Cell Ultrastructure of Kiwifruit (Actinidia chinensis) Shoot Tips During Cryopreservation

The changes in the cell ultrastructure of in vitro cultured shoot tips from dwarf genotype of kiwifruit （Actinidia chinensis Ganmi 5） during cryopreservation were investigated. Shoot tips were preserved in liquid nitrogen using vitrification, and the cell ultrastructure was examined using transmission electron microscopy （TEM）. The regular ultrastructure of the cell wall, cell membrane and nucleus of shoot tips could be damaged during the freezing and thawing associated with preservation using liquid nitrogen. The cell plasmolysis was increased and freezing tolerance was improved after precultufing and dehydrating in a preservation and vitrification solution （PVS2） （30% glycerol （Gly）＋ 15% ethylene glycol （EG）＋ 15% dimethylsulfoxide （DMSO） ＋ 0.4 mol L^-1 sucrose）. The structure of some cells with low degree of injury and reversible damage was similar to that of the control and they could undergo normal cell division and differentiation. Besides, they could recover automatically and regenerate after their reculture.

Response of carbohydrate metabolism-mediated sink strength to auxin in shoot tips of apple plants

Auxin(indole-3-acetic acid, IAA) has a considerable impact on the regulation of plant carbohydrate levels and growth, but the mechanism by which it regulates sugar levels in plants has received little attention. In this study, we found that exogenous IAA altered fructose(Fru), glucose(Glc), and sucrose(Suc) concentrations in shoot tips mainly by regulating MdSUSY1, MdFRK2, MdHxK1 and MdSDH2 transcript levels. Additionally, we used 5-year-old ’Royal Gala’ apple trees to further verify that these genes play primary roles in regulating sink strength. The results showed that MdSUSY1, MdFRK2, MdHxK1/3 and MdSDH2 might be major contributors to sink strength regulation. Taken together, these results provide new insight into the regulation of the carbohydrate metabolism mechanism, which will be helpful for regulating sink strength and yield.

Study on Shoot Tip Culture of Illciaceae Ornamental Plants

[Objective] This study aimed to establish a technology system for tissue culture and rapid propagation of Illciaceae ornamental plants. [Method] Effects of medium components and anti-browning agents on the survival and growth of shoot tips were investigated by using apical buds of IItciaceae plant Haierlian as experiment material and MS as basic medium. [Result] The results showed that apical buds at the early germination period in spring were the most suitable explants for tissue culture of IIIciaceae plant Haierlian. Sterilization with 0.1% HgCI2 for 6 min achieved the best effect, while conventional surface-sterilization with ethanol would affect the survival of explants. The optimal medium for primary culture was MS-D （with modifications in major elements and organic components） ＋ anti-browning agents （equa~ volume） ＋ 2.0 mg/L of 6-BA ＋ 0.5 mg/L of NAA. The optimal subculture medi- um was MS-F （with modifications in inorganic and organic components） ＋ anti-brown- ing agents （equal volume） ＋ 2.0 mg/L of 6-BA ＋ 0.1 mg/L of NAA. [Conclusion] This study laid the foundation for establishment of tissue culture and rapid propagation technology system for Haierlian.

Effect of Heat Treatment Combined with Shoot Tip Culture on the Virus-Free of Arena Strawberry

With Arena strawberry as experimental materials, heat treatments combined with shoot tip culture were used to explore their detoxification effects. The results indicated that if not heated, splitting shoot tips directly could not completely remove viruses. On the other hand, it will be obviously improved after shoot tips are heated. Incubators heat treatment （inconstant temperature） with shoot tip culture, constant temperature water bath incubation process with meristem-tip culture, hot air treatment （constant temperature） with apical meristem culture were three methods tested in searching for virus-free of Arena strawberry in this experiment. And their virus-free rates were 78.9%, 86.0% and 50.0% respectively. Of the three methods, the second, constant temperature water bath process with shoot tip culture, with 78.7% survival, is the best. Thus, it can be inferred that constant temperature water bath treatment combined with shoot tip culture might be the most appropriate method to remove viruses of Arena strawberry.

Effect of Cytokinins on the Micropropagation of Carob(Ceratonia siliqua L.)through Shoot Tip Culture

In order to determine the most suitable cytokinin for the micropropagation of carob (Ceratonia siliqua L.), the effect of four cytokinins: BAP, zeatin, kinetin and 2-iP, was tested on explants derived from young seedlings of seven days. Organogenesis is better in the presence of BAP (0.5 mg/l), while buds growth is favored by zeatin (0.5 mg/l). The combination of the most suitable cytokinin (BAP at 0.5 mg/l) with IBA improves the caulogenesis when the concentration of this latter is low (0.1 mg/l);high concentrations of IBA have an inhibitory effect on elongation and neoformation of shoots and leaves. The multiplication and growth of shoots are more favorable on WPM medium in the presence of BAP (0.5 and 1 mg/l) alone or combined with 0.5 mg/l with GA3, while rooting is mainly favored by IBA, especially at 2 mg/l.

