Morphactin-kinetin interaction on growth and shoot formation in tobacco callus cultures

Pith-derived calluses of Nicotiana tabacum L. cv. Wisconsinno. 38 were inoculated on an RM-1964 medium containing variousconcentrations of a morphactin, chlorflurenol (CFl) and kinetin(KIN). An addition of KIN (0.1–2 mg/liter) alone was effectivefor shoot formation from the calluses, but a high dose (10 mg/liter)resulted in the inhibition of growth and in no differentiation.The inhibitory effect of a high dose of KIN was counteractedwith CFl. Three combinations of KIN and CFl; CFl (0.1 mg/liter)$KIN(2.0 mg/liter); CFl (0.1 mg/liter)$KIN (10.0 mg/liter) and CFl(1.0 mg/liter) $KIN (2.0 mg/liter), were successful for 100%shoot redifferentiation in inoculated calluses. An appropriatebalance between CFl and KIN seems to be involved in shoot formation.The present results can best be interpreted by assuming thatCFl acts as an auxin in cultured tissues. (Received January 16, 1975; )     

Tissue culture inHaworthia

Effects of three different auxins and kinetin in various combinations on greening and redifferentiation of the callus ofHaworthia setata were investigated. All auxins at the concentration of 50 mg/l inhibited callus greening. NAA in combination with kinetin promoted both callus greening and production of redifferentiated shoots. Low concentrations of IAA without kinetin promoted redifferentiation of shoots, but not callus greening. Addition of 2,4-D completely inhibited both greening and redifferentiation regardless of the level of kinetin except for the effects on shoot formation in the medium with 0.1 mg/l 2,4-D added. The calluses with the highest chlorophyll content were observed in the medium containing 2.0 mg/l kinetin without any auxins or with 0.1 mg/l NAA added. Most frequent shoot redifferentiation was observed in the medium containing 0.1 mg/l IAA without kinetin (redifferentiation rate; 67%), followed by the medium containing 10 mg/l NAA with 2.0 mg/l kinetin (44%), and 0.1 mg/l 2,4-D with 0.2 mg/l kinetin (33%). Generally, higher degrees of greening were associated with better growth. However, the auxins (IAA, NAA and 2,4-D) given at concentrations optimal for growth did not exhibit the highest degree of callus greening. Differences of the three auxins in their actions and interaction with kinetin were disclosed. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 423     

Plant regeneration from leaf petioles in <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Summary Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production; however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium+8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf dise calluses, and cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf dise calluses regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2±0.5 versus 2.0±0.3, than did leaf dise calluses on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata.     

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.     

Thidiazuron Induced Regeneration in Cuminum cyminum L

In this communication we report shoot organogenesis from hypocotyl explants in cumin (Cuminum cyminum) genotype RZ-19 by the use of thidiazuron (TDZ). Various levels of TDZ were incorporated in MS basal medium to induce regeneration. Regeneration was achieved with a frequency up to 30% on 0.5 and 0.1 mg l^?1 concentration of TDZ. Shoots once produced could be multiplied on 0.5 mg l^?1 kinetin (KN) at the rate of approximately 8 shoots per regenerated shoot. These multiplied shoots could go through 3–4 multiplication cycles after which they root on 1.0 mg l^?1 IAA.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

Efficient in vitro regeneration of fertile plants from corm explants of Hypoxis hemerocallidea landrace Gaza—The “African Potato”

We present efficient protocols for the regeneration of fertile plants from corm explants of Hypoxis hemerocallidea Fisch. & C. A. Mey. landrace Gaza, either by direct multiple shoot formation or via shoot organogenesis from corm-derived calluses. The regeneration efficiency depended on plant growth regulator concentrations and combinations. Multiple direct shoot formation with high frequency (100% with 5–8 shoots/explant) was obtained on a basal medium (BM) supplemented with 3 mg/l kinetin (BM1). However, efficient indirect regeneration occurred when corm explants were first plated on callus induction medium (BM2) with high kinetin (3 mg/l) and naphthalene acetic acid (NAA 1 mg/l), and then transferred to shoot inducing medium (BM3) containing BA (1.5 mg/l) and NAA (0.5 mg/l). Shoot regeneration frequency was 100% and 30–35 shoots per explant were obtained. The regenerated shoots were rooted on a root inducing medium (BM4) containing NAA (0.1 mg/l). Rooted plantlets were transferred to the greenhouse. The regenerants were morphologically normal and fertile. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. Efficient cloning protocols described here, have the potential not only to substantially reduce the pressure on natural populations but also for wider biotechnological applications of Hypoxis hemerocallidea—an endangered medicinal plant.     

