Shoot Regeneration Rate Research Articles

Regeneration efficiency based on genotype, culture condition and growth regulators of eggplant (Solanum melongena L.)

Several experiments were carried out to establish an efficient regenerating protocol for cultivated eggplant varieties. Among the five varieties cultured on Murashige and Skoog (MS) medium with free plant growth regulator (PGR), Nayantara performed better considering the number of shoots/explant (2.48). Considering explant types and culture conditions, better performance was observed (3.68 shoots/explant) when seed germination in the dark was proceeded by bottom hypocotyl segments cultured under dark conditions. A higher rate of shoot regeneration was observed in Nayantara when cultured in Zeatin Riboside (ZR) and Thidizuron (TDZ) supplemented MS medium. The highest number of shoots per explant was produced on MS medium supplemented with 2.0 mg/L ZR and 0.1 mg/L indole acetic acid (6.65 shoots/explant). Proliferation and elongation of the regenerated shoots were obtained in the MS medium with free PGR. The best rooting performance was observed in MS medium supplemented with 2.0 mg/L indole butyric acid. Plantlets with well developed roots and shoots were successfully transferred to soil.

The Selection of Domestically Bred Cultivars for Spray-type Chrysanthemum Transformation

질전환에 적합한 국화 스프레이 타입 품종을 선발하기 위해서 국내 연구기관에서 육성된 39개 품종 및 일본품종인 스탠다드 타입 신마를 모아 재분화율과 아그로박테리움 감염도를 조사하였다. 잎과 마디 절편체로부터의 신초 재분화를 위하여 IAA <TEX>$0.5mg{\cdot}L^{-1}$</TEX>와 BAP <TEX>$1.0mg{\cdot}L^{-1}$</TEX>가 첨가된 MS 배지를 사용하였다. 잎 절편체로부터의 신초 재분화는 BRM 품종이 가장 높았고 VS, WW, YTM 품종순으로 분화되었다. 이전에 형질전환재료로 보고된 바 있는 신마는 PK, SPP 품종과 유사한 수준을 보였다. 마디 절편체로부터의 신초 재분화는 PA 품종에서 가장 높았으며 WW 품종이 다음으로 확인되었다. 40개의 국화 품종을 대상으로 아그로박테리움에 대한 감염도를 조사한 결과, 잎 절편체에서는 WPP, YNW, VS, PP, WW, FA, PA, YMN 품종이 가장 높았고 마디 절편체에서는 WPP, PA, PK, YNW 품종이 가장 높았다. 이상의 결과를 종합해 볼 때 국화의 잎 절편체를 이용한 형질전환은 VS와 WW 품종이 가장 적합한 반면에 마디형질전환에는 PA 품종이 가장 적합한 것으로 확인되었다. To select suitable spray chrysanthemum cultivars for Agrobacterium-mediated transformation, thirty-nine (39) spray cultivars bred in the National Institutes of Korea and a standard cultivar Jinba from Japan were collected and tested for regeneration rate and Agrobacterium infection assays. MS medium with <TEX>$0.5mg{\cdot}L^{-1}$</TEX> IAA and <TEX>$1.0mg{\cdot}L^{-1}$</TEX> BAP was used for shoot regeneration from leaf disks and internodes. The shoot regeneration rate in leaf disks was the highest in cultivar BRM, followed by cultivars VS, WW and YTM. The cultivar JB (Jinba) used as a transformation material in previous reports ranked similarly to cultivars PK and SPP. In shoot regeneration from internodes, the shoot regeneration rate was the highest for cultivar PA, followed by cultivar WW. The infection rate of leaves and internodes of 40 chrysanthemum cultivars with agrobacterium was investigated. Cultivars WPP, YNW, VS, PP, WW, FA, PA and YMN showed the highest infection levels in leaves, whereas cultivars WPP, PA, PK and YNW had the highest infection levels in internodes. Considering all of these results, cultivars VS and WW were the most appropriate for gene transformation of chrysanthemum using leaves, while cultivar PA was for internodes.

