Histo-Cytological Observations on Callus and Bud Formation in Cultures of Nicotiana tabacum L.

Leaf explants of tobacco were cultured on MS medium supplemented with 2 mg/ l NAA and 0.5 mg/l BA for induction of callus formation, or supplemented with 2 mg/l BA for bud formation. Histocytological observations on callus and bud formation were carried out. Three days after cultivation, mesophyll cells enlarged, the nuclei became more apparent and dark stained, and starch accumulated in the cells. Cell divisions began in the mesophyll cells at the cut ends, in the palisade cells near the vascular bundles and in the vascular parenchyma. Mitotic activity then spreaded over tbc explants, and was most active at the edges of leaf explants. Regular rows of cells appeared as a result of series of transverse divisions in the palisade. The number of chloroplast in the mesophyll cells decreased and degenerated gradually. A number of meristemoids ware initiated in the cultured leaf explants after 7 days of cultivation. They were originated from two kinds of tissues, the mesophyll and vascular bundle, including the phloem parenchyma and vascular sheath. On the medium with NAA and BA, callus formation was induced with vigorous divisions, whereas bud primordia were differentiated from the meristomoids on the medimn with 2 mg/l BA. The buds were developed from both the superficial meristemoids and the meristematic regions deep within the callused leaf explants. The accumulated starch in the cells gradually disappeared as bud formation proceeded.     

Induction Of Callus and Regeneration of Plantlets in Stem Segment Culture of Chinese Gooseberry

The stem segments of male and female plants of Actinidia chihensis Planch. var. chinensis C. F. Liang and A. chihensis Planch. var. hispida C. F. Liang were cultured in vitro. Callus and plantlets were obtained on MS basic medium supplemented with BAP (0.5, 2 ppm) + IAA (0.05 ppm) or zeatin (0.1–10 ppm). Best results were obtained on medium added with 1 ppm zeatin. It was possible to induce callus in both species but the frequency of callus redifferentiating into plantlet in A. chinensis Planch. var. hispida was far higher than that in A. chinensis Planch. var. chinensis and the frequency of plantlets derived from the female plant was higher than those from the male plant. The stem of plantlet was cut off when it reached 1 cm in height, then soaked in the solution with IBA 50 ppm for 2 hr. or with IBA 2 ppm for 24 hr. and the excised stem was transferred onto medium with 1/2 MS+ 1% sucrose +0.5% active carbon for rooting. It is possible that this technique of obtaining whole plants by stem segment culture provides a method for thes multiplication of female plants and the good individual plants of chinese gooseberry.     

Studies on Morphological Differentiation of Endosperm Plantlets of Chinese Gooseberry in Vitro

The present report deals with the process of embryoid induction and plantlet formation from cell-type endosperm of Actinidia chinensis var. chinensis and A. chinensis var. hispida. The callus was induced from endosperm on MS basic medium supplemented with zeatin 3 ppm, 2,4-D 0.5 ppm and CH 400 ppm and then transferred to differentiation medium of MS supplemented with zeatin 1 ppm and CH 400 ppm. After about half a month, the embryoid appeared from callus and then developed into plantlets. It could be seen from the histological figure 14—15, the embryoid of Chinese gooseberry is linear-shaped and consists of cells arranged in a long line in callus. When the cells regenerated at botk ends of the linear-shaped embryoid, the polarity of the embryoid is easily distinguished. The plantlets produced from embryoid appear rather stout at first and after some time, they changes gradually into normal plantlets.     

大果良种沙棘愈伤组织诱导及植株再生的研究

大果良种沙棘的幼嫩茎尖,茎段外植体接种在MS,1／2MS附加不同浓度配比的IAA,IBA,BA,NAA培养基上可诱导茎尖及腋芽生长,将诱导产生的无性系芽接种在MS或1／2MS附加BA0．3－0．5mg/L,NAA0．05mg/L的培养基上可形成丛生芽,同时在小叶片和嫩茎上诱导产生愈伤组织,继续培养愈伤组织表面形成大量的绿色突起,进一步分化成不定芽,在相同培养基上,不定芽上可直接产生不定芽,从而形成多达数百个的不定芽族,不定芽长至3cm时切下转至1／2MS附加IAA或IBA 0．2mg/L的培养基上可生根而形成完整 的再生植株。     

