Establishment of in vitro Rapid Propagation System for Ampelopsis grossedentata

[Objective] The research aimed to establish the fast and efficient rapid propagation system for Ampelopsis grossedentata by using the tissue culture method. [Method] Ampelopsis grossedentata stem with the bud was the material,and the influences of different sterilization methods,media and hormone combinations on the in vitro rapid propagation of Ampelopsis grossedentata were studied. [Result] The explant sterilization effect of 75% ethanol dipping 20 s ＋ 0.1% HgCl2 treating 8 min ＋ Tween-80 1-2 drops was better. The optimal medium for the axillary bud induction was B5 ＋ 1.00 mg/L BA ＋ 0.05 mg/L NAA. The optimal multiplication medium was MS/B5 ＋ 1.50 mg/L BA ＋ 0.05 mg/L NAA. 1/2MS ＋ 0.50 mg/L IBA was the best medium for the rooting. [Conclusion] The high frequency occurrence system of Ampelopsis grossedentata in vitro rapid propagation was established. It laid the technology basis for the callus regeneration system,genetic transformation and clonal mutation screening.

Studies on Electrical Activation of Porcine Oocytes Matured in vitro and Embryo Culture Systems

Conditions for electrical parthenogenetic activation of porcine oocytes matured in vitro and in vitro culture systems of porcine embryo were studied. The best results were achieved under the conditions of electrical field strength and the pulse duration at 130Vmm-1/80 us, with a blastocyst development rate of (20.12 ± 8.18) % (P > 0.05). No significant difference was found between treatments of multiple pulses and a single pulse (P > 0.05). Parthenogenetic embryos were cultured with different methods and air conditions for 7 days in vitro, blastocyst development rate of embryos with changed culture media [ (26.44 ± 8.35) % ] or changed media with 10% fetal bovine serum (FBS) [ (17.68 ± 5.39)% ] on the fifth day showing no significant difference from that of embryos without change of culture media [ (25.30 ± 7.55) % , P > 0.05 ], while cell numbers of blastocysts from embryos with changed culture media (15.78 + 5.46 and 14.55 ± 4.81) were significantly lower than number of blastocysts from embryos without change of culture media (18.01 ± 6.79, P<0.01). Blastocyst development rate and blastocyst cell number of embryos cultured in lower O2(5%CO2: 7%O2:88%N2) also showed no significant difference from those in high O2(5%CO2 in air) [(20.78 ± 8. 80)% and 17.0016.12 vs. (25.30 ± 7.55)% and 18.0116.79, P>0.05]. It is concluded that change of culture media with the same new one or changing over to media with 10% fetal bovine serum (FBS) on the fifth day and low O2 environment are not necessary for porcine embryos development.

In Vitro-Propagation of Agave tequilana Weber cv.azul in a Temporary Immersion System

In Mexico,there is a need to produce large quantities of plantlets for the establishment and replanting of blue(cv.azul)agave production areas.Most of these plots are within the origin denomination area(DOT,Spanish acronym)of the distilled product of this plant,known as tequila.The objective of this study was to develop an in vitropropagation protocol for Agave tequilana Weber cv.azul using segmented stems in both:solid and liquid media.A disinfection and in vitro technique were developed to obtain shoots,through plantlets collected in commercial plots,which attained 100%surface-disinfection and budding rate.At the multiplication stage,the effects of 6-Benzylaminopurine(BA)(0.0,4.4 and 13.2μM)and kinetin(0.0,9.4,18.8 and 37.6μM)were evaluated on lateralshoot production of segmented sagittal stems.These were cultivated on Murashige&Skoog(MS)medium,with the addition of 3.0%sucrose and 8 g L−1 agar.It was observed that BA and kinetin increased the number of shoots per explant,obtaining up to 18 and 26,respectively.Furthermore,it was found that just the sagittal segmentation of explants increased axillary budding.On the other hand,segmented-stem bases were grown in MS liquid medium with 3.0%sucrose,inside a RITA
system,programmed by a 5 min immersion step with a frequency of every 4 h.The effect of Indole−3-Acetic acid(IAA)(0.57,2.9,5.7μM)was evaluated,while maintaining a concentration of BA(13.2μM).It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant.These results offer a new methodology to increase the efficiency of A.tequilana Weber cv.azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.