In vitro plant regeneration of potato (Solanum tuberosum L.) at the rate of different hormonal concentration

One of the goals of the experiment is to standardization of HgCl2 treatment for explants sterilization. The objectives also include developing a reproducible cost effective protocol for large scale production of Solanum tuberosum of Cardinal variety plantlets from selectively better clones through plant in vitro propagation methods. Selection of growth regulators for proper multiple shoots regeneration, elongation and root induction. To produce genetically uniform plantlets within a short time capable surviving in natural condition raised in in vitro environment. Shoot tip and nodal segment explants from field grown plants were used as experimental materials in this investigation. All explants were cultured on Murashige and Skoog medium supplemented with various plant growth regulators. For surface sterilization of explants, HgCl2 (0.1%) for 2 minutes was found to be most effective for complete destroying of surface pathogens and getting healthy tissues. Shoot regeneration was observed from both shoot tips and nodal explants for the studied plant. Maximum number of shoot per culture (17) was recorded and it also obtained the highest average length of the shoot (5cm) in Murashige and Skoog medium containing no hormone. On the other hand 6-benzyl amino purine (0.2mg/l) in 3 media showed the highest rate of shoot multiplication (73%) and the highest average length (4cm). In case of Gibberellic acid (0.1mg/l) in Murashige and Skoog media showed its highest rate of shoot regeneration (82%) and the highest average length (4.5cm). From the overall experiment it was observed that shoot tips are more responsive for micro propagation. In root induction Murashige and Skoog medium supplemented with different concentration (0.5, 1, 1.5 and 2mg/l) of indol-3-acetic acid and kinetin. Indol-3-acetic acid and kinetin (1.5+1.5 mg/l) showed its lowest rate of root regeneration (40%) and the average length of the root (1.5 cm). On the contrary Murashige and Skoog medium with no hormone showed the rate of root regeneration (96%) and the highest average length of the root (2.5 cm). The supplemented Murashige and Skoog media with no hormone showed the best performance for root regeneration.Asian J. Med. Biol. Res. June 2015, 1(2): 297-303

Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos of Sapium sebiferum Roxb.

Sapium sebiferium Roxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) of S. sebiferium through direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence on in vitro morphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl−1 indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl−1 6-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement of S. sebiferium.

Production and characterization of transgenic Citrus plants carrying p35 anti-apoptotic gene

Transgenic plants of Citrus rootstock Carrizo citrange [Citrus sinensis (L.) Osbeck×Poncirus trifoliata (L.) Raf.] expressing a p35 gene from AcMNPV (Autographa californica moth Nuclear polyhedrosis virus) were produced. The p35 gene encodes a baculovirus protein which is suggested to be a negative regulator of apoptosis. Transgenic Carrizo plants carrying p35 gene in their genome had general phenotypic characteristics unchanged from wild-type (WT) plants. This includes tree height, shape, size of leaves, thorniness, levels of gas exchange, and levels/balance of major leaf plant hormones. Transgenic plants also retained their ability to respond to ethylene. Regarding the process of leaf senescence, all three transgenic lines were significantly slower as compared to the WT plants. Explants from two out of three transgenic lines sustained the treatment with Agrobacterium better than the explants from WT plants and exhibited higher shoot regeneration rate. For all three transgenic lines, transformation success rate was significantly lower than for the WT material. All these data taken together confirm that the viral p35 gene can exert its function when it is expressed in citrus plants and make them go through death-associated processes at a slower rate.

