| A new sporeling culture method using gametophyte clones was developed in the early nineties. In the course of extension, there exits two problems: commercial scale of gametophyte clones culture and high outgrowth rate in sporeling raising. The aim of this study, therefore, is to solve these two problems: I . Light etachment of egg from the tip of oogonium: In general, female and male gametes formed five to six days after the attachment of gametophyte fragments to the slides. In female gametophyte, the sign of approaching maturity was the erection of the swollen tip cell which is about 50?5 11 m in length and 19 20 l.A m in diameter, in male gametophyte, a transparent, greenish and papilla-like protrusion formed, through which sperms were eventually released. It was observed that under normal 11:13 LD photoperiod regime, egg discharge occurred five minutes after the beginning of the dark period and peaked 5-15 minutes later. The course of egg discharge took only a few seconds. The egg extruded out was firmly attached to the oogonium apex. The course of egg discharge took only a few seconds, but once it was disturbed by irradiance of as low as 5-6 i mol m2s necessary for microscopic observation, 95% of the discharged eggs separated from the oogonia. However, egg can not be detached by light once it has been discharged and attached to the oogonium apex. Even the detached eggs could be fertilized, but they died and decomposed finally. It was found that when the slides were transferred from the normal 11:13 photoperiod regime to continuous light, egg discharge occurred five minutes after the ending of the ormal light cycle and peaked 5-15 minutes later the first day after transfer. However, further observation revealed that 75% of the eggs separated from oogonia in a few seconds. The extension of the clone technique in Undaria pinnat fida sporeling culture carried out in Shandong Province since 1997 led to large-scale production of healthy sporelings for commercial cultivation. However, the following three years?practice showed that the sporeling outgrowth rates were uneven, in some cases, very low, even 50% lower than the usual rates. In sporeling culture practice, lights in the greenhouse were sometimes still on in the period 2-3 hours after dusk; consequently many empty oogonia were found the next morning. As loss of eggs was proved to be caused by the disturbance of light, darkness control in the period 2-3 hours after dusk was suggested as a means to enhance the outgrowth rate. II . Optimization of gametophyte clone mass culture and sporeling raising techniques 1. Fast propagation of gaznetophyte clones: (1). Clones grew best under irradiance of 80?20 l.mol m2s Culture density be better controlled between log/L. lg/L was considered as the suitable initial culture density. (2).Fast growth of clone consumes a lot of nitrogen. As clone can store nitrogen inside the cell, ammonium and nitrate nitrogen can be supplemented periodically to ensure fast growth of clone. Favorable ammonium itrogen concentration in culture medium is 20 .tmol/L which meets the demand of growth of clone in six days (clone density is 1 lOg/L). (3).Old culture medium had no apparent inhibitory effect on the growth of clone in 65 days. Other elements in PES medium exclude N were proved to be sufficient for growth of clone within this period. (4). Favorable temperature for growth of clone is 220C.
|
PDF Full Text Request