Embryogenic Suspension Cultures Of Pinus Armandii.: Growth Characteristics And Maturation Ability

Pinus armandii Franch. is one of the most important afforestation species in western mountains of China for its high growth speed. In addition, it is highly valued in high altitude area of southern China for yielding timer and turpentine. It is well known that it is one of the most valuable forest species with multiple uses. P. armandii. plants are normally propagated from seeds. However, it is difficult to maintain good traits from the female parent. And conventional asexual reproduction method such as cuttings, grafting is also very difficult. Therefore, it is very important to develop a new asexual reproduction approach for this species.In this paper, the immature zygotic embryos were used as explants to induct embryonic callus. Embryogenic cell suspension culture system was established and the mature culture of somatic embryos was studied. It will lay the foundation for development a protocol of regeneration plants via somatic embryogenesis for P. armandii..The results are as follows:1. Callus could be inducted by using two different explants of P.armandii’s: Embryos with endosperm and without endosperm. And the callus induction rate was much higher by using embryos with endosperm. High induction rate of 27% was achieved on mLV medium supplemented with 1.0mg/L 2,4-D and 1.0mg/L BA. Callus had a very small amount of yellow particles, loose and browning is not obvious.2. There were two types callus of Pinus armandii., embryogenic callus(EC)and non-embryogenic callus(NEC). EC was translucent,light white or white, relatively loose. And the callus which surface was wet or crystal, light yellow or yellow, relatively compact was NEC. Double staining technique was used to distinguish embryogenic cells from non-embryogenic cells. The embryonal head cells were stained bright red(acetocarmine) and suspensor cells were stained blue. If the mass was non-embryogenic callus, cells did not show any organization of headand suspensor and would stain blue with Evan’s blue.3. During P.armandii.’s embryonic cell suspension culture, the added fresh weight changed as "S" shaped curve, and the growth cycle was 12 d. The delay period after inoculation was 1-4d. Then 5-10 d was the rapid growth phase. 10 d later, cell viability decreased gradually into the stable; then cell were death which showed that the growth curve decreased.4. The best culture conditions for embryonic cell of P.armandii. was that: mLV+1.0mg/L 2,4-D+0.5mg/L 6-BA +30g/L sucrose,pH5.8, inoculum at 3%,rotation speed at 100 rpm, 25±1℃, dark under the conditions of culture shock.5. In the mature culture, there was not embryonic cotyledon tissue, but early embryo was observed on mLV maturation medium only. And a large number of 45 per gram of early embryo was achieved on mLV maturation medium supplemented with 80μM ABA +3.75% PEG4000 + 6% maltose.