Ex vitro and in vitro Paphiopedilum delenatii Guillaumin stem elongation under light-emitting diodes and shoot regeneration via stem node culture

Paphiopedilum spp. are high value horticultural crops; however, success in Paphiopedilum spp. culture is still limited. It is therefore necessary to find out adequate solutions to improve the culture techniques of these recalcitrant species. In the present study, ex vitro shoots exposed to LEDs for shoot elongation and in vitro multiplication of P. delenatii from single nodes were investigated. One-month-old ex vitro shoots with 1.5–2.0 cm length were grown under various light conditions including monochromatic blue and red LEDs, mixtures of blue and red LEDs, and the darkness for investigations of the shoot elongation. The results indicated that the best stem elongation was obtained under monochromatic blue LED (100B) after 4 months of culture. The highest shoot regeneration rate (45 %) was also recorded in shoots cultured on liquid SH medium containing 30 g l−1 sucrose, 1.0 mg l−1 TDZ and 0.3 mg l−1 NAA with cotton wool plug as the substrate and exposed to 100B after 45 days. P. delenatii young shoots, which had formed 5 leaves, were placed into SH medium supplemented with 0.5 mg l−1 BA, 0.5 mg l−1 NAA, 30 g l−1 sucrose, and 1 g l−1 activated charcoal in the darkness for stem node elongation before transferred into greenhouse. After 120 days, the average shoot length was 10.5 cm, and five new leaves and five single nodes per shoot were observed. These shoots then were maintained under fluorescent light for 60 days. Although the shoots regenerated new leaves, no new node was observed. After 180 days of culture, each shoot was cut into five single nodes, and cultured on fern fibers. These shoots showed to grow vigorously after 12 months.

Physiological and biochemical parameters influencing ex vitro establishment of the in vitro regenerants of Albizia lebbeck (L.) Benth.: an important soil reclaiming plantation tree

Present approach demonstrates a speedy and competent in vitro regeneration method for A. lebbeck. The hypocotyl explants (HP) excised from a 15-day-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-Benzyladenine (BA), Kinetin (Kn) and 2-Isopentenyl adenine (2-iP) singly as well as in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA; 0.1, 0.5, 1.0, 1.5 and 2.0 μM). The best shoot regeneration rate (88 %) with number of shoots (34.00 ± 1.15) and shoot length (6.30 ± 0.05 cm) per explant was obtained on MS + BA (7.5 μM) + NAA (0.5 μM) after 8 weeks of culture. While, the explants cultured onto the MS + TDZ (Thidiazuron; 0.5, 1.0, 2.5, 5.0 and 7.5 μM) singly showed maximum shoot proliferation response of 77 % when, TDZ exposed cultures were transferred on the BA (7.5 µM) + NAA (0.5 µM) supplied MS medium producing maximum no. of shoots (20.30 ± 0.32) and shoot length (5.15 ± 0.08 cm) after 8 weeks of culture. The regenerated shoots were rooted by using ex vitro rooting method and successfully acclimatized by using a simple acclimatization procedure of 70 days, primarily in soilrite for 28 days and finally in soilrite + garden soil (1:1) for 70 days under culture room conditions (150 PPFD). This acclimatization period under the optimized conditions of culture room ensures a high survival rate (80 %) of the plantlets in the field conditions. Photosynthetic pigments (Chlorophyll a, b and carotenoid content) evaluated during acclimatization period showed a decreasing trend in the initial 14 days afterwards a significant increase in the subsequent period of 21–70 days. While all the stress parameters MDA and H2O2 content showed decreasing trend all through the acclimatization, concurrently all the tested antioxidative enzymes (SOD, CAT, APX and GR) exhibited an increasing trend.

Direct and indirect in vitro plant regeneration and the effect of brassinolide on callus differentiation of Populus euphratica Oliv.

