Efficient Plant Regeneration Protocol Research Articles

True-to-typeness and phytomedicinal potential in somatic embryo-derived plants of Crinum malabaricum (Amaryllidaceae): A medicinally important source of pharmaceutical biomolecules

Crinum malabaricum, Lekhak & S.R. Yadav (Amaryllidaceae) is a critically endangered bulbous plant endemic to India. Establishing an efficient plant regeneration protocol is necessary for its conservation and large-scale propagation. In the present study, regeneration was achieved through somatic embryogenesis from callus produced on MS media supplemented with various different concentrations of 2,4-D alone and in combination with N6-benzyl-adenine (BA). Different advanced stages of embryo development (globular, torpedo and cotyledonary) and maturation were obtained on MS medium, with combinations of picloram and thidiazuron (TDZ). A high rate of somatic embryos (55.89 ± 0.60) was obtained after eight weeks. SEM examinations showed the occurrence of cell clusters (embryogenic) which converted to somatic embryos. Well-developed cotyledon embryos were successfully germinated on PGRs free full and half strength MS medium, however, 94.03% of somatic embryos germinated on half-strength MS medium supplemented with 1.0 mg L−1 gibberellic acid. The true-to-type genetic conformity of regenerated plants was examined by SCoT, ISSR and RAPD primers based molecular analysis. The amplification bands produced by SCoT, ISSR and RAPD primers applied were monomorphic across all the regenerated plants. This confirmed their genetic homogeneity compared to the mother plant and also demonstrated the reliability of our somatic embryogenesis system for C. malabaricum. A rapid method for phytochemical analysis based on LC-ESI/MS exhibited better separation and analysis of galanthamine and lycorine from methanolic extracts of in vitro raised plants derived from somatic embryogenesis. The protocol developed should be helpful in reintroduction, restoration and ex situ conservation of this valuable plant in the natural condition as well as in the pharmaceutical sectors.

Synergistic effects of Vermi-Co extracts mixture with cytokinins on shoot regeneration of Lindernia minima (Benth.) Mukerjee—An endangered medicinal plant

The present investigation illustrates the synergistic growth effect of Vermi-Co (Vermicast and Coconut-water) extracts mixture with cytokinins on in vitro shoot regeneration of Lindernia minima. Due to less distribution threat status, this plant species has been considered as an endangered plant by IUCN (International Union for Conservation of Nature). Hence, it is necessary to conserve and to develop an efficient plant regeneration protocol for the rapid multiplication of L. minima. The nodal explants were inoculated on (¼) quarter-strength MS (Murashige and Skoog) medium supplemented with different concentrations (0.5 – 2.5 mg L-1) of BA (6-Benzyladenine), KIN (Kinetin) and TDZ (Thidiazuron) combined with various percentages of (5%, 15%, and 25%) Vermi-Co extracts mixture. Among the concentrations tested, 1.5 mg L-1 TDZ with 15% of Vermi-Co extracts mixture produced the maximum percentage of response with the highest (19.5 ± 0.20 shoots/explant) number of shoots and (10.2 ± 0.14 cm) shoot length. Then, the well-elongated shoots were transferred to rooting medium (quarter-strength MS) supplemented with 0.5 – 3.0 mg L-1 of IBA (Indole -3- butyric acid) and IAA (Indole -3- acetic acid). The highest percentage of response (98%) and maximum number of roots were observed in 1.5 mg L-1 IBA with 11.5 ± 0.29 roots per shoot and 6.6 ± 0.28 cm root length. Finally, well-developed plants were successfully transferred to the natural field condition with a survival rate of 97%. The present research revealed that, the combined growth stimulating effect of Vermi-Co extracts mixture combined with cytokinins increased the number of shoots and shoot length on shoot regeneration of L. minima from nodal explants. Our findings will be very useful for regeneration of Lindernia minima through plant tissue culture.