Influence of Ethylene Inhibitor Silver Nitrate on Direct Shoot Regeneration from in Vitro Raised Shoot Tip Explants of Sphaeranthus indicus Linn.—An Important Antijaundice Medicinal Plant

In the present investigation, an attempt has been made to study the influence of ethylene inhibitor silver nitrate on direct shoot regeneration in Sphaeranthus indicus, an important antijaundice medicinal plant, by using in vitro raised shoot tip explants. The effect of various concentrations of kinetin, BAP (0.5 - 3.0 mg/l), and NAA (0.1 - 0.5 mg/l) along with AgNO3 (0.1 - 1.0 mg/l) was studied. Among the combinations tested MS medium augmented with kinetin (1.0 mg/l), NAA (0.1 mg/l) and AgNO3 (0.4 mg/l) was found to be optimum for production of multiple shoots (34.3 ± 0.36). Addition of AgNO3 to the media not only increases shoot number in all the concentrations tested but also shoot length. AgNO3 at the concentration of 0.4 mg/l produced 35% more number of multiple shoots when compared to multiple shoots (10.8 ± 0.12) produced in control. In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control. These in vitro raised shoots were transferred to the rooting medium containing different concentrations of auxins such as NAA and IAA along with AgNO3 (0.1 - 0.6 mg/l). Better rooting response (21.6) was observed on NAA (2.0 mg/l) and AgNO3 (0.4 mg/l) containing media. The healthy rooted plantlets were transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening. Finally the hardened plants were transferred to field environment for utmost survivability.

Establishment of Green Cotton Regeneration System

[Objective]The aimed to optimize transformation system for Agrobacterium-mediated green cotton shoot tip and provide reference for genetic engineering-assisted breeding of color cotton. [Method]With shoot tip of natural green cotton as receptor and by using Agrobacterium-mediated transformation method,the optimum conditions of regeneration system were obtained. [Result]Combining with vacuum infiltration,3-old-day shoot tips were infected for 10 minutes in Abarobacterium suspension with OD600 value about 0.5 followed by co-culture for 24 h on the medium containing MS＋l mg/L KT,pH value of 5.8. The regeneration culture was conducted in MS medium containing l mg/L KT and increasingly 30,50 and 70 mg/L Kan. The highest transformation frequency was 34.6%,and the positive resistant plantlets of green cotton 9804 were obtained. [Conclusion]Green cotton is more beneficial than white cotton to conduct genetic transformation.

Contributions of Chinese Botanists to Plant Tissue Culture in the 20th entury

This paper looks back to the development of plant tissue culture in China in the last century. Since 1934, tissue culture studies in China has kept up with the international development in the fields. Progress has been made by Chinese in nearly every branches of tissue culture, including in vitro organogenesis, shoot tip culture, anther culture, ovary culture, endosperm culture, protoplast culture as well as mass cell culture. On the basis of reviewing the articles written by Chinese on plant tissue culture, the internationally recognized contributions are specially mentioned. The applications of plant tissue culture to agriculture and industry in China are also introduced.

Tissue Culture and Rapid Multiplication Techniques of Apocynum L.

[Objective] This study aimed to investigate rapid multiplication of Apocynum by tissue culture so as to provide plantlet sources for its industrialized cultivation. [Method] The asepsis seedlings were obtained by dealing with Apocynum seeds. Its cotyledons, hypocotyls and shoot tips were cultured on the media containing different concentrations of hormones. Finally, the influence of different hormone combinations on differentiation of cotyledons and hypocotyls, rapid multiplication of shoot tips, rapid multiplication of regenerated shoots, and rooting of test-tube plantlets was com- pared. [Result] MS＋2.0 mg/L BA＋0.03 mg/L NAA and MS＋0.07 mg/L NAA were the optimum medium for inducing regenerated buds from cotyledons and hypocotyls re- spectively; MS＋2.0 mg/L BA＋0.02 mg/L NAA was the best medium for rapid multi- plication of shoot tips; MS＋1.9 mg/L BA＋I.7 mg/L NAA was the best medium for rapid multiplication of regenerated buds： and 1/2MS＋0.6 mg/L NAA was the best medium for inducing roots. [Conclusion] The optimum hormone combination was de- termined for Apocynum rapid multiplication by tissue culture, which provides technical support on Apocynum industrialized cultivation.

