In vitro Regeneration Culture of Pepper and Its Application in Breeding

This paper summarized the technology of haploid production, protoplast culture, organ regeneration culture of pepper and the key factors affecting in vitro regeneration culture of pepper, including explants, seedling age, medium,genotype and plant growth regulator, then pointed out several main problems, in order to provide the reference for building an efficient in vitro regeneration culture system of pepper and its application in breeding.

In vitro regeneration of Populus tomentosa from petioles

A reliable in vitro regeneration procedure for Populus tomentosa is a prerequisite for its trait improvement through genetic transformation. We established a systematic protocol for indirect regeneration of P. tomentosa using in vitro petioles of Chinese poplar cultivar ＇fasta-3＇. A high frequency of callus induction （〉97 %） was obtained from isolated petioles cultured on the modified 1/2MS basal medium supplemented with 0.5 mg/L ZT and 1.0 mg/L NAA, and the tested calli were subsequently plated on 1/2MS basal medium supplemented with 0.25 mg/L BA, 0.25 mg/L ZT, 0.25 mg/L NAA, 0.01 mg/L TDZ, and 0.5 mg/L KT for efficient regeneration of shoots after being cultured for 6 weeks. The regenerated shoots were vigorously rooted on the tested media supplemented with 1.0 mg/ L IBA and 0.5 mg/L NAA. These results can facilitate genetic transformation of P. tomentosa for trait improvements in future.

Development of High Efficient in vitro Regeneration and Transformation System in Strawberry

In this paper, several factors that affect the efficiency of in vitro adventitious bud regeneration and Agrobacterium tumefaciens-mediated transformation of F. vesca were studied. The results showed that F. vesca seeds germination rate was the highest while seeds were cultured in water, and the germination rate was the lowest while seeds were cultured on MS medium supplemented with hormone; the germination rates that seeds cultured on two and three layers filter paper were higher than that seeds cultured on four and five layers filter paper. In vitro adventitious regeneration efficiency was affected by different explants types. The significant difference was existed between petioles and leaves. When using the same type explants, in vitro adventitious buds regeneration rate and the average number of buds per explant between Ruegen （RE） and Yellow Wonder （YW） had no significant difference. RE to Agrobacterium tumefaciens was more sensitive than YW. Using seedling leaves of RE and YW as materials, an efficient Agrobacterium-mediated transformation system was developed. In this system, the concentration of bacteria was OD600=0.5, the explants were immersed in bacteria broth for 9 min, the co-cultured time was 2 days, and had no pre-cultured time. The percentage of explants with resistant buds of RE and YW was compared. The putative transformed plants were confirmed by PCR.

Study on Effects of Two Kinds of Auxinin vitro Regeneration of Cyclamen

Cyclamen leaves and petioles explants were cultured on MS media supplemented with different concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) or 1-naphthaleneacetic acid (NAA) to induce callus. The effect of 2, 4-D on shoot regeneration was also studied. Either in media containing 2, 4-D or in media containing NAA, callus was observed, but the quality or quantity of callus induced by 2, 4-D or NAA were different. The callus induced by 2, 4-D was white, compact and having powerful multiplication capacity. The callus was inclined to browning then was poorly organogenetic. While the callus induced by NAA was yellowish in appearance. It was pultaceous and proliferated bradytelicly. The callus usually can give rise to many shoots. But the frequency of inducing callus of 2, 4-D is higher than that of NAA. The regenerative plantlets derived from the callus respectively induced by 2, 4-D or NAA were transferred into rooting medium. The frequency of rooting were no difference.

Effects of Different Natural Organic Additives on in Vitro Shoot Regeneration of Raphanus sativus L. Var, Beeralu

