transplanting young seedlings to vermiculite.
Zygotic seedlings with trifoliate leaves obtained were about 40 to 60% of total seeds examined and such seedlings came only from embroys in Stage IV and V.