HORTSCIENCE 27 (11): 1222-1223. 1992. Micropropagation of texensis from Cotyledonary Node Explants Abba Upadhyaya1, Tim D. Davis2, Daksha Sankhla, and N. Sankhla Agricultural Experiment Station, Texas A&M University Research and Extension Center, 17360 Coit Road, Dallas, TX 75252-6599 Additional index words. auxins, cytokinins, rooting, Texas bluebonnet, tissue culture Abstract. Both kinetin and BA promoted in vitro shoot formation from hypocotyl explants of Lupinus texensis Hook. placed on Murashige and Skoog (MS) medium. With either cytokinin, shoot formation was best at »4.5 µM. Adventitious root for- mation was observed only on tissue culture-derived shoots placed in MS media con- taining 5.4 to 54 µM NAA. IAA and IBA, at concentrations ranging from 5 to 55 µM, failed to stimulate rooting. Even at the optimal concentration of NAA, only 14% of the shoots produced roots. Thus, although hypocotyl explants readily produced shoots, adventitious root formation on these shoots occurred with relatively low frequency. Chemical names used: 6-benzylaminopnrine (BA); indole-3-acetic acid (IAA); indole-3- butyric acid (IBA); 6-furfurylaminopurine (kinetin); 1-naphthaleneacetic acid (NAA).

Lupinus texensis (Texas bluebonnet) is an ment of ornamentals with novel character- attractive spring-flowering leguminous an- istics (Mol et al., 1989). For instance, anti- nual that has potential as a low-maintenance sense RNA technology and chalcone syn- bedding or for use in roadside plant- thase gene constructs have been used to alter ings. This species is adapted to a variety of flower pigmentation (van der Krol et al., environmental conditions and has been grown 1990). Such constructs could be valuable in successfully in many parts of the world (An- creating new flower colors in L. texenis. As drews, 1986). Thus, there is considerable in- a prerequisite for establishing a gene transfer terest in the commercial development of this system for this plant, we initiated studies species as an ornamental (Davis et al., 1991). aimed at developing a micropropagation pro- Recent advances in plant biotechnology tocol. For other leguminous species, coty- have opened new avenues for the develop- ledonary nodes have been useful for the production of transgenic (Chee et al., Received for publication 16 Mar. 1992. Accepted 1989; Jackson and Hobbs, 1990). We found for publication 17 June 1992. The cost of pub- no information in the literature pertaining to lishing this paper was defrayed in part by the pay- the in vitro culture of L. texensis. Sator (1985), ment of page charges. Under postal regulations, however, attempted to regenerate plants from this paper therefore must be hereby marked ad- vertisement solely to indicate this fact. several other Lupinus spp. (L. polyphyllus, 1Current address: U.S. Dept. of Agriculture, Ag- L. hartwegii, L. angustifolius, and L. luteus) ricultural Research Service, Climate Stress Lab:, but only obtained limited shoot formation. BARC-W, Bldg. 001, Beltsville, MD 20705. The present report describes in vitro shoot 2To whom reprint requests should be addressed. and root formation in L. terensis.