Rapid and Efficient Regeneration of Rhododendron decorum from Flower Buds

Rhododendron decorum is a woody species with high ornamental and medical value. Herein, we introduce a novel in vitro regeneration method for R. decorum. We used flower buds to develop an efficient and rapid plant regeneration protocol. Sterile flower buds of R. decorum of a 2 cm size were used as explants to study the effects of the culture medium and plant growth regulators on the callus induction and adventitious shoot differentiation, proliferation, and rooting. According to the results, the optimal medium combination for callus induction was WPM + 1 mg/L TDZ + 0.2 mg/L NAA, and its induction rate reached 95.08%. The optimal medium combination for adventitious shoot differentiation from the callus was WPM + 0.5 mg/L TDZ + 0.1 mg/L NAA, and its differentiation rate reached 91.32%. The optimal medium combination for adventitious shoot proliferation was WPM + 2 mg/L ZT + 0.5 mg/L NAA, for which the proliferation rate reached 95.32% and the proliferation coefficient reached 9.45. The optimal medium combination for rooting from adventitious shoots was WPM + 0.1 mg/L NAA + 1 mg/L IBA, and its rooting rate reached 86.90%. The survival rates of the rooted regenerated plantlets exceeded 90% after acclimatization and transplantation. This regeneration system has the advantages of being simple and highly efficient, and it causes little damage to the shoots of the mother plants, laying a foundation for the plantlet propagation, genetic transformation, and new-variety breeding of R. decorum.

Multiple Plant Regeneration from Embryogenic Calli of Paulownia tomentosa (Thunb.) Steud.

The aim of this paper was to study the effect of plant growth regulators on callus induction and in vitro morphogenesis using various explants of Paulownia tomentosa to develop an efficient plant regeneration protocol. Different plant organ sections (leaves, apical shoot tips, petals, nodes, and internodes) were cultured as explants to identify the best in vitro explants responsive to callus induction and plant regeneration. Explants were cultivated on MS media supplemented with different concentrations of plant growth regulators (TDZ (Thidiazuron), BAP (6-Benzylaminopurine), kinetin, and NAA (1-Naphthaleneacetic acid). It was discovered that the addition of TDZ and NAA stimulated the induction of somatic embryogenesis. It was discovered that the MS medium with the combination of plant growth regulators BAP (35.5 µM) and NAA (5.4 µM) with the addition of 30.0 g/L maltose, 500.0 mg/L casein hydrolysate, and 250.0 mg/L L-proline was optimal for callus induction and multiple plant regeneration. The study of the regenerative capacity of various explants of Paulownia tomentosa in vitro showed that plant regeneration depends on the type of explant, and occurs in both ways, indirectly, through the formation of callus tissues and directly on the explant, without callus formation. As a result of this study, the efficient reproducible protocol of embryogenic callus formation and multiple shoot induction in vitro of Paulownia tomentosa was developed. This system provides a clear increase in the frequency of plant regeneration from 36.3 ± 3.4% to 38.6 ± 2.3% per embryogenic callus from leaves and apical shoot tips, respectively.

Indirect somatic embryogenesis of Piper hispidinervum L. and evaluation of the regenerated plants by flow cytometry

BackgroundPiper hispidinervum is a species native from the Amazon region with great economic potential, given its scientifically proven insecticidal properties. In this study, an efficient protocol of plant regeneration via indirect somatic embryogenesis has been established for the first time. In a first experiment, for the induction of calluses, foliar explants of non-discriminated accesses of P. hispidinervum were inoculated in MS medium supplemented with α-naphtalenacetic acid (NAA) and 6-benzylaminopurine (BAP), in different combinations. For a second experiment, foliar explants from five different accesses of P. hispidinervum (PH17, PH21, PH28, PH37, and PH39) were analyzed regarding the formation of calluses when cultivated in MS medium with 5 mg L−1 NAA + 2.5 mg L−1 BAP. To obtain somatic embryos-like structures, calluses were cultivated in MS medium with 10 mg L−1 NAA + 2.5 mg L−1 of BAP. The somatic embryos-like structures obtained were inoculated in MS medium devoid of growth regulators and the plantlets were subjected to acclimatization. Calluses and somatic embryos-like structures were subjected to anatomical analysis and genetic stability of regenerated plants was analyzed by flow cytometry. ResultsThe treatments 2.5 mg L−1 BAP and 5 mg L−1 NAA + 2.5 mg L−1 BAP, after 60 days of cultivation, provided each 32% of primary callus, not being verified the formation of calluses in medium devoid of BAP. It was found that accesses differed among them with respect to the formation of primary calluses, with emphasis on accesses PH28, PH37, and PH39, with mean percentage of 95.3%. Regarding the percentage of embryogenic calluses and formation of somatic embryos-like structures, there were no statistical differences between accesses, with mean values of 90.6% and 77.3%, respectively. The somatic embryos-like structures of P. hispidinervum have conspicuous morphoanatomical similarities with the zygotic embryo, and flow cytometry analysis showed no significant variation in nuclear DNA size among plants regenerated in vitro and plants coming from seed germination, which indicates ploidy level stability. ConclusionThis protocol is the first cited in the literature that demonstrates an efficient micropropagation process by somatic embryogenesis of P. hispidinervum. It can be used either to enable large-scale vegetative production or to subsidize germplasm conservation or genetic engineering of P. hispidinervum.

