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1.
Plant regeneration via somatic embryogenesis in cotton 总被引:6,自引:0,他引:6
An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants
of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant
growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis
was modified MS medium supplemented with 0.1 mg l-1 ZT and 2 g l-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants
respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets
when cultured on modified MS medium supplemented with 0.1 mg l-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system
of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture
and genetic engineering on cotton genetic improvement.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
2.
Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA
1 naphthaleneacetic acid
- IAA
indole-3-acetic acid
- 2,4-D
2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid
- MS
Murashige and Skoog
- BA
6 benzylamino purine
- 2i P
N6-(2-isopentenyladenine 相似文献
3.
Kui Shin Voo Clayton L. Rugh Joe C. Kamalay 《In vitro cellular & developmental biology. Plant》1991,27(3):117-124
Summary We describe a tissue culture procedure for somatic embryogenesis and plantlet regeneration in cotton (Gossypium hirsutum L. cv. Coker 312). Callused explants or individual globular embryos were transferred to basal media to induce somatic embryogenesis.
To determine characteristic early indicators of successful germination and conversion, we identified six types of embryos
that developed on basal media. Two of the six embryo types, designated as tulip-shaped and trumpet-shaped, could undergo conversion
in preliminary tests, whereas the others had little or no developmental potential. Several media treatments designed to enhance
the maturation of globular somatic embryos failed to increase the fraction of embryos which matured to form recoverable types.
In efforts to improve plantlet recovery, tulip-shaped embryos were used in limited trials to contrast the effects of chemical
and physical desiccation treatments on germination and conversion. The selective use of tulip-shaped somatic embryos, coupled
with partial desiccation, seems to have augmented plant recovery. Growth habit, flowering, seed set, and lint production of
most of the regenerated plants were comparable to seed-derived plants grown under the same conditions.
Partial research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development
Center, The Ohio State University. 相似文献
4.
Fertile regenerated plants were obtained from protoplasts via somatic embryogenesis in Coker 201 (Gossypium hirsutum L.). Protoplasts were isolated from six different explantsleaves, hypocotyls, young roots, embryogenic callus, immature somatic embryos and suspension cultures and cultured in liquid thin layer KM8P medium. Callus-forming percentage of 20–50% was obtained in protoplast cultures from embryogenic callus, immature embryos and suspension cultures, and visible callus formed within 2 months. Callus-forming percentage of 5–20% in protoplast cultures from young roots, hypocotyls and leaves, and visible callus formed in 3 months. NAA 5.371 μM/kinetin 0.929 μM was effective to stimulate protoplast division and callus formation from six explants. Percentage of callus formation in the medium with 2,4-D 0.452 μM/kinetin 0.465 μM was over 40% from suspension cultures and immature embryos, 25% from embryogenic callus and 10% from hypocotyls. Callus from protoplasts developed into plantlets via somatic embryogenesis. Over 100 plantlets were obtained from protoplasts derived from 6 explants. Ten plants have been transferred to the soil, where they all have set seeds. 相似文献
5.
Bao-Hong Zhang Fang Liu Zhi-Hong Liu Hong-Mei Wang Chang-Bing Yao 《Plant Growth Regulation》2001,33(2):137-149
The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed. 相似文献
6.
Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone
on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B5 vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating
calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with
their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential
somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially
for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos
at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated
calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of
somatic embryos. 相似文献
7.
Summary The storage protein content of somatic embryos of Gossypium hirsutum L. cv. Coker 201 was determined using extinction level, antigen/antibody association detection methods. Mature storage protein was first detected in early globular-stage somatic embryos at a total concentration of 0.36% of the embryo protein mass. Tulip-stage and mature somatic embryos were comprised of 3.0% and 1.3% mature storage protein, respectively. Maximum storage protein synthesis was found to occur during early globular- and early heart-stages. During this period of development, significant levels of protein precursors were found also to accumulate. The pattern of storage protein synthesis, processing and accumulation paralleled the pattern that has been reported for the zygotic system, although somatic embryos accumulate storage protein at much earlier stages and to a lesser degree. The possibility of using complex biochemical pathways to monitor embryogenic systems in vitro is discussed. 相似文献
8.