Effects of Hormone Factors on the in vitro Culture Flowering Induction of Dendrobium officinate Kimura et Migo

[Objective]The aim was to select the suitable hormone factors for flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.[Method]The test-tube plantlets from the stems of Dendrobium officinate Kimura et Migo were used as the experimental materials and MS medium as the basic medium.Comparative tests have been done between single-factor hormone treatments（different concentrations of PP333 or TDZ） and multi-factor hormone treatments（different combinations of PP333,TDZ,6-BA and NAA） to research the effects of hormone factors on the flowering induction of the plantlets.[Result]Among the single-factor hormone treatments,the suitable concentration and the rate of flower buds formation of PP333 treatment were 0.2 mg/L and 8.5%,the that of TDZ treatment were 0.06 mg/L and 15.5%;the effects of multi-factor hormone treatments on the flowering induction were ordered as follow：（PP333 ＋ 6-BA ＋ NAA ＋ TDZ）〉 （PP333 ＋ 6-BA ＋ NAA）〉 （PP333 ＋ 6-BA） and（PP333 ＋ NAA） ;the most suitable treatment was PP333 0.3 mg/L ＋ 6-BA 0.5 mg/L ＋ NAA 0.5 mg/L ＋ TDZ 0.06 mg/L,the rate of flower bud formation and the rate of the blossomed flower were respectly reached to 80.4% and 90.3%.[Conclusion]PP333 and TDZ showed the important effect on the flowering induction in vitro culture of Dendrobium officinate Kimura et Migo.The effect of TDZ was better than that of PP333.It is much more conducive to the flower bud formation,when using appropriate concentration of TDZ combined with other hormones properly.

Rapid Propagation of Chirita ophiopogoides in Vitro

A method for in vitro culture and rapid propagation of Chirita ophio- pogoides was developed using leaves as explants in this study, The results indicat- ed that the medium MS＋6-BA 0.1 mg/L＋NAA 0.1 mg/L was suitable for bud induc- tion and seedling regeneration from leaves in primary culture. The media MS＋0.5 mg/L 6-BA＋0,1 mg/L NAA＋10% banana＋5% potato and MS＋0.5 mg/L 6-BA＋0.5 mg/L NAA＋2% banana were very suitable for callus multiplication and seedling hardening in subculture, and the proliferation coefficients were 7,9 and 5.6 per 60 d respec- tively. The optimal rooting medium was MS and the rooting rate was 100% on day 30 of culture. The rooted plantlets of C. ophiopogoides were transplanted in green- house with humus soil and 92.5% survived. Theoretically, using the rapid propaga- tion system, about 20 176 seedlings can be reproduced from a sterile plantlet in a year.

Culture Experiment in vitro of Eperythrozoon of Mustela lutreola

[Objective] The research aimed to make the culture experiment in vitro of eperythrozoon of Mustela lutreola. [Method] 30% newborn bovine serum or Mustela lutreola was added in RPMI-1640, DMEM, M-199 and HL culture solutions. Epery-throzoon of M. lutreola was cultured in vitro under the conditions of 37 ℃, 5% CO2. The culture solutions were replaced every 24 hours. Appropriate amount of healthy erythrocytes were supplemented to make subculture. [Result] The infection rate and infection intensity of eperythrozoon in RPMI-1640 and HL culture solutions were higher than that in DMEM and M-199 culture solutions. The infection rate and infec-tion intensity of eperythrozoon in RPMI-1640 and HL culture solutions with adding 30% serum of M. lutreola were higher than that with adding 30% newborn bovine serum. And the infection rate and infection intensity of eperythrozoon in RPMI-1640 culture solution were slightly higher than that in HL culture solution. The infection rate was over 80% after culturing 12 hours. 23 generations of subculture in vitro was made. [Conclusion] The research provided an effective approach for screening out the drugs that could effectively control eperythrozoon of M. lutreola, preparing diagnostic antigen and producing the relevant vaccine.

Study on Culturing Prunus salicina cv.Zaoshi Embryos

[Objective]The aim was to study the effective factors on culturing Prunus salicina cv.Zaoshi embryos in vitro.[Method]Different age,culture medium and GA3 which affected culturing of prumssalicina embryos were discussed.[Result]The result showed that on the part of 26-41 d,after the blooming period,the embryos remained tiny and retained endosperms and showed no signs of change after having cultured for three generations.On the part of 48 d,after the blooming period,the endosperms had disappeared,the embryos kept growing until they filled the seed cavity;when they were planted on the MS culture medium,their survival rate reached 77%,in its first generation,the response of embryos was discernible.On the part of 65 d,after the blooming period,4.5% of their embryos grew into shoots on the MS culture medium;with the age of embryos growing,the survival rate of shoots increased until it reached 26% when the fruits went into ripeness;the embryos produced calli in their first generation of culturing.On the part of 65-83 d,after the blooming period,the embryos produced calli through more than 2-3 generations.On the part of 88 d,after the blooming period,the survival rate of shoots on the WPM culturing base doubled compared with that on the MS culturing base;on the same culture medium,the embryos were inhibited from growing into shoots when BA,KT or 2,4-D was added on to the culture medium.The survival rate of shoots was increased remarkably when the seeds were treated in 1 000 mg/L GA3.[Conclusion] This study provided experimental basis for the establishment of Prunus salicina cv.Zaoshi,embryos rescue techniques and cross breading.