An efficient protocol for shoot organogenesis and plant regeneration in Jackfruit

The present investigation was carried out for the rapid clonal mass multiplication of disease free plants of jackfruit cv. Pant Garima. The nodal explants were collected in winter season proved to be the best with regard to maximum survival of explants. The effect of sterilants (HgCl2 and ethanol) in various combinations was also assessed on survival percentage of explants. The MS medium was supplemented with five concentrations of BAP (1.0, 2.0, 3.0, 4.0 and 5.0 mgL−1) along with control (0.0mgL−1) and their effects on shootlets regeneration, and length of shootlets were evaluated. Maximum shoot proliferation was obtained in the medium supplemented with 4.0 mgL−1 BAP. Further, GA3@ 1.0 mgL−1 added to MS medium supplemented with 4.0 mgL−1 BAP gave the best results for enhancing the length of the shootlets. For rooting, half strength of MS liquid medium fortified with 2.0 mgL1 IBA gave maximum rooting.

In vitro propagation of an economically important medicinal plant Lawsonia inermis L. through nodal segments

The present investigation aimed to standardize efficient plant regeneration protocol through in vitro culture by using nodal segment for mass multiplication of Lawsonia inermis an economically important medicinal plant species. Mass multiplication of shoots induced on Murashige and Skoog (MS) medium supplemented with different growth regulators like auxins and cytokinins separately and in different combinations. The medium fortified with 6-Benzylaminopurine ( BAP) 1.0 mg/l + kinetin (KN) 1.5mg/l explained best compared to all other combinations. In vitro raised plantlets were excised and transferred in half strength MS medium supplemented with different growth regulators like Indole Butyric acid ( IBA) and naphthalene acetic acid (NAA ) (0.5-3.0 mg/l) in an experiment that gave rise to rooting. The half strength of MS medium additive with IBA in separate and in different combinations with NAA concentrations (0.5-3.0 mg/l) supported root development. The best response of rooting was obtained on half MS medium fortified with 1.0 mg/l IBA. The regenerated plantlets were successfully transplanted to pots. Regenerants were transferred to the field conditions and recorded the survival rate.. Among all the carbon sources and gelling agents used, sucrose (3%) in combination with 0.8 per cent agar-agar has proved significantly better. Multiple shoots formation with longer shoots were achieved on medium with 1.0mg/l BAP and 1.5mg/l Kn. Thus, it is possible to develop a large number of plants of L. inermis through shoot bud regeneration which can cater for the need of pharmaceutical as well as other industries.

