Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

花生分子标记的研究进展

国内外对花生的研究特别是在分子水平上的研究相对水稻、油菜等农作物比较薄弱。近些年,分子标记技术迅速发展,在花生上也得到广泛的应用。本文从花生属起源、种质资源的遗传多样性、抗性基因的标记和指纹图谱等方面,综述了国内外花生分子标记的研究进展。     

花生成熟胚胚轴的植株再生和快繁

利用正交试验筛选出了以花生成熟胚胚轴为外植体获得较高频率的不定芽分化和植株再生的条件,并在此基础上对花生试管苗的快速繁殖进行了研究,研究表明,4d龄实生苗的花生胚轴,接种于MS NAA0.4mg/L TDZ0.2mg/L诱导培养基上培养28d后,转移到MSo可获得丛生的再生苗。将丛生苗分离,接种到P1培养基（MS NAA0.4mg/L BAP0.5mg/L）,小苗长大,把较大的试管苗切成带叶节的茎段,接种于含不同浓度的TDZ和BAP的快繁培养基,结果表明,TDZ0.2mg/L最适宜试管苗的快繁,繁殖系数可达4.8/月,试管苗经IBA诱导生根后,移栽田间,开花结果。     

In vitro regeneration of shoot buds and plantlets from seedling root segments of Brassica napus L.

Root segments obtained from aseptically germinated seedlings of Brassica napus cv. Westar were used to optimize conditions for high-frequency shoot bud differentiation. The presence of low kinetin (0.5 M) and relatively high indole-butyric acid (1.0 M) levels facilitated optimum shoot bud differentiation. Modified MS medium (MMS) was superior to the other three basal media tested (MS, B5 and White's). Elevated sodium dihydrogen phosphate levels increased the differentiation of shoot buds. Increasing or decreasing the level of sucrose from 3% reduced the frequency of explants forming shoot buds. Addition of glutamine enhanced both the frequency of responding explants, as well as the number of shoots per responding explant. Root segments from 13-day-old seedlings produced the highest response (58%) in the presence of 100 mg l^-1 glutamine. The position of the segment on the main root, size, and the presence or absence of lateral roots altered the morphogenic response. Sealing of the donor seedling cultures with Parafilm^® instead of Stretch' n seal^® resulted in a higher production of shoot buds, although root segment cultures were not affected by the type of sealing. Spontaneous rooting occurred on all developed shoots.Dedicated to Dr. Friedrch Constabel on the occasion of his 60th birthday     

High-frequency shoot regeneration from cotyledon explants of watermelon cv. sugar baby

Summary A protocol for in vitro shoot regeneration from cotyledon explants of Citrullus lanatus (Thunb.) Matsum. & Nakai cv. Sugar Baby is described. The cotyledons excised from 7-d-old aseptic seedlings showed the highest percentage of shoots on Murashige and Skoog (MS) + N⁶-benzyladenine (BA; 3.0 μM) + N⁶-[2-isopentenyl] adenine (2iP; 3.0 μM) and MS + BA (3.0 μM) + indole-3-acetic acid (IAA; 3.0 μM). Whereas the latter medium induced shoot regeneration after the callusing of the explant, the former stimulated direct shoot formation. The regenerated shoots were rooted and the resulting plants were established in earthen pots with 55% success.     

In vitro regeneration of peanut (Arachis hypogaea L.) through organogenesis: Effect of culture temperature and silver nitrate

Summary The effect of culture temperature on the morphogenetic response of Arachis hypogaea was studied. Cotyledons were cultivated on MS medium supplemented with 110 μM 6-benzyladenine. Leaf explants were cultivated in the presence of the same growth regulator at 22 μM. Cultures were incubated at temperatures of 25, 28, and 35±5° C. Both direct organogenesis from cotyledons and development of organogenic calluses from leaves showed optimal rates at 35±5° C. The highest frequency of elongation of buds into shoots from leaf-derived calluses occurred in the presence of 5 μM AgNO₃. At the best culture temperature, an average of 95% of shoots formed roots on growth-regulator-free MS medium. Plants were successfully transferred to soil, showing normal phenotypes.     

花生幼叶为外植体的植株再生系统的建立

本文报道利用花生成熟胚幼叶为外植体获得高频植株再生的方法,为花生转基因提供有效的受体系统。通过诱导培养基TDZ、BA、NAA的浓度以及种子萌发时问、继代培养基种类五个因素不同水平的正交试验,筛选出了分化高频发生的最佳组合为：MS培养基中应含有TDZ 1．0／μmol／L、BA0．4μmol／L、NAA5．0μmol／L,种子萌发4d,继代培养基为MS0。本研究表明,五因素中诱导培养基TDZ浓度为诱导花生幼叶分化的主要影响因素,其次为继代培养基、种子萌发时问,而诱导培养基中BA和NAA的浓度作用较小。试管苗生根后移栽田间,可正常开花结果。     

影响沙田柚叶片离体培养的因素研究

研究了离体培养条件下影响沙田柚叶片愈伤组织诱导和分化的一些因素。结果表明 ,2 ,4 D可以诱导愈伤组织的形成 ,高浓度的蔗糖 (6% )显著提高叶片愈伤诱导率与愈伤组织重量 ,且在 2 ,4 D和蔗糖之间存在着相互作用。外源GA3处理抑制愈伤组织的诱导和生长 ,而CCC与ABA处理显著提高叶片的愈伤诱导率和愈伤组织生长量。愈伤组织转移到附加 3 .0mg/LBA的MS分化培养基上可以分化出芽 ,0 .2 5mg/LGA3的加入可以进一步提高愈伤组织的分化率和每块愈伤组织的再生芽数。     

High-frequency plant regeneration via adventitious shoot formation from deembryonated cotyledon explants of Sesamum indicum L.