甜樱桃新品系13-38茎尖无性系建立及试管苗嫁接研究

本文报告通过茎尖培养及试管苗嫁接快速繁殖甜樱桃新品系13—38苗木。取新品系母树休眠芽制备成无菌外植体,分别在MS附加6-BA 0.1-0.5mg／L,IAA 0.1mg／L培养基及MS附加ZTl-1.5 mg／L,IAA 0.1-0.25 mg／L培养基中进行微型扦插及茎尖分化培养,建立茎尖无性系。试管苗年扩繁量为48和108。以新品系试管苗为接穗,采用切接,皮接两种方法嫁接于四种砧木,成活率为20一70%不等。实验表明13—88试管苗与砧木新品系1090试管幼苗的亲和力较好。接合部位预先施用200ppm的NAA可提高嫁接成活率。     

金桔组织培养中愈伤组织和芽形成的组织细胞学观察

用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。     

云南山楂茎尖的离体培养及其无性系快速繁殖

用云南山楂(Crataegus scabrifolia(Franch.)Rehd.)成年树茎尖和实生芽两种不同发育阶段的材料为外殖体,诱导它们休眠芽萌动,丛生芽条并诱导芽条生根。实验结果如下:1.以成年态的云南山楂侧芽为外植体,培养在附加IAA 0.1—0.5mg/l+6-BA 1-2mg/l的MS培养基上可诱导芽的萌发;将芽继代培养在附加0.5—1mg/l 6-BA的SH或MS培养基上,40天后芽数增殖4—6倍;将芽条截下置于1/2MS培养基上,附加不同浓度的IAA或IBA,可得到50—80％的生根率。2.以实生芽为外殖体,在相同条件下,则20天后芽数增殖便可获4—6倍;98％以上生根。结果表明:云南山楂的幼年态要比成年态易脱分化和再分化。     

Micropropagation of pitaya (<Emphasis Type="Italic">Hylocereus undatus</Emphasis> Britton et rose)

Summary A procedure for micropropagation of pitaya using thidiazuron (TDZ) and naphthaleneacetic acid (NAA) is described. Explants were excised from young joints of mature plants and cultured on Murashige and Skoog medium (MS) containing 0.5 μM NAA and 0.5 μM TDZ. Shoots produced from these first explants were cut up to produce secondary explants, either by decapitation or by longitudinal division into three parts. The decapitated and longitudinal explants were cultured on MS supplemented with 0.5 μM NAA and either 0.01, 0.09, 0.5, or 0.9 μM TDZ. Decapitated explants produced more shoots at higher frequency that the longitudinal explants. For both types of secondary explants, most shoots were developed from the distal parts. Shoots produced from secondary explants were rooted in MS and then transferred to soil where they produced normal plants.     

High Frequency Shoot Organogenesis in Sorghum bicolor (L) Moench

In vitro morphogenesis that includes enlargement of apical meristems and differentiation of shoot buds on the surface of enlarged meristemoids, have been observed in Sorghum bicolor. The enlargement of apical meristems occurred on the MS medium containing different auxins [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T) and parachlorophenoxyacetic acid (pCPA)] with or without 6-benzylaminopurine (BAP)]. Large number of shoot buds arose over the entire surface of enlarged, green, compact, nodular, hard and shiny meristemoids on transfer to medium containing BAP (2.0 mg l^?1) + (IAA) indole 3-acetic acid (0.5 mg l^-1). Histological observations revealed the origin of shoot apices from the surface of enlarged meristemoids. Efficient rooting was achieved onto half-strength MS medium supplemented with α-napthaleneacetic acid (NAA, 2.0 mg l^?1) and sucrose 2% (w/v). Plantlets were transferred to earthen pots under field conditions for the evalution of several agronomically important characters.     

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

Adventitious shoot regeneration from Sinningia speciosa leaf discs in vitro and stability of ploidy level in subcultures

Summary Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N⁶ benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26.     

Plant Eegeneration from Protoplasts of Brassica carinata Braun

Protoplasts of Brassica carinata Braun. (accession No. 84A165) were enzymatically isolated from hypocotyls and cotyledons of 3–5 day-old test-tube seedlings or first true leaves taken from greenhouse grown plants at a three-leaf stage. The protoplasts were suspended in a P-B liquid medium solidified with 0.15%–0.3% low melting agarose which formed a thin layer floating on the surface of the liquid medium. The optimum protoplast density was ranging from 5× l03 to 1 × 104/ml. As for the hypocotyl protoplasts, the first division was observed after 48 h in the culture. The division frequency reached 21% and 34% at day 3 and 6 respectively. The initiation of cell division in the case of cotyledon and mesophyll protoplast culture was late, usually at day 5, and the division frequency was also somewhat lower. One week after culture, the cultures were transferred to fluorescent light condition with an intensity of about 1000 lx. A dilution medium DPDK3 was then added and the dilution procedure was repeated at one week interval thereafter. One month after culture, microcalli with 300–500 μm in size were formed. It was also found that in some cases globular embryoid structure protruded on the callus surface. Totally, a 2%–3% plating efficiency was achieved. Shoot regeneration occurred when cotyledon and mesophyll protoplast-derived calli were transferred onto a modified MS medium supplemented with NAA 0.1, BA 3 mg/l. Individual shoots were rooted on a rooting medium supplemented with 0.2 mg/l of IAA. Intact plants with normal morphology were eventually produced.     