An efficient regeneration protocol for in vitro direct organogenesis in einkorn (Triticum monococcum L.) wheat

Coleoptile, leaf and root explants of the einkorn (Triticum monococcum ssp. monococcum) were cultured in vitro to obtain an efficient plant regeneration protocol through direct shoot formation by using different combination and concentration of various plant growth regulators. A total of 180 different auxin and cytokinin combinations were tested for regeneration. Shoot formation was not observed with the root and leaf explants. Shoot formation was obtained only from the coleoptile explants, with a mean of 1.20 ± 0.24 shoots/explant and 86.60% of shoot formation frequency and with a 1.20 ± 0.53 shoots/explant and 80.00% shoot formation frequency on medium supplemented with 0.5 mg L-1 TDZ and 1 mg L-1 TDZ plus 1 mg L-1 NAA, respectively. The shoots were subculture on the MS medium containing the most effective hormone combination concurrently continued to shoot and root formation for 45 days. It was noteworthy that 3.66 ± 0.66 shoots per explant were induced by MS, which contained 1 mg L-1 TDZ plus 1 mg L-1 NAA and 2.0 mg L-1 KIN plus 0.5 mg L-1 NAA during a 45 days. Of the different auxin concentrations tested for rooting, 2.0 mg L-1 IAA was predominant, with the greatest number of roots (12.33 ± 0.88) produced per regenerated shoot. Finally, these well-developed plantlets were acclimatized with a 100 % success rate and they transferred to the ex vitro conditions. A highly efficient regeneration protocol for einkorn wheat was developed using somatic tissue as an explant source for the first time.

In vitro regeneration system of Halogeton glomeratus: an important halophyte

Halogeton glomeratus (H. glomeratus) is one of the most important halophytes in Asia, and the research on the genes and salt-tolerant mechanisms of this species is limited because of the lack of an optimal and efficient in vitro regeneration system. Here, we developed an efficient plant regeneration protocol using H. glomeratus leaves as explants, which has not been previously reported. High-quality calli were successfully obtained from the H. glomeratus leaves at a frequency of 100% supplemented with 2.00 mg/L 2,4-dichlorophenoxyacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose; the optimum callus subculture medium is 1.00 mg/L 2,4-dichlorophenoxyacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose. Shoots were regenerated on shoot induction medium at a frequency of 100% added with 0.50 mg/L 6-benzylaminopurine, 2.00 mg/L kinetin, 0.20 mg/L naphthaleneacetic acid, 0.50 g/L PVP, 5.00 g/L agar, and 30.00 g/L sucrose; then, shoots grew up on shoot regeneration medium. Finally, roots were regenerated from the shoots on Murashige and Skoog medium with high efficiency. This study firstly offers a rapid and efficient system for plantlet regeneration from leaves of H. glomeratus. Whether our protocol applies well to other tissue regeneration of H. glomeratus needs further confirmation. This work will facilitate basic research and salt-tolerant mechanisms of this important halophyte species.

In vitro micropropagation of Basilicum polystachyon (L.) Moench and identification of endogenous auxin through HPLC

An efficient plant regeneration protocol was developed for Basilicum polystachyon (L.) Moench using shoot tip from in vitro germinated plant. Both shoot multiplication and root induction were initiated from shoot tip explants in Murashige and Skoog’s (MS) basal medium supplemented with N6-benzylaminopurine (BAP) and 6-furfurylaminopurine (Kin) combination with 1-naphthaleneacetic acid (NAA) and without any plant growth regulator. Among the different concentrations and combinations of growth regulators, the highest number of shoots per explants was induced on 13.32 μM BAP with 0.53 μM NAA. It was also found that the multiplication of shoots along with roots induced in MS medium without any plant growth regulators. The in vitro grown plants were successfully hardened and acclimatized in the field with a 99% survival rate. The results obtained from HPLC analysis established the presence of a significant amount of endogenous auxin, viz. indole-3-acetic acid acid and indole-3-butyric acid in the shoot and root tips of B. polystachyon. This is the first report of a successful multiplication of B. polystachyon in absence of plant growth regulators and the presence of an abundant quantity of endogenous auxin in root and shoot tips using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) coupled with ultraviolet–visible (UV–Vis) detector. This is the first report on identification of endogenous indole-3-acetic acid and indole-3-butyric acid from the shoot and root tips of B. polystachyon (L.) Moench.

