Low Regeneration Frequency Research Articles

Influences of Various Factors on Inducing Adventitious Shoot Regeneration From Stem Explants of Cassia Mimosoides Linn.

In the present study, an efficient plant regeneration method was established in Cassia mimosoides by using stem explants. Low regeneration frequency with poor quality advantageous shoot buds were obtained when the explants were grown only Kinetin (KN) or thidiazeuron (TDZ) enriched media. For improving the regeneration efficiency and production of good quality adventitious buds, Cu2+, Mg2+, Fe2+ and Ag+ were supplemented in the regeneration medium at different concentrations. The highest (63.96%) adventitious bud regeneration rate, the maximum number of buds (5.39) per explant and the longest regenerated buds (3.49 cm) were obtained by adding 26 mg/l Ag+ in the regeneration medium. In order to improve the rooting in shoot buds, diethyl aminoethyl hexanoate (DA-6) at different concentrations was added into rooting medium. The optimal rooting was obtained by supplementing 0.6 mg/l DA-6. The highest rooting rate (90.23%), the highest number of roots per bud (6.08) and the longest roots (4.26 cm) were developed within 20 days of culture in this medium. By applying this method culture cycle for obtaining complete regenerated plantlets was significantly shortened (in 60 days), Additional culture for proliferation and elongation of regenerated adventitious buds was not needed. Therefore, the method established in the present study was helpful to improve the efficiency of plant regeneration from stem explants in C. mimosoides. Besides the establishment of this culture method could lay a foundation for the future large-scale cultivation and genetic transformation of C. mimosoides. Bangladesh J. Bot. 51(4): 873-882, 2022 (December) Special

ФАК ТОРЫ ПРЯМОГО ПРОРАСТАНИЯ МИКРОСПОРОГЕННЫХ ЭМБРИОИДОВ BRASSICA NAPUS L.

Isolated microspore culture is the main method of producing doubled rapeseed haploids and is widely used in research institutions and commercial companies. The protocol of rapeseed embryo production is well developed and efficient for many genotypes, but some issues remain due to the low regeneration frequency of plantlets from embryos. When the standard protocol is applied, regeneration of plantlets from microspore-derived embryos usually involves a callus-forming stage followed by regeneration of adventitious shoots or secondary embryos, which prolong the period of plantlet regeneration and makes production of doubled haploids complicated. Identifying factors, which affect the frequency of direct embryo germination will increase the frequency of plantlet formation and reduce the period of DH plant production. In this work, we studied the effect of medium pH on the duration of plantlet regeneration from rapeseed microspore-derived embryos and effect of their low temperature treatment of +1 and +5℃ for 3, 6, 8, 9, 12 days in complete darkness on the embryo maturation and germination. Rising the pH of the nutrient medium from 5.8 to 6.1 increased the frequency of direct embryos germination up to 18% and the overall frequency of plantlet regeneration up to 76%. Culturing embryos at low temperatures effected the frequency of direct germination of embryos into plantlets. The maximum frequency of 44–53% direct embryo germination was observed when cultured at +1℃ for 6 and 9 days, when embryos were cultured at +5℃ the frequency of direct germination was 0–10%. In the control variant without cold treatment it was 16%.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing.

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1 (TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex (mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of mTaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, mTaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Effect of Forchlorfenuron on Somatic Embryo Proliferation and Plantlet Regeneration in Oil Palm SUP-PSU1

In vitro propagation of palm oil is still an obstacle for mass propagation due to the low plant regeneration frequency. The objective of this study was to investigate the effect of N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) on a somatic embryo (SE) proliferation and plant regeneration of oil palm SUP-PSU1. At an approximate size of 5 mm, SE from chopping with a sharp razor blade at 100 times was cultured on oil palm culture medium (OPCM) with 0.1 mg/L dicamba and different concentrations CPPU. The results showed that 0.3 mg/L CPPU gave the highest SE proliferation at 100 % and the number of SEs at 5.16 embryos/tube after 4 weeks of culture. SE was transferred to Murashige and Skoog (MS) medium supplemented with 0.2M sorbitol, a secondary somatic embryo (SSE), which was induced at 66.67 % with 21.2 embryos/tube after 8 weeks. Furthermore, cultured SSE on PGR-free MS medium for 8 weeks gave the highest germination rate at 65 %, with the number of shoots at 2.29 shoots. Thus, this study provides new information for improving the plantlet regeneration system through somatic embryogenesis in oil palm.

