PiantCeU Reports

Plant Cdl Reports (t991) 10:183-186

9 Springcr-Verlag1991

Regeneration of Cladrastis lutea (Fabaceae) via somatic embryogenesis Lori A. Weaver and Robert N. Trigiano Institute of Agriculture, Department of Ornamental Horticulture and Landscape Design, P.O, Box 1071, University of Tennessee, KnoxvilJe, TN 37901, USA Received January 3, 1991/Revised version received April 29, 1991 - Communicated by J. M. Widholm

I m m a t u r e e m b r y o s from 5 C ! a d r a s t i s lutea (Michx.) K. Koch (yel!owwood) trees w e r e i n i t i a l l y c u l t u r e d on m o d i f i e d S c h e n k and H i l d e b r a n d t m e d i u m (SH) c o n t a i n i n g e i t h e r 4.5, 9.0, 13.5 or 23 #M 2,4-D. O n e - t h i r d of the explants were transferred to SH medium s u p p l e m e n t e d w i t h 25.0 #M N A A after 2 and 3 weeks r e s p e c t i v e l y . The r e m a i n i n g e x p l a n t s w e r e i n c u b a t e d on the initial 2,4-D c o n t a i n i n g m e d i a for 6 weeks. Groups of s o m a t i c embryos formed d i r e c t l y only at the p r o x i m a l end of cotyledons; only a few formed as single embryos. The g r e a t e s t n u m b e r s w e r e formed from zygotic e m b r y o s e x p l a n t e d from 6-8 w e e k s p o s t - a n t h e s i s and i n i t i a l l y c u l t u r e d on m e d i u m c o n t a i n i n g 9 or 13 ~M 2,4-D. However, all treatments supported somatic e m b r y o g e ne s i s . In the s e c o n d year, e x p l a n t s were i n i t i a l l y c u l t u r e d on SH m e d i u m c o n t a i n i n g e i t h e r 9.0, 13.5, or 23 #M 2,4-D and then t r a n s f e r r e d to SH m e d i u m c o n t a i n i n g 4.0 #M A B A after 2 or 3 weeks. A B A did not a f f e c t the d e v e l o p m e n t of s o m a t i c embryos. Six of 21 s o m a t i c e m b r y o s g e r m i n a t e d on h a l f - s t r e n g t h SH m e d i u m w i t h o u t growth regulators. Three entire plantlets were formed, but only one was e s t a b l i s h e d in soil. summary.

Abbreviations. dich 1 orophenoxyacet

2 , 4-D,

acid ; NAA, naphthaleneacetic acid; ABA, a b s c i s i c CRAF III, chromium trioxide-acetic formalin. ic

2 , 4-

lacid; acid-

Introduction

The g e n u s C l a d r a s t i s (Fabaceae) encompasses only 4 species; 3 are n a t i v e to s o u t h e a s t e r n Asia and not c o m m o n l y p l a n t e d in the U n i t e d States. The n a t u r a l r a n g e of the fourth species, C. lutea (Michx.) K. Koch (yellowwood), is l i m i t e d to p o r t i o n s of the southcentral and s o u t h e a s t e r n U n i t e d States (see Harrar, 1971 for details). Y e l l o w w o o d is a i0 to 15 m e t e r o r n a m e n t a l tree and u t i l i z e d either as a s i n g l e specimen or in a v e n u e plantings. The tree b e a r s n u m e r o u s h a n g i n g p a n i c l e s of w h i t e or v e r y r a r e l y p i n k p e a - l i k e flowers in late A p r i l f o l l o w e d by light b r o w n pods in July. Y e l l o w w o o d is d i f f i c u l t to p r o p a g a t e by conventional vegetative propagation methods such as r o o t i n g c u t t i n g s and g r a f t i n g and is usually commercially produced from seed.