In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.) 总被引:5,自引:0,他引:5
D. C. Agrawal A. K. Banerjee R. R. Kolala A. B. Dhage A. V. Kulkarni S. M. Nalawade S. Hazra K. V. Krishnamurthy 《Plant cell reports》1997,16(9):647-652
Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations
BAP
6-Benzylaminopurine
- GA3
Gibberellic acid
- MS
Murashige and Skoog medium
-
NAA
-Napthaleneacetic acid 相似文献
9.
Yuqiang Sun Xianlong Zhang Shuangxia Jin Shaoguang Liang Yichun Nie 《Plant Cell, Tissue and Organ Culture》2003,75(3):247-253
A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance. 相似文献
10.
Transformation of cotton (Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration of transgenic plants 总被引:2,自引:0,他引:2
Ebrahim Firoozabady David L. DeBoer Donald J. Merlo Edward L. Halk Lorraine N. Amerson Kay E. Rashka Elizabeth E. Murray 《Plant molecular biology》1987,10(2):105-116
Cotton (Gossypium hirsutum L.) cotyledon tissues have been efficiently transformed and plants have been regenerated. Cotyledon pieces from 12-day-old aseptically germinated seedlings were inoculated with Agrobacterium tumefaciens strains containing avirulent Ti (tumor-inducing) plasmids with a chimeric gene encoding kanamycin resistance. After three days cocultivation, the cotyledon pieces were placed on a callus initiation medium containing kanamycin for selection. High frequencies of transformed kanamycin-resistant calli were produced, more than 80% of which were induced to form somatic embryos. Somatic embryos were germinated, and plants were regenerated and transferred to soil. Transformation was confirmed by opine production, kanamycin resistance, immunoassay, and DNA blot hybridization. This process for producing transgenic cotton plants facilitates transfer of genes of economic importance to cotton. 相似文献
11.
Somatic embryogenesis in cotton (Gossypium). II. Requirements for embryo development and plant regeneration 总被引:1,自引:0,他引:1
Calli of cotton (Gossypium hirsutum L.) initiated from seedling hypocotyl tissue were placed in liquid suspension and maintained by serial subculture in hormone-free Murashige and Skoog (MS) medium. Suspensions were sieved and globular embryos collected, washed, resuspended in basal medium and plated onto various semi-solid media. High inorganic salts (MS), low salt (2/3 MS), excess KNO3, and the growth regulators napthaleneacetic acid (NAA), gibberellic acid (GA3) and kinetin were tested for their effects on somatic embryo maturation. Long-term embryo proliferation and maturation were best on medium containing MS plus 1.9g/l KNO3. Embryos 3 mm to 10 mm in size were removed from this plating medium and placed on sterile vermiculite saturated with Stewart and Hsu's medium plus 0.1 mg/l indoleacetic acid (IAA). Plants were recovered from 10.6% of the embryos. When 5 mm embryos were placed on this medium, 30% of the embryos formed plants within six weeks. Smaller embryos required a longer period of development on the vermiculite and the addition of fresh medium supplemented with 0.1 mg/l GA3. Plants with an extensive root system and two true leaves were removed from sterile culture and potted in either one-to-one peat and sand, or vermiculite. Eighty percent of the regenerants were successfully hardened when glass beakers of increasing size (10 to 150 ml) were sequentially placed over the young plants during a two-week period. 相似文献
12.