High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

Effect of sucrose on calus induction and green plantlet regeneration in anther culture of Indica x Indica rice

Anther culture is an important biotechnological tool. By rice anther culture homozygous pure lines in the form of doubled haploid (DH) plants can be produced within a year as compared to the long inbreeding method, which might take 8-10 years. Application of anther culture technique in breeding and genetics research is limited due to the very low regeneration frequency of anthers of rice in general, and indica cultivars in particular. Therefore, the successful use of the technique depends on the adequate production of DH plants for selection and field evaluation. Effect of various concentrations of sucrose in callus induction media was investigated on callus induction and regeneration of green plantlets from anther culture of several F1s derived from indica x indica crosses. Cold pretreated anthers of five genotype of anther donor plants (F1s) at 5 °C for 8 days were cultured on N6 callus induction medium containing sucrose at a concentration of 6.0%, 6.5%, and 7.0%. Results revealed that all genotypes showed similar response to callus induction, but a significantly different responses to plant regeneration depended on sucrose level in the callus induction media. Two genotypes, i.e. IR78788 / Inpara 5 and Dendang/Inpari 30 only regenerated albino plantlets. In this experiment, 6.5% sucrose compared to two other sucrose treatments was suitable for inducing high frequency callus induction and high green plant regeneration in all genotypes. The high concentration of sucrose (7.0%) in the culture medium not only resulted in a decrease in the number and percentage of callus formation but also decreased the regeneration of green plantlets.

Efficient invitro plant regeneration from cotyledon-derived callus cultures of sesame (Sesamum indicum L.) and genetic analysis of True-to-Type regenerants using RAPD and SSR markers

Sesame (Sesamum indicum L.) is an important oilseed crop which is rich in unsaturated fatty acids, proteins and natural antioxidants. Reports on tissue culture methods involving regeneration through callus phase are scanty, genotype dependent and have been reported with low regeneration frequency. This recalcitrant nature will impede the application of modern biotechnological tools for genetic advancement studies in sesame. An efficient protocol for invitro shoot organogenesis from callus cultures has been established using cotyledon explants excised from 1-week-old seedlings of sesame cv. TMV 3. Maximum frequency of greenish white friable compact (GWFC) callus (72.0%) was obtained on Murashige and Skoog (MS) medium supplemented with 4.54 μM of 2, 4-dichlorophenoxyacetic acid (2, 4-D). Organogenic GWFC callus cultured on MS medium containing 4.4 μM of 6-benzylaminopurine (BAP) and 10 μM of silver nitrate (AgNO3) showed the optimum frequency of shoot regeneration (66%) and produced 4.6 shoots per 150 mg callus. Optimum rooting of 70.2%, an average of 6.02 ± 0.8 roots per shoot was achieved on MS medium fortified with 5.6 μM of indole-3-acetic acid (IAA). The rooted plantlets were acclimatized successfully in a greenhouse with a 65.8% survival rate. Non significant phenotypic aberrations were observed among the ex vitro transferred plantlets. The genetic stability of regenerants was assessed by using Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) markers. PCR amplification using 10 RAPD and 10 SSR primers generated 71 distinct and scorable DNA bands. DNA banding patterns reveal the absence of somaclonal variation among the plantlets regenerated through indirect organogenesis compared to that of mother plant and confirms the genetic purity of the invitro raised plants. The developed protocol could be effectively employed for obtaining genetic traits invitro through genetic transformation studies.

Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos

BackgroundSorghum (Sorghum bicolor L.) is one of the world’s most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency.ResultsWe report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5–2.0 mm in length) isolated 12–15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt-II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs.ConclusionsThis protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

In Vitro Regeneration of Iranian Melon (Cucumis Melo L. ‘Samsoori’) Using Antibiotic and Benzyl adenine Micropropagation of Cucumis Melo L. ‘Samsoori’

