Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

High frequency in vitro regeneration of<Emphasis Type="Italic">Kigelia pinnata</Emphasis> L. via organogenesis

An effective and reproducible protocol for the micropropagation ofKigelia pinnata L through high frequency callus regeneration is described. Seeds were surface sterilized before culturing on Gamborgs basal medium (B5 medium). After two weeks the cotyledonary node along with a portion of the hypocotyl were carefully excised from well-developed embryos and subcultured on B5 medium supplemented with different concentrations of 2,4-D and BAR The cultured cotyledonary node expiants showed callus formation at the base of the lower cut end of the hypocotyl. This callus showed shoot initiation after two weeks of subculture on the regeneration medium supplemented with various concentrations of BAP alone or in combination with NAA. The highest number of shoot regeneration occurred on medium containing 5 γM BAP and 0.1 γM NAA. The optimum rooting of the regenerated shoots was observed on 1/2B5 medium supplemented with 4 γM IBA. Micropropagated plants were successfully established in soil in field condition with a survival frequency of 100%.     

Biochemical and histological changes associated with <Emphasis Type="Italic">in vitro</Emphasis> responses in sunflower cotyledonary explants

In vitro shoot regeneration from sunflower cotyledonary explants can be obtained in the presence of kinetin and indole-3-acetic acid. In contrast, callus proliferation is obtained in the presence of 2,4-dichlorophenoxyacetic acid on culture medium. The purpose of this study was to investigate changes in protein profiles during callus and shoot development from cotyledonary explants and to correlate them with ontogenic stages during in vitro culture. Cotyledons cultured in the presence of 2,4-dichlorophenoxyacetic acid produced friable callus as a result of early division of parenchymatic cells associated with the vascular bundles of the explant. The callogenic ability was independent of the cotyledonary region used as starting explant. Direct shoot organogenesis was observed from the same type of cells growing in culture media supplemented with kinetin and indole-3-acetic acid. In this case, the regeneration potential varied among regions from which the explants were obtained. Protein profiles revealed differences associated with shoots or callus developmental programs. A 27-kDa polypeptide was uniquely detected in the explants undergoing shoot organogenesis. The amount of this polypeptide during the first 4 d of culture increased and was followed by the appearance of meristematic centers in histologically analyzed samples. This polypeptide could be used as a specific marker for in vitro shoot development in this species.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

A rapid and efficient regeneration system for pea (Pisum sativum), suitable for transformation

A method for plant regeneration via organogenesis in pea (Pisum sativum) using nodal thin cell layer segments has been developed.From 10 to 12 days old sterile pea seedlings, nodal expiants were excised from which leaves and axillary buds were removed. Shoot regeneration was consistently obtained from liquid cultures where the expiants were floated on the medium. Shoots could be harvested after two weeks and thereafter up to ten weeks and no important effect of the cultivar (Bodil, Puget, Rondo and Trille) used could be observed as far as shooting capacity was concerned.Rooting frequency of the regenerated shoots was cultivar dependent. Plantlets were obtained within 7 weeks after expiant excision. Agrobacterium tumefaciens carrying a disarmed Tiplasmid and a binary vector containing the ß-glucuronidase reporter gene, were used in cocultivation experiments on pea nodal expiants in order to obtain transgenic shoots.Abbreviations DHZ dihydrozeatin - IBA indole-3-butyric acid - GUS ß-glucuronidase - NPTII neomycin phosphotransferase II     

A genetic analysis of cell culture traits in tomato

Summary Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

Rapid micropropagation of a tree of arid forestryAnogeissus acuminata

Multiple shoots (16–20 shoots per expiant) were induced from cotyledonary node region ofAnogeissus acuminata (Roxb. ex DC.) Guill. & Perr. on Murashige and Skoog’s (MS) medium containing IAA 0.1 mg 1^-1 + BAP 1.5 mg 1^-1 and ascorbic acid 50 mg 1^-1, citric acid 25.0 mg 1^-1, arginine 25 mg 1^-1 and adenine sulphate 25 mg 1^-1. From the first node of seedling only 4–6 shoots per expiant were proliferated. Segments ofin vitro produced shoots were used as expiants for further multiplication of shoots upto 16 successive cultures at an interval of 4 week on MS medium with IAA 0.1 mg 1^-1 + BAP 1.0 mg 1^-1 and additives. The original cotyledonary expiant was repeatedly subcultured upto 4 times after harvesting crop of shoots, each time.In vitro produced shoots were rooted on half strength MS medium containing 0.5 mg 1^-1 IBA. Plantlets were transferred to pots. Other expiants (cotyledons, hypocotyl, and leaf) produced callus on medium containing auxins and cytokinins. The calluses differentiated into embryo like structures or roots on MS medium.     