Offprint requests to: R. N. Trigiano

However, seed s u p p l i e s may be limited b e c a u s e individual trees usually flower only in a l t e r n a t e years or every t h i r d y e a r (Dirr, 1975). In vitro culture techniques potentially offer alternative forms of production, but d e s p i t e recent a d v a n c e s in the tissue c u l t u r e of w o o d y legumes, p r o p a g a t i o n of y e l l o w w o o d has not been reported. Here, we d e s c r i b e somatic e m b r y o g e n e s i s from immature zygotic embryos and subsequent plant r e g e n e r a t i o n of C. !utea. Materials

and methods

D e v e l o p i n g seed pods w e r e c o l l e c t e d from 4, 25-30 y e a r old y e l l o w w o o d t r e e s l o c a t e d on the University of Tennessee Agricultural campus in Knoxville, TN once a w e e k b e g i n n i n g 4 w e e k s p o s t - a n t h e s i s (May 30, 1988) until i0 weeks p o s t - a n t h e s i s (July 15, 1988). Fruits were surface sterilized in 50% (v/v) commercial bleach (2.62% NaOCl) containing 0.1% (w/v) T r i t o n X-100 for 5 min and then rinsed 3 t i m e s in sterile d i s t i l l e d water. A l t h o u g h fruits t y p i c a l l y c o n t a i n e d 5-8 seeds, many h a d failed to d e v e l o p and only 1-3 w e r e suitable for culture. Immature zygotic e m b r y o s w e r e e x c i s e d from seeds and c o t y l e d o n s detached from hypocotyl-radicle axes. D i s s e c t e d e m b r y o s w e r e p l a c e d in 60 m m petri d i s h e s c o n t a i n i n g S c h e n k and H i l d e b r a n d t (SH) (1972) basal m e d i u m s u p p l e m e n t e d w i t h 5 ~M (NH4)2SO4, 15 #M thiamine, 0 . 5 m M m y o - i n o s i t o l , 88 m M s u c r o s e and 8 g / l i t e r Phytagar. Initial t r e a t m e n t s c o n s i s t e d of e i t h e r 4.5, 9.0, 13.5 or 23.0 #M 2,4-D. The pH of all m e d i a was adjusted to 5.7 with 0.i N KOH before sterilization. All the zygotic e m b r y o s from an i n d i v i d u a l pod w e r e c u l t u r e d in the same dish and considered a replication. Nine r e p l i c a t i o n s or d i s h e s for each of the initial 4 treatments were cultured/tree each week. C u l t u r e s w e r e i n c u b a t e d at 25C with 70 # m o l e sec -I m -2 of light provided by cool w h i t e f l u o r e s c e n t t u b e s for 16 hr/day. A f t e r 2 and 3 weeks, 3 c u l t u r e s from each tree and for each 2 , 4 - D t r e a t m e n t were t r a n s f e r r e d to SH basal m e d i u m a m e n d e d w i t h 25.0 #M NAA. The r e m a i n i n g 3 c u l t u r e s of each of the 4 trees were not t r a n s f e r r e d and r e m a i n e d on the initial 2,4-D treatments for 6 weeks. Cultures were monitored weekly for s o m a t i c embryo development and after 6 w e ek s the

184 n u m b e r of e x p l a n t s p r o d u c i n g somatic embryos and the n u m b e r of embryos per explant w e r e recorded. A l t h o u g h the i d e n t i t y of e x p l a n t s from individual trees was maintained, the data were p o o l e d for p r e s e n t a t i o n . A f t e r 9 weeks of culture, r e s u l t i n g calli were t r a n s f e r r e d to SH m e d i u m lacking g r o w t h regulators. Thereafter, cultures were t r a n s f e r r e d at 4 w e e k intervals a l t e r n a t i n g b e t w e e n SH m e d i u m c o n t a i n i n g 9.0 ~M 2,4-D and SH basal m e d i u m w i t h o u t g r o w t h regulators. All c u l t u r e s were i n c u b a t e d u n d e r the same c o n d i t i o n s as d e s c r i b e d previously. The e x p e r i m e n t s w e r e r e p e a t e d the next year with some m o d i f i c a t i o n s . The n u m b e r of pods from the same 4 trees was g r e a t l y reduced and sample c o l l e c t i o n was r e s t r i c t e d from 6 weeks (June 23, 1989) to i0 w e e k s posta n t h e s i s (July 21, 1989). Also, one of the o r i g i n a l 4 trees did not set fruit and was replaced with another tree. Initial t r e a t m e n t s c o n s i s t e d of e i t h e r 9.0, 13.5 or 23.0 ~M 2,4-D. O n e - h a l f of the e x p l a n t s from each t r e a t m e n t w e r e i n c u b a t e d for 6 weeks on