Populus euphratica Oliver is a poplar species with very strong tolerance to drought, high temperature, alkalinity and salinity, but it is difficult to propagate due to its intrinsic physiological defects. In this study, an efficient in vitro plant regeneration protocol for P. euphratica has been established by using the young leaves and stems as explants. With this protocol, among the six types of explants tested, five ones simultaneously produced shoots and calli on the MS medium supplemented with 0.5mg/L α-naphthalene acetic acid (NAA) and 0.5mg/L N6-benzyl aminopurine (BA). Besides, the calli produced further differentiated into adventitious buds or shoots on the MS medium supplemented with 0.1mg/L NAA, 0.5mg/L BA and 10−4–10−1mg/L brassinolide (BL). The regeneration rates of buds/shoots that differentiated from the explants and calli were up to 52.7% and 73.3% respectively. Up to 75% of the shoots rooted on the 1/2 MS medium with indole-3-butyric acid (IBA) and grew well in outdoor soil. Moreover, it was found that in the presence of BL, the shoot regeneration rate of callus was increased by 4–57%, and the time for shoot regeneration was shortened by 20–30days. This is the first report that describes direct and indirect organogenesis simultaneously occurring in P. euphratica, and that BL can significantly promote the differentiation of P. euphratica calli into shoots.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins (AGPs) and hydrogen peroxide (H2O2) have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 (KN199) and Xinchun 9 (XC9), together with low-frequency regeneration wheat line Chinese Spring (CS), presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L−1 AGP or 0.005 to 0.01 ‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L−1 AGP application level in callus induction medium plant regeneration rates of 8.49, 409.06 and 283.16% were achieved for Jimai 22 (JM22), Jingdong 18 (JD18) and Yangmai 18 (YM18), respectively; whereas at 100 mg L−1 AGP level, CS (105.44%), Chuannong 16 (CN16) (80.60%) and Ningchun 4 (NC4) (62.87%) acted the best. Moreover CS (79.05%), JM22 (7.55%), CN16 (101.87%), YM18 (365.56%), Yangmai 20 (YM20) (10.48%), and CB301 (187.40%) were more responsive to 0.005 ‰ of H2O2, and NC4 (35.37%) obtained the highest shoot regeneration rates at 0.01 ‰ of H2O2. Overall, these two methods, inter-culture and AGP (or H2O2) application, can be further applied to wheat transgenic research.

Direct Shoot Regeneration from Callus of &amp;lt;i&amp;gt;Melicope lunu-ankenda&amp;lt;/i&amp;gt;

Melicope lunu-ankenda is commonly used in traditional medicine. The conventional propagation method for this species is inefficient due to low propagation rate and its lengthy period to maturity. In addition, insufficient planting materials often pose a problem for the plantation sector. The tissue culture technique is best alternative to overcome the problems. The callus induction and direct shoot regeneration protocols for M. lunu-ankenda were established. Callus was successfully initiated from leaves explants cultured in MS medium added with 2,4-D at concentrations 0.5 to 5.0 mg/L singly or in combination with NAA at concentrations 1.0 to 10 mg/L. Shoot was regenerated from callus in phytohormone-free medium, BAP at concentrations 0.5 - 5.0 mg/L singly or in combination of BAP with NAA or 2,4-D at concentration 0.5 and 1.0 mg/L, respectively. BAP at 1.0 mg/L induced the highest shoot regeneration rate (80%) and number of plantlet per calli. The established methods might be used for production of phytochemicals and plantlets in large scale.

Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers.

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog's (MS) medium supplemented with 2.0mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25-0.5mg/l NAA (1-napthalene acetic acid) and 3% (w/v) sucrose within 4weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0mg/l BA, 0.5mg/l NAA, 40mg/l Ads (adenine sulphate) within 4-6weeks of subculture. Inclusion of 0.25-0.50mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25-1.0mg/l NAA or indole-3-butyric acid (IBA) and 2% (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5mg/l NAA with 3% (w/v) sucrose. About 80% of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.

수박(Citrullus lanatus (thunb.) Matsum. &amp; Nakai) 자엽 절편의 재분화에 미치는 생장조절물질의 영향

수박에서 과형, 과피, 과육 색이 서로 다른 중소과종인 3가지 계통의 재분화 조건을 확립하고자 각 계통의 유묘의 자엽절편을 배양하여 캘러스 및 신초 형성에 미치는 생장조절물질의 효과를 조사한 결과는 다음과 같다. 각 발아된 유묘 자엽의 캘러스 형성은 모든 계통에서 유묘의 자엽 부위 중 기부(proximal)의 끝부분에서 가장 높은 효율을 나타내었다. 또한 캘러스 형성에 미치는 생장조절제의 영향은 'B02'의 경우 1.0 mg/L BAP와 0.5 mg/L IAA를 혼합한 처리구에서, 'B05'의 경우 1.0 mg/L BAP 단독 처리구에서, 'D04'의 경우 3.0 mg/L BAP와 0.1 mg/L IAA를 혼합한 처리구에서 가장 높게 나타나 계통에 따라 생장조절제의 조건이 달라짐을 확인하였다. 신초 형성율은 'B02'와 'B05'의 경우 1.0 mg/L BAP와 0.5 ㎎/L IAA를 혼합한 처리구에서 'D04'의 경우 1.0 mg/L BAP 단독 처리구에서 가장 효과적인 것으로 확인되었다. 재분화된 식물체의 길이신장은 MS 기본 배지에 생장조절제로 1 mg/L BAP가 첨가된 배지에서 가장 효과적이었으며, 이들 식물체의 최적 발근은 'B02' 경우 0.5 mg/L IBA, 'B05' 경우 0.1 mg/L NAA, 'D04' 경우 0.5 mg/L IAA를 첨가된 배지 조건에서 이루어졌다. In this study, we developed a high frequency watermelon regeneration system using three breeding lines ('B02', 'B05' and 'D04') of watermelon (Citrullus lanatus (thunb.) Matsum. & Nakai) which are differed in their fruits in shape, color of pericarp and flesh. The highest frequency of explants with callus was observed by using explants that consist of cotyledon proximal part end in all breeding lines, and the highest rate of callus induction was obtained on MS medium containing 1.0 mg/L BAP + 0.5 mg/L IAA for 'B02' (94%), 3.0 mg/L BAP for 'B05' (95%), 3.0 mg/L BAP + 0.1 mg/L IAA for 'D04' (90%). The highest shoot regeneration rates from derived callus were obtained on MS medium containing 1.0 mg/L BAP + 0.5 mg/L IAA for 'B05' (94%), and then a 'B02' (81%) with a same culture conditions, and the lowest regeneration was obtained on MS medium containing 1.0 mg/L BAP for 'D04' (56%). Regenerated plants showed the best rates of root formation on MS containing 0.1 mg/L IBA for 'B02' (67%), 0.1 mg/L NAA for 'B05' (87%), 0.5 mg/L IAA for 'D04' (88%). The regenerated plants showed a 100% survival rate in soil condition. The tissue culture and regeneration conditions obtained from this study will be useful for regenerating plants in breeding applications, and will be a useful tool for further genetic transformation studies on watermelons.

잎 절편 배양을 이용한 배 '신고' (Pyrus pyrifolia cv. Niitaka)의 신초 재분화

남방형 동양배(Pyrus pyrifolia Nakai)의 형질전환은 적합한 재분화 체계가 구축되어 있지 않아 어려운 실정이다. 본 연구의 목적은 배 신고 품종(P. pyrifolia cv. Niitaka)의 효율적인 재분화 체계를 개발하기 위해 식물생장조절제의 농도, 탄소원, 지지체 등이 신초 재분화에 미치는 영향을 연구하기 위하여 수행하였다. 생장조절제의 영향을 알아보기 위하여 NAA 0.01, 0.05, 0.1, 0.5 mg/L과 BA 3, 5, 10 mg/L을 조합하여 처리한 결과 BA 5 mg/L와 NAA 0.01 mg/L의 조합에 Daishin agar 7 g/L과 Linsmaier and Skoog (LS) vitamin을 따로 첨가한 Murashige and Skoog (MS) 배지에서 재분화율이 20%로 나왔다. 효율적인 재분화 체계를 위한 탄소원으로는 sorbitol 30 g/L가 사용되었으며 배 '신고'의 재분화에는 sucrose보다 sorbitol이 더 적합한 것으로 판단된다. 재분화가 잘 되지 않는 배 '신고'의 신초 재분화율을 높이기 위해 지지체로 plant agar와 Daishin agar를 사용하였으며 Daishin agar에서 재분화율이 더 높게 나타났다. Genetic manipulation of pear (Pyrus pyrifolia Nakai) breeding is still difficult due to lack of reliable regeneration system. The aim of this research is to establish shoot regeneration system from leaf explants for pear (P. pyrifolia cv. Niitaka) using various concentrations of plant growth regulators and carbon source supplemented to medium. The highest regeneration rate of about 20% was found on a medium containing 4.4 g/L of Murashige and Skoog (MS) without vitamins, Linsmaier and Skoog (LS) vitamins were added separately. Leaf explants of pear were cultured on MS medium containing 7 g/L of Daishin agar supplemented with various concentrations of NAA (0.01, 0.05, 0.1, 0.5 mg/L) in combination with BA(3, 5, 10 mg/L) for shoot regeneration. In medium with 5 mg/L of BA and 0.01 mg/L of NAA, adventitious shoot regeneration rate was higher than others treated. The optimal results were observed using MS medium supplemented with 30 g/L sorbitol as carbon source on regeneration system. Sorbitol is considered better carbon source than sucrose for shoot regeneration of pear (P. pyrifolia cv. Niitaka). In order to increase of shoot regeneration in pear (P. pyrifolia cv. Niitaka), plant agar and Daishin agar used as gelling agents, Daishin agar is more efficient in shoot regeneration.

포도 (Vitis labrusca L.)의 직접 재분화 방법을 이용한 식물체 재분화와 형질전환

Abstract Efficient regeneration methods and transformation system are a priority for successful application of genetic engineering to vegetative propagated plants such as grape ( Vitis labrusca L.). This research is to establish shoot regeneration system from plant explants for ‘Campbell Early’, ‘Tamnara’, ‘Heukgoosul’, ‘Heukbosek’ using two types of plant growth regulators supplemented to medium. The highest adventitious shoot regeneration rate of 5% was achieved on a medium containing of Murashige and Skoog (MS) inorganic salts and Linsmaier and Skoog (LS) vitamins, 2 mg/L of TDZ and 0.1 mg/L of IBA. Leaf tissue of ‘Campbell Early’, was co-cultivated with Agrobacterium strains, LBA4404 containing the vector pBI121 carrying with CaMV 35S promoter, gus gene as reporter gene and resistance to kanamycin as selective agent, the other Agrobacterium strains, GV3101 containing the vector pB7 WG2D carrying with mPAP1-D gene. mPAP1-D is a regulatory genes of the anthocyanin biosynthetic pathway. ‘Campbell Early’ harboring

English

A plant regeneration system was established from cotyledon explants of chieh-qua (Benincasa hispida Cogn. var. Chieh-qua How). To obtain optimal conditions of adventitious shoot induction, the cotyledon explants were excised from seedlings of different genotypes as well as seed germination conditions, and then cultured on media containing different concentrations of 6-benzylaminopurine (6-BA)/1-naphthaleneactic acid (NAA). Among the eight genotypes, the highest rate of shoot regeneration was obtained from the cotyledons of inbred line A39. The adaxial portion of cotyledons of seedlings cultured for 3&nbsp;days in darkness and 1&nbsp;day in light was the appropriate explants for adventitious shoot organogenesis. The highest frequency of adventitious shoot organogenesis (52.2%) and mean number of shoots per explant (4.2) were achieved on Murashige and Skoog (MS) medium supplemented with 6&nbsp;mg l-1 6-BA and 0.2&nbsp;mg l-1 NAA. Adventitious shoots were observed to regenerate directly from cotyledons rather than from calli. A medium supplemented with AgNO3 was not beneficial for shoot induction. Adventitious shoots were elongated in MS medium supplemented with 3&nbsp;mg l-1 6-BA and 0.2&nbsp;mg l-1 NAA. Elongated shoots were rooted in &frac12; MS medium with 0.5&nbsp;mg l-1 indole-3-acetic acid (IAA). Regenerated plantlets were transferred to a greenhouse for about 1&nbsp;month, and subsequently transplanted into an open field. Key words: Benincasa hispida, chieh-qua, adventitious shoot, genotypes, plant regeneration.

