A Two-step Pretreatment Significantly Enhances Shoot Organogenesis from Leaf Explants of Highbush Blueberry cv. Bluecrop

As part of a program to improve highbush blueberry (Vaccinium corymbosum L.) cultivars via tissue culture and genetic engineering, studies were conducted to determine optimum conditions for organogenesis from leaf explants of the previously recalcitrant cv. Bluecrop. The effects of a pretreatment, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% explants regenerated shoots with a mean of 11 shoots per leaf explant after 62 days when explants of 2-week-old shoot cultures were incubated on the following regime: pretreatment medium #1 containing 5 μm TDZ and 2.6 μm NAA for 4 days, pretreatment medium #2 containing 7 μm zeatin riboside and 2.6 μm NAA for 3 days, regeneration medium containing 1 μm TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatment prior to incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on regeneration medium containing 1 μm TDZ was about three times that on 0.5 μm TDZ or 20 μm zeatin riboside, and nine times that on 5 μm TDZ. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(β-D-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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307 Growth Regulator Pretreatments Significantly Enhance the Efficiency of Shoot Organogenesis from Leaf Explants of Highbush Blueberry Cultivar Bluecrop

HortScience ◽

10.21273/hortsci.35.3.445a ◽

2000 ◽

Vol 35 (3) ◽

pp. 445A-445

Author(s):

X. Cao ◽

F. Hammerschlag

Keyword(s):

Shoot Regeneration ◽

Growth Regulators ◽

High Efficiency ◽

Shoot Organogenesis ◽

Leaf Explants ◽

Vaccinium Corymbosum ◽

Zeatin Riboside ◽

Highbush Blueberry ◽

Shoot Cultures ◽

Regeneration Medium

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high-efficiency shoot regeneration from leaf explants of in vitro propagated, commercially important, tissue culture-recalcitrant `Bluecrop' shoot cultures. The effects of pretreatments, growth regulators, and age of explant source on shoot organogenesis were investigated. A maximum of 98% shoot regeneration and 10 shoots regenerating per leaf explant occurred when explants of 2-week-old shoot cultures were incubated in the dark (for a total of 14 days) on pretreatment medium #1 containing 2.6 μM NAA and 5 μM TDZ for 4 days, next on pretreatment medium #2 containing 2.6 μM NAA and 7 μM zeatin riboside for 3 days, then on regeneration medium containing 1 μM TDZ for 6 weeks, and last on medium without growth regulators for 10 days. No shoot regeneration occurred if explants were incubated without exposure to pretreatments before incubation on regeneration medium. There were no significant differences in percentage of regeneration or the number of shoots regenerating per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures. Shoot production per explant on 1 μM TDZ was about three times that on either 0.5 μM TDZ or 20 μM zeatin riboside, and nine times that on 5 μM TDZ.

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Improved Shoot Organogenesis from Leaf Explants of Highbush Blueberry

HortScience ◽

10.21273/hortsci.35.5.945 ◽

2000 ◽

Vol 35 (5) ◽

pp. 945-947 ◽

Cited By ~ 25

Author(s):

X. Cao ◽

F.A. Hammerschlag

Keyword(s):

High Efficiency ◽

Shoot Organogenesis ◽

Leaf Explants ◽

Vaccinium Corymbosum ◽

Zeatin Riboside ◽

Photon Flux ◽

Highbush Blueberry ◽

Shoot Cultures ◽

Regeneration Medium ◽

Number Of Shoots

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of shoots propagated in vitro. The effects on shoot organogenesis of age of explant source, length of dark treatment, the addition of either thidiazuron (TDZ) at 1 or 5 μm, or zeatin riboside at 20 μm to the regeneration medium, and a photosynthetic photon flux (PPF) of either 18 ± 5 or 55 ± 5 μmol·m–2·s–1 were investigated. A maximum of 13.0, 13.0, 12.6, and 4.6 shoots regenerating per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occurred on regeneration medium with zeatin riboside and under a PPF of 55 ± 5 μmol·m–2·s–1. `Duke' regenerated equally well on medium with either zeatin riboside or 1 μm TDZ, whereas the number of shoots per explant for `Georgiagem' and `Sierra' was significantly higher on zeatin riboside. Regeneration of `Duke', `Jersey', and `Sierra' on zeatin riboside was significantly better under a PPF of 55 ± 5 μmol·m–2·s–1 than under 18 ± 5 μmol·m–2·s–1, but the higher PPF inhibited regeneration of `Duke' on 5 μm TDZ. There were no significant differences in percentage of regeneration or the number of shoots per explant from leaf explants derived from either 1-, 2-, or 3-week-old shoot cultures, or when either 1 week or 2 weeks of darkness preceded light treatments. Chemical names used: 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ); 9-(-β-ribofuranosyl)-6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin riboside).