Cucumis melo L. is one of the most economically important horticultural crops worldwide. However, low plant regeneration frequency of this plant during genetic transformation is the major hurdle for applying biotechnological approaches. Hence, this study aimed to evaluate the effect of 6-benzyladenine (BA), cefotaxime (CTX), kanamycin (KAN), and indole-3-acetic acid (IAA) on the regeneration of cotyledonary petioles generated from 6-day-old in vitro grown seedlings. Results showed that application of 1.5 mgl-1 BA plus 250 mgl-1 CTX and 1 mgl-1 BA with 1000 mgl-1 CTX formed the most efficient media for plant regeneration. The highest callus production was recorded on medium containing 1 mgl-1 BA with 250 mgl-1 CTX and 1.5 mgl-1 BA with 750 mgl-1 CTX. Medium containing 500 mgl-1 CTX plus 0.1 mgl-1 IAA efficiently induced both root and leaf formation. All regenerated plants were died by adding 100 mgl-1 kanamycin therefore this level considered as threshold level for kanamycin application.. Overall, the results indicated that presence of BA plays an essential role for melon regeneration and cefotaxime can be considered as an auxiliary agent.

Enhancement of Regeneration Efficiency through Callus Induction Media Using 2,4-Dicholorophenoxyacetic acid in Indica Rice (Oryza sativa L. var. Ciherang)

Indica rice variety Ciherang is the most planted variety in Indonesia, but the micropropagation technique is restricted because it is known has low regeneration frequency and included as recalcitrant cultivar for tissue culture and tranformation activities. One of solution to resolve that problem is developing a technique of somatic embryogenesis in callus of ciherang rice cultivar. The aims of study were to determine medium composition for inducting embryogenic callus in Ciherang rice and to know the effectivity of rice regeneration using callus as explant. The methods were included induction of embryogenic callus in callus induction media (CIM) containing MS basal, Proline 600 mg/l, Casein Hidrolisat 300 mg/l, phytagel 2,5 g/l, BAP 0.25 mg/l, sukrosa 30 %, and 2,4-D in different concentration, from 2,4-D 0 mg/l as control (CIM 1), 2,4-D 2 mg/l (CIM 2), 2,4-D 3 mg/l (CIM 3), and 2,4-D 4 mg/l (CIM 4). It then be continued to regenerate the calli in RM 1 medium containing MS basal, NAA 0,2 mg/l, Kinetin 2 mg/l, Agarose 10 g/l, and sukrosa 30 %, pH 5,8 for six days in dark and RM2 medium containing MS basal , NAA 0,2 mg/l, Kinetin 2 mg/l, Agarose 8 g/l, sukrosa 30 %, pH 5,8 in light room. Results showed CIM 4 medium using 2,4-D 4 mg/l gave optimum result in calli induction with procentage 57,63% and CIM 3 using 2,4-D 3 mg/l gave optimum result in embryonic calli induction with procentage 53,63%. Result of embryonic calli gave regeneration frequency procentage is 9,6%. The total planlet obtained after five weeks old in regeneration medum were 11 planlets ready for aclimatization.&#x0D; Keywords: Somatic embryo, Indica rice (Oryza sativa L. var. Ciherang), Callus&#x0D;

Efficient culture protocol for plant regeneration from cotyledonary petiole explants of Jatropha curcas L.

ABSTRACTA high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from cotyledonary petiole explants of Jatropha curcas L. has been developed. The cotyledonary petiole explants of J. curcas cultured directly on medium supplemented with thidiazuron (TDZ) induce regeneration of poor quality shoot buds that have a low regeneration frequency. However, treating the explants with high concentrations (10–60 mg/L) of TDZ solution for certain time periods (5–80 min) significantly increased the regeneration frequency and improved the quality of the regenerated shoot buds. The best shoot buds induction (88.42%) and number of shoot buds (12.67) per explant were observed when in vitro explants were treated with 20 mg/L TDZ solution for 20 min before being transferred on hormone-free medium after 30 days. Regeneration was also influenced by the orientation (horizontal or vertical) of the explants on the medium, and by the origin of the cotyledonary petioles (in vitro or in vivo) used for the preparation of explants. We performed subsequent experiments for elongation and rooting of the regenerated shoot buds. Addition of L-arginine to the medium was conducive to the elongation of the shoot buds. A concentration of 7.5 mg/L L-arginine yielded the best results. The elongated shoots could initiate roots to become intact plantlets in half-strength Murashige and Skoog medium containing 0.1 mg/L indole-3-butyric acid. After acclimatization, these plantlets could be transplanted to the soil and the growth was normal. Therefore, application of the methods described here helped to increase plant regeneration efficiency.