Micropropagation of the Moroccan Endemic Plant <i>Thymus broussonetii</i>Boiss. with Aromatic-Medicinal Value and Conservation Concern

Micropropagation from shoot tips and nodal segments was carried out for the conservation and domestication of spontaneous Moroccan thyme, Thymus broussonetii Boiss. subsp. broussonetii (endemic threatened). The mineral composition of the culture medium, as well as the succession of different growth regulators, influenced the in vitro growth of this species. Sterilized achenes of T. broussonetii were able to germinate on an agar medium containing Gautheret macronutrients with a rate of 25% and a degree of contamination of less than 4%. Shoot apices of 15-day seedlings (two cotyledon leaves) were cultivated on SD + 0.46 μM Kin medium and the explants obtained were transplanted every month. Six macronutrients (MS, B5, SH, SD, MSm and N30K) were tested and N30K was chosen for the following experiments. Seven cytokinins (Kin, BAP, 2iP, DPU, adenine, Zeat and TDZ) at 0.46, 0.93 and 2.32 μM/l were evaluated and the addition of 0.93 μM adenine to N30K medium favored significantly the induction of buds and the elongation of explants. Three polyamines (putrescine, spermidine and spermine) at 2, 5, 10 and 20 μM/l were tested. A better multiplication of buds, shoots and roots was noted for N30K + 10 μM spermine. Cytokinin-auxin combinations led to better root multiplication and an increase in the number of buds and the length of explants, particularly for 0.46 μM Kin + 2.85 μM IAA. Acclimatization was successfully carried out using vitroplants developing a good root system. One month after the start of acclimatization, 97% of T. broussonetii plantlets were healthy. Three months later, they were transplanted into larger pots. 100% of the acclimatized plants developed flowers in the 2nd year between June and August. Re-initiation of the in vitro culture was carried out from sterilized twig segments collected from the acclimatized plants of T. broussonetii with 1 - 2 nodes on the medium N30K + 0.46 μM Kin, and 52.1% of the explants healthily proliferated. Finally, two micropropagation prototypes were developed: shoot tip culture from seedlings obtained after germination of achenes and node culture from acclimatized plants.

Effect of Plant Hormones on Callus Induction from Fruit and Seedling Explants of Chilli Pepper （Capsicum annuum L.）

This experiment was conducted to optimize the culture conditions to induce calli from chilli pepper （Capsicum annuum L.） local cultivar. The mature seeds were surface sterilized for one min with 70% Ethanol followed by 20 min with （0%, 2%, 4% or 6%） NaOCI and were germinated on MS medium with 2 mg/L GA3. Seedlings and mature fruits were used as explants source. The placenta, pericarp, hypocotyls, cotyledonal leaves, shoot tips and roots were cultured on MS media supplemented with Kinetin （0.0 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L） and IAA （0.0 mg/L, 1.0 mg/L, 2.0 mg/L） in different combinations or NAA or 2,4-D （0.0 mg/L, 1.0 mg/L, 2.0 mg/L, 4.0 mg/L）. Callus fresh weight was recorded after 4 weeks in culture. The results showed that the best sterilizing method was with 70% Ethanol followed by 20 mints with （4% or 6%） NaOCI, however 6% NaOCI reduced seed＇s viability. Callus was induced from all explants cultured on MS media supplemented with IAA and Kinetin except the placenta and the pericarp. The results showed that the hypocotyls surpass all other explants in the mean callus fresh weight which was 160.58 mg compared with 147.81 mg, 134.95 mg, and 122.33 mg for cotyledonal leaves, shoot tips and roots respectively. Moreover the analysis of the interaction between the growth regulators and the explants showed that 2 mg/L IAA and Kinetin had significant effect on callus mean fresh weight which was （309.74, 339.14, 358.48, and 284.64） mg for the shoot tips, cotyledonal leaves, hypocotyls and roots, respectively. On the other hand, 2 mg/L 2,4-D or NAA was the best concentration for callus induction from the placenta and the pericarp. The pericarp gave a mean fresh weight of 276.90 mg in the presence of 2,4-D compared with 253.60 mg for the placenta. Moreover the pericarps gave significantly higher fresh weight than the placenta with an average of 210.3mg and 184.9 mg respectively in the presence of 2 mg/L NAA. In conclusion the best sterilization method of chilli pepper seeds is by 70% ethanol for one minute followed by 20 min in 4% （NaOCI）. The best explants for callus induction only is the Hypocotyls grown on MS medium supplemented with 2 mg/L oflAA and Kinetin under the conditions of the current experiment.