A. P. Tyagi 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1987,74(2):280-283
Summary Two diverse parents of upland cotton namely J.34 and I.C. 1926 were crossed. A comparison between biparental intermated progenies and F3 families indicated alteration of correlation coefficient between yield and halo length. The significant negative correlation in F3 population between these two attributes changed to a positive but non significant one in biparental intermated progenies. A change in correlation coefficients was expected due to breakage of linkage upon intermating. An increase in the correlation coefficients could also be expected when linkages are predominantly in the repulsion phase. It is suggested that intermating in early generations coupled with selection of desirable segregants may prove a useful method for improving yield and quality simultaneously. The diallel selective mating system may also supplement intermating to improve yield and quality in cotton.Part of Ph.D. Thesis submitted to the Haryana Agricultural University. Hissar-125004, India 相似文献
13.
Plant regeneration in cotton: A short-term inositol starvation promotes developmental synchrony in somatic embryogenesis 总被引:1,自引:0,他引:1
Summary Despite high commercial interest, the success of biotechnological applications in cotton (Gossypium hirsutum) has been limited due to difficulties in genetic transformation. Major problems have been genotype dependence and low frequency
of somatic embryogenesis, making it difficult to regenerate plants from transgenic tissue. This study reports an increase
in somatic embryogenesis efficiency and the induction of developmental synchrony in embryogenic callus cultures of cotton
by a single cycle of myo-inositol depletion in liquid culture. Calluses were initiated on hypocotyl or cotyledon explants of cultivar Coker 312 by
culturing these explants on callus-inducing solid medium [Murashige and Skoog salts plus vitamins of Gamborg's B5 medium, 30 g l−1 glucose, 100 mg l−1
myo-inositol, 2.2 μM 2,4-dichlorophenoxyacetic acid, and 0.88 μM 6-benzyladenine]. The calluses were transferred to an identical liquid basal medium devoid of plant growth regulators. This
induced the development of embryogenic cells. Friable clumps of cells formed after 20 d in the medium were selectively collected
over filter mesh 40 subjected to one cycle of myo-inositol starvation. This induced a highly synchronized embryogenesis in the culture. The optimized protocol gave 100% embryos
at the globular stage, out of which more than 80% developed into bipolar torpedo-stage embryos. About 68% of these were converted
to plantlets by subculturing onto a simplified solid medium, and finally grown into healthy, fertile plants. 相似文献
14.
Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers
Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM
autoclaved conditioned medium
- AFC
autoclaved feeder cells
- BM
basic medium
- BM+
basic medium with phytohormones
- CM
non-autoclaved conditioned medium
- FC
non-autoclaved feeder cells
- FDA
fluorescein diacetate
- MM
maturation medium
- NAA
1-naphtaleneacetic acid
- PCM
protoplast culture medium
- PCM+
protoplast culture medium with phytohormones
- SC
settled cells
- 2,4-D
2,4-dichlorophenoxyacetic acid
- 6-BAP
6-benzylamino purine 相似文献
15.
Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 105/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D
dichlorophenoxyacetic acid
- NAA
naphthaleneacetic acid
- BAP
6-benzylaminopurine
- IAA
indole-3-acetic acid
- KT
kinetin
- IBA
indole-3-butyric acid
- CH
casein hydrolysate
- CM
coconut milk
- ZT
zeatin 相似文献
16.
Improvement in somatic embryogenesis has been achieved in several cotton lines (Gossypium hirsutumL.) from the Georgia and Pee Dee germplasm with culture media containing various Putrescine concentrations. The best results were obtained with the -naphthalene acetic acid (NAA)-based treatments, S15 g.05 NAA and EMMS2, as compared to the 2,4-dichlorophenoxyacetic acid (2,4-D)-based culture medium, EMMS4. Inclusion of 0.5 mg l–1 Putrescine improved somatic embryo (SE) induction for most treatments and lines tested. An 8-and 2-fold improvement was achieved in SE production on the EMMS2-0.5 Putrescine treatment as compared to EMMS2 alone for cotton lines PD 97019 and GA 98033, respectively. A significant increase in SE number (53-fold) was obtained with the addition of 0.5 mg l–1Putrescine to EMMS2 for PD 97021, which was essentially recalcitrant without Putrescine treatment. Conversion of SEs into plants was both genotype- and culture medium-dependent. 相似文献
17.
A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was
applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the
origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from
in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level
of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated
shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation
step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This
BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other
high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date
have initiated flowers and set viable R1 seeds.
Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997 相似文献
18.
An efficient grafting system for transgenic plant recovery in cotton (Gossypium
hirsutum L.) 总被引:1,自引:0,他引:1
Shuangxia Jin Shaoguang Liang Xianlong Zhang Yichun Nie Xiaoping Guo 《Plant Cell, Tissue and Organ Culture》2006,85(2):181-185
A successful transformation program relies on the number of survival plants in soil that can be obtained. Low recovery of transgenic plants is still a key restrictive factor for transgenic cotton production. In order to utilize genetic transformation in cotton breeding program effectively, an efficient grafting system for recovering plants derived from somatic embryogenesis following Agrobacterium infection and kanamycin selection was developed. Various aspects of in vitro grafting were examined in efforts to improve the efficiency of transformant recovery. Using strong seedling rootstocks was the first important step to obtain high rate of successful grafts. Scion size >0.6 cm and seedling rootstock at age of 6–12 days were appropriate for grafting. The successful grafting ratio was higher when using hypocotyls without radicle. Shoot-tip and shoot stem with axillary bud were also suitable for in vitro grafting, which meant we could significantly improve the survival ratio of transgenic plantlets, because one plantlet has a shoot-tip but several axillary buds. Based on our data, the period from in vitro seedling rootstock germination to transplant of grafts to field usually takes one month. Over 90% successful grafting ratio could be obtained under optimal conditions, which represented a significant improvement over currently available methods for recovery of cotton plantlet from somatic embryogenesis after transformation. Ex vitro grafting could also be used for plant recovery, which gave an average of successful grafting ratio of 71.9%. However, this method was strongly affected by environmental factors. 相似文献
19.
L. S. Kaushik D. P. Singh R. S. Paroda 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1984,68(6):487-491
Summary The data from an experiment in cotton consisting of three testers and 12 lines selected deliberately have been analysed. The investigation showed higher specific combining ability variance for yield of seed cotton and number of bolls, indicating the predominance of non-additive gene action. Of parental lines, H777 was found to possess high g.c.a. effects for seed cotton yield, number of bolls and number of sympodes. Parent H842 contributed only for boll weight, whereas H655 was good general combiner for number of monopodes. There appeared to be better chances for increasing the yield by exploiting hybrid vigour for the number of bolls and boll weight. The presence of marked non-additive gene effects, in addition to additive gene effects, indicated the need for exploiting both the fixable and non-fixable components of genetic variance for increasing productivity in cotton. 相似文献
20.
Hypocotyl, cotyledon and zygotic embryo explants from two Tunisian Cucumis melo L. cultivars Beji and Maazoun, cultured on the MS medium added with 2,4-D (0.25–1 mg l−1) and BA (0.10–0.50 mg l−1), produce calluses with somatic embryos after 3 weeks of culture. For Beji c.v. the highest percentage (62.50%) of embryogenesis
was observed for cotyledons. The average embryo number per callus was 10.40. Embryogenesis induction for zygotic embryos reached
33.50% with 29 embryos per callus. The embryogenesis ability of hypocotyls did not exceed 12.50% (2.50 embryos per callus).
Somatic embryogenesis for Maazoun c.v. explants was less efficient. Embryos formation was observed only for cotyledons (29%)
and zygotic embryos (25%). Cotyledonary staged embryos, when transferred to hormone free MS medium, germinated. The maximum
germination rates were 51.50 and 44.50%, respectively for Maazoun and Beji c.v. The highest percentage (36.50%) of survival
plants was noted for Beji c.v. Regenerants were diploids (2n = 2x = 24) and morphologically similar to their parents issued from seeds. 相似文献