Development of secondary inflorescences and in vitro plantlets from inflorescence cultures of Amaranthus paniculatus

Immature inflorescences of Amaranthus paniculatus were used as explants for in vitro culture studies. When placed on a medium supplemented with 3–6 mg/l kinetin, explants developed into secondary inflorescences. Leaves and shoots developed following culture of inflorescence tissue on media containing 8–15 mg/l kinetin or 5–10 mg/l BAP. These shoots when subcultured on MS medium supplemented with 12 mg/l kinetin + 15% coconut milk, formed roots. These rooted plantlets later flowered in vitro.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine - Kn 6-furfurylaminopurine - CM coconut milk     

Plant regeneration from tissue cultures initiated from immature inflorescences of a grass,Echinochloa colonum (L.) link

Organised structures develop on a white and compact callus initiated from small segments of immature inflorescences ofEchinochloa colonum cultured on Murashige and Skoog's (MS) medium supplemented with 5.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 10% coconut milk. These develop into plantlets upon subculture onto MS medium containing 0 or 0.2 mg/l 2,4-D. Twelve out of 17 plantlets regenerated grew well on transfer to soil and eleven plants produced seeds.     

唐菖蒲花色突变株开花后子房组织培养与植株再生的研究

以唐菖蒲花色突变株开花后的子房为外植体,接种于MS+1.0mg/L BA+0.01-0.1mg/L NAA或MS+1.0mg/LBA+1.0mg/L KT+0.01-0.1mg/LNAA的培养基中,从膨大的组织表面直接诱导不定芽.膨大的组织或带不定芽的组织每隔周转入1/2MS_1.0mg/L BA+0.01-0.1mg/L ANN培养基中,不断地诱导产生不定芽,分化的不定芽转入不含激素的1/2MS培养基中,形成幼苗、然后生根,最后可形成小球茎.幼苗转入1/2MS+0.1mg/L NAA培养基中,幼苗生根,再可形成小球茎.1个唐葛蒲突变株子房,经6—7个月培养,获得了上千株试管苗.     

Plants obtained from the Khair tree (Acacia catechu Willd.) using mature nodal segments

An in vitro method for obtaining plants of Acacia catechu has been developed using nodal explants from mature `elite' trees growing in the field. Maximum shoot bud development (eight to ten) from a single explant was achieved on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) (4.0 mg/l) and α-naphthaleneacetic acid (0.5 mg/l). Addition of adenine sulphate (25.0 mg/l), ascorbic acid (20.0 mg/l) and glutamine (150.0 mg/l) to the medium was found beneficial for maximum shoot bud induction. The shoot buds developed into healthy and sturdy shoots on MS medium containing BAP and kinetin at 1.0 mg/l. Excised shoots were rooted on 1/4-strength MS medium with indole-3-acetic acid at 3.0 mg/l and 1.5% sucrose to obtain complete plants. Received: 17 June 1997 / Revision received: 11 September 1997 / Accepted: 27 September 1997     

In vitro propagation of Allium tuberosum Rottl. ex. Spreng. by shoot proliferation

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg11). Ia vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Plant regeneration from mesophyll protoplast culture of cabbage (Brassica oleracea var ‘capitata’)

Summary Protoplasts were enzymatically isolated from the first leaves of cabbage (Brassica oleracea var capitata, F1 hybrid Baochun). Sustained cell division and somatic embryogenesis were obtained after culturing the protoplasts in modified liquid DPD medium supplemented with CaCl₂ · 2H₂O 800 mg/l, 2,4-D 0.5 mg/l, kinetin 1 mg/l, 0.3 M mannitol and sucrose 20 g/l. Upon transferring cell colonies onto a modified Murashige and Skoog (MS) agar medium, small calli were gradually formed. Callus proliferated on MS medium supplemented with hormone combinations of 2,4-D 0.1–0.5 mg/l and kinetin 3–4 mg/l. Multiple shoots were induced on differentiation medium supplemented with 3 mg/l of kinetin and 0.1 mg/l of gibberellic acid GA₃. After transferring differentiated shoots onto MS medium supplemented with indoleacetic acid (IAA), kinetin, GA₃ at 0.1 mg/l each and 500 mg/l of N.Z. amine, intact plants were eventually produced.     