Regeneration of Heracleum candicans Wall Plants from Callus Cultures through Organogenesis

Callus was initiated from petiole explants of Heracleum candicans on MS medium fortified with BAP and 2,4-D ( 0.5 mg I^-1 each). Maximum shoot differentiation from callus occurred on MS medium containing 1 mg I^-1 BAP and 0.2 mg I^-1 NAA. The regenerated shoots were rooted on MS medium supplemented with 1 mg I^-1 IBA. The rooted plants were transferred to the field after successful hardening in pots containing vermiculite. All regenerated plants were diploid with 2n=22 chromosomes in their root tip cells.     

Embryology and Embryo Rescue of an Interspecific Cross Between Actinidia deliciosa cv. Haywardand A. eriantha

The study deals with an investigation of embryo and endosperm development in seeds of interspecific hybridization betwwn Actinidia deliciosa cv. Hayward (6X) and A. eriantha (2X) and an attempt to hybrid embryo rescure. The average seed number per fruit of hybrid com suitable culture media for in vitro embryo growth and subsequent growth of the seedlings are tested for hybrid embryos from normal and abortive seeds. The highest percentage of seed germination and the best growth of early seedlings are obtained op. MS medium supplemented with 0.5 ppm of IAA and 0.5 ppm of GA3; MS supplemented with 2 ppm of 2ip, 0.5 ppm of IAA and 0.5 ppm of GA3; MS supplemented with of 2ip and 0.5 ppm of GA3. When these embryos, as seedlings, were transferred onto MS supplemented with 0.5 ppm of GA3 and then MS without growth regulators, they readily develop into seedlings with normal leaves and roots, A lot of adventitious buds produced from hypocotyl of some normal and abortive embryos on MS supplemented with 2 ppm of BAP and Ms+ 0.5 ppm IAA+0.5 ppm GA3 further grow into hybrid plantlets. Although various percentage of embryos developed from abortive seeds also germinate, but when inoculated onto media as mentioned in Table l, a number of malformed seedlings form subsequently. The remaining embryos on all the media, however, show limited growth, and eventually either form callus or die. The nature of hybrid seedling is confirmed by cytological test. Half of them showed chromosome number as 4X=116 approx., the rest may have 19, 27, 30, 46, 120, 131, ect. In conclusion, according to embryological and cytological observation on hybrid seeds and seedlings: (I) the cross between Actinidia deliciosa cv. Hayward and A. eriantha appears interspecific incompatibility and compatibility to some extent, (2) normal hybrid seedlings are produced by normal hybrid embryos from normal seeds and abnormal embryos from abortive seeds, and (3) both may induce initiation of adventitious buds from hypocotyl. We suggested that these results may be useful for plant breeding program.     

Totipotency of coleoptile tissue in indica rice (Oryza sativa L. cv. ch 1039)

Embryogenic and non-embryogenic calluses were induced from 3,4,5 and 7d old coleoptile segments of indica rice (Oryza sativa L. cv. CH 1039). Compact, globular, yellow and creamy embryogenic and white friable non-embryogenic callus arose from the cut end and entire length of the coleoptile segments. Murashige and Skoog's (MS) medium supplemented with 2.5mg/1 2,4-D was used as callus induction medium. Plant regeneration from coleoptile segments occurred with the transfer of embryogenic callus to MS basal medium supplemented with 2.0mg/1 BAP and 0.5mg/1 NAA in combination. Average number of regenerated plants from one coleoptile ranged from9.1 to 14.0.Four day old coleoptiles showed the highest frequency of plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) - NAA 1-naphthalene acetic acid     

Production of 2-hydroxy-4-methoxybenzaldehyde in roots of tissue culture raised and acclimatized plants of Decalepis hamiltonii Wight & Arn., an endangered shrub endemic to Southern India and evaluation of its performance vis-a-vis plants from natural habitat