Plant regeneration via direct and indirect adventitious shoot formation and chromosome-doubled somaclonal variation in Titanotrichum oldhamii (Hemsl.) Solereder

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing 0.1 mg l⁻¹ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing 0.1 mg l⁻¹ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.     

Rapid and efficient regeneration in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill] from whole cotyledonary node explants

A rapid and efficient regeneration protocol was established for soybeans [Glycine max (L.) Merrill]. Whole cotyledonary node explants were collected from aseptic seedlings cultured on MSB₅ medium containing 0.4 mg l⁻¹ N⁶-benzyladenine (BA). The effects of the plant growth regulators BA, kinetin (KT), indole-3-butyric acid (IBA) as well as the explant genotype on shoot regeneration were evaluated by the orthogonal design [L₁₆(4⁵)]. The process of shoot development was also observed. The regenerated shoots were elongated on MSB₅ medium and sufficiently elongated shoots were rooted on MSB₅ medium containing 0.5 mg l⁻¹ IBA. The results showed that all three of the plant growth regulators significantly affected shoot regeneration, with BA exhibiting the greatest benefit. The best combination for shoot regeneration was MSB₅ medium supplemented with 3.0 mg l⁻¹ BA , 0.2 mg l⁻¹ IBA and 0.5 mg l⁻¹ KT on Hefeng 25 genotype. Under these most favorable conditions, one explant could regenerate 30–35 shoots. Plantlets could be obtained within 2 months. The result of histocytological analysis indicated that protein accumulated gradually and reached to peak at late shoot bud formation.     

Tissue culture inHaworthia

Effects of three auxins and kinetin on growth of the calluses of two species ofHaworthia, H. aristala andH. setata, were investigated. Stock calluses derived from the flower buds of these species were maintained for two years on RM-1964 agar medium containing 5 mg/l NAA. Small pieces of the stock calluses were transferred to the basal medium containing either auxin, IAA, NAA or 2,4-D in six different concentrations (0.1–50 mg/l) combined with three concentrations (0–2 mg/l) of kinetin; in total, 54 kinds of media were used. Fresh weight of the calluses was measured 0 to 30 days from transfer and transformed to the natural logarithm. The linearity of their growth curves against the culture period was tested. The growth curves of theH. aristata calluses grown in dark and under continuous light and that of theH. setata callus grown in dark gave similar regression coefficients of 0.07 to 0.11, indicating that the doubling time of the callus mass was about 6.3 to 10.1 days. After 42 to 50 days from inoculation, the fresh weight of each individual callus was recorded, and the data were statistically analyzed. All auxins at the concentration of 50 mg/l significantly inhibited callus growth. Kinetin did not affect growth of theH. aristata callus in dark, while its effect on theH. setata callus was detected under light. Interaction of kinetin was found with IAA and 2,4-D inH. aristata and with IAA and NAA inH. setata. REsponses of theH. aristata callus to auxins and kinetin, when grown in dark, were different in several points from those of theH. setata callus grown under light. The best callus growth was observed in the following media; 0.2 mg/l kinetin supplemented with 1 mg/l IAA, or 0.5 mg/l 2,4-D, and 2 mg/l kinetin with 0.5 mg/l NAA inH. aristata, and 0.2 mg/l kinetin supplemented with 1 mg/l IAA, 5 mg/l NAA or 0.1 mg/l 2,4-D inH. setata. Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 413.     

In Vitro Regeneration from Internodal Explants and Somaclonal Variation in Chickpea (Cicer arietinum L)

Protocols have been developed for the in vitro regeneration of plants from calli derived from internode explants of chickpea (Cicer arietinum L) cv Pusa-372. Callusing was induced on both B5 and MS media supplemented with different combinations and concentrations of auxins and cytokinins, but shoot regeneration was achieved only in B5 medium supplemented with 4.0 mg l^?1 IAA and 0.5 mg l^?1 BAP after serial subculture of callus on media with increasing concentration of IAA and constant concentration of BAP. Rooting could not be achieved in in vitro regenerated shoots on any one of the media tried. Complete plantlets were, therefore, developed through grafting of the in vitro regenerated shoot on established root stock. The grafting methodology was found to be highly efficient and reproducible. The somaclones developed produced viable seeds which showed variability in terms of seed colour and seed weight. Thus, the protocols developed in this study remove one important bottleneck in the development of transgenic chickpea.     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Effect of brassinolide on callus growth and regeneration in Spartina patens (Poaceae)