Abstract： Raphunus sativus L. commonly known as ＂radish＂ is a popular vegetable crop used by people all over the world for its culinary and medicinal properties. Enhancement of in vitro regeneration system for radish variety beeralu is needed to further tissue culture studied. Therefore, the present study was carried out to find out the effects of different organic additives on in vitro shoot regeneration of Radish （Raphanus sativus L.） Var. Beeralu. Hypocotyl explants of aseptic plantlets were cultured on MS basal medium supplemented with 2.5 mg/L BAP （benzyl adenine） and 0.1 mg/L NAA （1-nphthaleneacetic acid） with different natural additives; 20% coconut water, 20% coconut milk, 10% grind spinach leaves, 10% grind potato tubers, 10% grind carrot, 5% rice flour, 10% green gram, 10% grind pumpkin, 10% banana fruit, 10% orange and control （without any additives）. Complete randomized design （CRD） with five replicates was used. After one month the numbers of regenerated shoots were counted and statistical analysis was carried out using the Student Newman-Kuells Means Separation Test of SAS program （9.1.3）. The highest mean number of shoots （12 shoots/explant） from Radish （Raphanus sativus L.） Var. Beeralu observed in MS basal medium with 2.5 mg/L BAP and 0.1 mg/L NAA with 10% orange juice whereas the 2nd highest shoots were obtained with 20% coconut water. The lowest number of shoots （0 shoot/explant） was observed from medium with carrot juice and pumpkin juice, but they induced callus formation. Media with grind spinach leaves, rice flour, green gram, grind potato tubers and banana inhibit the shoot regeneration.

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.

In vitro Plant Regeneration from the Mature Tissue of Navel Orange (Citrus sinensis L. Osbeck) by Direct Organogenesis

An efficient in vitro regeneration system by direct organogenesis from mature nodal and internodal stem segments ofNewhall navel orange (Citrus sinensis L. Osbeck) was developed. Illuminating conditions together with plant growthregulators affected the adventitious bud regeneration frequency and efficiency. The initial 15 d darkness inoculation isbeneficial for the adventitious bud regeneration. The highest regeneration frequency (85.2%) and bud formationefficiency (3.7 per responsive internodal stem segment) were obtained in the media supplemented with 1.0 mg L-1 BAPand 0.5 mg L-1 NAA. ABA at 0.2 mg L-1 positively affected the bud formation efficiency, which amounted to 8.5 buds perinternodal segment in the presence of BAP at 1.0 mg L-1. The adventitious shoots successfully rooted and weretransferred to the soil.

Linking axon transport to regeneration using in vitro laser axotomy

Spinal cord injury has devastating consequences because adult central nervous system （CNS） neurons do not regenerate their axons after injury. Two key reasons for axon regeneration fail- ure are extrinsic inhibitory factors and a low intrinsic capacity for axon regrowth. Research has therefore focused on overcom- ing extrinsic growth inhibition, and enhancing intrinsic regeneration capacity. Both of these issues will need to be addressed to enable optimal repair of the injured sp＋inal cord.

Establishment of high frequency shoot regeneration system in Himalayan poplar(Populus ciliata Wall. ex Royle) from petiole explants using Thidiazuron cytokinin as plant growth regulator

Populus species are important resources for industry and in scientific study on biological and agricul- tural systems. Our objective was to enhance the frequency of plant regeneration in Himalayan poplar （Populus ciliata wall. ex Royle）. The effect of TDZ alone and in combi- nation with adenine and NAA was studied on the regen- eration potential of petiole explants. The explants were excised from Himalayan poplar plants grown in glass- houses. After surface sterilization the explants were cul- tured on shoot induction medium. High percentage shoot regeneration （86 %） was recorded on MS medium sup- plemented with 0.004 mg L-1 TDZ and 79.7 mg L-1 adenine. The regenerated shoots for elongation and multi- plication were transferred to MS ＋ 0.5 mg L-1 BAP ＋ 0.2 mg L-1 IAA ＋ 0.3 mg L-1 GA3. Root re- generation from shoots developed in vitro was observed on MS medium supplemented with 0.10 mg L-1 IBA. Hi- malayan poplar plantlets could be produced within 2 months after acclimatization in a sterile mixture of sand and soil. We developed a high efficiency plant regeneration protocol from petiole explants of P. ciliata.

Buds Reactivity and Factors Promoting Shoots Proliferation and Rooting of Cashew Seedlings Using in Vitro Tissue Culture Process

Tissue culture techniques are widely used for the mass propagation of many species. In cashew in vitro propagation, some protocols need to be established at this end. The present work was carried out to evaluate the conditions for in vitro regeneration of cashew seedlings from micropropagation by organogenesis on Benin genotypes. Nodal explants from one-month-old cashew seedlings in the greenhouse and cotyledonary nodes from in vitro germination were used for this purpose. BAP and kinetin were evaluated alone at 2.2 mg/L and then the combination of 2.2 mg/L BAP + 0.2 mg/L IBA was also evaluated. The response of axillary bud proliferation on explants was obtained with both cotyledonary nodes and axillary buds from different combinations of growth regulators. However, the best responses were recorded with cotyledonary nodes. When 2.2 mg/L BAP was used, 80% of the explants responded with numerous proliferation (5 to 8) buds (5.75 ± 0.12) with good shoot length (6.73 ± 0.3 cm) on MS medium containing 150 mL coconut water. Rooting was observed with the combination of NAA (2.5 mg/l) + IBA (2.5 mg/l) on 1/2 MS containing 40 g/l sucrose.