Axillary buds obtained from field grown plants of D. hamiltonii were used to initiate multiple shoots on Murashige and Skoog's medium (MS) supplemented with 2 mg L(-1) 6-benzyl aminopurine (BA) and 0.5 mg L(-1) indole-3-acetic acid (IAA). Profuse rooting was achieved when the actively growing shoots were cultured on MS medium supplemented with 1.0 mg l(-1) indole-3-butyric acid (IBA). Regenerated plants were grown successfully in the plains, in contrast to wild growth in high altitudes and rocky crevices of hilly regions. Roots of different sizes from one-year-old tissue culture raised field grown plants had the same profile of 2-hydroxy-4-methoxybenzaldehyde as that of wild plants. A maximum of 0.14% and 0.12% 2-hydroxy-4-methoxybenzaldehyde was produced in roots of one year old tissue culture derived plants and greenhouse grown plants respectively.     

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l⁻¹ IAA and 1.0–3.0 mg l⁻¹ BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l⁻¹ BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.     

Regeneration of haploid plants from anther cultures of the Asiatic hybrid lily ‘Connecticut King’

A system for producing haploid plants from anther cultures was developed for the Asiatic hybrid lily ‘Connecticut King’. Anthers containing microspores at the mid- to late-uninucleate stages were cultured on MS media supplemented with various plant growth regulators. Microspores containing 3 or 4 vegetative-like nuclei were observed 2 to 3 weeks later, and yellowish nodular calluses appeared within dehisced anthers 2 to 3 months after culture. Picloram was superior to 2,4-d for inducing nodular calluses. Anthers from greenhouse-grown plants required higher concentrations of both picloram and cytokinins than those from field-grown plants and most frequently produced nodular calluses (17.6%) on MS medium containing 2 mg 1⁻¹ picloram and 2 mg 1⁻¹ zeatin. The nodular calluses regenerated many bulblets following transfer to MS medium supplemented with 0.1 or 0.5 mg 1⁻¹ picloram and 0.01 mg 1⁻¹ BA, and the bulblets developed into plantlets (bulblets with scaly leaves and roots) after transfer to MS medium containing 0.1 mg 1⁻¹ NAA. Chromosome counts of root-tip cells of 11 plantlets revealed that five were haploids (2n = 12), two diploids (2n = 24), and four mixoploid. This result suggests that at least some plantlets were of gametophytic origin.     

三叶半夏叶片一步成苗离体培养技术

以药用植物三叶半夏叶片为材料,通过比较直接和间接器官发生两种途径,建立了半夏一步成苗的快速繁殖技术体系。结果表明,经过愈伤组织阶段的一步成苗培养基为MS+0.5mg/L2,4-D+1.0mg/LKT,90d左右方可得到再生植株,植株分化率为74%,每个外植体上分化的块茎数为5.61±1.04。附加NAA与BA两种激素对一步成苗培养基进行优化,筛选出一步成苗最佳培养基MS+0.5mg/LNAA+0.5mg/LBA,60d后就可直接发育成完整植株,植株分化率为76%,每个外植体上分化的块茎数高达9.97±0·81,对这种培养基上的再生小植株进行移栽,1个月后,移栽成活率达100%。     

Mesophyll Protoplast Culture and Plant Regeneration of Oriental Planetree (Platanus orientalis)

Large populations of mesophyll protoplasts were released from the leaves of 1.5–2 month old sterile seedlings, with a high protoplast yield (3.7× 10 6g-1FW) after protoplast purification. The purified protoplasts were cultured in a modified K8p liquid medium supplemented with 0.5 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L BA. Higher density (1× 106/ml) in the initial culture of protoplasts is favourable to the division of cultured mesophyll protoplasts of this woody species among the densities tested. The protoplasts started to divide after 6 days of culture, and achieved 26.8% division frequency by 14 days. Sustained divisions resulted in mass production of cell colonies and small calli in 8 weeks. The calli further grew to 2–3mm on the gelrite-solidified K8 medium supplemented with 0.2 mg/L NAA aud 0.5 mg/L BA. Then, they were transferred onto the MSB proliferation medium with 0.1 mg/L NAA and 0.25 mg/L BA, where compact and cream-coloured calli were formed. Shoot formation was initiated on MSB differentiation medium coraming 0.5 mg/L IAA, 1 mg/L each of BA and ZT. It was observed that the frequency of shoot formation was about 28.7%. Whole plantlets were regenerated upon transferring 3 cm shoots to 1/2MS medium with 0.5mg/L IBA and 0.1mg/L BA, from which they were already transplanted into pots and grew well in the phytotron of Shanghai Institute of Plant Physiology.