Response of in vitro strawberry to antibiotics

By identifying antibiotics that had the least phytotoxic effects on explants during genetic transformation, we evaluated the effect of various antibiotics on callus induction and morphogenesis from leaf explants and in vitro growth of Fragaria × ananassa Duch. cv. Toyonaka. Results showed that kanamycin (Kan) significantly inhibited callus induction, bud differentiation and root morphogenesis while carbenicillin (Carb), cefotaxime (Cef) and an equal concentration of Cef and Carb up to 500 mg L⁻¹ had no significant effects on callus induction and shoot growth. Kan, even at 2.5 mg L⁻¹, significantly inhibited callus induction, shoot regeneration and root formation, while no shoots regenerated at concentrations above 15 mg L⁻¹. Rooting was completely inhibited in the presence of 50 mg L⁻¹ Kan. Cef had negative effects on shoot regeneration from leaf explants and in vitro growth of strawberry. Compared to Cef, Carb at ≤300 mg L⁻¹ significantly promoted shoot and root organogenesis. However, an equal concentration of Carb plus Cef could alleviate the negative effect of Cef on strawberry. Results from relative electrolyte leakage, root and antioxidant activities, O₂^·− production rate, H₂O₂, proline and MDA contents showed that Kan, Cef and Carb caused electrolyte leakage and triggered active enzymatic processes and metabolism. This offers a possible mechanism for the inhibition or stimulation of strawberry growth caused by these antibiotics.     

壳寡糖对不结球白菜子叶离体培养再生体系的影响

以不结球白菜（Brassica campestris L．ssp．chinensis Makino）子叶为外植体,考察壳寡糖对不结球白菜子叶离体培养再生体系的影响。在添加外源激素6．BA和NAA的条件下,比较了不同浓度（0．5、1．0、2．0和10．0mg·L^-1）壳寡糖对不结球白菜子叶形成愈伤组织、再生芽和再生不定根的影响。实验结果表明,低浓度的壳寡糖能促进子叶形成愈伤组织、再生芽。壳寡糖促进子叶形成愈伤组织和再生芽的最适浓度为0．5mg·L^-1,与其他浓度壳寡糖处理组相比,该浓度壳寡糖促进了子叶愈伤组织的形成,使出愈率达到92％。此外,该浓度壳寡糖能提高子叶的芽再生频率,使再生率达到80％,同时再生芽长度、叶绿素含量及外植体总鲜重达到最大,均显著高于对照组。然而,壳寡糖对再生芽生根有抑制作用,形成的不定根数目、平均根长和最长根长度均小于对照组。     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

The role of auxins in differentiation of rice tissues culturein Vitro

Effects of various auxins on callus induction (dedifferentiation) and organ redifferentiation from the callus were studied by using various tissues of rice,Oryza sativa L. cv. Kyoto Asahi. 2,4-D, NAA and IAA were used as auxins for the test of their ability to induce callus. All of these were active. This callus induction by auxin was successful in all tissues used; seed, root, shoot nodule, anther and ovary. In all of the calluses induced by various auxins such as 2,4-D, NAA and IAA and derived from various tissues such as seed, root, shoot nodule, anther and ovary, organ redifferentiation, i.e., formation of shoots and roots was achieved by removing the auxins from the medium used for the callus calture. Cytokinins were not necessary for the organ redifferentiation in these calluses. These results suggest that auxin is the only exogenous factor that determines dedifferentiation and redifferentiation in rice plant tissues culturedin vitro.     

KT和2,4-D对知母愈伤组织再分化的影响

本文探讨KT和2,4-D组合对知母愈伤组织再分化的影响。结果表明：知母愈伤组织再生芽和促进再生苗长高以及愈伤组织增殖的最佳培养基均为MS + KT 4.0 mg/L。单独使用KT或KT与2,4-D组合均可抑制知母愈伤组织生根。     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

Ιn vitro plant regeneration from leaf explants of the cherry rootstocks CAB-6P,Gisela 6, and MxM 14 using sodium nitroprusside