Micropropagation of Origanum acutidens (HAND.‐MAZZ.) IETSWAART Using Stem Node Explants

Origanum acutidens (HAND.-MAZZ.) IETSWAART is a promising ornamental plant that can be widely used in landscape management. It is endemic to Eastern Anatolian region of Turkey. Tissue culture has not been used to micropropagate it. The study reports stem node explants from one-week-old seedlings of the plant for successful micropropagation. The stem nodes were cultured on MS medium containing 0.6, 1.2, 1.8, and 2.4 mg/L BAP with 0.2 mg/L NAA. Visible effects of culture media on shoot proliferation were recorded. Shoot regeneration rate was maximum on MS medium containing 1.80 mg/L BAP-0.2 mg/L NAA. The micropropagated shoots were rooted on MS medium containing 0.2 mg/L NAA. All microrooted plantlets survived during acclimatisation on peat moss. It was concluded that O. acutidens can be successfully micropropagated under in vitro conditions.

Regeneration of multiple shoots from petiole callus of Viola serpens Wall

Experiments were conducted to develop methodology for in vitro propagation and rapid multiplication of Viola serpens Wall. using petiole explant. The MS medium supplemented with 2, 4-D (6.78 μm) was found most suitable for callus induction in petiole explant. The best growth response and higher rate of shoot regeneration from petiole callus was observed on MS medium containing BAP (11.10 μm) as the average number of shoots could be increased to 36.4 on fourth successive subculturing. Higher rooting response with larger number of roots were observed in shoots inoculated on the half-strength MS medium supplemented with IBA (19.68 μm).

황금배(Pyrus pyrifolia Nakai) 잎 조직으로부터 기내 신초 재분화

본 연구는 '황금배'('Whangkeumbae')의 엽 절편체로부터 형질전환 체계 확립을 위한 효율적인 재분화 체계 확립을 위해 식물 생장조절제와 주요 탄소원인 sucrose, sorbitol이 식물체 재분화에 미치는 영향을 조사하기 위해 수행되었다. 잎 절편체를 MS 배지에 TDZ 농도를 각각 0.1, 0.25, 0.5, 1, 2.5, 5 mg/L으로 하여 IBA 0.3 mg/L을 혼용 처리한 결과 TDZ 0.25 mg/L 농도에서 61.1%의 가장 양호한 재분화율을 보였으며 TDZ 2.5 mg/L 이상의 농도에서는 신초의 재분화율이 급격히 감소하였다. IBA와 IAA 농도에 의한 신초의 재분화율은 TDZ 0.5 mg/L와 IAA 혼용 처리한 구에서 전체적으로 높은 신초의 재분화율을 보였으며 특히 TDZ 0.5 mg/L + IAA 0.3 mg/L 처리구에서 76.7%의 가장 높은 재분화율을 보였다. 서로 다른 탄소원으로써 sucrose와 sorbitol의 영향에 대하여 15 g/L, 30 g/L 농도로 처리한 결과 sorbitol 30 mg/L에서 가장 높은 재분화율을 보였고 또한 절편체 당 신초의 수도 3.5개로 가장 양호하였다. 따라서 sorbitol이 sucrose보다 '황금배'의 신초의 재분화에 있어서 효과적이었다. In order to establish an efficient adventitious shoot regeneration conditions from leaf explants for Asian pear 'Whangkeumbae', the effect of concentration and kinds of plant growth regulator and carbon source was investigated. Leaf explants of cultures grown on Murashige and Skoog (MS) medium containing 8 g/L plant agar were used. When the medium contained 0.25 mg/L thidiazuron (TDZ) and 0.3 mg/L indolebutyric acid (IBA), the adventitious shoot regeneration rate (ASRR) was greater as 61.1% than others treated and higher TDZ concentrations (2.5 and 5 mg/L) treatment significantly reduced the ASRR. As the effect of IBA and indoleacetic acid (IAA) concentration on the ASRR, 0.5 mg/L TDZ plus different concentration of IAA exhibited relatively high ASRR and 0.5 mg/L TDZ plus 0.3 mg/L IAA showed the highest ASRR of 76.7%. Also the effect of sucrose and sorbitol as carbon source on regeneration was examined. The highest ASRR and the most shoots per explants averaged 94.4% and 3.49 by treatment of 30 mg/L sorbitol, respectably. Sorbitol is considered better carbon source than sucrose for shoot regeneration of 'Whangkeumbae' pear.