Direct and indirect regeneration of plants from internodal and leaf expiants of<Emphasis Type="Italic">Hypencum bupleuroides</Emphasis> gris

Species of the genusHypencum are of considerable interest worldwide because of their medicinal properties.In- vitro culture is a useful tool for both multiplication of the genus and studying its economically important secondary metabolites. Here, we present an effectivein- vitro propagation method forH. bupleuroides. Leaf and internodal expiants excised from 9-week-old,in vitro-germinated seedlings were cultured on a Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 1.0 or 0.1 mg L^-1) and 2,4-dichlorophenoxyacetic acid (2,4-D; 1.0 or 0.1 mg L¹). Depending on the BA and 2,4-D combination used, these cultures produced adventitious shoot buds directly on the surfaces of both types of explants as well as excessive calli. Numerous shoots were obtained when the calli from both expiant types were cultured on an MS medium supplemented with 2 mg L^-1 BA. Internodal expiants were more responsive than leaf tissues to direct and indirect plant regeneration. After shoots that regenerated from either the calli or the expiant surface were excised, rooting was best on an MS medium lacking any growth hormones. These rooted plants were then acclimatized under greenhouse conditions, and 90% of regenerants had survived. Ours is the first report ofin- vitro plant regeneration fromH. bupleuroides.     

Organogenesis andin vitro flowering ofEchinochloa colona. Effect of growth regulators and explant types

Echinochloa colona regeneration via organogenesis in callus cultures derived from leaf base and mesocotyl expiants andin vitro flowering were achived. Shoot bud regeneration was achieved on Murashige and Skoog’s (MS) basal medium supplemented with 6.66 μM 6-benzylaminopurine (BAP), 2.68 μM 1-naphthalene acetic acid (NAA) and 3 % (m/v) saccharose. Regenerated shoots were rooted on half strength basal MS medium with 2 % (m/v) saccharose devoid of growth regulators. About 90 -95 % of rooted plantlets survived in the greenhouse.In vitro flowering was induced in the regenerated shoots derived from callus on half strength MS medium supplemented with 4.4 μM BAP, 74.07 μM adeninesulphate, 0.72 μM gibberellic acid, and 3 % (m/v) saccharose. The frequency ofin vitro flowering was 80 – 90 % in three repeated experiments. Fertile seeds were recovered fromin vitro grown plantlets which were subsequently germinated into plants. Acknowledgement: The authors wish to thank to the Department of Environment and Forests, Government of India for financial assistance to undertake this investigation.     

Regeneration of plants from callus cultures of Origanum vulgare L.

Investigations were undertaken to achieve rapid multiplication and improvement of Origanum vulgare (a herbaceous, ornamental plant well known for its aromatic and medicinal value) through plant regeneration from callus. The explants (cotyledons, hypocotyl and root segments) excised from 15 d old aseptic seedlings were cultured on Gamborg's B₅ medium supplemented with 2,4-D, NAA and BAP individually and in various combinations (at concentrations of 0,10^–7,10^–6 and 10^–5 M). Best callus induction was noted on medium with 10^–7 M 2,4-D alone. The cotyledonary expiants proved to be the best source for compact and nodulated callus. The subcultured cotyledonary calli showed shoot induction when transferred onto media supplemented with BAP alone orin combination with 10^–7M or 10^–6MNAA. However, 10^–5M NAA completely suppressed the shoot inducing ability of BAP. In general, NAA promoted root induction from all explants used including cotyledonary callus. Best shoot induction was obtained on medium supplemented with 10^–6M BAP+10^–6MNAA. Both IBA and NAA at 10^–6 M proved to be equally effective in induction of roots from the cut ends of 15–20 mm long shoots (excised from callus) in half-strength B₅ liquid medium. Rooted shoots were successfully re-established in soil under controlled conditions.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine     

广藿香叶盘法高效再生体系主要影响因素的研究

广藿香是重要的芳香药用植物,利用基因工程技术对广藿香进行品种改良,需要建立一个高效的广藿香植株再生体系。该研究以广藿香无菌苗叶片为材料,将叶盘外植体分别置于不同条件下培养,观察、统计其再生植株的数量及生长状况。通过研究15~50 d的苗龄、第2~4节上的叶及培养基中2,4-D、NAA、BA和KT的浓度和配比等因素对广藿香叶盘再生植株的影响,在此基础上优化培养条件,建立广藿香高效再生体系。结果表明:广藿香无菌苗的苗龄、叶片在茎上的着生位置以及培养基中的植物生长调节物质浓度和配比都对广藿香的植株再生有显著影响;优化培养条件为以培养30 d的广藿香无菌苗顶芽下第2对展开叶片切割的叶盘为外植体,在含0.1 mg·L-1NAA和0.5 mg·L-1BA的MS培养基中培养28 d,叶盘的不定芽发生频率达到100%,单个叶盘的平均再生芽数为96.5个,经生根培养及温室炼苗,再生植株的移栽成活率达到96%。广藿香叶盘植株再生体系的建立为其基因转化研究及优良品种的快速繁育奠定了基础。     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> roxb

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microtuttings on a peat moss, soil, and compost mixture (1∶1∶1). About 52% of plantlets were successfully acclimatized and established in pots.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree <Emphasis Type="Italic">Pterocarpus marsupium</Emphasis> Roxb.

Summary A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the orginal cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1∶1∶1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.     