Conventional multiplication of cherry (Prunus cerasus L.) rootstocks utilizes division, cuttings, and propagation through seed, which are relatively slow and labor intensive and result in genetic variability. Tissue culture, on the other hand, ensures rapid, large-scale, and low-cost production of genetically identical, physiologically uniform, and pathogen-free plants. In the cherry rootstocks CAB-6P, Gisela 6, and MxM 14, sodium nitroprusside (SNP) promoted callus induction, in vitro shoot proliferation, and rooting from leaf explants in a medium containing 17.6 μM benzyladenine and 2.68 μM α-naphthaleneacetic acid. CAB-6P explants treated with 10 μM SNP gave the maximum shoot number (5), whereas 30 μM SNP gave the longest shoots and the greatest shoot induction rate (26.67%). Best rooting was obtained with 50 μM SNP. In Gisela 6 rootstock, the shoot number (10) and shoot length (20.5 mm) were maximal in the control group without plant growth regulators. The shoot induction rate was enhanced (40%) with 40 μM SNP. SNP at 40 μM resulted in root formation, while 30 μM produced the largest callus size, and 10 μM SNP resulted in the maximum callus fresh weight. MxM 14 leaves treated with 30 μM SNP gave the maximum shoot number (3), root number (7.56), and shoot induction rate (40%), whereas 40 μM SNP gave the longest shoots (12 mm) and roots (20 mm). Best results for callus size, callus fresh weight, and callus induction rate (100%) in the CAB-6P and MxM 14 rootstocks were observed with 30 and 40 μM SNP, respectively. Rooted explants with shoots were gradually acclimatized to the external environment with a high survival percentage (85%). An efficient protocol of indirect organogenesis was established for the three cherry rootstocks using SNP.     

珍稀濒危植物蒙古扁桃的组织培养及植株再生

对珍稀濒危植物蒙古扁桃进行组织培养获得再生植株。实验结果表明,在MS培养基上蒙古扁桃幼苗茎尖,茎切段和叶片等外植体均可以脱分化形成愈伤组织,并进一步分化形成再生植株。器官的脱分化与再分化决定于培养基中的激素种类及其浓度。诱导愈伤组织形成的最适培养基为MS＋6－BA0.8mg/L NAA0.1mg/L,芽分化诱导最适培养基为MS＋6－BA0.8mg/L,诱导生根的最适培养基是MS＋IBA0.5mg/L。     

Changes in Isozyme Patterns During in Vitro Regeneration from Cotyledon Explants of Brassica Species

High frequency shoot regeneration from cotyledons excised from 4-d-old seedlings of Brassica campestris L. cv. M 27 and Brassica juncea (L.) Czern. cv. Pusabold was achieved on Murashige and Skoog's (MS) medium supplemented with 1.0 mg dm^-3 N⁶-benzyladenine (BA) and 3 % (m/v) saccharose. Rooting occurred simultaneously with shoot formation on the medium containing 1.0 mg dm^-3 BA and 0.5 mg dm^-3 1-naphthaleneacetic acid. Cultures of cotyledon, cotyledon derived non-differentiating calli and differentiated calli with shoots and/or roots were analysed at different intervals for isozyme patterns of esterase and peroxidase. With the BA-induced development of shoot and/or root from non-differentiating callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root and shoot development rather than in induction of morphogenesis in callus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biomolecular changes during in vitro organogenesis of Asteracantha longifolia (L.) Nees--a medicinal herb

High frequency plant regeneration in A. longifolia (L.) was achieved from leaf explant implanted on MS basal medium supplemented with NAA (0.5 mg/l) + BA (2.0 mg/l) through intervening callus phase. Well-developed shoots (>3cm) were successfully rooted on MS medium supplemented with NAA (0.1 mg/l). Protein and total soluble sugar contents were maximum during organogenesis and multiple shoot induction phase compared with non-organogenic callus and root induction phase. Esterase and catalase activities were maximum during organogenic differentiation, while activities were minimum at non-differentiated callus stages. Peroxidase activities were higher during rhizogenesis. Contradiction to peroxidase activity, acid phosphatase activities were high during organogenesis and declined during rhizogenesis. SDS-PAGE analysis of total soluble proteins revealed expression of non-organogenic callus (97.9 kDa), organogenic callus (77.2, 74.1, 21.9 kDa), multiple shoot induction phase (106.6, 26.9, 11.6 kDa) and root induction phase (15.9 kDa) specific polypeptides. Esterase zymogram revealed one band (Rm 0.204) appeared in both organogenic callus and multiple shoot induction phase. Peroxidase zymogram detected two stage specific bands, one band (Rm 0.42) was specific to root induction phase, while another (Rm 0.761) was specific to multiple shoot induction. Catalase and acid phosphatase zymogram resolved one band (Rm 0.752 and 0.435, respectively) in differentiated stages including both multiple shoot induction phase and root induction phase, but absent in undifferentiated phases.     