Pupae are excellent explants with low microbial contamination and high regeneration frequency for micropropagation of Freesia ×hybrida Bailey 'Argenta'

Two separate factorial experiments were conducted to study the effects of explant sources, plant growth regulators, sucrose concentrations, and light conditions on in vitro cormlet formation of freesia (Freesia ×hybrida Bailey 'Argenta'). Interestingly, it was observed that the pupae had lower contamination levels compared to mother corms. Using 40% sodium hypochlorite solution for 40 min, contamination levels of pupae and mother corms reduced to 19.80 and 46.40%, respectively. Moreover, pupae showed the highest regeneration frequency. In the first experiment, 6.67 cormlets were directly produced per pupa (cold storage-produced corm) on Murashige and Skoog (MS) medium supplemented with 6 mg L-1 1-naphthaleneacetic acid (NAA), 1 mg L-1 6-benzylaminopurine (BA), and 60 g L-1 sucrose, when cultures were stored in the dark. In the second experiment, on average, 5.67 shoots were proliferated per pupa explant in the presence of 4 mg L-1 BA and 2 mg L-1 Kinetin (Kin). Subculturing these shoots on MS medium containing 3 mg L-1 BA and 0.5 mg L-1 NAA led to production of 3.67 cormlets per shoot. Finally, in vitro derived cormlets showed the highest percentage of rooting (77.80%), root number (8.33), and root length (2.13 cm) on MS medium containing 1 mg L-1 indole-3-butyric acid (IBA).

A robust tissue culture system for sorghum [Sorghum bicolor (L.) Moench

Sorghum tissue culture has been challenged by three predominant obstacles for decades, namely toxic pigments (phenolics), low regeneration frequencies and short duration of callus regenerability. Here, we report a robust tissue culture system for sorghum, which has minimized these major impediments. To optimize media, different concentrations of various plant growth regulators, such as 2,4-dichlorophenoxyacetic acid (2,4-D), N6-benzyladenine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) were evaluated. Additional ingredients, including KH2PO4, CuSO4·5H2O, l-asparagine, l-proline and polyvinylpyrrolidone (PVP) were also assessed. Results showed that callus age had a conspicuous effect on its growth and regenerability, with callus weekly growth ratio and regenerability peaked at two weeks after induction. A callus induction rate up to 100% was achieved in inbred line Tx430, whereas regeneration rates up to 100% were obtained from SA281 and 91419R. This highly efficient system has been utilized for sorghum transformation for several years and has been proven to be reliable and reproducible.

Functional roles of three cutin biosynthetic acyltransferases in cytokinin responses and skotomorphogenesis.

Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an Arabidopsis mutant named grow fast on cytokinins 1 (gfc1), whose seedlings grew larger aerial parts on MS medium with CK. gfc1 is allelic to a previously reported cutin mutant defective in cuticular ridges (dcr). GFC1/DCR encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity in vitro and is necessary for normal cuticle formation on epidermis in vivo. Here we show that gfc1 was a CK-insensitive mutant, as revealed by its low regeneration frequency in vitro and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, gfc1 had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A Arabidopsis Response Regulator (ARR) genes were higher in the gfc1 mutant. The gfc1-associated phenotypes were also observed in the cutin-deficient glycerol-3-phosphate acyltransferase 4/8 (gpat4/8) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes GPAT4 and GPAT8, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant cytochrome p450, family 86, subfamily A, polypeptide 2/aberrant induction of type three 1 (cyp86A2/att1), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in Arabidopsis.

Somatic embryogenesis and genetic uniformity of regenerated cassava plants from low-temperature preserved secondary somatic cotyledons

Low plant regeneration and transformation frequencies in cassava have been attributed to recycling of cassava secondary somatic embryos for long periods. This study examined the suitability of low-temperature storage of dehydrated cassava secondary somatic embryos as an alternative short-term conservation approach to recycling. The study included two experiments: in the first one, somatic embryogenesis from cotyledons of dehydrated secondary somatic embryos stored at six temperatures for 4, 8 and 12 months was established; and in the second, plant regeneration via shoot organogenesis using somatic cotyledon explants obtained in the first experiment was examined. The growth, photosynthetic pigment contents and genetic stability of the regenerated plants were assessed. At each storage temperature, the survival of explants, somatic embryogenesis, shoot induction and elongation decreased with increases in storage period. At 4 months after storage (MAS), explant survival, frequency of somatic embryogenesis, shoot induction, elongation and rooting had the best responses at 16EC and 20EC. Similarly, the growth and photosynthetic pigment contents of plants regenerated from somatic embryos preserved at 16EC and 20EC recorded the best results at 4 MAS. However, at 8 and 12 MAS, the best explant survival, somatic embryogenesis, shoot induction and elongation were recorded at 16EC. Eight RAPD primers produced a total of 56 distinct and scorable bands, ranging from 250 bp to 3000 bp in size. An random amplified DNA (RAPD) analysis showed a uniform banding profile among regenerants and to that of the mother plant, indicating no genetic variation among regenerants and between the regenerants and the mother plant. The data presented here suggest that cassava secondary somatic embryos dehydrated to 50% moisture content should be preserved at 16-20EC for 4 months; for 8 and 12 months storage, the embryos should be stored at 16EC. No genetic alteration was detected among the regenerants. Therefore, the method is a suitable means for short-term conservation of cassava secondary somatic embryos.