the 2,4-D containing media; whereas, the r e m a i n i n g c u l t u r e s were t r a n s f e r r e d after 3 weeks to SH basal m e d i u m s u p p l e m e n t e d w i t h 4.0 #M filter s t e r i l i z e d ABA. s i m i l a r p l a t i n g and c u l t u r i n g t e c h n i q u e s d e s c r i b e d p r e v i o u s l y were followed. Data from individual trees w e r e p o o l e d for p r e s e n t a t i o n . Embrvoconversion. Twenty-three cotyledonary-stage s o m a t i c embryos were removed from e x p l a n t s and t r a n s f e r r e d to h a l f - s t r e n g t h SH basal m e d i u m w i t h o u t g r o w t h r e g u l a t o r s and g e l l e d w i t h 6.0 g / l i t e r P h y t a g a r c o n t a i n e d in 60 mm p l a s t i c petri plates. Germinated embryos were t r a n s f e r r e d to the same m e d i u m in GA7 boxes (Magenta Corp., Chicago) a f t e r the p l a n t l e t s had at least one true leaf and then e s t a b l i s h e d in s o i l - l e s s p o t t i n g m e d i u m (Fafard's Pro-Mix, Anderson, SC) in GA7 boxes. Beginning 1 week after transfer to the s o i l l e s s medium, the GA7 box cover was r e m o v e d for i n c r e a s i n g p e r i o d s of time over 2 weeks. Histologicalstudies. S o m a t i c embryos a n d / o r e x p l a n t t i s s u e s were e i t h e r i m m o b i l i z e d in 1% a g a r o s e

T a b l e i. E f f e c t of e x p l a n t date, time on i n d u c t i o n m e d i u m and 2,4-D c o n c e n t r a t i o n on somatic e m b r y o g e n e s i s from i m m a t u r e zygotic e m b r y o s of C l a d r a s t i s lutea after six w e e k s in c u l t u r e (1988). Weeks post anthesis

W e e k s on induction medium a

3 6

4(5.0) b 0(0) 0(0)

0(0) 0(0) 0(0)

2 3 6

3(1.0) 0(0) 4(1.0)

6(2.0) 3(1.0) 3(2.0)

0(0) 11(2.5) 0(0)

3(3.0) 3(3.0) 3(2.0)

2 3 6

11(2.5) 6(1.5) 6(1.0)

17(2.0) 4(1.0) 0(0)

0(0) 3(4.0) 3(i.0)

7(3.0) 0(0) 5(2.0)

2 3 6

0(0) 20(1.5) 8(2.0)

21(1.0) 9(1.0) 16(2.2)

4(2.0) 24(2.4) 9(3.5)

17(1.0) 25(1.4) 28(2.4)

2 3 6

7(1.0) 29(2.0) 0(0)

13(1.5) 46(2.6) 37(2.2)

22(1.8) 13(1.0) 17(2.0)

24(1.2) 35(2.2) 32(3.3)

2 3 6

0(0) 16(2.5) 20(4.0)

7(1.0) 9(2.0) !3(4.5)

20(2.0) 0(0) 0(0)

6(1.0) 6(1.0) 0(0)

2 3 6

0(0) 0(0) 0(0)

0(0) 0(0) 0(0)

0(0) 0(0) 0(0)