Induction of somatic embryos in pea,Pisum sativum L.

Using low concentrations of picloram (0.06 mg/l), embryoids were formed on the surface of leaf-derived callus of pea, Pisum sativum L. (c.v. Dippes Gelbe Victoria) upon transfer to liquid medium. After some days in culture, embryoids spontaneously separated from the calli, and developed into torpedo-shaped embryos, which were transferred to solid medium. In a second series of experiments, embryos were also formed by mutant 489C and a genetic line of Pisum arvense, which additionally exhibited embryogenesis also from epicotyl-derived callus. Some of the embryos showed root formation, but no shoot morphogenesis occurred. In a limited number of cases, an additional root was formed in the apparent shoot apical region after 2–5 days.     

Effects of calcium on nickel tolerance and accumulation in Alyssum species and cabbage grown in nutrient solution

Nickel (Ni) phytoextraction using hyperaccumulator plant species to accumulate Ni from mineralized and contaminated soils rich in Ni is undergoing commercial development. Serpentinite derived soils have a very low ratio of Ca/Mg among soils due the nature of the parent rock. In crop plants, soil Ca reduces Ni uptake and phytotoxicity, so it is possible that the low Ca of serpentine soils could limit hyperaccumulator plant tolerance of serpentine soils used for commercial phytomining. In this study, we investigated the effects of varied Ca concentration in the presence of high Mg characteristic of serpentine soils on Ni uptake and tolerance by serpentine-endemic species Alyssum murale Waldst. et Kit. and A. pintodasilvae T.R. Dudley in comparison with cabbage (Brassica oleracea L. var. capita) in a nutrient solution study. The levels of Ca and Mg used were based on serpentine and normal soils, and Ni was based on achieving over 1% Ni in Alyssum shoots in preliminary tests. Varied solution concentrations of Ni (31.6–1,000 μM for Alyssum, 1.0–10 μM for cabbage) and Ca (0.128–5 mM) were used in a factorial experimental design; 2 mM Mg was used to mimic serpentine soils. Alyssum spp. showed much greater tolerance to high Ni, high Mg, and low Ca solution concentrations than cabbage. For Alyssum spp., Ni induced phytotoxicity was only apparent at 1,000 μM Ni with relatively low and high Ca/Mg quotient. In the 1,000 μM Ni treatment, shoot Ni concentrations ranged from 8.18 to 22.8 g kg^?1 for A. murale and 7.60 to 16.0 g kg^?1 for A. pintodasilvae. Normal solution Ca concentrations (0.8–2 mM) gave the best yield across all Ni treatments for the Alyssum species tested. It was clear that solution Ca levels affected shoot Ni concentration, shoot yield and Ni translocation from root to shoot, but the relation was non-linear, increasing with increasing Ca up to 2 mM Ca, then declining at the highest Ca. Our results indicate that Ca addition to high Mg serpentine soils with very low Ca/Mg ratio may reduce Ni phytotoxicity and improve annual Ni phytoextraction by Alyssum hyperaccumulator species. Removal of shoot biomass in phytomining will require Ca application to maintain full yield potential.     

