A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Plantlet regeneration from mature zygotic embryos andembryonic explants of masson pine (Pinus massoniana Lamb.)

Excised zygotic embryos, cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones. BA (1.0 mg/ L) in combination with NAA (0.05 mg/L)in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos, but most of them were formed at the tips of embryonic cotyledons. Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L. Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal (0.5%). Root initiation was achieved with full or half strength DCR inedium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L. Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated (3-20h) with BA 50-100 mg/L, followed by transfer to hormone-free DCR medium. The maximum number of shoots obtained per explant within six months was 33.

Cryopreservation by Vitrification of Vitis vinifera cv. ＂Red Globe＂ Zygotic Embryos and Effect on the Expression of DNA Methyltransferase Genes

Mexico is a large producer of table grape （Vitis vinifera L.） and therefore it is important to develop protocols to store the grape varieties germplasm. The objective of the present work was to design a protocol for the cryopreservation by vitrification of zygotic embryos of V. vinifera cv. ＂Red Globe＂ and evaluate possible epigenetics changes. The plant vitrification solution 2 （PVS2） was utilized before the utilization of liquid nitrogen （LN）. The effect of this protocol on embryo viability was tested by the triphenyl-tetrazolium chloride solution, as well as by the in vitro development of grape embryos into plantlet. A cDNA expression library of grape zygotic embryos was created to isolate expressed sequence tags of several DNA methyltrasferases. Gene expression of domains rearranged methyltransferase type 1 （DMR1） and DNA （cytosine-5）-methyltransferase 1 （MET1-2） isozymes was analyzed by quantitative reverse transcriptase PCR. The optimal conditions for vitrification were 10 min in 50% PVS2, followed by I0 min in 100% PVS2. Under these conditions, about 30% of plantlet was obtained from embryos after cryopreservation. It was recorded a reduction in the MET1-2 gene expression, which plays a role in the maintenance of DNA methylation. It is possible to cryopreserve viable grape zygotic embryos, although the treatment seems to induce alterations in the normal DNA methylation pattern of the zygotic embryo genome.

Observation on the Embryonic Development in Citrus after Cross Pollination

Embryonic development was studied in six cross combinations ofCitrus sinensis x C. tangerina, C. sinensis x C. reticulata, C. sinensis x (C. tangerina + C.reticulata), C. sinensis x Poncirus trifoliate, C.reticulata x C grandis and C. grandis xPoncirus trifoliate. The results showed that on the 30th day after pollination thezygote remained a single cell. On the 40th day, the zygote began to divide. On the50th day, zygotic embryo became globular-shaped while nucellar embryos had notinvaded the embryo sac. On the 55th day, a few nucellar embryos began to invadethe embryo sac. On the 60th day, the zygotic embryo became heart-shaped, and atthe same time, a large number of nucellar embryos invaded the embryo sac. On the80th day after pollination, the zygotic embryo was surrounded by nucellar embryosand it was not easy to distinguish these embryos. The cross combination affected thedevelopments of zygotic embryos, ovules and fruits, which were mainly determined bythe cross parents. As compared with interspecies crossing, the zygotic division ofintergenus crossing began later, the zygotic embryos developed slowlier and theinvading time of nucellar embryos was also delayed.

Embryo culture is an efficient way to conserve a medicinally important endangered forest tree species Strychnos potatorum

The present study reports a protocol for germination of Strychnos potatorum （ver. Tel. Chilla） using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog＇s medium （MS） containing 20 g/L sucrose in comparison to McCown and Lloyd＇s Woody Plant Medium （WPM）. Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L-1 . WPM/MS medium containing glucose at levels 30, 20, 15 g·L-1 showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0 2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L-1 sucrose. Germination rates improved when the embryos were placed upright （vertically） in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65% 75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture.