用组培法培养马铃薯—倍体和双单倍体进行染色体加倍的研究

分别取来自普通栽培种(S.tuberosum)的两个一倍体品系和两个双单倍体品系的茎段和叶片,用两步法进行组织培养来加倍其体细胞染色体数目。在以MS+2.25mg/1 BAP+5mg/1 NAA 为第一步的培养基上,从一倍体和双单倍体的茎段和叶片中获得了许多再生植株,其中约60%的加倍成植株(2n=2x=24或2n=4x=48),此结果表明此法是把单单倍体(以下简称一倍体)和双单倍体转化为纯合同源四倍体的有效方法。     

Chromosome number doubling of 2x potato lines with diverse genetic background through tissue culture

Summary Twelve 2x potato clones of diverse genetic origin were tested for doubling of their chromosome number by placing stem and petiole segments in the regeneration medium (Hermsen et al., 1981). Eleven of the 12 clones yielded doubled plants (2n=4x=48). The percentages of regenerated tetraploid plants varied from 0 to 77% among the genotypes tested. The high rates of successful doubling from many genetically different clones, demonstrates the applicability of this method as a means to convert valuable 2x potato germplasm into the 4x level.     

Screening and quantification of an antiseptic alkylamide, spilanthol from in vitro cell and tissue cultures of Spilanthes acmella Murr.

The study revealed, for the first time, accumulation of spilanthol, an antiseptic alkylamide, in in vitro cultures of Spilanthes acmella Murr., a medicinal plant of immense commercial value. To achieve this, in vitro shoots were regenerated via direct organogenesis from leaf-disc explants of Spilanthes. Shoots were induced in the presence of N⁶-benzylaminopurine (BAP) alone or in combination with either α-naphthalene acetic acid (NAA) or Indole-3-acetic acid (IAA) in Murashige and Skoog medium. The best treatment for shoot regeneration was MS + BAP (5.0 μM) + IAA (5.0 μM), which promoted adventitious shoot proliferation in >82% cultures with an average of 5.3 shoots per explant. Regenerated shoots rooted spontaneously with a frequency of 100% on half strength MS medium (major salts reduced to half strength) containing 50 g l⁻¹ sucrose. The plantlets were acclimatized successfully with 90% survival rate. Additionally, ploidy stability of the regenerated plants was assessed by flow cytometry which showed that all investigated plants had the similar ploidy as that of the mother plant. For spilanthol identification, peaks eluted from HPLC were analyzed by mass spectrometry with its characteristic fragmentation pattern. For quantification studies, calibration curve was generated, which revealed a higher amount of spilanthol content (3294.36 ± 12.4 μg/g DW) in the leaves of in vitro plants compare to those of in vivo plants (2703.66 ± 9.6 μg/g DW of spilanthol). An efficient multiplication frequency, ploidy stability and enhanced spilanthol accumulation ensure the efficacy of the protocol developed for this industrially important medicinal plant.     

Morphogenic Response of Leaf and Petiole Explants of Broccoli Using Thidiazuron

Broccoli (Brassica oleracea var. italica) is an important nutritionally rich vegetable cole crop grown in the world. Environmental stress, pests, and diseases cause enormous yield losses because of a limited gene pool. Genetic manipulation is becoming an important method for broccoli improvement. The objective of present study was to evaluate the potency of thidiazuron (TDZ) as a plant growth regulator in evoking morphogenic responses in leaf and petiole explants of broccoli. An efficient, reproducible, and high frequency plant regeneration protocol has been standardized in broccoli cv. Solan green head. Leaf and petiole explants were cultured on Murashige-Skoog (MS) medium, supplemented with a wide range of TDZ concentrations. The following treatments were designed for efficient in vitro shoot regeneration: TDZ alone, TDZ with adenine, TDZ with naphthalene acetic acid (NAA), and TDZ with indole acetic acid (IAA). Among the 36 combinations of growth regulators used, the highest percentage of leaf explants producing shoot (89.25%) was recorded on MS medium containing 1.0 μM TDZ and 0.107 μM NAA. The multiple shoot regeneration response of petiole explant producing shoots (91.55%) was obtained on MS medium containing 2.0 μM TDZ and 0.107 μM NAA. Shoot multiplication and elongation were obtained on the same medium. For root regeneration in in vitro regenerated shoots, different concentrations of NAA were applied. High frequency (100%) root regeneration response with healthy and vigorous roots was observed on MS medium supplemented with 0.54 μM NAA. The regenerated plantlets with well-developed shoots and root system were transferred to pots containing cocopeat and successfully acclimatized. We recommend 1.0 μM TDZ with 0.107 μM NAA and 2.0 μM TDZ and 0.107 μM NAA combinations for adventitious shoot regeneration from leaf and petiole explants in broccoli cv. Solan green head respectively. This is the first report on high frequency organogenesis from leaf and petiole explants of broccoli cv. Solan green head using thidiazuron.     