A protoplast to plant system for the chrysanthemum Dendranthema zawadskii x D. grandiflora

Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - MS Murashige and Skoog (1962) - NAA 1-naphthylacetic acid - Pfr Photon fluence rate - V-KM Binding and Nehls (1977)     

Sodium chloride resistant cell line from haploidDatura innoxia mill

Summary A cell line resistant to sodium chloride was selected from callus cultures of haploidDatura innoxia by cloning under selective pressure. Cells of the resistant cell line retained their resistance even after subculture in absence of NaCl. Plantlets could be regenerated from resistant cells in the presence as well as absence of NaCl. In contrast, regeneration of plantlets was not possible from normal cells in the presence of NaCl, although regeneration readily occurred in the absence of NaCl.To examine the stability of the resistance in the long-term, callus cultures were initiated in presence of NaCl from stem expiants of the differentiated plantlets. All expiants of plantlets derived from resistant cells showed callus formation. This callus, derived from resistant explants, retained the trait of resistance upon subculture.     

Competence for regeneration of cucumber cotyledons is restricted to specific developmental stages

The total duration of the plant regeneration process from cucumber (Cucumis sativus L.) cotyledonary explants was only six weeks, which included the induction of buds and their development into plants. Regeneration of shoots from cotyledons from three to five day-old seedlings ranged up to 100%. The regenerated plants were morphologically normal, flowered and set seed. The regeneration capacity of cotyledons from seven days-old and older seedlings was lowered dramatically. Most of those regenerated plants were polyploid/mixoploid and had an abnormal morphology. During seedling development (3 to 13 days), the DNA content of cotyledonary cells changed from 2C to 4C and more. The results show that the decrease in regeneration competence correspond with the change in DNA content of the cotyledonary cells.     

Genotypic variability for callus formation and plant regeneration in rice (Oryza sativa L.)

Summary Sixty rice varieties (Oryza sativa L.), belonging to three subspecies, japonica, indica and javanica (some japonicaXindica hybrids were included), were compared for their capacity for callus growth and plant regeneration. Tissue cultures initiated from mature seeds on Murashige and Skoog's (1962) medium with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were transferred to a medium containing 0.02 mg/l 2,4-D and 10 mg/l kinetin, from which plantlets were regenerated. Large variabilities in callus growth and plant regeneration potentials were revealed among the varieties tested. Most japonica varieties formed a callus that weighed more than 100 mg per seed 30 days after inoculation, and showed a relatively high regenerative potential, whereas indica varieties, japonica-indica hybrids and javanica varieties showed poor callus growth and plant regeneration, although considerable varietal variation was observed in each subspecies. The callus growth potential was not correlated with the plant regeneration potential. Histological observations revealed that the epithelium cells of the scutellum mainly proliferated to form a callus, from which shoot and root primordia were differentiated independently from each other. The shoot primordia developed into plantlets when roots were formed adventitiously. In a few cases, shoots and roots were bilaterally initiated from a single primordium.     

Regeneration of Indian ginseng plantlets from stem callus

The development of stem callus mediated plant regeneration system for Withania somnifera is described. Maximum callus proliferation was obtained on Murashige and Skoog medium supplemented with 2.26 μM 2,4-D. Three-week-old, white, friable callus was used for shoot regeneration. The maximum shoot regeneration (6.2 ± 0.34 shoots/explant) was achieved in four weeks when callus was cultured on MS medium fortified with 4.44 μM BA and 0.57 μM IAA. Regenerated shoots were excised and multiplied (8.4 ± 0.43 shoots/explant) on MS medium supplemented with 4.44 μM of BA. Multiple shoots were divided into single shoots and were rooted (5.1 ± 0.49 rootlets/shoot) on half strength MS medium supplemented with 9.84 μM of IBA. After a hardening phase of 3 weeks the plantlets were transferred to the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis,encapsulation, and plant regeneration of <Emphasis Type="Italic">Rotula aquatica</Emphasis> lour., a rare rhoeophytic woody medicinal plant

Summary Indirect somatic embryogenesis, encapsulation, and plant regeneration was achieved with the rare rhoeophytic woody medicinal plant Rotula aquatica Lour. (Boraginaceae). Friable callus developed from leaf and internode explants on Murashige and Skoog (MS) medium with 0.45 μM 2,4-dichlorophenoxyacetic, acid (2,4-D) was most effective for the induction of somatic embryos. Subculture of the callus onto half-strength MS medium with the same concentration of 2,4-D resulted in highly embryogenic callus. Suspension culture was superior to solid medium culture for somatic embryogenesis. Embryogenic callus.during subsequent transfer to suspension cultures of half-strength MS medium having 0.23 μM 2,4-D induced the highest number of somatic embryos (a mean of 25.6 embryos per 100 mg callus) and the embryos were grown up to the torpedo stage. Transfer of embryos to half-strength MS basal solid medium allowed development, of 50% of the embryos to the cotyledonary stage. Of the cotyledonary embryos, 90% underwent conversion to plantlets on the same medium. Encapsulated cotyledonary embryos exhibited 100% conversion to plantlets. Ninety-five percent of the plantlets established in field conditions survived, and were morphologically identical to the mother plant.