Somatic embryogenesis and plant regeneration in diploid Allium fistulosum × A. cepa F1 hybrid onions

Procedures were developed for disinfestation of non-dormant basal plate tissue excised from field grown basal plate tissue of diploid Allium fistulosum × A. cepa F₁ hybrid onions. Contamination levels varied with the season and vegetative development of plant material. Callus initiated from basal plate tissue and immature inflorescences of the F₁ hybrids was maintained on a BDS-based medium containing 0.75 mg/l picloram and 2.0 mg/l BA. When this medium was supplemented with vitamins and glycine, and with proline at 2.5 gm/1, somatic embryos began to form. Their development continued on a BDS-based shoot promotion medium containing 0.03 mg/l picloram and 0.32 mg/l 2iP supplemented with vitamins, glycine and proline. Genotypes differed significantly in the numbers of structures regenerated. Plantlets from somatic embryos were rooted into BDS or half-strength BDS medium without growth substances and were successfully transferred to sterilized potting mix in plastic commercial corsage boxes.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - 2iP N6-(2-isopentenyl)adenine - NAA 1-naphthylacetic acid - BDS Gamborg's B5 medium modified by Dunstan and Short (1977a)     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg/l). In vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.Abbreviations B5 Gamborg et al. (1968) medium - BAP 6-benzylaminopurine - 2ip 2isopentenyladenine - NAA 1-naphthalene acetic acid     

Callus-mediated organogenesis and effect of growth regulators on production of different valepotriates in Indian valerian (Valeriana jatamansi Jones.)

A reproducible and efficient callus-mediated shoot regeneration system was developed for the large-scale production of Valeriana jatamansi Jones., a highly medicinal plant species of global pharmaceutical importance. Effect of Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) on callus induction and production of valepotriates accumulation was studied by using different explants. In V. jatamansi, the degree of callus induction varied significantly depending on explants type and the growth regulators used. Among different explants used, rhizomes have the highest callus induction potential followed by leaf. The callus induction frequency was found to be optimum in rhizome explants on media supplemented with 0.5 mg/l 2,4-D. The regenerative ability of proliferated compact calli was studied by the application of cytokinins alone and in combination with auxin. MS medium fortified with 0.75 mg/l thidiazuron in combination with 0.5 mg/l NAA showed the highest regeneration frequency (88.6 %) and produced the maximum number of shoot buds (15.20 ± 0.20) capable of growing into single plants. Vigorous callus obtained from MS medium supplemented with different concentrations of 2,4-D, NAA and IBA were used for industrially important valepotriates [acevaltrate (ACE), valtrate (VAL) and didrovaltrate (DID)] analysis. High performance liquid chromatography analysis of callus revealed that medium with 2,4-D (1 mg/l) was found responsible for increasing ACE and DID yield, whereas VAL production was higher in case of medium supplemented with NAA (1 mg/l). However, the accumulation of valepotriates in callus decreased in logarithmic phase after 8 weeks. IBA was not beneficial for the valepotriate synthesis, as it helped to accumulate significantly lower concentration of ACE, VAL and DID. Micropropagated plantlets with well-developed root system were successfully acclimatized in greenhouse condition, in root trainers containing garden soil with a survival frequency of 100 %. As Indian valerian is a highly traded medicinal plant due to extensive use of its industrially important secondary metabolites, the present system can be utilized to obtain mass multiplication of the species as well as for the stable biomass and continuous valepotriate production for the pharmaceutical industries.     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION IN TISSUE CULTURES OF PANICUM MAXIMUM JACQ.

Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.     

Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences

Summary Transgenic sorghum plants (Sorghum bicolor L. Moench, cv. SRN39) were obtained by microprojectile-mediated DNA delivery (Bio-Rad PDS 1000/He Biolistic Delivery System) to explants derived from immature inflorescences. Explants were precultured on medium supplemented with 2.5 mg/l (11.31 μM) 2,4-D, 0.5 mg/l (2.32 μM) kinetin, and 60 g/l sucrose for 1 to 2 wk prior to bombardment. Bialaphos selectron pressure was imposed 2 wk after bombardment and maintained throughout all the culture stages leading to plant regeneration. More than 2500 explants from 1.5 to 3.0 cm inflorescences were bombarded and subjected to bialaphos selection. Out of more than 190 regenerated plants, 5 were determined to be Ignite resistant. Southern analyses confirmed the likelihood that the 5 herbicide resistant plants derived from two independent transformation events. The phosphinothricin acetyltransferase gene (bar) was inherited by and functionally expressed in T₁ progeny. However, no β-glucuronidase (GUS) activity could be detected in T₁ plants that contained uidA restriction fragments. Histological analyses indicated that in the absence of bialaphos morphogenesis was primarily via embryogenesis while organogenesis was more predominant in callus maintained with herbicide selection.     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64

Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.     