Multiple shoot induction and plant regeneration from axillary buds of Magnolia ‘Vulcan’

An efficient protocol for multiple shoot induction and plant regeneration from axillary bud culture of Magnolia ‘Vulcan’ was developed in the present study. Primary shoots were obtained from axillary bud explants cultured on Murashige and Skoog (MS) medium containing 1.0 mg/L 6-benzylaminopurine (BA). To induce multiple shoots effectively, primary shoot tips were cultured on MS medium supplemented with different concentrations of BA and zeatin at 0, 0.2, 0.5, and 1.0 mg/L. Of these treatments, the MS medium with 0.5 mg/L BA resulted in the highest number of shoots per explant with an average value of 5.9, and it produced the greatest shoot height at 4.8 cm after 12 weeks of culturing. In the rooting of in vitro produced shoots, the greatest percentage of explants forming roots (91.3%), number of roots per explant (9.7), and root length (2.8 cm) were obtained in half-strength MS medium supplemented with 6.0 mg/L indole-3-butyric acid (IBA). Regenerated plantlets were successfully acclimatized and hardened off inside the culture room with 87.5% survival rate. Plants were transferred to a greenhouse with a 97.2% survival rate. The highly efficient shoot multiplication and plant regeneration system reported herein can be used for large-scale clonal propagation of valuable Magnolia species or cultivars.

Emetine and cephaeline production and regulation by in vitro propagation of Psychotria ipecacuanha (Brot.) Stokes in semi-solid media and temporary immersion bioreactor

Psychotria ipecacuanha, is a plant species with known medicinal properties that is critically endangered due to overexploitation of natural populations. Although the difficulties in conventional propagation by seed and by vegetative propagation are generally understood, the present study enhances our knowledge by describing efficient plant regeneration and root induction protocols for P. ipecacuanha while comparing alkaloid content (emetine and cephaeline) in in vitro-derived tissues. Stem node explants were cultured on MS medium MS supplemented with indolbutiric acid (IBA) in semi-solid media and the RITA® temporary immersion bioreactor. The highest root formation (81%) was in MS + 1.5 mg L−1 IBA in the bioreactor. After 24 months of acclimatization, the plants cultivated in MS + 0.50 and 1.0 mg L-1 of IBA had the highest number of roots (3), with mean values of 10.47 and 9.40 cm, respectively. The cultures coming from 1.0 mg L−1 and 0.5 mg L−1 IBA in the bioreactor contained higher cephaeline content, with a relative area of 14.2 and 14.9%, respectively. For emetine, the 1.0 mg L−1 IBA cultures in the bioreactor, 0.5 mg L−1 IBA and MS0 cultures contained higher content than the other treatments, with a relative area of 10.2, 10.2 and 10.1%, respectively.

In Vitro Regeneration Potential of White Lupin (Lupinus albus) from Cotyledonary Nodes.

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, 1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L−1 + NAA 0.1 mg L−1) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L−1 + NAA 0.1 mg L−1 + BAP 1.67 mg L−1), and (KT 2.0 mg L−1 + NAA 0.1 mg L−1) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L−1 activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L−1 + KT 0.1 mg L−1 (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes.

Optimization of Plant Regeneration in Different Pepper (Capsicum annuum L.) Lines

Development of an efficient plant regeneration protocol is essential for vegetable crop advancement by biotechnological methods. In this study, regeneration protocols of four pepper lines of different pepper types were optimized. Different protocols for organogenesis were investigated in regeneration experiments. Optimum plant regeneration was obtained in different combinations of 6-Benzylaminopurine (BAP) and Indole-3-acetic acid (IAA) in organogenesis assays. In organogenesis experiment, the highest shoot development was determined as 80% from hypocotyl explant of Demre pepper in 4 mg L-1 BA+0.5 mg L-1 IAA, 80% from hypocotyl explant of Charleston pepper in 3 mg L-1 BA+0.5 mg L-1 IAA, 80% from hypocotyl explant of capia pepper in 5 mg L-1 BA+0.5 mg L-1 and 84% from hypocotyl explant of bell pepper in 5 mg L-1 BA+0.5 mg L-1 IAA. Afterward, shoots were rooted and whole plants were obtained.

Development of an In Vitro Propagation Protocol and a Sequence Characterized Amplified Region (SCAR) Marker of Viola serpens Wall. ex Ging.