1222 HORTSCIENCE, VOL. 27(11), NOVEMBER 1992 Table 1. Effect of kinetin and BA on in vitro and Skoog, 1962). Seedlings were cultured regenerated shoots of L. hartwegii and L. shoot count and length of Lupinus ferensis after in cylindrical glass culture vessels (50-ml ca- luteus. 4 weeks. pacity) at 25 ± 1C under a 16-h photoperiod The results of this study indicate that shoot and a photosynthetic photon flux of 40 to 50 formation from cotyledonary node explants µmol·m-2·s-1 provided by cool-white flu- of L. texensis occurs readily on MS medium orescent lamps. All subsequent experiments supplemented with cytokinins. Adventitious were conducted under these conditions. root formation on the microshoots, however, Explants containing the cotyledonary node occurs only in the presence of relatively high (10 ± 1 mm long with cotyledons removed) concentrations of NAA and even then only from 8- to 12-day-old seedlings (first true at low frequency. About half of our rooted just becoming visible at the apex) were plantlets survived transfer to ex vitro con- placed on MS medium supplemented with ditions. More research is needed to over- various concentrations of kinetin or BA. One come the recalcitrant nature of L. texensis explant was used per culture vessel. The shoots with regard to rooting and to improve number of shoots per culture and shoot lengths acclimatization so that a higher frequency of were determined after 4 weeks. For the root- plantlet regeneration can be achieved. ing experiments, 3- to 4-week-old micro- z± Values indicate SE of the mean (n ³ 60); shoots (50 ± 5 mm long) that developed on Literature Cited regression analyses indicated shoot count response the medium containing 4.4 µM BA were to either kinetin or BA was cubic and significant placed on basal MS medium supplemented Andrews, J. 1986. The Texas bluebonnet. Univ. at P = 0.05; regression analysis indicated shoot Texas Press, Austin. length response to either kinetin or BA was qua- with various concentrations of IAA, IBA, or Ball, E. 1946. Development in sterile culture of dratic and significant at P = 0.05. NAA. The percentage of microcuttings ex- stem tips and subjacent regions of Tropaeolum hibiting adventitious roots and the number majus L. and Lupinus albus L. Amer. J. Bot. Table 2. Effect of NAA on in vitro rooting of of roots per culture were determined after 28 33:301-318. tissue culture-derived shoots of Lupinus texen- days. There was a minimum of 20 cultures Chee, P.P., K.A. Fober, and J.L. Slightom. 1989. sis. Data obtained 28 days after placement of per treatment and each experiment was re- Transformation of soybean (Glycine max) by 3-week-old shoots (developed on MS medium peated at least three times. infecting germinating seeds with Agrobacter- containing 4.4 µM BA) in rooting medium. Kinetin (³4.6 µM) and BA (at all con- ium tumefaciens. Plant Physiol. 91:1212-1218. centrations used) increased the number of Davis, T.D., S.W. George, A. Upadhyaya, and J. Parsons. 1991. Improvement of seedling shoots formed at the cotyledonary node (Ta- emergence in Lupinus texensis Hook. following ble 1). For both cytokinins, shoot formation seed scarification treatments. J. Environ. Hort. was greatest at »4.5 µM. With BA, this 9:17-21. concentration resulted in »15 shoots per ex- Jackson, J.A. and S.L.A. Hobbs. 1990. Rapid plant after 4 weeks. With kinetin or BA >4.5 shoot production from cotyledonary node ex- µM, shoot formation declined but was still plants of pea. In Vitro Cell Dev. Biol. 26:835- higher than in the control. Both cytokinins 838. inhibited shoot growth, with severe stunting Lee, A.E. 1955. Growth in culture of excised por- tion of lupin embryos. Bot. Gaz. 116: 354-364. z occurring at the higher concentrations. ± Values indicate SE of the mean. Mol, J.N.M., A.R. Stuitje, A. Gerats, A.R. van y P = 0.05 determined by regression analysis. Adventitious root formation was only ob- der Krol, and R. Jorgenson. 1989. Saying it served on shoots placed in culture media with genes! Molecular flower breeding. Trends containing NAA (Table 2). Both IAA and Biotechnol. 7:148-153. Seeds were scarified in concentrated sul- IBA, at concentrations ranging from 5 to 54 Murashige, T. and F. Skoog. 1962. A revised me- furic acid for 35 to 40 min (Davis et al., µM, failed to induce rooting (data not shown). dium for rapid growth and bioassays with to- 1991) and rinsed with distilled water. Fol- The highest rooting percentage (14%) and bacco tissue cultures. Physiol. Plant. 15:473- lowing scarification, the seeds were dipped the most roots (24) were produced in the 497. in 70% ethanol for 30 sec and then rinsed medium containing 27 µM NAA. Compared Sator, C. 1985. Studies on shoot regeneration of four to five times with distilled water. There- with most other herbaceous species, L. tex- lupins (Lupinus spp.) Plant Cell Rptr. 4:126- 128. after, the seeds were treated with 5.25% so- ensis appears to be difficult to root. Adven- van der Krol, A.R., L.A. Mur, P.D. Lange, J.N.M. dium hypochlorite for 15 min and 0.2% HgCl2 titious root formation has been observed from Mol, and A.R. Stuitje. 1990. Inhibition of flower for 5 min. After being rinsed with sterile apical stem cuttings of L. albus and L. har- pigmentation by antisense CHS gene: Promoter distilled water, seeds were placed on basal twegii (Ball, 1946; Lee, 1955), but Sator and minimal sequence requirement for antisense MS medium adjusted to pH 5.8 (Murashige (1985) had little success in rooting in vitro- effect. Plant Mol. Biol. 14:457-466.

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