2

i0

Induction medium 2,4-D c o n c e n t r a t i o n (~M) 4.5 9.0 13.5 23.0

0(0) 0(0) 0(0)

0(0) 0(0) 0(0)

0(0) 0(0) 0(0)

a t r a n s f e r r e d to SH m e d i u m c o n t a i n i n g 25~M NAA a f t e r the n u m b e r of w e e k s indicated; 6 w e e k s = no transfer. b First n u m b e r equals the p e r c e n t of e x p l a n t s that p r o d u c e d s o m a t i c embryos; n u m b e r in p a r e n t h e s e s equals the m e a n n u m b e r of somatic embryos p r o d u c e d per e x p l a n t that responded.

Figures 1 and 2. S o m a t i c embryos of C l a d r a s t i s lutea, i. Callus (CA) and a pair of somatic e m b r y o s (arrows) formed d i r e c t l y from the base of an e x c i s e d zygotic e m b r y o c o t y l e d o n (ZC). Bar = 1.5 mm. 2. Longitudinal section through the pair of somatic e m b r y o s (arrows) shown in Figure i. N o t e the m a l f o r m e d c o t y l e d o n s (C). Bar = 0.6 mm.

185 (Trigiano et al., 1987) first or d i r e c t l y fixed in CRAF III (Sass, 1958) for histological examination. Embryos were aspirated, d e h y d r a t e d in a g r a d e d series of ethanol: t - b u t a n o l and e m b e d d e d in P a r a p l a s t Plus (Sherwood Medical, St. Louis). Ten ~mt h i c k s e c t i o n s w e r e cut on a rotary microtome, and stained w i t h safranin, crystal v i o l e t and fast g r e e n (Johansen, 1940). Results

and D i s c u s s i o n

D e v e l o p m e n t of somatic embryos was g e n e r a l l y limited to c u l t u r e s i n i t i a t e d from 5-9 or 6-9 w e e k s p o s t - a n t h e s i s zygotic embryos for the first (Table i) and second year (Table 2), respectively. V e r y few somatic embryos formed from the 4 weeks post-anthesis zygotic embryos; only one explant p r o d u c e d 5 embryos. The d e v e l o p m e n t a l / p h y s i o l o g i c a l stage of the explant s t r o n g l y i n f l u e n c e d the ability of an e x p l a n t to r e s p o n d m o r p h o g e n e t i c a l l y (Williams and Maheswaran, 1986). Somatic e m b r y o g e n e s i s in two other w o o d y legumes, Cercis c a n a d e n s i s L. (redbud) (Trigiano et al., 1988; Geneve and Kester, 1990) and R o b i n i a p s e u d o a c a c i a L.

(black locust) (Merkle and Wiecko, 1989) were only i n i t i a t e d from zygotic embryos w i t h i n discrete time intervals of development. E x p l a n t s h a r v e s t e d prior to this d e v e l o p m e n t a l time p e r i o d p r o d u c e d little callus and u s u a l l y became n e c r o t i c after several weeks of culture regardless of the m e d i u m used. Zygotic embryos h a r v e s t e d from i0 weeks p o s t - a n t h e s i s ovules did not form somatic embryos; however, immature cotyledons expanded and formed copious amounts of y e l l o w friable callus and o c c a s i o n a l l y roots. These cultures have been m a i n t a i n e d for over a year, but have never p r o d u c e d somatic embryos. Somatic embryos formed only from c o t y l e d o n s that were d e t a c h e d from h y p o c o t y l root axes and h y p o c o t y l explants only p r o d u c e d small amounts of callus. Embryogenesis limited to s p e c i f i c organs has been r e p o r t e d for other w o o d y species and the r e s p o n s e of y e l l o w w o o d appears to be similar to Cornus florida L. (Trigiano et al., 1989). However with other leguminous trees, either m a t u r e zygotic embryos or seedling h y p o c o t y l explants were capable of p r o d u c i n g somatic embryos (Gharyal and Maheshwari, 1981; Skolmen, 1986).