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112 The Influence of Plant Growth Regulators and Light Levels on Shoot Morphogenesis from Leaf Explants of Highbush Blueberry

HortScience ◽

10.21273/hortsci.34.3.460f ◽

1999 ◽

Vol 34 (3) ◽

pp. 460F-461

Author(s):

Xiaoling Cao ◽

F.A. Hammerschlag

Keyword(s):

Light Intensity ◽

Shoot Regeneration ◽

High Efficiency ◽

Leaf Explants ◽

Vaccinium Corymbosum ◽

Zeatin Riboside ◽

Highbush Blueberry ◽

Shoot Cultures ◽

Regeneration Medium

As part of a program to develop transgenic highbush blueberry (Vaccinium corymbosum L.) cultivars, studies were conducted to determine optimum conditions for high efficiency shoot regeneration from leaf explants of in vitro-propagated shoot cultures. The effect of either thidiazuron at 1 or 5 μM, or zeatin riboside at 20 μM, and two lit levels (18 ± 5 or 55 ± 5 μmol·m-2·s-1) on shoot organogenesis were investigated. With the exception of `Bluecrop', which did not regenerate shoots, maximum shoot regeneration of 13, 12.7, 12.6 and 4.6 shoots per explant for cultivars Duke, Georgiagem, Sierra, and Jersey, respectively, occured on regeneration medium with zeatin riboside and under a light intensity of 55 μmol·m-2·s-1. Whereas `Duke' regenerated equally well on regeneration medium with either zeatin riboside or 5 μM thidiazuron, regeneration frequencies for `Georgiagem' and `Sierra' were significantly higher on zeatin riboside. A light intensity of 55 μmol·m-2·s-1 significantly increased regeneration of cultivars Duke, Jersey, and Sierra on zeatin riboside, but inhibited regeneration of Duke on 5 μM thidazuron.

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Influences of Growth Regulators and Cultivars on Callus and Shoot Production of Alfalfa

HortScience ◽

10.21273/hortsci.33.3.461f ◽

1998 ◽

Vol 33 (3) ◽

pp. 461f-462

Author(s):

Guochen Yang ◽

Marihelen Kamp-Glass

Keyword(s):

Growth Regulators ◽

Culture Medium ◽

Shoot Organogenesis ◽

Multiple Shoots ◽

Apparent Difference ◽

Shoot Production ◽

Callus Initiation ◽

Dark Green ◽

Number Of Shoots ◽

Alfalfa Seeds

Alfalfa seeds of Cimarron VR, CW1446, CW2440, C94-118, C94-785, and WL311 were used as explants. BA, zeatin, and TDZ were evaluated on callus initiation, development, and shoot production. Callus initiation and development toward shoot organogenesis were enhanced when BA was added in the culture medium. Calli produced from BA treatments were compact, solid, and dark green. Similar results were obtained when zeatin was added in the culture medium. However, no shoots were produced from such calli. Multiple shoots were produced directly from each individual explant when TDZ was added to culture medium. However, when higher concentration of TDZ was used, number of shoots per explant was decreased, and dwarf shoots were produced. No apparent difference on shoot production was observed among the cultivars tested so far. Data on number of shoots per explant from two of these cultivars need to be statistically analyzed.