Duration of sucrose preculture is critical for shoot regrowth of in vitro-grown apple shoot-tips cryopreserved by encapsulation-dehydration

A simple and efficient cryopreservation protocol using encapsulation-dehydration was established for in vitro-grown shoot-tips of apple ‘Gala’ (Malus × domestica Borkh.). Shoot-tips, of 2.0 mm in length and with 5–6 leaf primordia, excised from 4-week-old shoot stock cultures, without cold-hardening, were encapsulated into beads, each being about 5 mm in diameter and containing a single shoot-tip. The beads were precultured on MS medium containing 0.5 M sucrose for 7 days. The precultured beads were dehydrated by air-drying to reduce the water content of the beads to about 22–20 % in 5–7 h, followed by a direct immersion in liquid nitrogen for 1 h. Frozen shoot-tips were re-warmed in a water bath at 38 °C for 2 min and post-cultured on a recovery medium for shoot regrowth. This protocol was successfully applied to four Malus species and one hybrid, among which M. micromalus and M. robusta are wild species native to China. The highest and lowest shoot regeneration rates were found in ‘Gala’ (75 %) and ‘Wangshanhong’ (36 %), with a mean shoot regrowth rate of 61 % attained for the seven Malus genotypes tested. Histological studies revealed that shoots could be regenerated in cryopreserved shoot-tips only when many cells in the leaf primordia and most of the cells in the apical dome survived following cryopreservation. Morphologies of the regenerated plantlets were identical to those from the in vitro stock cultures. Therefore, the encapsulation-dehydration procedure developed in the present study should provide a technical support for setting-up Malus cryo-banking in China.

In vitro propagation of Stemona hutanguriana W.Chuakul, an endangered medicinal plant

In vitro propagation of Stemona hutanguriana W.Chuakul, an endangered species of Stemonaceae, was established. Nodal and internodal explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) alone or in combination with 1-naphthaleneacetic acid (NAA). When cultured on MS medium supplemented with 18.16μM TDZ with 0.54μM NAA for 8weeks and then on MS medium without plant growth regulator (PGR) for 8weeks, nodal explants demonstrated a responding frequency of 91.67% and a shoot regeneration rate of 5.46 shoots/responding explant. Furthermore, internodal explants demonstrated a responding frequency of 17.17% with 11.17 shoots/responding explant. The regenerated shoots were rooted using a two-step protocol by culturing for 4weeks on MS medium supplemented with various concentrations of NAA and Indole-3-butyric acid (IBA) and 8weeks on MS medium without PGR. When 5.40μM NAA was used as a PGR, the maximum root induction rate was 69.45%. Regenerated plantlets were successfully transferred to soil.