T a b l e 2. E f f e c t s of explant date, 2,4-D and a b s c i s i c acid on somatic e m b r y o g e n e s i s from immature zygotic embryos of C l a d r a s t i s lutea after six w e e k s in c u l t u r e (1989).

Weeks p o s t anthesis

ABA a

Induction medium 2,4-D c o n c e n t r a t i o n ~ M } 13.5 23.0 9.0

+

9(7.0

-

lO(1.o

8(1.0) 11(3.0)

20(1.5) 11(3.5)

+

10(2.0

14(3.0)

i0(2.0)

-

8(1.0

8

+ -

o(o.o 9(2.0)

9

+

0(0.0)

0(0.0)

0(0.0)

-

5(1.0)

4(1.0)

4(1.0)

+

0(0.0)

0(0.0)

0(0.0)

-

0(0.0)

0(0.0)

6(2.0)

6

7

10

5(4.0) 0(0.0) 12(2.6)

13(3.3) 8(2.0) 15(3.6)

a + equals c u l t u r e s t r a n s f e r r e d to SH m e d i u m c o n t a i n i n g 4 ~M ABA after 3 weeks; - equals c u l t u r e s that r e m a i n e d on SH m e d i u m c o n t a i n i n g 2,4-D for 6 weeks. b First n u m b e r equals the p e r c e n t a g e of e x p l a n t s that p r o d u c e d somatic embryos; n u m b e r in p a r e n t h e s i s equals the m e a n n u m b e r of somatic embryos p r o d u c e d per explant that responded.

Figures 3 and 4. Somatic embryos of C l a d r a s t i s lutea. 3. A single somatic embryo with fully e x p a n d e d c o t y l e d o n s formed from the base of an excised zygotic embryo c o t y l e d o n (ZC). CA = Callus; Bar = 1.0 mm. 4. Median longitudinal section t h r o u g h the s o m a t i c embryo shown in Figure 3. The v a s c u l a r tissue (V) and cotyledons are welldeveloped. CA= callus; Bar = 1.3 mm.

186 Somatic embryos were formed asynchronously and various stages of development, including globular, cordate and cotyledonary, were observed after 6 weeks of culture. The gross developmental sequence of the somatic embryos was assumed to be similar to their zygotic counterparts, although abnormalities were noted. Most of the embryos formed as fused clusters from the proximal ends of zygotic cotyledons and were morphologically and anatomically abnormal, often lacking one of the apices and having fasciated cotyledons. However, in some cases, two or more somatic embryos developed singly (Figures 1 and 2) ; formation of a single embryo with well-developed cotyledons was rare (Figure 3) . Histological evaluation demonstrated that the embryos were formed directly (Figure 4). Anatomically, the somatic embryos were bipolar, possessed procambium or vascular tissue (Figures 2 and 4) and were not connected to the explant by vascular tissue (Figure 4). All treatments used in both experiments supported somatic embryogenesis (Tables 1 and 2). The greatest number of somatic embryos for all 5 trees occurred from 6-8 weeks postanthesis zygotic embryos. Approximately onehalf of the zygotic embryos initially cultured on 9 ~M 2,4-D from 8 weeks post-anthesis explants and transferred after 3 weeks to medium containing 25 ~M NAA produced somatic embryos (Table i). Cultures which remained on the 2,4-D induction medium for the entire 6 weeks also produced somatic embryos, but at a lower frequency and number. Although the rate and frequency of embryogenesis were greater in cultures transferred to NAA containing medium, these treatments had little effect on embryo ontogeny. Most embryos, regardless of the growth regulators used had abnormal morphology compared to the zygotic embryo. In an attempt to normalize ontogeny, zygotic embryos explanted during 1989 were transferred to SH medium containing 4.0 ~M ABA after initial culture on 2,4-D medium (cf. Ammirato, 1977). ABA did not affect ontogeny and somatic embryos were induced by all treatments (Table 2). The ability to produce somatic embryos has been reported to be genotype specific for some leguminous species (Oelck and Schieder, 1983) . Immature zygotic embryos from all 5 yellowwood trees used in this study were capable of forming somatic embryos, although at slightly different rates (data not shown). The origin of these trees is unknown, but based on commercial production methods, they probably are of heterozygous seed origin. Therefore, the ability to produce somatic embryos does not appear to be genotype dependent in yellowwood; however, individual trees may have different embyogenic capacities. Twenty-one embryos with distinctive cotyledons similar to the one shown in Figure 3 were placed on germination medium and radicles emerged from six of them within 2 weeks. Three embryos formed both shoots and roots and were transferred to potting medium 3 weeks later. Only 1 of these plantlets was successfully established in potting medium and was planted in the field 9 months after embryo germination. Lack of conversion of somatic embryos to plantlets may be due to physiological immaturity or anatomical