High–frequency in vitro plantlet regeneration from apical bud as a novel explant of Carum copticum L.

To develop an in vitro regeneration system to increase the recovery of Carum copticum L. plantlets as a part of developing a metabolic engineering program. The efficacy of different concentrations and combinations of 6-benzyladenine, indole-3-acetic acid and indole butyric acid on direct shoot regeneration and rooting of ajowan from apical bud explants were assessed. All explants were cultured on Murashige and Skoog (MS) medium supplemented with different combinations of 6-benzyl amino purine (BAP) (0, 2.2, 4.4, 8.8 µmol/L) and indole-3-acetic acid (IAA) (0, 0.5, 1.1, 2.2 µmol/L). The maximum shoot regeneration frequency (97.5%) and the highest number of shoots produced from apical buds (34 shoots per explant) were obtained on MS medium fortified with BAP (4.4 µmol/L) and IAA (0.5 µmol/L). Low shoot regeneration frequency was observed in BAP free treatments. The effects of different strengths of MS medium and various concentrations of IAA and indole-3- butyric acid on rooting rate, length and average number of roots were also investigated. Application of indole-3- butyric acid (6 µmol/L) in full-strength MS medium, was more effective than IAA and resulted in highest shoot regeneration frequency with the rooting rate of 100% and highest mean number of roots per shoot (41.8). The rooted plantlets were acclimatized successfully in greenhouse conditions with a survival rate of 90%. In this study, a simple and reliable regeneration and acclimatization protocol for Carum copticum has been presented. This protocol can be found very advantageous for a variety of purposes, including mass multiplication of Carum species, medicinal plant breeding studies and transgenic plant production.

PmTCP1 encodes a putative TCP transcription factor and is differentially expressed during in vitro organogenesis in Passiflora

The genus Passiflora includes economically important passion fruits and over 600 other wild species. Micropropagation of Passiflora species is far from routine due to low regeneration frequencies and multiplication rates, so there is great interest in understanding the molecular control of the in vitro regeneration processes of these species. Here, we report the characterization of a Passiflora morifolia gene encoding PmTCP1, a putative TCP transcription factor that showed high sequence similarity to Arabidopsis class I TCPs. The expression patterns of the PmTCP1 gene during in vitro organogenesis and callus growth revealed differential expression of PmTCP1 modulated by different combinations of auxin and cytokinin concentrations in the culture medium. At a constant auxin concentration, cytokinin increased PmTCP1 expression, and at a constant cytokinin concentration, auxin repressed PmTCP1 expression. We also observed a correlation between PmTCP1 expression and the in vitro organogenesis of roots and shoots. We expect that these results will increase our understanding of the molecular networks and environmental signals that modulate the processes of organogenesis during plant development.

EFFECT OF SINGLE-WALL CARBON NANOTUBE ON SOYBEAN (GLYCINE MAX) REGENERATION FROM MATURE COTYLEDONARY NODE EXPLANTS

One of the major limitations in producing transgenic soybeans using the agrobacterium-mediated cotyledonary-node method is low regeneration frequency. An improved highly efficient regeneration system of soybean was established herein. Cotyledonary node explants were placed in shoot initiation medium with single-wall carbon nanotube (SWNT) for adventitious shoots regeneration, and adventitious shoots were subcultured in shoot elongation medium with SWNT for shoot elongation and rooting. 40 mg/L SWNT supplemented in shoot initiation medium was found to be the optimal concentration with shoots regeneration frequency significantly increased by 21.5% compared with the control treatment, while for 4 mg/L and 400 mg/L, the increase was 4.6% and 6.5%, respectively. Faster elongation and rooting of adventitious shoots was observed in shoot elongation medium with 40 mg/L SWNT. Soybean plantlet formation frequency within the limited four weeks supplemented with 40 mg/L SWNT reached 48.4% while in the other three treatments: 4 mg/L, 400 mg/L and control, 0% were observed. These results indicate that supplement of SWNT in the soybean medium can efficiently promote adventitious shoots formation frequency, increase plantlet formation frequency and shorten the regeneration period.