In vitro Culture of Unfertilized Ovary of Strawberry(Fragaria spp.)

[Objective] This study was to investigate the in vitro culture of unfertilized ovary of strawberry.[Methods] Employing single factor experiment,we investigated six key factors including genotype,extrinsic hormone,sucrose concentration,low temperature pretreatment,growth environment and development status,and illumination condition on induction of gynogenesis in vitro of unfertilized ovary,on the induction of gynogenesis in vitro of unfertilized ovary.[Results] The optimal conditions for in vitro culture of unfertilized ovary of strawberry were as follows：the primary flower buds cultured on bare land as explants,selection of appropriate genotype,2,4-D as external hormone,sucrose at the concentration of 6%,low temperature pretreatment for 48 hours and dark culture under alternated temperature.[Conclusion] The research provided reference for ploidy breeding in strawberry.

Differentiation of murine male germ cells to spermatozoa n a soft agar culture system

Establishment of an in vitro system that allows the development of testicular germ cells to sperm will be valuable for studies of spermatogenesis and future treatments for male infertility. In the present study, we developed in vitro culture conditions using three-dimensional agar culture system （SACS）, which has the capacity to induce testicular germ cells to reach the final stages of spermatogenesis, including spermatozoa generation. Seminiferous tubules from testes of 7-day-old mice were enzymatically dissociated, and intratubular cells were cultured in the upper layer of the SACS in RPMI medium supplemented with fetal calf serum （FCS）. The lower layer of the SACS contained only RPMI medium supplemented with FCS. Colonies in the upper layer were isolated after 14 and 28 days of culture and were classified according to their size. Immunofluorescence and real-time PCR were used to analyse specific markers expressed in undifferentiated and differentiated spermatogonia （Vasa, Dazl, OCT-4, C-Kit, GFR- a-l, CD9 and a-6-integrin）, meiotic cells （LDH, Crem-1 and Boule） and post-meiotic cells （Protamine-1, Acrosin and SP-IO）. Our results reveal that it is possible to induce mouse testicular pre-meiotic germ cell expansion and induce their differentiation to spermatozoa in SACS. The spermatozoa showed normal morphology and contained acrosomes. Thus, our results demonstrate that SACS could be used as a novel in vitro system for the maturation of pre-meiotic mouse germ cells to post-meiotic stages and morphologically-normal spermatozoa.

Effect of pre-culture on virus elimination from in vitro apple by thermotherapy coupled with shoot tip culture

We evaluated the role of pre-culture on survival rate of in vitro apple plants treated by thermotherapy. Two apple cultivars, Malusxdomestica cv. Pink Lady and Huafu, were used in the experiment and both have widely grown in China and infected with Apple chlorotic leafspot virus （ACLSV） and Apple stem grooving virus （ASGV）. Results in growth and virus titer of apple plants did not exhibit clear trends during five different periods of pre-culture. Whilst, pre-culture increased the survival rate of the two cultivars during thermotherapy. The survival rate of plants pre-cultured for 13 d （P-13d） was 14 and 51% higher than that of P-ld plants for Pink Lady and Huafu, respectively. Moreover, pre-culture positively influenced regeneration of Huafu plants. The average survival rate of plants regenerated from P-ld and P-4d was 20% lower than that regenerated from P-7d, P-10d, and P-13d. The efficiency of virus eradication was determined by reverse-transcription PCR with two primer pairs for each virus, and the detection results showed that pre-culture scarcely affected apple virus elimination. Despite the fact that the two viruses were hardly detected at 5 d of thermotherapy, no virus-free plants were found in the two cultivars of regenerated apple plantlets after 30-d treatment.