abnormalities, especially at the shoot apex (Ammirato, 1987). The lack of conversion of C. canadenesis somatic embryos was attributed to abnormal shoot apex development (Trigiano et al., 1988). The type of auxin used and duration that the explant is exposed to the growth regulator can affect not only the initiation of somatic embryos, but embryo development. A greater percentage of morphologically normal somatic embryos were formed from Glycine max L. (soybean) cotyledons using NAA instead of 2,4-D (Lazzeri et al., 1987). A greater number of morphologically normal somatic embryos of C. canadensis were formed using pulse treatments compared to a long exposure to 2,4-D (Geneve and Kester, 1990) and somatic embryos of R. pseudoacacia which were formed on explants exposed to 2,4-D for a week differentiated normally and were capable of conversion. This research indicates that yellowwood can be regenerated from somatic embryos. Somatic embryos were formed directly from tissue at the base of immature cotyledons and this mode of embryogenesis appears to be most similar to that described for C. canadenesis and R. pseudoacacia. Future research will focus on increasing the frequency of morphologically normal embryos formed and converting embryos to plants.

References

Ammirato, PV. (1977). Plant Physiol. 59:579586. Ammirato, PV. (1987) In: Plant Tissue and Cell Culture. (Green, CD, Somers, DA, Hackett, W P and Biesboer, DD, eds.). AR Liss, Inc., p. 57-81. Dirr, MA. (1975) Manual of Woody Landscape Plants, 3rd edition. Stipes Publishing, Chicago. p. 181-182. Geneve, RL and ST Kester. (1990) Plant Cell Tissue Organ Culture 22:71-76. Gharyal, PK and Maheshwari, SC. (1981) Naturwissenschaften 68:379-380. Harrar, EC. (1971). In: Hugh's Encyclopedia of American Woods. Speller and Sons, New York. p. 95-100. Johansen, DA. (1940) Plant Microtechnique. McGraw Hill Co., New York. Merkle, SA and Weicko, AT. (1989) Can J. For. Res. 19:285-288. Lazzeri, PA, Hildebrand, DF and Collins, GB. (1987) Plant Cell Tissue Organ Culture 10:197-208. Oelck, MM and Schieder O. (1983) Z. Pflanzenzuchtg. 91:312-321. Sass, JE. (1958) Botanical Microtechnique, 3rd. Ed. Iowa State Univ. Press. Ames, Iowa. Schenk, RU and Hildebrandt, AC. (1972) Can. J. Bot. 50:199-204. Skolmen, RG. (1986) In: Biotechnology In Forestry and Agriculture. Vol. i. ( Bajaj, YPS, ed.) Springer-Verlag, Berlin. p. 375384. Trigiano, RN, Beaty, RM and Graham, ET. (1988) Plant Cell Rpt. 7:148-150. Trigiano, RN, Beaty, RM and Dietrich, JT. (1989) Plant Cell Rpt. 8:270-273. Trigiano, RN, Conger, BV and Songstad, DD. (1987) Plant Growth Regul. 6:133-146. Williams, EG and Maheswaran, G. (1986) Ann. Bot. 57:443-462.