The influence of ethylene and ethylene modulators on shoot organogenesis in tomato

The influence of ethylene and ethylene modulators on the in vitro organogenesis of tomato was studied using a highly regenerating accession of the wild tomato Solanum pennellii and an F1 plant resulting from a cross between Solanum pennellii and Solanum lycopersicum cv. Anl27, which is known to have a low regeneration frequency. Four ethylene-modulating compounds, each at four levels, were used, namely: cobalt chloride (CoCl2), which inhibits the production of ethylene; AgNO3 (SN), which inhibits ethylene action; and Ethephon and the precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which both promote ethylene synthesis. Leaf explants of each genotype were incubated on shoot induction medium supplemented with each of these compounds at 0, 10 or 15 days following bud induction. The results obtained in our assays indicate that ethylene has a significant influence on tomato organogenesis. Concentrations of ethylene lower than the optimum (according to genotype) at the beginning of the culture may decrease the percentage of explants with buds (B), produce a delay in their appearance, or indeed inhibit bud formation. This was observed in S. pennellii and the F1 explants cultured on media with SN (5.8–58.0 μM) as well as in the F1 explants cultured on medium with 21.0 μM CoCl2. The percentage of explants with shoots (R) and the mean number of shoots per explant with shoots (PR) also diminished in media that contained SN. Shoots isolated from these explants were less developed compared to those isolated from control explants. On the other hand, ethylene supplementation may contribute to enhancing shoot development. The number of isolable shoots from S. pennellii explants doubled in media with ACC (9.8–98.0 μM). Shoots isolated from explants treated with ethylene releasing compounds showed a higher number of nodes when ACC and Ethephon were added at 10 days (in F1 explants) or at 15 days (in S. pennellii) after the beginning of culture. Thus, the importance of studying not only the concentration but also the timing of the application of regulators when developing regeneration protocols has been made manifest. An excess of ethylene supplementation may produce an inhibitory effect, as was observed when using Ethephon (17.2–69.0 μM). These results show the involvement of ethylene in tomato organogenesis and lead us to believe that ethylene supplementation may contribute to enhancing regeneration and shoot development in tomato.

Androgenesis-inducing stress treatments change phytohormone levels in anthers of three legume species (Fabaceae)

Legumes are recalcitrant to androgenesis and induction protocols were only recently developed for pea (Pisum sativum L.) and chickpea (Cicer arietinum L.), albeit with low regeneration frequencies. Androgenesis is thought to be mediated through abscisic acid (ABA) but other phytohormones, such as auxins, cytokinins, and gibberellins, have also been implicated. In view of improving induction protocols, the hormone content of pea, chickpea, and lentil anthers was measured after exposure to cold, centrifugation, electroporation, sonication, osmotic shock, or various combinations thereof using an analytical mass spectrometer. Indole-3-acetic acid (IAA) had a key function during the induction process. In pea, high concentrations of IAA-asparagine (IAA-Asp), a putative IAA metabolite, accumulated during the application of the different stresses. In chickpea, the IAA-Asp concentration increased 30-fold compared to pea but only during the osmotic shock treatment and likely as a result of the presence of exogenous IAA in the medium. In contrast, no treatment in lentil (Lens culinaris) invoked such an increase in IAA-Asp content. Of the various cytokinins monitored, only cis zeatin riboside increased after centrifugation and electroporation in pea and possibly chickpea. No bioactive gibberellins were detected in any species investigated, indicating that this hormone group is likely not linked to androgenesis in legumes. In contrast to the other stresses, osmotic shock treatment caused a reduction in the levels of all hormones analyzed, with the exception of IAA-Asp in chickpea. A short period of low hormone content might be a necessary transition phase for androgenesis induction of legumes. Five androgenesis-inducing stress treatments changed content of ABA, auxin and cytokinin in anthers of three legumes. Osmotic shock treatment differed because it reduced hormone content to very low levels.