A regeneration system using cotyledons and cotyledonary node explants of Toona ciliata

We used the cotyledons and cotyledonary nodes of Toona ciliata(Chinese mahogany)as explants to examine callus and adventitious shoot induction when exposed to different ratios of hormones.We also investigated the effects of seedling age,inoculation method,and genotype on the efficient regeneration of T.ciliata.The results showed that different genotypes exhibited significantly different callus induction efficiency.The cotyledons and cotyledonary nodes of 20-day seedlings inoculated onto MS medium with 0.5 mg/L 6-benzylaminopurine(6-BA),0.5 mg/L kinetin(KT)and 0.05 mg/L 1-naphthylacetic acid(NAA)achieved a greater regeneration rate than did other concentrations of cytokinin and auxin.The numbers of shoots per cotyledon and cotyledonary node explant were 7.33 and 6.67.The optimal inoculation method for cotyledons was that the distal end of the explants was placed in contact with the medium.The optimal adventitious shoot differentiation medium for cotyledon explants was MS medium containing 0.3 mg/L 6-BA and 0.2 mg/L NAA,producing a 3.4 cm height of shoot on average.This study established an efficient regeneration system for T.ciliata with cotyledons and cotyledonary nodes as explants.

Strain Cultivation Stage and Infectious Concentration on Soybean Cotyledonary Node via Agrobacterium–mediated Transformation System

In soybean cotyledonary transformation,Agrobacterium infection is influenced by many factors.The cultivation stage of Agrobacterium strain and infectious concentration were studied in this research.Results showed that the highest rate of resistant shoots was obtained when the strain cultivation concentration was OD 600 =0.6,and the infectious concentration was OD 600 =0.5.Seven soybean cultivars in North China were also screened for high susceptibility to Agrobacterium and three genotypes were found to be s...

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (Arachis hypogaea L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits.A combination of 6-benzylaminopurine(BAP)and thidiazuron(TDZ)plant growth regulators was applied to evaluate shoot regeneration capacity whereasα-naphthalene acetic acid(NAA)combination with 6-benzylaminopurine(BAP),and 2,4-dichlorophenoxyacetic acid(2,4-D)with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars.The result showed combination(BAP with TDZ)was found to be effective in promoting shoot.The highest shoot regeneration frequency(93%)was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency(87%)was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ.The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased.The highest rooting rate(93%)was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars.The rooting rate increased as the concentration of auxin to cytokinin ratio increased.The maximum rooting rate(83%)was obtained on MS medium supplemented with 0.3 mg/L 2,4-D with 0.2 mg/L BAP for the cultivar N3.The result indicated that BAP with NAA was much better than BAP with 2,4-D in rooting rate.Thus,the protocol developed was genotype independent and effective for peanut tissue culture.

Direct Organogenesis from Cotyledonary Node Explants of Cucurbita pepo (L.)—An Important Zucchini Type Vegetable Crop

Cotyledonary node explants from 3 - 5-d-old seedling derived explants of Cucurbita pepo (L.) produced multiple shoots in Murashige and Skoog (MS) salts B5 vitamins containing medium in the presence of N6-benzylamino-purine BAP 1 mg/L + Thidiazuron TDZ 0.05 mg/L. After 4 weeks explants were subcultured to medium containing MS salts B5 Vitamins BAP 0.5 mg/L, Gibberellic acid GA3 1 mg/L and L-Glutamine 15 mg/L. Periodic excision of regenerated shoots from the explants increased shoot efficiency during subculture. The combination of L-Glutamine with BA and GA3 significantly increased the shoot proliferation. Elongated shoots were excised from shoot clumps and transferred to rooting medium containing indole-3-butyric acid (IBA, 1.0 mg/L). The rooted plants were hardened in small pot containing standardized garden soil, well developed plant transferred to greenhouse and survival rate was 85%.

Evaluation of Beta-Lactam Antibiotics on the Regeneration of Peanut Plants and Their Inhibitory Effect on Agrobacterium Growth

The effect of beta-lactam antibiotics on shoot induction and plantlet regeneration from cotyledonary nodes was tested using two peanut cultivars.The culture media contained 4 mg/L 6-benzylaminopurine(BAP)as the main growth regulator.Various concentrations(100–600 mg/L)of cefotaxime,carbenicillin,and timentin were applied in the culture media.In all the tested media,there were no significant differences in the shoot induction as compared to the control.However,little phytotoxic effect was observed at higher concentrations of these antibiotics in the shoot elongation media.Under shoot elongation medium,shoots turned brownish and partly died at higher concentrations where shooting rates were not affected by the treatments.In cefotaxime,timentin,and carbenicillin-containing media,levels of antibiotics greater than 400,300,and 200 mg/L,respectively resulted in the brown coloration of plantlets.Moreover,the mean shoot number and shoot weight significantly decreased as their dosage increased.The results indicate that maximum levels of antibiotics have an adverse effect on the growth and development of peanuts.Also,cefotaxime(100–300 mg/L)and timentin(100–300 mg/L)will be sufficient in controlling Agrobacterium growth in the culture media with the least phytotoxic effect on the peanut plants.