In vitro direct plant regeneration and Agrobacterium‐mediated transformation of lucerne (Medicago sativa L.)

In vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron (TDZ) and 6‐benzyladenine (BA) in Murashige and Skoog (MS) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA, and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L^?1 TDZ and 1 mg L^?1 BA. Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T₀ plants and T₁ progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.     

Plant regeneration from leaf tissues of four North Dakota genotypes of potato (Solanum tuberosum L.)

An efficient plant regeneration system was developed fromin vitro leaf tissues of four North Dakota potato genotypes. The best medium for genotype ND860-2 was Murashige and Skoog medium with 20.0 μM IAA and 11.4 μM zeatin riboside. Two to three weeks of initial dark treatments had a significant effect in reducing the time for shoot regeneration and increasing the number of regenerated shoots. Four antibiotics commonly used inAgrobacterium- mediated transformation were tested for their effects on shoot regeneration. Shoot induction was completely inhibited by kanamycin at 15 mg/L and hygromycin at 4 mg/L or higher, but not by carbenicillin (except at 1000 mg/L) and cefotaxime. Hygromycin significantly stimulated shoot induction at 1 mg/L.     

Electrotherapy and shoot tip culture eliminate potato virus X in potatoes

Potato stems infected with potato virus X (PVX) were exposed to either 5, 10, or 15 miliampers (mA), for 5 or 10 minutes, followed by immediate planting the axillary buds tipin vitro. Temperature increased from 4 to 10 C in the tissues during the exposure to the electricity. After a 60 days growing period, therapy efficiency (TE, TE = % plant regeneration X% virusfree resulting plants) was influenced by the severity of treatment, since organogenesis and virus elimination were both stimulated by the electricity. The highest TE values were obtained at 15 mA for 5 minutes. Under these conditions, 40% to 80% of the buds regenerated, and 60% to 100% of the regenerated plantlets tested virus negative.     

An improved and efficient micropropagation of Eclipta alba through transverse thin cell layer culture and assessment of clonal fidelity using RAPD analysis

An improved and efficient in vitro regeneration system has been developed for Eclipta alba, a medicinally important plant, through transverse thin cell layer culture (tTCL). The transverse section of the nodal segment of field grown plants was used as tTCL explants for plant regeneration. Shoot multiplication from tTCL nodal explants was influenced by BAP and their interaction with Kin or NAA. MS medium containing 13.2 μM BAP and 4.6 μM Kin was most effective for shoot multiplication from tTCL nodal explants. Upon this medium, percent response for shoot proliferation was 100% with an average of 32.6 shoot buds per tTCL nodal explant. Regenerated shoots from tTCL nodal explants were rooted on the growth regulator free MS medium. The rooted plantlets were successfully acclimatized and established in soil with a survival frequency of 90-100%. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic fidelity of the micropropagated plants. RAPD profile analysis indicated that micropropagated plants were genetically similar to mother plant.     

Tissue culture for the international transfer of potato genetic resources

The use of tissue culture techniques for the international movement of potato clonal germplasm was studied. “Multi-meristem” cultures were established from virus-free plants by procedures described earlier by the authors. Well developed plantlets, regenerated aseptically from “nodal cuttings” of shoots produced in multi-meristem cultures, were used for international transfer. The medium contained the Murashige-Skoog mineral and vitamin components, with 2% sucrose, and was supplemented with 2 mg/l calcium pantothenate and 0.2 mg/l gibberellic acid. The cultures survived the mail shipment when 1% agar medium and small test tubes were used; polystyrene containers reduced damage due to abrupt temperature changes. At the receiving end, higher survival rates during the recovery of plants was achieved through potting of plantlets regenerated from nodal cutting cultures. A total of 43 pest and disease-free potato clones have been distributedin vitro from CIP to 10 different countries. The training of technicians from developing countries played an important role in the application of these methods.     

Diploid and tetraploidSolanum chacoense genotypes that synthesize leptine glycoalkaloids and deter feeding by Colorado potato beetle

A selection (8380-1) fromSolatium chacoense Bitter (2n=2x=24) accession PI 458310 that synthesizes the leptine glycoalkaloids was compared in growth chambers with tetraploid (2n=4x=48) genotypes derived from tissue culture of 8380-1 leaf expiants for plant growth habit, leaf glycoalkaloid content, and effect on the development of Colorado potato beetle [Leptinotarsa decemlineata (Say)] larvae. The plants of the 4x regenerant genotypes were more vigorous with larger, more oval shaped leaflets than 8380-1 plants. The leaf concentrations of leptines and total glycoalkaloids were significantly lower (about 34%) in the 4x genotypes than in 8380-1. The proportion of leptines in the total glycoalkaloid content was nearly the same (about 80%) in both ploidy groups. In leaf-disk feeding tests, the development of Colorado potato beetle neonate larvae was not significantly different for the 2x and 4x genotypes. Both groups significantly slowed development compared with development on cv. Kennebec leaf disks. The 8380-1 selection and a group of 4x 8380-1 regenerant genotypes are maintained in the Vegetable Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705 and are available for distribution.     

Haploid extraction inSolanum tuberosum group Andigena

Haploids were extracted from 66 differentSolanum tuberosum Group Andigena introductions usingS. tuberosum Group Phureja clone 1.22 as pollinator. The overall frequency of haploids was 13 per 100 fruit, with a high of 106. The overall average number of seeds per fruit was 1.4. Some Group Andigena clones produced primarily haploid, others primarily triploid, and still others primarily tetraploid progeny. The “triploid block” is still evident in these materials even though some clones produce primarily triploid progeny. It is suggested that the ploidy level of progeny produced in 4x - 2x crosses may be under relatively simple genetic control.     

Haploids of tetraploid (2n=4x=48) Mexican potato species—Their extraction,cytology and crossability

Successful isolation of haploids (2n=2x=24) from 4x(2EBN) Mexican species was achieved by using Modified Wenzel (MW) and Nitsch and Nitsch (NN) media for anther culture. Both media, MW and NN, gave consistent regeneration of plantlets from 4x(2EBN) Mexican species, and responses to the media were both species and accession (PI) specific. Plantlets regenerated directly from microspores, by-passing the callus cycle, were mostly haploid (2n=2x=24). Haploid plants appeared smaller and weaker than their parents with a drastic reduction in both male and female fertility. Abnormalities observed during meiosis and the lack of success in crossing confirmed the effect of the meiotic irregularities observed in the haploids. A total of 290 seeds was obtained from the 4x × 2x crosses of 4x Mexican species with irradiated pollen of 2xS. cardiophyllum and 2xS. chancayense; however, no haploid (2n=2x=24) plants resulted. Extraction of haploids from colchicinedoubled 4x (2EBN) or natural 6x(4EBN) Mexican species using a Phureja pollinator was examined to provide clues to the potential of using 2x (1EBN) pollinators for haploid induction in 4x(2EBN) × 2x(1EBN) crosses.     

Effect of leaves on microtubers produced from potato single-node cuttingsIn Vitro

Microtubers are used to propagate, to store, and to transport potato clones. Culturing single-node explants from potato plantletsin vitro without subtending leaves was reported to result in plantlets with lower vigor and a higher coefficient of variation. The effect on microtuber productionin vitro of leaf area and the presence or absence of leaves on potato single-node cuttings was investigated as an extension of the above study. Stock plantlets of potato cvs Atlantic, Kennebec, Russet Burbank, and Shepody were cultured under a 16-h photoperiod. Single-node cuttings were excised and grown in a high-sucrose tuberization medium in darkness. Leaf area did not affect the frequency, size, or weight of microtubers of cvs Katahdin and Russet Burbank. The absence of leaves reduced microtuber diameter for Russet Burbank; whereas Atlantic, Kennebec, and Shepody were unaffected. Mean fresh weight of microtubers was reduced when leaves were removed for all cvs except Atlantic. No effect of the removal of the leaf was observed for mean dry weights of microtubers from all cvs, although microtubers from single-node cuttings without leaves accumulated significantly more percent dry matter than those with leaves. Rapid multiplication facilities may therefore wish to consider conserving resources such as media, vessels, and growth room space by culturing explants without leaves for the production of microtubers.     