An efficient protocol of plant regeneration through indirect organogenesis in Viola serpens was developed in the present study. Culture of leaf explants on MS (Murashige and Skoog) medium supplemented with 2.0 mg/L 6-benzyladenine and 0.13 mg/L 2,4-dichloro phenoxy acetic acid. Adventitious shoot formation was observed when calli were transferred on to MS medium containing 0.5 mg/L α-naphthalene acetic acid and 2.25 mg/L kinetin, which showed the maximum 86% shoot regeneration frequency. The highest root frequency (80.92%) with the 5.6 roots per explant and 1.87 cm root length was observed on MS medium supplemented with 2 mg/L indole-3-butyric acid. The plantlets were transferred to the mixture of sand, coffee husk and soil in the ratio of 1:2:1 in a pot, and placed under 80% shade net for one month. It was then transferred to 30% shade net for another one month, prior to transplantation in the field. These plantlets successfully acclimatized under field conditions. A Sequence Characterized Amplified Region (SCAR) marker was also developed using a 1135 bp amplicon that was obtained from RAPD (Random Amplification of Polymorphic DNA) analysis of six accessions of V. serpens. Testing of several market samples of V. serpens using the SCAR marker revealed successful identification of the genuine samples of V. serpens. This study, therefore, provides a proficient in vitro propagation protocol of V. serpens using leaf explants and a SCAR marker for the authentic identification of V. serpens. This study will be helpful for conservation of authentic V. serpens.

An effective method for Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. using cotyledon explants

ABSTRACT Jatropha curcas is one of oilseed crops and has been considered as an energy crop. In the present study, efficient plant regeneration protocol and transformation method were developed for J. curcas. Because the regeneration efficiency of adventitious bud from cotyledon explants of J. curcas induced by traditional methods is low, and it takes a long time to get complete plants. It is necessary to establish a new regeneration system to improve regeneration efficiency. Cotyledon explants were dipped into TDZ solution at different concentrations respectively for various times to obtain higher efficiency of adventitious bud regeneration. This new regeneration method was then applied to genetic transformation of J. curcas. Cotyledon explants were precultured for 1 day after treated with high concentration of Thidiazuron (TDZ) solution (20 mg/L for 40 min), followed by Agrobacterium tumefaciens infection. After co-cultured for 2 days, the explants were placed on the induction hormone-free media for bud regeneration and resistant screening. After 30 days, selected shoot buds were transferred onto elongation medium for 15 days. Young leaf sections of the regenerated shoots were used for PCR (Polymerase chain reaction) detection of the transgenic shoots. The PCR positive shoots were isolated and used for in vitro grafting. The intact plants were obtained within 20 days. GUS (β-Glucosidase) staining and Southern analysis confirmed the transformation events. Briefly, a transformation efficiency of 34.32% was achieved and an intact transgenic plant could be obtained within 65 days.

In vitro Multiple Shoot Regeneration from Petunia hybrida

An efficient plant regeneration protocol was developed from in vitro germinated seeds of Petunia hybrida an ornamentally important plant in the family Solanaceae. Shoot tip and node explants of Petunia hybrida were cultured on MS basal medium supplemented with different concentrations and combinations of Benzyl amino purine (BAP), 1-Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Gibberellic acid (GA3). The highest shoot length was obtained from MS medium supplemented with 1 mg/l BAP + 1 mg/l NAA. The highest shoot number (3 shoots/explant) were obtained from MS medium supplemented with 0.6 mg/l BAP + 0.5 mg/l IBA. The isolated shoots were transferred to MS basal medium supplemented with different concentrations of GA3 ranging from 0.05, 0.2, 0.5 and 1 mg/l for shoot elongation. The highest shoot length (5.75 cm) was recorded from the MS medium supplemented with 0.2 mg/l GA3 +0.2 mg/l BAP. Rooting of regenerated shoots were achieved on MS medium supplemented with 0.1-1 mg/1 IBA and NAA. The regenerated shoots with well developed roots were successfully acclimatized and established in pots containing sterilized peat moss and grown under laboratory conditions with 70% survival rates.