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Particle Bombardment of Apple Leaf Explants Influences Adventitious Shoot Formation

HortScience ◽

10.21273/hortsci.29.12.1536 ◽

1994 ◽

Vol 29 (12) ◽

pp. 1536-1538 ◽

Cited By ~ 6

Author(s):

P. Gercheva ◽

R.H. Zimmerman ◽

L.D. Owens ◽

C. Berry ◽

F.A. Hammerschlag

Keyword(s):

Shoot Regeneration ◽

Particle Bombardment ◽

Dark Period ◽

Leaf Explants ◽

Adventitious Shoot ◽

Shoot Formation ◽

Regeneration Medium ◽

Adventitious Shoot Formation ◽

Shoot Production ◽

And Control

Shoot regeneration from apple (Malus domestica Borkh.) leaf explants following particle bombardment at various acceleration pressures was studied. Basal leaf segments of micropropagated `Royal Gala' apple were bombarded with 1 μm gold particles, accelerated using helium pressures of 4.5, 6.2, 7.6, 9.3, or 13.8 MPa (650–2000 psi), and cultured on shoot regeneration medium consisting of N6 salts supplemented with 10 μM TDZ for 5, 10, or 20 days in darkness. Bombarded and control explants exhibited 63% to 100% shoot regeneration. With a 5-day dark period, average shoot production per explant ranged from 6.1 to 14; bombardments of 4.5 and 6.2 MPa significantly increased shoot production over the controls. With a 10-day dark period, average shoot production per explant ranged from 9.1 to 22 following bombardment at 9.3 and 6.2 MPa, respectively. Following bombardment at 6.2 MPa, 75% of the explants produced more than 20 regenerants per explant. With a 20-day dark period, average shoot production per explant ranged from 8.9 to 19 following bombardment at 13.8 MPa and following no bombardment, respectively. Shoot production per explant was significantly less than the controls following bombardments ranging from 6.2 to 13.8 MPa. Shoot production was highest per explant with particle bombardment at 6.2 MPa followed by incubation in darkness for 10 days. Chemical name used: thidiazuron (TDZ).

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Use of a Cytokinin Conjugate for Efficient Shoot Regeneration from Leaf Sections of Highbush Blueberry

HortScience ◽

10.21273/hortsci.27.10.1127 ◽

1992 ◽

Vol 27 (10) ◽

pp. 1127-1129 ◽

Cited By ~ 26

Author(s):

Lisa J. Rowland ◽

Elizabeth L. Ogden

Keyword(s):

Tissue Culture ◽

Shoot Regeneration ◽

Culture Medium ◽

Vaccinium Corymbosum ◽

Zeatin Riboside ◽

Tissue Culture Medium ◽

Highbush Blueberry ◽

Leaf Section ◽

Number Of Shoots

Conditions for improving the efficiency of shoot regeneration from leaf sections of highbush blueberry (Vaccinium corymbosum L.) were investigated. Effectiveness of tissue culture medium supplemented with the cytokinin conjugate zeatin riboside or the cytokinin zeatin at 10, 20, or 30 μm was compared with medium supplemented with the optimum 2iP concentration of 15 μm. Use of 20 μm zeatin riboside resulted in the most shoots per leaf section, » 6-fold higher than the number of shoots produced on 2iP medium. The number of shoots produced on medium supplemented with zeatin was not significantly higher than the number of shoots produced on 2iP medium. Consequently, we concluded that the cytokinin conjugate zeatin riboside was more effective than either of the free cytokinins, 2iP or zeatin, in promoting shoot regeneration from leaf sections of highbush blueberry. Chemical names used: 6-(y,y-dimethylallylamino)-purine (2iP); 6-(4-hydroxy-3-methyl-but-2-enylamino)purine (zeatin).

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Adventitious Shoot Organogenesis and Plant Regeneration from Cotyledons of Tetraploid Watermelon

HortScience ◽

10.21273/hortsci.29.3.211 ◽

1994 ◽

Vol 29 (3) ◽

pp. 211-213 ◽

Cited By ~ 15

Author(s):

Michael E. Compton ◽

D.J. Gray

Keyword(s):