荷兰鸢尾(Iris xiphium L. var. hybridum)的组织培养

取荷兰鸢尾（ＩｒｉｓｘｉｐｈｉｕｍＬ．ｖａｒ．ｈｙｂｒｉｄｕｍ）鳞茎片不同部位外植体块,接种于附加不同激素配比的基本培养基上,其中取自鳞茎片基部外植体块２ｍｍ×２ｍｍ×２ｍｍ,培养基为ＭＳ＋ＢＡ１．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ的不定芽诱导率为最高（７０％）;最理想的增殖培养基为ＭＳ＋ＢＡ２．０ｍｇ／Ｌ＋ＮＡＡ０．２ｍｇ／Ｌ。不定芽直径４～５ｍｍ,培养基为ＭＳ＋ＢＡ０．２ｍｇ／Ｌ＋ＮＡＡ０．５ｍｇ／Ｌ有利于不定根的发生,诱导生根率达８３３％。试管苗不经练苗可直接出瓶,移栽于泥炭∶田土∶河沙＝１∶１∶１（Ｖ／Ｖ）的基质中。     

Shoot Regeneration from Cultured Leaf Explants of Lycium barbarumand Agrobacterium-Mediated Transformation1

A method for fast plant regeneration via organogenesis directly from Lycium barbarumleaf explants has been developed. The key factor for shoot regeneration was the presence of benzyladenine (BA) in the medium. NAA could only induce root formation and explant callusing. Murashige and Skoog (MS) medium supplemented with 2 mg/l BA and 0.5 mg/l NAA is the most efficient condition for shoot formation, with up to 92.6% shoot regeneration and no callus formation. All adventitious shoots cultured on MS medium supplemented with 1 mg/l IAA formed an extensive root system. Regenerated plants were morphologically normal and were also proved to be diploid (2n = 24). Using the optimized regeneration system, the genetic transformation of L. barbarumwas carried out mediated by Agrobacterium tumefaciensEHA101(pIG121Hm). 11.8% leaf explants produced kanamycin-resistant shoots after infection by A. tumefaciens.The putative transgenic nature of plants was confirmed by GUS assay and PCR analysis. Expression of the nptIIgene in the regenerated plants was also detected by observing the callus formation by leaf pieces on MS medium containing 0.2 mg/l 2,4-D and 0–100 mg/l kanamycin.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

Micropropagation of 21 species of Mexican cacti by axillary proliferation

Summary We have developed micropropagation systems for 21 species of Mexican cacti using explants from seedlings germinatedin vitro or shoot segments of juvenile 2–3-yr-old greenhouse plants. The species propagated belong to the generaAstrophytum, Cephalocereus, Coryphantha, Echinocactus, Echinocereus, Echinofossulocactus, Ferocactus, Mammillaria, Nyctocereus, andStenocactus. Multiple shoot formation from areoles was achieved in Murashige and Skoog (MS) medium supplemented with either 1 or 2 mg N⁶-benzyladenine (BA) per 1 (4.44 or 8.87 μM) or BA at 1 or 2 mg/l plus naphthaleneacetic acid at 0.1 or 1 mg/l (0.54 or 5.37 μM). The requirements of growth regulators for optimal shoot proliferation, the velocity of the response, and the number of buds produced by explant were different among the genera and species studied. Rooting of the shoots generatedin vitro was achieved in MS medium supplemented with indoleacetic acid at 0.5–1 mg/l (2.85–5.71 μM) or indolebutyric acid at 0.5–1 mg/l (2.46–4.90 μM). Finally, 70–95% of the rooted plants transferred to potting medium survived.     

Direct somatic embryogenesis and plant regeneration from single cell suspension cultures of Elymus giganteus Vahl

Summary A yellowish, nodular callus was induced from mature embryos of Elymus giganteus Vahl on MS medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l kinetin, from which a cell suspension culture was initiated in liquid MS medium supplemented with 0.5 mg/l 2,4-D, 1.0 mg/l kinetin and 0.2 mg/1 naphthaleneacetic acid (NAA). By filtering through a series of sieves with decreasing mesh sizes and collecting the resultant filtrate, a suspension culture composed mainly of single embryogenic cells was established. In a medium containing 0.3 mg/l 2,4-D, 1.0 mg/l 6-benzylaminopurine (6-BAP) and 500 mg/l casein hydrolysate (CH), the single cells underwent direct somatic embryogenesis resulting in the formation of proembryos. These proembryos developed into mature embryos when placed in a double-layer liquid overlay culture. Intact plants were developed from somatic embryos when they were transferred onto solidified MS medium without added growth regulators.     

Fertile transgenic pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.