The effect of brassinolide (BL) on cultured calluses of Spartina patens (Ait.) Muhl. (Poaceae), a halophyte monocot was studied. BL at 0.03–0.04 mg l^–1 at fixed concentrations of IAA (0.2 mg l^–1) and BA (3.0 mg l^–1) added in MS medium increased the ratio for fresh weight (CIRFW) to dry weight (CIRDW) by 96–111% and 235–326%. Similarly, in callus regeneration capacity, BL at 0.03 mg l^–1 was most effective, increasing the shoot regeneration ratio (SRR) by 425%. BL at 0.04 mg l^–1 had not such an increasing effect as BL at 0.03 mg l^–1, which increased SRR by 79%. However, BL at 0.005 mg l^–1 promoted regenerated shoot growth most significantly, increasing the shoot height increasing ratio (SHIR) by 395% after a 40-day culture. BL at 0.05 mg l^–1 was least effective in the callus regeneration and regenerated shoot growth, decreasing SRR by 27% and SHIR by 52%. Present results suggest that BL at 0.03 mg l^–1 is suitable for the callus growth and shoot regeneration, while BL at 0.005 mg l^–1 effectively enhanced the regenerated shoot growth.     

Plant regeneration from hairy roots and calluses of periwinkle Vinca minor L. and foxglove purple Digitalis purpurea L.

Plants regenerated from hairy root culture and calluses of foxglove purple and periwinkle have been obtained. It was found that organogenesis in hairy root culture occurs spontaneously on hormone-free medium but with varying efficiency. The frequency of direct shoot formation from root cultures was up to 60% in Digitalis and 3.7% in Vinca. The addition of 1 mg/L BA, 0.1 mg/L NAA, and 5% sucrose to B5 medium increases regenerative capacity of periwinkle up to 19.1%. Regenerated plants showed morphological features typically seen in Ri-transgenic plants. They include growth and plagiotropism of the root system, increased shoot formation, changed leaf morphology, and short internodes.     

In vitro regeneration from excised leaves of Flaveria trinervia (Sprengel) C. Mohr

Flaveria trinervia (Compositae) leaves are used for the treatment of jaundice and fever. From the leaf callus cultures regeneration of plantlets has been achieved. The results showed that BAP greatly stimulated the bud formation in concentrations ranging from 2–5 mg l^–1 than at very low concentrations (0.2–1.0 mg l^–1). Roots developed on the regenerated shoots, over a range of treatments, but were most prolific in the medium containing 1 mg l^–1 IAA. Histological observations revealed that cultured spongy cells of the mesophyll were greatly enlarged and underwent repeated cell divisions leading to the formation of hard nodular callus from which shoot buds differentiated. The shoots obtained were readily rooted and transplanted into glass houses. Cytological studies of the callus showed abnormalities such as bridges, endomitosis and multinucleolate conditions. Root tip squashes of the regenerated plants showed no variations and were diploid in chromosome number.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA napthalene acetic acid - IAA indole acetic acid - BAP 6-benzyl aminopurine - Kn kinetin     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

One step shoot tip multiplication and rooting of Digitalis thapsi L.

Shoot tips from seedlings of Digitalis thapsi L. were cultured on Murashige and Skoog's medium and the effect of various auxins (2,4-D, NAA and IAA) were analyzed alone or in combination with cytokinis (BA and kinetin). Shoot multiplication and direct rooting of the new shoots were obtained after four weeks of culture in MS medium without hormones, but callus formation and the appearance of abnormal phenotypes were frequent. The addition of auxins to the cultures prevented the formation of callus but not the appearance of variant phenotypes. Both drawbacks could be avoided by combination of NAA or IAA with BA or kinetin. The best results for shoot multiplication and direct rooting were obtained with 0.5 mg l^-1 NAA and 0.1 or 0.5 mg l^-1 kinetin.Abbreviations BA 6-benciladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin kinetin - NAA naphtalene acetic acid - MS Murashige and Skoog     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses

Panax ginseng calluses were cultured for 5 weeks on solid MS medium supplemented with kinetin 0.46 mM (0.1 mg l^–1) and 2 mg l^–1 of 2,4-D (9.05 mM), IBA (9.98 mM) or NAA (10.74 mM). In the conditions studied, 2,4-D inhibited the organogenic capacity of the calluses, whereas IBA or NAA increased this capacity. IBA induced the formation of a high number of buds and roots, but the roots were thin and necrotized. Calluses grown with NAA produced fewer buds and roots than those grown in IBA medium, but the roots were thick and showed good growth. The highest ginsenoside content was found in root forming calluses grown in the presence of NAA.In calluses forming roots or buds, 2,4-D, NAA and especially IBA increased the Rb group of ginsenosides rather than that of the Rg group.