Shoot Organogenesis ◽

Citrullus Lanatus ◽

Adventitious Shoot ◽

Shoot Elongation ◽

Mature Seed ◽

Regeneration Medium ◽

Breeding Lines ◽

Cotyledon Explants ◽

Explant Source ◽

Adventitious Shoot Organogenesis

Cotyledon explants of four watermelon [Citrullus lanatus (Thunb.) Mataum. & Nakai] breeding lines (F92U8, SP90-1, SP90-2, and SP90-4) were prepared from mature seed or from 2-, 4-, 6-, 8-, or 10-day-old seedlings. Explants were incubated on shoot regeneration medium for 8 weeks followed by 4 weeks on shoot elongation medium. The four genotypes differed in their ability to produce shoots at each explant age. The highest frequency with which F92U8 (66%) and SP90-2 (60%) explants produced shoots was for 2-day-old seedlings. Fewer explants formed shoots when established from mature seed or seedlings older than 2 days. In contrast, the percentage of SP90-4 explants that produced shoots was highest when cotyledons were obtained from 4-day-old seedlings (40%), but the response was less than the optimum for F92U8 and SP90-2. SP90-1 cotyledon explants exhibited the poorest response of the four breeding lines (<11% produced shoots), with little difference in response among the explant ages tested. The number of shoots per responding explant also depended on the age of the explant source. Explants from 2- to 4-day-old seedlings produced the most shoots. Fewer shoots formed on cotyledons from mature seed or seedlings older than 4 days.

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Effects of different cytokinins on the shoot regeneration from apple leaves of 'Royal Gala' and 'M.26'

International Journal of Horticultural Science ◽

10.31421/ijhs/10/1/442 ◽

2004 ◽

Vol 10 (1) ◽

Author(s):

J. Dobránszky ◽

I. Hudák ◽

K. Magyar-Tábori ◽

E. Jámbor-Benczúr ◽

Zs. Galli ◽

...

Keyword(s):

Shoot Regeneration ◽

Chemical Structure ◽

Shoot Organogenesis ◽

Leaf Explants ◽

Similar Relationship ◽

Regeneration Medium ◽

Apple Rootstock ◽

Apple Leaves ◽

Shoot Number ◽

Different Types

The effects of different types of cytokinins on the shoot regeneration from leaf explants of apple scion 'Royal Gala' and apple rootstock 'M.26' were evaluated. Regeneration media contained either thidiazuron, or 6-benzylaminopurine, or meta-topolin, or zeatin, or kinetin, or their N9-ribosides, respectively, in the concentration range 0.5 to 8.0 mg 1-1. Effects of 'these cytokinins were evaluated on the percentage of regeneration (R%) and that of vitrification (V%) and on the number of regenerated shoots per explant (SN). Organogenetic index (0I) calculated from these data was used for the evaluation of efficacy of cytokinins. The course of shoot organogenesis also was followed using stereomicroscope. Types and concentrations of cytokinins applied in the regeneration media influenced each parameter significantly and the regeneration answer was strongly genotype-dependent. The best regeneration (SN: 11.08, 01: 7.5) was achieved in `Royal Gala' by using TDZ in concentration of 0.5 mg 1-1 (2.271,1M). There was a clear relationship between the effect on the regeneration efficacy and the chemical structure of cytokinins considering classical cytokinins, namely N9-ribosides applied in less concentration than nonribosides have the same or best regeneration effects except for 6-benzylaminopurine riboside. However, similar relationship could not be detected in the case of 'M.26'. SN was the highest (3.22) using 6.5 mg 1-1 (18.2011M) 6-benzylaminopurine riboside or 8.0 mg 1-1 (21.44 µM) meta-topolin riboside (3.18). SN was not significantly lower (3.12) by using 2.0 mg 1-1 (9.08 1M) TDZ, however, OI was about half as big (0.63 compared to 1.29 or 1.74 with 6-benzylaminopurine riboside or meta-topolin riboside, respectively). 'Royal Gala' had higher organogenetic ability, than `M.26': 3.5-fold higher shoot number per explant and more than 4-fold higher organogenetic index was reached with this cultivar than with 'M.26'. Moreover, the similar developmental stage of shoots could be observed 3-5 days earlier than in 'M.26' and if explants of 'Royal Gala' were further cultured with 3 weeks, SN increased from 11.08 to 24.42 on TDZ-containing regeneration medium, which might suggest higher organogenetic ability, too.