Segregation of leptines and other glycoalkaloids inSolanum tuberosum (4x) ×S. chacoense (4x) crosses

A clone, 8380-1, selected fromSolanum chacoense (PI 458310) for its high foliage content of the leptine glycoalkaloids, a factor in resistance to Colorado potato beetle, was doubled in chromosome number from 2n=2x=24 to 2n=4x=48. Three 4x clones were crossed with sixS. tuberosum (4x) clones. Foliage glycoalkaloid contents were measured for 452 F1 hybrids from 15 crosses. The 4xchacoense parental clones were not different in respect to glycoalkaloid contents and were similar to the original 2x clone. All F1 hybrids synthesized foliage leptines ranging from 9 to 369 mg/100 g fresh weight (fw) with a mean content of 113 mg/100 g fw. The proportion of leptines in the total glycoalkaloid content ranged from 1% to 62% with a mean of 25%. The 4xchacoense parent mean leptine content was 1482 mg/ 100 g fw which was 90% of the total glycoalkaloid content. Tubers from 136 hybrids and the threechacoense parental clones were tested for glycoalkaloid contents. The tuber solanine + chaconine contents of the 136 hybrids ranged from 30 to 180 mg/100 g fw with a mean of 79. The mean tuber content of the threechacoense parental clones was 157 mg/100 g fw. Leptines were not found in any of the tubers.     

Somatic embryogenesis and regeneration of five multipurpose cassava landraces extensively integrated in African cropping system

Successful development and adoption of transgenic cassava (Manihot esculenta Crantz) varieties in Africa depend on in vitro regeneration of landraces because of their agronomic value and amenability to genetic modification. The study investigated somatic embryogenesis from axillary bud and immature leaf-lobe explant and regeneration via shoot organogenesis in five cassava landraces. Landraces exhibited significant (P < 0.05) differences in frequencies of primary somatic embryo production, number of embryos per explant, and frequency of secondary embryos. The frequency of primary somatic embryogenesis ranged from 7.8 to 14.5%, whereas the number of embryos per explant varied from 5.3 to 10.6. However, only frequency of primary somatic embryos and number of embryos per explant showed significant (P < 0.05) differences when immature leaf lobe was used as explant. The frequency of primary somatic embryogenesis ranged from 42.7 to 49.2%, whereas number of embryos per immature leaf-lobe explant varied from 9.5 to 15.2 per explant. Secondary embryogenesis was cultivar-independent in the case of immature leaf-lobe explant. Cyclic and green (mature) embryogenesis showed no significant (P < 0.05) landrace differences in both axillary bud and immature leaf-lobe explants. Shoot regeneration from cotyledon of somatic embryos had significant (P < 0.05) landrace differences. The mean shoot-bud formation frequency of axillary bud and immature leaf-lobe explants was 51.4% and 37.2%, respectively. Root formation was efficient, with greater than 70% of shoots forming root in all landraces. Similarly, landrace differences were detected for the survival of acclimatized regenerated plants, with a mean of 94.7% for both explants. In conclusion, somatic embryos were produced from immature leaf and axillary bud explants of the landraces and the embryos were converted to plantlets via shoot organogenesis.     

Production and characterization of “second generation” somatic hybrids derived from protoplast fusion between interspecific somatohaploid and dihaploidSolanum tuberosum L.

Protoplasts were fused to produce somatic hybrids between a triploid (2n=3x=32-34) interspecific somatohaploid betweenSolanum brevidens Phil. andS. tuberosum L., and a dihaploid (2n=2x=24) anther-derived line ofS. tuberosum cv. Van Gogh. A total of 265 plants were regenerated from protoplast fusion derived calli and their hybridity was verified using fusion partner specific RAPD markers. These “second generation” somatic hybrids were aneuploid pentaploids (2n=5x=51-65) with a 2C DNA content ranging from 3.36 to 4.43 pg, which corresponded to the sum of the 2C values of each of the fusion partners (somatohaploid: 2.22 pg; and the dihaploid line of cv. Van Gogh: 1.87 pg). Most of the “second generation” somatic hybrids were vigorous, but variable in morphology. They were extremely resistant to PLRV and they had tolerance to PVY infection derived from the somatohaploid fusion partner. Even though most of the “second generation” hybrids tuberized, the tuber morphology was variable and most were poorly shaped. InErwinia soft rot resistance tests, the tubers showed higher level of resistance than the tetraploidS. tuberosum cultivars, the dihaploidS. tuberosum fusion partners and the hexaploid somatic hybrids betweenS. brevidens andS. tuberosum. The “second generation” somatic hybrids were all male sterile and failed to produce berries or seeds.     