Enhanced shoot organogenesis in Bixa orellana L. in the presence of putrescine and silver nitrate

An efficient protocol for direct shoot organogenesis in Bixa orellana, known as achiote or annatto or Latkhan (India), has been developed. Using nodal shoot-tip explants, significant organogenetic responses, mean shoot number and shoot elongation were observed when these were incubated on Murashige and Skoog (MS) medium containing 6.66 ??M 6-benzyladenine (BA) and 4.9 ??M indole-3-butyric acid (IBA), and supplemented with either 200?C1,500 ??M putrescine or 40 ??M silver nitrate (AgNO₃). Putrescine at 800 and 1,000 ??M promoted the highest mean shoot length and mean shoot number per explant, respectively. Moreover, various concentrations of putrescine induced callus development. Incorporation of a polyamine biosynthesis inhibitor Difluro-Methyl Ornithine (DFMO) inhibited in vitro shoot multiplication and also altered the endogenous polyamine pool of B. orellana shoots. The field survival of in vitro-derived plants of putrescine and AgNO₃ treatments was 70%. This protocol can be used for improving the in vitro regeneration of B. orellana for transformation studies.     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

Haploid plants from anther cultures of poplar (Populus × beijingensis)

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial woody plants using conventional breeding techniques is currently a challenge due to a long juvenile period, high heterozygosity, and substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In the present study, we report the regeneration of haploid lines of poplar (Populus × beijingensis) via anther culture. Anthers at the uninucleate stage were induced to produce callus using three basic media. Two auxins (naphthalene acetic acid [NAA] and 2,4-dichloro-phenoxyacetic acid [2,4-D]), and two cytokinin (kinetin [KT] and 6-benzyladenine [BA]) were tested to explore the influence of plant growth regulators on callus response. H medium (Bourgin and Nitsch 1967) supplemented with 1.0 mg/L NAA and 1.0 mg/L KT induced the highest rate of callus formation. When callus obtained from anthers were subcultured in MS medium containing 1.0 mg/L BA and 0.2 mg/L NAA, followed by transfer to half-strength MS medium supplemented with indole-3-butyric acid (0.2–0.5 mg/L), the formation of regenerated plantlets increased dramatically. Inclusion of gibberellic acid (0.02–0.2 mg/L) in addition to a combination of BA (0.6 mg/L)-NAA (0.2 mg/L) in the culture medium resulted in enhanced frequency of shoot development, as well as greater internode elongation. Ploidy analysis of 580 regenerated plants, using both flow cytometry and chromosome counting, revealed 10.3 % haploid and 1.0 % triploid plantlets. The remaining plantlets were all diploid.     

植物生长调节剂对白头翁叶片培养的影响

以白头翁试管苗叶片为外植体,以MS为基础培养基,探讨不同细胞分裂素和生长素对叶片不定芽诱导、愈伤组织的诱导、增殖、再分化的影响。实验结果表明：细胞分裂素TDZ适合于白头翁叶片培养,与生长素搭配使用使叶片发生分化;2,4-D对愈伤组织的诱导能力相对较强。叶片直接诱导不定芽的激素组合为TDZ0．3mg／L＋NAA0．1mg／L;愈伤组织增殖的激素组合为TDZ0．2mg／L＋2,4-D0．2mg／L,将愈伤组织转接到TDZ和NAA组合的培养基上时会再分化。     

Induced Caulogenesis in Long-term Callus Cultures of Rosrnarinus officinalis L

The callus formed in Rosmarinus officinalis L in association with shoot tip proliferation was isolated and subjected to different treatments for good growth. Two basal media, namely, Murashige and Skoog (MS) and Schenk and Hildebrdndt (SH) and their modifications supplemented with 0.25 mg I^-1 6-benzylaminopurine (BAP), 0.5 mg I^-1 indole-3-acetic acid (IAA) and 1.0 mg I^-1 2,4-dichlorophenoxyacetic acid (2,4-D) were used. Callus in MS medium, was compact and remained fresh and green upto 30 days but grew slowly. Whereas, in SH medium callus growth was rapid but it turned brown within 15 days.The browning of callus could be checked with the addition of 1500 mg I^-1 NH,NO, to the medium, in which callus grew 15 fold in fresh weight during 21 days and remained fresh upto 45 days of incubation.The shoot buds differentiated in this somatic callus with the addition of 0.5 mg I^-1 each of BAP, 2-isopentenyl adenine (2ip), IAA and 10 mg I^-1 gibberellic acid (GA₃), within 15 days of incubation provided the callus remained floating on the liquid medium. Histological investigations revealed both peripheral and occasionally internal differentiation of shoot buds. Differentiated shoot buds were proliferated, rooted and transplanted in the soil.     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.