One Rapid and Efficient Method for Isolation of Total RNA from Shoots Regenerated in vitro of Populus suaveolens

Total RNA was isolated from shoots regenerated in vitro of Populus suaveolens by the modified method of CTAB, and two clear bands of rRNA (28S and 18S) were observed in agarose electrophoresis. In addition, the values of OD260/OD280 and OD260/OD230 of extracted RNA were 2.12 and 2.23 respectively. The results show that RNA is little decomposed and the purity of RNA is high. Moreover, RNA isolated by the modified method of CTAB reagent had been successfully used for reverse transcription of P. suaveolens cDNAs and ideal special band was observed.

Nerve growth factor promotes in vitro proliferation of neural stem cells from tree shrews

Neural stem cells promote neuronal regeneration and repair of brain tissue after injury,but have limited resources and proliferative ability in vivo.We hypothesized that nerve growth factor would promote in vitro proliferation of neural stem cells derived from the tree shrews,a primate-like mammal that has been proposed as an alternative to primates in biomedical translational research.We cultured neural stem cells from the hippocampus of tree shrews at embryonic day 38,and added nerve growth factor（100 μg/L） to the culture medium.Neural stem cells from the hippocampus of tree shrews cultured without nerve growth factor were used as controls.After 3 days,fluorescence microscopy after DAPI and nestin staining revealed that the number of neurospheres and DAPI/nestin-positive cells was markedly greater in the nerve growth factor-treated cells than in control cells.These findings demonstrate that nerve growth factor promotes the proliferation of neural stem cells derived from tree shrews.

miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Differential expression of mi RNAs occurs in injured proximal nerve stumps and includes mi RNAs that are firstly down-regulated and then gradually up-regulated following nerve injury.These mi RNAs might be related to a Schwann cell phenotypic switch.mi R-30 c,as a member of this group,was further investigated in the current study.Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1,4,7,14,21,and 28 days post injury for analysis.Following sciatic nerve injury,mi R-30 c was down-regulated,reaching a minimum on day 4,and was then upregulated to normal levels.Schwann cells were isolated from neonatal rat sciatic nerve stumps,then transfected with mi R-30 c agomir and co-cultured in vitro with dorsal root ganglia.The enhanced expression of mi R-30 c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells.We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of mi R-30 c agomir on myelin sheath regeneration.Fourteen days after surgery,sciatic nerve stumps were harvested and subjected to immunohistochemistry,western blot analysis,and transmission electron microscopy.The direct injection of mi R-30 c stimulated the formation of myelin sheath,thus contributing to peripheral nerve regeneration.Overall,our findings indicate that mi R-30 c can promote Schwann cell myelination following peripheral nerve injury.The functional study of mi R-30 c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

Agrobacterium tumefaciens-mediated transformation of Eucalyptus urophylla clone BRS07-01

Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.

Engineered olfactory system for in vitro artificial nose

The engineered biomimetic sensors can not only realize the action of organs,but also combine functional materials as in vitro organs by simulating the response of biological organs to different environmental signals.Artificial nose is a concept proposed by imitating biological olfactory system,simulating olfactory nerve cells,olfactory bulb and olfactory cortex through different materials to realize olfactory function.The sensor array used to sense external gas stimulation can be analyzed based on different recognition principles through different original signals such as optics,electricity,electrochemistry and bioelectricity.Furthermore,combined with pattern recognition and microarray technology,artificial nose can be highly integrated with biocompatible and other important properties to achieve in vitro application.The design principle and necessary components of artificial nose are introduced in this paper including sensing structure,recognition system and functional module.At the same time,the potential development prospects of molecular recognition technology,polymer-based materials and microarray integration in artificial nose are prospected.