In vitro plant regeneration from decapitated embryonic axes of Clitoria ternatea L.—An important medicinal plant

In vitro clonal propagation of Clitoria ternatea has been achieved by employing decapitated embryonic axes (DEAs) explants. The explants induced multiple shoots on cytokinin-containing medium. Several cytokinins [6-benzylaminopurine (BAP), 6-furfuryl aminopurine (KIN) and thidiazuron (TDZ)] were assayed. The best response was achieved with 2 mg l⁻¹ BAP in which 100% of cultures produced 6.0 ± 0.14 shoots per explant. MS + 1 mg l⁻¹ gibberellic acid (GA₃) was the most suitable for shoot elongation. Regenerated shoots were rooted in half-strength Murashige and Skoog (MS) medium with 0.2 mg l⁻¹ indole-3-butyric acid (IBA). Plantlets were successfully acclimatized and established in soil, and they were morphologically indistinguishable from the source plant. The plantlets attained maturity and flowered normally. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for genetic transformation of this valuable medicinal plant for its further improvement.     

Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion,regeneration and isozyme analysis

Mesophyll protoplasts ofSolanum commersonii, a frost tolerant wild species not crossable with the cultivated potato, were fused with either dihaploid or tetraploid S.tuberosum. Protoplasts were aggregated by means of alternating current (AC) or polyethylene glycol (PEG), and electrofused with three direct current (DC) pulses. The treatments with PEG/DC generally resulted in very low heterofusion frequency and protoplast viabiity. On the other hand, AC/DC fusion conditions were optimized by increasing the fusion density of protoplasts and adding CaCl₂ to fusion medium. When a density of 4.8 × 10⁵ protoplasts ml^?1 was used in the fusion medium containing 0.2 mM Ca⁺⁺, AC/DC treated protoplasts showed heterofusion frequencies and plating efficiencies of about 10 and 3%, respectively. Fast growing calli from AC/DC fusion experiments were further cultured for regeneration. Fifty-seven plants were regenerated and clonedin vitro as shoot cultures. Compared to parents they showed heterotic vigor and could be identified as hybrids, based on isozyme analysis.     

Genetic heterogeneity estimated by RAPD polymorphism of four tuber-bearing potato species differing by breeding system

Most wild potato germplasm in genebanks is collected, preserved, and evaluated as botanical seed populations that may be highly heterozygous and heterogeneous. However, some species are selfers so potentially very homozygous, perhaps also homogeneous. Intrapopulation heterogeneity increases sampling error that can undermine consistency in seed regeneration in the genebank, screening results, germplasm collecting, and estimates of taxonomic relationships. Thus, knowledge of genetic heterogeneity (GH) can predict the need to commit more resources for larger sample sizes or replication when populations of a given species are being regenerated, evaluated, collected, and classified. This study investigated withinpopulation GH in 32 potato populations comprising four different breeding systems observed inSolatium species:S. fendleri (2n=4x=48, disomic selfer),S. jamesii (2n=2x=24, outcrosser),S. sucrense (2n=4x=48, tetrasomic outcrosser), andS. verrucosum (2n=2x=24, selfer). RAPD markers were used to estimate heterogeneity among 24 individuals per population. Populations ofS. verrucosum were quite homogeneous with an average GH of 6.0%. Similarly low heterogeneity was detected among the eight populations ofS. fendleri (average GH=7.1%). In contrast,S. jamesii andS. sucrense had a much higher GH of 29.4% and 44.1%, respectively. These results demonstrate and quantify the great difference in intrapopulation heterogeneity among wild potato species. Calculations based on intrapopulation heterogeneity indicate that samples should be composed of 25 to 30 random plants for low sample variation that is uniform for all species.     

In vitro propagation and regeneration of an industrial plant kenaf (Hibiscus cannabinus L.)

The present study report a protocol for the efficient in vitro propagation of kenaf (Hibiscus cannabinus L., an industrial crop having high cellulosic fiber content) on hormone free MS medium using the shoot apex and nodal explants. Shoot tips and nodes were isolated from 15 days old seedlings cultivated on MS medium. Different combinations and concentrations of auxin/cytokinin were used and added to the MS medium to assess the shoot and root induction of theses explants. Several subcultures were drived in order to enhance the multiplication rate. Healthy and well developed in vitro propagated shoots were transferred for acclimatization under greenhouse conditions in pots filled with different substrates (sand + compost or perlite). Our results showed that shoots could elongate and root within 4-6 weeks on MS basal medium without any callus formation. However, addition of growth regulators to the MS medium leaded to a decrease in shoot and root induction rates. Indeed, the highest shoot regeneration frequency (90.5%) was obtained on MS control medium. Elongated shoots were transferred onto the same hormone free MS medium using five subcultures where the multiplication rate reached the highest value (3.66) at the fifth and last step. The in vitro rooted plantlets were acclimatized in greenhouse and successfully transplanted to natural conditions with 70% survival.