Toxicity Evaluation of in vitro Cultures of Freshwater Cyanobacterium Microcystis aeruginosa:Ⅰ.Hepatotoxic and Histopathological Effects in Rats

Laboratory cultures of freshwater cyanobacterium (blue-green alga) Microcystis aeruginosa PCC 7806 was cvaluated for its hepatotoxic effects in rats. The lyophilized cell extract injected intraperitoneally at 1 and 2 LD50 (15.8 and 31.6 mg/kg, respectively) produced significant increase in liver-specific enzymes viz. plasma alkaline phosphatase,γ-glutamyl transferase, lactate dehydrogenase with a concomitant decrease in hepatic glutamic pyruvic transaminase. A corresponding increase in liver body weight index and histopathological changes in liver (degeneration of hepatocytes, congestion and hemorrhage etc.) are indicative of a dose and time dependent hepatotoxic nature of the algal extract

Echinococcus Granulosus: Suitable in vitro Protoscolices Culture Density

The present study is to determine the suitable protoscolices （PSCs） density for long-time culturing in vitro. The PSCs were divided into eight groups with different densities and the viability tests were carried out with 0.1% methylene blue staining. Then the infection ability of cultured PSCs was assessed by the mean cyst weight of mice inoculated intraperitoneally with PSCs after 8 months post-infection.

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

To investigate the effect of clenbuterol hydrochloride on the in vitro development of both 1 cell and 2 cell mouse embryos. Methods The cultural systems of both 1 cell and 2 cell mouse embryo were used to determine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and 10 ng/mL on developmental rates of mouse embryos. Results When 1 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, developmental rates from the 4 cell stage to blastocyst stage were significantly lower than those in the control group (P<0.05), but on dosages of 3 ng/mL and 10 ng/mL, the inhibiting effects on embryo development were significantly increased (P<0.01). When 2 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious differences in developmental rates were not found between the 2 cell embryo group and the control (P>0.05). However, at levels of 3 ng/mL and 10 ng/mL, significant decrease of developmental rates in 2 cell embryos was observed from the 4 cell and from the 8 cell stage, respectively (P<0.05). Embryos cultured with clenbuterol hydrochloride appeared to have more granules, fragments and degeneration than those in the control. Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and the effect is in a dose dependent. 1 cell mouse embryos cultured with clenbuterol hydrochloride could be easily inhibited at 2 cell stage, but the effect of clenbuterol hydrochloride on development of the late 2 cell embryos would be reduced.

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that： 1） germination rate of grade 3 embryos in immature seeds with 0.6-0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4-0.6 cm diameter was 77,8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2） The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3） The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata vat. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.

The Key Factors Affecting Tuber Development of Potato in vitro and the Relation with Protein Fractions

According to previous analysis, some properties bounding up with tuber yield were investigated. The results showed that tuber average weight, plastid Mg2 + -ATPase activity, plastid Ca2 + -ATPase activity, mitochondria Mg2 + -ATPase activity, total soluble protein content, tuber average diameter, and Q-enzyme activity were important factors determining the tuber yield. The linear regression equation was:Y = 0.5211 + 0.0595X(1)+0.8389X(2) +0.0882X(3) -0.0073X(4) +0.1449X(5) +0.3510X(6) +0.0031X(7) -0.00003X(8) + 0.3412X(9) + 0.0127X(10) + 0.2904X(ll) + 0.0570X(12) + 0.0159X(13) + 0.3585X(14) + 0.0134X(15) - 0.1012X(16). At the same time, the relation between several important properties and soluble protein fractions were analyzed.

Normal and Degenerated Rabbit Nucleus Pulposus Cells in in vitro Cultures: A Biological Comparison

This study examined the biological characteristics of normal and degenerated rabbit nucleus pulposus （NP） cells in vitro in order to provide seed cells for intervertebral disc （IVD） tissue engineering. A total of 8 adult New Zealand white rabbits underwent annulus puncture to establish models ofintervertebral disc degeneration （IDD）. Four weeks later, normal and degenerated NP cells were obtained. Cell morphology was observed by light and electron microscopy. Cell viability was measured by MTT assay. Cell cycle and expression of extracellular matrix （ECM）-related genes （aggrecan and type II col- lagen） were determined by using flow cytometry and RT-PCR respectively. The growth curve of normal NP cells showed that the cells at passage 4 tended to slowly grow on the fifth day of culture. The density of normal NP cells at passages 5 to 7 was significantly less than that of the first-passage cells 2 or 3 days after seeding （P〈0.05）. The degenerated NP cells at passage 3 showed slow growth at 4th day. After 5 passages, the degenerated NP cells assumed stagnant growth and the growth seemed to stop at passage 7. The MTT assay revealed that for both normal and degenerated NP cells, the absorbance （.4） value at passages 4-7 was obviously decreased as compared with that at passage 1 （P〈0.05）. Cell cycle analysis showed that the proportion of normal NP cells at G1 phase was 65.4%-3.5%, significantly lower than that of degenerated NP cells at the same cell cycle phase With the value being 77.6%-4.8%. The degen- erated NP cells were predominantly arrested at Gt phase and failed to enter S phase. The expression of type II collagen and aggrecan was significantly decreased with passaging. It was concluded that normal NP cells possessed good viability and proliferative capacity by the third passage, and they could secrete large amounts of ECM within this period. The normal NP cells may serve as seed cells for IVD tissue engineering.