Effect of 6-BA and 2, 4-D on Soybean Transformation

Regeneration from cotyledonary nodes and embryonic tips of soybean ＂Peking＂ was studied. The disinfectant ways of the mercuric chloride and chlorine gas were used and the concentrations of 6-BA and 2, 4-D were studied in the culture medium The results showed that the sterilization effect of chlorine gas was better than that of mercuric chloride. The best concentration of 6-BA was 1.0 mg·L^-1 and the best concentration of 2, 4-D was 2.0 mg·L^-1 in the germinating medium. The number of buds of each explant was 3.56 and 2.98, respectively. The best concentrations of 6-BA and 2, 4-D were 3.0 mg·L^-1 and 3.5 mg·L^-1 in regeneration of embryonic tips. The best inducing time was 16-20 h and the mean shoots per explant was 2.69 and 2.78, respectively.

Seed germination and seedling growth of Pycnanthus angolensis (Welw.) Warb., African false nutmeg

This study investigated the germination behavior and seedling growth of Pycnanthus angolensis seeds.The germination study was carried out in the laboratory and included pretreatment studies and observation of the seed-germination process.For each treatment,three replications of 20 seeds were sown in a transparent plastic germination box(12cm×22cm×5cm)lined with moistened filter paper at room temperature.To monitor seedling growth,seedlings were transplanted into pots filled with topsoil and laid out in a completely randomized design.Ten seedlings replicated thrice were measured monthly;and the shoot height,leaf area,and root length,as well as the fresh and dry weights of the seedlings,were recorded.Results showed that mechanically scarified seeds exhibited the significantly highest germination percent(83.33%,P<0.005),followed by control seeds(70.83%),whereas seeds treated with 98%sulphuric acid(H2SO4)displayed the lowest germination percent(1.67).The endospermous seeds exhibited cryptogeal germination,while seeds stored for a month(with or without arils)failed to germinate.During seed germination,radicle protrusion continued with a pseudo-opening of the root,through which the brownish cotyledonary petiole was emitted,thus releasing the plumule at the posterior position.Moreover,P.angolensis exhibited a slow growth rate,attaining a shoot height of 73 cm within a year.The highest positive change in leaf number and area was recorded in the fourth month,a period during which the least change in shoot height occurred.The study concluded that mechanical scarification of the seeds ensured significant and faster germination than chemical scarification or no treatment at all.Additionally,P.angolensis displayed a cryptogeal germination,with the seedling growth of the tree species observed to be slow.

Preliminary Study on Tissue Culture Technique of Brassica campestris L. ssp. chinensis (L.) Makino var. utilis

In order to establish a rapid propagation system for Brassica campestris L. ssp. chinensis var. utilis, the anther and cotyledon-cotyledonary petioles were used as explants to conduct tissue culture research. The results showed that not bloomed flower buds with higher styles than petals were appropriate for anther cul- ture. Moreover, most microspores were at the mid-late uninucleate stage at that time. The pollen germination rates of Brassica campestris L. ssp. chinensis var. u- tills were low and the germination rates in autumn and winter were higher than that in summer. The induction medium of anther callus of Brassica campestris L. ssp. chinensis var. utilis was MS＋1.0 mg/L KT＋1.0 mg/L 2, 4-D＋3% sugar＋6 g/L agar＋ 8% coconut milk and the induction media of adventitious bud were MS＋2.0 mg/L 6-BA＋ 0.5 mg/L NAA＋I.0 g/L activated carbon＋2% sugar＋6 g/L agar or MS＋2.0 mg/L ZT＋ 0.5 mg/L IAA＋0.5 g/L AgNO3＋1.0 g/L activated carbon＋2% sugar＋6 g/L agar. The induction percentage of adventitious bud by anther culture was 36.7%. Browning phenomenon appeared during the culture of adventitious bud and regeneration plant could not be formed. The plant regeneration rate reached 80% when cotyledon- cotyledonary petioles were used as explant.