Sesamum indicum L. was used as an important oil crop in the world. An efficient protocol for in vitro plant regeneration via adventitious shoot formation from deembryonated cotyledon explants isolated from mature seeds of sesame is developed. Optimal medium for direct adventitious shoot formation was Murashige and Skoog (MS) medium with 22.2 μM 6-benzylaminopurin (BA) and 5.7 μM indole-3-acetic acid (IAA). Abscisic acid (3.8 μM ABA) and AgNO₃ (29.4 μM) were effective in enhancing the frequency of adventitious shoot formation. Preculture of cotyledon explants on high sucrose concentration (6–9%) for 2 wk and subsequent transfer to 3% sucrose enhanced the frequency of adventitious shoot induction. Root formation from the adventitious shoots was easily achieved on MS medium containing 2.7 μM of α-naphthalene acetic acid (NAA). Regenerated plantlets were acclimatized on sand and peat moss (1:1), showing 95% survival with subsequent flowering and seed set. We established the high-frequency plant regeneration via adventitious shoot formation in S. indicum L.     

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 M TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 M BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 M BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 M NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 M NAA.     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

In vitro plant regeneration of fig (Ficus carica L. cv. gular) using apical buds from mature trees

A reliable procedure for multiple-shoot induction and plantlet regeneration was developed with apical buds collected from 7- to 8-year-old trees of Ficus carica L. using Murashige and Skoog's (MS) medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid. The in-vitro-regenerated shoots were further multiplied on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine and 0.2 mg/l 1-naphthaleneacetic acid and an average multiplication rate of four per subculture was established with 90% success. Excised shoots were rooted in liquid half strength MS medium supplemented with 2.0 mg/l indole-3-butyric acid and 0.2% activated charcoal. Regenerated plantlets were successfully established in soil, with a success rate of 68%. Received: 28 July 1997 / Revision received: 15 January 1998 / Accepted: 7 February 1998     

In nature dormant buds and in vitro dormant-like buds ofFraxinus excelsior L.

Summary Microcuttings ofFraxinus excelsior, sampled from adult trees during the period of cell cycle blockage of bud in G_0–1, developed long rejuvenated sprouts on the Woody Plant Medium (WPM) with benzylaminopurine (BAP) (4.0 mg/l) and indolebutyric acid (IBA) (0.03 mg/l). These sprouts had the ability to enter a resting period, building dormant-like buds when maintained on the original WPM. Sprouts developed from subcultures also entered a resting period without any transfer. Comparison of in nature buds in active growth and dormancy with buds of growing sprouts and in vitro dormant-like buds revealed similarity in behaviour at the shoot apical level. In particular, in dormant-like buds in a constant environment, shoot apical functioning was suppressed while the cell cycle of the shoot apex was blocked at the G_0–1 phase, like in nature dormant buds.Abbreviations a.u. arbitrary unit - BAP benzylaminopurine - CF cumulative frequency - d₁, d₂ diameters of the shoot apex - IBA indolebutyric acid - P_n last opposite primordia of range n - WPM woody plant medium     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.     

In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants

Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1-naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-buds proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l: mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 ∶ 5.0, 0.0 ∶ 5.0, and 0.1 ∶ 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explants and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/l IAA or 0.4 mg/l NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

Somatic embryogenesis in peanut (Arachis hypogaea L.): stimulation of direct differentiation of somatic embryos by forchlorfenuron (CPPU)

Summary The ability of forchlorfenuron (CPPU), a substituted phenylurea compound, for inducing somatic embryogenesis in peanut (Arachis hypogaea L.) seedlings has been demonstrated. CPPU promoted somatic embryogenesis at a range of concentrations in all three peanut cultivars tested. Embryogenic response was dependent on applied CPPU concentrations. Exposure of seedlings for only two days to CPPU induced somatic embryogenesis, but the most effective treatment was to induce seed germination on media supplemented with either 2.5 or 4.0 M CPPU and to maintain the seedlings on the same medium. Number of somatic embryos and the frequency of embryogenesis was higher for younger seedlings (up to 9 days), regardless of the CPPU concentrations and seedlings older than 21 days failed to produce somatic embryos. Removal of cotyledons from the seeds drastically reduced the embryogenic potential of the seedlings. Somatic embryos developed into whole plants following their separation and subculture on a medium lacking growth regulators. The induction of somatic embryos using CPPU as a sole growth regulator may provide a useful system to study the role of this compound in plant morphogenesis.Abbreviations CPPU N-(2-chloro-4-pyridyl)-N'-phenylurea - DPU N,N'-diphenylurea - IAA Indole-3-acetic acid