Study on in vitro Cultural Behaviors of Spermatogonium Stem Cells of New Born Calves

Three methods were adopted in culture spermatogoniums of newly born calf in vitro,such as enzymatic digestion and percoll density gradient centrifugation（MethodⅠ）,tubular fragments culture（MethodⅡ）and tissue culture（MethodⅢ）,and cultural behaviors of cells were observed.The results showed that typical spermatogonium colonies appeard at 144 h of culture by enzymatic digestion-percoll density gradient centrifugation method and tubular fragments culture method,2.5%FBS kept the characteristics of spermatogonium stem cell better than others,produced more mass clones,and FBS of more than 2.5%concentration benefited spermatogonium differentiation and the number of colonies was significantly affected by FBS concentration.After 1 week of culture in method Ⅲ,the diameter of lumens and quantity of sertoli’s cells in tubal wall increased obviously,lumen of seminiferous tubules appeared.Sertoli’s cells kept constant and the number of spermatogoniums decreased obviously after 2 weeks of culture.

Direct Regeneration of Plants Derived from in vitro Cultured Shoot Tips and Leaves of Poplar （Populus×euramericana ＇Neva＇）

The purpose of the present study was to establish a regeneration procedure for Populus × euramericana ＇Neva＇ by using in vitro shoots tips and leaves. For sterilization, 0.1% （w/v） mercuric chloride （HgCl2） solution for 8 to 10 min was the optimal treatment for this poplar cultivation. The effects of benzyladenine （BA） and α-naphthaleneacetic acid （NAA） added to Murashige and Skoog （MS） medium were tested on organogenesis. The highest regeneration rate and numbers of shoots/explant from shoot tips （96.7%, 9.8） and leaves （90.0%, 8.7） were obtained on the half-strength MS medium supplemented with 0.5 mg/L BA and 0.1 mg/L NAA. The optimal medium for in vitro rooting of shoots was on a half-strength MS medium containing 1 mg/L indolebutyric acid （IBA） with the highest rooting frequency （93.3%） and numbers of roots/explant （8.2）. For acclimatization, in vitro rooted plantlets were transferred to plastic cups containing vermiculite and peat （1： 1）. After acclimatization, transplanted plantlets grew well in a shade house. Therefore, we believe that this efficient plant regeneration protocol especially by leaf explants is very important for in vitro clonal propagation of Populus×euramericana ＇Neva＇.

Studies on Single Cell Culture in vitro in Wheat——The variation of grain protein content and its fractions from regenerated plants

On the basis of previous studies dealing with the variation of major agronomic and yield characteristics of regenerated plants derived from single cell culture in vitro of common wheat (Triticum aestivum L.Cultivar NE 7742), the grain protein content and its fractions from regenerated plants with stable agronomic characteristics were studied from 1992 to 1995. The results showed that the variation of grain protein content and its fractions in somaclones from single cell culture in vitro were very significant and the range was very wide (11531770%). Several types of variation were found in the studies, especially the type with higher protein content than that of cultivar NE 7742 (non-culture parent). Among them, -2069% of lines the grain protein content was significantly higher than that of NE 7742 and combined with high yielding potential. The tendency of variation of the four protein fractions showed that the variation of albumin was not obvious and maintained the same level as NE774 increased in some somaclones and decreased in others. However, the percentages both globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and glutenin tended to increase. The variation of total amount of structural protein and the ratio between globulin and albumm was mainly influenced by globulin under the condition of culture in vitro. The variation of total amount of storage protein and the ratio between gliadin and glutenin was mainly affected by glutenin. The results mentioned above demonstrated that the induction and screening of somaclonal variation could be an effective way in wheat improvement in combining high protein content with high yield.

In vitro reproduction of Kidney Tea （Orthosiphon stamineus Bents）

The stages of introduction in vitro culture of the local population of the kidney tea （Orthosiphon stamineus Bents） cultivated in the Georgia’s medicinal plant farm and the microclonal propagation, in particular, have been elaborated. The cultivation of explants was carried out on the Gamborg （B5） feeding area. The hormonal （BAP; Zn; NAA） composition of the feeding area and their concentrations have been selected; proliferation of buds in the basal part of the sprout has been achieved from the formed morphogenic tissue. The microclones by activating axillary meristem have been received.