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Response of highbush blueberry seed germination to gibberellin A3 and 6N-benzyladenine

Canadian Journal of Botany ◽

10.1139/b89-412 ◽

1989 ◽

Vol 67 (11) ◽

pp. 3391-3393 ◽

Cited By ~ 4

Author(s):

I. M. Dweikat ◽

P. M. Lyrene

Keyword(s):

Seed Germination ◽

Gibberellic Acid ◽

Acid Concentration ◽

Growth Regulators ◽

Vaccinium Corymbosum ◽

Germination Percentage ◽

Highbush Blueberry ◽

Gibberellin A3 ◽

The Relationship ◽

Germinated Seeds

Open-pollinated highbush blueberry (Vaccinium corymbosum L.) seeds were treated with 0, 2.6, 5.2, 10.4, or 20.8 mM gibberellic acid, 0, 0.4, 2.2, or 4.4 mM 6N-benzyladenine, or the two growth regulators in combination. Seeds were treated for 24 h, then germinated under intermittent mist during summer with temperatures fluctuating between 21 and 40 °C. Germinated seeds were counted weekly from day 15 to day 30, using hypocotyl exposure to indicate germination. Treatment with gibberellic acid at 10.4 mM in combination with 6N-benzyladenine at 0.4 to 2.2 mM gave the highest germination percentage. The relationship between gibberellic acid concentration and seed germination was cubic. Seedlings from the germinated seed flowered and fruited the following spring, making it possible for highbush blueberry to complete a generation in 1 year.

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Optimizing the concentrations of plant growth regulators for in vitro shoot cultures, callus induction and shoot regeneration from calluses of grapes

OENO One ◽

10.20870/oeno-one.2015.49.1.95 ◽

2015 ◽

Vol 49 (1) ◽

pp. 37 ◽

Cited By ~ 6

Author(s):

Nadra Khan ◽

Maqsood Ahmed ◽

Ishfaq Hafiz ◽

Nadeem Abbasi ◽

Shaghef Ejaz ◽

...

Keyword(s):

Shoot Regeneration ◽

Growth Regulators ◽

Callus Induction ◽

Leaf Explants ◽

Shoot Cultures ◽

Ms Medium ◽

Grape Cultivar ◽

Half Strength ◽

Number Of Shoots

Aim: To optimize the concentrations of growth regulators in the media for the proficient micropropagation of grapevine (Vitis vinifera L.) cv. King’s Ruby.Methods and results: Apical meristems of the grape cultivar were used to establish in vitro shoot cultures. Nodal explants, each containing an axillary bud, taken from in vitro grown shoots were inoculated in shoot proliferation medium, i.e., half strength Murashige and Skoog (MS) medium supplemented with benzyl aminopurine (BAP), kinetin, glycine and gibberellic acid (GA3). A higher number of shoots (5.33) with greater shoot length (2.75 cm) was produced in the medium supplemented with 1.0 mg L-1 BAP and 0.1 mg L-1 GA3. Calluses were induced from leaf explants taken from in vitro grown shoots. Callus induction was greater (73.00%) on the medium containing 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.3 mg L-1 BAP and 0.2 mg L-1 α-naphthaleneacetic acid (NAA). The maximum frequency of shoot regeneration (53.33%) was achieved on the medium supplemented with 1.5 mg L-1 BAP and 0.5 mg L-1 NAA, and the regenerated shoots successfully formed roots on growth regulator-free half strength MS medium.Conclusion: Optimizing the concentration of BAP and GA3 and omitting the glycine and kinetin in the culture medium increased the number and length of shoots. Similarly, for inducing the callus of the leaf explants, taken from in vitro grown shoots, it is recommended to adjust the medium with the higher concentration of 2,4-D and lower concentrations of BAP. Moreover, the maximum number of shoots was regenerated on a medium supplemented with relatively high levels of both BAP and NAA (1.5 and 0.5 mg L-1, respectively). Finally, we suggest the half strength MS medium that is free from growth regulators for the root formation of the regenerated shoots.Significance and impact of the study: Optimizing the concentration of growth regulators is crucial for the efficient micropropagation of a grape cultivar. Knowing the specific balance between the growth regulators is necessary to establish in vitro shoot cultures, callus induction and shoot regeneration and, hence, to propagate disease-free true to type grape cultivars in a short time.

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