Establishment of efficient adventitious shoots induction system and ex vitro rooting in Vaccinium corymbosum (Ericaceae)

Background: The extension of the blueberry industry in China is restricted by the low performance of proliferation coefficient, transplanting survival rate and the long cycle production.Hypothesis: We hypothesized the suitable medium with optimal concentration and type combination would improve the survival rate of Vaccinium corymbosum. Species studied: Vaccinium corymbosum L. cultivar ‘O’Neal.Study site and years of study: Our study was conducted in Yunnan Breeding and Cultivation Research and Development Center of Endangered and Daodi Chinese medicinal materials, Yunnan University of Chinese Medicine since November 2015.Methods: Efficient protocol of plant regeneration about Southern highbush blueberry (cultivar ‘O’Neal) was established using annual shoots via single factor and orthogonal experiments.Results: Olive medium with 2.0 mg·L-1 zeatin was found to be most suitable for initiation culture. The highest callus induction and bud formation rate were determined with 93.67 % and 95.57 %, respectively. Furthermore, as the germination of axillary buds, numerous shoots were formed with the proliferation coefficient (> 60). Well-developed shoots were obtained using rejuvenation culture on half-strength Olive medium supplemented with combination of 2.0 mg·L-1 indole-3-butyric acid, 1.0 mg·L-1 naphthaleneacetic acid, 0.01 mg·L-1 chlormequat chloride and 0.5 g·L-1 activated charcoal. The rooting rate could reach to 100 % using 2 cm length of well-developed shoots transferred into the 5:1 sterilized peat:perlite, which was enhanced from 27.61 up to 95 % after transplanting to the field.Conclusions: An efficient system for plant regeneration from bud induction to transplanting to the field was established to maintain the characteristics of southern highbush blueberry varieties.

High frequency plant regeneration from leaf culture of Neolamarckia cadamba.

Neolamarckia cadamba is a miracle tree species with considerable economic potential uses as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish a highly efficient and robust protocol of plant regeneration for N. cadamba. Greenish callus was induced from very young leaf explants of sterile in vitro plantlets cultured on Murashige and Skoog's (MS) medium supplemented with 3 mg l-1 thidiazuron (TDZ), 0.1 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l-1 α-naphthaleneacetic acid (NAA). The callus could differentiate into nodular embryogenic structures or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. The micro-shoots developed roots in MS supplemented with 0.05 mg l-1 NAA and 0.05 mg l-1 indole-3-butyric acid (IBA), while the nodular embryogenic structures germinated directly and developed into plantlets on induction medium contained with 0.5 mg l-1 (or 1 mg l-1) 6-benzyladenine (6-BA) and 0.05 mg l-1 NAA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 92.0% survival. This regeneration protocol can be used in large scale cultivation needs and may be useful for future genetic modifications of N. cadamba.

Plant regeneration and Agrobacterium-mediated transformation of the miracle tree Neolamarckia cadamba

Neolamarckia cadamba is a miracle tree species with considerable economic potential used as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish an efficient protocol of plant regeneration and Agrobacterium-mediated genetic transformation for N. cadamba. Greenish callus were induced from different type explants when cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) and kinetin (KT). The callus could differentiate into somatic embryos or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. It was found that MS medium supplemented with 2 mg/L TDZ, 0.2 mg/L KT provided the most suitable medium for leaf explants to induce adventitious shoot, delivering a maximum regeneration frequency with a mean of 8.2 shoots per explant. The micro-shoots developed roots in MS supplemented with 0.05 mg/L α-naphthaleneacetic acid (NAA), and could be successfully acclimatized to a greenhouse with more than 90% survival. Based on this regeneration protocol, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for N. cadamba. Genetic transformation was obtained through the infection of leaf with A. tumefaciens strain GV3101 harboring a modified binary vector pCAMBIA1305 containing the marker genes β-glucuronidase (GUS) and fluorescent protein YFP. The presence of GUS in transformed plants was confirmed using PCR and histological stain, while the expression of YFP was confirmed through fluorescent microscopy and RT-PCR. Out of 276 putative transformants, 15 plants were found to be positive transgenic. Nevertheless, by adopting this protocol, positive transformed plants could be obtained within three months.